US20060260777A1 - Surface-modified microparticles and methods of forming and using the same - Google Patents

Surface-modified microparticles and methods of forming and using the same Download PDF

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US20060260777A1
US20060260777A1 US11380556 US38055606A US2006260777A1 US 20060260777 A1 US20060260777 A1 US 20060260777A1 US 11380556 US11380556 US 11380556 US 38055606 A US38055606 A US 38055606A US 2006260777 A1 US2006260777 A1 US 2006260777A1
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microparticle
monolayer
charged
surface
method
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US11380556
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Julia Rashba-Step
Terrence Scott
Ramin Darvi
Yuri Lvov
Tatsiana Shutava
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Baxter Healthcare SA
Baxter International Inc
Louisiana Tech University
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Baxter Healthcare SA
Baxter International Inc
Louisiana Tech University
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088Compounds having three or more nucleosides or nucleotides
    • A61K31/7105Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/0012Galenical forms characterised by the site of application
    • A61K9/0019Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/0012Galenical forms characterised by the site of application
    • A61K9/0019Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0024Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/10Dispersions; Emulsions
    • A61K9/127Liposomes
    • A61K9/1271Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1272Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
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    • A61K9/14Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
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    • A61K9/16Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605Excipients; Inactive ingredients
    • A61K9/1629Organic macromolecular compounds
    • A61K9/1635Organic macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K9/16Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605Excipients; Inactive ingredients
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    • A61K9/1682Processes
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    • A61K9/5026Organic macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
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    • A61K9/50Microcapsules 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
    • A61K9/5005Wall or coating material
    • A61K9/5021Organic macromolecular compounds
    • A61K9/5036Polysaccharides, e.g. gums, alginate; Cyclodextrin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K9/5021Organic macromolecular compounds
    • A61K9/5036Polysaccharides, e.g. gums, alginate; Cyclodextrin
    • A61K9/5042Cellulose; Cellulose derivatives, e.g. phthalate or acetate succinate esters of hydroxypropyl methylcellulose
    • A61K9/5047Cellulose ethers containing no ester groups, e.g. hydroxypropyl methylcellulose
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    • A61K9/48Preparations in capsules, e.g. of gelatin, of chocolate
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    • A61K9/50Microcapsules 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
    • A61K9/5073Microcapsules 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 having two or more different coatings optionally including drug-containing subcoatings
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/48Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50Microcapsules 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
    • A61K9/5089Processes
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K17/00Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02Peptides being immobilised on, or in, an organic carrier
    • C07K17/08Peptides being immobilised on, or in, an organic carrier the carrier being a synthetic polymer

Abstract

Surface-modified microparticles and methods of making and using such particles are disclosed. The surface modified microparticles include a preformed or core microparticle that contains at least one active agent. The outer surface of the preformed or core microparticle carries a net surface charge. A monolayer is associated with the outer surface of the preformed or core microparticle. The monolayer includes at least one charged compound that has a charge that is different from the net surface charge of the preformed or core microparticle,

Description

    CROSS-REFERENCE TO RELATED APPLICATIONS
  • This application claims the benefit of U.S. Provisional Patent Application Ser. No. 60/675,372 filed Apr. 27, 2005 and U.S. Provisional Patent Application Ser. No. 60/750,903 filed Dec. 16, 2005.
  • FIELD AND BACKGROUND
  • The present disclosure is generally directed to microparticles containing one or more active agents and delivery methods of such microparticles to subjects. More particularly, the present disclosure is directed to surface modifications of the microparticles such that they are capable of controlled release of one or more active agents. The present disclosure is further directed to methods for making and using such surface-modified microparticles.
  • Microparticles have been used in many different applications, including the controlled delivery and/or release of active agents. Controlling or modifying the release profile of an active agent can, if desired, prolong the levels (such as therapeutic levels) of the active agent in the blood stream of the recipient, improve pharmacokinetics and pharmacodynamics, and result in greater convenience to the recipient.
  • SUMMARY
  • The present disclosure is generally directed to methods for preparing a surface-modified microparticle. In one example, the method includes providing an amorphous and solid preformed microparticle including at least one active agent. The preformed microparticle has an outer surface carrying a net surface charge. The method further includes exposing at least the outer surface of the preformed microparticle to at least one charged compound having a net charge that is opposite in sign to the net surface charge of the preformed microparticle outer surface. A monolayer of the charged compound is formed whereby the monolayer is associated with the preformed microparticle outer surface.
  • The present disclosure is also directed to a method for preparing a surface-modified microparticle including providing a liquid continuous phase system containing a solvent, at least one active agent, and one or more phase-separation enhancing agents. The method includes inducing a liquid-solid phase separation at, optionally, a controlled rate to cause a liquid-solid separation, and forming a solid phase that includes a solid and amorphous microparticle containing the active agent, the microparticle having an outer surface carrying a net surface charge, while the solvent and the phase-separation enhancing agents remain in the liquid phase. After forming the microparticle, at least the outer surface of the formed microparticle is exposed to at least one charged compound having a net charge that is opposite in sign to the net surface charge of microparticle outer surface. The method further includes forming a monolayer including at least one charged compound on the formed microparticle whereby the formed monolayer is associated with the microparticle outer surface.
  • The present disclosure is also directed to a method for preparing a surface modified microparticle that includes providing an amorphous and solid preformed microparticle including at least one active agent, the preformed microparticle having an outer surface carrying a net surface charges In this example, the method further includes exposing at least the outer surface of the preformed microparticle to at least one charged compound having a net charge that is opposite in sign to the net surface charge of the preformed microparticle. An intermediate microparticle is formed that includes the preformed microparticle and a formed monolayer including the at least one charged compound wherein the formed monolayer is associated with the preformed microparticle outer surface. The formed monolayer is then exposed to at least a different charged compound to form a surface modified microparticle that includes the intermediate microparticle and a subsequent monolayer including at least the one different charged compound. The surface modified microparticle has a release profile for release of the at least one active agent that is different from the release profile of the intermediate microparticle.
  • The present disclosure is also directed to a microparticle that includes a solid, amorphous core microparticle that includes between 80% or greater than by weight, of at least one active agent. The outer surface of the core microparticle carries a selected net surface charge. A monolayer of at least one charged compound carrying a net surface charge that is sufficiently different from the surface charge of the core microparticle outer surface to allow for association therewith, is associated with the core microparticle outer surface at least by, but not limited to, electrostatic interaction with the outer surface.
  • A solid microparticle including at least 80%, by weight, of at least one active agent where the outer surface of the microparticle includes at least one charged compound associated with the active agent may display a 1-hour percentage of cumulative release of the active agent that is 50% or less when subjected to in vitro release in a suitable buffer at selected pH and temperature,
  • Furthermore, a solid, preformed microparticle having at least 80%, by weight, of at least one proteinaceous compound where the outer surface of the preformed microparticle has at least one charged compound associated with the proteinaceous compound are suitable for in vivo administration Upon such administration, the microparticle provides a Cmax and tmax that is different from the Cmax and tmax of the core microparticle.
  • Further details of the above-described microparticles, methods of making microparticles and methods for controlling the release of active agents from the microparticles are discussed below.
  • BRIEF DESCRIPTION OF THE FIGURES
  • FIG. 1 is a flow chart showing an exemplary method set forth in the present disclosure;
  • FIG. 2 is a schematic illustration of the fabrication of monolayers of charged compounds on a preformed microparticle;
  • FIG. 3 is a graph that shows the change in zeta potential of insulin microparticles with alternatingly charged monolayers (Examples 1A & 1B);
  • FIG. 4 is a graph that shows the change in zeta potential of insulin microparticles, each, with one monolayer of different polyanionic compounds (Example 2A);
  • FIG. 5 is a graph that shows the change in zeta potential of insulin microparticles, each with one monolayer of different polycationic compounds (Example 2B);
  • FIG. 6 is a laser scanning confocal (LSC) micrograph of insulin microparticles with one FITC-labeled protamine monolayer (Example 2B);
  • FIG. 7 is a graph showing surface electric charge alteration of insulin microparticles, each with a first monolayer of different polyanionic compounds and a second monolayer of poly-L-lysine (Example 3A);
  • FIG. 8 is a LSC micrograph of insulin microparticles with a first monolayer of PSS and a second monolayer of FITC-labeled PLL (Example 3A);
  • FIG. 9 is a graph showing progressive film thicknesses after each successive deposition of alternatingly charged polyelectrolyte monolayers (e.g., PLL and chondroitin sulfate) on the QCM electrode (Example 3A);
  • FIG. 10 is a graph showing changes in zeta-potential of insulin microparticles following subsequent depositions of a chondroitin sulfate monolayer and a gelatin A monolayer (Example 3B);
  • FIG. 11 is a graph comparing zeta-potential of insulin microparticles with monolayers of protamine sulfate and chondroitin sulfate in the presence and absence of zinc cations in the PEG-containing reaction medium (Example 4A);
  • FIG. 12 is a graph showing zeta-potential of insulin microparticles with monolayers of protamine sulfate and chondroitin sulfate in presence of zinc cations in a PEG-free reaction medium (Example 4B);
  • FIG. 13 is a graph showing LSC micrograph of insulin microparticles with one monolayer of Rodamine B-labeled protamine (top left) and one monolayer of FITC-labeled DAP (top right) (Example 5);
  • FIG. 14 is a graph showing the zeta-potential of insulin microparticles following deposition of each monolayer as described in Example 5;
  • FIG. 15 is a graph showing insulin release profiles from microparticles coated with a monolayer of protamine sulfate with various concentrations of the polycation in the reaction medium (Example 6);
  • FIG. 16 is a graph showing insulin release profiles from microparticles with a first monolayer of protamine sulfate and a second monolayer of carboxymethyl cellulose at various concentrations of the polyanion in the reaction medium (Example 7);
  • FIG. 17 is a graph showing release profiles of insulin from insulin microparticles coated with three monolayers (Example 7);
  • FIG. 18A is a graph showing the serum human insulin (hINS) concentration versus time profiles in rats that have received a single subcutaneous injection of uncoated insulin microparticles, or protamine-coated insulin microparticles (Example 8);
  • FIG. 18B is a graph showing the serum glucose depression versus time profiles of rats treated with single subcutaneous injection of uncoated insulin microparticles, or protamine-coated insulin microparticles Example 8);
  • FIG. 19 is a graph showing alteration in the surface charge of insulin microparticle by deposition of one monolayer of protamine in various solubility-reducing media at pH 7.0 (Example 9);
  • FIG. 20A is a graph showing the effect of protamine concentration in the reaction medium on the surface charge of insulin microparticles, and the extent of the dissolution after 48 h from initiation of the in vitro release (Example 10);
  • FIG. 20B is a graph showing the effect of CMC concentration in the reaction medium on the surface charge of protaimne-coated insulin microparticles, and the extent of their dissolution after 48 h from initiation of the in vitro release (Example 10);
  • FIG. 20C is a graph showing the effect of protamine concentration in the reaction medium on the surface charge of insulin microparticles coated with one monolayer of protamine and one monolayer of CMC, and the extent of their dissolution after 48 h from initiation of the in vitro release (Example 10);
  • FIG. 21A is a graph showing the alteration of zeta-potential of hGH microparticles following subsequent depositions of protamine sulfate and chondroitin sulfate monolayers (Example 11);
  • FIG. 21B is a graph showing profiles of hGH release from microparticles coated with one, two, or three monolayers of alternating protamine and condroitin sulfate (Example 11);
  • FIG. 22 is a graph showing the alteration of zeta-potential of intravenous immunoglobulin (IVIG) microparticles following subsequent depositions of chondroitin sulfate and protamine sulfate monolayers (Example 12);
  • FIG. 23 is a graph showing net surface charge characteristics of insulin microspheres in 16% PEG solution across a pH range of 4 to 7.5 (Example 13);
  • FIG. 24 is a flow chart showing another exemplary method of preparing microparticles set forth in the present disclosure;
  • FIG. 25 is a graph showing the effect of reaction pH on the zeta potential of insulin microparticles that have been surface-modified with one monolayer of protamine sulfate, poly-l-lysine, or poly-l-arginine (Example 14);
  • FIG. 26 is a graph showing the effect of reaction pH on the in vitro 1-hour percentage of cumulative insulin release of insulin microparticles surface-modified with one monolayer of protamine sulfate, poly-l-lysine, or poly-l-arginine (Example 14);
  • FIG. 27 is a laser scanning confocal (LSC) micrograph of nucleic acid microparticles surface-modified with one monolayer of rodamine B-labeled poly-l-lysine (Example 15);
  • FIG. 28 is a graph showing the in vitro release profiles of PLL-insulin microparticles thermally treated at different temperatures; and
  • FIG. 29A is a graph showing the serum insulin concentration versus time profiles in rats that have received a single subcutaneous injection of uncoated insulin microparticles, PLA-modified insulin microparticles treated at 28° C., and PLA-modified insulin microparticles treated at 4° C. (Example 18); and
  • FIG. 29B is a graph showing the serum glucose depression concentration versus time profiles in rats that have received a single subcutaneous injection of uncoated insulin microparticles, PLA-modified insulin microparticles treated at 28° C., and PLA-modified insulin microparticles treated at 4° C. (Example 18).
  • DETAILED DESCRIPTION
  • Unless otherwise defined herein, scientific and technical terminologies employed in the present disclosure shall have the meanings that are commonly understood and used by one of ordinary skill in the art. Unless otherwise required by context, it will be understood that singular terms shall include plural forms of the same and plural terms shall include the singular. Specifically, as used herein and in the claims, the singular forms “a” and “an” include the plural reference unless the context clearly indicates otherwise. Thus, for example, the reference to a particular microparticle is a reference to one such microparticle or a plurality of such microparticles, including equivalents thereof known to one skilled in the art. Also, as used herein and in the claims, the terms “at least one” and “one or more” have the same meaning and include one, two, three or more. The following terms, unless otherwise indicated, shall be understood to have the following meanings when used in the context of the present disclosure.
  • “Active agent” refers to naturally occurring, synthetic, or semi-synthetic materials (e.g., compounds, fermentates, extracts, cellular structures) capable of eliciting, directly or indirectly, one or more physical, chemical, and/or biological effects, in vitro and/or in vivo. The active agent may be capable of preventing, alleviating, treating, and/or curing abnormal and/or pathological conditions of a living body, such as by destroying a parasitic organism, or by limiting the effect of a disease or abnormality by materially altering the physiology of the host or parasite. The active agent may be capable of maintaining, increasing, decreasing, limiting, or destroying a physiologic body function The active agent may be capable of diagnosing a physiological condition or state by an in vitro and/or in vivo test. The active agent may be capable of controlling or protecting an environment or living body by attracting, disabling, inhibiting, killing, modifying, repelling and/or retarding an animal or microorganism. The active agent may be capable of otherwise treating (such as deodorizing, protecting, adorning, grooming) a body. Depending on the effect and/or its application, the active agent may further be referred to as a bioactive agent, a pharmaceutical agent (such as a prophylactic agent, a therapeutic agent), a diagnostic agent, a nutritional supplement, and/or a cosmetic agent, and includes, without limitation, prodrugs, affinity molecules, synthetic organic molecules, polymers, molecules with a molecular weight of 2 kD or less (such as 1.5 kD or less, or 1 kD or less), macromolecules (such as those having a molecular weight of 2 kD or greater, preferably 5 kD or greater), proteinaceous compounds, peptides, vitamins, steroids, steroid analogs, lipids, nucleic acids, carbohydrates, precursors thereof and derivatives thereof. Active agents may be ionic or non-ionic, may be neutral, positively charged, negatively charged, or zwitterionic, and may be used singly or in combination of two or more thereof. Active agents may be water insoluble, but more preferably are water soluble. Active agents may have an isoelectric point of 7.0 or greater, but preferably less than 7.0.
  • “Microparticle” refers to a particulate that is solid (including substantially solid or semi-solid, but excluding gel, liquid and gas), having an average geometric particle size (sometimes referred to as diameter) of less than 1 mm, preferably 200 microns or less, more preferably 100 microns or less, most preferably 10 microns or less. In one example, the particle size may be 0.01 microns or greater, preferably 0.1 microns or greater, more preferably 0.5 microns or greater, and most preferably from 0.5 microns to 5 microns. Average geometric particle size may be measured by dynamic light scattering methods (such as photocorrelation spectroscopy, laser diffraction, low-angle laser light scattering (LALLS), medium-angle laser light scattering (MALLS)), light obscuration methods (such as Coulter analysis method), or other methods (such as rheology, light or electron microscopy). Particles for pulmonary delivery will have an aerodynamic particle size determined by time of flight measurements or Andersen Cascade Impactor measurements. Microparticles may have a spherical shape (sometimes referred to as microspheres) and/or may be encapsulated (sometimes referred to as microcapsules). Certain microparticles may have one or more internal voids and/or cavities. Other microparticles may be free of such voids or cavities. Microparticles may be porous or, preferably non-porous. Microparticles may be formed from, in part or in whole, one or more non-limiting materials, such as the active agents, carriers, polymers, stabilizing agents, and/or complexing agents disclosed herein.
  • “Peptides” refer to natural, synthetic, or semi-synthetic compounds formed at least in part from two or more of the same or different amino acids and/or imino acids. Non-limiting examples of peptides include oligopeptides (such as those having less than 50 amino/imino acid monomer units, including dipeptides and tripeptides and the like), polypeptides, proteinaceous compounds as defined herein, as well as precursors and derivatives thereof (e.g., glycosylated, hyperglycosylated, PEGylated, FITC-labeled, salts thereof). Peptides may be used singly, or in combination of two or more thereof. Peptides may be neutral, positively charged, negatively charged, or zwitterionic, and may be used singly or in combination of two or, more thereof
  • “Proteinaceous compounds” refer to natural, synthetic, semi-synthetic, or recombinant compounds of or related structurally and/or functionally to proteins, such as those containing or consisting essentially of a-amino acids covalently associated through peptide linkages. Non-limiting proteinaceous compounds include globular proteins (edge, albumins, globulins, histones), fibrous proteins (e.g., collagens, elastins, keratins), compound proteins (including those containing one or more non-peptide component, e.g., glycoproteins, nucleoproteins, mucoproteins, lipoproteins, metalloproteins), therapeutic proteins, fusion proteins, receptors, antigens (such as synthetic or recombinant antigens), viral surface proteins, hormones and hormone analogs, antibodies (such as monoclonal or polyclonal antibodies), enzymes, Fab fragments, cyclic peptides, linear- peptides, and the like. Non-limiting therapeutic proteins include bone morphogenic proteins, drug resistance proteins, toxoids, erythropoietins, proteins of the blood clotting cascade (e.g., Factor VII, Factor VIII, Factor IX, et al.), subtilisin, ovalbumin, alpha-1-antitrypsin (AAT), DNase, superoxide dismutase (SOD), lysozyme, ribonuclease, hyaluronidase, collagenase, human growth hormone (hGH), erythropoietin, insulin and insulin-like growth factors or their analogs, interferons, glatiramer, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, desmopressin, leutinizing hormone release hormone (LHRH) agonists (e.g., leuprolide, goserelin, buserelin, gonadorelin, histrelin, nafarelin, deslorelin, fertirelin, triptorelin), LHER antagonists, vasopressin, cyclosporine, calcitonin, parathyroid hormone, parathyroid hormone peptides, insulin, glucogen-like peptides, and analogs thereof Proteinaceous compounds may be neutral, positively charged, negatively charged, or zwitterionic, and may be used singly or in combination of two or more thereof.
  • “Nucleic acids” refer to natural, synthetic, semi-synthetic, or recombinant compounds formed at least in part from two or more of the same or different nucleotides, and may be single-stranded or double-stranded. Non-limiting examples of nucleic acids include oligonucleotides (such as those having 20 or less base pairs, e.g., sense, anti-sense, or missense), aptamers, polynucleotides (e g., sense, anti-sense, or missense), DNA (e.g., sense, anti-sense, or missense), RNA (erg., sense, anti-sense, or missense), siRNA, nucleotide acid constructs, single-stranded or double-stranded segments thereof, as well as precursors and derivatives thereof (e.g., glycosylated, hyperglycosylated, PEGylated, FITC-labeled, nucleosides, salts thereof). Nucleic acids may be neutral, positively charged, negatively charged, or zwitterionic, and may be used singly or in combination of two or mole thereof.
  • “Carbohydrates” refer to natural, synthetic, or semi-synthetic compounds formed at least in part from monomeric sugar units. Non-limiting carbohydrates include polysaccharides, sugars, starches, and celluloses, such as carboxymethylcellulose, dextrans, hetastarch, cyclodextrins, alginates, chitosans, chondroitins, heparins, as well as precursors and derivatives thereof (e.g., glycosylated, hyperglycosylated, PEGylated, FITC-labeled, salts thereof). Carbodydrates may be ionic or non-ionic, may be neutral, positively charged, negatively charged, or zwitterionic, and may be used singly or in combination of two or, more thereof.
  • “Lipids” refer to natural, synthetic, or semi-synthetic compounds that are generally amphiphilic. The lipids typically comprise a hydrophilic component and a hydrophobic component. Non-limiting examples include fatty acids, neutral fats, phosphatides, oils, glycolipids, surfactants, aliphatic alcohols, waxes, terpenes and steroids. Lipids may be ionic or non-ionic, may be neutral, positively charged, negatively charged, or zwitterionic, and may be used singly or in combination of two or more thereof
  • “Complexing agent” refers to a material capable of forming one or more non-covalent associations with the active agent. Through such associations, the complexing agent is capable of facilitating the loading of one or more active agents into the microparticle, retaining the active agent(s) within the microparticle, and/or otherwise modifying the release of the active agent(s) from the microparticle. Complexing agents may be ionic or non-ionic, may be neutral, positively charged, negatively charged, or zwitterionic, and may be used singly or in combination of two or more thereof.
  • “Stabilizing,” used especially in conjunction with an agent (e.g., compound), a process, or a condition, refers to the capability of such agent, process or condition to, at least in part, form the microparticles (or a composition or formulation or kit containing such microparticles), facilitate the formation thereof, and/or enhance the stability thereof (e.g., the maintenance of a relatively balanced condition, like increased resistance against destruction, decomposition, degradation, and the like). Non-limiting stabilizing processes or conditions include thermal input/output (e.g., heating, cooling), electromagnetic irradiation (e.g., gamma rays, X rays, UV, visible light, actinic, infrared, microwaves, radio waves), high-energy particle irradiation (e.g., electron beams, nuclear), and ultrasound irradiation. Non-limiting stabilizing agents include lipids, proteins, polymers, carbohydrates, surfactants, salts (erg., organic, inorganic, with cations that are monovalent or polyvalent, metallic, organic, or organometallic, and anions that are monovalent or polyvalent, organic, inorganic, or organometallic), as well as certain of the carriers, the active agents, the crosslinkers, the co-agents, and the complexing agents disclosed herein. The stabilizing agents may be ionic or non-ionic, may be neutral, positively charged, negatively charged, or zwitterionic, and may be used singly or in combination of two or more thereof.
  • “Macromolecule” refers to a material capable of providing a three-dimensional (e.g., tertiary and/or quaternary) structure, and includes carriers and certain active agents of the present disclosure. Non-limiting macromolecules used to form the microparticles include, inter alia, polymers, copolymers, proteins (edge, enzymes, recombinant proteins, albumins like human serum albumin), peptides, lipids, carbohydrates, polysaccharides, nucleic acids, vectors (e.g., virus, viral particles), complexes and conjugates thereof (egg, by covalent and/or non-covalent associations, between two macromolecules like carbohydrate-protein conjugates, between an active agent and a macromolecule like hapten-protein conjugates, the active agent may or may not be capable of having a tertiary and/or quaternary structure), and mixtures of two or more thereof, preferably having a molecular weight of 1,500 or greater. Macromolecules may be neutral, positively charged, negatively charged, or zwitterionic, and may be used singly or in combination of two or more thereof.
  • “Spherical” refers to a geometric shape that is at least “substantially spherical.” “Substantially spherical” means that the ratio of the longest length (i.e., one between two points on the perimeter and passes the geometric center of the shape) to the shortest length on any cross-section that passes through the geometric center is about 1.5 or less, preferably about 1.33 or less, more preferably 1.25 or less. spherical does not require a line of symmetry. Further, the microparticles may have surface texturing (such as continuous or discrete lines, islands, lattice, indentations, channel openings, protuberances that are small in scale when compared to the overall size of the microparticles) and still be spherical. Surface contact there between is minimized in microparticles that are spherical, which minimizes the undesirable agglomeration of the microparticles. In comparison, microparticles that are crystals or flakes typically display significant agglomeration through ionic and/or non-ionic interactions at relatively large flat surfaces.
  • “Monodisperse size distribution” refers to a preferred microparticle size distribution in which the ratio of the volume diameter of the 90th percentile (i.e., the average particle size of the largest 10% of the microparticles) to the volume diameter of the 10th percentile (i.e., the average particle size of the smallest 10% of the microparticles) is about 5 or less, preferably about 3 or less, more preferably about 2 or less, most preferably about 1.5 to 1. Consequently, “polydisperse size distribution” refers to one where the diameter ratio described above is greater than 5, preferably greater than 8, more preferably greater than 10. In microparticles having a polydisperse size distribution, smaller microparticles may fill in the caps between larger microparticles, thus possibly displaying large contact surfaces and significant agglomeration there between A Geometric Standard Deviation (GSD) of 2.5 or less, preferably 1.8 or less, may also be used to indicate a monodisperse size distribution. Calculation of GSD is known and understood to one skilled in the art.
  • “Amorphous” refers to materials and constructions that are “substantially amorphous,” such as microparticles having multiple non-crystalline domains (or lacking crystallinity altogether) or otherwise non-crystalline. Substantially amorphous microparticles of the present disclosure are generally random solid particulates in which crystalline lattices constitute less than 50% by volume and/or weight of the microparticles, or are absent, and include semi-crystalline micropaticles and non-crystalline microparticles as understood by one skilled in the art.
  • “Solid” refers to a state that includes at least substantially solid and/or semi-solid, but excludes gel, liquid, and gas.
  • “Preformed microparticle” refers to a microparticle fabricated using one or more non-limiting methods, such as those known to one skilled in the art, without surface modification as described herein, having or capable of having on its outer surface a net surface electric charge that is positive, negative, or neutral. A preformed microparticle is also referred to herein as “core microparticle” or “core.” The preformed or core microparticle typically comprises one or more active agents and, optionally, one or more carriers, which, independently, may be compartmentalized in a portion of the preformed or core microparticle or preferably be distributed substantially homogeneously throughout the preformed microparticles. The net surface charge, preferably being non-zero, may be contributed primarily or at least, substantially, by the active agent(s) and/or the optional carrier(s).
  • “Carrier” refers to a compound, typically a macromolecule, having a primary function to provide a three-dimensional structure (including tertiary and/or quaternary structure) The carrier may be unassociated or associated with the active agent (such as conjugates or complexes thereof) in forming microparticles as described above. The carrier may further provide other functions, such as being an active agent, modify release profile of the active agent from the microparticle, and/or impart one or more particular properties to the microparticle (such as contribute at least in part to the net surface charge). In one example, the carrier is a protein (such as albumins, preferably human serum albumin) having a molecular weight of 1500 Daltons or greater,
  • “Polymer” or “polymeric” refers to a natural, synthetic, or semi-synthetic molecule having in a main chain or ring structure two or more repeating monomer units. Polymers broadly include dimers, trimers, tetramers, oligomers, higher molecular weight polymer, adducts, homopolymers, random copolymers, pseudo-copolymers, statistical copolymers, alternating copolymers, periodic copolymer, bipolymers, terpolymers, quaterpolymers, other forms of copolymers, substituted derivatives thereof, and mixtures thereof, and narrowly refer to molecules having 10 or more repeating monomer units. Polymers may be linear, branched, block, graft, monodisperse, polydisperse, regular, irregular, tactic, isotactic, syndiotactic, stereoregular, atactic, stereoblock, single-strand, double-strand, star, comb, dendritic, and/or ionomeric, may be ionic or non-ionic, may be neutral, positively charged, negatively charged, or zwitterionic, and may be used singly or in combination of two or more thereof
  • “Suspension” or “dispersion” refers to a mixture, preferably finely divided, of two or more phases (e g., solid, liquid, gas), such as solid in liquid, liquid in liquid, gas in liquid, solid in solid, solid in gas, liquid in gas, and the like. The suspension or dispersion may preferably remain stable for extended periods of time (e.g., minutes, hours, days, weeks, months, years).
  • “Resuspending” refers to changing microparticles from a non-flowable (e.g., solid) state to a flowable (e.g., liquid) state by adding a flowable medium (e.g., a liquid), while retaining most or all of the characteristics of the microparticles. The liquid may be, for example, aqueous, aqueous miscible, or organic.
  • “Charged” and “electrically charged” refer interchangeably to the capability of providing one, two, three, or more formal units of electrical charges of the same or opposite sign and/or the presence of such charges (i.e., “charged” refers to chargeable and/or charged). The electrical charges may be provided by and/or be present in tile form of one or more of the same or different organic and/or organometallic moieties (e.g., ionic groups, ionizable groups, precursors thereof) in the compound (e.g., polyelectrolytes, proteins) or the structure (e.g., the preformed microparticle, the monolayer's) of interest, preferably when subjected to certain conditions (such as in solution or suspension).
  • “Charged compound” and “electrically charged compound” refer interchangeably to a single compound that is charged as described above, or a combination of two or more different compounds in unassociated and/or associated forms (e.g., conjugates, aggregates, and/or complexes thereof), each of which independently has and/or is capable of having a net charge of the same sign.
  • “Monolayer” refers to a single layer formed over a three-dimensional substrate, from a composition of one or more compounds (such as a charged compound as described above). The monolayer may be a continuous and nonporous monolayer, a continuous and porous monolayer (such as a lattice network), a non-continuous monolayer of a plurality of discrete elements (e.g., islands, strips, clusters, etch), or a combination thereof. Typically, the monolayer will be decomposable or degradable, such as biodegradable, enzymatically or hydrolytically degradable and the like, to allow for non-diffusional release of an active agent (from the microparticle) over which the monolayer is deposited The monolayer may have a thickness of 100 nm or less, preferably 50 nm or less, more preferably 20 nm or less, most preferably 10 nm or less. In one example, the monolayer is formed through self-assembly of a charged compound.
  • “Saturated monolayer” refers to a monolayer as defined above that is incapable of further incorporating, cumulatively, an excess amount of the composition forming the monolayer when subjected to the same set of conditions under which the monolayer is formed. Saturated monolayers are preferred monolayers used in surface-modified microparticles.
  • “Net charge” and “net electric charge” are used interchangeably and refer to the sum of all formal units of electric charge a charged compound is capable of having or has, such as in a flowable medium under certain conditions (preferably in a solution of certain pH). The net charge may be positive, negative, or zero (such as in zwitterionic compounds), and is condition-dependent (erg., solvent, pH).
  • “Net surface charge” and “net surface electric charge” are used interchangeably and refer to an overall cumulative electric charge on an outermost surface of a three-dimensional structure (e.g., a microparticle, a monolayer). The net surface charge may be positive, negative, or zero, and is condition-dependent (e.g., solvent, pH).
  • “Ambient temperature” refers to a temperature of around room temperature, typically in a range of about 20° C. to about 40° C.
  • “Subject” or “patient” refers to animals, including vertebrates like mammals, preferably humans.
  • “Region of a subject” refers to a localized internal or external area or portion of the subject (e.g., an organ), or a collection of areas or portions throughout the entire subject (e.g., lymphocytes). Non-limiting examples of such regions include pulmonary region (e.g., lung, alveoli, gastrointestinal region (e.g., regions defined by esophagus, stomach, small large intestines, and rectum), cardiovascular region (e.g., myocardial tissue), renal region (e.g., the region defined by the kidney, the abdominal aorta, and vasculature leading directly to and from the kidney), vasculature (i.e., blood vessels, e.g., arteries, veins, capillaries, and the like), circulatory system, healthy or diseased tissues, benign or malignant (e.g., tumorous or cancerous) tissues, lymphocytes, receptors, organs and the like, as well as regions to be imaged with diagnostic imaging, regions to be administered and/or treated with an active agent, regions to be targeted for the delivery of an active agent, and regions of elevated temperature,
  • “Therapeutic” refers to any pharmaceutic, drug, prophylactic agent, contrast agent, or dye useful in the treatment (including prevention, diagnosis, alleviation, suppression, remission, or cure) of a malady, affliction, disease or injury in a subject. Therapeutically useful peptides and nucleic acids may be included within the meaning of the term “pharmaceutic” or “drug.”
  • “Affinity molecule” refers to any material or substance capable of promoting binding and/or targeting of regions in vivo and/or tissues/receptors in vitro. Affinity molecules, including receptors and targeting ligands, may be natural, synthetic, or semi-synthetic, may be ionic or non-ionic, may be neutral, positively charged, negatively charged, or zwitterionic, and may be used singly or in combination of two or more thereof. Non-limiting affinity molecules include proteinaceous compounds (e.g., antibodies, antibody fragments, hormones, hormone analogues, glycoproteins and lectins), peptides, polypeptides, amino acids, sugars, saccharides (egg monosaccharides, polysaccharides, carbohydrates), vitamins, steroids, steroid analogs, cofactors, active agents, nucleic acids, viruses, bacteria, toxins, antigens, other ligands, precursors thereof, and derivatives thereof.
  • “Precursor” refers to any material or substance capable of being converted to a desired material or substance, preferably through a chemical and/or biochemical reaction or pathway, such as anchoring a precursor to a material. Non-limiting precursor moieties include maleimide groups, disulfide groups (e.g., ortho-pyridyl disulfide), vinylsulfone groups, azide groups, and α-iodo acetyl groups
  • “Derivative” refers to any material or substance formed from a parent material or substance, preferably through a chemical and/or biochemical reaction or pathway considered routine by one of ordinary skill in the art. Non-limiting examples of derivatives include glycosylated, hyperglycosylated, PEGylated, FITC-labelled, protected with protecting groups (e.g., benzyl for alcohol or thiol, t-butoxycarbonyl for amine), as well as salts, esters, amides, conjugates, complexes, manufacturing related compounds, and metabolites thereof. Salts may be organic or inorganic, with cations that are monovalent or polyvalent, metallic, organic, or organometallic, and anions that are monovalent or polyvalent, organic, inorganic, or organometallic. Preferred salts are pharmaceutically acceptable, and include, without limitation, mineral or organic acid salts of basic residues (e.g., amines), alkali or organic salts of acidic residues (e.g., carboxylic acids), and the like, such as conventional nontoxic salts or the quaternary ammonium salts of the parent compound formed from non-toxic inorganic acids (e.g., hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric) and organic acids (e.g., acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, famaric, toluensulfonic, methanesulfonic, ethane dislfonic, oxalic, isethionic).
  • “Analog” refers to a compound having a chemically modified form of a principle compound or class thereof, which maintains the pharmaceutical and/or pharmacological activities characteristic of the principle compound or class
  • “Prodrug” refers to any covalently bonded carriers that release an active agent in vivo when administered to a subject. Prodrugs are known to enhance numerous desirable qualities (e.g., solubility, bioavailability, manufacturing) of the active agents Prodrugs may be prepared by modifying functional groups (e.g., hydroxy, amino, carboxyl, and/or sulfhydryl groups) present in the active agent in such a way that the modifications are reversed (e g., modifier group cleaved), either in routine manipulation or in vivo, to afford the original active agent. The transformation in vivo may be, for example, as the result of some metabolic process, such as chemical or enzymatic hydrolysis of a carboxylic, phosphoric or sulphate ester, or reduction or oxidation of a susceptible functionality.
  • “Metabolite” refers to a form of a compound obtained in a subject body by action of the body on the administered form of the compounds. For example, a demethylated metabolite may be obtained in the body after administration of a methylated compound bearing a methyl group. Metabolites may themselves have biological, preferably therapeutic, activities.
  • “Diagnostic agent” refers to any material or substance useful in connection with methods for perceptually observing (e.g., imaging) a normal or abnormal biological condition or state, or detecting the presence or absence of a pathogen or a pathological condition. Non-limiting diagnostic agents include contrast agents and dyes for use in connection with radiography imaging (e.g., X-ray imaging), ultrasound imaging, magnetic resonance imaging, computed tomography, positron emission tomography imaging, and the like. Diagnostic agents further include any other agents useful in facilitating diagnosis in vivo and/or in vitro, whether or not imaging methodology is employed.
  • “Cross-link,” “cross-linked” and “cross-linking” generally refer to the lining of two or more materials and/or substances, including any of those disclosed herein, through one or more covalent and/or non-covalent (e.g., ionic) associations. Cross-linking may be effected naturally (e.g., disulfide bonds of cystine residues) or through synthetic or semi-synthetic routes, for example, optionally in the presence of one or more cross-linkers (i.e., a molecule X by itself capable of reacting with two or more materials/substances Y and Z to form a cross-link product Y—X-Z, where the associations of Y—X and X-Z are independently covalent and/or non-covalent), initiators (i-e., a molecule by itself capable of providing reactive species like flee radicals for the cross-link reaction, e g., thermally decomposable initiators like organic peroxides, azo initiators, and carbon-carbon initiators, actinically decomposable initiators like photoinitiators of various wavelengths), activators (i e., a molecule A capable of reacting with a first material/substance Y to form an activated intermediate [A-Y], which in turn reacts with a second material/substance Z to form a cross-link product Y-Z, while A is chemically altered or consumed during the process), catalysts (i.e., a molecule capable of modifying the kinetics of the cross-link reaction without being chemically modified during the process), co-agents (i.e., a molecule that, when co-present with one or more of the initiators, activators, and/or catalysts, is capable of modifying the kinetics of the cross-link reaction and/or being incorporated into the cross-link product of the two or more materials/substances, but otherwise is non-reactive to the materials/substances), and/or energy sources (edge, heating; cooling; high-energy radiations like electromagnetic, e-beam, and nuclear; acoustic radiations like ultrasonic; etc.).
  • “Covalent association” refers to an intermolecular interaction (e.g., a bond) between two or more individual molecules that involves the sharing of electrons in the bonding orbitals of two atoms.
  • “Non-covalent association” refers to an intermolecular interaction between two or more individual molecules without involving a covalent bond. Intermolecular interaction depends on, for example, polarity, electric charge, and/or other characteristics of the individual molecules, and includes, without limitation, electrostatic (e.g., ionic) interactions, dipole-dipole interactions, van der Waal's forces, and combinations of two or more thereof.
  • “Electrostatic interaction” refers to an intermolecular interaction between two or more positively or negatively charged moieties/groups, which may be attractive when two are oppositely charged (i.e., one positive, another negative), repulsive when two charges are of the same sign (i.e., two positive or two negative), or a combination thereof.
  • “Dipole-dipole interaction” refers an intermolecular attraction between two or more polar molecules, such as a first molecule having an uncharged, partial positive end δ+ (e.g., electropositive head group like the choline head group of phosphatidylcholine) and a second molecule having an uncharged, partial negative end δ (e.g., an electronegative atom like heteroatom O, N, or S in a polysaccharide). Dipole-dipole interaction also refers to intermolecular hydrogen bonding in which a hydrogen atom serves as a bridge between electronegative atoms on separate molecules and in which a hydrogen atom is held to a first molecule by a covalent bond and to a second molecule by electrostatic forces.
  • “Hydrogen bond” refers to an attractive force or bridge between a hydrogen atom covalently bonded to a first electronegative atom (e.g., O, N, S) and a second electronegative atom, where the first and second electronegative atoms may be in two different molecules (intermolecular hydrogen bonding) or in a single molecule (intramolecular hydrogen bonding).
  • “Van der Waal's forces” refers to the attractive forces between non-polar molecules that are accounted for by quantum mechanics. Van der Waal's forces are generally associated with momentary dipole moments induced by neighboring molecules undergoing changes in electron distribution.
  • “Hydrophilic interaction” refers to an attraction toward water molecules, where a material/compound or a portion thereof may bind with, absorb, and/or dissolve in water. This may result in swelling and/or the formation of reversible hydrogels.
  • “Hydrophobic interaction” refers to a repulsion against water molecules, where a material/compound or a portion thereof do not bind with, absorb, or dissolve in water.
  • “Biocompatible” refers to materials/substances that are generally not injurious to biological functions and do not result in unacceptable toxicity (e.g., allergenic responses or disease states).
  • “In association with” and “associated with” refer in general to the one or more interactions between, and/or incorporation of, different materials (typically those that are part of the microparticles), one or more of such materials and one or more structures (or portions thereof) of the microparticles, and different structures (or portions thereof) of the microparticles. The materials of the microparticles include, without limitation, ions such as monovalent and polyvalent ions disclosed herein, as well as compounds such as active agents, stabilizing agents, cross-link agents, charged or uncharged compounds, the various polymers disclosed herein, and combinations of two or more thereof. The structures of the microparticles and portions thereof include, without limitation, core, core microparticle, preformed microparticle, monolayer, intermediate microparticle, surface-modified microparticle, portions of such structures (such as outer surfaces, inner surfaces), domains between such structures and portions thereof, and combinations of two or more thereof. Various associations, being reversible or irreversible. Migratory or non-migratory, may be present singly or in combination of two or more thereof. Non-limiting associations include, without limitation, covalent and/or non-covalent associations (e.g., covalent bonding, ionic interactions, electrostatic interactions, dipole-dipole interactions, hydrogen bonding, van der Waal's forces, cross-linking, and/or any other interactions), encapsulation in layer/membrane, compartmentalization in center or vesicles or between two layers/membranes, homogeneous integration throughout the microparticle or in a portion thereof (e.g., containment in, adhesion to, and/or affixation to center or layer or vesicle or an inner and/or outer surface thereof; interspersion, conjugations, and/or complexation between different materials).
  • “Tissue” refers generally to an individual cell or a plurality or aggregate of cells specialized and capable of performing one or more particular functions. Non-limiting, tissue examples include membranous tissues, (e.g., endothelium, epithelium), blood, laminae, connective tissue (e.g., interstitial tissue), organs (e.g., myocardial tissue, myocardial cells, cardiomyocites), abnormal cell(s) (e.g., tumors).
  • “Receptor” refers to a molecular structure within a cell or on its surface, generally characterized by its selective binding of a specific substance, e.g., ligand. Non-limiting receptors include cell-surface receptors for peptide hormones, neurotransmitters, antigens, complement fragments, and immunoglobulins, and cytoplasmic receptors for steroid hormones.
  • “Controlled release” refers to a predetermined in vivo and/or in vitro release (e.g., dissolution) profile of an active agent, as compared to the release profile of the active agent in its native form. The active agent is preferably associated with a microparticle or a composition or formulation containing such a microparticle, as disclosed herein, such that one or more aspects of its release kinetics (e.g., initial burst, quantity and/or rate over a specified time period or phase, cumulative quantity over a specific time period, length of time for total release, pattern and/or profile, etc.) are increased, decreased, shortened, prolonged, and/or otherwise modified as desired. Non-limiting examples of controlled release include immediate/instant release (i.e., initial burst or rapid release), extended release, sustained release, prolonged release, delayed release, modified release, and/or targeted release, occurring individually, in combination of two or more thereof or in the absence of one or more thereof (e. g. extended or sustained release in the absence of an initial burst).
  • “Extended release” refers to the release of an active agent, preferably in association with a microparticle or a composition or formulation containing such a microparticle, as disclosed herein, over a time period longer than the free aqueous diffusion period of the active agent in its native form. The extended release period may be hours (e.g., at least about 1, 2, 5, or 10 hours), days (e.g., at least about 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, 30, 40, 45, 60, or 90 days), weeks (at least about 1, 2, 3, 4, 5, 6, 10, 15, 20, 30, 40, or 50 weeks), months (at least about 1, 2, 3, 4, 6, 9, or 12 months), about 1 or more years, or a range between any two of the time periods. The pattern of an extended release may be continuous, periodic, sporadic, or a combination thereof.
  • “Sustained release” refers to an extended release of an active agent such that a functionally significant level of the active agent (i.e., a level capable of bring about the desired function of the active agent) is present at any time point of the extended release period, preferably with a continuous and/or uniform release pattern. Non-limiting examples of sustained release profiles include those, when displayed in a plot of release time (x-axis) versus cumulative release (y-axis), showing at least one upward segment that is linear, step-wise, zig-zagging, curved, and/or wavy, over a time period of 1 hour or longer.
  • Other than in the operating examples, or unless otherwise expressly specified, all of the numerical ranges, amounts, values and percentages such as those for quantities of materials, times, temperatures, reaction conditions, ratios of amounts, values for molecular weight (whether number average molecular weight Mn or weight average molecular weight Mw), and others disclosed herein should be understood as modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the present disclosure and attached claims are approximations that may vary as desired. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
  • Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Furthermore, when numerical ranges of varying scope are set forth herein, it is contemplated that any combination of these values inclusive of the recited values may be used.
  • “Formed from” and “formed of” denote open, language. As such, it is intended that a composition “formed from” or “formed of” a list of recited components be a composition comprising at least these recited components, and can further include other non-recited components during formulation of the composition
  • Examples provided herein, including those following “such as” and “e.g.,” are considered as illustrative only of various aspects of the present disclosure and embodiments thereof, without being specifically limited thereto. Any suitable equivalents, alternatives, and modifications thereof (including materials, substances, constructions, compositions, formulations, means, methods, conditions, etc.) known and/or available to one skilled in the art may be used or carried out in place of or in combination with those disclosed herein, and are considered to fall within the scope of the present disclosure,
  • In one example, each of the surface-modified microparticles of the present disclosure preferably contains an amorphous (e.g., such as free of crystalline structures) and solid preformed microparticle associated, at least at its outer surface, with at least one monolayer containing at least one charged compound. The preformed microparticle contains at least one active agent and/or at least one macromolecule having a molecular weight of 4,500 Daltons or greater. The macromolecule may be the active agent or may be different from the active agent. The macromolecule may be a carrier, a stabilizing agent, or a complexing agent (e.g., proteinaceous compounds, polyelectrolytes). The active agent and/or the macromolecule may constitute 40% to 100% or less, and typically at least 80%, such as 90% or more or 95% or more, by weight of the preformed microparticle. Preferably, the active agent and/or the macromolecule is/are distributed homogeneously throughout the core microparticle. An outer surface of the preformed microparticle carries a net surface charge, which may be attributed, at least in part, and more typically in large part, to the active agent and/or the macromolecule, especially when the outer surface is formed of the active agent and/or the macromolecule. The preformed microparticle may be free of covalent crosslinking, hydrogel, lipids, and/or encapsulation. Alternatively, the preformed microparticle may contain one or more charged compounds, covalent crosslinking, and/or encapsulation. The one or more charged compounds in the preformed microparticle may be distributed homogeneously throughout the preformed microparticle, or compartmentalized in specific portions thereof, such as in a layer. The preformed microparticle may preferably have a particle size of 10 μm or less, and may have a monodisperse or polydisperse size distribution.
  • Methods of pre-forming the preformed microparticle are not particularly limiting, and include those disclosed in U.S. Pat. No. 6,458,387 and U.S. Patent Publication No. 2005/0142206, which are incorporated herein by reference in their entirety. In one example, a single flowable continuous phase system (such as liquid, gas, or plasma, preferably a solution or suspension) is formulated to contain one or more active agents, a medium, and one or more phase-separation enhancing agents (PSEAs) The medium is preferably a liquid solvent (e.g., hydrophilic or hydrophobic organic solvents, water, buffers, aqueous-miscible organic solvents, and combinations of two or more thereof), more preferably an aqueous or aqueous-miscible solvent. Suitable organic solvents include, without limitation, methylene chloride, chloroform, acetonitrile, ethylacetate, methanol, ethanol, pentane, the likes thereof, and combinations of two or more thereof (such as a 1:1 mixture of methylene chloride and acetone). The active agent and the PSEA may independently be dissolved, suspended, or otherwise homogeneously distributed within the medium. When subjecting the flowable system to certain conditions (such as a temperature below the phase transition temperature of the active agent in the medium), the active agent undergoes a liquid-solid phase separation and forms a discontinuous, preferably solid, phase (such as a plurality of core microparticles suspended in the medium), while the PSEA remains in the continuous phase (such as being dissolved in the medium).
  • The medium can be organic, containing an organic solvent or a mixture of two or more inter-miscible organic solvents, which may independently be aqueous-miscible or aqueous-immiscible. The solution can also be an aqueous-based solution containing an aqueous medium or an aqueous-miscible organic solvent or a mixture of aqueous-miscible organic solvents or combinations thereof. The aqueous medium can be water, a buffer (e.g., normal saline, buffered solutions, buffered saline), and the like. Suitable aqueous-miscible organic solvents may be monomers or polymers, and include, but are not limited to, N-methyl-2-pyrrolidinone (N-methyl-2-pyrrolidone), 2-pyrrolidinone (2-pyrrolidone), 1,3-dimethyl-2-imidazolidinone (DMI), dimethylsulfoxide, dimethylacetamide, acetic acid, lactic acid, acetone, methyl ethyl ketone, acetonitrile, methanol, ethanol, n-propanol, isopropanol, 3-pentanol, benzyl alcohol, glycerol, tetrahydrofuran (THF), polyethylene glycol (PEG, e.g., PEG-4, PEG-8, PEG-9, PEC-12, PEG-14, PEG-16, PEG-120, PEG-75, PEG-150), PEG esters (e.g., PEG-4 dilaurate, PEG-20 dilaurate, PEG-6 isostearate, PEG-8 palmitostearate, PEG-150 palmitostearate), PEG sorbitans (such as PEG-20 sorbitan isostearate), PEG ethers (such as monoalkyl and dialkyl ethers, e.g., PEG-3 dimethyl ether, PEG-4 dimethyl ether, and glycofurol), polypropylene glycol (PPS), PPG esters (such as polypropylene glycol alginate (PGA), PPG dicaprylate, PPG dicaprate, PPG laurate), alkoxylated linear alkyl diols (such as PPG-10 butanediol), alkoxylated alkyl glucose ether (e.g., PPG-10 methyl glucose ether, PPG-20 methyl glucose ether), PPG alkyl ethers (such as PPG-15 stearyl ether), alkanes (e.g., propane, butane, pentane, hexane, heptane, octane, nonane, decane), and combinations of two or more thereof.
  • In a preferred example, a solution of the PSEA in a first solvent is provided, in which the PSEA is soluble in or miscible with the first solvent. The active agent is mixed in, directly or as a second solution in a second solvent, with the first solution. The first and second solvent may be the same or at least miscible with each other. Preferably the active agent is added at a temperature equal to or lower than ambient temperature, particularly when the active agent is a heat labile molecule such as certain proteinaceous compounds. However, the system may be heated to increase solubility of the active agent in the system, as long as the activity of the active agent is not adversely affected.
  • When the mixture is brought to phase separation conditions, the PSEA, while remaining in the liquid continuous phase, enhances and/or induces a liquid-solid phase separation of the active agent from the solution (such as by reducing solubility of the active agent), forming the core microparticles (the solid discontinuous phase), which may preferably be microspheres. Suitable PSEA compounds include, but are not limited to, natural and synthetic polymers, linear polymers, branched polymers, cyclo-polymers, copolymers (random, block, grafted, such as poloxamers, particularly PLURONIC® F127 and F68), terpolymers, amphiphilic polymers, carbohydrate-based polymers, polyaliphatic alcohols, poly(vinyl)polymers, polyacrylic acids, polyorganic acids, polyamino acids, polyethers, polyesters, polyimides, polyaldehydes, polyvinylpyrrolidone (PVP), and surfactants. Suitable or exemplary PSEAs include, without limitation, polymers acceptable as pharmaceutical additives, such as PEGs (e.g., PEG 200, PEG 300, PEG 3350, PEG 8000, PEG 10000, PEG 20000, etc,), poloxamers, PVP, hydroxyethylstarch, amphiphilic polymers, as well as non-polymers (such as mixtures of propylene glycol and ethanol).
  • Conditions capable of enhancing, inducing, promoting, controlling, suppressing, retarding, or otherwise affecting the liquid-solid phase separation include, without limitation, changes in temperature, pressure, pH, ionic strength and/or osmolality of the solutions, concentrations of the active agent and/or the PSEA, the likes thereof, as well as rates of such changes, and combinations of two or more thereof. Such conditions may desirably be applied before and up to the phase separation, or even during the phase separation. In one example, the system is exposed to a temperature below the phase transition temperature of the active agent therein, alone or in combination with adjustments to the concentrations of the active agent and/or the PSEA, as described in U.S. Patent Application Publication 2005/0142206, the entire disclosure of which is incorporated herein by reference. The rate of temperature drop may be held constant or altered in any controlled manner, as long as it is within a range of 0.2° C./minute to 50° C./minute, preferably 0.2° C./minute to 30° C./minute. Freezing point depressing agents (FPDAs), used individually or in combination of two or more thereof, may be mixed in the system directly or in solutions (such as aqueous solutions) thereof, particularly for systems in which the freezing point is higher than the phase transition temperature of the active agent. Suitable FPDAs include, without limitation, propylene glycol, sucrose, ethylene glycol, alcohols (e.g., ethanol, methanol), and aqueous mixtures thereof.
  • In one example, the preformed microparticles may further comprise one or more excipients that negligibly affect the phase separation. The excipient may imbue the core microparticles and/or the compounds therein (e.g., the active agent, the optional carrier) with additional characteristics such as increased stability, controlled release of the active agent from the preformed microparticles, and/or modified permeation of the active agent through biological tissues. Suitable excipients include, but are not limited to, carbohydrates (e.g., trehalose, sucrose, mannitol), polyvalent cations (preferably metal cations, e.g., Zn2+, Mg2+, Ca2+, Cu2+, Fe2+, Fe3+), anions (e.g., CO3 2−, SO4 2−), amino acids (such as glycine), lipids, phospholipids, fatty acids and esters thereof, surfactants, triglycerides, bile acids and conjugates and salts thereof (e.g., cholic acid, deoxycholic acid, glycocholate, taurocholate, sodium cholate), and any polymers disclosed herein.
  • The preformed microparticles may optionally be separated from the solution and washed prior to the surface modification as disclosed herein, or be surface-modified without separation or washing. Separation means include, without limitation, centrifugation, dialysis, sedimentation (creaming), phase separation, chromatography, electrophoresis, precipitation, extraction, affinity binding, filtration, and diafiltration. For active agents with relatively low aqueous solubility, the washing medium may be aqueous, optionally containing one or more solubility reducing agents (SRAs) and/or excipients as disclosed herein Preferred SRAs are capable of forming insoluble complexes with the active agents and/or carriers in the microparticles, and include, without limitation, compounds such as salts that comprise divalent or polyvalent cations (such as those disclosed herein). For active agents with relatively high aqueous solubility (such as proteinaceous compounds), the washing medium may be organic, or aqueous but containing at least one SRA or precipitating agent (such as ammonium sulfate). In one example, the washing medium is the same solution used in the phase separation reaction, such as an aqueous solution including approximately 16% (w/v) PEG and 0.7% (w/v)
  • It is preferred that the washing medium has a low boiling point for easy removal by, for example, lyophilization, evaporation, or drying The washing medium may be a supercritical fluid or a fluid near its supercritical point, used alone or in combination with a co-solvent. Supercritical fluids may be solvents for the PSEAs, but not for the preformed microparticles. Non-limiting examples of supercritical fluids include liquid CO2, ethane, and xenon. Non-limiting examples of co-solvents include acetonitrile, dichloromethane, ethanol, methanol, water, and 2-propanol.
  • As indicated above, active agents with varying degrees of solubility in water may be employed in the microparticles described herein. While water insoluble active agents may be used, water soluble active agents are preferred.
  • The active agent may be a pharmaceutical agent. Depending on its effect and/or application, the pharmaceutical agent includes, without limitation, adjuvants, adrenergic agents, adrenergic blocking agents, adrenocorticoids, adrenolytics, adrenomimetics, alkaloids, alkylating agents, allosteric inhibitors, anabolic steroids, analeptics, analgesics, anesthetics, anorexiants, antacids, anthelmintics, anti-allergic agents, antiangiogenesis agents, anti-arrhythmic agents, anti-bacterial agents, antibiotics, antibodies, anticancer agents. anticholinergic agents, anticholinesterases, anticoagulants, anticonvulsants, antidementia agents, antidepressants, antidiabetic agents, antidiarrheals, antidotes, antiepileptics, antifolics, antifungals, antigens, antihelmintics, antihistamines, antihyperlipidemics, antihypertensive agents, anti-infective agents, anti-inflammatory agents, antimalarials, antimetabolites, antimuscarinic agents, antimycobacterial agents, antineoplastic agents, antiosteoporosis agents, antipathogen agents, antiprotozoal agents, adhesion molecules, antipyretics, antirheumatic agents, antiseptics, antithyroid agents, antiulcer agents, antiviral agents, anxioltic sedatives, astringents, beta-adrenoceptor blocking agents, biocides, blood cloning factors, calcitonin, cardiotonics, chemotherapeutics, cholesterol lowering agents, cofactors, corticosteroids, cough suppressants, cytokines, diuretics, dopaininergics, estrogen receptor modulators, enzymes and cofactors thereof enzyme inhibitors, growth differentiation factors, growth factors, hematological agents, hematopoietics, hemoglobin modifiers, lemostatics, hormones and hormone analogs, hypnotics, hypotensive diuretics, immunological agents, immunostimulants, immunosuppressants, inhibitors, ligands, lipid regulating agents, lymphokines, muscarinics, muscle relaxants, neural blocking agents, neurotropic agents, paclitaxel and derivative compounds, parasympathomimetics, parathyroid hormone, piromotors, prostaglandins, psychotherapeutic agents, psychotropic agents, radio-pharmaceuticals, receptors, sedatives, sex hormones, sterilants, stimulants, thrombopoietics, trophic factors, sympathonmimetics, thyroid agents, vaccines, vasodilators, vitamins, xanthines, as well as conjugates, complexes, precursors, and metabolites thereof. The active agent may be used individually or in combinations of two or more thereof In one example, the active agent is a prophylactic and/or therapeutic agent that includes, but is not limited to, peptides, carbohydrates, nucleic acids, other compounds, precursors and derivatives thereof, and combinations of two or more thereof
  • As discussed above, the active agent may be a cosmetic agent. Non-limiting cosmetic agents include inter-alia emollients, humectants, free radical inhibitors, anti-inflammatories, vitamins, depigmenting agents, anti-acne agents, antiseborrhoeics, keratolytics, slimming agents, skin coloring agents and sunscreen agents. Non-limiting compounds useful as cosmetic agents include linoleic acid, retinol, retinoic acid, ascorbic acid alkyl esters, polyunsaturated fatty acids, nicotinic esters, tocopherol nicotinate, unsaponifiables of rice, soybean or shea, ceramides, hydroxy acids such as glycolic acid, selenium derivatives, antioxidants, beta-carotene, gamma-orizanol and stearyl glycerate. The cosmetic agents may be commercially available and/or prepared by known techniques.
  • As discussed above, the active agent may be a nutritional supplement. Non-limiting nutritional supplements include proteins, carbohydrates, water-soluble vitamins (e.g., vitamin C, B-complex vitamins, and the like), fat-soluble vitamins (e.g., vitamins A, D, E, K, and the like), and herbal extracts. The nutritional supplements may be commercially available and/or prepared by known techniques.
  • As discussed above, the active agent may be a compound having a molecular weight of 2 kD or less. Non-limiting examples of such compounds include steroids, beta-agonists, anti-microbials, antifungals, taxanes (antimitotic and antimicrotubule agents), amino acids, aliphatic compounds, aromatic compounds, and urea compounds
  • In one example, the active agent may be a therapeutic agent for prevention and/or treatment of pulmonary disorders. Non-limiting examples of such agents include steroids, beta-agonists, anti-fungals, anti-microbial compounds, bronchial dialators, anti-asthmatic agents, non-steroidal anti-inflammatory agents (NSAIDS), AAT, and agents to treat cystic fibrosis. Non-limiting examples of steroids include beclomethasone (such as beclomethasone dipropionate), fluticasone (such as fluticasone propionate), budesonide, estradiol, fludrocortisone, flucinonide, triamcinolone (such as triamcinolone acetonide), flunisolide, and salts thereof. Non-limiting examples of beta-agonists include salmeterol xinafoate, formoterol fumarate, levo-albuterol, bambuterol, tulobuterol, and salts thereof. Non-limiting examples of anti-fungal agents include itraconazole, fluconazole, amphotericin B, and salts thereof.
  • As discussed above, the active agent may be a diagnostic agent. Non-limiting diagnostic agents include x-ray imaging agents and contrast media. Non-limiting examples of x-ray imaging agents include ethyl 3,5-diacetamido-2,4,6-triiodobenzoate (WIN-8883, ethyl ester of diatrazoic acid); 6-ethoxy-6-oxohexyl-3,5-bis(acetamido)-2,4,6-triiodobenzoate (WIN 67722); ethyl-2-(3,5-bis(acetamido)-2,4,6-triiodobenzoyloxy)-butyrate (WIN 16318); ethyl diatrizoxyacetate (WIN 12901); ethyl 2-(3,5-bis(acetamido)-7,4,6-triiodobenzoyloxy)propionate (WIN 16923); N-ethyl 2-(3,5-bis(acetamido)-2,4,6-triiodobenzoyloxy-acetamide (WIN 65312); isopropyl 2-(3,5-bis(acetamide)-2,4,6-triiodobenzoyloxy)acetamide (WIN 12855); diethyl 2-(3,5-bis(acetamido)-2,4,6-triiodobenzoyloxymalonate (WIN 67721); ethyl 2-(3,5-bis(acetamido)-2,4,6-triiodobenzoyloxy)phenyl-acetate (WIN 67585); propanedioic acid, [[3,5-bis(acetylamino)-2,4,5-triodobenzoyl]oxy]bis(1-methyl)ester (WIN 68165); and benzoic acid, 3,5-bis(acetylamino)-2,4,6-triodo-4-(ethyl-3-ethoxy-2-butenoate)ester (WIN 68209). Preferred contrast agents desirably disintegrate relatively rapidly under physiological conditions, thus minimizing any particle associated inflammatory response. Disintegration may result from enzymatic hydrolysis, solubilization of carboxylic acids at physiological pH, or other mechanisms. Thus, poorly soluble iodinated carboxylic acids such as iodipamide, diatrizoic acid, and metrizoic acid, along with hydrolytically labile iodinated species such as WIN 67721, WIN 12901, WIN 68165, and WIN 68209 or others may be preferred.
  • As discussed above, the active agents may be used in a combination of two or more thereof. Non-limiting examples include a steroid and a beta-agonist, e.g., fluticasone propionate and salmeterol, budesonide and formeterol, etc
  • Preformed microparticles may be substantially free of internal voids and/or cavities (such as being free of vesicles), substantially free of encapsulation, substantially free of lipids, substantially free of hydrogel or swelling, substantially and non-porous, amorphous solid, and/or spherical as those terms are defined herein. Preformed microparticles may have multiple surface channel openings, the diameter of which are generally 100 nm or less, preferably 10 nm or less, more preferably 5 nm or less, most preferably 1 nm or less. Preformed microparticles may have an overall density of 0.5 g/cm3 or greater, preferably 0.75 g/cm3 or greater, more preferably 0-85 g/cm3 or greater. The density may be generally up to about 2 g/cm3, preferably 1.75 g/cm3 or less, more preferably 1.5 g/cm3 or less.
  • Preformed microparticles may exhibit a high payload of the at least one active agent. Depending on the formulation and the physical/chemical nature of the compounds, there are typically at least 1000 or more, such as a few million to hundreds of millions of the active agent molecules in each of the preformed microparticles. The weight percentage of the active agent in the preformed microparticle may be any of the amount below or greater, or any ranges there between, but less than 100%: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%. While incorporation of a significant amount of hulking agents and/or other excipients is not required in the preformed microparticles, one or more of such compounds may be present therein. In any event, the desired integrity and/or activity are retained for a majority (50% or greater, preferably 75% or greater, more preferably 90% or greater, most preferably 95% or greater) of the active agent, if not 100%.
  • Surface modification of the preformed mictoparticles is achieved, without limitation, by forming, in a controlled manner, at least one monolayer containing at least one charged compound about the preformed microparticle. When two or more such monolayers are formed, each contains different charged compounds, and preferably each carries on its outer surface a net surface charge that is different in sign and/or value from that of the preceding one and/or the subsequent one, if present. Deposition of such monolayers one at a time allows for optimal control over various properties of the resulting microparticles, allowing one to tailor or “fine-tune” the microparticles to achieve a desired result.
  • Preferably, the monolayer immediately about the performed microparticle (“formed monolayer”) contains one or more charged compounds, each independently having a net charge that is opposite in sign to the net surface charge of the core microparticle. The preformed micropartiele may at least, in part, be penetrable by the charged compound in the formed monolayer An outer surface of the formed monolayer may carry a net surface charge that is different from, preferably opposite in sign to, that of the preformed microparticle outer surface, especially when the formed monolayer is a saturated monolayer as defined herein. The charged compounds may include one or more of polyelectrolytes, charged polyaminoacids, charged polysaccharides, polyionic polymers, charged proteinaceous compounds, charged peptides, charged lipids optionally in combination with uncharged lipids, charged lipid structures, and derivatives thereof.
  • The surface-modified microparticle may further contain one or more additional alternatingly charged monolayers, such that the surface-modified microparticle has a desired release profile of the active agent. This number is not particularly limited, but may typically be between 1 to 7, such as 2, 3, 4, 5, or 6. Optionally, one or more of such charged monolayers may independently have one or more of the same or different active agents, such as an affinity molecule, especially a targeting ligand, associated covalently and/or non-covalently thereto, preferably on their respective outer surfaces. Alternatively or in combination, the core microparticle may have one or more portions, such as a center or an underlying layer (a charged monolayer, for example), containing at least one such active agent, preferably on the outer surface of the portion.
  • The preformed microparticle, the surface-modified microparticle, and any intermediates there between, if any, may be and/or have one or more of the following characteristics: spherical as defined herein, free of covalent crosslinking, free of hydrogel and/or swelling, and have a polydisperse or, preferably, monodisperse, size distribution. The preformed microparticle, may be free of lipids and/or encapsulation.
  • Preferably, the surface-modified microparticle is capable of controlled release, especially sustained release, of the active agent, with a non-limiting release profile such as an initial burst and a linear release profile, and may be provided as a suspension or a dry powder in compositions or formulations for pharmaceutical, therapeutic, diagnostic, cosmetic, and/or nutritional applications As discussed above, the controlled release may occur within a selected pH environment. In that regard, preferably the controlled release may occur within a pH range of approximately 2 to 10, and more preferably approximately 5 to 7.5, such as a physiological pH of 7 to 7.4 or endosomal pH of 5 to 6.5.
  • Controlled deposition of the one or more monolayers may further involve alteration of the net surface charge of the microparticle (such as the preformed microparticle onto which one or more of the monolayers have been deposited) through a controlled manipulation of one or more conditions, such as changes in temperature, pressure, pH, ionic strength and/or osmolality of the reaction medium, concentrations of components within the reaction medium, the likes thereof, as well as rates of such changes, and combinations of two or more thereof. Such controlled manipulations may desirably be applied before and up to the deposition of the one or more monolayers, or even during the monolayer formations. In one example, the net surface charge of the microparticle is capable of being positive, neutral, and negative. The net surface change is selected through, for example, a controlled change in one or more of the conditions described above, such as a controlled change in pH. In one example, the pH of the solution is selected such that the net surface charge of the microparticle is negative, and the difference between the pH of the solution and the surface-neutral point of the microparticle is less than 0.3, alternatively equal to or greater than 0.3, preferably 0.5 or greater, more preferably 0.8 or greater, most preferably 1 or greater.
  • FIG. 1 illustrates one exemplary method of providing surface-modified microparticles in accordance with the present disclosure. is A suspension of a plurality of preformed microparticles used as three-dimensional substrates for the deposition is first provided. Non-limiting methods of forming the preformed microparticle include those disclosed herein and any other methods known to those of skill in the art. One such method is illustrated on the top portion of FIG. 24, which involves providing a solution containing, the active agent and the phase-separation enhancing agent, inducing a liquid-solid phase separation through, for example, controlled cooling, and forming the preformed microparticle. In one example, any one, two, or more, or all of the compounds used to form the preformed microparticles may preferably be distributed homogeneously throughout each preformed microparticle (e.g., being present at similar concentrations in the center, on the surface, and anywhere else therein). It will be understood that methods of surface modification as disclosed herein may be incorporated in whole or in part into the underlying methods of fabricating the preformed microparticles or made to be a continuation thereof, as illustrated in FIG. 24. Between the pre-formation of the unmodified microparticle and the surface modification, the preformed microparticle may be separated from the liquid phase and, optionally, washed, preferably in the presence of the phase-separation enhancing agent. For example, the washing medium may be the same solution used during phase separation, containing the phase-separation enhancing agent. Alternatively, the preformed microparticle is not separated from the liquid phase or washed. In any event, as shown in FIGS. 1 and 24, the suspension or a re-suspension of the preformed microparticle is combined and mixed with a solution that includes at least one suitable charged compound.
  • As described above, the preformed microparticles may have a weight percent (wt. %) loading of the active agent of 40% or more, preferably 60% or more, or 80% or more, or 90% or more, or 95% or more, and less than 100%, typically 98% or less. The preformed microparticles may further have, or are capable of being induced (such as from a neutral state) to have, a net surface electric charge. In one example, the net surface charge is contributed primarily or essentially by the active agent and/or the carrier, if any, present in the preformed microparticles; the compound(s) may preferably be homogeneously distributed therein. Alternatively, the active agent is compartmentalized in one or more portions of the preformed microparticle, such as a center or an underlying layer (a charged monolayer, for example), preferably distributed substantially homogeneously within the portion or primarily on an outer surface thereof. The preformed microparticles may be exposed to (such as mixed with) at least one charged compound having or capable of having a net electrical charge that is, preferably, opposite in sign to the net surface charge of the preformed microparticle, thereby forming the formed monolayer of the charged compound about the preformed microparticle. The formed monolayer or the surface modified microparticle has a net surface electric charge that may be the same in sign as that of the preformed microparticle, zero or, preferably, opposite in sign to that of the preformed microparticle. In other words, if the outer surface of the preformed microparticle has a negative net surface charge (such as determined by zeta-potential measurements), then the formed monolayer may preferably have on its outer surface a positive net surface charge. Alternatively, if the preformed microparticle has a positive net surface charge, then the formed monolayer may preferably have a negative net surface charge. Deposition of the monolayer can take place in an aqueous medium (e.g., water, buffer, or aqueous solution containing some water miscible organic solvent of the type previously described, or one that may be present in the manufacture of the preformed microparticle).
  • To prepare the surface-modified microparticle, a non-limiting method includes pre-forming or otherwise providing the unmodified microparticle, exposing it to the one or more charged compounds, which may be provided in a solution into which the microparticle may be immersed, and forming the monolayer. The solution may contain one or more of water, a buffer, and a water-miscible organic solvent, and one or more solubility reducing agents (e.g., alcohols, carbohydrates, non-ionic aqueous-miscible polymers, and/or inorganic ionic compounds containing monovalent or polyvalent cations), with a concentration in weight-to-volume percentage of 5% to 50%, preferably 10% to 90%. A non-limiting example of the solution contains about 16% (w/v) polyethylene glycol and 0.7% (w/v) NaCl. The pH of the solution, typically in a range of 4 to 10, may be adjusted to be same as or close to the surface-neutral point of the core microparticle (such as with a difference of 0 to less than 0.3), or away from that (such as with a difference of 0.3 pH units or greater). The charged compound may be present in the solution at a concentration of 0.05 mg/mL to 10 mg/mL. The preformed microparticle and the charged compound are co-incubated in the solution, preferably at a temperature of 2° C. to 5° C. or up to ambient temperature over a period of 1 second to 10 hours. The formation of the monolayer may be carried out in a controlled manner. The resulting surface-modified microparticle or an intermediate thereof may be separated from the solution with optional washing. The washing medium may be the same as the solution described above. The procedure may be repeated using alternatingly charged compounds to form the alternatingly charged monolayers, if desired
  • As indicated above, the reaction system can include one or more solubility reducing and/or viscosity increasing agents (SRA/VIA), as well as one or more PSEAs. Suitable SRA/VIAs and PSEAs include, without limitation, those known to one skilled in the art and those disclosed herein, such as alcohols (e.g., ethanol, glycerol), carbohydrates (such as sucrose), non-ionic aqueous-miscible polymers (e.g., PEG, PVP, block copolymers of polyoxyethylene and polyoxypropylene(poloxamers), hetastarch, dextran, etc.), and inorganic ionizable compounds containing polyvalent (e.g., divalent, trivalent) cations (e.g., metal and organic cations such as those disclosed herein), such as ZnCl2.
  • Thus, in one example, deposition of the formed monolayer may take place in a solution that includes buffered saline (that is, 0.7% NaCl buffer) and 8% or more by weight or volume of a SRA/VIA such as PEG, preferably 12% or more, more preferably 15% or more; typically 30% or less, preferably 25% or less, more preferably 20% or less, most preferably about 16% or more. The amount of SRA/VIA required in the solution will depend, in part, on the stability of the active agent, as well as the dissolution profile of the monolayer(s). Certain charged compounds (such as the polycations gelatin B and chitosan) may worlk in solutions containing 16% or less SRA/VIA.
  • The pH of the solution at which the net surface charge of the microparticle is zero is referred herein as the surface-neutral point of the microparticle in the particular solution. In certain examples, the pH of the solution may be adjusted to be at or near, the surface-neutral point of the microparticle in the solution with a difference there between of less than 0.3 (pH units), preferably 0.25 or less, more preferably 0.2 or less. In other examples, the pH of the solution may preferably be adjusted away from the surface-neutral point of the microparticle in the solution, with a difference there between of 0.3 (pH units) or greater, preferably 0.5 or greater, more preferably 0.8 or greater, most preferably 1 or greater. It has been observed that in certain examples, adjusting the solution pH away from the surface-neutral point of the microparticle can affect dissolution kinetics of the active agent therein. Incubation of the microparticles in the solution can be performed at or, preferably, below ambient temperature, but preferably above the freezing temperature of the solution, to minimize disintegration of the microparticles. Incubation temperature may even be lower than the freezing temperature of the solution when one or more FPDAs disclosed herein are used. For example, the incubation temperature may be between 0° C. and 15° C., preferably between 1° C. and 10° C., more preferably between 2° C. and 5° C., most preferably less than 5° C. In general, the concentration of the charged compound in the solution for each monolayer fabrication may be equal to, less than, and/or greater than one of the following, or in a range between any two thereof: 0.05 mg/mL, 0.1 mg/mL, 0.5 mg/mL, 1 mg/mL, 10 mg/mL, 5 mg/mL, 3 mg/mL. When the preformed microparticle is co-incubated with the charged compound in the solution, a weight ratio of the preformed microparticle to the charged compound may be 1:1 or greater, preferably 2:1 to 10:1, more preferably 2.5:1 to 7:1
  • Incubation time may be adjusted to achieve the desired charge modification (such as neutralization or charge reversal), monolayer coverage, and/or monolayer thickness. Depending on the particular reaction (such as ingredients and/or conditions), the incubation time may be equal to, shorter than, and/or longer than one of the following, or in a range between any two thereof: 10 hours, 5 hours, 3 hours, 10 minutes, 30 minutes, 100 minutes, 75 minutes, 60 minutes, 15 minutes, 5 minutes, 1 minute, 30 seconds, 10 seconds, 5 seconds, 1 second. Each monolayer may have a thickness that is equal to, less than, and/or greater than one of the following, or in a range between any two thereof: 100 nm, 50 nm, 20 nm, 5 nm, 1 nm, 0.5 nm, 0.1 nm, 2 nm, 10 nm. A typical monolayer of the present disclosure is less than 100 nm in thickness, preferably less than 10 nm.
  • Without wishing to be bound by any particular theory, it is believed that a factor in controlling release of the active agent from the microparticles may be the type and/or degree of interaction and/or association(e.g., non-covalent association, ionic complexation) that occurs at or near the outer surface of the preformed microparticle (such as the interface with the formed monolayer), which may involve the active agent, the charged compound, and/or other components, if any. In some cases, a strong interaction or association at this interface slows down, delays, and/or otherwise hinders dissolution of the active agent, and is believed to stabilize the surface-modified microparticle and facilitate fabrication of additional alternatingly charged monolayers, if desired. In addition, as described in greater detail below, the interaction can be further affected by the subsequent formation of additional alternatingly charged monolayers.
  • Thus, turning briefly to FIG. 2, co-incubation of the preformed microparticles 10 and a charged compound 20, preferably in a solution, results in intermediate microparticles 40 with a single monolayer of the charged compound 20 formed on and associated with at least the outer surface of the preformed microparticle 10. Following incubation, the suspension of the intermediate microparticles 40 may then be separated from the solution through centrifugation, filtration, diafiltration, and/or other separation methods. The intermediate microparticles 40 are optionally washed with a washing solution (preferably an aqueous medium, such as the SRA-containing buffer described above, as generally shown in FIG. 1). The temperature during the incubation and the optional washing is optimized based on the solubility of the active agent and the charged compound 20.
  • If further surface modification is desired or required, after the optional washing, the intermediate microparticle 40 may be further exposed to (such as mixed with) a different charged compound 30, preferably in a solution, to form a subsequent monolayer of the charged compound 30 about and associated with the formed monolayer of the intermediate microparticle 40. Charged compound 30 preferably has a net electric charge opposite in sign to that of the charged compound 20. The subsequent monolayer may be formed immediately about the formed monolayer. The intermediate microparticle 50 may have a net surface electric charge that is same in sign as that of the intermediate microparticle 40, neutral or, preferably, opposite in sign to that of the intermediate microparticle 40. As shown in FIG. 1, the monolayer formation procedure may be repeated as in the previous cycle, to form microparticles 50 and 60 that have additional and, preferably but not necessarily, adjacent alternatingly charged monolayers, each associated with the preceding monolayer (FIG. 2). The total number of the monolayers to be formed may be selected or predetermined such that controlled release of the active agent with a desired release profile is achievable in the surface-modified microparticle. As set forth above, this number can be an integer of 1, 2, 3, 4, 5, 6, 7, or greater, preferably 100 or less, more preferably 20 or less, and most preferably 10 or less,
  • In another example, one or more of the charged compounds forming the monolayers may be active agent(s) identical to or different from the one in the preformed microparticle. For example, one or more of the odd numbered (e.g., first, third) monolayers may independently be formed of the same or different charged active agent(s), having net electric charge(s) opposite in sign to the net surface charge of the preformed microparticle. Alternatively or in combination, one or more of the even numbered (e.g., second, forth) monolayers may independently be formed of the same or different charged active agent(s), having net electric charge(s) same in sign as the net surface charge of the preformed microparticle. With reference to FIG. 2, charged compound 20 or 30 may be an active agent that is the same as or different from the one in the preformed microparticle 10, and charged compound 30 or 20, respectively, may be an otherwise inert charged compound or a charged active agent different from the one in the preformed microparticle.
  • In another example, one or more active agents, charged and/or uncharged, may be incorporated into one or more of the monolayers through covalent and/or non-covalent associations. Such monolayer-bound active agent(s) may be the same as that of the preformed microparticle, or different therefrom. Such a construction may allow controlled release (e.g., extended release, sustained release) of the monolayer-bound active agent(s). Alternatively or in combination, one of more of such monolayer-bound active agent(s) may be affinity molecules, such as targeting ligands, which may selectively bring the underlying microparticle to a predetermined region to achieve targeted delivery of the active agent within the core microparticle.
  • In a further example, the surface-modified microparticles described above having one or more monolayers of charged compounds, preferably in a suspension, may undergo one or more physical and/or chemical treatments to further modify one or more characteristics of the surface-modified microparticles, such as, but not limited to, the release profile of the active agent therein. The treatments may be carried out immediately after the formation of the surface-modified microparticles and prior to any optional washing, or immediately following the optional washings. The treatment may involve manipulation of one or more parameters of the reaction mixture, such as, without limitation, temperature, pH, and/or pressure. Typically the one or more parameters may be adjusted (such as increased or decreased) from an initial value to a second value and held for a period of time, and then adjusted (such as decreased or increased) to a third value or returned or allowed to return to the initial value and held for another period of time.
  • The thermal treatment, for example, may involve a heating stage and a cooling stage. Prior to the additional treatment, the suspension may be kept at a relatively low temperature below ambient temperature to at least minimize dissolution of the microparticles therein, preferably the temperature at which the surface-modified microparticles are formed, more preferably 2° C. to 10° C., such as 4° C. During the heating stage, the suspension may be heated to a temperature and incubated at this elevated temperature for an incubation period of 1 minute to 5 hours, preferably 15 minutes to 1 hour, such as 30 minutes. The elevated temperature may be higher than the relatively low temperature at which the suspension was kept prior to the additional treatment, and lower than a degradation temperature of the surface-modified microparticles in the suspension, preferably between 5° C. and 40° C., more preferably between 10° C. and 30° C. The heating stage may optionally be immediately followed with a cooling stage, during which the suspension may be chilled at a temperature, rapidly or gradually in a controlled manner and optionally incubated at this depressed temperature for an incubation period of 1 minute to 5 hours, preferably 15 minutes to 1 hour, such as 30 minutes. In one example, chilling is achieved by washing with a chilled washing solution. Alternatively, the suspension may be allowed to return to or close to its original temperature or to a selected temperature below the temperature to which the suspension was heated. The depressed temperature may be lower than the elevated temperature, and higher than a freezing temperature of the suspension, preferably at or below ambient temperature, optionally equal to or different from the relatively low temperature at which the suspension was kept prior to the additional treatment, more preferably 15° C. or lower, most preferably 10° C. or lower, such as 4° C. The resulting mixture may further undergo optional washings as described herein to yield additionally treated, surface-modified, microparticles.
  • Surface-modified microparticles suitable for the additional treatment described above include those formed from amorphous, solid, and homogenous preformed microparticles having 40% to less than 100%, or more typically 80% or greater, by weight, of an active agent as described herein. Non-limiting examples of suitable suspensions include microparticles (such as insulin microspheres) in a buffer, such as a PEG buffer containing 16% PEG, 0.7% NaCl, 67 nM Na acetate, and having a pH in the range of 5 to 8 (e.g., 5.7, 5.9, 6.5, 7.0). The microparticles may have a concentration in the buffer of 0.01 mg/ml to 50 mg/ml, preferably 0.1 mg/ml to 10 mg/ml, such as 1 mg/ml. A charged compound or a mixture of two or more thereof, such as protamine sulfate, poly-L-lysine, and/or poly-L-arginine, may be mixed into the suspension to provide a concentration of 0,01 mg/ml to 10 mg/ml, preferably 0.1 mg/ml to 1 mg/ml, such as 0.3 mg/ml. The mixture may be incubated at the relatively low temperature, such as 4° C., and under agitation for an incubation period of 10 seconds to 5 hours, such as 1 hour, to ensure the formation of a monolayer of the charged compound on the outer surface of each of the preformed microparticles. Then the suspension may be subjected to the thermal treatment as described above. Optional washings may be carried out on the suspension prior to the additional treatment.
  • The additional treatments may be carried out immediately after the formation of any one or more of the monolayers as disclosed herein. In one example, the additional treatment may be carried out immediately after the formation of a single monolayer on the preformed microparticles, the monolayer being formed of positively charged compounds or negatively charged compounds. When optionally one or more additional monolayers are formed on the first monolayer, the additional treatment may or may not be carried out immediately following the formation of such additional monolayers. In another example, two or more monolayers may be formed sequentially on the core microparticles, and the additional treatment may be carried out only immediately after a single predetermined monolayer (such as the last monolayer; the first monolayer, or any other monolayer there between) is formed. In a further example, two or more monolayers may be formed sequentially on the core microparticles, and the additional treatment may be carried out immediately after the formation of each and every monolayer having one or more predetermined characteristics, such as containing positively charged or negatively charged compounds, or containing a particular compound (e.g., active agent, affinity molecule, derivative) or moiety (e.g., functional group, label), or being a particular monolayer from the core (e.g., first, second, third, fourth, fifth). In a further example, the additional treatment may be carried out immediately after the formation of each monolayer of a predetermined set, which may be all of the monolayers or a subset thereof
  • As described in Example 16 below, the surface-modified microparticles following the additional treatment may display modifications in net surface charge (zeta potential) and/or release profile of the active agent therein. With certain charged compounds (such as PLL and PLA, but not protamine sulfate), a change (such as an increase) in the surface charge of the surface-modified microparticles may be observed. When subjected to the in vitro release protocol as disclosed herein, the additionally treated, surface-modified microparticles are capable of displaying a reduction in the 1-hour percentage of cumulative release (% CR1 h) of the active agent therein, as compared to the surface-modified microparticles without the additional treatment. Inasmuch as it is believed that the initial burst of the active agent release typically occurs within the first hour, the example demonstrates that the initial burst of the active agent release may be significantly reduced by the additional treatment. The same additionally treated, surface-modified microparticles may be capable of continued, preferably sustained, release beyond 1 hour, preferably beyond 24 hours, more preferably beyond 48 hours, most preferably beyond 7 days, having a 24-hour percentage of cumulative release (% CR24 hr) that is greater than % CR1 h. As a result of the additional treatment, the surface-modified microparticles of the present disclosure, when subjected to in vitro release in a release buffer (10 mM Tris, 0.05% Brij 35, 0.9% NaCl, pH 7.4, free of divalent cation) at 37° C., may be capable of displaying a % CR1 h of 50% or less and/or a ratio of % CR24 h to % CR1 h of greater than 1:1. The % CR1 h may preferably be 40% or less, more preferably 30% or less, further preferably 20% or less, most preferably 10% or less. The ratio of % CR24 h, to % CR1 h may preferably be 1.05:1 or greater, more preferably 1.1:1 or, greater, but not more than 10:1, preferably 5:1 or less, more preferably 2:1 or less, most preferably 1.5:1 or less.
  • Without being bound to any particular theory, it is believed that the additional treatment following the monolayer formation as disclosed herein allows the charged compound in the monolayer and the molecules (e.g., the active agent, the optional carrier molecules in the preformed microparticle, the charged compound in the preceding monolayer) that comprises the outer surface of the substrate (e.g., the preformed microparticle, the preceding monolayer) to rearrange and form an association that is much stronger than the electrostatic interaction between the monolayer and the outer surface of the substrate prior to the additional treatment. It is believed that through the additional treatment a modified shell is formed on the outer surface of the surface-modified microparticle, the modified shell containing a homogenous mixture of the charged compound and the molecules that form the outer surface of the substrate.
  • Deposition of additional alternatingly charged monolayers of charged compounds beyond the formed monolayer may further affect, among other things, the release profile of the active agent in the preformed microparticle. As previously described, depending on the attractive forces at the interface between the preformed microparticle and the formed monolayer; strong association between the two may be observed. This may result in retarding the quantity and/or rate of release of the active agent. The release profile may be further modified by forming one or more additional alternatingly charged monolayers about the formed monolayer. Without being restricted to any particular theory, it is believed that addition of a second oppositely charged monolayer may ease the association between the formed monolayer and the preformed microparticle, thereby enhancing the release of the active agent. Subsequent application of the alternatingly charged monolayers, arranged consecutively with optional interleaving layers of active agents, if desired, can allow fine-tuning of active agent release from the surface-modified microparticles, as shown in some of the examples disclosed herein.
  • Suitable charged compounds that may be used in accordance with the present invention may be charged compounds capable of associating with any substrate, preferably by, but not limited to non-covalent association and, more preferably, electrostatic interactions. Thus, suitable charged compounds include positively charged, negatively charged, or zwitterionic, and include, but are not limited to, polyelectrolytes, charged polyaminoacids, polysaccharides, polyionic polymers, ionomers, charged peptides, charged proteinaceous compounds, charged lipids optionally in combination with uncharged lipids, charged lipid structures such as liposomes, precursors and derivatives thereof, and combinations of two or mote thereof. Non-limiting examples include negatively charged polyelectrolytes such as polystyrene sulfonate (PSS) and polyacrylic acid (PAA), negatively charged polyaminoacids such as polyaspartic acid, polyglutamic acid, and alginic acid, negatively charged polysaccharides such as chondroitin sulfate and dextran sulfate, positively charged polyclectrolytes such as polyallyl amine hydrochloride (PAH) and poly(diallyldimethyl ammonium chloride (PDDA), positively charged polyaminoacids such as poly(L-lysine) hydrochloride, polyornithine hydrochloride, and polyarginine hydrochloride, and positively charged polysaccharides such as chitosan and chitosan sulfate. Also useful as charged compounds in the present invention are, without limitation, biocompatible polyionic polymers (e.g., ionomers, polycationic polymers such as polycationic polyurethanes, polyethers, polyesters, polyamides; polyanionic polymers such as polyanionic polyurethanes, polyethers, polyesters, polyamides), charged proteins (e.g., protamine, protamine sulfate, xanthan gum, human serum albumin, zein, ubiquitins, and gelatins A & B), and charged lipids (e.g., phosphatidyl choline, phosphatidyl serine). Also included are derivatives (e.g., glycosylated, hyperglycosylated, PEGylated, FITC-labeled, salts thereof), conjugates, and complexes of the charged compound disclosed herein. More specifically, suitable positively charged lipids (that is, polyanionic lipids), negatively charged lipids (that is, polycationic lipids), and zwitterionic lipids include 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(carboxyfluorescein) (FITC-EA), 1,2-distaeroyl-sn-glycero-3[phospho-rac-O(1-glycerol)](sodium salt) (DSPG), 1,2-dipalmitoyl-sn-glyceuo-3-phosphate (monosodium salt) (DPPA) 1,2-dioleoyl-3-dimethylammonium-propane (D
  • Furthermore, lipid structures (such as liposomes) can be used in alternate deposition with charged compounds. Uncharged (such as non-ionic) lipids may be used in combination with electrically charged lipids to form one or more of the monolayers, and the molar ratio there between can be optimized to achieve minimum permeability of the active agent through the monolayer.
  • The surface-modified microparticle disclosed herein, typically containing a preformed microparticle and one or more monolayers, preferably has a release profile of the active agent that is different from that of the core microparticle. Non-limiting examples of the differences in release profile include a reduction in the initial burst, an extension of release time, a display of linear/constant release over a time period, and/or a reduction in rate of release over a prolonged time period. The surface-modified microparticles may be present, preferably in a functionally (e.g., therapeutically, pharmaceutically, diagnostically) effective amount, as a suspension or dry powder in a liquid or solid composition or formulation, in the presence or absence of one or more of preservatives, isotonicity agents, pharmaceutically acceptable carriers, and stabilizing agents. Such compositions and formulations may be administered in an effective amount to a subject for prevention or treatment of a condition or state, or as a nutritional supplement, or for the purpose of physical enhancement or psychological well-being. Such compositions and formulations may be incorporated into a diagnostic method, tool, or kit for in vitro and/or in vivo detection of a substance, condition, or disorder being present or absent, or a disposition for such a condition or disorder. For example, the substance, upon contact, may form an association (e.g., conjugate, complex) with the surface-modified microparticle or a portion thereof (such as the core microparticle), which is capable of providing one or more signals for detection. The one or more signals may be one or more moieties labeled on one or more portions of the association (e.g., the substance, the microparticles), or may be elicited upon the formation of the association (e.g., emission of light, discharge of another substance). Additionally, the surface-modified microparticles may be incorporated into a nutritional and/or dietary supplement or a food composition, or used as a food additive, for prevention and/or treatment of a condition or disorder in a subject.
  • Set forth below are analytical methods and several examples of surface-modified microparticles fabricated in accordance with the present disclosure. All of the charged monolayers formed in the examples are believed to be saturated monolayers as described herein. Readings and measurements reported were recorded using instruments and methods described below.
  • Quartz Crystal Microbalance (QCM) Measurements
  • The QCM method was used to confirm the presence of layer-by-layer assembly of electrically charged compounds in the presence of an SRA-containing solution. Precursor films of multiple (e.g., 2, 3, or 4) PAH/PSS bilayers (i.e., each bilayer includes a PAH monolayer immediately adjacent to a PSS monolayer) were deposited on 9 MHz silver resonators of a QCM (JJSI QCM System, Model 260303, Sanwa Tsusho Co., Ltd, Japan). To form each monolayer, the resonators were incubated in 0.25 M NaCl aqueous buffer with a charged compound concentration of 1.5 mg/mL, at room temperature for 15 minutes, washed three times with deionized (DI) water, and dried. Instead of the aqueous buffer, the monolayers can also be formed using DI water solution having a charged compound concentration of 3 mg/mL.
  • To form each monolayer of the electrically charged compound of interest on the precursor film, the coated resonators were further incubated in aqueous buffers containing the respective charged compounds at +2° C. for about 1 hour, followed by washings with DI water. For polyelectrolytes and charged proteins, the concentration of the charged compound in the buffer was in a range of 0.1 mg/mL to 3 mg/mL, preferably 1 mg/mL. For charged lipids, the concentration of the charged compound in the buffer (suspension) was 0.1 mg/mL to 3 mg/mL, preferably 0.25 mg/mL to 1 mg/mL. Different buffers were used, including (expressed in w/v percentages): a) 16% PEG-0.7% NaCl, pH 5.8; b) 16% PEG-0.7% NaCl pH 7.0; c)0.16% acetic acid-0.026% ZnCl2. Concentrations of PEG, NaCl and ZnCl2 in the buffers for assembly may be varied for optimization.
  • The monolayers, after forming, were dried in a stream of nitrogen gas. The frequency change of the resonators following formation of each monolayer was monitored and converted into thickness as understood by one skilled in the art, the results of which are shown in FIG. 9,
  • Microparticle Net Surface Charge Measurements
  • For microparticle net surface charge (zeta potential) measurements a Zeta Potential Analyzer (Model ZetaPALS, Brookhaven Instruments Corp., Holtsville, N.Y.) was used A 40 μL aliquot of each sample under investigation was added to 1.5 mL of the corresponding salt-free PEG solution, mixed, and the resulting suspension was immediately used for the measurements. The temperature of the suspension was equilibrated to 8° C. to minimize microparticle disintegration.
  • In Vitro Release (IVR)
  • To generate the IVR profile of the active agent (such as insulin), a 10 mL aliquot of a releasing buffer (10 mM Tris, 0.05% Brij 35, 0.9% NaCl, pH 7.4) was added into a glass vial containing 0.5 mL of the concentrated particle suspension (equivalent to 3 mg of insulin), mixed, and incubated at 37° C. At designated time intervals 400 μL of the IVR medium was transferred into a microfuge tube and centrifuged for 2 minutes at 1.3 k rpm. A 300 μL aliquot of the supernatant was removed and stored at −80° C. until analyzed by Bicinchoninic Acid (BCA) assay as understood by one skilled in the art. A 300 μL aliquot of flesh releasing buffer was added to the microfuge tube to reconstitute the pellet. The 400 μL suspension was transferred back to the IVR. Total active agent content of the microparticle was determined by BCA assay after complete dissolution of the microparticle in an aqueous alkaline solution containing dimetbyl sulfoxide (DMSO) and a surfactant and pH neutralization.
  • EXAMPLES Example 1A Microspheres with Polystyrene Sulfonate Monolayer
  • Insulin microspheres formed using a phase separation method disclosed herein (i.e., preformed microparticles) were incubated in an aqueous solution of 16% (w/v) PEG and 0.7% (w/v) NaCl in the presence of 0.3 mg/mL polyion PSS at 2° C. and pH 4.8 for 1 hr. To remove the unassociated PSS centrifugal washing (at 3000 rpm for 15 minutes) was applied twice, each using the initial volume of the aqueous solution described above as the washing medium to re-suspend the microspheres. Comparison of zeta-potential values of the unmodified and modified microspheres confirms the formation of the PSS monolayer (FIG. 3).
  • Example 1B Microspheres with Multiple Alternating Monolayers of Polystyrene Sulfonate and Polyallylamine Hydrochloride
  • The PSS-modified microparticles of Example 1A were used as intermediate microparticles to form a subsequent monolayer of polycation PAH, Similar formation and washing procedures were used as described in Example 1A, except that PAH was substituted for PSS at the same concentration. The procedures were repeated to form desired number of alternating monolayers, FIG. 3 illustrates the zeta-potential values of four consecutive depositions of the PSS/PAH bilayer assembly after formation of each monolayer.
  • Example 2A Microspheres Surface-Modified at a pH Below the Surface-Neutral Point of the Microspheres
  • The procedures of Example 1A were used to form a polyanion monolayer on insulin microspheres at pH 418 (below surface-neutral point of the microspheres, which was observed to be about 5.6) FIG. 4 depicts zeta-potential values of insulin microspheres with a monolayer of polyacrylic acid (model polyanion), dextran sufate, polyaspartic acid, polyglutamic acid, and alginate. The zeta-potential of the preformed insulin microspheres at pH of 4.8 showed a positive net surface charge. Following the formation of the respective polyanion monolayers, the net surface charges of the resulting surface-modified microspheres were negative.
  • Example 2B Microspheres Surface-Modified at a pH Above the Surface-Neutral Point of the Microspheres
  • The procedures of Examples 1A & 1B were used to form a polycation monolayer on insulin microspheres at pH 7.0 (above surface-neutral point of the microspheres). FIG. 5 depicts zeta-potential values of insulin microspheres with a monolayer of polydiallyldimethylammonium chloride (PDDA, model polycation), protamine sulfate (ProtS), poly-l-arginine (PLA), and poly-l-lysine (PLL). The zeta-potential of the preformed insulin microspheres at pH of 7.0 showed a negative net surface charge. Following the formation of the respective polycation monolayers, the net surface charges of the resulting surface-modified microspheres were positive LSC micrograph of insulin microspheres with FITC-labeled protamine monolayer, as shown in FIG. 6, confirmed formation of the polycation monolayer.
  • Example 3A Microspheres with Multiple Monolayers of Oppositely Charged Polyions
  • The resulting microspheres of Examples 1A & 2A were used as intermediate microspheres, re-suspended in the aqueous solution (16% PEG, 0.7% NaCl, pH 4.8) in the presence of 0.3 mg/mL. polycation PLL, and incubated for 1 hr at 2° C. to form a subsequent monolayer of PLL over the formed polyanion monolayer. Net surface charge reversal of the microspheres, shown in FIG. 7, confirmed formation of the polyion monolayers. LSC micrograph of insulin microspheres with a formed PSS monolayer and a subsequent FITC-labeled PLL monolayer, shown in FIG. 8, demonstrated surface deposition of the polycation PLL.
  • A QCM as described above was used to measure the thickness of each polyion monolayer. The reaction medium contained PLL or chondroitin sulfate at 1 mg/mL in 16% PEG, 0.7% NaCl The film assembly was constructed by consecutive incubation of the QCM resonators in the reaction media containing the polyions for 15 minutes each, followed intermediately with DI water washes and drying with nitrogen stream. FIG. 9 illustrates the progressive film thickness following formation of each monolayer. Depending on the polyion, each monolayer was estimated to increase the total thickness by about 1 nm or less, with an averaged increase of about 0.5 nm.
  • Example 3B Microspheres with Multiple Monolayers of Oppositely Charged Biocompatible Polyions
  • Condroitin sulfate and gelatin A were used to form multiple monolayers of oppositely charged polyions about preformed insulin microspheres, in an aqueous solution of 16% PEG & 0.7% NaCl, at pH 4,8 and 2° C. Formation of the condroitin sulfate monolayer, according to the procedures of Example 1A, was followed by subsequent formation of the gelatin A monolayer. The procedures were repeated to form a total of 6 alternatively charged monolayers. Reversal of net surface charge of the microspheres following formation of each monolayer is shown in FIG. 10.
  • Example 4A Monolayer Formation in the Presence of Polyvalent Cation and PEG
  • The procedures of Example 1B were used to form monolayers of protamine and condroitin sulfate about preformed insulin microspheres, with the exception that the aqueous solution had a pH1 of 6.4 and contained 16% PEG, 0.7% NaCl, 0.16% (w/v) acetic acid, and 0.026% (w/v) ZnCl2. Comparative examples were formed using a Zn-free & acetate-flee aqueous solution with a pH of 6.4 containing 16% PEG and 0 7% NaCl. Zeta-potentials of the resulting microspheres are shown in FIG. 11
  • Example 4B Monolayer Formation in the Presence of Polyvalent Cation and Absence of PEG
  • The procedures of Example 1B were used to form monolayers of protamine and condroitin sulfate about preformed insulin microspheres, with the exception that the aqueous solution was PEG-free, had a pH of 7.0, and contained 0.7% NaC], 0.16% acetic acid, and 0.026% ZnCl12 Zeta-potentials of the resulting microspheres are shown in FIG. 12.
  • Example 5 Microspheres Surface-modified with Liposomes
  • Liposomes containing 60% cationic lipid 1,2-dioleoyl-3-dimethylanimonium-piopane (DAP) and 20% zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), suspended in an aqueous solution of 16% PEG, 0.7% NaCl, 0.16% acetic acid and 0,026% ZnCl2 at pH 7.0 were used to co-incubate with preformed insulin microspheres (pre-washed with the same aqueous solution) at 2° C. for 1 hr. The procedures of Example 41B were applied to form a subsequent monolayer of chondroitin sulfate.
  • Alternatively, liposomes containing anionic 1,2-distearoyl-sn-glycero-3[phosphor-rac-(1-glycero)]) (DSPG, sodium salt), DOPC and cholestrol were deposited on protamine-modified insulin microspheres, using the procedures of Example 4B. FIG. 13 illustrates LSC micrographs of microspheres containing Rodamine B-labeled protamine monolayer (top right), microspheres containing FITC-labeled DAP (top left), and microspheres containing both (bottom left), Zeta-potential values of the microspheres following each deposition are shown in FIG. 14.
  • Example 6 Sustained Release of Insulin from Protamine-Modified Microspheres
  • Protamine-modified insulin microspheres were formed using the procedures of Example 21B at various concentrations of the polyion in the reaction medium The IVR profiles of resulting protamine-modified insulin microspheres are shown in FIG. 15. An increase in concentration of polyion reduced the initial burst and subsequent release rate of insulin from the surface-modified microspheres.
  • Example 7 Release Modification with Multiple Monolayers
  • The resulting microspheres of Example 2B were used as intermediate microparticles, about which carboxymethyl cellulose (CMC) was deposited at various concentrations in the reaction medium. The IVR profiles shown in FIG. 16 demonstrated the capability of the subsequent monolayer in further modifying the release of active agents from the surface-modified microspheres. Deposition of an additional protamine monolayer was able to partially or fully restore the release profile (FIG. 17).
  • Example 8 In Vivo Release of Insulin from Protamine-Modified Insulin Microspheres
  • In vivo release of insulin from protamine-modified insulin microspheres was investigated in chemically induced Sprague-Dawley rats. The surface-modified microspheres prepared according to Example 2B were administered as a suspension in 16% PEG 3350, pH 7.0. The preformed insulin microspheres free of surface modification were administered in the PEG solution, or in phosphate-buffered saline, pH 7,4, as control. The animals received an initial subcutaneous dose of 1 IU/kg of the microspheres. An ELISA assay was used to determine the recombinant human insulin (rhINS) serum levels in the collected samples. The results, as illustrated in Table 1 and FIGS. 18A and 18B, demonstrate the significant effect of the surface-modification on pharmacokinetics of the administered dose. Specifically, the surface-modification increased the maximum serum concentration of rhINS (Cmax) and the time to achieve Cmax (tmax), as well as the area under the rhINS concentration-time curve (AUC) and the mean residence time (MRT) of the protein. The serum glucose depression (FIG. 18B) also was in agreement with the corresponding serum rhINS. As shown below, the increase in Cmax greater in the surface modified microparticles as compared to Cmax and tmax of the unmodified, preformed microparticles. As demonstrated by this Example, the Cmax of the surface modified microparticle was 2.5 fold greater than the Cmax of the preformed microparticle. In other examples, the Cmax of the surface modified microparticle may be increased 1.1 fold or greater, 1.25 fold or greater, 1.5 fold or greater, 2.0 fold or greater than the Cmax of the preformed microparticle.
    TABLE 1
    Parameter Preformed Microparticles Modified Microparticles
    AUC0-7 h 203.3 ± 46.5  780.9 ± 81.3 
    MRT0-7 h 1.7 ± 0.2 2.9 ± 0.2
    Cmax 103.5 ± 27.3  259.0 ± 52.9 
    tmax 0.55 ± 0.41 2.60 ± 0.55
  • Example 9 Surface Modification in Presence of Various Solubility Reducing Agents
  • Aqueous media of protamine sulfate (0.15 mg/mL) used to incubate the preformed insulin microparticles contained one of PLURONICOR F-68 or F-127 (10% or 16% w/v), glycerol (20%, 40%, or 60% v/v), and ethanol (10% v/v). Procedures as described in Example 1A were followed. Zeta-potential values of the microspheres before and after surface modification, shown in FIG. 19, indicated formation of protamine monolayer.
  • Example 10 Effect of Concentration of Charged Compound on Release Profile of Surface-Modified Microspheres
  • Procedures of Example 1A were followed, in which the concentration of protamine sulfate was varied in a range of 0.1 mg/mL to 1.5 mg/mL. FIG. 20A illustrates the relationship between zeta-potential of the microspheres and the cumulative release of insulin at 48 hrs. An increase in protamine concentration in the reaction mediumeorrelated with reduction of insulin release, with an observed maximum effective concentration of about 0.3 mg/mL.
  • The protamine-modified microspheres prepared at a protamine concentration of 1.5 mg/mL were further modified with polyanion carboxymethyl cellulose or chondroitin sulfate in the concentration range of 0.05-1.2 mg/mL or 0.1-1.2 mg/mL, respectively Formation of the subsequent monolayer significantly reversed the release reduction effect of the protamine monolayer, as shown in FIGS. 20B and 20C. The results suggested the ability of a few monolayers in fine-adjusting the release profile of the microparticles in a controlled manner.
  • Example 11 Surface Modification of hGH Microspheres
  • Preformed hGH microspheres were incubated, in alternating sequence, in aqueous media (16% PEG 3350, 0.7% NaCl, pH 6.0) containing 0.3 mg/mL prolamine sulfate and chondroitin sulfate, respectively, at 2° C. for 1 hr each, to form the alternatingly charged monolayers. FIG. 21A depicts the zeta-potential of the microspheres after deposition of each monolayer. The IVR profiles of surface-modified hGH microspheres with one, two, or three monolayers are compared with that of the unmodified preformed hGH microspheres in FIG. 21B.
  • Example 12 Surface Modification of Microspheres of Intravenous Immunoglobulin
  • Preformed intravenous immunoglobulin (IVIG) microspheres were modified with alternating monolayers of chondroitin sulfate and protamine sulfate. For each monolayer, the incubation was carried out in a pH 7.0 aqueous medium containing 12.5% PEG 8000, 50 mM ammonium acetate, and 0.15 mg/mL of the respective polyion at 4° C. for 1 hr. Centrifugal washing was used to remove excess polyions. FIG. 22 depicts the zeta-potential of the microspheres after deposition of each monolayer.
  • Example 13 Surface Charge Characteristics of Microspheres in Aqueous PEG Media
  • In order to determine surface charge characteristics of preformed insulin microspheres in solubility reducing media containing 16% PEG, pH of tie media was adjusted in a range of 4-7.5. Zeta-potential of the microspheres were determined in each medium, and plotted versus the corresponding pH, as shown in FIG. 23. The surface-neutral point for the preformed insulin microspheres was estimated to be 5.6. As the pH of the medium decreased below or increased above the surface-neutral point, the net surface charge of the preformed insulin microspheres became more and more positive or negative, respectively.
  • Example 14 Effect of Reaction pH on Zeta Potential and Release Profile of Surface-Modified Microspheres
  • At 4° C., insulin microspheres (20 mg) formed using a phase separation method disclosed herein were suspended in 19 ml of a buffer [containing 16% (w/v) PEG, 0.7% (w/v) NaCl, and 67 mM sodium acetate] at one of the following pH values: 5.7, 5.9, 6.5, and 7.0. Zeta potential of the unmodified microspheres in the buffer of different pH was measured as described herein above. Protamine sulfate, poly-l-lysine, or poly-l-arginine was added to the suspension as 1 ml of a 6 mg/ml solution in the same buffer at the same pH as that of the suspension. The resulting reaction mixtures each had a microsphere concentration of 1 mg/ml and a polycation concentration of 0.3 mg/ml. The reaction mixtures were incubated at 4° C. for one hour and then centrifugally washed (3000 rpm for 15 minutes) three times with 20 ml fresh aliquots of the buffer at the respective pH values of the reaction mixtures. Zeta potentials of the resulting surface-modified microspheres in the resuspensions of the last washing were measured as described above. The surface-modified microspheres were then subjected to in vitro release following the protocol disclosed herein.
  • As shown in FIG. 25, the formation of the polycation monolayer qualitatively reversed (from negative to positive) the surface charge of insulin microspheres at the different reaction pH values described above. The zeta potential of the surface-modified microspheres and the magnitude of the charge reversal appeared to depend at least in part on the reaction pH and/or the polycation. Specifically, the zeta potential of the PLL-modified insulin microspheres was higher (in a general range of 15 mV or greater, such as about 20 mV) at reaction pH values (e.g., 5.7, 5.9) close to the surface-neural point of the unmodified insulin microspheres (insulin SNPcore, about 5.6), and lower (in a general range of less than 15 mV, such as about 8 mV) at reaction pH values (e.g., 6.5, 7.0) away from insulin SNPcore. The magnitude (about 30 mV) of the charge reversal following the formation of the PLL monolayer was the same across the different reaction pH values specified above.
  • The zeta potential of the PLA-modified insulin microspheres was higher (above 20 mV) at reaction pH values close to insulin SNPcore, and lower (below 20 mV) at reaction pH values away from insulin SNPcore. The magnitude of the charge reversal following the formation of the PLA monolayer was less (about 30 mV) at reaction pH values close to insulin SNPcore, and greater (about 40 mV) at reaction pH values away from insulin SNPcore. The zeta potential (about 20 mV) of the ProtS-modified insulin microspheres was the same across the different reaction pH values specified above. The magnitude of the charge reversal following the formation of the protamine sulfate monolayer was less (about 30 mV or less) at reaction pH values close to insulin SNPcore, and greater (about 40 mV or greater) at reaction pH values away from insulin SNPcore.
  • As shown in FIG. 26, the in vitro 1-hour percentage of cumulative release (% CR1 h) of insulin from the surface-modified insulin microspheres was affected by the reaction pH and/or the polycation used in the surface modification reaction. Specifically, insulin % CR1 h was generally greater at reaction pH values close to insulin SNPcore, than that at reaction pH values away from insulin SNPcore, with the difference there between ranging from greater than 5% to 10% to 20% to less than 30%. At the same reaction pH, PLA-modified microspheres and ProtS-modified microspheres had comparable insulin % CR1 h, the level of which was less than that of PLL-modified microspheres, with the differences there between ranging generally from 20% to 30% or more.
  • In cases where it may be desired to have a % CR1 h, of less than 50%, preferably 40% or less, more preferably 30% or less, most preferably 20% or less, the preformed microparticles of the present disclosure (such as the unmodified insulin microspheres) may be surface-modified at reaction pH away from SNPcore using certain charged compounds (e.g., protamine sulfate, PLA) In cases where it may be desired to have a % CR1 h, of 50% or greater, preferably 60% or greater, more preferably 70% or greater, most preferably 75% or greater, the preformed microparticles of the present disclosure (such as the unmodified insulin microspheres) may be surface-modified at reaction pH close to SNPcore using certain charged compounds (such as PLL).
  • Example 15 Surface-Modified Nucleic Acid Microspheres
  • Nucleic acid microspheres were formed according to the disclosures of U.S. Patent Application Publication Nos. 2006-0018971 and 2006-0024240, the entirety of which are incorporated herein by express reference thereto. Each of the microspheres had a homogenous mixture containing at least 80% (such as 85% to 90%) by weight of a CD40 siRNA and 15% or less (such as 6% to 10%) by weight of poly-l-lysine. The nucleic acid microspheres were suspended in 100 μl of nuclease-free deionized water (pH 7.0) containing 1 mg/ml Rodamine B-labeled PLL (70 kD). The suspension was incubated with agitation at 4° C. for 45 minutes to form the surface-modified microspheres, which were then centrifugally washed with nuclease-flee deionized water (pH 7.0). The zeta-potentials of the unmodified microspheres and the surface-modified microspheres were measured to be −24 mV and 34 mV, respectively. Clearly, surface modification of the nucleic acid microspheres through formation of a polycation monolayer is capable of reversing their surface electric charge. Laser scanning confocal micrograph of Rodamine B-labeled PLL is shown in FIG. 27, confirming successful formation of the monolayer on the outer surface of the nucleic acid microspheres.
  • Example 16 Thermal Treatment of Surface-Modified Microparticles
  • Preformed, unmodified insulin microspheres (12 mg), such as those formed from controlled phase separation as disclosed herein, were suspended in 1.5 ml of a buffer containing 16% (w/v) PEG, 0.7% (w/v) NaCl, and 67 mM sodium acetate at pH 7.0 and 4° C. At a concentration of 6 mg/ml, a 1.5 ml aqueous solution of a polycation (ProtS, PLL, or PLA) dissolved in the same buffer was mixed with the suspension, resulting in reaction mixtures having a microsphere concentration of 4 mg/ml and a polycation concentration of 3 mg/ml. The reaction mixtures were incubated at 4° C. with continuous agitation for 30 minutes to form the surface-modified insulin microspheres having the respective polycation monolayer. Next, the reaction mixtures were further incubated for another 30 minutes at a temperature of 4° C., 15° C., 28° C., or 37° C. Then the thermally-treated insulin microspheres were centrifugally (3000 rpm at 4° C. for 10 minutes) collected and washed three times with fresh aliquots of the buffer at pH 7.0 and 4° C. Zeta potential data and in vitro release profiles of the thermally-treated, surface-modified insulin microspheres were generated as described herein above.
  • It was found, unexpectedly, that certain thermal treatments as described above (such as incubation at a temperature of 15° C., 28° C., or 37° C., but not limited thereto) were capable of selectively and differentially altering certain characteristics (such as zeta potential and release profile) of the surface-modified microparticles without adversely affecting other properties thereof (such as particle size, extended release phase). As illustrated in FIG. 28, the incubation at different elevated temperatures as described above results in different levels of initial insulin release from the polycation-modified insulin microspheres (insulin % CR1 h being lower following incubation at 28° C. than that following incubation at 15° C.), which are consistently less than that following incubation at non-elevated temperature (i.e., 4° C.).
  • As exemplified in Table 2, the initial release or “burst” phase in the in vitro release profiles, as represented by % CR1 h, of the thermally-treated, surface-modified microparticles were reduced as compared to that of the surface-modified microparticles that did not undergo the thermal treatment (such as those incubated at a second temperature of 4° C.). The amount of reduction in % CR1 h, of the active agent released in vitro from the thermally-treated, surface-modified microparticles, with respect to the control, can be 10% or greater, such as 15% or greater, 25% or greater, or 40% or greater,
    TABLE 2
    Percentage of Reduction in % CR1 h of PLL-modified Insulin Microspheres
    Sample (2nd % Reduction (in % CR1 h as compared to
    Incubation Temp) % CR1 h Control)
    Control (4° C.) 57.4
    Thermal 1 (15° C.) 48.3 16
    Thermal 2 (28° C.) 33.1 42
    Thermal 3 (37° C.) 34.1 40
  • The reduction effect of the thermal treatment (at 28° C.) on the initial release phase of the in vitro release of insulin was consistently observed in the polycations tested above. Unexpectedly, different polycations exert different levels of reduction on the initial release of insulin. As illustrated in FIGS. 26 and 28, and summarized in Table 3, the reduction effect observed in PLA-modified insulin microspheres of the present disclosure is greater than that in PLL-modified insulin microspheres, while the reduction effect observed in ProtS-modified insulin microspheres is less than that in PLL-modified insulin microspheres,
    TABLE 3
    Effect of Thermal Treatment (at 28° C.) on % CR1 h of Surface-modified
    Insulin Microspheres
    % CR1 h without % CR1 h with Thermal
    Thermal Treatment Treatment (Example % Reduction in
    Polycation (Example 14) 16) % CR1 h
    PLL 50.1 28.4 43
    ProtS 28.9 15.1 48
    PLA 19.5 5.1 74
  • Example 17 Thermal Treatment of Surface-Modified Microparticles
  • Preformed, unmodified hGH microspheres, such as those formed from controlled phase separation as disclosed herein, underwent the same thermal treatments as described in Example 16. Zeta potential data and in vitro release profiles of the thermally-treated, surface-modified hGH microspheres were generated as described herein above. As exemplified in Table 4, % CR1 h, of the surface-modified hOH microspheres following incubation at 28° C. was reduced as compared to that of the surface-modified hGH-microspheres following incubation at 4° C.
    TABLE 4
    Percentage of Reduction in % CR1 h of PLA-modified hGH Microspheres
    Sample (2nd % Reduction (in % CR1 h as compared to
    Incubation Temp) % CR1 h Control)
    Control (4° C.) 24.9
    Thermal 1 (15° C.) 10.8 57
    Thermal 2 (28° C.) 9.6 61
    Thermal 3 (37° C.) 13.0 48
  • Example 18 Effect of Thermally-Treated, Surface-Modified Microparticles In Vivo
  • Unmodified insulin microspheres were prepared using the controlled phase separation method disclosed herein. Two portions of the unmodified insulin microspheres were surface-modified with PLA, with one portion thermally-treated (at 28° C), and the other portion incubated at 4° C. as control, according to the procedures described in Example 16. Injectable compositions each containing one of the three different insulin microspheres suspended in a buffer [16% (w/v) PEG, 0.7% (w/v) NaCl, at pH 7.0] were prepared. The compositions were administered subcutaneously to normal Spague-Dawley rats at a dose of 4 IU/kg. An ELISA assay was used to determine the insulin serum levels in the collected samples. The results are illustrated in Table 5 and FIGS. 29A and 29B. As shown in FIG. 29A, CR1 h was likewise reduced following heat treatment.
  • PLA-modified insulin microspheres treated at 4° C. provided comparable serum insulin concentration profile, serum glucose depression profile, Cmax, and to Imax comparison, PLA-modified insulin microspheres treated at 28° C. provided flattened and right-shifted serum insulin concentration profile, right-shifted serum glucose depression profile, depressed Cmax, and prolonged tmax.
    TABLE 5
    In Vivo Insulin Release from Different Insulin Microspheres
    PLA-
    Parameter Unmodified PLA-modified, 4° C. modified, 28° C.
    Cmax 479.1 ± 147.0 463.5 ± 136.9 256.8 ± 95.8 
    tmax 1.1 ± 0.5 1.1 ± 0.5 2.0 ± 0.7
  • FIG. 29A is a graph showing the serum insulin concentration versus time profiles in rats that have received a single subcutaneous injection of uncoated insulin microspheres, PLA-modified insulin microspheres treated at 28° C., or PLA-modified insulin microspheres treated at 4° C. (Example 18);
  • FIG. 29B is a graph showing the serum glucose depression versus time profiles of rats treated with single subcutaneous injection of uncoated insulin microspheres, PLA-modified insulin microspheres treated at 28° C., or PLA-modified insulin microspheres treated at 4° C. (Example 18);
  • It is to be understood that the embodiments disclosed herein are merely exemplary of aspects of the disclosure, which may be embodied in various different forms. Therefore, specific details and preferred embodiments disclosed herein are not to be interpreted as limiting, but merely as a basis for the claims and as a representative basis for teaching one skilled in the art to variously employ the subject matter disclosed herein in any appropriate manner. The embodiments which have been described are illustrative of some of the applications of the principles of the present disclosure, and modifications may be made, including those combinations of features that are individually disclosed or claimed herein.

Claims (55)

  1. 1. A method for preparing a surface-modified microparticle, comprising:
    providing a solid and amorphous preformed microparticle comprising at least one active agent, said microparticle having an outer surface carrying a net surface charge;
    exposing at least said outer surface of said preformed microparticle to at least one charged compound having a net charge that is opposite in sign to said net surface charge; and
    forming a monolayer comprising said at least one charged compound, whereby said formed monolayer is associated with said outer surface,
  2. 2. The method of claim 1, wherein said formed monolayer has an outer surface carrying a net surface charge that is different from that of said preformed microparticle.
  3. 3. The method of claim 1, wherein said outer surface of said preformed microparticle consists substantially of said at least one active agent.
  4. 4. The method of claim 2, wherein said formed monolayer is a saturated monolayer and said net surface charge is opposite in sign to that of said preformed microparticle.
  5. 5. The method of claim 1, wherein said preformed microparticle comprises, by weight, approximately 40% to less than 100% of said at least one active agent distributed substantially homogeneously throughout said preformed microparticle.
  6. 6. The method of claim 1 wherein said preformed microparticle comprises, by weight, 80% or greater of said at least one active agent distributed substantially homogenously throughout said preformed microparticle.
  7. 7. The method of claim 1, wherein said exposing further comprises:
    providing a solution comprising said at least one charged compound and one or more of water, a buffer, and a water-miscible organic solvent and one or more solubility reducing agents; and
    contacting said preformed microparticle in said solution.
  8. 8. The method of claim 1, wherein said net surface charge is contributed substantially by said at least one active agent.
  9. 9. The method of claim 7, wherein said one or more solubility reducing agents comprise one or more of alcohols, carbohydrates, non-ionic aqueous-miscible polymers, and inorganic ionic compounds comprising polyvalent cations.
  10. 10. The method of claim 7, wherein said solution comprises, in weight-to-volume percentage, 16% polyethylene glycol and 0.7% sodium chloride and has a pH of between 4and 10.
  11. 11. The method of claim 7, wherein said solution has a pH that differs from a surface-neutral point of said preformed microparticle by 0 to less than 0.3.
  12. 12. The method of claim 7, wherein said solution has a pH that differs from a surface-neutral point of said preformed microparticle by 0.3 or greater.
  13. 13. The method of claim 1, wherein said exposing is carried out at a temperature between 2° C. and 5° C.
  14. 14. The method of claim 1, further comprising carrying out one or more treatments to the surface-modified microparticle, the treatments comprising manipulations of temperature, pressure, pH or a combination thereof.
  15. 15. The method of claim 14, wherein the treatment comprises heating a suspension including the microparticle at an elevated temperature.
  16. 16. The method of claim 15 further comprising allowing said suspension to arrive at a depressed temperature that is lower than said elevated temperature.
  17. 17. The method of claim 7, further comprising separating said surface-modified microparticle from said solution.
  18. 18. The method of claim 1, wherein said at least one charged compound comprises one or more of polyelectrolytes, charged polyaminoacids, charged polysaccharides, polyionic polymers, charged peptides, charged proteinaceous compounds, charged lipids optionally in combination with uncharged lipids, charged lipid structures, and derivatives thereof.
  19. 19. The method of claim 1, further comprising:
    exposing at least said outer surface of a formed monolayer to at least one different charged compound having a net charge that is opposite in sign to said net surface charge of said formed monolayer; and
    forming a subsequent monolayer comprising said at least one different charged compound, wherein said subsequent monolayer is associated with said formed monolayer
  20. 20. The method of claim 19, wherein said subsequent monolayer has an outer surface carrying a net surface charge that is opposite in sign to that of said formed monolayer.
  21. 21. The method of claim 20, further comprising forming one to five additional alternatingly charged monolayers.
  22. 22. The method of claim 20, further comprising forming an odd number of additional alternatingly charged monolayers.
  23. 23. The method of claim 1, further comprising forming said surface-modified microparticle capable of controlled release of said at least one active agent.
  24. 24. The method of claim 23, wherein said controlled release comprises an initial burst and a substantially linear release profile.
  25. 25. The method of claim 1, wherein said preformed microparticle is spherical.
  26. 26. The microparticle of claim 1 wherein said at least one active agent is a proteinaceous compound.
  27. 27. The method of claim 1, wherein said preformed microparticle is free of covalent crosslinking and free of hydrogel.
  28. 28. The method of claim 1, wherein said preformed microparticle is free of lipids and free of encapsulation.
  29. 29. A method for preparing a surface-modified microparticle, comprising:
    providing a liquid continuous phase system comprising at least one solvent, at least one active agent, at least one phase-separation enhancing agent;
    inducing a phase change at optionally a controlled rate in said system to cause a liquid-solid phase separation;
    forming a solid phase that comprises an amorphous and solid microparticle comprising said at least one active agent and having an outer surface carrying a net surface charge, and a liquid phase that comprises said solvent and said at least one phase-separation enhancing agents;
    exposing at least said outer surface of said formed microparticle to at least one charged compound having a net charge that is opposite in sign to said net surface charge; and
    forming a monolayer comprising said at least one charged compound, wherein said formed monolayer is associated with said outer surface of said formed microparticle.
  30. 30. The method of claim 29, wherein said method is free of washing said formed microparticle prior to said exposing.
  31. 31. The method of claim 29, further comprising washing said formed microparticle in the presence of at least one phase-separation enhancing agents prior to said exposing.
  32. 32. The method of claim 29, wherein said exposing is carried out in the presence of at least one phase separation enhancing agents.
  33. 33. The method of claim 29 wherein said preformed microparticle comprises, by weight, 80% or greater of said at least one active agent distributed substantially homogenously throughout said preformed microparticle.
  34. 34. The method of claim 34, wherein said exposing comprises:
    providing a solution that comprises said at least one charged compound, optionally a polyvalent cation, and, in weight-to-volume percentage, 16% polyethylene glycol and 0.7% sodium chloride, and
    incubating said formed microparticle in said solution at a temperature of 2° C. to 5° C. over a period of 1 second to 10 hours.
  35. 35. The method of claim 29, wherein said at least one charged compound is provided in a solution having a pH that differs from a surface-neutral point of said formed microparticle by 0 to less than 0.3.
  36. 36. The method of claim 29, wherein said at least one charged compound is provided in a solution having a pH that differs from a surface-neutral point of said formed microparticle by 0.3 or greater.
  37. 37. The method of claim 29, further comprising forming said surface-modified microparticle having a monodisperse or polydisperse size distribution.
  38. 38. A method for preparing a surface-modified microparticle, comprising:
    providing an amorphous and solid preformed microparticle comprising at least one active agent, said preformed microparticle having an outer surface carrying a net surface charge;
    exposing at least said outer surface of said preformed microparticle to at least one charged compound having a net charge that is opposite in sign to said net surface charge of said preformed microparticle;
    forming an intermediate microparticle that comprises said preformed microparticle and a formed monolayer comprising said at least one charged compound, wherein said formed monolayer is associated with said outer surface of said preformed microparticle;
    exposing at least said formed monolayer to at least one different charged compound;
    forming said surface-modified microparticle that comprises said intermediate microparticle and a subsequent monolayer comprising said at least one different charged compound, wherein said surface-modified microparticle has a release profile for release of said at least one active agent that is different from the release profile of said intermediate microparticle.
  39. 39. The method of claim 38 further comprising carrying out one or more treatments to said intermediate microparticle, the treatment comprising manipulation of temperature, pressure, pH or a combination thereof.
  40. 40. A method of claim 39 wherein the treating comprises heating a suspension including the microparticle at an elevated temperature.
  41. 41. A microparticle comprising:
    a solid, amorphous core microparticle comprising 80% or greater, by weight, of at least one active agent, said core microparticle comprising an outer surface carrying a net surface charge; and
    a monolayer comprising at least one charged compound associated at least by electrostatic interaction with said outer surface of said core microparticle, said charged compound having a net charge that is sufficiently different from said net surface charge of said core microparticle,
  42. 42. The microparticle of claim 41 wherein said at least one active agent is homogeneously distributed across said core microparticle.
  43. 43. The microparticle of claim 41 wherein said core microparticle is spherical.
  44. 44. The microparticle of claim 41 wherein said monolayer has a thickness of less than 50 nm.
  45. 45. The microparticle of claim 41 wherein said charge of said charged compound is opposite in sign to said net surface charge of said microparticle outer surface.
  46. 46. The microparticle of claim 41 further comprising another monolayer comprising at least one different charged compound, said other monolayer carrying a net charge that is different from said net charge of said monolayer associated with said core microparticle.
  47. 47. The microparticle of claim 46 wherein said net surface charge of said other monolayer is opposite in sign to that of said monolayer associated with said core microparticle.
  48. 48. The microparticle of claim 41 wherein said active agent is insulin or human growth hormone.
  49. 49. A microparticle comprising at least 80% by weight of at least one active agent, the microparticle being solid, and an outer surface of the microparticle comprising at least one charged polymer associated with the active agent, wherein the microparticle is capable of displaying a 1-hour percentage of cumulative release of the active agent of 50% or less when subjected to in vitro release in a release buffer at pH 7.4 and 37° C., the release buffer consisting 10 mM Tris, 005% (w/v) Brij 35, and 0.9% (w/v) NaCl in water.
  50. 50. A microparticle for in vivo administration comprising a solid, microparticle having at least 80%, by weight, of at least one proteinaceous compound homogeneously distributed across said microparticle, wherein the outer surface of said microparticle comprises at least one charged compound associated with said outer surface,
    wherein upon administration, said microparticle provides a Cmax and tmax that is than the Cmax and tmax of said core microparticle.
  51. 51. The microparticle of claim 50 wherein said monolayer associated with said microparticle outer surface comprises a positively charged compound selected from the group consisting essentially of positively charged polyelectrolytes, positively charged polyaminoacids and positively charged polysaccharides.
  52. 52. The microparticle of claim 50 wherein said monolayer associated with said core comprises a negatively charged compound selected from the group consisting essentially of negatively charged polyelectrolytes, negatively charged polyaminoacids and negatively charged polysaccharides.
  53. 53. The microparticle of claim 51 wherein said another monolayer comprises a negatively charged compound selected from the group consisting essentially of negatively charged polyelectrolytes, negatively charged polyaminoacids and negatively charged polysaccharides.
  54. 54. The microparticle of claim 52 wherein said another monolayer comprises a positively charged compound selected from the group consisting essentially of positively charged polyelectrolytes, positively charged polyaminoacids and positively charged polysaccharides.
  55. 55. The microparticle of claim 50 wherein said proteinaceous compound is insulin or human growth hormone.
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