WO2018222840A1 - Poly(amine-co-disulfide ester) nanoparticles and methods of use - Google Patents

Poly(amine-co-disulfide ester) nanoparticles and methods of use Download PDF

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WO2018222840A1
WO2018222840A1 PCT/US2018/035348 US2018035348W WO2018222840A1 WO 2018222840 A1 WO2018222840 A1 WO 2018222840A1 US 2018035348 W US2018035348 W US 2018035348W WO 2018222840 A1 WO2018222840 A1 WO 2018222840A1
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substituted
peg
polymer
unsubstituted
alkyl
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Zhaozhong Jiang
Jie Liu
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Yale University
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
    • C08G63/68—Polyesters containing atoms other than carbon, hydrogen and oxygen
    • C08G63/685—Polyesters containing atoms other than carbon, hydrogen and oxygen containing nitrogen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1641—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
    • C08G63/02—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds
    • C08G63/60—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds derived from the reaction of a mixture of hydroxy carboxylic acids, polycarboxylic acids and polyhydroxy compounds
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
    • C08G63/66—Polyesters containing oxygen in the form of ether groups
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
    • C08G63/68—Polyesters containing atoms other than carbon, hydrogen and oxygen
    • C08G63/688—Polyesters containing atoms other than carbon, hydrogen and oxygen containing sulfur

Definitions

  • This invention is generally in the field of stimuli-responsive polymeric nanoparticles, particularly dual pH-responsive and redox- responsive polymeric nanoparticles for delivering therapeutic, diagnostic, and/or prophylactic agents to diseased tissues, particularly those releasing enzymes and oxidants leading to pH changes.
  • Polymeric nanoparticles are important platforms for delivering cargo (e.g. therapeutic, diagnostic, and/or prophylactic agents) to subjects. They are known to protect these cargoes from premature biodegradation and to increase their accumulation in some diseased tissues via the enhanced permeability and retention (EPR) effect (Torchilin, et al, Adv Drug Deliv Rev. 2011, 63, 131-135; Davis, et al., Nat Rev Drug Discov. 2008, 7, 771- 782; Ullrich, et al, Chem Rev. 1999, 99, 3181-3198).
  • cargo e.g. therapeutic, diagnostic, and/or prophylactic agents
  • GSH glutathione
  • GSH concentration is approximately 1 to 10 mM in normal cells, but is at least four times higher in many tumor cells (Wu, et al, J Nutr. 2004, 134, 489- 492; Kuppusamy, et al, Cancer Res. 2002, 62, 307-312).
  • GSH concentration is only around 2 to 20 ⁇ (Wu, et al, J Nutr. 2004, 134, 489-492).
  • polymeric nanoparticles still exhibit problems, such as inefficient release of cargo under disease-relevant conditions, non-specific uptake by tissues, premature release of significant quantities of cargo, premature degradation during systemic circulation, and a combination thereof. There remains a need for the development of improved polymeric nanoparticles that circumvent these existing problems.
  • nanoparticles with efficient release of therapeutic, diagnostic, and/or prophylactic agents in acidic pH extracellular and intracellular environments, and low redox potential environments, wherein the polymeric nanoparticles possess dual pH-responsive and redox-responsive properties.
  • Polymers containing one or more poly(amine-co-disulfide ester) polymeric units, and polymeric nanoparticles formed therefrom, have been developed which are stable under physiological conditions (such as pH 7.4), but have dual pH-responsive, and redox-responsive properties, as shown by changes in the sizes of the polymeric nanoparticles in environments having pH less than 7 and/or low redox potentials. These properties give rise to enhanced release of cargo from the nanoparticles, in diseased tissues whose microenvironments have pH less than 7 and/or low redox potential, such as tumors.
  • the nanoparticles also show selective uptake by diseased tissues, compared to healthy tissues.
  • the nanoparticles have a diameter between 50 nm and 500 nm, inclusive. In some forms, the zeta potential of the nanoparticle is between -10 mV and +10 mV, inclusive.
  • the polymers include a polymeric unit that can be represented by the general formula: wherein:
  • A, B, C, and D independently comprise monomeric units derived from a lactones, a polyfunctional molecule containing an amine group and a hydroxyl group, a molecule containing a disulfide bond, or hydrophilic polymer; the monomeric units comprise the lactone, the polyfunctional molecule containing an amine group and a hydroxyl group, and the molecule containing the disulfide bond;
  • a, b, c, and d are independently integers between 0 and 1000, inclusive, with the proviso that the sum (a + b + c + d) is greater than one;
  • h is an integer between 1 and 1000, inclusive.
  • the polymeric unit has the formula:
  • x, y, and z are independently integers between 1 and 1000, inclusive; preferably, m is 3 or 12, or a combination thereof;
  • p, q, n, and t are 2;
  • R 8 and R 10 are O;
  • the polymers can further include a block of a hydrophilic polyalkylene oxide, such as polyethylene glycol.
  • the molecular weight of the polymers can be between 1 kDa and 50 kDa, preferably between 5 kDa and 15 kDa.
  • the polymers are synthesized by mixing reactants such as a lactone, a molecule containing a disulfide bond, and a polyfunctional molecule containing an amine group, a hydroxyl group, preferably both, with an enzyme catalyst, such as a lipase, under conditions in which the polymers are formed.
  • the reactants can further include a hydrophilic polymer, such as polyalkylene oxide (e.g. polyethylene glycol).
  • compositions including nanoparticles having an effective amount of a therapeutic agent are also provided, which can be used, for example, for in vitro and in vivo delivery of the therapeutic agents.
  • a therapeutic agent e.g. chemotherapeutic agent
  • Figures 1A and IB are NMR spectral data of carbonyl C-13 resonance absorptions of different diads in polyethylene glycol (2 kDa)- poly( ⁇ -pentadecalactone (70%)-co-N-methyldiethyleneamine-co-3,3'- dithioproprionate) (PEG2K-PPMD-70% PDL) ( Figure 1A), and polyethylene glycol (2 kDa)-poly( ⁇ -caprolactone (70%)-co-N- methyldiethyleneamine-co-3,3'-dithioproprionate) (PEG2K-PCMD-70% CL) ( Figure IB).
  • the solvent in each case was CDCI 3 .
  • Figures 2A and 2B are line graphs of the intensity ratios (I3/I1) as a function of the logarithm of polymer concentration (Log C) for PEG-PPMD ( Figure 2A) and PEG-PCMD ( Figure 2B) copolymers.
  • Figures 3A and 3B are column graphs of the nanoparticle size distributions of docetaxel (DTX)-loaded nanoparticles containing PEG- PPMD-12% PDL ( Figure 3A) and PEG-PCMD-13% CL ( Figure 3B).
  • the scale bar 100 nm.
  • the sizes were determined using transmission electron microscopy (TEM).
  • Figures 5A-5F are line graphs of the size distributions of blank nanoparticles incubated with different PBS media for 48 h: PEG-PPMD-12% PDL (Figure 5A); PEG-PPMD-43% PDL ( Figure 5B); PEG-PPMD-70% PDL (Figure 5C); PEG-PCMD-13% CL ( Figure 5D); PEG-PCMD-41% CL ( Figure 5E); and PEG-PCMD-70% CL ( Figure 5F).
  • the sizes of the micelles were determined using dynamic light scattering (DLS).
  • Figures 6A-6F are line graphs of the in vitro drug release from DTX- loaded micelles of the following polymers incubated in PBS under different pH and redox conditions: PEG-PPMD-12% PDL (Figure 6A); PEG-PPMD- 43% PDL ( Figure 6B); PEG-PPMD-70% PDL ( Figure 6C); PEG-PCMD- 13% CL ( Figure 6D) ; PEG-PCMD-41 % CL ( Figure 6E) ; and PEG-PCMD- 70% CL ( Figure 6F).
  • FIG 7 is a column graph showing uptake of free coumarin-6 (C6) and C6-loaded PEG-PPMD and PEG-PCMD micelles by HeLa cells.
  • C6 free coumarin-6
  • MFI mean fluorescence intensity
  • Figures 12A and 12B are line graphs showing the measured tumor volumes (Figure 12A) and body weights (Figure 12B) of Balb/C mice treated with free DTX (Duopafei ® ), DTX-loaded PEG-PPMD- 12% PDL and PEG-PCMD-13% CL micelle formulations at 4 x 10 mg/kg DTX dose for 21 days. 0.9% NaCl solution was used as a control. Arrows indicate the dates when the formulations were administered.
  • Figure 13 is a column graph showing in vivo distribution of DiR- loaded PEG-PPMD- 12% PDL and PEG-PCMD-13% CL micelles injected intravenously through the tail vein of mice.
  • Figure 14 is an illustration of some of the steps involving cargo (e.g. DTX) delivery by PEG-PPMD and PEG-PCMD nanoparticles to cells (e.g. cancer cells).
  • cargo e.g. DTX
  • PEG-PPMD and PEG-PCMD nanoparticles e.g. cancer cells.
  • lactone and "lactone unit” are used to describe define a chemical compound that includes a cyclic ester, or the open chain chemical structure that results from the cleavage of the ester bond in the cyclic ester.
  • lactone is used to describe the cyclic ester shown below, and the corresponding lactone-derived open chain structure;
  • n being an integer, such as between 1 and 30, inclusive.
  • the open chain structure is formed via methods known in the art, including but not limited to, solvolysis, such as hydrolysis, and enzymatic cleavage.
  • “Positively ionizable atom” refers to an atom that can be protonated under acidic conditions, resulting in the atom having a. positive formal charge.
  • An example of a positively ionizable atom is nitrogen.
  • Acidic diseased tissue refers to a tissue in a diseased state, which has a pH, particularly extracellular matrix pH, of less than 7.
  • Exemplary diseases include cancer, inflammation, stroke, arthritis, and ischemia.
  • amphiphilic refers to a property where a molecule has both a hydrophilic portion and a hydrophobic portion. Often, an amphiphilic compound has a hydrophilic portion covalently attached to a hydrophobic portion. In some forms, the hydrophilic portion is soluble in water, while the hydrophobic portion is insoluble in water. In addition, the hydrophilic and hydrophobic portions may have either a formal positive charge, or a formal negative charge. However, overall they will be either hydrophilic or
  • An amphiphilic compound can be an amphiphilic polymer, such that the hydrophilic portion can be a hydrophilic polymer, and the hydrophobic portion can be a hydrophobic polymer.
  • Hydrophilic refers to the property of having affinity for water.
  • hydrophilic polymers or hydrophilic polymer segments
  • hydrophilic polymer segments are polymers (or polymer segments) that are primarily soluble in aqueous solutions and/or have a tendency to absorb water.
  • Hydrophilicity can be quantified by measuring its partition coefficient between water (or a buffered aqueous solution) and a water-immiscible organic solvent, such as octanol, methylene chloride, or methyl i ⁇ ?ri-butyl ether.
  • Hydrophilic may also refer to a material that when applied to a surface, such as glass, forms a contact angle with water, which is less than the contact angle of water on a surface of glass without the material.
  • Hydrophobic refers to the property of lacking affinity for or repelling water. For example, the more hydrophobic a polymer (or polymer segment), the more that polymer (or polymer segment) tends to not dissolve in, not mix with, or not be wetted by water.
  • Hydrophobicity can be quantified by measuring its partition coefficient between water (or a buffered aqueous solution) and a water-immiscible organic solvent, such as octanol, methylene chloride, or methyl tert-butyl ether. If after equilibration a greater concentration of the compound is attained in the organic solvent than in water, the compound is considered hydrophobic. For example, if the organic solvent is octanol, then a positive log P value indicates that the compound is hydrophobic. "Hydrophobic" may also refer to a material that when applied to a surface, such as glass, forms a contact angle with water, which is greater than the contact angle of water on a surface of glass without the material. Hydrophilicity and hydrophobicity can also be quantitated in relative terms, such as, but not limited to, a spectrum of
  • hydrophilicity/hydrophobicity within a group of polymers or polymer segments.
  • hydrophobic polymer can be defined based on the polymer's relative hydrophobicity when compared to another, more hydrophilic polymer.
  • Nanoparticle generally refers to a particle having a diameter, such as an average diameter, greater than or equal to 10 nm and less than 1 micron.
  • the particles can have any shape. Nanoparticles having a spherical shape are generally referred to as “nanospheres.”
  • Mean particle size generally refers to the statistical mean particle size (diameter) of the particles in a population of particles.
  • the diameter of an essentially spherical particle may refer to the physical or hydrodynamic diameter.
  • the diameter of a non- spherical particle may refer preferentially to the hydrodynamic diameter.
  • the diameter of a non- spherical particle may refer to the largest linear distance between two points on the surface of the particle.
  • Mean particle size can be measured using methods known in the art, such as dynamic light scattering, and transmission electron microscopy.
  • Analog as relates to a given compound, refers to another compound that is structurally similar, functionally similar, or both, to the specified compound.
  • Structural similarity can be determined using any criterion known in the art, such as the Tanimoto coefficient that provides a quantitative measure of similarity between two compounds based on their molecular descriptors.
  • the molecular descriptors are 2D properties such as fingerprints, topological indices, and maximum common substructures, or 3D properties such as overall shape, and molecular fields. Tanimoto coefficients range between zero and one, inclusive, for dissimilar and identical pairs of molecules, respectively.
  • a compound can be considered an analog of a specified compound, if it has a Tanimoto coefficient with the specified compound between 0.5 and 1.0, inclusive, preferably between 0.7 and 1.0, inclusive, most preferably between 0.85 and 1.0, inclusive.
  • a compound is functionally similar to a specified, if it induces the same pharmacological effect, physiological effect, or both, as the specified compound.
  • "Analog” can also refer to a modification including, but not limited to, hydrolysis, reduction, or oxidation products, of the disclosed compounds. Hydrolysis, reduction, and oxidation reactions are known in the art.
  • inhibitor and reduction means to reduce or decrease in activity or expression. This can be a complete inhibition or reduction of activity or expression, or a partial inhibition or reduction. Inhibition or reduction can be compared to a control or to a standard level.
  • Inhibition can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%.
  • parenteral administration and “administered parenterally” are art-recognized terms, and include modes of administration other than enteral and topical administration, such as injections, and include without limitation intravenous, intramuscular, intrapleural, intravascular, intrapericardial, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradennal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion.
  • Small molecule generally refers to an organic molecule that is less than about 2000 g/mol in molecular weight, less than about 1500 g/mol, less than about 1000 g/mol, less than about 800 g/mol, or less than about 500 g/mol. In some forms, small molecules are non-polymeric and/or non- oligomeric.
  • the terms "subject,” “individual,” and “patient” refer to any individual who is the target of treatment using the disclosed compositions.
  • the subject can be a vertebrate, for example, a mammal.
  • the subject can be a human.
  • the subjects can be symptomatic or asymptomatic.
  • the term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered.
  • a subject can include a control subject or a test subject.
  • sustained release refers to release of a substance over an extended period of time in contrast to a bolus type administration in which the entire amount of the substance is made biologically available at one time.
  • Effective amount and “therapeutically effective amount,” used interchangeably, as applied to the nanoparticles, therapeutic agents, and pharmaceutical compositions described herein, mean the quantity necessary to render the desired therapeutic result.
  • an effective amount is a level effective to treat, cure, or alleviate the symptoms of a disease for which the composition and/or therapeutic agent, or pharmaceutical composition, is/are being administered.
  • Amounts effective for the particular therapeutic goal sought will depend upon a variety of factors including the disease being treated and its severity and/or stage of development/progression; the bioavailability and activity of the specific compound and/or antineoplastic, or pharmaceutical composition, used; the route or method of administration and introduction site on the subject; the rate of clearance of the specific composition and other pharmacokinetic properties; the duration of treatment; inoculation regimen; drugs used in combination or coincident with the specific composition; the age, body weight, sex, diet, physiology and general health of the subject being treated; and like factors well known to one of skill in the relevant scientific art. Some variation in dosage will necessarily occur depending upon the condition of the subject being treated, and the physician or other individual administering treatment will, in any event, determine the appropriate dosage for an individual patient.
  • Substituted refers to all permissible substituents of the compounds or functional groups described herein.
  • the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds.
  • Illustrative substituents include, but are not limited to, halogens, hydroxyl groups, or any other organic groupings containing any number of carbon atoms, preferably 1-14 carbon atoms, and optionally include one or more heteroatoms such as oxygen, sulfur, or nitrogen grouping in linear, branched, or cyclic structural formats.
  • substituents include alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio, phenylthio, substituted phenylthio, arylthio, substituted arylthio, cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl, polyaryl
  • Heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. It is understood that “substitution” or “substituted” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, i.e. a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.
  • Alkyl refers to the radical of saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl, cycloalkyl (alicyclic), alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl.
  • a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C 1 -C30 for straight chains, C3-C30 for branched chains), preferably 20 or fewer, more preferably 15 or fewer, most preferably 10 or fewer.
  • Alkyl includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, i-butyl, pentyl, hexyl, heptyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the like.
  • Preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure.
  • alkyl (or “lower alkyl) as used throughout the specification, examples, and claims is intended to include both “unsubstituted alkyls” and “substituted alkyls,” the latter of which refers to alkyl moieties having one or more substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone.
  • substituents include, but are not limited to, halogen, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, a phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamide, sulfonyl, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety.
  • carbonyl such as a carboxyl, alkoxycarbonyl, formyl, or an acyl
  • thiocarbonyl such as a thioester, a thi
  • lower alkyl as used herein means an alkyl group, as defined above, but having from one to ten carbons, more preferably from one to six carbon atoms in its backbone structure. Likewise, “lower alkenyl” and “lower alkynyl” have similar chain lengths. Throughout the application, preferred alkyl groups are lower alkyls. In preferred forms, a substituent designated herein as alkyl is a lower alkyl.
  • Alkyl includes one or more substitutions at one or more carbon atoms of the hydrocarbon radical as well as heteroalkyls. Suitable substituents include, but are not limited to, halogens, such as fluorine, chlorine, bromine, or iodine; hydroxyl; -NRR', wherein R and R' are independently hydrogen, alkyl, or aryl, and wherein the nitrogen atom is optionally quaternized; -SR, wherein R is hydrogen, alkyl, or aryl; -CN; - N0 2 ; -COOH; carboxylate; -COR, -COOR, or -CON(R) 2 , wherein R is hydrogen, alkyl, or aryl; azide, aralkyl, alkoxyl, imino, phosphonate, phosphinate, silyl, ether, sulfonyl, sulfonamido, heterocyclyl, aromatic or heteroaromatic moieties
  • the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate.
  • the substituents of a substituted alkyl may include halogen, hydroxy, nitro, thiols, amino, azido, imino, amido, phosphoryl (including phosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls (including ketones, aldehydes, carboxylates, and esters), haloalkyls, -CN and the like. Cycloalkyls can be substituted in the same manner.
  • Heteroalkyl refers to straight or branched chain, or cyclic carbon-containing radicals, or combinations thereof, containing at least one heteroatom. Suitable heteroatoms include, but are not limited to, O, N, Si, P and S, wherein the nitrogen, phosphorous and sulfur atoms are optionally oxidized, and the nitrogen heteroatom is optionally quaternized.
  • alkoxyl or “alkoxy,” “aroxy” or “aryloxy,” generally describe compounds represented by the formula -OR v , wherein R v includes, but is not limited to, substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, arylalkyl, heteroalkyls, alkylaryl, alkylheteroaryl.
  • alkoxyl or “alkoxy” as used herein refer to an alkyl group, as defined above, having an oxygen radical attached thereto.
  • alkoxyl groups include methoxy, ethoxy, propyloxy, tert- butoxy and the like.
  • An "ether" is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as can be represented by one of -O- alkyl, -O-alkenyl, and -O-alkynyl.
  • alkoxy also includes cycloalkyl, heterocyclyl, cycloalkenyl, heterocycloalkenyl, and arylalkyl having an oxygen radical attached to at least one of the carbon atoms, as valency permits.
  • a "lower alkoxy” group is an alkoxy group containing from one to six carbon atoms.
  • substituted alkoxy refers to an alkoxy group having one or more substituents replacing one or more hydrogen atoms on one or more carbons of the alkoxy backbone.
  • substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), silyl, ether, ester, thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino (or quarternized amino), amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sul
  • alkenyl as used herein is a hydrocarbon group of from 2 to 24 carbon atoms and structural formula containing at least one carbon- carbon double bond.
  • alkynyl group as used herein is a hydrocarbon group of 2 to 24 carbon atoms and a structural formula containing at least one carbon- carbon triple bond.
  • aryl as used herein is any C5-C26 carbon-based aromatic group, fused aromatic, fused heterocyclic, or biaromatic ring systems.
  • aryl includes 5-, 6-, 7-, 8-, 9-, 10-, 14-, 18-, and 24-membered single-ring aromatic groups, including, but not limited to, benzene, naphthalene, anthracene, phenanthrene, chrysene, pyrene, corannulene, coronene, etc.
  • Aryl further encompasses polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings (i.e., "fused rings") wherein at least one of the rings is aromatic, e.g., the other cyclic ring or rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or he terocycles.
  • the aryl group can be substituted with one or more groups including, but not limited to, alkyl, alkynyl, alkenyl, aryl, halide, nitro, amino, ester, ketone, aldehyde, hydroxy, carboxylic acid, or alkoxy.
  • substituted aryl refers to an aryl group, wherein one or more hydrogen atoms on one or more aromatic rings are substituted with one or more substituents including, but not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxy, carbonyl (such as a ketone, aldehyde, carboxyl, alkoxycarbonyl, formyl, or an acyl), silyl, ether, ester, thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino (or quarternized amino), amido, amidine, imine, cyano, nitro, azido, sulfhydryl, imino, alkylthio, sulfate,
  • Heterocycle refers to a cyclic radical attached via a ring carbon or nitrogen atom of a monocyclic or bicyclic ring containing 3- 10 ring atoms, and preferably from 5-6 ring atoms, consisting of carbon and one to four heteroatoms each selected from the group consisting of non-peroxide oxygen, sulfur, and N(Y) wherein Y is absent or is H, O, C 1 - C 10 alkyl, phenyl or benzyl, and optionally containing 1-3 double bonds and optionally substituted with one or more substituents.
  • Heterocyclyl are distinguished from heteroaryl by definition. Examples of heterocycles include, but are not limited to piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl,
  • heteroaryl refers to C5-C26-membered aromatic, fused aromatic, biaromatic ring systems, or combinations thereof, in which one or more carbon atoms on one or more aromatic ring structures have been substituted with a heteroatom.
  • Suitable heteroatoms include, but are not limited to, oxygen, sulfur, and nitrogen.
  • heteroaryl includes 5-, 6-, 7-, 8-, 9-, 10-, 14-, 18-, and 24-membered single-ring aromatic groups that may include from one to four heteroatoms, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, tetrazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.
  • the heteroaryl group may also be referred to as “aryl heterocycles" or "heteroaromatics”.
  • Heteroaryl further encompasses polycyclic ring systems having two or more rings in which two or more carbons are common to two adjoining rings (i.e. , “fused rings") wherein at least one of the rings is heteroaromatic, e.g., the other cyclic ring or rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heterocycles, or combinations thereof.
  • heteroaryl rings include, but are not limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2- dithiazinyl, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H- indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isat
  • substituted heteroaryl refers to a heteroaryl group in which one or more hydrogen atoms on one or more heteroaromatic rings are substituted with one or more substituents including, but not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxy, carbonyl (such as a ketone, aldehyde, carboxyl, alkoxycarbonyl, formyl, or an acyl), silyl, ether, ester, thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino (or quarternized amino), amido, amidine, imine, cyano, nitro, azidome, thiophene, phosphinate, amino (or quarternized amino), amido, amidine, im
  • CN aryl, heteroaryl, and combinations thereof.
  • substituted alkenyl refers to alkenyl moieties having one or more substituents replacing one or more hydrogen atoms on one or more carbons of the hydrocarbon backbone.
  • substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), silyl, ether, ester, thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino (or quarternized amino), amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate,
  • substituted alkynyl refers to alkynyl moieties having one or more substituents replacing one or more hydrogen atoms on one or more carbons of the hydrocarbon backbone.
  • substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), silyl, ether, ester, thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino (or quarternized amino), amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate
  • cycloalkyl as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms.
  • examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.
  • heterocycloalkyl group is a cycloalkyl group as defined above where at least one of the carbon atoms of the ring is substituted with a heteroatom such as, but not limited to, nitrogen, oxygen, sulphur, or phosphorus.
  • aralkyl as used herein is an aryl group having an alkyl, alkynyl, or alkenyl group as defined above attached to the aromatic group.
  • An example of an aralkyl group is a benzyl group.
  • hydroxyalkyl group as used herein is an alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, halogenated alkyl, or heterocycloalkyl group described above that has at least one hydrogen atom substituted with a hydroxyl group.
  • alkoxyalkyl group is defined as an alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, halogenated alkyl, or heterocycloalkyl group described above that has at least one hydrogen atom substituted with an alkoxy group described above.
  • Carbonyl as used herein, is art-recognized and includes such moieties as can be represented by the general formula:
  • R represents a hydrogen, a substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, -(CH 2 ) m -R", or a pharmaceutical acceptable salt
  • R' represents a hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl,
  • substituted carbonyl refers to a carbonyl, as defined above, wherein one or more hydrogen atoms in R, R' or a group to which the moiety
  • substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), silyl, ether, ester, thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino (or quarternized amino), amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamide, sulfonyl, heterocyclyl, alkylaryl, haloalkyl, -CN
  • Carboxyl is as defined above for the formula and is defined more specifically by the formula -R lv COOH, wherein R 1V is an alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, alkylaryl, arylalkyl, aryl, or heteroaryl.
  • a straight chain or branched chain alkyl, alkenyl, and alkynyl have 30 or fewer carbon atoms in its backbone (e.g., C 1 - C30 for straight chain alkyl, C3-C30 for branched chain alkyl, C2-C30 for straight chain alkenyl and alkynyl, C3-C30 for branched chain alkenyl and alkynyl), preferably 20 or fewer, more preferably 15 or fewer, most preferably 10 or fewer.
  • preferred cycloalkyls, heterocyclyls, aryls and heteroaryls have from 3-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure.
  • substituted carboxyl refers to a carboxyl, as defined above, wherein one or more hydrogen atoms in R 1V are substituted.
  • substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), silyl, ether, ester, thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino (or quarternized amino), amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl,
  • phenoxy is art recognized, and refers to a compound of the formula -OR v wherein R v is (i.e., -O-C6H5).
  • R v is (i.e., -O-C6H5).
  • a phenoxy is a species of the aroxy genus.
  • substituted phenoxy refers to a phenoxy group, as defined above, having one or more substituents replacing one or more hydrogen atoms on one or more carbons of the phenyl ring.
  • substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), silyl, ether, ester, thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino (or quarternized amino), amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sul
  • aromatic radical and aryloxy are represented by -O-aryl or -O-heteroaryl, wherein aryl and heteroaryl are as defined herein.
  • substituted aroxy and "substituted aryloxy,” as used interchangeably herein, represent -O-aryl or -O-heteroaryl, having one or more substituents replacing one or more hydrogen atoms on one or more ring atoms of the aryl and heteroaryl, as defined herein.
  • substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), silyl, ether, ester, thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino (or quarternized amino), amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamide, sulfonyl, heterocyclyl, alkylaryl, haloalkyl, -CN, aryl, heteroaryl
  • alkylthio refers to an alkyl group, as defined above, having a sulfur radical attached thereto.
  • the "alkylthio” moiety is represented by -S-alkyl.
  • Representative alkylthio groups include methylthio, ethylthio, and the like.
  • alkylthio also encompasses cycloalkyl groups having a sulfur radical attached thereto.
  • substituted alkylthio refers to an alkylthio group having one or more substituents replacing one or more hydrogen atoms on one or more carbon atoms of the alkylthio backbone.
  • substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), silyl, ether, ester, thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino (or quarternized amino), amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, s
  • substituted phenylthio refers to a phenylthio group, as defined above, having one or more substituents replacing a hydrogen on one or more carbons of the phenyl ring.
  • substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), silyl, ether, ester, thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino (or quarternized amino), amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate,
  • Arylthio refers to -S-aryl or -S-heteroaryl groups, wherein aryl and heteroaryl as defined herein.
  • substituted arylthio represents -S-aryl or -S-heteroaryl, having one or more substituents replacing a hydrogen atom on one or more ring atoms of the aryl and heteroaryl rings as defined herein.
  • substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), silyl, ether, ester, thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino (or quarternized amino), amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, alkylaryl, haloalkyl, -CN, aryl, hetero
  • amide or “amido” are used interchangeably, refer to both “unsubstituted amido” and “substituted amido” and are represented by the general formula:
  • E is absent, or E is substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aralkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclyl, wherein independently of E, R and R' each independently represent a hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbonyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl,
  • R and R' can be a carbonyl, e.g., R and R' together with the nitrogen do not form an imide.
  • R and R' each independently represent a hydrogen atom, substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, or -(CH2) m -R' ' ' .
  • E oxygen
  • a carbamate is formed. The carbamate cannot be attached to another chemical species, such as to form an oxygen-oxygen bond, or other unstable bonds, as understood by one of ordinary skill in the art.
  • E is absent, or E is alkyl, alkenyl, alkynyl, aralkyl, alkylaryl, cycloalkyl, aryl, heteroaryl, heterocyclyl, wherein independently of E, R represents a hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted amine, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, -(CH 2 ) m -R' " , or E and R taken together with the S atom to which they are attached complete a heterocycle having from 3 to 14 atoms in the
  • substituted sulfonyl represents a sulfonyl in which E, R, or both, are independently substituted.
  • substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), silyl, ether, ester, thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino (or quarternized amino), amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfamoyl
  • sulfonic acid refers to a sulfonyl, as defined above, wherein R is hydroxyl, and E is absent, or E is substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
  • sulfate refers to a sulfonyl, as defined above, wherein E is absent, oxygen, alkoxy, aroxy, substituted alkoxy or substituted aroxy, as defined above, and R is independently hydroxyl, alkoxy, aroxy, substituted alkoxy or substituted aroxy, as defined above.
  • E oxygen
  • the sulfate cannot be attached to another chemical species, such as to form an oxygen- oxygen bond, or other unstable bonds, as understood by one of ordinary skill in the art.
  • sulfonate refers to a sulfonyl, as defined above, wherein E is oxygen, alkoxy, aroxy, substituted alkoxy or substituted aroxy, as defined above, and R is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted amine, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, -(CH 2 ) m -R" ⁇ R' " represents a hydroxy group, substituted or unsubstituted carbonyl group, an aryl, a cycloalkyl ring
  • sulfamoyl refers to a sulfonamide or sulfonamide represented by the formula
  • E is absent, or E is substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aralkyl, substituted or unsubstituted alkylaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclyl, wherein independently of E, R and R' each independently represent a hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbonyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted alkylaryl, substituted or
  • E is absent, or E is substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aralkyl, substituted or unsubstituted alkylaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclyl, wherein, independently of E, R vi and R vii are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbonyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted alkylaryl, substituted
  • substituted phosphonyl represents a phosphonyl in which
  • R vi and R vii are independently substituted.
  • substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), silyl, ether, ester, thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino (or quartemized amino), amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamide, sulfonyl, heterocyclyl, alkylaryl, halo
  • phosphoryl defines a phosphonyl in which E is absent, oxygen, alkoxy, aroxy, substituted alkoxy or substituted aroxy, as defined above, and independently of E, R vi and R vii are independently hydroxyl, alkoxy, aroxy, substituted alkoxy or substituted aroxy, as defined above.
  • E oxygen
  • the phosphoryl cannot be attached to another chemical species, such as to form an oxygen-oxygen bond, or other unstable bonds, as understood by one of ordinary skill in the art.
  • the substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), silyl, ether, ester, thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino (or quartemized amino), amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamide, sulfonyl, heterocyclyl, alkylaryl, halo
  • polyaryl refers to a chemical moiety that includes two or more aryls, heteroaryls, and combinations thereof.
  • the aryls, heteroaryls, and combinations thereof, are fused, or linked via a single bond, ether, ester, carbonyl, amide, sulfonyl, sulfonamide, alkyl, azo, and combinations thereof.
  • the chemical moiety can be referred to as a "polyheteroaryl.”
  • substituted polyaryl refers to a polyaryl in which one or more of the aryls, heteroaryls are substituted, with one or more substituents including, but not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), silyl, ether, ester, thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino (or quartemized amino), amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl
  • C3-C20 cyclic refers to a substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or
  • cyclic structures are formed from single or fused ring systems.
  • substituted cycloalkyls, cycloalkenyls, cycloalkynyls and heterocyclyls are substituted as defined above for the alkyls, alkenyls, alkynyls and
  • ether as used herein is represented by the formula AOA 1 , where A and A 1 can be, independently, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.
  • R and R' can be, independently, hydrogen, an alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, halogenated alkyl, or heterocycloalkyl group described above.
  • R, R', and R" can be, independently, hydrogen, an alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, halogenated alkyl, alkoxy, or heterocycloalkyl group described above.
  • hydroxyl and “hydroxy” are used interchangeably and are represented by -OH.
  • thiol and “sulfhydryl” are used interchangeably and are represented by -SH.
  • cyano and "nitrile” are used interchangeably to refer to -CN.
  • nitro refers to -N0 2 .
  • phosphate refers to -O-PO3.
  • Polymers containing one or more poly(amine-co-disulfide ester) polymeric units, and polymeric nanoparticles formed therefrom, are provided.
  • the nanoparticles are stable under physiological conditions (such as pH 7.4), but have dual pH-responsive, and redox-responsive properties, as shown by changes in the sizes of the polymeric nanoparticles in
  • the polymeric nanoparticles swell under low pH conditions and show enhanced degradation under reducing conditions (such as intracellular reduction potential).
  • the swelling of the polymeric nanoparticles and enhanced degradation in environments having pH less than 7 and/or low redox potentials give rise to enhanced release of cargo in diseased tissues whose microenvironments display these conditions, such as tumors.
  • the stability, dual pH-responsive and redox-responsive properties of the polymeric nanoparticles are as a result of chemical functionalities that are included in the polymers that are included the nanoparticles.
  • nanoparticles also provide for a sustained release of therapeutic, diagnostic, or prophylactic agent.
  • the therapeutic agent is a chemotherapeutic agent, i.e. , an anti-cancer agent, such as docetaxel.
  • the nanoparticles also show selective uptake by diseased tissues, compared to healthy tissues.
  • nanoparticles refers to polymeric nanoparticles, i.e. , nanoparticles formed from a population of one or more of the polymers described herein.
  • the polymers forming the nanoparticles include at least a lactone unit, a polyfunctional molecule containing an amine group, a hydroxyl group, preferably both, and a molecule containing a disulfide bond.
  • the distributions of the units within the polymer can be ordered or random.
  • the polyfunctional molecule contains one or more cations, one or more positively ionizable atoms, or combinations thereof. The one or more cations are formed from the protonation of a basic nitrogen atom, or from quaternary nitrogen atoms.
  • Protonation of the amine group at low pH causes the nanoparticles to swell due to increased electrostatic charge-charge repulsions between the protonated amine groups.
  • the ester bond facilitates degradation of the nanoparticles, via enzymatic degradation, hydrolysis, or a combination thereof.
  • the disulfide bond confers stability to the nanoparticles during circulation at physiological pH (e.g. 7.4), but enhances the degradation of the nanoparticles in low redox potential environments, due to reductive cleavage of the disulfide bonds upon exposure to a reductant, such as glutathione.
  • the polyfunctional molecule containing the amine group and the molecule containing the disulfide bond are
  • the polyfunctional molecule containing the amine group and the molecule containing the disulfide bond are in the main chain of the polymer.
  • the polymers are amphiphilic.
  • the polymers can include a block of a hydrophilic polymer, such as polyethylene glycol.
  • the hydrophilic block can be located at one terminus of the polymer, i.e. , a diblock copolymer, or it can be located at both termini of the polymer, i. e. , a triblock copolymer.
  • the hydrophilic polymer constitutes between about 30 wt/wt% and about 50 wt/wt% of the polymer, preferably between about 35 wt/wt% and about 45 wt/wt% of the polymer.
  • the weight average molecular weight (Mw) of the polymers can be between 1 kDa and 50 kDa, inclusive, preferably between 1 kDa and 30 kDa, inclusive, most preferably between 5 kDa and 15 kDa, inclusive. In some forms, Mw is about 8.1 kDa, 8.3 kDa, 8.7 kDa, 9.4 kDa, 11 kDa, 11.3 kDa, 12.5 kDa, 13.3 kDa, or 15.2 kDa. Mw can be measured using any means in the art, such as by gel permeation chromatography (GPC).
  • GPC gel permeation chromatography
  • the polymers have the general formula:
  • A, B, C, and D independently include monomeric units derived from lactones (e.g. ⁇ -pentadecalactone, ⁇ -caprolactone), a polyfunctional molecule containing an amine group, a hydroxyl group, preferably both (e.g. N-methyldiethanolamine), a molecule containing a disulfide bond (e.g. dimethyl 3,3'-dithiodipropionate), or hydrophilic polymer, such as a hydrophilic polyalkylene oxide (e.g. polyethylene glycol).
  • the polymers include at least a lactone; a polyfunctional molecule containing an amine group, a hydroxyl group, preferably both; and a molecule containing a disulfide bond.
  • the polyfunctional molecule contains one or more cations, one or more positively ionizable atoms, or combinations thereof.
  • the polyfunctional molecule containing the amine group and the hydroxyl group as well as the molecule containing the disulfide bond are in the main chain of the polymer.
  • a, b, c, and d are independently integers between 0 and 1000, inclusive, with the proviso that the sum (a + b + c + d) is greater than one.
  • h is an integer between 1 and 1000, inclusive.
  • the polymers include a polymeric unit having the formula:
  • x, y, and z are independently integers between 1 and 1000, inclusive; each occurrence of m is an integer between 1 and 30, inclusive, preferably between 2 and 15, inclusive. In some forms, m is 3 ( ⁇ - caprolactone), 12 ( ⁇ -pentadecalactone), or a combination thereof;
  • p, q, n, and t are independently integers between 1 and 20, inclusive, preferably between 1 and 10, inclusive, most preferably between 2 and 5, inclusive. In some forms, p, q, n, and t are each 2;
  • R 8 and R 10 are independently O or NR' , wherein R' is hydrogen, substituted alkyl, unsubstituted alkyl, substituted aryl, or unsubstituted aryl. In some forms, at least one of R 8 and R 10 is O. In some forms, R 8 and R 10 are O; and
  • R 9 is unsubstituted alkyl, substituted alkyl, hydrogen, substituted aryl, or unsubstituted aryl. In some forms, R 9 is unsubstituted C 1 -C 10 alkyl, substituted C 1 -C 10 alkyl. Preferably, R 9 is unsubstituted C 1 alkyl, such as methyl.
  • R 8 and R 10 are O, and m is an integer between 1 and 30, inclusive, such as 3 (e.g. ⁇ -caprolactone), 12 (e.g. ⁇ -pentadecalactone), or a combination thereof.
  • R 8 and R 10 are O; and m is an integer between 1 and 30, inclusive, such as 3 (e.g. ⁇ -caprolactone), 12 (e.g. ⁇ -pentadecalactone), or a combination thereof; p and q are integers between 1 and 20, such as 2.
  • R 8 and R 10 are O; and m is an integer between 1 and 30, inclusive, such as 3 (e.g. ⁇ -caprolactone), 12 (e.g. ⁇ -pentadecalactone), or a combination thereof; p and q are integers between 1 and 20, such as 2; and n and t are integers between 1 and 20, such as 2.
  • R 8 and R 10 are O; and m is an integer between 1 and 30, inclusive, such as 3 (e.g. ⁇ -caprolactone), 12 (e.g. ⁇ -pentadecalactone), or a combination thereof; p and q are integers between 1 and 20, such as 2; n and t are integers between 1 and 20, such as 2; and R 9 is unsubstituted C 1 -C 10 alkyl or substituted C 1 -C 10 alkyl, such as ethyl, n-propyl, isopropyl, n-butyl, or t-butyl, or unsubstituted aryl, such as phenyl.
  • m is an integer between 1 and 30, inclusive, such as 3 (e.g. ⁇ -caprolactone), 12 (e.g. ⁇ -pentadecalactone), or a combination thereof; p and q are integers between 1 and 20, such as 2; n and t are integers between
  • m is 12 (e.g. ⁇ -pentadecalactone, PDL); R 8 and R 10 are O, p and q are 2, R 9 is methyl (e.g. N-methyldiethanolamine, MDEA); and n and t are 2 (e.g. 3,3'-dithiodipropionate, DTDP); i.e., poly(ro- pentadecalactone-co-N-methyldiethyleneamine-co-3,3'-dithiodipropionate).
  • m is 3 (e.g. ⁇ -caprolactone, CL); R 8 and R 10 are O, p and q are 2, R 9 is methyl (e.g. N-methyldiethanolamine, MDEA); and n and t are 2 (e.g. 3,3'-dithiodipropionate, DTDP); i.e., poly ⁇ -caprolactone-co-N- methyldiethyleneamine-co-3,3'-dithiodipropionate).
  • the polymers can be diblock copolymers that include a block of a hydrophilic polymer, preferably a polyalkylene oxide such as polyethylene glycol (PEG) at one terminus.
  • the diblock copolymer can have the formula:
  • R 8 , R 9 , and R 10 are as described above;
  • r is an integer between 1 and 1000, inclusive;
  • T is O or absent
  • R 6 is hydrogen, unsubstituted alkyl, substituted alkyl, unsubstituted aryl, substituted aryl, unsubstituted cycloalkyl, substituted cycloalkyl, maleimide, amine, thiol, N-hydroxysuccinimide ester, azide, acrylate, methacrylate, alkyne, hydroxide, or isocynate.
  • T is O and R 6 is unsubstituted alkyl, such as methyl.
  • the diblock copolymer is polyethylene glycol-poly(ro- pentadecalactone-co-N-methyldiethyleneamine-co-3,3'-dithiodipropionate) (PEG-PPMD) or polyethylene glycol-poly(s-caprolactone-co-N- methyldiethyleneamine-co-3,3'-dithiodipropionate) (PEG-PCMD).
  • the polymers can be triblock copolymers that include a block of a hydrophilic polymer, preferably a polyalkylene oxide such as polyethylene glycol at both termini.
  • the triblock copolymer can have the
  • x, y, z, m, p, q, n, t, and r are as described above;
  • R 8 , R 9 , and R 10 are as described above;
  • the triblock copolymer is PEG-PPMD-PEG or PEG-PCMD-PEG.
  • hydrophilic polymers that can be used include polysaccharides such as celluloses; hydrophilic polypeptides and poly(amino acids) such as poly-L-glutamic acid (PGS), gamma-polyglutamic acid, poly- L-aspartic acid, poly-L-serine, and poly-L-lysine; poly(oxyethylated polyol); poly(olefinic alcohol); polyvinylpyrrolidone;
  • poly(hydroxyalkylmethacrylamide) poly(hydroxyalkylmethacrylate) ;
  • the nanoparticles possess dual pH-responsive and redox-responsive properties, ascertained by variations in the sizes of the nanoparticles under different conditions.
  • the average sizes of the nanoparticles remain fairly constant under physiological conditions, such as pH 7.4, for periods of time such as one day, two days, one week, one month, etc.
  • the nanoparticles swell under acidic pH conditions and degrade in low redox potential environments, as commonly found in tumor tissues, or in the lysosomal and endosomal vesicles within cells.
  • the nanoparticles are formed from the polymers described above.
  • the polymers are amphiphilic and include the polymeric unit of Formula I, as described above.
  • the nanoparticles are micelles, i.e. , the nanoparticles have a core-shell structure formed by amphiphilic block copolymers containing a hydrophobic polymer and a hydrophilic polymer.
  • the hydrophobic polymer and the hydrophilic polymer form the core and the shell, respectively, of the nanoparticles.
  • nanoparticles formed from polymers containing ⁇ -caprolactone, m 3 (CL), (between 140 nm and 203 nm).
  • This effect of lactone molecular weight on nanoparticle size can be attributed to more hydrophobic interactions in the cores of nanoparticles containing PDL with respect to nanoparticles containing CL, which significantly reduces the water absorption in the nanoparticle cores, thereby giving rise to the smaller size.
  • the zeta potential of the nanoparticles can be between -50 mV and +50 mV, inclusive, between -25 mV and +25 mV, inclusive, or between -10 mV and +10 mv, inclusive.
  • the zeta potential is slightly negative, such as between 0 mV and -10 mV.
  • the hydrophilic polymers are selected such that the nanoparticle has a near-neutral zeta potential, such as between -10 mV and + 10 mV, inclusive.
  • the nanoparticles can include cargo (e.g. therapeutic, diagnostic, and/or prophylactic agents) to be delivered to a desired site of subject.
  • the site of delivery can be intracellular, extracellular, the extracellular matrix, a tissue, or organ.
  • the site of delivery is associated with a disease.
  • the nanoparticles can display sustained release of the cargo over a period of hours, days, weeks, etc.
  • the cargo can be encapsulated within the nanoparticles, dispersed within the polymeric matrix that forms the nanoparticles, on the surface of the nanoparticles, non-covalently or covalently bound to a polymer from which the nanoparticles are formed, or a combination thereof.
  • the cargo can be encapsulated non- covalently within the nanoparticles.
  • the therapeutic agent is a chemotherapeutic agent, i.e. , an anti-cancer agent, such as docetaxel.
  • these cargoes are covalently conjugated to the polymer prior to the formation of the nanoparticle.
  • the nanoparticles can include targeting moieties that selectively target the nanoparticles to a specific site, by specifically recognizing and binding to a target molecule specific for a cell type, a tissue type, or an organ.
  • targeting moieties include, but are not limited to, antibodies and antigen binding fragments thereof, aptamers, peptides, and small molecules.
  • the binding moiety can be conjugated, covalently or non-covalently, to a polymer that forms the nanoparticle.
  • the binding moiety is displayed on the surface of the nanoparticle.
  • the targeting moieties should have an affinity for a cell-surface receptor or cell- surface antigen on the target cells.
  • the targeting moieties may result in internalization of the nanoparticles within the target cell.
  • the target molecule is associated with a disease or preferentially over-expressed in a diseased tissue or cell compared to a non- diseased tissue or cell.
  • the target molecule can be a cell surface polypeptide, lipid, or glycolipid.
  • the target molecule can be a receptor that is selectively expressed on a specific cell surface, a tissue or an organ.
  • Specific markers can be for specific types of cells including, but not limited to stem cells, skin cells, blood cells, immune cells, muscle cells, nerve cells, cancer cells, virally infected cells, and organ specific cells.
  • the cell specific markers include, but are not limited to cancer specific markers.
  • the polymers are synthesized via catalytic polymerization of reactants in a one-step process, wherein an enzyme serves as the catalyst.
  • the enzyme catalyst is a lipase.
  • the lipase is lipase B from Candida antartica.
  • the reactants include a lactone, a molecule containing a disulfide bond, and a polyfunctional molecule containing an amine group, a hydroxyl group, preferably both.
  • the reactants can further include a hydrophilic polymer, such as polyalkylene oxide (e.g. polyethylene glycol).
  • the molar feed ratios of the lactone/molecule containing a disulfide bond/polyfunctional molecule containing an amine group and hydroxyl group can be between 5:20: 10 and 80:90:90.
  • the molar feed ratios of the lactone/molecule containing a disulfide bond/polyfunctional molecule containing an amine group/hydrophilic polymer can be between 5:20: 10:3 and 80:90:90: 15.
  • the method of synthesizing the polymers includes a first step of incubating the reactants and enzyme catalyst at a temperature from between 60 °C and 95 °C, inclusive, preferably between 80 °C and 90 °C, inclusive, at about 1 atm N 2 .
  • the method further includes, after the first step, incubating the reactants and enzyme catalyst at a temperature from between 60 °C and 95 °C, inclusive, preferably between 80 °C and 90 °C, inclusive, under high vacuum (e.g. 2 mmHg).
  • the weight average molecular weight (Mw) of the polymers can be between 1 kDa and 50 kDa, inclusive, preferably between 1 kDa and 30 kDa, inclusive, most preferably between 5 kDa and 15 kDa, inclusive. In some forms, Mw is about 8.1 kDa, 8.3 kDa, 8.7 kDa, 9.4 kDa, 11 kDa, 11.3 kDa, 12.5 kDa, 13.3 kDa, or 15.2 kDa.
  • the polymers are prepared, as shown in Scheme 1.
  • m is an integer between 1 and 30, inclusive, preferably between 2 and 15, inclusive. In some forms, m is 3 ( ⁇ - caprolactone), 12 ( ⁇ -pentadecalactone), or a combination thereof. In some forms, p, q, n, and t are independently integers between 1 and 20, inclusive, preferably between 1 and 10, inclusive, most preferably between 2 and 5, inclusive. In some forms, p, q, n, and t are each 2. In some forms, r is an integer between 1 and 1000.
  • R 1 and R 2 are independently hydrogen, substituted alkyl, unsubstituted alkyl, substituted aryl, or unsubstituted aryl.
  • Ri and R2 are unsubstituted C 1 -C 10 alkyl, substituted C 1 -C 10 alkyl.
  • Ri and R2 are unsubstituted C 1 alkyl, such as methyl.
  • R3 and R5 are independently OH or N(R') 2 , wherein each R' is independently hydrogen, substituted alkyl, unsubstituted alkyl, substituted aryl, or unsubstituted aryl.
  • R3 and R5 are OH, and at least one R' is hydrogen. In some forms, R3 and R5 are OH.
  • R 4 is unsubstituted alkyl, substituted alkyl, hydrogen, substituted aryl, or unsubstituted aryl. In some forms, R 4 is unsubstituted C 1 -C 10 alkyl, substituted C 1 -C 10 alkyl. Preferably, R 4 is unsubstituted C 1 alkyl, such as methyl.
  • the reactants are 00- pentadecalactone (PDL) or ⁇ -caprolactone, with N-methyldiethanolamine, (MDEA), 3,3'-dithiodipropionate (DTDP), and polyethylene glycol.
  • PDL pentadecalactone
  • MDEA N-methyldiethanolamine
  • DTDP 3,3'-dithiodipropionate
  • polyethylene glycol polyethylene glycol
  • x, y, and z are independently integers between 1 and 1000, inclusive.
  • R 8 and R 10 are independently O or NR' , wherein R' is hydrogen, substituted alkyl, unsubstituted alkyl, substituted aryl, or unsubstituted aryl.
  • at least one of R 8 and R 10 is O.
  • R 8 and R 10 are O.
  • R 9 is unsubstituted alkyl, substituted alkyl, hydrogen, substituted aryl, or unsubstituted aryl.
  • R 9 is unsubstituted C 1 -C 10 alkyl, substituted C 1 -C 10 alkyl.
  • R 9 is unsubstituted C 1 alkyl, such as methyl.
  • T is O or absent.
  • R 6 hydrogen, unsubstituted alkyl, substituted alkyl,
  • T is O and R 6 is unsubstituted alkyl, such as methyl.
  • the product is poly(ro-pentadecalactone-co-N-methyldiethyleneamine-co-3,3'- dithiodipropionate) (PPMD), poly(s-caprolactone-co-N- methyldiethyleneamine-co-3,3'-dithiodipropionate) (PCMD), or PEG- poly(ro-pentadecalactone-co-N-methyldiethyleneamine-co-3,3'- dithiodipropionate) (PEG-PPMD), PEG-poly(s-caprolactone-co-N- methyldiethyleneamine-co-3,3'-dithiodipropionate) (PEG- PCMD), PEG- PCMD-PEG, PEG-PPMD-PEG, or a combination thereof.
  • PPMD poly(ro-pentadecalactone-co-N-methyldiethyleneamine-co-3,3'- dithiodipropionate)
  • PCMD poly(
  • the desired amphiphilic products can be isolated using the methods described below in Example 1.
  • polyethylene glycol is included as a hydrophilic block in the polymers.
  • suitable hydrophilic polymers can be used as well. These include polysaccharides such as celluloses; hydrophilic polypeptides and poly(amino acids) such as poly-L-glutamic acid (PGS), gamma-polyglutamic acid, poly- L-aspartic acid, poly-L-serine, and poly-L-lysine; poly(oxyethylated polyol); poly(olefinic alcohol); polyvinylpyrrolidone;
  • poly(hydroxyalkylmethacrylamide) poly(hydroxyalkylmethacrylate) ;
  • hydrophilic poly hydroxy acids
  • poly vinyl alcohol
  • copolymers thereof hydrophilic poly (hydroxy acids)
  • the nanoparticles are formed by self-assembly of amphiphilic block copolymers in an aqueous solution.
  • the amphiphilic copolymers can spontaneously self-assemble to form nanoparticles with a hydrophobic core and a hydrophilic outer shell.
  • a solution containing the amphiphilic polymers is mixed with another solution containing a therapeutic, diagnostic, and/or prophylactic agent to be encapsulated.
  • the amphiphilic polymers and therapeutic, diagnostic, and/or prophylactic agent to be delivered are dissolved in a suitable solvent, such as tetrahydrofuran, DMSO, or methylene chloride.
  • the therapeutic agent is a chemotherapeutic agent, i.e. , an anti-cancer agent, such as docetaxel.
  • concentrations of the amphiphilic polymer and therapeutic, diagnostic, and/or prophylactic agent in the solvent can be varied as needed.
  • the solution can be added continuously to an aqueous solution, such as PBS (10 mM, pH 7.4) using syringe to induce nanoparticle formation (micellization).
  • the nanoparticle solutions can be stirred at room
  • the nanoparticles described herein can be formed using a variety of techniques known in the art.
  • the technique to be used can depend on a variety of factors including the polymer used to form the nanoparticles, the desired size range of the resulting nanoparticles, and suitability for the therapeutic, diagnostic, and/or prophylactic agent to be incorporated.
  • Suitable techniques include, but are not limited to:
  • water-soluble or water-miscible organic solvents are used to dissolve the polymer and form emulsion upon mixing with the aqueous phase.
  • the quick diffusion of the organic solvent into water leads to the formation of nanoparticles immediately after the mixing.
  • the polymer is dissolved in a volatile organic solvent.
  • the drug (either soluble or dispersed as fine particles) is added to the solution, and the mixture is suspended in an aqueous solution that contains a surface active agent such as poly(vinyl alcohol).
  • a surface active agent such as poly(vinyl alcohol).
  • the resulting emulsion is stirred until most of the organic solvent evaporated, leaving solid nanoparticles.
  • the resulting nanoparticles are washed with water and dried overnight in a lyophilizer. Nanoparticles with different sizes and morphologies can be obtained by this method.
  • Solvent removal solvent removal
  • the drug is dispersed or dissolved in a solution of the selected polymer in a volatile organic solvent.
  • This mixture is suspended by stirring in an organic oil (such as silicon oil) to form an emulsion.
  • an organic oil such as silicon oil
  • this method can be used to make nanoparticles from polymers with high melting points and different molecular weights.
  • the external morphology of spheres produced with this technique is highly dependent on the type of polymer used.
  • the polymer is dissolved in organic solvent.
  • a known amount of the active drug is suspended (insoluble drugs) or co-dissolved (soluble drugs) in the polymer solution.
  • the solution or the dispersion is then spray-dried.
  • Nanospheres can be formed from polymers using a phase inversion method wherein a polymer is dissolved in a "good" solvent, fine particles of a substance to be incorporated, such as a drug, are mixed or dissolved in the polymer solution, and the mixture is poured into a strong non solvent for the polymer, to spontaneously produce, under favorable conditions, polymeric microspheres, wherein the polymer is either coated with the particles or the particles are dispersed in the polymer.
  • the method can be used to produce nanoparticles in a wide range of sizes, including, for example, about 100 nanometers to about 10 microns.
  • Substances which can be incorporated include, for example, imaging agents such as fluorescent dyes, or biologically active molecules such as proteins or nucleic acids.
  • the polymer is dissolved in an organic solvent and then contacted with a non solvent, which causes phase inversion of the dissolved polymer to form small spherical particles, with a narrow size distribution optionally incorporating an antigen or other substance.
  • the microfluidic device comprises at least two channels that converge into a mixing apparatus.
  • the channels are typically formed by lithography, ⁇ ?iching, embossing, or molding of a polymeric surface.
  • a source of fluid is attached to each channel, and the application of pressure to the source causes the flow of the fluid in the channel.
  • the pressure may be applied by a syringe, a pump, and/or gravity.
  • nanoparticles having the desired size and density of moieties on the surface.
  • pressure and flow rate in the inlet channels and the nature and composition of the fluid sources nanoparticles can be produced having reproducible size and structure.
  • the dual pH responsive and redox-responsive nanoparticles can be used to deliver therapeutic, diagnostic, and/or prophylactic agents to a subject.
  • these agents are delivered to an acidic diseased tissue, resulting from a diseased state such as cancer, inflammation, stroke, arthritis, or ischemia.
  • the therapeutic agent is a chemotherapeutic agent, i.e. , an anti-cancer agent, such as docetaxel.
  • the therapeutic agent e.g.
  • chemotherapeutic agent constitutes between 0.5 wt/wt% and 25 wt/wt%, inclusive, between 1 wt/wt% and 20 wt/wt%, inclusive, between 1 wt/wt% and about 15 wt/wt%, inclusive, between 1 wt/wt% and 10 wt/wt%, inclusive, or between 1 wt/wt% and 5 wt/wt%, inclusive of the nanoparticles.
  • Methods of use typically involve administering to a subject, in need thereof, a composition containing the nanoparticles having an effective amount of a therapeutic agent to inhibit progression of a disease.
  • the composition inhibits the proliferation of tumor cells, induce cell- cycle arrest, and/or induce senescence in tumor cells, in a subject.
  • methods include administering to a subject in a need thereof an effective amount of the composition to reduce or inhibit proliferation of tumor cells, induce cell-cycle arrest or tumor cells, and/or induce senescence in tumor cells.
  • chemotherapeutic agents that can be included as cargo in the nanoparticles include, but are not limited to, docetaxel and analogs thereof, paclitaxel and analogs thereof, alkylating agents (such as cisplatin, carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, dacarbazine, lomustine, carmustine, procarbazine, chlorambucil and ifosfamide), actinomycin, bleomycin, daunorubicin, doxorubicin, epirubicin, mitomycin, methotrexate, fluorouracil, carboplatin, methyl- chloroethylnitrosoureas, etoposide, camptothecin and analogs thereof, phenesterine, piposulfan, altretamine, asparaginase, busulfan, chlorambucil, cladribine, cytarabine, daca
  • the nanoparticles can encapsulate functional nucleic acids.
  • Functional nucleic acids are nucleic acid molecules that have a specific function, such as binding a target molecule or catalyzing a specific reaction.
  • Functional nucleic acid molecules can be divided into the following non-limiting categories: antisense molecules, siRNA, miRNA, aptamers, ribozymes, triplex forming molecules, RNAi, and external guide sequences.
  • the functional nucleic acid molecules can act as effectors, inhibitors, modulators, and stimulators of a specific activity possessed by a target molecule, or the functional nucleic acid molecules can possess a de novo activity independent of any other molecules.
  • the nucleic acid is less than 1,000 base pairs, less than 500 base pairs, less than 250 base pairs, or less than 100 base pairs.
  • compositions can be administered by a number of routes including, but not limited to, oral, intravenous, intraperitoneal, intramuscular, transdermal, subcutaneous, topical, sublingual, rectal, intranasal, pulmonary, and other suitable means.
  • routes including, but not limited to, oral, intravenous, intraperitoneal, intramuscular, transdermal, subcutaneous, topical, sublingual, rectal, intranasal, pulmonary, and other suitable means.
  • routes including, but not limited to, oral, intravenous, intraperitoneal, intramuscular, transdermal, subcutaneous, topical, sublingual, rectal, intranasal, pulmonary, and other suitable means.
  • routes including, but not limited to, oral, intravenous, intraperitoneal, intramuscular, transdermal, subcutaneous, topical, sublingual, rectal, intranasal, pulmonary, and other suitable means.
  • the administration may be localized (i.e., to a particular region, physiological system, tissue, organ, or cell type) or systemic, depending on the condition being treated.
  • Suitable parenteral administration routes include intravascular administration (e.g. , intravenous bolus injection, intravenous infusion, intra- arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature); peri- and intra-tissue injection (e.g. , intraocular injection, intra-retinal injection, or sub-retinal injection);
  • subcutaneous injection or deposition including subcutaneous infusion (such as by osmotic pumps); direct application by a catheter or other placement device (e.g. , an implant comprising a porous, non-porous, or gelatinous material).
  • a catheter or other placement device e.g. , an implant comprising a porous, non-porous, or gelatinous material.
  • compositions can be administered in a single dose or in multiple doses. Certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. It will also be appreciated that the effective dosage of the composition used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays.
  • Dosing is dependent on severity and responsiveness of the disease condition to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of disease state is achieved.
  • Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual therapeutic agent, and can generally be estimated based on EC50S found to be effective in in vitro and in vivo animal models.
  • Dosage levels on the order of about lmg/kg to 100 mg/kg of body weight per administration are useful in the treatment of a disease.
  • the dosage levels are about lOmg/kg -50 mg/kg of body weight per administration.
  • One skilled in the art can also readily determine an appropriate dosage regimen for administering the disclosed compositions to a given subject.
  • the compositions can be administered to the subject once, e.g. , as a single injection, infusion or bolus.
  • the formulation can be administered once or twice daily to a subject for a period of from about three to about twenty-eight days, or from about seven to about ten days.
  • a number of stimuli-responsive nanoparticles have previously been formed from synthetic polymers that are produced via chemical
  • PEG-PPMS PEG-poly( ⁇ -pentadecalactone-co-N- methyldiethyleneamine-co-sebacate)
  • PEG-PPBD PEG-poly(ro-pentadecalactone-co-butylene-co-3,3'- dithiodipropionate)
  • PEG-poly( ⁇ -pentadecalactone-co- N-methyldiethyleneamine-co-3,3'-dithiodipropionate) PEG-PPMD
  • PEG-poly(s-caprolactone-co-N-methyldiethyleneamine-co-3,3'- dithiodipropionate) PEG-PCMD
  • amphiphilic block copolymers were produced in one step via lipase-catalyzed copolymerization of lactone ( ⁇ - pentadecalactone or ⁇ -caprolactone), iV-methyldiethanolamine (MDEA), dimethyl 3,3'-dithiodipiOpionate (DTDP) and polyfethylene glycol) methyl ether (MeO-PEG-OH).
  • lactone ⁇ - pentadecalactone or ⁇ -caprolactone
  • MDEA iV-methyldiethanolamine
  • DTDP dimethyl 3,3'-dithiodipiOpionate
  • MeO-PEG-OH polyfethylene glycol) methyl ether
  • FIG. 14 illustrates steps that are involved in the therapeutic actions of the pH and redox-responsive nanoparticles in delivering the drug to tumor cells.
  • PEG-PPMD and PEG-PCMD represent the first examples of enzymatic polymers with pH and redox dual-responsive properties
  • ⁇ -Pentadecalactone (PDL, >98%), ⁇ -caprolactone (CL, 99%), N- methyldiethanolamine (MDEA, 99%), diphenyl ether (99%), poJy(ethylene glycol) methyl ether (2000 Da, MeO-PEG2K-OH) and L-buthionine-iS.i?)- sulfoximine (BSO) were purchased from Sigma-AJdrich Chemical Co. and were used as received.
  • Immobilized CALB Candida antarctica lipase B supported on acrylic resin
  • Novozym 435 3-(4,5-dimethyl-2- thiazoIyl)-2,5-diphenyI-2H-tetrazoliuni bromide ( M T T ).
  • chloroform HPLC grade
  • chloroform-d, «-hexane 9+% were also purchased from Aldrich Chemical Co.
  • the lipase catalyst was dried at 40 °C under 2.0 mmHg for 20 h prior to use. Dimethyl 3,3 * -tjithiodipropionate (DTDP, 98%) was obtained from.
  • DMEM and RPMI-1640 (from Gibco, both containing 10% (v/v) fetal bovine serum (FBS) and 1% (w/v) penicillin-streptomycin) were used as the culture media for the HeLa cell line and CT-26 cell line, respectively.
  • the block copolymers were prepared via copolymerization of 00- pentadecalactone (PDL), N-methyldiethanolamine (MDEA), dimethyl 3,3'- dithiodipropionate (DTDP) with MeO-PEG2K-OH using Novozym 435 as the catalyst.
  • PDL pentadecalactone
  • MDEA N-methyldiethanolamine
  • DTDP dimethyl 3,3'- dithiodipropionate
  • the amount of MeO-PEG2K-OH was selected to allow formation of the block copolymers with 40 wt% PEG upon complete conversion of the substrates to polymer products.
  • PDL, MDEA, DTDP and MeO-PEG2K-OH in different ratios were mixed with Novozym 435 (10 wt% vs total substrate) and diphenyl ether (200 wt% vs total substrate).
  • the resultant reaction mixtures were stirred at 90 °C initially under 1 atm nitrogen gas for 21 h and subsequently under a reduced pressure of 1.8 mmHg for 70 h.
  • the formed product was purified, as described previous (Liu, et al. , Polym. Chem. 2015, 6, 1997-2010). Briefly, n-hexane was added to the product mixtures to precipitate the formed copolymers.
  • the copolymers were washed with n-hexane three times to extract residual diphenyl ether solvent, dissolved in chloroform, and then filtered to remove the enzyme catalyst. Complete evaporation of the chloroform solvent from the filtrates at 30 °C under high vacuum ( ⁇ 1.0 mmHg) for 20 h, yielded the purified PEG2K- PPMD block copolymers.
  • Each PEG-PPMD copolymer is denoted as PEG- PPMD-x% PDL indicating molar percentage content of PDL units vs. (PDL + diester) units in the PPMD segments of the copolymer.
  • PEG-PPMD block copolymer PEG-PPMD block copolymer.
  • PEG-PPMD-x%PDL mol% of PDL units vs (PDL + DTDP) units, which is calculated from the peak intensities in the NMR spectra.
  • the data shown below are the peak positions in the spectra and are same for all PEG-PPMD samples with different PDL contents.
  • the PEG-PCMD copolymers were synthesized following procedures analogous to those used for the preparation of PEG-PPMD polymers above except that ⁇ -caprolactone (CL) was employed instead of PDL as the lactone comonomer.
  • CL ⁇ -caprolactone
  • MDEA MDEA
  • DTDP MeO-PEG2K-OH in different ratios (shown in Table 1) were blended with Novozym 435 (10 wt% vs total substrate) and diphenyl ether (200 wt% vs total substrate).
  • the resultant reaction mixtures were stirred at 80 °C initially under 1 atm nitrogen gas for 20 h and subsequently under 2.0 mmHg vacuum for 71 h.
  • PEG- PCMD copolymers were purified using the same method as described above for isolation of the PEG-PPMD copolymers.
  • Each PEG-PCMD copolymer is denoted as PEG-PCMD-x% CL indicating molar percentage content of CL units vs (CL + diester) units in the PCMD segments of the copolymer.
  • PEG- PCMD block copolymer The data shown below are the peak positions in the spectra and are same for all PEG-PCMD samples with different CL contents.
  • the PEG2K-PPMD copolymers and PEG2K-PCMD copolymers synthesized above were amphiphilic block copolymers.
  • the amphiphilic block copolymers containing tertiary amino and disulfide functional groups were synthesized via CALB-catalyzed copolymerization of lactone (PDL or CL), N-methyldiethanolamine (MDEA), dimethyl 3,3'-dithiodipropionate (DTDP) with polyethylene glycol) methyl ether (MeO-PEG2K-OH) in two stages: the first stage oligomerization under 1 atm nitrogen gas, followed by the second stage polymerization under ⁇ 2 mmHg vacuum (Scheme 3).
  • the comonomer feed ratios employed, and the composition and properties of the resultant polymer products are shown in Table 1.
  • All purified PEG-PPMD and PEG-PCMD copolymers contain 40 ( ⁇ 1) wt% PEG (calculated from the proton NMR spectra) and were obtained in 75% to 94% yield.
  • the M w values range from 11000 to 15000 Da for PEG-PPMD copolymers and from 8100 to 9400 Da for PEG-PCMD copolymers.
  • the PEG-PPMD and PEG-PCMD copolymers act as a chain terminator, the PEG-PPMD and PEG-PCMD copolymers have or can contain two possible types of block structures: PEG-polyester diblock chains and PEG-polyester- PEG triblock chains where polyester is PPMD or PCMD (Scheme 3).
  • PEG-PPMD and PEG-PCMD copolymer chains contain both PEG blocks and polyester blocks consisting of lactone (PDL or CL), N- methyldiethyleneamine (MDEA), and 3,3'-dithiodipropionate (DTDP) repeating units.
  • PDL or CL lactone
  • MDEA N- methyldiethyleneamine
  • DTDP 3,3'-dithiodipropionate
  • compositions (Table 2).
  • the results reveal that the repeat unit arrangements in the polyester chain blocks of the polymers (e.g. PEG-PPMD and PEG- PCMD copolymers) are nearly random.
  • lactone unit represents co-pentadecalactone for PEG-PPMD or ⁇ -caprolactone for PEG-PCMD.
  • b Measured by carbon- 13 NMR spectroscopy.
  • the blank and DTX-loaded micelles were fabricated using a dialysis method.
  • PEG-PPMD or PEG-PCMD copolymers 38 mg
  • DTX 2 mg
  • the resultant solutions were continuously added into 5 ml of PBS (10 mM, pH 7.4) using a0 syringe to induce micellization.
  • the micelle solutions were stirred for 30 min at room temperature and dialyzed of PBS (10 mM, pH 7.4) overnight using 3500 Da cutoff size dialysis bag.
  • dialyzed micelle solutions were then centrifuged for 20 min at 8000 rpm using MWCO 100 kDa ultrafiltration centrifuge tubes. Finally, an aliquot of the5 concentrated micelle solutions was lyophilized, and the yield of each micelle sample was calculated and recorded.
  • CMC critical micelle concentration
  • the resultant mixtures were incubated overnight at room temperature.
  • the emission spectra were recorded from 350 nm to 420 nm using an excitation wavelength of 334 nm.
  • the slit- width was 2 nm for both excitation and emission beams.
  • the CMC value was estimated as the micelle0 concentration at the cross point in the curves of hfii (the third peak/the first peak) intensity ratio vs logarithm of micelle concentration.
  • PEG-PPMD and PEG-PCMD micelles were evaluated by DLS. Briefly, freshly prepared micelle solution was added into PBS (pH 7.4, 10 mM) containing 10% fetal bovine serum (FBS), and incubated at 37 °C in a shaking bed with a rotation speed of 100 rpm.
  • PBS pH 7.4, 10 mM
  • FBS fetal bovine serum
  • Samples were withdrawn at predetermined time intervals for DLS analysis on average micelle size to evaluate in vitro stability of the micelles.
  • the content of DTX encapsulated in PEG-PPMD or PEG-PCMD micelles was measured by high performance liquid chromatography (HPLC, Agilent 1260) equipped with an Eclipse XDB-C 1 8 column.
  • the mobile phase (a 1 : 1 (v/v) acetonitrile/water mixture) was used at 1 niL/min flow rate.
  • the UV absorption at 230 nm was used for detection.
  • an aliquot of micelle samples was dissolved in 950 ⁇ iL THF, and the organic solutions were centrifuged (13000 rpm, 10 min) and filtered using 0.2 ⁇ syringe filters. The filtered solutions (10 ⁇ iL) were then injected to the HPLC for DTX content analysis.
  • the drug e.g. DTX
  • DL DL
  • entrapment efficiency were calculated according to the following equations:
  • the PEG-PPMD and PEG-PCMD copolymers self-assemble readily in aqueous medium to form micelle nanoparticles.
  • the formation of the micelle nanoparticles was monitored by fluorometry using pyrene as a fluorescent probe and the critical micelle concentration (CMC) values were calculated.
  • the CMC values for PEG-PPMD with 12%, 43% and 70% PDL content are shown in Figure 2 A, and those for PEG-PCMD with 13%, 41% and 70% CL content are shown in Figure 2B.
  • Figures 2 A and 2B show variations in fluorescence intensity ratio (I3/I1) of pyrene as a function of logarithm of polymer concentration for different PEG-PPMD and PEG- PCMD copolymers in PBS.
  • the CMC values of the copolymers with 13%, 41% and 70% CL are 15.5 ⁇ g/mL, 14.7 ⁇ g/mL and 9.7 ⁇ g/mL, correspondingly.
  • the higher lactone content in the polymer chains improves the stability of the copolymer micelles in aqueous medium by increasing the hydrophobicity in the micelle cores.
  • PDL is a stronger hydrophobicity- enhancer than CL, leading to lower CMC values for PEG-PPMD vs PEG- PCMD at the same lactone content.
  • the drug-loaded PEG-PPMD micelles had an average size between 84 and 121 nm, which is smaller than the average sizes between 140 and 203 nm observed for the DTX-encapsulated PEG-PCMD micelles. This likely due to the fact that the presence of PDL units in PEG- PPMD chains render the copolymer micelle cores significantly more hydrophobic than those of the PEG-PCMD micelles, thus substantially reducing the water absorption in the nanoparticle cores (Theerasilp, et al., J Microencapsul. 2013, 30, 390-397; Riley, et al., Langmuir 2001, 17, 3168- 3174).
  • the sizes of the micelles measured by TEM are smaller than those measured by DLS. Possibly, the removal of water from the micelle samples during the TEM analysis may shrink the micelle particles (Fontana, et al., Biomaterials 2001, 22, 2857-2865). All DTX-loaded micelle samples were slightly negative-charged on surface (Table 3). This is beneficial for in vivo drug delivery because previous studies indicate that nanoparticles with zeta potential values ranging from -10 to +10 mV can decrease serum protein binding and increase particle circulation time in the blood (Li, et al., Mol Pharm. 2008, 5, 496-504; Levchenko, et al, Int J Pharm. 2002, 240, 95- 102).
  • the drug loading and entrapment efficiency for the DTX- loaded micelles were determined by HPLC analysis (Table 3).
  • the DTX entrapment efficiencies (EE) are 65% -67% for the PEG-PPMD micelles and are in the range between 65% and 76% for the PEG-PCMD micelles.
  • the drug loading (DL) amounts in all micelle samples are comparable (3.3-3.8 wt%).
  • Example 3 pH and redox- triggered disassembly of micelles
  • the size change of the PEG-PPMD and PEG-PCMD micelles in response to acidic or reductive conditions in PBS solution was analyzed by dynamic light scattering (DLS).
  • DLS dynamic light scattering
  • aliquots of blank micelle solutions were added into six different media: (i) PBS buffer (10 mM, pH 7.4), (ii) PBS buffer (10 mM, pH 7.4) containing 10 mM D,L-dithiothreitol (DTT), (iii) PBS buffer (10 mM, pH 7.4) containing 50 mM DTT, (iv) PBS buffer (10 mM, pH 5.0), (v) PBS buffer (10 mM, pH 5.0) containing 10 mM DTT (D,L-dithiothreitol), and (vi) PBS buffer (10 mM, pH 5.0) containing 50 mM DTT.
  • Functional nanoparticles responsive to acidic pH and intracellular reduction potential are useful drug carriers that can selectively deliver and release a drug at controllable rates to specific disease sites (e.g., acidic tumors and tumor cells).
  • specific disease sites e.g., acidic tumors and tumor cells.
  • the micelles were incubated for 48 h in different PBS buffers with pH of 7.4 or 5.0 containing various amount of D,L-dithiothreitol (DTT, 0 to 50 mM), and the size variations of the micelle particles were measured by DLS analysis.
  • DTT D,L-dithiothreitol
  • Example 4 In vitro drug release from DTX-loaded micelles
  • DTX-loaded PEG-PPMD and PEG- PCMD micelles were studied using a dialysis method.
  • Each DTX-loaded micelle sample was placed into four dialysis bags (MWCO 3500 Da) which respectively were immersed into four different PBS solutions (10 mM) containing 0.5% (w/v) Tween 80: (i) PBS buffer with pH 7.4, (ii) PBS buffer with pH 7.4 and 10 mM DTT, (iii) PBS buffer with pH 5.0, and (iv) PBS buffer with pH 5.0 and 10 mM DTT.
  • the micelle samples were incubated at 37 °C in a shaking bed with a rotation speed of 100 rpm.
  • PEG-PPMD and PEG-PCMD micelles e.g., PEG-PPMD- 12% PDL, PEG-PPMD- 43% PDL, PEG-PCMD-41% CL, PEG-PCMD-70% CL
  • PEG-PCMD-70% CL also respond predictably to DTT that was added to the media, and are triggered by the reductant to release the drug at an accelerated rate.
  • the minimal response to DTT for the PEG-PPMD-70% PDL micelles is presumably due to their highly hydrophobic, PDL-rich micelle cores that prohibit the diffusion of water-soluble DTT from the media to react with and cleave the low abundant disulfide bonds in the micelles ( Figure 6C).
  • the current PEG-PPMD and PEG- PCMD nanoparticles with synergistic pH and redox-responsive properties are expected to be significantly more potent nanocarriers for intracellular delivery and release of chemotherapeutic agents to cancer cells since their drug delivery efficiency can be boosted by both acidic tumor or endosomal pH and the unusually high reduction potential (due to high GSH level) in cancer cells.
  • Example 5 Cellular uptake and intracellular distribution of PEG- PPMD and PEG-PCMD nanoparticles
  • fluorescence probe molecule coumarin-6 (C6) was encapsulated in PEG-PPMD- 12%, 43%, and 70% PDL and PEG-PCMD- 13%, 41%, and 70% CL micelles according to the protocol employed for preparation of DTX-loaded micelles (Example 2, A) and the cellular uptake of the C6-loaded micelles was examined by flow cytometry using HeLa cells. Specifically, HeLa cells in 500 ⁇ medium at a density of 4.0 x 10 5 cells/mL were seeded in a 24- well plate overnight at 37 °C under 5% CO2.
  • the cellular internalization efficiency of the micelle samples was analyzed by FACSCalibur at an excitation wavelength of 488 nm and an emission wavelength of 585 nm (10000 cells per group).
  • C6-loaded micelles The intracellular location of C6-loaded micelles was visualized using confocal laser scanning microscopy (CLSM).
  • CLSM confocal laser scanning microscopy
  • HeLa cells (1 x 10 5 cells/well) were seeded on 15 mm glass-bottom dishes in a 6-well plate overnight. The cells were incubated with free C6, C6-loaded PEG-PPMD-12% PDL micelles or C6-loaded PEG-PCMD-13% CL particles at a C6 concentration of 0.2 ⁇ g/mL. After incubation for 2 and 6 h, the media were removed and the cells were washed three times with cold PBS solution.
  • the cell lysosomes were stained with 75 nM Lysotracker-red and the cell nuclei were stained with 10 ⁇ g/mL Hoechst 33342.
  • the cells were then rinsed, fixed with paraformaldehyde (PFA), washed by PBS and then observed by CLSM.
  • the excitation wavelength for detecting Hoechst 33342, Lysotracker-red, and C6 was 405 nm, 577 nm and 467 nm, respectively.
  • nanoparticles encapsulating anticancer drugs can be triggered by tumor- relevant acidic pH and intracellular GSH to rapidly release the drug molecules for efficient killing of cancer cells.
  • cytotoxicity of both blank micelles and DTX- loaded micelles were evaluated against HeLa cells and CT-26 cells using an MTT method described previously (Liu, et al, Polym. Chem. 2015, 6, 1997-2010.). Briefly, cells (3.0 x 10 3 cells/well) were seeded in 96- well plates and allowed to adhere overnight. Then the culture medium was removed and 200 of the fresh medium containing different concentrations of the blank or DTX- loaded micelles were added to each well.
  • the culture media with different pH (7.4 or 6.5) and different reduction potential (10 mM GSH or 0.2 mM BSO) were used to mimic different intracellular environments. After incubation with micelles under different medium environments for additional 48 h and then subjected to MTT assay. The absorbance of the solutions at 570 nm was measured on a microplate reader (BioTek Synergy4). Cell viabilities were calculated as the values in percentage of (absorbance of cells treated)/(absorbance of cells without micelle treatment).
  • cytotoxicity of DTX-loaded PEG-PPMD and PEG-PCMD micelles were also evaluated by MTT assay against HeLa cells and CT-26 cells. Similarly, cells were seeded into 96-well plates and kept at 37 °C in 5% CO2 atmosphere.
  • the culture medium was replaced by the medium containing one of the following four components: (i) various amount of DTX in the micelles with medium pH of 7.4, (ii) various amount of DTX in the micelles with medium pH of 6.5, (iii) various amount of DTX in the micelles and 10 mM glutathione (GSH) with medium pH of 7.4, (iv) various amount of DTX in the micelles and 0.2 mM L-buthionine-(5,R)-sulfoximine (BSO) with medium pH of 7.4.
  • GSH glutathione
  • BSO L-buthionine-(5,R)-sulfoximine
  • the PEG-PPMD and PEG-PCMD micelles were incubated with erythrocytes (RBCs) to determine blood compatibility of the copolymers.
  • Human blood was centrifuged at 2500 rpm for 5 min at 4 °C and the RBC cells were washed three times with PBS (10 mM, pH 7.4).
  • the blank micelles (400 ⁇ g/mL) and 1 x 108 RBCs were mixed in PBS and the mixtures were then incubated for 2, 12 and 24 h at 37 °C. Thereafter, the samples were centrifuged for 10 min at 1000 rpm and the supernatants were analyzed by a microplate reader to measure their absorbance at 413 nm. Additionally, Triton- 100 (1%, w/v) was tested as a positive control and isotonic PBS was tested as a negative control. Hemolysis value was calculated by following equation:
  • Asampie, APBS, and Amton represent the absorbance intensity values of the supernatants from RBCs treated with the micelle samples, PBS and Triton X-100, respectively.
  • PEG-PCMD micelles was evaluated on HeLa cells and CT-26 cells. All blank micelles exhibited minimal cytotoxicity and the viabilities of the cells treated with the micelles were over 80% at various polymer concentrations up to 400 ⁇ g/mL ( Figures 8A-8D). Further, these PEG-PPMD and PEG- PCMD micelle samples are compatible with human blood, showing essentially no hemolytic activity ( ⁇ 3% hemolysis value, Figure 9) and no tendency to induce erythrocyte agglutination even at a high polymer concentration of 400 ⁇ g/mL.
  • FIG. 10A-10F show the cell viability values after treating the cells with the PEG-PPMD micelles at pH of 7.4 and 6.5 under a controlled intracellular reduction potential.
  • the cytotoxicity of the micelles is dependent on the composition of PEG-PPMD copolymers, medium pH and intracellular reduction potential.
  • the micelles formed from PEG-PPMD copolymer with a low PDL content appear to possess higher efficacy than those formed from the PDL-rich copolymer.
  • the DTX-loaded micelles of PEG-PPMD- 12% PDL, PEG-PPMD-43% PDL and PEG-PPMD-70% PDL against HeLa cells at pH of 7.4 yielded ICso values of 0.535, 0.671 and 2.94 ⁇ g/mL, respectively.
  • the viability of the HeLa cells and CT-26 cells was lower upon treatment at pH of 6.5 vs pH of 7.4 ( Figures 10A-10F).
  • the ICso values of DTX-loaded PEG- PPMD- 12% PDL micelles against HeLa and CT-26 cell lines are respectively 0.535 and 0.604 ⁇ g/mL at pH of 7.4, which decrease correspondingly to 0.248 and 0.171 ⁇ g/mL at a pH of 6.5.
  • the intracellular reduction potential of both cell types can be increased by feeding the cells with free reductant GSH or reduced by feeding the cells with BSO (an inhibitor for cellular GSH synthesis).
  • BSO an inhibitor for cellular GSH synthesis
  • Example 7 In vivo antitumor efficiency
  • CT-26 cells (lxlO 6 cells) were injected subcutaneously in the right back of Balb/C mice (5 weeks, 16 g). When tumors reached 50— 100 mm 3 in volume, treatments were started and the initial treatment day was designated as day 0.
  • mice were randomly assigned to one of the following 4 groups (at least 4 mice in each group): 0.9% NaCl (control), free DTX (Duopafei ® ), DTX-loaded PEG-PPMD-12% PDL micelles and DTX-loaded PEG-PCMD-13% CL micelles.
  • mice were injected intravenously through the tail vein with free DTX, DTX-loaded PEG-PPMD-12% PDL micelles and PEG-PCMD-13% CL micelles (at 10 mg/kg DTX dose) every three days for four times.
  • the control group of mice was administered via injection with 0.9% NaCl following the same procedure.
  • mice The tumor volume was measured every other day using a vernier caliper and the body weight of the mice was recorded at the same time. At day 27, the mice were sacrificed to collect the tumors and important organs (heart, liver, spleen, lung and kidney), whose tissues were analyzed by hematoxylin-eosin (H&E) staining to determine the antitumor effects of the micelles.
  • H&E hematoxylin-eosin
  • Figures 12A and 12B illustrate variations of the tumor volume and body weight of the mice during the 21 -day treatment period. The results clearly show that the DTX-loaded micelles are substantially more potent than free DTX in inhibiting growth of the tumor xenografts.
  • the average tumor volume of the mice treated with 0.9% NaCl, free DTX, DTX-loaded PEG-PPMD-12% PDL and DTX-loaded PEG- PCMD-13% CL micelles were 2300, 994, 524 and 658 mm 3 , respectively ( Figure 12A).
  • the antitumor potency follows the order: DTX-loaded PEG-PPMD-12% PDL micelles > DTX-loaded PEG-PCMD-13% CL micelles > free DTX > 0.9% NaCl.
  • DTX-loaded PEG-PPMD-12% PDL and DTX-loaded PEG-PCMD-13% CL micelles > free DTX > 0.9% NaCl.
  • CT-26 tumor-bearing mouse models were used to investigate the biodistribution of DiR- loaded polymeric micelles.
  • Fluorescence probe DiR was encapsulated in PEG-PPMD-12% PDL and PEG-PCMD-13% CL micelles according to the protocol employed for preparation of DTX- loaded micelles (Example 2, A).
  • CT-26 cells (lxlO 6 cells/0.1 mL) were injected subcutaneously in the right back of Balb/C mice (5 weeks, 16 g). When tumors reached 200-300 mm 3 in volume, the DiR-loaded PEG-PPMD-12% PDL and DiR-loaded PEG-PCMD- 13% CL micelles (0.5 mg/kg DiR) were injected intravenously through the tail vein.
  • DiR-loaded PEG-PPMD-12% PDL and PEG-PCMD-13% CL micelles were injected intravenously into the CT26 tumor-bearing mice, and in vivo biodistribution was monitored using IVIS imaging system. The accumulation of the DiR-loaded micelles in tumor was time-dependent.
  • PEGylation was remarkably effective in improving the in vivo circulation time and stability of both PEG-PPMD-12% PDL and PEG- PCMD-13% CL micelles.
  • the intensity of fluorescence signals measured from the ex vivo organs and tumors confirmed the results of in vivo observation.
  • the micelle samples were largely distributed in the liver, lung, spleen and tumor.
  • the PEG-PPMD- 12% PDL and PEG-PCMD-13% CL micelles are more abundantly present in the tumor than in the other organs examined.
  • nanoparticle drug delivery systems have been constructed which are stable under physiological conditions and responsive to tumor-relevant pH and intracellular reduction potential.
  • the nanoparticles were fabricated from new enzymatic PEG-PPMD and PEG- PCMD block copolymers via a self-assembly process in aqueous solution.
  • a reductant e.g., DTT or GSH
  • the nanosized micelle particles rapidly swelled and disintegrated due to the protonation of amino groups and reductive cleavage of disulfide bonds in the micelle cores.
  • the DTX-loaded PEG-PPMD and PEG-PCMD micelles were triggered synergistic ally by both acidic endosomal pH and a high

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Abstract

Polymers, containing one or more poly(amine-co-disulfide ester) polymeric units, and polymeric nanoparticles formed therefrom, are provided. The nanoparticles possess dual pH-responsive and redox- responsive properties, giving rise to enhanced release of cargo in disease- relevant microenvironments. The nanoparticles also show selective uptake by diseased tissues, compared to healthy tissues. The polymers are synthesized via enzyme-catalyzed polymerization reactions. Also provided are pharmaceutical compositions including nanoparticles having an effective amount of chemotherapeutic agents for inhibit the growth of tumor tissue.

Description

POLY(AMINE-CO-DISULFIDE ESTER) NANOPARTICLES AND METHODS OF USE
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of and priority to U.S. Application Nos. 62/513,737 filed June 1, 2017 and 62/585,915 filed November 14, 2017, the contents of which are incorporated by reference in their entirety.
FIELD OF THE INVENTION
This invention is generally in the field of stimuli-responsive polymeric nanoparticles, particularly dual pH-responsive and redox- responsive polymeric nanoparticles for delivering therapeutic, diagnostic, and/or prophylactic agents to diseased tissues, particularly those releasing enzymes and oxidants leading to pH changes.
BACKGROUND OF THE INVENTION
Polymeric nanoparticles are important platforms for delivering cargo (e.g. therapeutic, diagnostic, and/or prophylactic agents) to subjects. They are known to protect these cargoes from premature biodegradation and to increase their accumulation in some diseased tissues via the enhanced permeability and retention (EPR) effect (Torchilin, et al, Adv Drug Deliv Rev. 2011, 63, 131-135; Davis, et al., Nat Rev Drug Discov. 2008, 7, 771- 782; Ullrich, et al, Chem Rev. 1999, 99, 3181-3198).
It is generally known that the extracellular pH of normal tissues and blood is different from that of tumor tissues: the pH of normal tissues and blood is slightly basic (pH of -7.4), while that of tumors are weakly acidic (pH of 5.7-7.0) (Potineni, et al., J Control Release 2003, 86, 223-234; Yin, et al., J Control Release 2008, 126, 130-138; Bae, et al., Angewandte Chemie. 2003, 42, 4640-4643; Stubbs, et al, Mol Med Today. 2000, 6, 15-19). In addition to acidic pH, glutathione (GSH, a tripeptide containing cysteine) is also overexpressed in tumor tissues compared to normal tissues. GSH concentration is approximately 1 to 10 mM in normal cells, but is at least four times higher in many tumor cells (Wu, et al, J Nutr. 2004, 134, 489- 492; Kuppusamy, et al, Cancer Res. 2002, 62, 307-312). In contrast, in a normal extracellular environment (e.g., plasma), GSH concentration is only around 2 to 20 μΜ (Wu, et al, J Nutr. 2004, 134, 489-492).
Recently, substantial efforts have been made to improve the cargo- delivering efficiency of polymeric nanoparticle formulations by using functional nanoparticles that are responsive to stimuli (e.g., acidic pH or glutathione) present at tumor sites (Du, et al, J Am Chem Soc. 2011, 133, 17560-17563; Zugates, et al., J Am Chem Soc. 2006, 128, 12726-12734; Wu, et al., J Am Chem Soc. 2014, 136, 3145-3155; Karimi, et al., Chem Soc Rev. 2016, 45, 1457-1501). Upon arrival at the disease sites after administration, the polymeric nanoparticles are triggered by the stimuli to rapidly disintegrate and unload encapsulated therapeutic agents for efficient killing of cancer cells.
Among stimuli-responsive nanoparticles reported, (Karimi, et al., Chem Soc Rev. 2016, 45, 1457-1501; Bahadur, et al, Adv Mater. 2012, 24, 6479-6483; Cheng, et al, Biomaterials 2013, 34, 3647-3657; Chen, et al, Small 2014, 10, 2678-2687) only a few (Yoon, et al, Small 2013, 9, 284- 293; Cheng, et al, Macromol Biosci. 2014, 14, 347-358; Chen, et al, Biomacromolecules 2011, 12, 3601-3611; Chen, et al, Nanoscale 2015, 7, 15763-15779; Lu, et al, Chem Commun. 2014, 50, 15105-15108; Yi, et al, Nanoscale 2016, 8, 5985-5995) are biodegradable drug nanocarriers capable of responding to tumor relevant pH and intracellular reduction potential and only two such nanocarrier systems Chen, et al, Nanoscale 2015, 7, 15763- 15779; Yi, et al, Nanoscale 2016, 8, 5985-5995) have been shown to be effective in vivo.
Despite all of these efforts, polymeric nanoparticles still exhibit problems, such as inefficient release of cargo under disease-relevant conditions, non-specific uptake by tissues, premature release of significant quantities of cargo, premature degradation during systemic circulation, and a combination thereof. There remains a need for the development of improved polymeric nanoparticles that circumvent these existing problems.
Therefore, it is an object of the invention to provide polymeric nanoparticles with efficient release of therapeutic, diagnostic, and/or prophylactic agents under disease-relevant conditions compared to physiological pH (-7.4).
It is another object of the invention to provide polymeric
nanoparticles with efficient release of therapeutic, diagnostic, and/or prophylactic agents in acidic pH extracellular and intracellular environments, and low redox potential environments, wherein the polymeric nanoparticles possess dual pH-responsive and redox-responsive properties.
It is a further object of the invention to provide polymers with dual pH-responsive and redox-responsive properties.
It is also an object of the invention to provide methods of making polymers with pH-responsive and redox-responsive chemical units.
SUMMARY OF THE INVENTION
Polymers containing one or more poly(amine-co-disulfide ester) polymeric units, and polymeric nanoparticles formed therefrom, have been developed which are stable under physiological conditions (such as pH 7.4), but have dual pH-responsive, and redox-responsive properties, as shown by changes in the sizes of the polymeric nanoparticles in environments having pH less than 7 and/or low redox potentials. These properties give rise to enhanced release of cargo from the nanoparticles, in diseased tissues whose microenvironments have pH less than 7 and/or low redox potential, such as tumors. The nanoparticles also show selective uptake by diseased tissues, compared to healthy tissues. The nanoparticles have a diameter between 50 nm and 500 nm, inclusive. In some forms, the zeta potential of the nanoparticle is between -10 mV and +10 mV, inclusive.
The polymers include a polymeric unit that can be represented by the general formula: wherein:
Figure imgf000005_0001
A, B, C, and D independently comprise monomeric units derived from a lactones, a polyfunctional molecule containing an amine group and a hydroxyl group, a molecule containing a disulfide bond, or hydrophilic polymer; the monomeric units comprise the lactone, the polyfunctional molecule containing an amine group and a hydroxyl group, and the molecule containing the disulfide bond;
a, b, c, and d are independently integers between 0 and 1000, inclusive, with the proviso that the sum (a + b + c + d) is greater than one; and
h is an integer between 1 and 1000, inclusive.
In some forms, the polymeric unit has the formula:
Figure imgf000006_0001
wherein:
x, y, and z are independently integers between 1 and 1000, inclusive; preferably, m is 3 or 12, or a combination thereof;
preferably, p, q, n, and t are 2;
preferably, R8 and R10 are O; and
preferably R9 methyl.
The polymers can further include a block of a hydrophilic polyalkylene oxide, such as polyethylene glycol. The molecular weight of the polymers can be between 1 kDa and 50 kDa, preferably between 5 kDa and 15 kDa.
Also described are methods of making the polymers. Preferably, the polymers are synthesized by mixing reactants such as a lactone, a molecule containing a disulfide bond, and a polyfunctional molecule containing an amine group, a hydroxyl group, preferably both, with an enzyme catalyst, such as a lipase, under conditions in which the polymers are formed. The reactants can further include a hydrophilic polymer, such as polyalkylene oxide (e.g. polyethylene glycol).
Pharmaceutical compositions including nanoparticles having an effective amount of a therapeutic agent (e.g. chemotherapeutic agent) are also provided, which can be used, for example, for in vitro and in vivo delivery of the therapeutic agents.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1A and IB are NMR spectral data of carbonyl C-13 resonance absorptions of different diads in polyethylene glycol (2 kDa)- poly(ω-pentadecalactone (70%)-co-N-methyldiethyleneamine-co-3,3'- dithioproprionate) (PEG2K-PPMD-70% PDL) (Figure 1A), and polyethylene glycol (2 kDa)-poly(ε-caprolactone (70%)-co-N- methyldiethyleneamine-co-3,3'-dithioproprionate) (PEG2K-PCMD-70% CL) (Figure IB). The solvent in each case was CDCI3.
Figures 2A and 2B are line graphs of the intensity ratios (I3/I1) as a function of the logarithm of polymer concentration (Log C) for PEG-PPMD (Figure 2A) and PEG-PCMD (Figure 2B) copolymers.
Figures 3A and 3B are column graphs of the nanoparticle size distributions of docetaxel (DTX)-loaded nanoparticles containing PEG- PPMD-12% PDL (Figure 3A) and PEG-PCMD-13% CL (Figure 3B). The scale bar = 100 nm. As shown in the examples, nanoparticles formed from polymers containing ω-pentadecalactone, m = 12 (PDL), generally had smaller average sizes (between 84 nm and 121 nm), compared to nanoparticles formed from polymers containing ε-caprolactone, m = 3 (CL), (between 140 nm and 203 nm). The sizes were determined using transmission electron microscopy (TEM).
Figures 4A and 4B are line graphs of the stabilities of the sizes of selected PEG-PPMD (Figure 4A) and PEG-PCMD (Figure 4B) blank micelles in PBS (10 mM, pH 7.4) with 10% FBS, as a function of time (days). Data are given as the mean + SD (n = 3).
Figures 5A-5F are line graphs of the size distributions of blank nanoparticles incubated with different PBS media for 48 h: PEG-PPMD-12% PDL (Figure 5A); PEG-PPMD-43% PDL (Figure 5B); PEG-PPMD-70% PDL (Figure 5C); PEG-PCMD-13% CL (Figure 5D); PEG-PCMD-41% CL (Figure 5E); and PEG-PCMD-70% CL (Figure 5F). The sizes of the micelles were determined using dynamic light scattering (DLS). Figures 6A-6F are line graphs of the in vitro drug release from DTX- loaded micelles of the following polymers incubated in PBS under different pH and redox conditions: PEG-PPMD-12% PDL (Figure 6A); PEG-PPMD- 43% PDL (Figure 6B); PEG-PPMD-70% PDL (Figure 6C); PEG-PCMD- 13% CL (Figure 6D) ; PEG-PCMD-41 % CL (Figure 6E) ; and PEG-PCMD- 70% CL (Figure 6F).
Figure 7 is a column graph showing uptake of free coumarin-6 (C6) and C6-loaded PEG-PPMD and PEG-PCMD micelles by HeLa cells. The intracellular C6 mean fluorescence intensity (MFI) values were measured by flow cytometry after 1-8 h incubation. Data are given as mean + SD (n = 3).
Figures 8A-8D are column graphs showing the viabilities of HeLa cells (Figures 8A and 8C) and CT-26 cells (Figures 8B and 8D) after incubation for 48 h with various doses of PEG-PPMD (Figures 8A and 8B) and PEG-PCMD (Figures 8C and 8D) blank micelles. Data are given as the mean + SD (n = 3).
Figure 9 is a column graph showing the hemolytic activities of various blank PEG-PPMD and PEG-PCMD micelles after incubation with erythrocytes for 2, 12 and 24 h. Data are given as the mean + SD (n = 3).
Figures 10A-10F are column graphs showing the viabilities of HeLa cells (Figures 10A, IOC, and 10E) and CT-26 cells (Figures 10B, 10D, and 10F) after incubation for 48 h with various doses of DTX-loaded micelles of PEG-PPMD-12% PDL (Figures 10A and 10B), PEG-PPMD-43% PDL (Figures IOC and 10D), PEG-PPMD-70% PDL (Figures 10E and 10F). Data are given as the mean + SD (n = 3). * represents <0.05, **represents <0.01.
Figures 11A-11F are column graphs showing the viabilities of HeLa cells (Figures HA, 11C, HE) and CT-26 cells (Figures 11B, 11D, 11F) after incubation for 48 h with various doses of DTX-loaded micelle of PEG- PCMD- 13% CL (Figures HA and 11B), PEG-PCMD-41% CL (Figures llC and 11D), PEG-PCMD-70% CL (Figures HE and 11F). Data are given as the mean + SD (n = 3). * represents <0.05, ** represents <0.01
Figures 12A and 12B are line graphs showing the measured tumor volumes (Figure 12A) and body weights (Figure 12B) of Balb/C mice treated with free DTX (Duopafei®), DTX-loaded PEG-PPMD- 12% PDL and PEG-PCMD-13% CL micelle formulations at 4 x 10 mg/kg DTX dose for 21 days. 0.9% NaCl solution was used as a control. Arrows indicate the dates when the formulations were administered.
Figure 13 is a column graph showing in vivo distribution of DiR- loaded PEG-PPMD- 12% PDL and PEG-PCMD-13% CL micelles injected intravenously through the tail vein of mice.
Figure 14 is an illustration of some of the steps involving cargo (e.g. DTX) delivery by PEG-PPMD and PEG-PCMD nanoparticles to cells (e.g. cancer cells).
DETAILED DESCRIPTION1 OF THE INVENTION
L Definitions
The terms "lactone" and "lactone unit" are used to describe define a chemical compound that includes a cyclic ester, or the open chain chemical structure that results from the cleavage of the ester bond in the cyclic ester. For example, lactone is used to describe the cyclic ester shown below, and the corresponding lactone-derived open chain structure;
Figure imgf000009_0001
m being an integer, such as between 1 and 30, inclusive. The open chain structure is formed via methods known in the art, including but not limited to, solvolysis, such as hydrolysis, and enzymatic cleavage.
"Positively ionizable atom" refers to an atom that can be protonated under acidic conditions, resulting in the atom having a. positive formal charge. An example of a positively ionizable atom is nitrogen.
"Acidic diseased tissue" refers to a tissue in a diseased state, which has a pH, particularly extracellular matrix pH, of less than 7. Exemplary diseases include cancer, inflammation, stroke, arthritis, and ischemia.
"Amphiphilic" refers to a property where a molecule has both a hydrophilic portion and a hydrophobic portion. Often, an amphiphilic compound has a hydrophilic portion covalently attached to a hydrophobic portion. In some forms, the hydrophilic portion is soluble in water, while the hydrophobic portion is insoluble in water. In addition, the hydrophilic and hydrophobic portions may have either a formal positive charge, or a formal negative charge. However, overall they will be either hydrophilic or
hydrophobic. An amphiphilic compound can be an amphiphilic polymer, such that the hydrophilic portion can be a hydrophilic polymer, and the hydrophobic portion can be a hydrophobic polymer.
"Hydrophilic," refers to the property of having affinity for water. For example, hydrophilic polymers (or hydrophilic polymer segments) are polymers (or polymer segments) that are primarily soluble in aqueous solutions and/or have a tendency to absorb water. In general, the more hydrophilic a polymer is, the more that polymer tends to dissolve in, mix with, or be wetted by water. Hydrophilicity can be quantified by measuring its partition coefficient between water (or a buffered aqueous solution) and a water-immiscible organic solvent, such as octanol, methylene chloride, or methyl i<?ri-butyl ether. If after equilibration a greater concentration of the compound is attained in water than in the organic solvent, then the compound is considered hydrophilic. For example, if the organic solvent is octanol, then a negative log P value indicates that the compound is hydrophilic. "Hydrophilic" may also refer to a material that when applied to a surface, such as glass, forms a contact angle with water, which is less than the contact angle of water on a surface of glass without the material.
"Hydrophobic," as used herein, refers to the property of lacking affinity for or repelling water. For example, the more hydrophobic a polymer (or polymer segment), the more that polymer (or polymer segment) tends to not dissolve in, not mix with, or not be wetted by water.
Hydrophobicity can be quantified by measuring its partition coefficient between water (or a buffered aqueous solution) and a water-immiscible organic solvent, such as octanol, methylene chloride, or methyl tert-butyl ether. If after equilibration a greater concentration of the compound is attained in the organic solvent than in water, the compound is considered hydrophobic. For example, if the organic solvent is octanol, then a positive log P value indicates that the compound is hydrophobic. "Hydrophobic" may also refer to a material that when applied to a surface, such as glass, forms a contact angle with water, which is greater than the contact angle of water on a surface of glass without the material. Hydrophilicity and hydrophobicity can also be quantitated in relative terms, such as, but not limited to, a spectrum of
hydrophilicity/hydrophobicity within a group of polymers or polymer segments. In some forms wherein two or more polymers are being discussed, the term "hydrophobic polymer" can be defined based on the polymer's relative hydrophobicity when compared to another, more hydrophilic polymer.
"Nanoparticle" generally refers to a particle having a diameter, such as an average diameter, greater than or equal to 10 nm and less than 1 micron. The particles can have any shape. Nanoparticles having a spherical shape are generally referred to as "nanospheres."
"Mean particle size" generally refers to the statistical mean particle size (diameter) of the particles in a population of particles. The diameter of an essentially spherical particle may refer to the physical or hydrodynamic diameter. The diameter of a non- spherical particle may refer preferentially to the hydrodynamic diameter. As used herein, the diameter of a non- spherical particle may refer to the largest linear distance between two points on the surface of the particle. Mean particle size can be measured using methods known in the art, such as dynamic light scattering, and transmission electron microscopy.
"Analog" as relates to a given compound, refers to another compound that is structurally similar, functionally similar, or both, to the specified compound. Structural similarity can be determined using any criterion known in the art, such as the Tanimoto coefficient that provides a quantitative measure of similarity between two compounds based on their molecular descriptors. Preferably, the molecular descriptors are 2D properties such as fingerprints, topological indices, and maximum common substructures, or 3D properties such as overall shape, and molecular fields. Tanimoto coefficients range between zero and one, inclusive, for dissimilar and identical pairs of molecules, respectively. A compound can be considered an analog of a specified compound, if it has a Tanimoto coefficient with the specified compound between 0.5 and 1.0, inclusive, preferably between 0.7 and 1.0, inclusive, most preferably between 0.85 and 1.0, inclusive. A compound is functionally similar to a specified, if it induces the same pharmacological effect, physiological effect, or both, as the specified compound. "Analog" can also refer to a modification including, but not limited to, hydrolysis, reduction, or oxidation products, of the disclosed compounds. Hydrolysis, reduction, and oxidation reactions are known in the art.
The terms "inhibit" and "reduce" means to reduce or decrease in activity or expression. This can be a complete inhibition or reduction of activity or expression, or a partial inhibition or reduction. Inhibition or reduction can be compared to a control or to a standard level. Inhibition can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%.
The phrases "parenteral administration" and "administered parenterally" are art-recognized terms, and include modes of administration other than enteral and topical administration, such as injections, and include without limitation intravenous, intramuscular, intrapleural, intravascular, intrapericardial, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradennal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion.
"Small molecule" generally refers to an organic molecule that is less than about 2000 g/mol in molecular weight, less than about 1500 g/mol, less than about 1000 g/mol, less than about 800 g/mol, or less than about 500 g/mol. In some forms, small molecules are non-polymeric and/or non- oligomeric.
The terms "subject," "individual," and "patient" refer to any individual who is the target of treatment using the disclosed compositions. The subject can be a vertebrate, for example, a mammal. Thus, the subject can be a human. The subjects can be symptomatic or asymptomatic. The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered. A subject can include a control subject or a test subject.
"Sustained release" as used herein refers to release of a substance over an extended period of time in contrast to a bolus type administration in which the entire amount of the substance is made biologically available at one time.
"Effective amount" and "therapeutically effective amount," used interchangeably, as applied to the nanoparticles, therapeutic agents, and pharmaceutical compositions described herein, mean the quantity necessary to render the desired therapeutic result. For example, an effective amount is a level effective to treat, cure, or alleviate the symptoms of a disease for which the composition and/or therapeutic agent, or pharmaceutical composition, is/are being administered. Amounts effective for the particular therapeutic goal sought will depend upon a variety of factors including the disease being treated and its severity and/or stage of development/progression; the bioavailability and activity of the specific compound and/or antineoplastic, or pharmaceutical composition, used; the route or method of administration and introduction site on the subject; the rate of clearance of the specific composition and other pharmacokinetic properties; the duration of treatment; inoculation regimen; drugs used in combination or coincident with the specific composition; the age, body weight, sex, diet, physiology and general health of the subject being treated; and like factors well known to one of skill in the relevant scientific art. Some variation in dosage will necessarily occur depending upon the condition of the subject being treated, and the physician or other individual administering treatment will, in any event, determine the appropriate dosage for an individual patient.
"Substituted" refers to all permissible substituents of the compounds or functional groups described herein. In the broadest sense, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, but are not limited to, halogens, hydroxyl groups, or any other organic groupings containing any number of carbon atoms, preferably 1-14 carbon atoms, and optionally include one or more heteroatoms such as oxygen, sulfur, or nitrogen grouping in linear, branched, or cyclic structural formats. Representative substituents include alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio, phenylthio, substituted phenylthio, arylthio, substituted arylthio, cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl, polyaryl, substituted polyaryl, C3-C20 cyclic, substituted C3-C20 cyclic, heterocyclic, substituted heterocyclic, amino acid, poly(lactic-co-glycolic acid), peptide, and polypeptide groups. Such alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio, phenylthio, substituted phenylthio, arylthio, substituted arylthio, cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl, polyaryl, substituted polyaryl, C3-C20 cyclic, substituted C3-C20 cyclic, heterocyclic, substituted heterocyclic, amino acid, poly(lactic-co- glycolic acid), peptide, and polypeptide groups can be further substituted.
Heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. It is understood that "substitution" or "substituted" includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, i.e. a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. "Alkyl," as used herein, refers to the radical of saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl, cycloalkyl (alicyclic), alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl. In preferred forms, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straight chains, C3-C30 for branched chains), preferably 20 or fewer, more preferably 15 or fewer, most preferably 10 or fewer. Alkyl includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, i-butyl, pentyl, hexyl, heptyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the like.
Preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure. The term "alkyl" (or "lower alkyl") as used throughout the specification, examples, and claims is intended to include both "unsubstituted alkyls" and "substituted alkyls," the latter of which refers to alkyl moieties having one or more substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents include, but are not limited to, halogen, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, a phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamide, sulfonyl, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety.
Unless the number of carbons is otherwise specified, "lower alkyl" as used herein means an alkyl group, as defined above, but having from one to ten carbons, more preferably from one to six carbon atoms in its backbone structure. Likewise, "lower alkenyl" and "lower alkynyl" have similar chain lengths. Throughout the application, preferred alkyl groups are lower alkyls. In preferred forms, a substituent designated herein as alkyl is a lower alkyl.
"Alkyl" includes one or more substitutions at one or more carbon atoms of the hydrocarbon radical as well as heteroalkyls. Suitable substituents include, but are not limited to, halogens, such as fluorine, chlorine, bromine, or iodine; hydroxyl; -NRR', wherein R and R' are independently hydrogen, alkyl, or aryl, and wherein the nitrogen atom is optionally quaternized; -SR, wherein R is hydrogen, alkyl, or aryl; -CN; - N02; -COOH; carboxylate; -COR, -COOR, or -CON(R)2, wherein R is hydrogen, alkyl, or aryl; azide, aralkyl, alkoxyl, imino, phosphonate, phosphinate, silyl, ether, sulfonyl, sulfonamido, heterocyclyl, aromatic or heteroaromatic moieties, haloalkyl (such as -CF3, -CH2-CF3, -CCI3); -CN; -
NCOCOCH2CH2; -NCOCOCHCH; -NCS; and combinations thereof.
It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. For instance, the substituents of a substituted alkyl may include halogen, hydroxy, nitro, thiols, amino, azido, imino, amido, phosphoryl (including phosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls (including ketones, aldehydes, carboxylates, and esters), haloalkyls, -CN and the like. Cycloalkyls can be substituted in the same manner.
"Heteroalkyl," as used herein, refers to straight or branched chain, or cyclic carbon-containing radicals, or combinations thereof, containing at least one heteroatom. Suitable heteroatoms include, but are not limited to, O, N, Si, P and S, wherein the nitrogen, phosphorous and sulfur atoms are optionally oxidized, and the nitrogen heteroatom is optionally quaternized.
The terms "alkoxyl" or "alkoxy," "aroxy" or "aryloxy," generally describe compounds represented by the formula -ORv, wherein Rv includes, but is not limited to, substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, arylalkyl, heteroalkyls, alkylaryl, alkylheteroaryl.
The terms "alkoxyl" or "alkoxy" as used herein refer to an alkyl group, as defined above, having an oxygen radical attached thereto.
Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert- butoxy and the like. An "ether" is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as can be represented by one of -O- alkyl, -O-alkenyl, and -O-alkynyl. The term alkoxy also includes cycloalkyl, heterocyclyl, cycloalkenyl, heterocycloalkenyl, and arylalkyl having an oxygen radical attached to at least one of the carbon atoms, as valency permits. A "lower alkoxy" group is an alkoxy group containing from one to six carbon atoms.
The term "substituted alkoxy" refers to an alkoxy group having one or more substituents replacing one or more hydrogen atoms on one or more carbons of the alkoxy backbone. Such substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), silyl, ether, ester, thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino (or quarternized amino), amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamide, sulfonyl, heterocyclyl, alkylaryl, haloalkyl, -CN, aryl, heteroaryl, and combinations thereof.
The term "alkenyl" as used herein is a hydrocarbon group of from 2 to 24 carbon atoms and structural formula containing at least one carbon- carbon double bond. Asymmetric structures such as (AB)C=C(CD) are intended to include both the E and Z isomers. This may be presumed in structural formulae herein wherein an asymmetric alkene is present, or it may be explicitly indicated by the bond symbol C.
The term "alkynyl group" as used herein is a hydrocarbon group of 2 to 24 carbon atoms and a structural formula containing at least one carbon- carbon triple bond.
The term "aryl" as used herein is any C5-C26 carbon-based aromatic group, fused aromatic, fused heterocyclic, or biaromatic ring systems.
Broadly defined, "aryl," as used herein, includes 5-, 6-, 7-, 8-, 9-, 10-, 14-, 18-, and 24-membered single-ring aromatic groups, including, but not limited to, benzene, naphthalene, anthracene, phenanthrene, chrysene, pyrene, corannulene, coronene, etc. "Aryl" further encompasses polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings (i.e., "fused rings") wherein at least one of the rings is aromatic, e.g., the other cyclic ring or rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or he terocycles. The aryl group can be substituted with one or more groups including, but not limited to, alkyl, alkynyl, alkenyl, aryl, halide, nitro, amino, ester, ketone, aldehyde, hydroxy, carboxylic acid, or alkoxy.
The term "substituted aryl" refers to an aryl group, wherein one or more hydrogen atoms on one or more aromatic rings are substituted with one or more substituents including, but not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxy, carbonyl (such as a ketone, aldehyde, carboxyl, alkoxycarbonyl, formyl, or an acyl), silyl, ether, ester, thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino (or quarternized amino), amido, amidine, imine, cyano, nitro, azido, sulfhydryl, imino, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, alkylaryl, haloalkyl (such as CF3, -CH2-CF3, -CCI3), -CN, aryl, heteroaryl, and combinations thereof.
"Heterocycle," "heterocyclic" and "heterocyclyl" are used interchangeably, and refer to a cyclic radical attached via a ring carbon or nitrogen atom of a monocyclic or bicyclic ring containing 3- 10 ring atoms, and preferably from 5-6 ring atoms, consisting of carbon and one to four heteroatoms each selected from the group consisting of non-peroxide oxygen, sulfur, and N(Y) wherein Y is absent or is H, O, C1- C10 alkyl, phenyl or benzyl, and optionally containing 1-3 double bonds and optionally substituted with one or more substituents. Heterocyclyl are distinguished from heteroaryl by definition. Examples of heterocycles include, but are not limited to piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl,
dihydrofuro[2,3-£>]tetrahydrofuran, morpholinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pyranyl, 2H-pyrrolyl, 4H-quinolizinyl, quinuclidinyl, tetrahydrofuranyl, 6H-l,2,5-thiadiazinyl. Heterocyclic groups can optionally be substituted with one or more substituents as defined above for alkyl and aryl.
The term "heteroaryl" refers to C5-C26-membered aromatic, fused aromatic, biaromatic ring systems, or combinations thereof, in which one or more carbon atoms on one or more aromatic ring structures have been substituted with a heteroatom. Suitable heteroatoms include, but are not limited to, oxygen, sulfur, and nitrogen. Broadly defined, "heteroaryl," as used herein, includes 5-, 6-, 7-, 8-, 9-, 10-, 14-, 18-, and 24-membered single-ring aromatic groups that may include from one to four heteroatoms, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, tetrazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. The heteroaryl group may also be referred to as "aryl heterocycles" or "heteroaromatics". "Heteroaryl" further encompasses polycyclic ring systems having two or more rings in which two or more carbons are common to two adjoining rings (i.e. , "fused rings") wherein at least one of the rings is heteroaromatic, e.g., the other cyclic ring or rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heterocycles, or combinations thereof. Examples of heteroaryl rings include, but are not limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2- dithiazinyl, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H- indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl,
naphthyridinyl, octahydroisoquinolinyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, pyrrolyl, quinazolinyl, quinolinyl, quinoxalinyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1 ,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl and xanthenyl. One or more of the rings can be substituted as defined below for "substituted heteroaryl". The term "substituted heteroaryl" refers to a heteroaryl group in which one or more hydrogen atoms on one or more heteroaromatic rings are substituted with one or more substituents including, but not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxy, carbonyl (such as a ketone, aldehyde, carboxyl, alkoxycarbonyl, formyl, or an acyl), silyl, ether, ester, thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino (or quarternized amino), amido, amidine, imine, cyano, nitro, azido, sulfhydryl, imino, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamide, sulfonyl, heterocyclyl, alkylaryl, haloalkyl (such as CF3, -CH2-CF3, -CCI3), -
CN, aryl, heteroaryl, and combinations thereof.
The term "substituted alkenyl" refers to alkenyl moieties having one or more substituents replacing one or more hydrogen atoms on one or more carbons of the hydrocarbon backbone. Such substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), silyl, ether, ester, thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino (or quarternized amino), amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamide, sulfonyl, heterocyclyl, alkylaryl, haloalkyl, -CN, aryl, heteroaryl, and combinations thereof.
The term "substituted alkynyl" refers to alkynyl moieties having one or more substituents replacing one or more hydrogen atoms on one or more carbons of the hydrocarbon backbone. Such substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), silyl, ether, ester, thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino (or quarternized amino), amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamide, sulfonyl, heterocyclyl, alkylaryl, haloalkyl, -CN, aryl, heteroaryl, and combinations thereof. The term "cycloalkyl" as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc. The term "heterocycloalkyl group" is a cycloalkyl group as defined above where at least one of the carbon atoms of the ring is substituted with a heteroatom such as, but not limited to, nitrogen, oxygen, sulphur, or phosphorus.
The term "aralkyl" as used herein is an aryl group having an alkyl, alkynyl, or alkenyl group as defined above attached to the aromatic group. An example of an aralkyl group is a benzyl group.
The term "hydroxyalkyl group" as used herein is an alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, halogenated alkyl, or heterocycloalkyl group described above that has at least one hydrogen atom substituted with a hydroxyl group.
The term "alkoxyalkyl group" is defined as an alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, halogenated alkyl, or heterocycloalkyl group described above that has at least one hydrogen atom substituted with an alkoxy group described above.
"Carbonyl," as used herein, is art-recognized and includes such moieties as can be represented by the general formula:
Figure imgf000022_0001
wherein X is a bond, or represents an oxygen or a sulfur, and R represents a hydrogen, a substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, -(CH2)m-R", or a pharmaceutical acceptable salt, R' represents a hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl or -(CH2)m-R" ; R" represents a hydroxy group, substituted or unsubstituted carbonyl group, an aryl, a cycloalkyl ring, a cycloalkenyl ring, a heterocycle, or a polycycle; and m is zero or an integer ranging from 1 to 8. Where X is oxygen and R is defines as above, the moiety is also referred to as a carboxyl group. When X is oxygen and R is hydrogen, the formula represents a 'carboxylic acid'. Where X is oxygen and R' is hydrogen, the formula represents a 'formate'. Where X is oxygen and R or R' is not hydrogen, the formula represents an "ester". In general, where the oxygen atom of the above formula is replaced by a sulfur atom, the formula represents a 'thiocarbonyl' group. Where X is sulfur and R or R' is not hydrogen, the formula represents a 'thioester.' Where X is sulfur and R is hydrogen, the formula represents a 'thiocarboxylic acid.' Where X is sulfur and R' is hydrogen, the formula represents a 'thioformate.' Where X is a bond and R is not hydrogen, the above formula represents a 'ketone. ' Where X is a bond and R is hydrogen, the above formula represents an 'aldehyde.'
The term "substituted carbonyl" refers to a carbonyl, as defined above, wherein one or more hydrogen atoms in R, R' or a group to which the moiety
Figure imgf000023_0001
is attached, are independently substituted. Such substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), silyl, ether, ester, thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino (or quarternized amino), amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamide, sulfonyl, heterocyclyl, alkylaryl, haloalkyl, -CN, aryl, heteroaryl, and combinations thereof.
The term "carboxyl" is as defined above for the formula
Figure imgf000023_0002
and is defined more specifically by the formula -RlvCOOH, wherein R1V is an alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, alkylaryl, arylalkyl, aryl, or heteroaryl. In preferred forms, a straight chain or branched chain alkyl, alkenyl, and alkynyl have 30 or fewer carbon atoms in its backbone (e.g., C1- C30 for straight chain alkyl, C3-C30 for branched chain alkyl, C2-C30 for straight chain alkenyl and alkynyl, C3-C30 for branched chain alkenyl and alkynyl), preferably 20 or fewer, more preferably 15 or fewer, most preferably 10 or fewer. Likewise, preferred cycloalkyls, heterocyclyls, aryls and heteroaryls have from 3-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure.
The term "substituted carboxyl" refers to a carboxyl, as defined above, wherein one or more hydrogen atoms in R1V are substituted. Such substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), silyl, ether, ester, thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino (or quarternized amino), amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, alkylaryl, haloalkyl, -CN, aryl, heteroaryl, and combinations thereof.
The term "phenoxy" is art recognized, and refers to a compound of the formula -ORv wherein Rv is (i.e., -O-C6H5). One of skill in the art recognizes that a phenoxy is a species of the aroxy genus.
The term "substituted phenoxy" refers to a phenoxy group, as defined above, having one or more substituents replacing one or more hydrogen atoms on one or more carbons of the phenyl ring. Such substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), silyl, ether, ester, thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino (or quarternized amino), amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, alkylaryl, haloalkyl, -CN, aryl, heteroaryl, and combinations thereof.
The terms "aroxy" and "aryloxy," as used interchangeably herein, are represented by -O-aryl or -O-heteroaryl, wherein aryl and heteroaryl are as defined herein.
The terms "substituted aroxy" and "substituted aryloxy," as used interchangeably herein, represent -O-aryl or -O-heteroaryl, having one or more substituents replacing one or more hydrogen atoms on one or more ring atoms of the aryl and heteroaryl, as defined herein. Such substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), silyl, ether, ester, thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino (or quarternized amino), amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamide, sulfonyl, heterocyclyl, alkylaryl, haloalkyl, -CN, aryl, heteroaryl, and combinations thereof.
The term "alkylthio" refers to an alkyl group, as defined above, having a sulfur radical attached thereto. The "alkylthio" moiety is represented by -S-alkyl. Representative alkylthio groups include methylthio, ethylthio, and the like. The term "alkylthio" also encompasses cycloalkyl groups having a sulfur radical attached thereto.
The term "substituted alkylthio" refers to an alkylthio group having one or more substituents replacing one or more hydrogen atoms on one or more carbon atoms of the alkylthio backbone. Such substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), silyl, ether, ester, thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino (or quarternized amino), amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamide, sulfonyl, heterocyclyl, alkylaryl, haloalkyl, -CN, aryl, heteroaryl, and combinations thereof. The term "phenylthio" is art recognized, and refers to -S-C6H5, i.e. , a phenyl group attached to a sulfur atom.
The term "substituted phenylthio" refers to a phenylthio group, as defined above, having one or more substituents replacing a hydrogen on one or more carbons of the phenyl ring. Such substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), silyl, ether, ester, thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino (or quarternized amino), amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamide, sulfonyl, heterocyclyl, alkylaryl, haloalkyl, -CN, aryl, heteroaryl, and combinations thereof.
"Arylthio" refers to -S-aryl or -S-heteroaryl groups, wherein aryl and heteroaryl as defined herein.
The term "substituted arylthio" represents -S-aryl or -S-heteroaryl, having one or more substituents replacing a hydrogen atom on one or more ring atoms of the aryl and heteroaryl rings as defined herein. Such substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), silyl, ether, ester, thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino (or quarternized amino), amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, alkylaryl, haloalkyl, -CN, aryl, heteroaryl, and combinations thereof.
The terms "amide" or "amido" are used interchangeably, refer to both "unsubstituted amido" and "substituted amido" and are represented by the general formula:
Figure imgf000026_0001
wherein, E is absent, or E is substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aralkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclyl, wherein independently of E, R and R' each independently represent a hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbonyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, -(CH2)m-R'", or R and R' taken together with the N atom to which they are attached complete a heterocycle having from 3 to 14 atoms in the ring structure; R' " represents a hydroxy group, substituted or unsubstituted carbonyl group, an aryl, a cycloalkyl ring, a cycloalkenyl ring, a heterocycle, or a polycycle; and m is zero or an integer ranging from 1 to 8. In preferred forms, only one of R and R' can be a carbonyl, e.g., R and R' together with the nitrogen do not form an imide. In preferred forms, R and R' each independently represent a hydrogen atom, substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, or -(CH2)m-R' ' ' . When E is oxygen, a carbamate is formed. The carbamate cannot be attached to another chemical species, such as to form an oxygen-oxygen bond, or other unstable bonds, as understood by one of ordinary skill in the art.
term "sulfonyl" is represented by the formula
Figure imgf000027_0001
wherein E is absent, or E is alkyl, alkenyl, alkynyl, aralkyl, alkylaryl, cycloalkyl, aryl, heteroaryl, heterocyclyl, wherein independently of E, R represents a hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted amine, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, -(CH2)m-R' " , or E and R taken together with the S atom to which they are attached complete a heterocycle having from 3 to 14 atoms in the ring structure; R' ' ' represents a hydroxy group, substituted or unsubstituted carbonyl group, an aryl, a cycloalkyl ring, a cycloalkenyl ring, a heterocycle, or a polycycle; and m is zero or an integer ranging from 1 to 8. In preferred forms, only one of E and R can be substituted or unsubstituted amine, to form a "sulfonamide" or "sulfonamido" The substituted or unsubstituted amine is as defined above.
The term "substituted sulfonyl" represents a sulfonyl in which E, R, or both, are independently substituted. Such substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), silyl, ether, ester, thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino (or quarternized amino), amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamide, sulfonyl, heterocyclyl, alkylaryl, haloalkyl, -CN, aryl, heteroaryl, and combinations thereof.
The term "sulfonic acid" refers to a sulfonyl, as defined above, wherein R is hydroxyl, and E is absent, or E is substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
The term "sulfate" refers to a sulfonyl, as defined above, wherein E is absent, oxygen, alkoxy, aroxy, substituted alkoxy or substituted aroxy, as defined above, and R is independently hydroxyl, alkoxy, aroxy, substituted alkoxy or substituted aroxy, as defined above. When E is oxygen, the sulfate cannot be attached to another chemical species, such as to form an oxygen- oxygen bond, or other unstable bonds, as understood by one of ordinary skill in the art. The term "sulfonate" refers to a sulfonyl, as defined above, wherein E is oxygen, alkoxy, aroxy, substituted alkoxy or substituted aroxy, as defined above, and R is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted amine, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, -(CH2)m-R" \ R' " represents a hydroxy group, substituted or unsubstituted carbonyl group, an aryl, a cycloalkyl ring, a cycloalkenyl ring, a heterocycle, or a polycycle; and m is zero or an integer ranging from 1 to 8. When E is oxygen, sulfonate cannot be attached to another chemical species, such as to form an oxygen- oxygen bond, or other unstable bonds, as understood by one of ordinary skill in the art.
The term "sulfamoyl" refers to a sulfonamide or sulfonamide represented by the formula
Figure imgf000029_0001
wherein E is absent, or E is substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aralkyl, substituted or unsubstituted alkylaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclyl, wherein independently of E, R and R' each independently represent a hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbonyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, -(CH2)m-R' " , or R and R' taken together with the N atom to which they are attached complete a heterocycle having from 3 to 14 atoms in the ring structure; R' " represents a hydroxy group, substituted or unsubstituted carbonyl group, an aryl, a cycloalkyl ring, a cycloalkenyl ring, a heterocycle, or a polycycle; and m is zero or an integer ranging from 1 to 8. In preferred forms, only one of R and R' can be a carbonyl, e.g., R and R' together with the nitrogen do not form an imide.
The term "phosphonyl" is represented by the formula
Figure imgf000030_0001
wherein E is absent, or E is substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aralkyl, substituted or unsubstituted alkylaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclyl,, wherein, independently of E, Rvi and Rvii are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbonyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, - (CH2)m-R' " , or R and R' taken together with the P atom to which they are attached complete a heterocycle having from 3 to 14 atoms in the ring structure; R' " represents a hydroxy group, substituted or unsubstituted carbonyl group, an aryl, a cycloalkyl ring, a cycloalkenyl ring, a heterocycle, or a polycycle; and m is zero or an integer ranging from 1 to 8.
The term "substituted phosphonyl" represents a phosphonyl in which
E, Rvi and Rvii are independently substituted. Such substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), silyl, ether, ester, thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino (or quartemized amino), amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamide, sulfonyl, heterocyclyl, alkylaryl, haloalkyl, -CN, aryl, heteroaryl, and combinations thereof.
The term "phosphoryl" defines a phosphonyl in which E is absent, oxygen, alkoxy, aroxy, substituted alkoxy or substituted aroxy, as defined above, and independently of E, Rvi and Rvii are independently hydroxyl, alkoxy, aroxy, substituted alkoxy or substituted aroxy, as defined above. When E is oxygen, the phosphoryl cannot be attached to another chemical species, such as to form an oxygen-oxygen bond, or other unstable bonds, as understood by one of ordinary skill in the art. When E, Rvi and Rvii are substituted, the substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), silyl, ether, ester, thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino (or quartemized amino), amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamide, sulfonyl, heterocyclyl, alkylaryl, haloalkyl, -CN, aryl, heteroaryl, and combinations thereof.
The term "polyaryl" refers to a chemical moiety that includes two or more aryls, heteroaryls, and combinations thereof. The aryls, heteroaryls, and combinations thereof, are fused, or linked via a single bond, ether, ester, carbonyl, amide, sulfonyl, sulfonamide, alkyl, azo, and combinations thereof. When two or more heteroaryls are involved, the chemical moiety can be referred to as a "polyheteroaryl."
The term "substituted polyaryl" refers to a polyaryl in which one or more of the aryls, heteroaryls are substituted, with one or more substituents including, but not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), silyl, ether, ester, thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino (or quartemized amino), amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamide, sulfonyl, heterocyclyl, alkylaryl, haloalkyl, -CN, aryl, heteroaryl, and combinations thereof. When two or more heteroaryls are involved, the chemical moiety can be referred to as a "substituted polyheteroaryl."
The term "C3-C20 cyclic" refers to a substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or
unsubstituted cycloalkynyl, substituted or unsubstituted heterocyclyl that have from three to 20 carbon atoms, as geometric constraints permit. The cyclic structures are formed from single or fused ring systems. The
substituted cycloalkyls, cycloalkenyls, cycloalkynyls and heterocyclyls are substituted as defined above for the alkyls, alkenyls, alkynyls and
heterocyclyls, respectively.
The term "ether" as used herein is represented by the formula AOA1, where A and A1 can be, independently, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.
The term "urethane" as used herein is represented by the
formula -OC(0)NRR', where R and R' can be, independently, hydrogen, an alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, halogenated alkyl, or heterocycloalkyl group described above.
The term "silyl group" as used herein is represented by the
formula -SiRR'R", where R, R', and R" can be, independently, hydrogen, an alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, halogenated alkyl, alkoxy, or heterocycloalkyl group described above.
The terms "hydroxyl" and "hydroxy" are used interchangeably and are represented by -OH.
The terms "thiol" and "sulfhydryl" are used interchangeably and are represented by -SH.
The term "oxo" refers to =0 bonded to a carbon atom.
The terms "cyano" and "nitrile" are used interchangeably to refer to -CN. The term "nitro" refers to -N02.
The term "phosphate" refers to -O-PO3.
The term "azide" or "azido" are used interchangeably to refer to -N3. II. Compositions
Polymers, containing one or more poly(amine-co-disulfide ester) polymeric units, and polymeric nanoparticles formed therefrom, are provided. The nanoparticles are stable under physiological conditions (such as pH 7.4), but have dual pH-responsive, and redox-responsive properties, as shown by changes in the sizes of the polymeric nanoparticles in
environments having pH less than 7 and/or low redox potentials: the polymeric nanoparticles swell under low pH conditions and show enhanced degradation under reducing conditions (such as intracellular reduction potential). The swelling of the polymeric nanoparticles and enhanced degradation in environments having pH less than 7 and/or low redox potentials, give rise to enhanced release of cargo in diseased tissues whose microenvironments display these conditions, such as tumors. The stability, dual pH-responsive and redox-responsive properties of the polymeric nanoparticles are as a result of chemical functionalities that are included in the polymers that are included the nanoparticles. The polymeric
nanoparticles also provide for a sustained release of therapeutic, diagnostic, or prophylactic agent. Preferably, the therapeutic agent is a chemotherapeutic agent, i.e. , an anti-cancer agent, such as docetaxel. The nanoparticles also show selective uptake by diseased tissues, compared to healthy tissues.
Unless the context clearly indicates otherwise, the phrase "nanoparticles," as used herein, refers to polymeric nanoparticles, i.e. , nanoparticles formed from a population of one or more of the polymers described herein.
The polymers forming the nanoparticles include at least a lactone unit, a polyfunctional molecule containing an amine group, a hydroxyl group, preferably both, and a molecule containing a disulfide bond. The distributions of the units within the polymer can be ordered or random. The molecular weight of the lactone unit in the polymer, the lactone unit's content of the polymer, or both, influence the stability of the nanoparticles. In general, the polyfunctional molecule contains one or more cations, one or more positively ionizable atoms, or combinations thereof. The one or more cations are formed from the protonation of a basic nitrogen atom, or from quaternary nitrogen atoms. Protonation of the amine group at low pH causes the nanoparticles to swell due to increased electrostatic charge-charge repulsions between the protonated amine groups. The ester bond facilitates degradation of the nanoparticles, via enzymatic degradation, hydrolysis, or a combination thereof. The disulfide bond confers stability to the nanoparticles during circulation at physiological pH (e.g. 7.4), but enhances the degradation of the nanoparticles in low redox potential environments, due to reductive cleavage of the disulfide bonds upon exposure to a reductant, such as glutathione. In some forms, the polyfunctional molecule containing the amine group and the molecule containing the disulfide bond are
independently in the main chain, side chain, or both, of the polymer.
Preferably, the polyfunctional molecule containing the amine group and the molecule containing the disulfide bond are in the main chain of the polymer.
Preferably, the polymers are amphiphilic. The polymers can include a block of a hydrophilic polymer, such as polyethylene glycol. The hydrophilic block can be located at one terminus of the polymer, i.e. , a diblock copolymer, or it can be located at both termini of the polymer, i. e. , a triblock copolymer. In some forms, the hydrophilic polymer constitutes between about 30 wt/wt% and about 50 wt/wt% of the polymer, preferably between about 35 wt/wt% and about 45 wt/wt% of the polymer.
The weight average molecular weight (Mw) of the polymers can be between 1 kDa and 50 kDa, inclusive, preferably between 1 kDa and 30 kDa, inclusive, most preferably between 5 kDa and 15 kDa, inclusive. In some forms, Mw is about 8.1 kDa, 8.3 kDa, 8.7 kDa, 9.4 kDa, 11 kDa, 11.3 kDa, 12.5 kDa, 13.3 kDa, or 15.2 kDa. Mw can be measured using any means in the art, such as by gel permeation chromatography (GPC).
A. Polymers
The polymers have the general formula:
Figure imgf000034_0001
wherein A, B, C, and D independently include monomeric units derived from lactones (e.g. ω-pentadecalactone, ε-caprolactone), a polyfunctional molecule containing an amine group, a hydroxyl group, preferably both (e.g. N-methyldiethanolamine), a molecule containing a disulfide bond (e.g. dimethyl 3,3'-dithiodipropionate), or hydrophilic polymer, such as a hydrophilic polyalkylene oxide (e.g. polyethylene glycol). In some forms, the polymers include at least a lactone; a polyfunctional molecule containing an amine group, a hydroxyl group, preferably both; and a molecule containing a disulfide bond. The polyfunctional molecule contains one or more cations, one or more positively ionizable atoms, or combinations thereof. Preferably, the polyfunctional molecule containing the amine group and the hydroxyl group as well as the molecule containing the disulfide bond are in the main chain of the polymer.
In general, a, b, c, and d are independently integers between 0 and 1000, inclusive, with the proviso that the sum (a + b + c + d) is greater than one. h is an integer between 1 and 1000, inclusive.
In some forms, the polymers include a polymeric unit having the formula:
Figure imgf000035_0001
wherein:
x, y, and z are independently integers between 1 and 1000, inclusive; each occurrence of m is an integer between 1 and 30, inclusive, preferably between 2 and 15, inclusive. In some forms, m is 3 (ε- caprolactone), 12 (ω-pentadecalactone), or a combination thereof;
p, q, n, and t are independently integers between 1 and 20, inclusive, preferably between 1 and 10, inclusive, most preferably between 2 and 5, inclusive. In some forms, p, q, n, and t are each 2;
R8 and R10 are independently O or NR' , wherein R' is hydrogen, substituted alkyl, unsubstituted alkyl, substituted aryl, or unsubstituted aryl. In some forms, at least one of R8 and R10 is O. In some forms, R8 and R10 are O; and
R9 is unsubstituted alkyl, substituted alkyl, hydrogen, substituted aryl, or unsubstituted aryl. In some forms, R9 is unsubstituted C1-C10 alkyl, substituted C1-C10 alkyl. Preferably, R9 is unsubstituted C1 alkyl, such as methyl.
In some forms, R8 and R10 are O, and m is an integer between 1 and 30, inclusive, such as 3 (e.g. ε-caprolactone), 12 (e.g. ω-pentadecalactone), or a combination thereof.
In some forms, R8 and R10 are O; and m is an integer between 1 and 30, inclusive, such as 3 (e.g. ε-caprolactone), 12 (e.g. ω-pentadecalactone), or a combination thereof; p and q are integers between 1 and 20, such as 2.
In some forms, R8 and R10 are O; and m is an integer between 1 and 30, inclusive, such as 3 (e.g. ε-caprolactone), 12 (e.g. ω-pentadecalactone), or a combination thereof; p and q are integers between 1 and 20, such as 2; and n and t are integers between 1 and 20, such as 2.
In some forms, R8 and R10 are O; and m is an integer between 1 and 30, inclusive, such as 3 (e.g. ε-caprolactone), 12 (e.g. ω-pentadecalactone), or a combination thereof; p and q are integers between 1 and 20, such as 2; n and t are integers between 1 and 20, such as 2; and R9 is unsubstituted C1-C10 alkyl or substituted C1-C10 alkyl, such as ethyl, n-propyl, isopropyl, n-butyl, or t-butyl, or unsubstituted aryl, such as phenyl.
In some forms, m is 12 (e.g. ω-pentadecalactone, PDL); R8 and R10 are O, p and q are 2, R9 is methyl (e.g. N-methyldiethanolamine, MDEA); and n and t are 2 (e.g. 3,3'-dithiodipropionate, DTDP); i.e., poly(ro- pentadecalactone-co-N-methyldiethyleneamine-co-3,3'-dithiodipropionate).
In some forms, m is 3 (e.g. ε-caprolactone, CL); R8 and R10 are O, p and q are 2, R9 is methyl (e.g. N-methyldiethanolamine, MDEA); and n and t are 2 (e.g. 3,3'-dithiodipropionate, DTDP); i.e., poly^-caprolactone-co-N- methyldiethyleneamine-co-3,3'-dithiodipropionate).
In some forms, the polymers can be diblock copolymers that include a block of a hydrophilic polymer, preferably a polyalkylene oxide such as polyethylene glycol (PEG) at one terminus. The diblock copolymer can have the formula:
Figure imgf000037_0001
wherein:
x, y, z, m, p, q, n, and t are as described above;
R8, R9, and R10 are as described above;
r is an integer between 1 and 1000, inclusive;
T is O or absent; and
R6 is hydrogen, unsubstituted alkyl, substituted alkyl, unsubstituted aryl, substituted aryl, unsubstituted cycloalkyl, substituted cycloalkyl, maleimide, amine, thiol, N-hydroxysuccinimide ester, azide, acrylate, methacrylate, alkyne, hydroxide, or isocynate.
In some forms, T is O and R6 is unsubstituted alkyl, such as methyl. In some forms, the diblock copolymer is polyethylene glycol-poly(ro- pentadecalactone-co-N-methyldiethyleneamine-co-3,3'-dithiodipropionate) (PEG-PPMD) or polyethylene glycol-poly(s-caprolactone-co-N- methyldiethyleneamine-co-3,3'-dithiodipropionate) (PEG-PCMD).
In some forms, the polymers can be triblock copolymers that include a block of a hydrophilic polymer, preferably a polyalkylene oxide such as polyethylene glycol at both termini. The triblock copolymer can have the
Figure imgf000037_0002
wherein:
x, y, z, m, p, q, n, t, and r are as described above;
R8, R9, and R10 are as described above; and
T is as described above. In some forms, the triblock copolymer is PEG-PPMD-PEG or PEG-PCMD-PEG.
Other suitable hydrophilic polymers that can be used include polysaccharides such as celluloses; hydrophilic polypeptides and poly(amino acids) such as poly-L-glutamic acid (PGS), gamma-polyglutamic acid, poly- L-aspartic acid, poly-L-serine, and poly-L-lysine; poly(oxyethylated polyol); poly(olefinic alcohol); polyvinylpyrrolidone;
poly(hydroxyalkylmethacrylamide) ; poly(hydroxyalkylmethacrylate) ;
poly(saccharides); hydrophilic poly(hydroxy acids); poly(vinyl alcohol), and copolymers thereof.
B. Nanoparticles
The nanoparticles possess dual pH-responsive and redox-responsive properties, ascertained by variations in the sizes of the nanoparticles under different conditions. Preferably, the average sizes of the nanoparticles remain fairly constant under physiological conditions, such as pH 7.4, for periods of time such as one day, two days, one week, one month, etc. Preferably, the nanoparticles swell under acidic pH conditions and degrade in low redox potential environments, as commonly found in tumor tissues, or in the lysosomal and endosomal vesicles within cells.
The nanoparticles are formed from the polymers described above. Preferably, the polymers are amphiphilic and include the polymeric unit of Formula I, as described above. In some forms, the nanoparticles are micelles, i.e. , the nanoparticles have a core-shell structure formed by amphiphilic block copolymers containing a hydrophobic polymer and a hydrophilic polymer. In some forms, the hydrophobic polymer and the hydrophilic polymer form the core and the shell, respectively, of the nanoparticles.
The sizes or diameters of the nanoparticles can be between 50 nm and 500 nm, inclusive, preferably between 50 nm and 300 nm, inclusive. In some forms, the sizes of the nanoparticles can be controlled by altering the composition of the hydrophobic polymer, such as by changing the molecular weight of the lactone unit. As shown in the examples, nanoparticles formed from polymers containing ω-pentadecalactone, m = 12 (PDL), generally had smaller average sizes (between 84 nm and 121 nm), compared to
nanoparticles formed from polymers containing ε-caprolactone, m = 3 (CL), (between 140 nm and 203 nm). This effect of lactone molecular weight on nanoparticle size can be attributed to more hydrophobic interactions in the cores of nanoparticles containing PDL with respect to nanoparticles containing CL, which significantly reduces the water absorption in the nanoparticle cores, thereby giving rise to the smaller size.
In some forms, the zeta potential of the nanoparticles can be between -50 mV and +50 mV, inclusive, between -25 mV and +25 mV, inclusive, or between -10 mV and +10 mv, inclusive. Preferably, the zeta potential is slightly negative, such as between 0 mV and -10 mV. Generally, it is beneficial to have a near-neutral zeta potential, such as between 10 mV and +10 mv, inclusive, because in in vivo applications this can lead to a decrease in serum protein binding, thereby increase systemic circulation of the nanoparticle (Li, et al., Mol Pharm. 2008, 5, 496-504; Levchenko, et al., Int J Pharm. 2002, 240, 95-102). Preferably, the hydrophilic polymers are selected such that the nanoparticle has a near-neutral zeta potential, such as between -10 mV and + 10 mV, inclusive.
The nanoparticles can include cargo (e.g. therapeutic, diagnostic, and/or prophylactic agents) to be delivered to a desired site of subject. The site of delivery can be intracellular, extracellular, the extracellular matrix, a tissue, or organ. Preferably, the site of delivery is associated with a disease. The nanoparticles can display sustained release of the cargo over a period of hours, days, weeks, etc. The cargo can be encapsulated within the nanoparticles, dispersed within the polymeric matrix that forms the nanoparticles, on the surface of the nanoparticles, non-covalently or covalently bound to a polymer from which the nanoparticles are formed, or a combination thereof. In some forms, the cargo can be encapsulated non- covalently within the nanoparticles. Preferably, the therapeutic agent is a chemotherapeutic agent, i.e. , an anti-cancer agent, such as docetaxel. In some forms, when the therapeutic, diagnostic, and/or prophylactic agents are covalently bound to a polymer from which the nanoparticles are formed, these cargoes are covalently conjugated to the polymer prior to the formation of the nanoparticle.
Optionally, the nanoparticles can include targeting moieties that selectively target the nanoparticles to a specific site, by specifically recognizing and binding to a target molecule specific for a cell type, a tissue type, or an organ. Representative targeting moieties include, but are not limited to, antibodies and antigen binding fragments thereof, aptamers, peptides, and small molecules. The binding moiety can be conjugated, covalently or non-covalently, to a polymer that forms the nanoparticle.
Typically the binding moiety is displayed on the surface of the nanoparticle. The targeting moieties should have an affinity for a cell-surface receptor or cell- surface antigen on the target cells. The targeting moieties may result in internalization of the nanoparticles within the target cell.
Preferably, the target molecule is associated with a disease or preferentially over-expressed in a diseased tissue or cell compared to a non- diseased tissue or cell. The target molecule can be a cell surface polypeptide, lipid, or glycolipid. In some forms, the target molecule can be a receptor that is selectively expressed on a specific cell surface, a tissue or an organ.
Specific markers can be for specific types of cells including, but not limited to stem cells, skin cells, blood cells, immune cells, muscle cells, nerve cells, cancer cells, virally infected cells, and organ specific cells. Preferably, the cell specific markers include, but are not limited to cancer specific markers. III. Methods of making
A. Polymers
Also provided are methods of synthesizing poly(amine-co-disulfide ester) polymers. Preferably, the polymers are synthesized via catalytic polymerization of reactants in a one-step process, wherein an enzyme serves as the catalyst. In some forms, the enzyme catalyst is a lipase. In some forms, the lipase is lipase B from Candida antartica.
The reactants include a lactone, a molecule containing a disulfide bond, and a polyfunctional molecule containing an amine group, a hydroxyl group, preferably both. The reactants can further include a hydrophilic polymer, such as polyalkylene oxide (e.g. polyethylene glycol). In some forms, the molar feed ratios of the lactone/molecule containing a disulfide bond/polyfunctional molecule containing an amine group and hydroxyl group can be between 5:20: 10 and 80:90:90. In some forms, the molar feed ratios of the lactone/molecule containing a disulfide bond/polyfunctional molecule containing an amine group/hydrophilic polymer can be between 5:20: 10:3 and 80:90:90: 15. In some forms, the method of synthesizing the polymers includes a first step of incubating the reactants and enzyme catalyst at a temperature from between 60 °C and 95 °C, inclusive, preferably between 80 °C and 90 °C, inclusive, at about 1 atm N2. In some forms, the method further includes, after the first step, incubating the reactants and enzyme catalyst at a temperature from between 60 °C and 95 °C, inclusive, preferably between 80 °C and 90 °C, inclusive, under high vacuum (e.g. 2 mmHg).
The weight average molecular weight (Mw) of the polymers, as measured by gel permeation chromatography (GPC) can be between 1 kDa and 50 kDa, inclusive, preferably between 1 kDa and 30 kDa, inclusive, most preferably between 5 kDa and 15 kDa, inclusive. In some forms, Mw is about 8.1 kDa, 8.3 kDa, 8.7 kDa, 9.4 kDa, 11 kDa, 11.3 kDa, 12.5 kDa, 13.3 kDa, or 15.2 kDa.
In some forms, the polymers are prepared, as shown in Scheme 1.
Figure imgf000042_0001
In some forms, m is an integer between 1 and 30, inclusive, preferably between 2 and 15, inclusive. In some forms, m is 3 (ε- caprolactone), 12 (ω-pentadecalactone), or a combination thereof. In some forms, p, q, n, and t are independently integers between 1 and 20, inclusive, preferably between 1 and 10, inclusive, most preferably between 2 and 5, inclusive. In some forms, p, q, n, and t are each 2. In some forms, r is an integer between 1 and 1000.
With regard to the reactants, in some forms, R1 and R2 are independently hydrogen, substituted alkyl, unsubstituted alkyl, substituted aryl, or unsubstituted aryl. In some forms, Ri and R2 are unsubstituted C1-C10 alkyl, substituted C1-C10 alkyl. In some forms, Ri and R2 are unsubstituted C1 alkyl, such as methyl. In some forms, R3 and R5 are independently OH or N(R')2, wherein each R' is independently hydrogen, substituted alkyl, unsubstituted alkyl, substituted aryl, or unsubstituted aryl. In some forms at least one of R3 and R5 is OH, and at least one R' is hydrogen. In some forms, R3 and R5 are OH. In some forms, R4 is unsubstituted alkyl, substituted alkyl, hydrogen, substituted aryl, or unsubstituted aryl. In some forms, R4 is unsubstituted C1-C10 alkyl, substituted C1-C10 alkyl. Preferably, R4 is unsubstituted C1 alkyl, such as methyl. In some forms, the reactants are 00- pentadecalactone (PDL) or ε-caprolactone, with N-methyldiethanolamine, (MDEA), 3,3'-dithiodipropionate (DTDP), and polyethylene glycol.
With regard to the products (diblock and triblock copolymers), in some forms, x, y, and z are independently integers between 1 and 1000, inclusive. In some forms, R8 and R10 are independently O or NR' , wherein R' is hydrogen, substituted alkyl, unsubstituted alkyl, substituted aryl, or unsubstituted aryl. In some forms, at least one of R8 and R10 is O. In some forms, R8 and R10 are O. In some forms, R9 is unsubstituted alkyl, substituted alkyl, hydrogen, substituted aryl, or unsubstituted aryl. In some forms, R9 is unsubstituted C1-C10 alkyl, substituted C1-C10 alkyl. Preferably, R9 is unsubstituted C1 alkyl, such as methyl. In some forms, T is O or absent. In some forms, R6 hydrogen, unsubstituted alkyl, substituted alkyl,
unsubstituted aryl, substituted aryl, unsubstituted cycloalkyl, substituted cycloalkyl, maleimide, amine, thiol, N-hydroxysuccinimide ester, azide, acrylate, methacrylate, alkyne, hydroxide, or isocynate. Preferably, T is O and R6 is unsubstituted alkyl, such as methyl. In some forms, the product is poly(ro-pentadecalactone-co-N-methyldiethyleneamine-co-3,3'- dithiodipropionate) (PPMD), poly(s-caprolactone-co-N- methyldiethyleneamine-co-3,3'-dithiodipropionate) (PCMD), or PEG- poly(ro-pentadecalactone-co-N-methyldiethyleneamine-co-3,3'- dithiodipropionate) (PEG-PPMD), PEG-poly(s-caprolactone-co-N- methyldiethyleneamine-co-3,3'-dithiodipropionate) (PEG- PCMD), PEG- PCMD-PEG, PEG-PPMD-PEG, or a combination thereof.
The desired amphiphilic products can be isolated using the methods described below in Example 1.
As shown in Scheme 1, polyethylene glycol is included as a hydrophilic block in the polymers. However, it should be noted that other suitable hydrophilic polymers can be used as well. These include polysaccharides such as celluloses; hydrophilic polypeptides and poly(amino acids) such as poly-L-glutamic acid (PGS), gamma-polyglutamic acid, poly- L-aspartic acid, poly-L-serine, and poly-L-lysine; poly(oxyethylated polyol); poly(olefinic alcohol); polyvinylpyrrolidone;
poly(hydroxyalkylmethacrylamide) ; poly(hydroxyalkylmethacrylate) ;
hydrophilic poly (hydroxy acids); poly (vinyl alcohol), and copolymers thereof.
B. Nanoparticles
i. Self-assembly
In some forms, the nanoparticles are formed by self-assembly of amphiphilic block copolymers in an aqueous solution. In an aqueous environment, the amphiphilic copolymers can spontaneously self-assemble to form nanoparticles with a hydrophobic core and a hydrophilic outer shell. In some forms, a solution containing the amphiphilic polymers is mixed with another solution containing a therapeutic, diagnostic, and/or prophylactic agent to be encapsulated. In some forms, the amphiphilic polymers and therapeutic, diagnostic, and/or prophylactic agent to be delivered are dissolved in a suitable solvent, such as tetrahydrofuran, DMSO, or methylene chloride. Preferably, the therapeutic agent is a chemotherapeutic agent, i.e. , an anti-cancer agent, such as docetaxel. The concentrations of the amphiphilic polymer and therapeutic, diagnostic, and/or prophylactic agent in the solvent can be varied as needed. After forming a solution containing the amphiphilic polymer and therapeutic, diagnostic, and/or prophylactic agent, the solution can be added continuously to an aqueous solution, such as PBS (10 mM, pH 7.4) using syringe to induce nanoparticle formation (micellization). The nanoparticle solutions can be stirred at room
temperature, followed by dialysis, placement in an ultrafiltration centrifuge tube, and centrifuging to obtain the nanoparticles.
ii. Other methods of forming nanoparticles
The nanoparticles described herein can be formed using a variety of techniques known in the art. The technique to be used can depend on a variety of factors including the polymer used to form the nanoparticles, the desired size range of the resulting nanoparticles, and suitability for the therapeutic, diagnostic, and/or prophylactic agent to be incorporated.
Suitable techniques include, but are not limited to:
a. Solvent diffusion/displacement
In this method, water-soluble or water-miscible organic solvents are used to dissolve the polymer and form emulsion upon mixing with the aqueous phase. The quick diffusion of the organic solvent into water leads to the formation of nanoparticles immediately after the mixing.
b. Solvent evaporation
In this method the polymer is dissolved in a volatile organic solvent.
The drug (either soluble or dispersed as fine particles) is added to the solution, and the mixture is suspended in an aqueous solution that contains a surface active agent such as poly(vinyl alcohol). The resulting emulsion is stirred until most of the organic solvent evaporated, leaving solid nanoparticles. The resulting nanoparticles are washed with water and dried overnight in a lyophilizer. Nanoparticles with different sizes and morphologies can be obtained by this method. c. Solvent removal
In this method, the drug is dispersed or dissolved in a solution of the selected polymer in a volatile organic solvent. This mixture is suspended by stirring in an organic oil (such as silicon oil) to form an emulsion. Unlike solvent evaporation, this method can be used to make nanoparticles from polymers with high melting points and different molecular weights. The external morphology of spheres produced with this technique is highly dependent on the type of polymer used.
d. Spray-drying
In this method, the polymer is dissolved in organic solvent. A known amount of the active drug is suspended (insoluble drugs) or co-dissolved (soluble drugs) in the polymer solution. The solution or the dispersion is then spray-dried.
e. Phase inversion
Nanospheres can be formed from polymers using a phase inversion method wherein a polymer is dissolved in a "good" solvent, fine particles of a substance to be incorporated, such as a drug, are mixed or dissolved in the polymer solution, and the mixture is poured into a strong non solvent for the polymer, to spontaneously produce, under favorable conditions, polymeric microspheres, wherein the polymer is either coated with the particles or the particles are dispersed in the polymer. The method can be used to produce nanoparticles in a wide range of sizes, including, for example, about 100 nanometers to about 10 microns. Substances which can be incorporated include, for example, imaging agents such as fluorescent dyes, or biologically active molecules such as proteins or nucleic acids. In the process, the polymer is dissolved in an organic solvent and then contacted with a non solvent, which causes phase inversion of the dissolved polymer to form small spherical particles, with a narrow size distribution optionally incorporating an antigen or other substance.
f. Microfluidics
Methods of making nanoparticles using microfluidics are known in the art. Suitable methods include those described in U.S. Patent Application Publication No. 2010/0022680 Al by Karnik, et al. In general, the microfluidic device comprises at least two channels that converge into a mixing apparatus. The channels are typically formed by lithography, <?iching, embossing, or molding of a polymeric surface. A source of fluid is attached to each channel, and the application of pressure to the source causes the flow of the fluid in the channel. The pressure may be applied by a syringe, a pump, and/or gravity. The inlet streams of solutions with polymer, targeting moieties, lipids, drug, payload, etc. converge and mix, and the resulting mixture is combined with a polymer non-solvent solution to form the nanoparticles having the desired size and density of moieties on the surface. By varying the pressure and flow rate in the inlet channels and the nature and composition of the fluid sources nanoparticles can be produced having reproducible size and structure.
IV. Methods of using
The dual pH responsive and redox-responsive nanoparticles can be used to deliver therapeutic, diagnostic, and/or prophylactic agents to a subject. In general, these agents are delivered to an acidic diseased tissue, resulting from a diseased state such as cancer, inflammation, stroke, arthritis, or ischemia. Preferably, the therapeutic agent is a chemotherapeutic agent, i.e. , an anti-cancer agent, such as docetaxel. In some forms, the therapeutic agent (e.g. chemotherapeutic agent) constitutes between 0.5 wt/wt% and 25 wt/wt%, inclusive, between 1 wt/wt% and 20 wt/wt%, inclusive, between 1 wt/wt% and about 15 wt/wt%, inclusive, between 1 wt/wt% and 10 wt/wt%, inclusive, or between 1 wt/wt% and 5 wt/wt%, inclusive of the nanoparticles.
Methods of use typically involve administering to a subject, in need thereof, a composition containing the nanoparticles having an effective amount of a therapeutic agent to inhibit progression of a disease. In some forms, the composition inhibits the proliferation of tumor cells, induce cell- cycle arrest, and/or induce senescence in tumor cells, in a subject. Thus, methods include administering to a subject in a need thereof an effective amount of the composition to reduce or inhibit proliferation of tumor cells, induce cell-cycle arrest or tumor cells, and/or induce senescence in tumor cells.
A. Therapeutic agents to be delivered
i. Chemotherapeutic agents
Representative chemotherapeutic agents that can be included as cargo in the nanoparticles include, but are not limited to, docetaxel and analogs thereof, paclitaxel and analogs thereof, alkylating agents (such as cisplatin, carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, dacarbazine, lomustine, carmustine, procarbazine, chlorambucil and ifosfamide), actinomycin, bleomycin, daunorubicin, doxorubicin, epirubicin, mitomycin, methotrexate, fluorouracil, carboplatin, methyl- chloroethylnitrosoureas, etoposide, camptothecin and analogs thereof, phenesterine, piposulfan, altretamine, asparaginase, busulfan, chlorambucil, cladribine, cytarabine, dacarbazine, diethylstilbestrol, ethinyl estradiol, mitotane, mitoxantrone, pentastatin, pipobroman, plicamycin, prednisone, procarbazine, streptozocin, tamoxifen, teniposide, vinblastine, and vincristine.
ii. Nucleic acids
In some forms, the nanoparticles can encapsulate functional nucleic acids. Functional nucleic acids are nucleic acid molecules that have a specific function, such as binding a target molecule or catalyzing a specific reaction. Functional nucleic acid molecules can be divided into the following non-limiting categories: antisense molecules, siRNA, miRNA, aptamers, ribozymes, triplex forming molecules, RNAi, and external guide sequences. The functional nucleic acid molecules can act as effectors, inhibitors, modulators, and stimulators of a specific activity possessed by a target molecule, or the functional nucleic acid molecules can possess a de novo activity independent of any other molecules.
In some forms, the nucleic acid is less than 1,000 base pairs, less than 500 base pairs, less than 250 base pairs, or less than 100 base pairs. B. Methods of administration
The compositions can be administered by a number of routes including, but not limited to, oral, intravenous, intraperitoneal, intramuscular, transdermal, subcutaneous, topical, sublingual, rectal, intranasal, pulmonary, and other suitable means. Such administration routes and appropriate formulations are generally known to those of skill in the art.
The administration may be localized (i.e., to a particular region, physiological system, tissue, organ, or cell type) or systemic, depending on the condition being treated. Suitable parenteral administration routes include intravascular administration (e.g. , intravenous bolus injection, intravenous infusion, intra- arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature); peri- and intra-tissue injection (e.g. , intraocular injection, intra-retinal injection, or sub-retinal injection);
subcutaneous injection or deposition including subcutaneous infusion (such as by osmotic pumps); direct application by a catheter or other placement device (e.g. , an implant comprising a porous, non-porous, or gelatinous material).
The compositions can be administered in a single dose or in multiple doses. Certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. It will also be appreciated that the effective dosage of the composition used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays.
Dosing is dependent on severity and responsiveness of the disease condition to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual therapeutic agent, and can generally be estimated based on EC50S found to be effective in in vitro and in vivo animal models.
Dosage levels on the order of about lmg/kg to 100 mg/kg of body weight per administration are useful in the treatment of a disease. In preferred embodiments, the dosage levels are about lOmg/kg -50 mg/kg of body weight per administration. One skilled in the art can also readily determine an appropriate dosage regimen for administering the disclosed compositions to a given subject. For example, the compositions can be administered to the subject once, e.g. , as a single injection, infusion or bolus. Alternatively, the formulation can be administered once or twice daily to a subject for a period of from about three to about twenty-eight days, or from about seven to about ten days.
The methods, compounds, and compositions herein described are further illustrated in the following examples, which are provided by way of illustration and are not intended to be limiting. It will be appreciated that variations in proportions and alternatives in elements of the components shown will be apparent to those skilled in the art and are within the scope of disclosed forms. Theoretical aspects are presented with the understanding that Applicants do not seek to be bound by the theory presented. All parts or amounts, unless otherwise specified, are by weight.
Examples
A number of stimuli-responsive nanoparticles have previously been formed from synthetic polymers that are produced via chemical
polymerization processes (Karimi, et al, Chem Soc Rev. 2016, 45, 1457- 1501; Cheng, et al., Biomaterials 2013, 34, 3647-3657). Another alternative to chemical polymerization has been explored, such as the environmentally benign, enzyme-catalyzed polymerization procedures for preparation of functional biodegradable polyesters for drug and gene delivery applications (Jiang, ACS Sym Ser. 2013, 1144, 29-42; Zhou, et al , Nat Mater. 2012, 11, 82-90; Zhang, et al, J Mater Chem B. 2014, 2, 4034-4044; Yang, et al,
Nanoscale 2014, 6, 10193-10206; Chen, et al, ACS Appl Mater Inter. 2016, 8, 490-501; Zhang, et al, ACS Biomater-Sci Eng. 2016, 2, 2080-2089; Mazzocchetti, et al, Macromolecules 2009, 42, 7811-7819; Liu, et al., Biomaterials 2009, 30, 5707-5719; Liu, et al., Biomaterials 2011, 32, 6646- 6654; Yang, et al., Macromolecules 2013, 46, 1743-1753; Han, et al., ACS Nano 2016, 10, 4209-4218). Because of the high activity and selectivity of enzyme catalysts and their extraordinary tolerance toward organic functional groups (Gross, et al., Trends Biotechnol. 2010, 28, 435-443; Kobayashi and Makino, Chem Rev. 2009, 109, 5288-5353; Gross, et al., Chem Rev. 2001, 101, 2097-2124; Kobayashi, et al., Chem Rev. 2001, 101, 3793-3818; Kumar and Gross, /. Am. Chem. Soc. 2000, 122, 11767-11770; Jiang, et al, Macromolecules, 2008, 41, 4671-4680), copolyesters with diverse chain structures and functionalities were successfully synthesized, typically in one step from readily available monomers (Jiang, Biomacromolecules 2008, 9, 3246-3251; Jiang, Biomacromolecules 2010, 11, 1089-1093; Jiang,
Biomacromolecules 2011, 12, 1912-1919; Martino, et al., Polymer 2012, 53, 1839-1848; Jiang, et al, Polymer 2013, 54, 6105-6113; Feng, et al,
Macromol. Rapid Commun. 1999, 20, 88-90. Feng, et al, Macromol. Biosci. 2001, 1, 66-74.). Further, the enzymatic polyesters possess high purity and are metal-free, which render them particularly suitable for biomedical uses.
Recently, the synthesis of PEG-poly(ω-pentadecalactone-co-N- methyldiethyleneamine-co-sebacate) (PEG-PPMS) copolymers that are responsive to acidic endosomal pH (Zhang, et al, Colloid Surface B. 2014, 115, 349-358), and PEG-poly(ro-pentadecalactone-co-butylene-co-3,3'- dithiodipropionate) (PEG-PPBD) copolymers that are responsive to intracellular glutathione (Liu, et al, Polym. Chem. 2015, 6, 1997-2010) have been described. Anticancer drug-loaded nanoparticles formed from PEG- PPMS and PEG-PPBD exhibited a potency that can be enhanced
correspondingly by acid and glutathione. Herein, the successful synthesis of functional polyesters bearing both tertiary amino groups and disulfide groups in the polymer main chain are described: PEG-poly(ω-pentadecalactone-co- N-methyldiethyleneamine-co-3,3'-dithiodipropionate) (PEG-PPMD) and PEG-poly(s-caprolactone-co-N-methyldiethyleneamine-co-3,3'- dithiodipropionate) (PEG-PCMD). The amphiphilic block copolymers were produced in one step via lipase-catalyzed copolymerization of lactone (ω- pentadecalactone or ε-caprolactone), iV-methyldiethanolamine (MDEA), dimethyl 3,3'-dithiodipiOpionate (DTDP) and polyfethylene glycol) methyl ether (MeO-PEG-OH). These polymers contain dithio diester, amino diol and lactone repeat units, and are structurally different from previously reported pH and redox-responsive materials. pj£G-PPMD and PEG-PCMD were designed to possess following structural components and
functionalities: PEG for nanoparticle colloidal stability, lactone units for tuning micelle stability and improving cellular uptake (Chen, et at., ACS Appl Mater Inter, 2016, 8, 490-501 ), representative anticancer drug docetaxel (DTX, a common commercial anti-mifotic chemotherapy medicine) for evaluation of their drug delivery efficiency. Figure 14 illustrates steps that are involved in the therapeutic actions of the pH and redox-responsive nanoparticles in delivering the drug to tumor cells. The examples demonstJ'ate the construction of stable nanoparticles with desirable sizes from PEG-PPMD and PEG-PCMD copolymers, their excellent biocompatibility and cellular uptake properties, swift responses of the particles to tumor relevant acidic pH and intracellular reduction potential, in vitro efficacy of the DTX-loaded nanoparticles in killing cancer cells, as well as their high in vivo antitumor efficiency. To the best of our knowledge, PEG-PPMD and PEG-PCMD represent the first examples of enzymatic polymers with pH and redox dual-responsive properties,
Materials
ω-Pentadecalactone (PDL, >98%), ε-caprolactone (CL, 99%), N- methyldiethanolamine (MDEA, 99%), diphenyl ether (99%), poJy(ethylene glycol) methyl ether (2000 Da, MeO-PEG2K-OH) and L-buthionine-iS.i?)- sulfoximine (BSO) were purchased from Sigma-AJdrich Chemical Co. and were used as received. Immobilized CALB (Candida antarctica lipase B supported on acrylic resin) catalyst or Novozym 435, 3-(4,5-dimethyl-2- thiazoIyl)-2,5-diphenyI-2H-tetrazoliuni bromide ( M T T ). chloroform (HPLC grade), chloroform-d, «-hexane (97+%) were also purchased from Aldrich Chemical Co. The lipase catalyst was dried at 40 °C under 2.0 mmHg for 20 h prior to use. Dimethyl 3,3*-tjithiodipropionate (DTDP, 98%) was obtained from. TCI Co., Ltd; Docetaxel (DTX) was purchased from Beijing Norzer Pharmaceutical Co., Ltd.; Duopafei® (commercial DTX injection) for in vivo treatment was manufactured by Qilu Pharm Co., Ltd (Jinan, China). DiR ( l , r-dioctadecyl-3,3,3\3'-tetrainethy]indotricai-bocyanine iodide) was purchased from Thermo Fisher Scientific Inc. Both HeLa cells and CT-26 cells were acquired from Shanghai cell bank of Chinese Academy of Science
(Shanghai, China) and were maintained at 37 °C under 5% CO2 humidified atmosphere. DMEM and RPMI-1640 (from Gibco, both containing 10% (v/v) fetal bovine serum (FBS) and 1% (w/v) penicillin-streptomycin) were used as the culture media for the HeLa cell line and CT-26 cell line, respectively.
Instrumentation methods
Proton and carbon- 13 NMR spectra were recorded on an Agilent 500 spectrometer. The chemical shifts reported were referenced to internal tetramethylsilane (0.00 ppm). The number and weight average molecular weights (Mn and Mw, respectively) of polymers were measured by gel permeation chromatography (GPC) using chloroform as the mobile phase and narrow polydispersity polystyrenes as the standards according to previously reported procedures (Jiang, et al., Biomacromolecules 2010, 11, 1089-1093). The average size, size distribution, and zeta potential of micelles were measured by dynamic light scattering (DLS) using a Zetasizer Nano ZS90 (Malvern Instruments). Transmission electron microscope (TEM) was used to observe the morphology of micelle particles after staining with phosphotungstic acid.
Example 1: Synthesis and characterization of polymers
Methods
A. Synthesis of PEG2K-poly( ω-pentadecalactone-co-N- methyldiethyleneamine-co-3, 3 ' -dithiodipropionate ) ( PEG2K-PPMD ) copolymers
The block copolymers were prepared via copolymerization of 00- pentadecalactone (PDL), N-methyldiethanolamine (MDEA), dimethyl 3,3'- dithiodipropionate (DTDP) with MeO-PEG2K-OH using Novozym 435 as the catalyst. The amount of MeO-PEG2K-OH was selected to allow formation of the block copolymers with 40 wt% PEG upon complete conversion of the substrates to polymer products. Typically, PDL, MDEA, DTDP and MeO-PEG2K-OH in different ratios (shown in Table 1) were mixed with Novozym 435 (10 wt% vs total substrate) and diphenyl ether (200 wt% vs total substrate). The resultant reaction mixtures were stirred at 90 °C initially under 1 atm nitrogen gas for 21 h and subsequently under a reduced pressure of 1.8 mmHg for 70 h. At the end of the reactions the formed product was purified, as described previous (Liu, et al. , Polym. Chem. 2015, 6, 1997-2010). Briefly, n-hexane was added to the product mixtures to precipitate the formed copolymers. The copolymers were washed with n-hexane three times to extract residual diphenyl ether solvent, dissolved in chloroform, and then filtered to remove the enzyme catalyst. Complete evaporation of the chloroform solvent from the filtrates at 30 °C under high vacuum (<1.0 mmHg) for 20 h, yielded the purified PEG2K- PPMD block copolymers. Each PEG-PPMD copolymer is denoted as PEG- PPMD-x% PDL indicating molar percentage content of PDL units vs. (PDL + diester) units in the PPMD segments of the copolymer. PEG-PPMD block copolymer. x% in "PEG-PPMD-x%PDL" is mol% of PDL units vs (PDL + DTDP) units, which is calculated from the peak intensities in the NMR spectra. The data shown below are the peak positions in the spectra and are same for all PEG-PPMD samples with different PDL contents. ¾ NMR (CDCI3; ppm) 1.26 (br.), 1.61 (m), 2.27-2.32 (m), 2.36 (s), 2.71-2.77 (m), 2.92 (t), 3.64 (s), 4.04-4.10 (m), 4.16-4.22 (m), plus a singlet at 3.38 ppm; 13C NMR (CDCI3; ppm) 24.92, 25.02, 25.89, 25.93, 28.58, 28.65, 29.16, 29.26, 29.28, 29.47, 29.52, 29.58-29.63 (m), 33.01, 33.05, 33.15, 33.20, 34.06, 34.14, 34.27, 34.39, 42.84, 55.82, 55.91, 61.89, 62.42, 63.34, 63.89, 64.38, 64.99, 70.55, 171.60, 171.72, 173.78, 173.98; plus small peaks at 59.02, 69.03 and 71.92 ppm due to terminal -OCH3 and -COO-CH2-CH2- O- groups.
B. Synthesis of PEG2K-poly( ε-caprolactone-co-N- methyldiethyleneamine-co-3, 3 '-dithiodipropionate ) ( PEG2K-PCMD ) copolymers
The PEG-PCMD copolymers were synthesized following procedures analogous to those used for the preparation of PEG-PPMD polymers above except that ε-caprolactone (CL) was employed instead of PDL as the lactone comonomer. Thus, CL, MDEA, DTDP and MeO-PEG2K-OH in different ratios (shown in Table 1) were blended with Novozym 435 (10 wt% vs total substrate) and diphenyl ether (200 wt% vs total substrate). The resultant reaction mixtures were stirred at 80 °C initially under 1 atm nitrogen gas for 20 h and subsequently under 2.0 mmHg vacuum for 71 h. The formed PEG- PCMD copolymers were purified using the same method as described above for isolation of the PEG-PPMD copolymers. Each PEG-PCMD copolymer is denoted as PEG-PCMD-x% CL indicating molar percentage content of CL units vs (CL + diester) units in the PCMD segments of the copolymer. PEG- PCMD block copolymer. The data shown below are the peak positions in the spectra and are same for all PEG-PCMD samples with different CL contents. ¾ NMR (CDCI3; ppm) 1.39 (br.), 1.66 (m), 2.29-2.37 (m), 2.70- 2.77 (m), 2.92 (t), 3.64 (s), 4.05-4.11 (m), 4.16-4.21 (m), plus a singlet at 3.38 ppm; 13C NMR (CDCI3; ppm) 24.48, 24.50, 24.53, 24.56, 25.46, 25.48, 25.51, 28.27, 28.33, 33.02, 33.04, 33.11, 33.13, 34.05, 34.09, 42.83, 55.79, 55.87, 61.96, 62.41, 64.11, 64.59, 70.56, 171.56, 171.63, 173.36, 173.48; plus small peaks at 59.01, 69.02 and 71.92 ppm due to terminal -OCH3 and - COO-CH2-CH2-O- groups.
Results
The PEG2K-PPMD copolymers and PEG2K-PCMD copolymers synthesized above were amphiphilic block copolymers. The amphiphilic block copolymers containing tertiary amino and disulfide functional groups were synthesized via CALB-catalyzed copolymerization of lactone (PDL or CL), N-methyldiethanolamine (MDEA), dimethyl 3,3'-dithiodipropionate (DTDP) with polyethylene glycol) methyl ether (MeO-PEG2K-OH) in two stages: the first stage oligomerization under 1 atm nitrogen gas, followed by the second stage polymerization under ~2 mmHg vacuum (Scheme 3). The comonomer feed ratios employed, and the composition and properties of the resultant polymer products are shown in Table 1.
Figure imgf000057_0001
All purified PEG-PPMD and PEG-PCMD copolymers contain 40 (±1) wt% PEG (calculated from the proton NMR spectra) and were obtained in 75% to 94% yield. The Mw values range from 11000 to 15000 Da for PEG-PPMD copolymers and from 8100 to 9400 Da for PEG-PCMD copolymers.
Figure imgf000059_0001
The experimental results in Table 1 show that the composition of the copolymers can be readily controlled by varying the monomer feed ratio during the copolymerization reactions. GPC analyses revealed mono-model molecular weight distributions for both PEG-PPMD and PEG-PCMD block copolymers, and no unreacted, i.e. , free PEG2K was detected in the products. Because MeO-PEG-OH can only link to the ends of PPMD or PCMD chains, i.e. , act as a chain terminator, the PEG-PPMD and PEG-PCMD copolymers have or can contain two possible types of block structures: PEG-polyester diblock chains and PEG-polyester- PEG triblock chains where polyester is PPMD or PCMD (Scheme 3).
PEG-PPMD and PEG-PCMD copolymer chains contain both PEG blocks and polyester blocks consisting of lactone (PDL or CL), N- methyldiethyleneamine (MDEA), and 3,3'-dithiodipropionate (DTDP) repeating units. The molecular structure and composition of the copolymers were determined by proton and carbon- 13 NMR spectroscopy. Structural assignments for the resonance absorptions of the polymers were facilitated by observing changes in signal intensities versus copolymer composition. As an example, the ¾ NMR spectra of PEG2K-PPMD-70% PDL and PEG2K- PCMD-70% CL along with the structural assignments for their proton resonance absorptions were analyzed (spectral data shown above in the Methods section). The small singlet peak at 3.38 ppm in both spectra was attributed to the -OCH3 terminal groups linked to the PEG chain blocks. The PEG and lactone contents of the copolymers were calculated from the integrations of relevant proton resonance peaks in their spectra, and the results are summarized in Table 1.
The chain structures of PEG-PPMD and PEG-PCMD copolymers were further supported by their carbon- 13 NMR spectra. Both copolymers exhibited four carbonyl carbon- 13 resonance peaks due to the presence of four ester diads in the polymer chains. They appear at 171.60, 171.72, 173.78 and 173.98 ppm for DTDP*-MDEA, DTDP*-PDL, PDL*-MDEA and
PDL*-PDL diads in PEG-PPMD chains, and at 171.56, 171.63, 173.36 and 173.48 ppm for DTDP*-MDEA, DTDP*-CL, CL*-MDEA and CL*-CL diads in PEG-PPCD chains. To determine repeat unit distributions in the polyester blocks of the copolymers, carbonyl carbon- 13 absorptions or carbon- 13 absorbance of the polymers were determined quantitatively. Both PEG-PPMD and PEG- PCMD copolymers show four carbonyl carbon- 13 absorbances attributed to lactone* -lactone, lactone* -MDEA, DTDP*-lactone and DTDP* -MDEA
diads (Figures 1A and IB). For PEG-PPMD-43% PDL, PEG-PPMD-70%
PDL, PEG-PCMD-41% CL and PEG-PCMD-70% CL, the measured
abundances of the four diads were compared to the values calculated,
respectively, for random PPMD chains or PCMD chains at same
compositions (Table 2). The results reveal that the repeat unit arrangements in the polyester chain blocks of the polymers (e.g. PEG-PPMD and PEG- PCMD copolymers) are nearly random.
Table 2. Diad Distributions in the Polyester Segments
Figure imgf000061_0001
3,3'-dithiodipropionate unit. The lactone unit represents co-pentadecalactone for PEG-PPMD or ε-caprolactone for PEG-PCMD. b Measured by carbon- 13 NMR spectroscopy. c Calculated for random poly(lactone-co-MDEA-co-DTDP) chains. Abundance of lactone*-lactone diad = L xfu abundance of lactone*-MDEA = L X (2 x fu)- abundance of DTDP*-lactone = (2 x fD) xfu abundance of DTDP*- MDEA = (2 X /D) X (2 X /M). The symbols /L, /M and/b represent respectively molar fractions of lactone, MDEA and DTDP units in the copolymer chains. Example 2: Preparation and characterization of blank and docetaxel (DTX)-loaded PEG-PPMD and PEG-PCMD micelles
Methods
A. Preparation of blank and docetaxel (DTX)-loaded PEG¬S ' PPMD and PEG-PCMD micelles
The blank and DTX-loaded micelles were fabricated using a dialysis method. PEG-PPMD or PEG-PCMD copolymers (38 mg) with or without DTX (2 mg) were dissolved in 1 mL of tetrahydrofuran (THF). The resultant solutions were continuously added into 5 ml of PBS (10 mM, pH 7.4) using a0 syringe to induce micellization. Subsequently, the micelle solutions were stirred for 30 min at room temperature and dialyzed of PBS (10 mM, pH 7.4) overnight using 3500 Da cutoff size dialysis bag. Finally, the dialyzed micelle solutions were then centrifuged for 20 min at 8000 rpm using MWCO 100 kDa ultrafiltration centrifuge tubes. Finally, an aliquot of the5 concentrated micelle solutions was lyophilized, and the yield of each micelle sample was calculated and recorded.
B. Critical micelle concentration measurement
The critical micelle concentration (CMC) values of PEG-PPMD copolymers with 12%, 43% and 70% PDL content, and PEG-PCMD
0 copolymers with 13%, 41% and 70% CL content, were evaluated by
fluorescence spectroscopy using pyrene as the probe molecule according to previously reported procedures (Liu, et al , Polym. Chem. 2015, 6, 1997). Briefly, 60 of 6.0 x 10"5 M pyrene solution in THF were added to empty containers, and 6 mL of micelle solutions in PBS (10 mM, pH 7.4) at various5 concentrations (0.0001-1.0 mg/mL) were added to each container.
Thereafter, the resultant mixtures were incubated overnight at room temperature. The emission spectra were recorded from 350 nm to 420 nm using an excitation wavelength of 334 nm. The slit- width was 2 nm for both excitation and emission beams. The CMC value was estimated as the micelle0 concentration at the cross point in the curves of hfii (the third peak/the first peak) intensity ratio vs logarithm of micelle concentration. C. In vitro micelle stability tests
The in vitro stabilities of PEG-PPMD and PEG-PCMD micelles were evaluated by DLS. Briefly, freshly prepared micelle solution was added into PBS (pH 7.4, 10 mM) containing 10% fetal bovine serum (FBS), and incubated at 37 °C in a shaking bed with a rotation speed of 100 rpm.
Samples were withdrawn at predetermined time intervals for DLS analysis on average micelle size to evaluate in vitro stability of the micelles.
D. Measurements ofDTX contents in micelles
The content of DTX encapsulated in PEG-PPMD or PEG-PCMD micelles was measured by high performance liquid chromatography (HPLC, Agilent 1260) equipped with an Eclipse XDB-C1 8 column. The mobile phase (a 1 : 1 (v/v) acetonitrile/water mixture) was used at 1 niL/min flow rate. The UV absorption at 230 nm was used for detection. In a typical procedure, an aliquot of micelle samples was dissolved in 950 \iL THF, and the organic solutions were centrifuged (13000 rpm, 10 min) and filtered using 0.2 μιη syringe filters. The filtered solutions (10 \iL) were then injected to the HPLC for DTX content analysis. The drug (e.g. DTX) loading (DL) and entrapment efficiency (EE) were calculated according to the following equations:
Figure imgf000063_0001
Results
The PEG-PPMD and PEG-PCMD copolymers self-assemble readily in aqueous medium to form micelle nanoparticles. The formation of the micelle nanoparticles was monitored by fluorometry using pyrene as a fluorescent probe and the critical micelle concentration (CMC) values were calculated. The CMC values for PEG-PPMD with 12%, 43% and 70% PDL content are shown in Figure 2 A, and those for PEG-PCMD with 13%, 41% and 70% CL content are shown in Figure 2B. Figures 2 A and 2B show variations in fluorescence intensity ratio (I3/I1) of pyrene as a function of logarithm of polymer concentration for different PEG-PPMD and PEG- PCMD copolymers in PBS. With increasing polymer concentration from 0.0001 mg/mL to 1.0 mg/mL, the curves show cross points yielding the CMC values of the polymeric micelles. Above CMC values, stable micelles encapsulating an increased amount of pyrene are formed (Huh, et al. , Langmuir 2000, 16, 10566- 10568). The CMC values calculated for the PEG- PPMD copolymers with 12%, 43% and 70% PDL are respectively 10.9 μg/mL, 10.6 μg/mL and 8.5 μg/mL. A similar trend is found for PEG-PCMD micelle samples. The CMC values of the copolymers with 13%, 41% and 70% CL are 15.5 μg/mL, 14.7 μg/mL and 9.7 μg/mL, correspondingly. Thus, the higher lactone content in the polymer chains improves the stability of the copolymer micelles in aqueous medium by increasing the hydrophobicity in the micelle cores. It is also notable that PDL is a stronger hydrophobicity- enhancer than CL, leading to lower CMC values for PEG-PPMD vs PEG- PCMD at the same lactone content.
Table 3. Characterization Data of DTX-loaded PEG-PPMD and PEG-PCMD
Micelles in PBS 10 mM H 7.4 .
Figure imgf000064_0001
standard formulations (Taxotere® and Duopafei®) require Tween 80 and ethanol vehicle for parenteral administration (Li, et al., Chem Pharm Bull. 2012, 60, 1146-1154). Here, we use biodegradable PEG-PPMD and PEG- PCMD copolymers for encapsulation of DTX to improve its solubility and cellular uptake. The average size, polydispersity index (PDI), and zeta potential of DTX-loaded PEG-PPMD and PEG-PCMD micelles measured by DLS are shown in Table 3. The drug-loaded PEG-PPMD micelles had an average size between 84 and 121 nm, which is smaller than the average sizes between 140 and 203 nm observed for the DTX-encapsulated PEG-PCMD micelles. This likely due to the fact that the presence of PDL units in PEG- PPMD chains render the copolymer micelle cores significantly more hydrophobic than those of the PEG-PCMD micelles, thus substantially reducing the water absorption in the nanoparticle cores (Theerasilp, et al., J Microencapsul. 2013, 30, 390-397; Riley, et al., Langmuir 2001, 17, 3168- 3174). The morphologies of DTX-loaded micelles formed from PEG-PPMD- 12% PDL and PEG-PCMD-13% CL copolymers were examined by TEM. The size distributions of these two micelles are shown in Figures 3 A and 3B. The TEM images of both micelle samples show uniform, spherical shape with comparable average sizes and narrow size distributions.
The sizes of the micelles measured by TEM are smaller than those measured by DLS. Possibly, the removal of water from the micelle samples during the TEM analysis may shrink the micelle particles (Fontana, et al., Biomaterials 2001, 22, 2857-2865). All DTX-loaded micelle samples were slightly negative-charged on surface (Table 3). This is beneficial for in vivo drug delivery because previous studies indicate that nanoparticles with zeta potential values ranging from -10 to +10 mV can decrease serum protein binding and increase particle circulation time in the blood (Li, et al., Mol Pharm. 2008, 5, 496-504; Levchenko, et al, Int J Pharm. 2002, 240, 95- 102).
The results for the in vitro stabilities for selected PEG-PPMD and PEG-PCMD micelles are shown in Figures 4 A and 4B. Consistently, the average size of PEG-PPMD and PEG-PCMD micelles remained fairly constant upon incubation for 7 d in PBS (10 mM, pH 7.4) with 10% FBS.
These results demonstrate that the PEG-PPMD and PEG-PCMD micelles are stable under the physiological conditions and the PEG shells in the micelle particles are efficient in preventing the nanoparticles from forming large agglomerates in PBS solution with 10 vol% FBS.
The drug loading and entrapment efficiency for the DTX- loaded micelles were determined by HPLC analysis (Table 3). The DTX entrapment efficiencies (EE) are 65% -67% for the PEG-PPMD micelles and are in the range between 65% and 76% for the PEG-PCMD micelles. The drug loading (DL) amounts in all micelle samples are comparable (3.3-3.8 wt%).
Example 3: pH and redox- triggered disassembly of micelles
Methods
The size change of the PEG-PPMD and PEG-PCMD micelles in response to acidic or reductive conditions in PBS solution was analyzed by dynamic light scattering (DLS). Typically, aliquots of blank micelle solutions were added into six different media: (i) PBS buffer (10 mM, pH 7.4), (ii) PBS buffer (10 mM, pH 7.4) containing 10 mM D,L-dithiothreitol (DTT), (iii) PBS buffer (10 mM, pH 7.4) containing 50 mM DTT, (iv) PBS buffer (10 mM, pH 5.0), (v) PBS buffer (10 mM, pH 5.0) containing 10 mM DTT (D,L-dithiothreitol), and (vi) PBS buffer (10 mM, pH 5.0) containing 50 mM DTT. After the resultant mixtures were incubated in a shaking bed at 37 °C and 100 rpm for 48 h, the micelle sizes, size distributions and count rates were determined by DLS.
Results
Functional nanoparticles responsive to acidic pH and intracellular reduction potential are useful drug carriers that can selectively deliver and release a drug at controllable rates to specific disease sites (e.g., acidic tumors and tumor cells). Thus, the responsive behaviors of blank PEG- PPMD and PEG-PCMD micelles upon exposure to acidic and reductive conditions, either individually or synergistically, were investigated. The micelles were incubated for 48 h in different PBS buffers with pH of 7.4 or 5.0 containing various amount of D,L-dithiothreitol (DTT, 0 to 50 mM), and the size variations of the micelle particles were measured by DLS analysis.
The DLS plots of six PEG-PPMD and PEG-PCMD micelle samples (PEG-PPMD with 12%, 43% and 70% PDL and PEG-PCMD with 13%, 41% and 70% CL) are shown in Figures 5A-5F, and the data for representative PEG-PPMD-12% PDL and PEG-PCMD-13% CL
nanoparticles are summarized in Table 4. At pH of 7.4, the mean particle size of PEG-PPMD and PEG-PCMD micelle samples remains essentially constant (Figures 4A and 4B). In general, upon decreasing the pH to 5.0, the sizes of the micelles increase significantly due to protonation of the tertiary amino groups in the micelle cores which become more hydrophilic to absorb extra water molecules from the media (Table 4). There is an exceptional case where the average size of PEG-PCMD-13% CL micelles was smaller in PBS (without DTT) at pH of 5.0 vs 7.4 (Table 4). Possibly, some of the large micelle particles become completely soluble in the medium after excessive swelling at pH of 5.0, thus reducing the average size of the whole sample. This is consistent with the count rate results showing that the count rate value is substantially lower for the PEG-PCMD-13% CL micelles upon decreasing the pH of PBS from 7.4 to 5.0 (Table 4). Similar phenomena were observed previously for other cationic polymers (Zhang, et al., Colloid Surface B. 2014, 115, 349-358; Liu, et al, Colloid Polym Sci. 2004, 282, 387-393). It is also notable from Table 4 that during protonation, the size distribution of the micelles tends to be broader and the count rate (in proportion to micelle concentration) tends to drop, confirming that protonation decreases the stability of the PEG-PPMD and PEG-PCMD micelles in aqueous medium.
Table 4. The size variations of PEG-PPMD and PEG-PCMD micelles incubated for 48 h in PBS solutions with different pH and DTT Contents
Figure imgf000068_0001
When DTT concentration is increased from 0 to 50 mM in the PBS media at a constant pH, PEG-PPMD-12% PDL and PEG-PCMD-13% CL micelles also swell remarkably with average particle size increase between 150% and 230%, corresponding to 320 nm and 270 nm, respectively (Table 4). In addition to protonation, the presence of DTT further increases the micelle size, broadens the micelle PDL value and decreases the micelle count rate, which is attributable to the cleavage of -S-S- bonds in the micelles by DTT to form more hydrophilic and looser micelle inner cores (Zhang, et al., Polym Chem- Uk. 2014, 5, 5124-5138) . Therefore, reductants such as DTT can effectively trigger disintegration of the PEG-PPMD and PEG-PCMD nanoparticles for controlled drug release and delivery applications.
Importantly, the response of both PEG-PPMD and PEG-PCMD micelles to pH and reduction potential are markedly synergistic (Table 4), which should render these nanocarriers especially effective for drug delivery to target sites with acidic pH and high reduction potential such as tumors.
Example 4: In vitro drug release from DTX-loaded micelles
Methods
The drug release behavior of DTX-loaded PEG-PPMD and PEG- PCMD micelles was studied using a dialysis method. Each DTX-loaded micelle sample was placed into four dialysis bags (MWCO 3500 Da) which respectively were immersed into four different PBS solutions (10 mM) containing 0.5% (w/v) Tween 80: (i) PBS buffer with pH 7.4, (ii) PBS buffer with pH 7.4 and 10 mM DTT, (iii) PBS buffer with pH 5.0, and (iv) PBS buffer with pH 5.0 and 10 mM DTT. The micelle samples were incubated at 37 °C in a shaking bed with a rotation speed of 100 rpm. At each time interval, 1 mL of the external PBS solutions was withdrawn and the same amount of fresh PBS was then added. The harvested PBS solutions were analyzed by HPLC and the amount of DTX released from the micelles was calculated. All experiments were performed in triplicate.
Results
The in vitro release of DTX from PEG-PPMD and PEG-PCMD micelles were investigated at 37 °C under following four different conditions: (i) in PBS with pH of 7.4, (ii) in PBS with pH of 7.4 and 10 mM DTT, (iii) in PBS with pH of 5.0, and (iv) in PBS with pH of 5.0 and 10 mM DTT. The drug release rate is dependent on the composition of the copolymers and is affected also by the medium pH and reduction potential. The accumulative release of DTX from the PEG-PPMD and PEG-PCMD micelles during a period of 168 h are depicted in Figures 6A-6F. Generally, the drug release rate of all micelle followed biphasic release kinetics. For example, at physiological pH of 7.4, the DTX-loaded PEG-PPMD micelles released 35-38% drug during the initial 12 h, then a gradual release of additional 25-32% drug was found for the subsequent 156 h (Figures 6A- 6C). The accumulated DTX released from the micelles of PEG-PPMD- 12% PDL, PEG-PPMD-43% PDL and PEG-PPMD-70% PDL copolymers were respectively 70%, 66% and 69% at the end of the incubation period (168 h). Upon decreasing the medium pH from 7.4 to 5.0, all micelle samples respond to the acidic pH to exhibit accelerated drug release rates (Figures 6A-6F). In particular for the PEG-PCMD- 13% CL, PEG-PCMD-41% CL and PEG-PCMD-70% CL micelles over the 168 h incubation period, the total released drug is respectively 67%, 70% and 67% at pH 7.4, which increases correspondingly to 89%, 95% and 88% at pH 5.0 (Figures 6D-6F). This acid- triggered fast drug release is consistent with our previous results obtained from PEG-poly(PDL-co-MDEA-co-sebacate) (PEG-PPMS) drug delivery system (Zhang, et al., Colloid Surface B. 2014, 115, 349-358) and is attributable to the swelling of the micelle particles due to protonation of the micelle cores. In addition to being responsive to pH, many of the PEG- PPMD and PEG-PCMD micelles (e.g., PEG-PPMD- 12% PDL, PEG-PPMD- 43% PDL, PEG-PCMD-41% CL, PEG-PCMD-70% CL) also respond predictably to DTT that was added to the media, and are triggered by the reductant to release the drug at an accelerated rate. The minimal response to DTT for the PEG-PPMD-70% PDL micelles is presumably due to their highly hydrophobic, PDL-rich micelle cores that prohibit the diffusion of water-soluble DTT from the media to react with and cleave the low abundant disulfide bonds in the micelles (Figure 6C). The abnormal DTT-responsive drug release behaviors were also observed for DTX-loaded PEG-PCMD- 13% CL micelles (Figure 6D). This is likely due to the rapid reduction of the copolymer by DTT, causing precipitation of bulky polymer that traps DTX with a minimal drug release rate. Importantly, the drug release rate from the PEG-PPMD and PEG-PCMD micelles is responsive to and regulated by both medium pH and reductant DTT. Further, the accelerated drug-release effects triggered by pH and DTT are substantially synergistic, and the fastest DTX release rates occurred at acidic pH of 5.0 and in the presence of 10 mM DTT (Figures 6D-6F). Compared to the previously reported PEG-PPMS micelles that are solely pH-responsive (Zhang, et al., Colloid Surface B. 2014, 115, 349-358) and PEG-PPBD micelles that are solely redox-responsive (Liu, et al, Polym. Chem. 2015, 6, 1997-2010), the current PEG-PPMD and PEG- PCMD nanoparticles with synergistic pH and redox-responsive properties are expected to be significantly more potent nanocarriers for intracellular delivery and release of chemotherapeutic agents to cancer cells since their drug delivery efficiency can be boosted by both acidic tumor or endosomal pH and the unusually high reduction potential (due to high GSH level) in cancer cells.
Example 5: Cellular uptake and intracellular distribution of PEG- PPMD and PEG-PCMD nanoparticles
Methods
To evaluate the cellular uptake capacities of PEG-PPMD and PEG- PCMD micelles, fluorescence probe molecule coumarin-6 (C6) was encapsulated in PEG-PPMD- 12%, 43%, and 70% PDL and PEG-PCMD- 13%, 41%, and 70% CL micelles according to the protocol employed for preparation of DTX-loaded micelles (Example 2, A) and the cellular uptake of the C6-loaded micelles was examined by flow cytometry using HeLa cells. Specifically, HeLa cells in 500 μΐ medium at a density of 4.0 x 105 cells/mL were seeded in a 24- well plate overnight at 37 °C under 5% CO2. Subsequently, free C6, C6-loaded PEG-PPMD micelles and C6-loaded PEG- PCMD micelles were respectively added to each well at a C6 concentration of 0.2 μg/mL. The experimental methods described here is a general method that was used for all tested samples. After incubation for 1 to 8 h, the culture media were removed and the cells were washed three times with cold PBS solution (10 mM, pH 7.4). The washed cells were detached with trypsin, harvested and centrifuged at 2000 rpm for 5 min to remove the supernatants. Upon resuspension of the cells in 500 μΐ PBS, the cellular internalization efficiency of the micelle samples was analyzed by FACSCalibur at an excitation wavelength of 488 nm and an emission wavelength of 585 nm (10000 cells per group).
The intracellular location of C6-loaded micelles was visualized using confocal laser scanning microscopy (CLSM). HeLa cells (1 x 105 cells/well) were seeded on 15 mm glass-bottom dishes in a 6-well plate overnight. The cells were incubated with free C6, C6-loaded PEG-PPMD-12% PDL micelles or C6-loaded PEG-PCMD-13% CL particles at a C6 concentration of 0.2 μg/mL. After incubation for 2 and 6 h, the media were removed and the cells were washed three times with cold PBS solution. Thereafter, the cell lysosomes were stained with 75 nM Lysotracker-red and the cell nuclei were stained with 10 μg/mL Hoechst 33342. The cells were then rinsed, fixed with paraformaldehyde (PFA), washed by PBS and then observed by CLSM. The excitation wavelength for detecting Hoechst 33342, Lysotracker-red, and C6 was 405 nm, 577 nm and 467 nm, respectively.
Results
The cellular uptake efficiency of the DTX-loaded PEG-PPMD and PEG-PCMD micelles was evaluated with both flow cytometry and confocal laser scanning microscopy (CLSM). To facilitate the cellular uptake study, the DTX drug was replaced by fluorescent probe molecule coumarin-6 (C6) and C6-loaded micelles of PEG-PPMDPDL and PEG-PCMD were used instead. Figure 7 shows the mean fluorescence intensity (MFI) values of HeLa cells incubated with free C6 or the C6-encapsulated micelle samples for up to 8 h. It is evident that the cellular uptake process is time-dependent with an increasing number of the micelles being internalized by the cells during the first six hours. Over this period, C6-loaded PEG-PPMD-12% PDL and PEG-PCMD-13% CL micelles exhibit higher cellular uptake efficiency than other micelle samples, and are the best performers in their own micelle sample groups (Figure 7). Among the PEG-PPMD micelle samples, the cellular uptake efficiency decreases with increasing PDL content in the copolymer. The free C6 showed fastest cellular uptake due to its high hydrophobicity and quick diffusion through the cell membrane.
To determine if the micelles can escape from endosomes and lysosomes, the locations of the C6-loaded PEG-PPMD-12% PDL and PEG- PCMD- 13% CL micelles in HeLa cells relative to the positions of acidic endosomes/lysosomes (stained with Lysotracker Red) were visualized using CLSM. Clearly, the C6-loaded micelles were internalized by the cells and distributed over the whole cytoplasm after 2 h incubation. Importantly, the green fluorescence of both micelle samples exhibited minimal co- localization with the Lysotracker Red-stained organelles at 6 h of incubation, indicating their rapid endosomal escaping capability. It is possible that the pH buffer capacity of the tertiary amino groups present in the PPMD and PCMD segments of the copolymer chains facilitates the endosomal escape of the internalized micelles due to proton sponge effects. Further, the intracellular GSH is anticipated to react with the disulfide bonds in the micelles to cause polymer chain cleavage and disintegration of the micelle particles, thus promoting C6 release in the cells. These results strongly show that the pH and redox-responsive PEG-PPMD and PEG-PCMD
nanoparticles encapsulating anticancer drugs can be triggered by tumor- relevant acidic pH and intracellular GSH to rapidly release the drug molecules for efficient killing of cancer cells.
Example 6: In vitro cytotoxicity of micelles
Methods
A. Cytotoxicity towards cancer cell lines
The cytotoxicity of both blank micelles and DTX- loaded micelles were evaluated against HeLa cells and CT-26 cells using an MTT method described previously (Liu, et al, Polym. Chem. 2015, 6, 1997-2010.). Briefly, cells (3.0 x 103 cells/well) were seeded in 96- well plates and allowed to adhere overnight. Then the culture medium was removed and 200 of the fresh medium containing different concentrations of the blank or DTX- loaded micelles were added to each well. In order to investigate the stimuli- responsive properties of the DTX-loaded PEG-PPMD and PEG-PCMD micelles, the culture media with different pH (7.4 or 6.5) and different reduction potential (10 mM GSH or 0.2 mM BSO) were used to mimic different intracellular environments. After incubation with micelles under different medium environments for additional 48 h and then subjected to MTT assay. The absorbance of the solutions at 570 nm was measured on a microplate reader (BioTek Synergy4). Cell viabilities were calculated as the values in percentage of (absorbance of cells treated)/(absorbance of cells without micelle treatment).
The cytotoxicity of DTX-loaded PEG-PPMD and PEG-PCMD micelles were also evaluated by MTT assay against HeLa cells and CT-26 cells. Similarly, cells were seeded into 96-well plates and kept at 37 °C in 5% CO2 atmosphere. After the cells were allowed to adhere overnight, the culture medium was replaced by the medium containing one of the following four components: (i) various amount of DTX in the micelles with medium pH of 7.4, (ii) various amount of DTX in the micelles with medium pH of 6.5, (iii) various amount of DTX in the micelles and 10 mM glutathione (GSH) with medium pH of 7.4, (iv) various amount of DTX in the micelles and 0.2 mM L-buthionine-(5,R)-sulfoximine (BSO) with medium pH of 7.4. The cells treated only with the media (without the micelles) were used as the control. The viabilities of the cells after 48 h incubation were measured using the standard MTT assay. The results are reported as mean values + standard deviation (n = 3).
B. Erythrocyte agglutination and hemolysis assay
The PEG-PPMD and PEG-PCMD micelles were incubated with erythrocytes (RBCs) to determine blood compatibility of the copolymers. Human blood was centrifuged at 2500 rpm for 5 min at 4 °C and the RBC cells were washed three times with PBS (10 mM, pH 7.4). The blank micelles (400 μg/mL) and 1 x 108 RBCs were mixed in PBS and the mixtures were then incubated for 2, 12 and 24 h at 37 °C. Thereafter, the samples were centrifuged for 10 min at 1000 rpm and the supernatants were analyzed by a microplate reader to measure their absorbance at 413 nm. Additionally, Triton- 100 (1%, w/v) was tested as a positive control and isotonic PBS was tested as a negative control. Hemolysis value was calculated by following equation:
Figure imgf000074_0001
where Asampie, APBS, and Amton represent the absorbance intensity values of the supernatants from RBCs treated with the micelle samples, PBS and Triton X-100, respectively.
Results
The in vitro cytotoxicity of blank and DTX-loaded PEG-PPMD and
PEG-PCMD micelles was evaluated on HeLa cells and CT-26 cells. All blank micelles exhibited minimal cytotoxicity and the viabilities of the cells treated with the micelles were over 80% at various polymer concentrations up to 400 μg/mL (Figures 8A-8D). Further, these PEG-PPMD and PEG- PCMD micelle samples are compatible with human blood, showing essentially no hemolytic activity (<3% hemolysis value, Figure 9) and no tendency to induce erythrocyte agglutination even at a high polymer concentration of 400 μg/mL.
To elucidate the effects of pH and redox-promoted drug release on the micelle cytotoxicity, HeLa cells and CT-26 cells were treated with DTX- loaded PEG-PPMD and PEG-PCMD micelle samples and their viabilities were measured by MTT assay. Figures 10A-10F show the cell viability values after treating the cells with the PEG-PPMD micelles at pH of 7.4 and 6.5 under a controlled intracellular reduction potential. The cytotoxicity of the micelles is dependent on the composition of PEG-PPMD copolymers, medium pH and intracellular reduction potential. At a constant pH and reduction potential, the micelles formed from PEG-PPMD copolymer with a low PDL content appear to possess higher efficacy than those formed from the PDL-rich copolymer. Thus, the DTX-loaded micelles of PEG-PPMD- 12% PDL, PEG-PPMD-43% PDL and PEG-PPMD-70% PDL against HeLa cells at pH of 7.4 yielded ICso values of 0.535, 0.671 and 2.94 μg/mL, respectively. In general, at a constant reduction potential, the viability of the HeLa cells and CT-26 cells was lower upon treatment at pH of 6.5 vs pH of 7.4 (Figures 10A-10F). For example, the ICso values of DTX-loaded PEG- PPMD- 12% PDL micelles against HeLa and CT-26 cell lines are respectively 0.535 and 0.604 μg/mL at pH of 7.4, which decrease correspondingly to 0.248 and 0.171 μg/mL at a pH of 6.5. As reported previously (Liu, et al., Polym. Chem. 2015, 6, 1997-2010), the intracellular reduction potential of both cell types can be increased by feeding the cells with free reductant GSH or reduced by feeding the cells with BSO (an inhibitor for cellular GSH synthesis). As shown, increasing intracellular GSH concentration enhances the cytotoxicity of DTX-loaded PEG-PPMD micelles. Thus, for both HeLa and CT-26 cell lines treated with the micelle formulations at a same DTX dose, the cell group pretreated with GSH mostly had lower viability and those pretreated with BSO mostly exhibited higher viability than the group without GSH or BSO treatment (Figures 10A-10F). Specifically, the ICso values of the drug-loaded PEG-PPMD- 12% PDL micelles were respectively 0.136, 0.535 and 0.612 μg/mL against three HeLa cell groups that were pretreated with GSH, without pretreatment and pretreated with BSO. This trend also applies to CT-26 cell line. For CT-26 cell groups that were pretreated with GSH, without pretreatment and pretreated with BSO, the ICso values of the PEG-PPMD- 12% PDL micelles were correspondingly 0.334, 0.604 and 0.915 μg/mL. These results demonstrate that both acidic pH and a high reduction potential elevate the potency of the DTX-encapsulated PEG-PPMD micelles against cancer cells. Similar effects of pH and reduction potential on micelle cytotoxicity were also observed for the drug-loaded PEG-PCMD micelle samples (Figures 11A-11F). The cytotoxicity data in Figures 10A-10F, and 11A-11F are consistent with the in vitro drug release and cellular uptake results described above, further supporting our hypothesis that the PEG-PPMD and PEG- PCMD micelles encapsulating DTX can be triggered by tumor-relevant acidic pH and high intracellular reduction potential to rapidly unload the drug for efficient killing of cancer cells.
Example 7: In vivo antitumor efficiency
Methods
The antitumor efficiency of DTX-loaded PEG-PPMD- 12% PDL micelles and PEG-PCMD-13% CL micelles was evaluated using Balb/C mice bearing mouse colon carcinoma CT-26 xenograft. CT-26 cells (lxlO6 cells) were injected subcutaneously in the right back of Balb/C mice (5 weeks, 16 g). When tumors reached 50— 100 mm3 in volume, treatments were started and the initial treatment day was designated as day 0.
On day 0, the mice were randomly assigned to one of the following 4 groups (at least 4 mice in each group): 0.9% NaCl (control), free DTX (Duopafei®), DTX-loaded PEG-PPMD-12% PDL micelles and DTX-loaded PEG-PCMD-13% CL micelles. Mice were injected intravenously through the tail vein with free DTX, DTX-loaded PEG-PPMD-12% PDL micelles and PEG-PCMD-13% CL micelles (at 10 mg/kg DTX dose) every three days for four times. The control group of mice was administered via injection with 0.9% NaCl following the same procedure.
The tumor volume was measured every other day using a vernier caliper and the body weight of the mice was recorded at the same time. At day 27, the mice were sacrificed to collect the tumors and important organs (heart, liver, spleen, lung and kidney), whose tissues were analyzed by hematoxylin-eosin (H&E) staining to determine the antitumor effects of the micelles.
To verify anticancer efficacy of the micelle formulations in vivo, free DTX (Duopafei®), 0.9% NaCl (as a control), DTX-loaded PEG-PPMD-12% PDL and PEG-PCMD-13% CL micelles were intravenously injected into Balb/C mice bearing subcutaneous CT-26 tumors.
Results
Figures 12A and 12B illustrate variations of the tumor volume and body weight of the mice during the 21 -day treatment period. The results clearly show that the DTX-loaded micelles are substantially more potent than free DTX in inhibiting growth of the tumor xenografts. At day 13 during the treatment, the average tumor volume of the mice treated with 0.9% NaCl, free DTX, DTX-loaded PEG-PPMD-12% PDL and DTX-loaded PEG- PCMD-13% CL micelles were 2300, 994, 524 and 658 mm3, respectively (Figure 12A). Thus, the antitumor potency follows the order: DTX-loaded PEG-PPMD-12% PDL micelles > DTX-loaded PEG-PCMD-13% CL micelles > free DTX > 0.9% NaCl. To further evaluate the antitumor activity of the DTX-loaded PEG- PPMD-12% PDL and DTX-loaded PEG-PCMD-13% CL micelles, tumor specimens from the mice receiving different treatments were collected at the end of the treatment period and prepared for hematoxylin and eosin (H&E) staining. All tumor tissue samples stained with H&E, showed cracked nuclear membranes and condensed chromatins that were marginalized and divided into blocks or apoptotic bodies, indicating apoptosis of the tumor cells. The rate of necrosis of pieces was in the order: 0.9% NaCl (60-70%) > DTX-loaded PEG-PCMD-13% CL micelles (50-60%) > DTX-loaded PEG- PPMD-12% PDL micelles (40-50%) > free DTX (10-20%). The highest rate of necrosis of pieces obtained for 0.9 % NaCl is primarily due to the extremely big size of its treated tumors which causes self-necrosis. On the other hand, the lowest rate of necrosis of pieces obtained for the free DTX is possibly attributed to the instability and fast biodegradation of the unprotected drug molecules. These experimental results demonstrate the advantages of using intracellular stimuli-responsive nanoparticles for controlled delivery and release of chemotherapeutic drugs to kill cancer cells. Finally, while increasing efficacy of anticancer drug formulations is important, it is also beneficial to minimize their effects on normal organ tissues during treatment. H&E staining images were made of various organs including heart, liver, spleen, lung and kidney, which were harvested from the mice on day 27 after the treatment with DTX-loaded PEG-PPMD-12% PDL and PEG-PCMD-13% CL micelles at DTX dose of 4 x 10 mg/kg. Consistent with the minimal body weight loss (Figure 12B), no apparent histological damage in these organs was observed following the treatment courses. Therefore, the DTX formulations based on PEG-PPMD or PEG- PCMD micelles are not only therapeutically efficient, but also potentially safe for antitumor treatment in vivo. Example 8: In vivo biodistribution
Methods
CT-26 tumor-bearing mouse models were used to investigate the biodistribution of DiR- loaded polymeric micelles. Fluorescence probe DiR was encapsulated in PEG-PPMD-12% PDL and PEG-PCMD-13% CL micelles according to the protocol employed for preparation of DTX- loaded micelles (Example 2, A). CT-26 cells (lxlO6 cells/0.1 mL) were injected subcutaneously in the right back of Balb/C mice (5 weeks, 16 g). When tumors reached 200-300 mm3 in volume, the DiR-loaded PEG-PPMD-12% PDL and DiR-loaded PEG-PCMD- 13% CL micelles (0.5 mg/kg DiR) were injected intravenously through the tail vein. In vivo fluorescence images were taken at 4 h, 12 h, and 24 h after injection using the IVIS Lumina XR imaging system (the excitation wavelength and emission wavelength of DiR are respectively 750 nm and 780 nm). The mice were sacrificed for further observation of organ accumulation of DiR after its administration for 24 h. The heart, lung, spleen, liver, kidney and tumor were harvested, washed with cold saline and photographed using the IVIS Lumina XR imaging system. Results
To determine the biodistribution of the PEG-PPMD and PEG-PCMD micelles, DiR-loaded PEG-PPMD-12% PDL and PEG-PCMD-13% CL micelles were injected intravenously into the CT26 tumor-bearing mice, and in vivo biodistribution was monitored using IVIS imaging system. The accumulation of the DiR-loaded micelles in tumor was time-dependent.
At 4 h of post-injection, strong fluorescence was detected throughout the body (including the tumor region) of the mice, indicating that the DiR- loaded polymeric micelles entered the blood circulation and partially accumulated in the tumor. With increasing circulation time, the fluorescence signal at tumor sites became stronger, which is clearly observable for the mouse injected with PEG-PPMD-12% PDL micelles. Further, it is notable that the fluorescence signal persisted for 24 h over the whole body.
PEGylation was remarkably effective in improving the in vivo circulation time and stability of both PEG-PPMD-12% PDL and PEG- PCMD-13% CL micelles. The intensity of fluorescence signals measured from the ex vivo organs and tumors confirmed the results of in vivo observation. As shown in Figure 13, the micelle samples were largely distributed in the liver, lung, spleen and tumor. Importantly, the PEG-PPMD- 12% PDL and PEG-PCMD-13% CL micelles are more abundantly present in the tumor than in the other organs examined. On the basis of these observations, it can be concluded that the PEG-PPMD-12% PDL and PEG- PCMD-13% CL micelle samples possess excellent in vivo circulation and passive tumor-targeting properties which are highly beneficial for achieving effective in vivo antitumor treatments.
In summary, multifunctional nanoparticle drug delivery systems have been constructed which are stable under physiological conditions and responsive to tumor-relevant pH and intracellular reduction potential. The nanoparticles were fabricated from new enzymatic PEG-PPMD and PEG- PCMD block copolymers via a self-assembly process in aqueous solution. At acidic pH and in the presence of a reductant (e.g., DTT or GSH), the nanosized micelle particles rapidly swelled and disintegrated due to the protonation of amino groups and reductive cleavage of disulfide bonds in the micelle cores. The DTX-loaded PEG-PPMD and PEG-PCMD micelles were triggered synergistic ally by both acidic endosomal pH and a high
intracellular reduction potential to rapidly release the drug for efficient killing of cancer cells. The drug formulations based on PEG-PPMD and PEG-PCMD copolymers exhibited a significantly higher potency than free DTX in inhibiting tumor growth in mice, while their therapeutic effects on important organ tissues were minimal. These results demonstrate that PEG- PPMD and PEG-PCMD nanoparticles serve as site specific, pH and redox- responsive drug nanocarriers for safe and efficient antitumor treatment.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

We claim:
1. A polymer comprising a polymeric unit having the formula: wherein:
Figure imgf000082_0002
A, B, C, and D independently comprise monomeric units derived from a lactones, a polyfunctional molecule containing an amine group and a hydroxyl group, a molecule containing a disulfide bond, or hydrophilic polymer;
the monomeric units comprise the lactone, the polyfunctional molecule containing an amine group and a hydroxyl group, and the molecule containing the disulfide bond;
a, b, c, and d are independently integers between 0 and 1000, inclusive, with the proviso that the sum (a + b + c + d) is greater than one; and
h is an integer between 1 and 1000, inclusive.
2. The polymer of claim 1, wherein the polymeric unit has the formula:
Figure imgf000082_0001
wherein:
x, y, and z are independently integers between 1 and 1000, inclusive; each occurrence of m is an integer between 1 and 30, inclusive;
p, q, n, and t are independently integers between 1 and 20, inclusive;
R8 and R10 are independently O or NR' , wherein R' is hydrogen, substituted alkyl, unsubstituted alkyl, substituted aryl, or unsubstituted aryl, wherein at least one of R8 and R10 is O; and
R9 is unsubstituted alkyl, substituted alkyl, hydrogen, substituted aryl, or unsubstituted aryl.
3. The polymer of claim 2, wherein m is an integer between 2 and 15, inclusive.
4. The polymer of claim 3, wherein n, p, q, and t are independently integers between 2 and 5, inclusive.
5. The polymer of claim 4, wherein R8 and R10 are O.
6. The polymer of claim 5, R9 is unsubstituted C1-C10 alkyl, or substituted C1-C10 alkyl.
7. The polymer of claim 6, wherein m is 3, 12, or a combination thereof; and p and q are 2.
8. The polymer of claim 7, wherein n and t are 2.
9. The polymer of claim 8, wherein R9 is methyl, ethyl, n-propyl, isopropyl, n-butyl, or t-butyl, or phenyl.
10. The polymer of claim 9, wherein the polymeric unit selected from the group consisting of poly(ro-pentadecalactone-co-N-methyldiethyleneamine- co-3,3'-dithiodipropionate) and poly(s-caprolactone-co-N- methyldiethyleneamine-co-3,3'-dithiodipropionate).
11. The polymer of any one of claims 2-10, having the formula:
Figure imgf000083_0001
wherein:
r is an integer between 1 and 1000, inclusive;
T is O or absent; and
R6 is hydrogen, unsubstituted alkyl, substituted alkyl, unsubstituted aryl, substituted aryl, unsubstituted cycloalkyl, substituted cycloalkyl, maleimide, amine, thiol, N-hydroxysuccinimide ester, azide, acrylate, methacrylate, alkyne, hydroxide, or isocynate.
12. The polymer of claim 11, wherein T is O and R6 is methyl.
13. The polymer of claim 12, wherein the polymers are selected from the group consisting of polyethylene glycol-poly(ω-pentadecalactone-co-N- methyldiethyleneamine-co-3,3'-dithiodipropionate) (PEG-PPMD) and polyethylene glycol-poly(s-caprolactone-co-N-methyldiethyleneamine-co-
3 ,3'-dithiodipropionate) (PEG-PCMD). The polymer of any one of claims 11-13, having the formula:
Figure imgf000084_0001
wherein:
r is an integer between 1 and 1000, inclusive;
T is O or absent; and
R6 is hydrogen, unsubstituted alkyl, substituted alkyl, unsubstituted aryl, substituted aryl, unsubstituted cycloalkyl, substituted cycloalkyl, maleimide, amine, thiol, N-hydroxysuccinimide ester, azide, acrylate, methacrylate, alkyne, hydroxide, or isocynate.
15. The polymer of claim 14, wherein T is O and R6 is methyl.
16. The polymer of any one of claims 1-15, wherein the weight average molecular weight of the polymer is between 1 kDa and 50 kDa, inclusive.
17. A nanoparticle comprising a polymer of any one of claims 1 to 16, wherein the diameter of the nanoparticle is between 50 nm and 500 nm, inclusive.
18. The nanoparticle of claim 17, having a zeta potential between -10 mV and +10 mV.
19. The nanoparticle of claim 17 or 18, further comprising a therapeutic, diagnostic, and or prophylactic agent encapsulated within the nanoparticles, dispersed within the polymeric matrix that forms the nanoparticles, on the surface of the nanoparticles, non-covalently or covalently bound to a polymer from which the nanoparticles are formed, or a combination thereof.
20. The nanoparticle of any one of claims 17-19, formed by self- assembly of the polymers.
21. The nanoparticle of claim 19, wherein the therapeutic agent constitutes between 0.5 wt/wt% and 25 wt/wt% of the nanoparticle.
22. The nanoparticle of any one of claims 19-21, wherein the therapeutic agent is a chemotherapeutic agent.
23. The nanoparticle of any one of claims 17-22, further comprising a targeting moiety.
24. A method of making polymers, the method comprising:
mixing an enzyme catalyst with monomeric units under conditions forming a polymer of Formula I:
Figure imgf000085_0001
wherein:
the monomers are selected from the group consisting of one or more lactones having the formula:
Figure imgf000085_0002
one or more polyfunctional molecules containing an amine group and a hydroxyl group, having the formula:
Figure imgf000085_0003
and one or more compounds containing a disulfide bond, having the formula:
Figure imgf000085_0004
x, y, and z are independently integers between 1 and 1000, inclusive; each occurrence of m is an integer between 1 and 30, inclusive;
p, q, n, and t are independently integers between 1 and 20, inclusive;
Ri and R2 are independently hydrogen, substituted alkyl,
unsubstituted alkyl, substituted aryl, or unsubstituted aryl;
R3 and R5 are independently OH or N(R')2, wherein each R' is independently hydrogen, substituted alkyl, unsubstituted alkyl, substituted aryl, or unsubstituted aryl, wherein at least one of R3 and R5 is OH, and at least one R' is hydrogen;
R4 is unsubstituted alkyl, substituted alkyl, hydrogen, substituted aryl, or unsubstituted aryl;
R8 and R10 are independently O or NR' , wherein R' is hydrogen, substituted alkyl, unsubstituted alkyl, substituted aryl, or unsubstituted aryl, wherein at least one of R8 and R10 is O; and
R9 is unsubstituted alkyl, substituted alkyl, hydrogen, substituted aryl, or unsubstituted aryl.
25. The method of claim 24, wherein the enzyme catalyst is a lipase.
26. The method of claim 25, wherein the lipase is lipase B from Candida antarctica.
27. The method of claim 24, wherein the molar feed ratios of the lactone/molecule containing the disulfide bond/polyfunctional molecule containing the amine group and hydroxyl group is between 5 :20: 10 and 80:90:90.
28. The method of any one of claims 24-27, wherein the weight average molecular weight of the polymer is between 1 kDa and 50 kDa, inclusive.
29. The method of claim 24, comprising
(i) incubating the monomer units and enzyme catalyst at a temperature from between about 80 °C and about 90 °C, at about 1 atm N2.
30. The method of claim 29, further comprising, after step (i),
(ii) incubating the monomer units and enzyme catalyst at a temperature from between about 80 °C and about 90 °C, at about 2 mm Hg.
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