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  • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
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  • C08G65/32—Polymers modified by chemical after-treatment
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  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
  • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
  • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• • A—HUMAN NECESSITIES
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  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
  • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/06—Antiasthmatics
• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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  • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
  • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
  • C08G65/32—Polymers modified by chemical after-treatment
  • C08G65/329—Polymers modified by chemical after-treatment with organic 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
  • C08G83/00—Macromolecular compounds not provided for in groups C08G2/00 - C08G81/00
  • C08G83/002—Dendritic macromolecules
  • C08G83/003—Dendrimers
  • C08G83/004—After treatment of dendrimers
• • C—CHEMISTRY; METALLURGY
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  • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
  • C08L71/00—Compositions of polyethers obtained by reactions forming an ether link in the main chain; Compositions of derivatives of such polymers
  • C08L71/02—Polyalkylene oxides
• • A—HUMAN NECESSITIES
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  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
  • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
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  • Y10T428/29—Coated or structually defined flake, particle, cell, strand, strand portion, rod, filament, macroscopic fiber or mass thereof
  • Y10T428/2982—Particulate matter [e.g., sphere, flake, etc.]

Definitions

• the final size of the nanocarriers ( 10 to 100 nm) is tunable by using various, or a combination of, different cholane-PEG preparations.
• the nanocarrier components, PEG and cholic acid are all biocompatible and largely non-toxic. Indeed, the PTX nanotherapeutics exhibited safe profile in in vivo administration for anticancer treatment in mouse models and companion dogs. However, some nanocarriers exhibited some hemolytic activity both in vitro and in vivo, as well as reduced stability and loading capacity for certain drugs.
• the present invention is based on the surprising discovery that certain crosslinkable functional groups can be introduced into telodendrimers, therefore crosslinking the nanoparticles reversibly to minimize premature drug release and increase in vitro and in vivo stability of the nanotherapeutics.
• the crossl inked nanotherapeutics improve the therapeutic properties without disrupting nanocarrier assembly and drug loading capacity and stability, therefore addressing the needs described above.
• Each R of formula I can be the end group of the dendritic polymer, a hydrophobic group, a hydrophilic group, an amphiphilic compound or a drug, such that when R is not an end group then each R is linked to one of the end groups.
• Each Y 1 and Y 2 of formula I is a crosslinkable group that can be any of boronic acid, dihydroxybenzene or a thiol.
• Subscript m of formula I is an integer from 0 to 20.
• Subscript n of formula I is an integer from 2 to 20, wherein subscript n is equal to the number of end groups on the dendritic polymer, and wherein at least half the number n of R groups that can each be a hydrophobic group, a hydrophilic group, an amphiphilic compound or a drug. And, each of subscripts p and q are 0 or from 2 to 8, such that one of subscripts p and q is from 2 to 8.
• the present invention provides a method of reversing the cross-linking of the reversibly crosslinked nanocarrier of the present invention, by contacting the reversibly crosslinked nanocarrier with a bond cleavage component suitable for cleaving the cross-linked bond, thereby reversing the cross-linking of the reversibly crosslinked nanocarrier.
• the present invention provides a method of treating a disease, including administering to a subject in need of such treatment, a therapeutically effective amount of a nanocarrier of the present invention, wherein the nanocarrier includes a drug.
• the drug can be a covalently attached to a conjugate of the nanocarrier.
• the present invention provides a method of delivering a drug to a subject in need thereof by administering a nanocarrier of the present invention to the subject, wherein the nanocarrier includes the drug and a plurality of cross-linked bonds.
• the method also includes cleaving the cross-linked bonds using a bond cleavage component, such that the drug is released from the nanocarrier, thereby delivering the drug to the subject.
• Figure 1 shows a schematic presentation of the disulfide cross-linked micelles formed by oxidation of thiolated telodendrimer PEG 5k -Cys 4 -Ls-CA 8 after self-assembly.
• Figure 2 shows the absorbance of PEG 5k -Cys4-Lg-CA 8 micelle solutions in Ellman's test (A) and the thiol conversions (B) as a function of oxidation time.
• the volume of the final micelle solution was kept at 1 mL and the final concentration of the polymers at 20 mg/mL.
• Figure 3 shows the particle size of PTX loaded non-crosslinked micelles (NCMs) (PTX loading: 5.0 mg/mL) in human plasma 50% (v/v) for 1 min (A), 24 h (B) and PTX loaded disulfide crosslinked micelles (DCMs) (PTX loading: 4.6 mg/mL) in plasma 50% (v/v) for 1 min (C) and 24 h (D) at 37 °C, respectively.
• NCMs non-crosslinked micelles
• DCMs disulfide crosslinked micelles
• Figure 4 shows (A) the stability in particle size of NCMs and DCMs in the presence of 2.5 mg/mL SDS measured by DLS.
• TEM images of NCMs B), DCMs (C) and DCMs treated with 10 mM GSH for 30 min (D) in the presence of 2.5 mg/mL SDS (scale bar: 50 nm).
• Figure 5 shows (A) PTX release profiles of DCMs at different GSH concentrations.
• Figure 6 shows MTT assays showing the viability of S OV-3 cells after 2 h incubation with (A) different concentrations of empty NCMs and DCMs; and (B) Taxol®,. PTX-NCMs and PTX-DCMs with and without pre-treatment of 20 mM GSH-OEt.
• C In vitro red blood cell (RBC) lysis of empty NCMs and DCMs. Values reported are the mean ⁇ SD for triplicate samples.
• Figure 7 shows the fluorescence signal of BODIPY labeled (A) and DiD loaded (B) DCMs and NCMs in the blood collected at different time points after i.v. injection in the nude mice.
• Figure 8 shows in vivo and ex vivo near infra-red fluorescence (NIRF) optical imaging.
• NIRF near infra-red fluorescence
• Figure 10 shows the chemical structure (A) and schematic representation (B) of PEG 5k -Cys 4 -L CA 8 .
• Figure 1 1 shows the MALDI-TOF MS of PEG 5k -Cys 4 -L s -C A 8 telodendrimer comparing with the starting PEG 5000 and PEG 5k -CA g telodendrimer.
• Figure 12 shows ⁇ NMR spectra of PEG 5k -Cys 4 -L 8 -CA 8 telodendrimer recorded in CDCls and D 2 0.
• Figure 14 shows cumulative PTX release profile from Taxol®, PTX loaded NCMs and DCMs.
• Figure 15 shows particle size of NCM-VCR (A), DCM-VCR (B) and DCM-VCR after cross-linking (C).
• TEM image of DCM-VCR after cross-linking (D) (scale bar: 50 nm).
• Vincristine (VCR) loading was 20: 1 telodendrimer to VCR (w/w).
• Figure 16 shows particle size of NCM-VCR and DCM-VCR under micelle disrupting conditions. NCM-VCR (A) and DCM-VCR (C) were incubated in 50% human plasma (v/v) for 24 h at 37°C.
• NCM-VCR (B) and DCM-VCR (D) were diluted to 2 mg/mL and incubated with 2.5 mg/mL SDS for 30 min.
• DCM-VCR (E) was incubated with both SDS and 20 raM N-acerylcysteine (NAC).
• FIG 17 shows drug release profile of conventional VCR, NCM-VCR and DCM- VCR (A).
• VCR formulations were dialyzed against 1 L PBS at 37°C in the presence of 10 g/L charcoal to maintain sink conditions.
• the in vitro cytotoxicity of conventional VCR, NCM-VCR and DCM-VCR was assessed in Raji cells treated for 72 h continuously (B) or 2 h, washed and then incubated for 70 h (C). Cell viability was measured using an MTS assay.
• Figure 18 shows ex vivo near-infrared optical imaging of Raji tumor bearing mice intravenously injected with free DiD or DCM co-loaded with VCR and DiD (DCM- VCR DiD). 72 h post injection, tumors and major organs were excised and imaged using an excitation/emission filter of 625/700 iim.
• Figure 19 shows in vivo anti-tumor efficacy (A) and body weight loss (B) of Raji tumor bearing nude mice treated with PBS, conventional VCR ( 1 mg/kg), DCM-VCR (1 mg/kg) plus or minus 100 mg/kg NAC or DCM-VCR (2.5 mg/kg). Arrows indicate the days when mice were treated. *, p ⁇ 0.05; **, p ⁇ 0.005.
• Figure 21 shows a schematic representation of the telodendrimer pair [PEG 5k - (boronic acid/catechol ⁇ -CAs] and the resulting boronate crosslinked micelles (BCM) in response to mannitol and/or acidic pH.
• Figure 26 shows the chemical structure of the catechol (A) containing
• telodendrimers and boronic acid (B and C) containing telodendrimers.
• Figure 27 shows the MALDI-TOF S of the starting PEG and the representative telodendrimer pair (PEG 5k -NBA 4 -CA 8 / PEG 5k -Catechol 4 -CA 8 ) comparing with PEG 5k -CA 8 telodendrimer.
• the pinacol ester form of PEG 5k -NBA 4 -CAg was shown.
• the present invention provides telodendrimers having crosslinking groups such that the nanocarrier micelles formed from the telodendrimers are crosslinked to improve stability of the nanocarrier micelle.
• the crosslinking groups can be on the dendritic polymer itself, or on the linking portion between the dendritic polymer and the PEG group. Any suitable crosslinking group can be used, such as those capable of reacting with themselves, or a complementary pair of functional groups that react with each other.
• telodendrimer refers to a dendrimer containing a hydrophilic PEG segment and one or more chemical moieties covalently bonded to one or more end groups of the dendrimer. These moieties can include, but are not limited to, hydrophobic groups, hydrophilic groups, amphiphilic compounds, and drugs. Different moieties may be selectively installed at a desired end group using orthogonal protecting group strategies.
• the term “nanocarrier” refers to a micelle resulting from aggregation of the dendrimer conjugates of the invention.
• the nanocarrier has a hydrophobic core and a hydrophilic exterior.
• dihydroxy carboxylic acid groups of the present invention include, but are not limited to, glyceric acid, 2,4-dihydroxybutyric acid, glyceric acid, 2,4-dihydroxybutyric acid, 2,2- Bis(hydroxymethyl)propionic acid and 2,2-Bis(hydroxymethyl)butyric acid.
• hydroxyl amino carboxylic acids include, but are not limited to, serine and homoserine.
• linker refers to a chemical moiety that links one segment of a dendrimer conjugate to another.
• bonds used to link the linker to the segments of the dendrimers include, but are not limited to, amides, amines, esters, carbamates, ureas, thioethers, thiocarbamates, thiocarbonate and thioureas.
• amides, amines, esters, carbamates, ureas, thioethers, thiocarbamates, thiocarbonate and thioureas One of skill in the art will appreciate that other types of bonds are useful in the present invention.
• oligomer refers to five or fewer monomers, as described above, covalently linked together.
• the monomers may be linked together in a linear or branched fashion.
• the oligomer may function as a focal point for a branched segment of a telodendrimer.
• hydrophobic group refers to a chemical moiety that is water-insoluble or repelled by water.
• hydrophobic groups include, but are not limited to, long-chain alkanes and fatty acids, fluorocarbons, silicones, certain steroids such as cholesterol, and many polymers including, for example, polystyrene and polyisoprene.
• hydrophilic group refers to a chemical moiety that is water-soluble or attracted to water. Examples of hydrophilic groups include, but are not limited to, alcohols, short-chain carboxylic acids, quaternary amines, sulfonates, phosphates, sugars, and certain polymers such as PEG.
• cholic acid refers to (R)-4-((3R, 55, 7R, 8R, 95, 105, 125, 13R, 145, 17R)-3, 7, 12-trihydroxy- 10, 13-dimethylhexadecahydrp- 1 H- cyclopenta[a]phenanthren- l 7-yl)pentanoic acid.
• Cholic acid is also known as 3 ⁇ ,7 ⁇ , 12 ⁇ - trihydroxy-5P-cholanoic acid; 3-a,7-a, 12-a-Trihydroxy-5- -cholan-24-oic acid; 17- ⁇ -(1 - methyl-3-carboxypropyl)etiocholane-3 a,7 a, 12 a -triol; cholalic acid; and cholalin.
• Cholic acid derivatives and analogs such as allocholic acid, pythocholic acid, avicholic acid, deoxycholic acid, chenodeoxycholic acid, are also useful in the present invention.
• Cholic acid derivatives can be designed to modulate the properties of the nanocarriers resulting from telodendrimer assembly, such as micelle stability and membrane activity.
• the cholic acid derivatives can have hydrophilic faces that are modified with one or more glycerol groups, aminopropanediol groups, or other groups.
• drug or “therapeutic agent” refers to an agent capable of treating and/or ameliorating a condition or disease.
• a drug may be a hydrophobic drug, which is any drug that repels water.
• Hydrophobic drugs useful in the present invention include, but are not limited to, paclitaxel, doxorubicin, etoposide, irinotecan, SN-38, cyclosporin A, podophyllotoxin, Carmustine, Amphotericin, Ixabepilone, Patupilone
• drugs of the present invention also include prodrug forms.
• prodrug forms One of skill in the art will appreciate that other drugs are useful in the present invention.
• crosslinkable group refers to a functional group capable of binding to a similar or complementary group on another molecule, for example, a first crosslinkable group on a first dendritic polymer linking to a second crosslinkable group on a second dendritic polymer.
• Groups suitable as crosslinkable and crosslinking groups in the present invention include thiols such as cysteine, boronic acids and 1 ,2-diols including 1 ,2-dihydroxybenzenes such as catechol. When the crosslinkable and crosslinking groups combine, they form cross-linked bonds such as disulfides and boronic esters. Other crosslinkable and crosslinking groups are suitable in the present invention.
• bond cleavage component refers to an agent capable of cleaving the cross-linked bonds formed using the crosslinkable and crosslinking groups of the present invention.
• the bond cleavage component can be a reducing agent, such as glutathione, when the cross-linked bond is a disulfide, or mannitol when the cross-linked bond is formed from a boronic acid and 1 ,2-diol.
• imaging agent refers to chemicals that allow body organs, tissue or systems to be imaged.
• imaging agents include paramagnetic agents, optical probes, and radionuclides.
• treat refers to any indicia of success in the treatment or amelioration of an injury, pathology, condition, or sy mptom (e.g., pain), including any objective or subjective parameter such as abatement; remission;
• the treatment or amelioration of symptoms can be based on any objective or subjective parameter; including, e.g., the result of a physical examination.
• the term "subject” refers to animals such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice and the like. In certain embodiments, the subject is a human.
• therapeutically sufficient amount or dose or “effective or sufficient amount or dose” refer to a dose that produces therapeutic effects for which it is administered.
• the exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g. , Lieberman, Pharmaceutical Dosage Forms (vols. 1 -3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding ( 1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins). In sensitized cells, the therapeutically effective dose can often be lower than the conventional therapeutically effective dose for non-sensitized cells.
• the present invention provides crosslinkable telodendrimer conjugates having a hydrophilic poly( ethylene glycol) (PEG) segment and a hydrophobic segment.
• the PEG segment can have a branched or linear architecture including one or more PEG chains.
• the hydrophobic segment of the telodendrimer can be provided by cholic acid, which has a hydrophobic face and a hydrophilic face.
• the cholic acid and the PEG are connected by oligomers and/or polymers that can contain a variety of acid repeats units. Typically, the oligomers and polymers comprise a diamino carboxylic acid, lysine.
• the telodendrimers are also functionalized with a crosslinkable group.
• the telodendrimers can aggregate in solution to form micelles with a hydrophobic interior and a hydrophilic exterior, and can be used as nanocarriers to deliver drugs or other agents having low water solubility. Following micelle formation, the telodendrimers can be crosslinked using the crosslinkable groups, forming a more stable micelle.
• the present invention provides a PEGylated dendrimer, referred to as a telodendrimer, containing cholic acid groups and other moieties at the dendrimer periphery, and crosslinkable groups.
• the invention provide a compound of formula 1: (PEG) m -A(Y' )p-L-D(Y 2 ) q -(R)n (1) wherein radical A of formula I is linked to at least one PEG group.
• Radical D of formula I is a dendritic polymer having a single focal point group, a plurality of branched monomer units X and a plurality of end groups.
• Radical L of formula 1 is a bond or a linker linked to the focal point group of the dendritic polymer.
• Each PEG of formula I is a polyethyleneglycol (PEG) polymer, wherein each PEG polymer has a molecular weight of 1 - 100 kDa.
• Each R of formula 1 can be the end group of the dendritic polymer, a hydrophobic group, a hydrophilic group, an amphiphilic compound or a drug, such that when R is not an end group then each R is linked to one of the end groups.
• Each Y 1 and Y 2 of formula I is a crosslinkable group that can be any of boronic acid, dihydroxybenzene or a thiol.
• Subscript m of formula I is an integer from 0 to 20.
• Subscript n of formula I is an integer from 2 to 20, wherein subscript n is equal to the number of end groups on the dendritic polymer, and wherein at least half the number n of R groups that can each be a hydrophobic group, a hydrophilic group, an amphiphilic compound or a drug.
• each of subscripts p and q are 0 or from 2 to 8, such that one of subscripts p and q is from 2 to 8.
• Radical A can be any suitable group capable of linking the PEG to the linker or dendritic polymer D. Suitable A groups include the monomer units X described below for the dendritic polymer.
• telodendrimers of the present invention include, but are not limited to, fluorescent dyes, chelates and radiometals.
• the dendritic polymer can be any suitable dendritic polymer.
• the dendritic polymer can be made of branched monomer units including amino acids or other bifunctional AB2-type monomers, where A and B are two different functional groups capable of reacting together such that the resulting polymer chain has a branch point where an A-B bond is formed.
• each branched monomer unit X can be a diamino carboxylic acid, a dihydroxy carboxylic acid and a hydroxyl amino carboxylic acid.
• each diamino carboxylic acid can be 2,3-diamino propanoic acid, 2,4- diaminobutanoic acid, 2,5-diaminopentanoic acid (ornithine), 2,6-diaminohexanoic acid (lysine), (2-Aminoethyl)-cysteine, 3-amino-2-aminomethyl propanoic acid, 3-amino-2- aminomethyl-2-methyl propanoic acid, 4-amino-2-(2 ⁇ aminoethyl) butyric acid or 5-amino-2- (3-aminopropyl) pentanoic acid.
• each dihydroxy carboxylic acid can be glyceric acid, 2,4-dihydroxybutyric acid, 2,2-Bis(hydi xymethyl)propionic acid, 2,2- Bis(hydroxymethyl)butyric acid, serine or threonine.
• each hydroxyl amino carboxylic acid can be serine or homoserine.
• the diamino carboxylic acid is an amino acid.
• each branched monomer unit X is lysine.
• the dendritic polymer of the telodendrimer can be any suitable generation of dendrimer, including generation 1 , 2, 3, 4, 5, or more, where each "generation" of dendrimer refers to the number of branch points encountered between the focal point and the end group following one branch of the dendrimer.
• the dendritic polymer of the telodendrimer can also include partial-generations such as 1.5, 2.5, 3.5, 4.5, 5.5, etc., where a branch point of the dendrimer has only a single branch.
• the various architectures of the dendritic polymer can provide any suitable number of end groups, including, but not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 3 1 or 32 end groups.
• the R groups installed at the telodendrimer periphery can be any suitable chemical moiety, including hydrophilic groups, hydrophobic groups, or amphiphilic compounds.
• hydrophobic groups include, but are not limited to, long-chain alkanes and fatty acids, fluorocarbons, silicones, certain steroids such as cholesterol, and many polymers including, for example, polystyrene and polyisoprene.
• hydrophilic groups include, but are not limited to, alcohols, short-chain carboxylic acids, amines, sulfonates, phosphates, sugars, and certain polymers such as PEG.
• amphiphilic compounds include, but are not limited to, molecules that have one hydrophilic face and one hydrophobic face.
• Amphiphilic compounds useful in the present invention include, but are not limited to, cholic acid and cholic acid analogs and derivatives.
• “Cholic acid” refers to (R)-4-((3R, 5S, 1R, 8R, 9S, 10S, ⁇ 2S, ⁇ 3R, 145, 17R)-3,7, 12-trihydroxy-10, 13-dimethylhexadecahydro- l H- cyclopenta[fl]phenanthren- 17-yl)pentanoic acid, having the structure:
• Cholic acid derivatives and analogs include, but are not limited to, allocholic acid, pythocholic acid, avicholic acid, deoxycholic acid, and chenodeoxycholic acid.
• Cholic acid derivatives can be designed to modulate the properties of the nanocarriers resulting from telodendrimer assembly, such as micelle stability and membrane activity.
• the cholic acid derivatives can have hydrophilic faces that are modified with one or more glycerol groups, aminopropanediol groups, or other groups.
• Telodendrimer end groups may also include drugs such as paclitaxel, doxorubicin, etoposide, irinotecan, SN-38, cyclosporin A, podophyllotoxin, carmustine, amphotericin, ixabepilone, patupilone (epothelone class), rapamycin and platinum drugs.
• drugs such as paclitaxel, doxorubicin, etoposide, irinotecan, SN-38, cyclosporin A, podophyllotoxin, carmustine, amphotericin, ixabepilone, patupilone (epothelone class), rapamycin and platinum drugs.
• drugs such as paclitaxel, doxorubicin, etoposide, irinotecan, SN-38, cyclosporin A, podophyllotoxin, carmustine, amphotericin, ixabepil
• each R can be cholic acid, (3a, 5 ⁇ , 7a, 12a)-7,12-dihydroxy- 3-(2,3-dihydroxy-l -propoxy)-cholic acid, (3a, 5 ⁇ , 7a, 12a)-7-hydroxy-3, 12-di(2,3- dihydroxy-l-propoxy)-cholic acid, (3a, 5 ⁇ , 7a, 12a)-7, 12-dihydroxy-3-(3-amino-2-hydroxy- l-propoxy)-cholic acid, cholesterol formate, doxorubicin, or rhein.
• each R can be cholic acid.
• PEG polymers of any size and architecture are useful in the nanocarriers of the present invention.
• the PEG is from 1 - 100 kDa. In other embodiments, the PEG is from 1 -10 kDa. In some other embodiments, the PEG is about 3kDa.
• additional PEG polymers are linked to the amphiphilic compounds. For example, when the amphiphilic compound is cholic acid, up to 3 PEG polymers are linked to each cholic acid.
• the PEG polymers linked to the amphiphilic compounds are from 200- 10,000 Da in size. In yet other embodiments, the PEG polymers linked to the amphiphilic compounds are from 1 -5 kDa in size.
• PEG can be any suitable length.
• Crosslinkable groups suitable in the compounds of the present invention include any functional group capable of forming a covalent bond with the same functional group on another telodendrimer, or with a complementary functional group on another telodendrimer.
• Functional groups capable of forming a covalent bond with the same functional group include thiols.
• Thiols useful in the compounds of the present invention include any thiols, such as cysteine.
• Complementary functional groups capable of forming a covalent bond include boronic acid and a 1 ,2-diol.
• Boron ic acids useful in the compounds of the present invention include, but are not limited to, phenylboronic acid, 2-thienylboronic acid, methylboronic acid, and propenylboronic acid.
• Suitable 1,2-diols include alkyl- 1 ,2-diol and phenyl- 1 ,2-diols such as catechol.
• each crosslinkable group Y 1 and Y 2 can be a thiol. In some embodiments, each crosslinkable group Y 1 and Y 2 can be cysteine. In some embodiments, each crosslinkable group Y " can be cysteine.
• the compound of formula I has the structure:
• subscript p is an integer from 2 to 8 and subscript q is 0.
• the compound of formula has the structure:
• each L' is a linker Ebes
• PEG is PEG5k
• each R is cholic acid
• each branched monomer unit X is lysine
• Y 1 can be carboxyphenylboronic acid, carboxynitropheny boronic acid and 3,4-dihydroxybenzoic acid.
• the compound of formula I has the structure:
• the compound of formula has the structure:
• A is lysine
• each L' is a linker Ebes
• PEG is PEG5k
• each R is cholic acid
• each branched monomer unit X is lysine
• each Y 2 is cysteine.
• the compounds and conjugates of the present invention can be prepared by methods known to one of skill in the art.
• the well-established stepwise Fmoc peptide chemistry method was employed in the preparation of the compounds and conj gates of the present invention, with the resulting thiolated telodendrimers having well-defined polymer structure.
• the compound and conjugate is designated as PEG 5k -CyS4-Lg-CAg corresponding to length of PEG and the number of cysteines, hydrophilic spacers and cholic acids in the structure.
• PEG 5k -Cys4-Lg-CAg includes a dendritic oligomer of cholic acids attached to one terminus of the linear PEG through a poly(lysine- cysteine-Ebes) backbone.
• the thiol free telodendrimer, PEG 5k -CAg was also synthesized for comparison as described previously.
• Fluorescent dyes such as BODIPY can be attached to the ⁇ -amino group of the lysine at the junction between the PEG and the oligo-cholic acid chains after removal of Dde protecting group.
• telodendrimers of the present invention aggregate to form nanocarriers with a hydrophobic core and a hydrophilic exterior, where the crosslinkable groups are subsequently crosslinked to provide additional stability to the resulting nanocarrier.
• the crosslinking groups can be any suitable crosslinking group, as described above.
• the crosslinking groups can be thiol, boronic acid or
• Drugs that can be sequestered in the nanocarriers or linked to the conjugates of the present invention include, but are not limited to, cytostatic agents, cytotoxic agents (such as for example, but not limited to, DNA interactive agents (such as cisplatin or doxorubicin)); taxanes (e.g.
• topoisomerase II inhibitors such as etoposide
• topoisoinerase I inhibitors such as irinotecan (or CPT- 1 1 ), camptostar, or topotecan
• tubulin interacting agents such as paclitaxel, docetaxel or the epothilones
• hormonal agents such as tamoxifen
• thymidilate synthase inhibitors such as 5-fluorouracil
• anti-metabolites such as methotrexate
• alkylating agents such as temozolomide (TEMODARTM from Schering- Plough Corporation, enilworth, N.J.), cyclophosphamide); aromatase combinations; ara-C, adriamycin, Cytoxan, and gemcitabine.
• Other drugs useful in the nanocarrier of the present invention include but are not limited to Uracil mustard, Chlormethine, Ifosfamide,
• Imaging agents include paramagnetic agents, optical probes and radionuclides.
• Paramagnetic agents include iron particles, such as iron nanoparticles that are sequestered in the hydrophobic pocket of the nanocarrier.
• the imaging agent can include organic fluorescent dyes, quantum dots (QDs), super paramagnetic iron oxide nanoparticles (SPlO-NPs), or gold nanoparticles.
• the imaging agent is a radionuclide.
• each amphiphilic compound can be any of cholic acid, allocholic acid, pythocholic acid, avicholic acid, deoxycholic acid, or chenodeoxycholic acid.
• each amphiphilic compound can be cholic acid.
• the crosslinking groups of the nanocarrier telodendrimers can be crosslinked by any suitable means known to one of skill in the art.
• the crosslinkable group is a thiol
• a disulfide bond can be formed between two conjugates of the present invention by oxidation of the thiols.
• Any suitable oxidation agent can be used, such as oxygen.
• the crosslinking groups such as the complementary groups boronic acid and 1 ,2-diols such as dihydroxybenzene, the crosslinking occurs spontaneously and without the need for additional reactants.
• the crosslinking in the nanocarriers of the present invention is reversible to facilitate delivery of a drug, for example, to a target site. Reversing the crosslinking of the crosslinked nanocarrier requires contacting the crosslinked nanocarrier with a suitable bond cleavage component.
• the present invention provides a method of reversing the cross-linking of the reversibly crosslinked nanocarrier of the present invention, by contacting the reversibly crosslinked nanocarrier with a bond cleavage component suitable for cleaving the cross-linked bond, thereby reversing the cross-linking of the reversibly crosslinked nanocarrier. In some embodiments, the contacting is performed in vivo.
• any suitable bond cleavage component can be used in the present invention.
• the bond cleavage component can be N-acetyl cysteine (NAC), glutathione, 2-inercaptoethane sulfonate sodium (MESNA), mannitol or acid.
• NAC N-acetyl cysteine
• MESNA 2-inercaptoethane sulfonate sodium
• mannitol or acid.
• any disulfide reducing agent is suitable.
• the nanocarrier also includes an imaging agent.
• the imaging agent can be a covalently attached to a conjugate of the nanocarrier, or the imaging agent can be sequestered in the interior of the nanocarrier.
• both a hydrophobic drug and an imaging agent are sequestered in the interior of the nanocarrier.
• both a drug and an imaging agent are covalently linked to a conjugate or conjugates of the nanocarrier.
• the nanocarrier can also include a radionuclide.
• nanocarriers of the present invention include: (I) inflammatory or allergic diseases such as systemic anaphylaxis or
• hypersensitivity responses drug allergies, insect sting allergies; inflammatory bowel diseases, such as Crohn's disease, ulcerative colitis, ileitis and enteritis; vaginitis; psoriasis and inflammatory dermatoses such as dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, urticaria; vasculitis; spondyloarthropathies; scleroderma; respiratory allergic diseases such as asthma, allergic rhinitis, hypersensitivity lung diseases, and the like, (2) autoimmune diseases, such as arthritis (rheumatoid and psoriatic), osteoarthritis, multiple sclerosis, systemic lupus erythematosus, diabetes mellitus, glomerulonephritis, and the like,
• graft rejection including allograft rejection and graft-v-host disease
• other diseases in which undesired inflammatory responses are to be inhibited e.g., atherosclerosis, myositis, neurological conditions such as stroke and closed-head injuries, neurodegenerative diseases, Alzheimer's disease, encephalitis, meningitis, osteoporosis, gout, hepatitis, nephritis, sepsis, sarcoidosis, conjunctivitis, otitis, chronic obstructive pulmonary disease, sinusitis and Behcet's syndrome).
• the disease is asthma.
• the nanocarriers of the present invention are useful for the treatment of infection by pathogens such as viruses, bacteria, fungi, and parasites. Other diseases can be treated using the nanocarriers of the present invention.
• nanocarriers of the present invention can be formulated in a variety of different manners known to one of skill in the art.
• Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there are a wide variety of suitable formulations of pharmaceutical compositions of the present invention (see, e.g., Remington 's Pharmaceutical Sciences, 20 th ed., 2003, supra). Effective formulations include oral and nasal formulations, formulations for parenteral administration, and compositions formulated for with extended release.
• Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of a compound of the present invention suspended in diluents, such as water, saline or PEG 400; (b) capsules, sachets, depots or tablets, each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; (d) suitable emulsions; and (e) patches.
• the liquid solutions described above can be sterile solutions.
• Lozenge forms can comprise the active ingredient in a flavor, e.g., sucrose, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art.
• a flavor e.g., sucrose
• an inert base such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art.
• the pharmaceutical preparation is preferably in unit dosage form.
• the preparation is subdivided into unit doses containing appropriate quantities of the active component.
• the unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules.
• the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.
• the composition can, if desired, also contain other compatible therapeutic agents.
• Preferred pharmaceutical preparations can deliver the compounds of the invention in a sustained release formulation.
• extended-release formulations useful in the present invention are described in U.S. Patent No. 6,699,508, which can be prepared according to U.S. Patent No. 7, 125,567, both patents incorporated herein by reference.
• the pharmaceutical preparations are typically delivered to a mammal, including humans and non-human mammals.
• Non-human mammals treated using the present methods include domesticated animals (i.e., canine, feline, murine, rodentia, and lagomorpha) and agricultural animals (bovine, equine, ovine, porcine).
• compositions can be used alone, or in combination with other therapeutic or diagnostic agents.
• the nanocarriers of the present invention can be administered as frequently as necessary, including hourly, daily, weekly or monthly.
• the compounds utilized in the pharmaceutical method of the invention are administered at the initial dosage of about 0.0001 mg/kg to about 1000 mg/kg daily.
• a daily dose range of about 0.01 mg/kg to about 500 mg/kg, or about 0.1 mg/kg to about 200 mg/kg, or about 1 mg/kg to about 100 mg/kg, or about 10 mg kg to about 50 mg kg, can be used.
• the dosages may be varied depending upon the requirements of the patient, the severity of the condition being treated, and the compound being employed. For example, dosages can be empirically determined considering the type and stage of disease diagnosed in a particular patient.
• Optical probes useful in the present invention include, but are not limited to, Cy5.5, Alexa 680, Cy5, DiD (l, -dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate) and DiR (l, -dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide).
• Other optical probes include quantum dots. Radionuclides are elements that undergo radioactive decay.
• Radionuclides useful in the present invention include, but are not limited to, 3 H, "C, l3 N, l 8 F, 19 F, 60 Co, 64 Cu, 67 Cu, 68 Ga, 82 Rb, 0 Sr, 90 Y, 99 Tc, 99m Tc, 1 1 'in, ,23 I, 124 I, , 25 I, ,29 I, ,31 1, 137 Cs, ,77 Lu, , 86 Re, , 88 Re, 21 1 At, Rn, Ra, Th, U, Pu and 241 Am.
• telodendrimer was dissolved in acetonitrile/water and lyophilized.
• the PEG 5k -CAg thiol free telodendrimer was synthesized to prepare the non- cross-linked micelles according to our previously reported method.
• the mono-dispersed mass traces were detected for the starting PEG and the telodendrimers, and the molecular weights of the telodendrimers from MALDI-TOF MS were almost identical to the theoretical value.
• the chemical shift of PEG chains (3.5-3.7 ppm) and cholic acid (0.6-2.4 ppm) could be observed in the ⁇ NMR spectra of the PEG 5k -Cys 4 -Lg-CAs in CDCI 3 .
• the integration of these peaks can be used to calculate the chemical compositions of the telodendrimers.
• the number of cholic acids determined by ⁇ -NMR for the telodendrimers was consistent with the molecular formula of the target telodendrimers.
• the micelles retained the similar particle size and 100% PTX loading efficiency at a PTX loading of 5.0 mg/mL and lower after cross-linking. However, beyond 5.0 mg/mL, the particle sizes of the cross-linked micelles increased ( Figure 2D) while the loading efficiency decreased to 81 %.
• the stability of PTX-loaded NCMs and DCMs was further studied in 50% (v/v) plasma from healthy human volunteers.
• the mixture was incubated at physiological body temperature (37 °C) followed by size measurements at predetermined time intervals up to 96 h.
• PTX-NCMs PTX loaded non-cross-linked micelles
• PTX-DCMs disulfide cross- linked micelles
• the PTX-NCMs and PTX-DCMs were incubated with 50% human plasma, and the particle sizes of micelles were monitored by DLS over time.
• Both of the DCMs and NCMs micelles with similar PTX loading retained the average particle size around 30 nm in human plasma for 24 hours ( Figure 3).
• the PTX-DCMs still kept the uniformity and narrow distribution in size while the PTX-NCMs showed broader size distribution and population of size over 100 nm, indicating the formation of aggregates (Figure 3).
• SDS sodium dodecyl sulfate
• SDS a strong ionic detergent
• the exchange rate between polymeric micelles and unimers is accelerated by low concentrations of SDS while at higher concentrations, the presence of SDS micelles solubilize the amphiphilic block copolymers resulting in destabilization of the polymeric micelles.
• the stability of NCMs and DCMs was also tested in the presence of the reported micelle-disrupting SDS concentration of 2.5 mg/mL.
• the size of SDS background is below the detection limit of DLS analysis, showing a 0.9 nm population in the spectra.
• the GSH concentration inside cells (-10 mM) is known to be substantially higher than the extracellular level ( ⁇ 2 ⁇ ).
• the DCMs were stable in SDS solution with a cellular exterior level of GSH ( ⁇ 2 ⁇ ).
• the disulfide cross-linked micelle particle size signal remained unchanged for 30 min until it decreased suddenly (within 10 sec), indicating that rapid dissociation of the micelle when a critical number of disulfide bonds were reduced (Figure 4A, Figure 13F).
• SKOV-3 ovarian cancer cells were seeded at a density of 50000 cells per well in eight-well tissue culture chamber slides (BD Biosciences, Bedford, MA, USA), followed by 24 h of incubation in McCoy's 5a Medium containing 10% FBS. The medium was replaced, and DiD labeled micelles (100 ⁇ g/mL) were added to each well. After 30 min, l h, 2h and 3h, the cells were washed three times with PBS, fixed with 4% paraformaldehyde and the cell nuclei were stained with DAPI. The slides were mounted with cover slips and observed under confocal laser scanning microscope (Olympus, FV1000).
• SKOV-3 cells were seeded in 96-well plates at a density of 10000 cells/well 24 h prior to the treatment.
• the cells were first treated with or without GSH-OEt (20 mM) for 2 h and then washed 3 times with PBS. Empty micelles and various formulations of PTX with different dilutions were added to the plate and then incubated for 2 h.
• the cells were washed with PBS and incubated for another 22 h in a humidified 37 °C, 5% C ⁇ 3 ⁇ 4 incubator. MTT was added to each well and further incubated for 4 h.
• the hemolysis of NCMs and DCMs was investigated using fresh citrated blood from healthy human volunteers.
• the red blood cells (RBCs) were collected by centrifugation at 1000 rpm for 10 m in, washed three times with PBS, and then brought to a final concentration of 2% in PBS.
• 200 of erythrocyte suspension was mixed with different concentrations (0.2 and 1.0 mg/mL) of NCMs and DCMs, respectively, and incubated for 4 h at 37°C in an incubator shaker.
• the mixtures were centrifuged at 1000 rpm for 5 min, and 100 ⁇ of supernatant of all samples was transferred to a 96-well plate. Free hemoglobin in the supernatant was measured by the absorbance at 540 nm using a micro-plate reader
• NIRF signal of blood background was found to be very low.
• BODIPY signal of NCMs was rapidly eliminated from circulation and fell into the background level within 8 hours post injection. It should be mentioned that BODIPY signal of DCMs in blood was 8 times higher than that of NCMs at 8 hours post injection and sustained up to 24 hours (Figure 7A).
• PTX-loaded cross-linked micelle solution was prepared to determine the in vitro drug release profile.
• the initial PTX concentration was 4.6 mg/mL.
• Aliquots of PTX-loaded cross-linked micelle solution were injected into dialysis cartridges (Pierce Chemical Inc.) with a 3.5 kDa MWCO.
• the cartridges were dialyzed against 1 L PBS with various GSH concentrations (0, 2 ⁇ , 1 mM, and 10 mM) at 37 °C. In order to make an ideal sink condition, 10 g charcoal was added in the release medium.
• the concentration of PTX remaining in the dialysis cartridge at various time points was measured by HPLC.
• PTX concentration 5.0 mg/mL
• GSH or NAC 10 mM
• the PTX release profiles of the lyophilized and rehydrated micelle solution were evaluated under the same conditions. Values were reported as the means for each triplicate sample.
• NAC can be applied in vivo as an on-demand cleavage reagent via systemic i.v. injection to trigger drug release after nanotherapeutics have accumulated in tumor sites.
• Example 6 Treatment of Ovarian Cancer with PTX Loaded Disulfide Cross-Linked Micelles
• the subcutaneous xenograft model of ovarian cancer was established by injecting 7x l 0 6 SKOV-3 ovarian cells in a 100 ⁇ of mixture of PBS and Matrigel (1 : 1 v/v) subcutaneously into the right flank of female nude mice.
• Nude mice bearing SKOV-3 ovarian cancer xenografts were used to evaluate the therapeutic efficacy of the different formulations of PTX.
• the treatments were initiated when tumor xenograft reached a tumor volume of 100-200 mm 3 and this day was designated as day 0.
• day 0 these mice were randomly divided into seven groups and injected intravenously via the tail vein with the formulations and repeated every 3 days for total 6 doses. Injection volume was 0.1 mL for each 10 g of mouse body weight.
• MTD maximum tolerated dose
• NAC N-acetylcysteine
• the Burkitt's B-cell lymphoma cell line, Raji was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were cultured in ATCC formulated RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin G and 100 ⁇ . streptomycin at 37 °C using a humidified 5% CO 2 incubator. 3 days before tumor cell implantation, mice received 400 rads of whole body radiation. To establish tumors, 5x10 6 Raji cells resuspended in PBS were subcutaneously implanted on to the flank of each mouse.
• ATCC American Type Culture Collection
• FBS fetal bovine serum
• penicillin G 100 U/mL
• streptomycin 100 ⁇ .
• mice received 400 rads of whole body radiation.
• 5x10 6 Raji cells resuspended in PBS were subcutaneously implanted on to the flank of each mouse.
• VCR is known to cause axonal degeneration and demyelination of nerve fibers which can be observed with either light or electron microscopy. Histological analysis revealed no obvious damage to the nerve fibers from the groups treated with conventional VCR ( 1 mg/kg) and DCM-VCR (2.5 mg/kg) and no differences compared to the PBS control group ( Figure 20).
• boronic acid-containing telodendrimer and catechol-containing telodendrimer were first dissolved in anhydrous chloroform in a 10 mL round bottom flask. The chloroform was evaporated under vacuum to form a thin film. PBS buffer (1 mL) was added to re-hydrate the thin film, followed by 30 min of sonication. Boronate ester bonds formed between boronic acids and catechols of adjacent telodendrimers, upon self-assembly in PBS, resulted in the formation of boronate cross-linked micelles (BCM). The micelle solution was filtered with 0.22 ⁇ filter to sterilize the sample.
• BCM boronate cross-linked micelles
• the PTX loaded micelles on the filters were recovered with PBS.
• the amount of drug loaded in the micelles was analyzed on a HPLC system (Waters) after releasing the drugs from the micelles by adding 9 times of acetonitrile and 10 min sonication.
• the drug loading was calculated according to the calibration curve between the HPLC area values and concentrations of drug standard.
• the loading efficiency is defined as the ratio of drug loaded into micelles to the initial drug content.
• Hydrophobic dye (DiO or DiD) was loaded into the micelles using the same strategy.
• the amount of dye loaded in the micelles was analyzed on a fluorescence spectrometry (SpectraMax M2, Molecular Devices, USA) after releasing the drugs from the micelles by adding 9 times of acetonitrile and 10 min sonication.
• the dye loading was calculated according to the calibration curve between the fluorescence intensity and concentrations of dye standard in acetonitrile.
• the size and size distribution of the micelles were measured by dynamic light scattering (DLS) instruments (Microtrac).
• the micelle concentrations were kept at 1.0 mg mL for DLS measurements. All measurements were performed at 25 °C, and data were analyzed by Microtrac FLEX Software 10.5.3.
• the morphology of micelles was observed on a Philips CM-120 transmission electron microscope (TEM).
• TEM transmission electron microscope
• the aqueous micelle solution 1.0 mg/mL
• the aqueous micelle solution 1.0 mg/mL
• the apparent critical micelle concentration (CMC) of the NCM and BCMs was measured through fluorescence spectra by using pyrene as a hydrophobic fluorescent probe as described previously.
• ⁇ Number of phenylboronic acids calculated based on the average integration ratio of the peaks of the phenyl protons of phenylboronic acids (6.7-7.2 ppm) and methylene proton of PEG to in'H-NMR spectra in DMSO-d6 (90° pulse).
• ARS is a catechol dye displaying dramatic changes in color and fluorescence intensity upon binding to boronic acid.
• ARS also displays a dramatic change in fluorescence intensity in response to the binding of boronic acids.
• Boronic acid containing telodendrimers solutions (boronic acid concentrations: 0-5 mM) were mixed ARS solution in PBS at pH 7.4 and the fluorescence signal of the mixtures was measured by fluorescence spectrometry (Nanodrop3000, Microtrac). The final concentration of ARS was fixed at 0.1 mM.
• ARS fluorescence assay was further used to characterize the binding between the boronic acid containing telodendrimers and catechol containing telodendrimers. In this experiment, the final concentration of ARS and boronic acid of boronic acid containing telodendrimers were fixed at O.
• telodendrimers Different molar ratio of catechol containing telodendrimers was premixed with boronic acid containing telodendrimers (0.1 mM) in anhydrous chloroform. The chloroform was evaporated and the thin film on the inner surface of flask was re-hydrated with PBS buffer to generate boronate crosslinked micelles. ARS solution was then mixed with the above micelle solutions and the fluorescence signal of the mixtures was measured by fluorescence spectrometry (Nanodrop 000, Microtrac).
• the stability study was performed to monitor the change in particle size of the NCM and BCMs in the presence of sodium dodecyl sulfate (SDS), which was reported to be able to efficiently break down polymeric micelles.
• SDS sodium dodecyl sulfate
• An SDS solution (7.5 mg/mL) was added to aqueous solutions of micelles (1.5 mg/mL). The final SDS concentration was 2.5 mg/mL and the micelle concentration was kept at 1.0 mg/mL.
• the size and size distribution of the micelle solutions was monitored continuously via dynamic light scattering (DLS) instruments (Microtrac) for 2 days.
• DLS dynamic light scattering
• the stability of the micelles was also evaluated in PBS at different pH levels or in presence of mannitol and glucose (0, 10 mM, 50 mM, and 100 mM), together with SDS. Hydrogen chloride and sodium hydroxide solutions were used to prepare PBS at different pH levels. The pH values of the buffer were determined by a digital pH meter ( ⁇ 350 pH/Temp/mV meter, Beckman Coulter, USA) which gave pH values within 0.01 units. During the stability study, a small portion of the samples were taken out and further observed under TEM. The stability of NCM and BCMs was further studied in 50% (v/v) plasma from healthy human volunteers. The mixture was incubated at physiological body temperature (37 °C) followed by size measurements at predetermined time intervals up to 96 h.
• BCM3 and BCM4 containing double the number of boronate esters retained their structural integrity significantly longer in the presence of SDS, when compared to BCM1 and BCM2, respectively.
• BCM2 and BCM4 crosslinked via nitro phenyl boronate esters were more stable than the corresponding phenyl boronate esters crosslinked micelles BCM 1 and BCM3.
• SKOV-3 ovarian cancer cells were seeded at a density of 50000 cells per well in eight-well tissue culture chamber slides (BD Biosciences, Bedford, MA, USA), followed by 24 h of incubation in McCoy's 5a Medium containing 10% FBS. The medium was replaced, and DiD labeled micelles (100 ⁇ g mL) were added to each well. After 30 min, l h, 2h and 3h, the cells were washed three times with PBS, fixed with 4% paraformaldehyde and the cell nuclei were stained with DAPI. The slides were mounted with cover slips and observed under confocal laser scanning microscope (Olympus, FV1000). For the DiD channel, the excitation was set to 625 nm while the emission was set to 700 nm.
• SKOV-3 ovarian cancer cells were seeded in 96-well plates at a density of 5000 cells/well 24 h prior to the treatment.
• the culture medium was replaced with fresh medium containing various formulations of PTX with different dilutions at pH 7.4 or 5.0, in the absence or in the presence of 100 mM mannitol.
• the cells were washed with PBS and incubated for another23 h in a humidified 37 °C, 5% C0 2 incubator. MTT was added to each well and further incubated for 4 h.
• the absorbance at 570 nm and 660 nm was detected using a micro-plate ELISA reader (SpectraMax M2, Molecular Devices, USA). Untreated cells served as a control. Results were shown as the average cell viability [(OD tre a t
• PTX-NCM showed comparable in vitro anti-tumor effects against SKOV-3 cells as Taxol® (free drug of paclitaxel).
• Taxol® free drug of paclitaxel.
• PTX-BCM4 was found to be considerably less cytotoxic than Taxol ® and PTX-NCM at equal dose levels. There were minimal changes in the toxicity profile of PTX-NCM and free drug triggered with acidic pH and mannitol.
• PTX-BCM4 showed significantly enhanced cancer cell inhibition at pH 5.0 in the presence of mannitol (100 mM).
• Rhodamine B labeled NCM and BCMs were prepared for the blood elimination study.
• concentration of rhodamine B conjugated micelles was 2.0 mg/mL.
• the absorbance and fluorescence spectra of these micelles diluted 20 times by PBS were characterized by fluorescence spectrometry (SpectraMax M2, Molecular Devices, USA).
• 100 of Rhodamine B conjugated NCM and BCMs were injected into tumor free nude mice via tail vein. 50 ⁇ , blood was collected at different time points post- inject ion to measure the fluorescence signal of Rhodamine B.
• Rhodamine B signal of NCM was rapidly eliminated from blood circulation and fell into the background level within 10 hr post injection ( Figure 24F). Rhodamine B signal of BCM4 in blood was 6 times higher than that of NCM at 10 hr post injection and sustained for more than 24 hr.
• telodendrimer loaded NCM have been safely applied for in vivo cancer treatment.
• the single treatment MTD in mice was observed to be 75 mg PTX/kg, the corresponding telodendrimer dosage was 200 mg/kg.
• both empty non-cross-linked and cross- linked micelles were injected in tumor free nude mice at the single dose of 200 mg/kg via tail vein.
• PBS was injected into the mice as a control. Mice were checked for possible signs of toxicity and the survival situation was monitored daily for two weeks. At day 7 after injection, blood samples were obtained from all the mice for the measurement of blood cell counts, serum chemistry including alanine aminotransferase (ALT), aspartate
• AST aminotransferase
• BUN blood urea nitrogen
• PTX-loaded NCM and BCMs was prepared to determine the in vitro release profile.
• the PTX loading for NCM, BCM 1 , BCM2, BCM3 and BCM4 were 9.9%, 9.8%, 9.8%, 9.9%, 10.0% (w/w, PTX/micelle) in the presence of total 20 mg telodendemers measured.
• Aliquots of PTX-loaded micelle solution were injected into dialysis cartridges (Pierce Chemical Inc.) with a 3.5 kDa MWCO. In order to make an ideal sink condition, 10 g charcoal was added in the release medium.
• the cartridges were dialyzed against PBS at different pH levels (pH6.5, pH6.0, pH5.5 and pH5.0) or in the presence of various concentrations of glucose or mannitol (0, 1 0mM, 50 mM, and 1 00 mM) at 37 °C.
• the release medium was stirred at a speed of 1 00 rpm.
• the concentration of PTX remaining in the dialysis cartridge at various time points was measured by HPLC.
• the release medium pH7.4 was replaced with fresh medium at different pH levels (pH6.5, pH6.0, pH5.5 and pH5.0) and/or in the presence of mannitol or glucose (10 and 100 raM) at a specific release time (5 h). Values were reported as the means for each duplicate samples.
• PTX release from NCM was rapid with almost 30% of PTX released within the first 9 h independently from the pH of the release medium or the presence of diols.
• PTX release from BCM3 crosslinked via phenyl boronate was significantly slower than NCM but faster than BCM4 with nitro- phenyl boronate crosslinking at pH7.4.
• PTX release from BCM3 was promoted when decreasing the pH of the medium from 7.4 to 6.5 while that of BCM4 was accelerated at pH 5.5.
• PTX release from BCM3 and BCM4 was similar to that in the release media without glucose. It was noted that PTX release was not sensitive to 10 mM mannitol but could be gradually facilitated as the concentration of mannitol increased up to the range of 50- 100 mM.
• the PTX release from BCM4 was first incubated under psychological pH for a period of time (e.g. 5hr) and then was triggered with acidic pH and/or mannitol.
• the PTX release from BCM4 was significantly slower than that from NCMs at the initial 5 h.
• 100 mM mannitol was added or the pH of the medium was adjusted to 5.0 at the 5 hr time point, there was a burst of drug release from the BCM4. It should be noted that the PTX release can be further accelerated via the combination of 100 mM of mannitol and pH 5.0.
• This two-stage release strategy can be exploited so that premature drug release can be minimized during circulation in vivo followed by rapid drug release triggered by the acidic tumor microenvironment, or upon micelle exposure to the acidic compartments of cancer cells or by the additional administration of mannitol.

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