US20120288537A1 - Active self-healing biomaterial system - Google Patents

Active self-healing biomaterial system Download PDF

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US20120288537A1
US20120288537A1 US13/512,913 US201113512913A US2012288537A1 US 20120288537 A1 US20120288537 A1 US 20120288537A1 US 201113512913 A US201113512913 A US 201113512913A US 2012288537 A1 US2012288537 A1 US 2012288537A1
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self
plga
polymer
microspheres
agent
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Steven P. Schwendeman
Kashappa-Goud Desai
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University of Michigan
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1682—Processes
    • A61K9/1694—Processes resulting in granules or microspheres of the matrix type containing more than 5% of excipient
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    • A61K38/08—Peptides having 5 to 11 amino acids
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    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/38—Albumins
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    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
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Definitions

  • the present technology relates to a delivery system with high encapsulation efficiency for an agent such as a biomolecule.
  • Injectable biodegradable polymeric particles such as microspheres, provide a means to deliver and control the release of molecules such as drugs, proteins, peptides, and vaccine antigens. Once injected, the biodegradable polymeric particles can release the molecule over the course of hours, days, or even weeks to months, providing a distinct advantage over daily injections in terms of patient acceptability and compliance. For example, controlled release of a protein antigen can reduce the number of doses in an immunization schedule and can optimize the desired immune response via selective targeting of antigen to antigen presenting cells.
  • biodegradable polymers have been explored for the microencapsulation and delivery of macromolecules.
  • Copolymers of lactic acid and glycolic acid are one type of biodegradable polymer used in pharmaceutical products or medical devices including several approved by the U.S. Food and Drug Administration.
  • PLGAs are used in commercially available controlled-release peptide delivery systems, including the Lupron DepotTM (leuprolide acetate), Sandostatin LARTM (octreotide acetate), and ZoladexTM implant (goserelin acetate).
  • hydrophilic macromolecules including many proteins, cannot readily diffuse through a hydrophobic polymer phase, such as PLGA.
  • the release of encapsulated protein drugs from PLGA requires at some point the diffusion of the macromolecules through water-filled pores and channels.
  • protein release from PLGA microspheres can exhibit tri-phasic behavior.
  • protein on the surface or having access to the surface of microspheres i.e., in open pores
  • a time lag commonly exists as the protein cannot diffuse through the polymer phase.
  • Third, a continuous release of protein occurs following polymer erosion so that more pores and channels are formed allowing protein in previously isolated pores to be released.
  • the polymer may also sorb peptides of moderate MW deep into the polymer phase itself.
  • Encapsulation methods employing self-healing polymers have been developed to form biodegradable polymeric particles loaded with various macromolecules; e.g., peptides, proteins, DNA, siRNA, etc.
  • U.S. Pat. Appl. Pub. 2008/0131478 to Schwendeman et al. describes methods that obviate damaging stresses during microencapsulation, which include forming a porous polymer, ideally with a percolating pore network, incubating an aqueous solution of the macromolecules below the glass transition temperature (T g ) of the polymer so that the macromolecule is taken up into the pores of the polymer, and raising the temperature above the T g so that the polymer pores close irreversibly encapsulating the macromolecule.
  • Other methods can be used to close the pores besides temperature change. For example, exposure to solvent, such as alcohol vapor, can be used to facilitate self-healing of the polymer. Using this methodology, loadings of about 10% w/w or more can be achieved
  • Passive encapsulation methods rely on equilibration of the protein between the solution outside the polymer and the aqueous pores inside the polymer. Such methods may not provide high encapsulation efficiency; i.e., mass macromolecule encapsulated/mass of macromolecule charged to the system. As a result, a significant proportion of the macromolecule to be loaded remains in solution outside the polymer as the polymer pores close. The macromolecule solution may have to be reused multiple times in this case to avoid wasting the macromolecule. Furthermore, passive encapsulation typically requires very high concentrations of the macromolecule (e.g., >100 mg/mL) in order to achieve elevated loading. Some macromolecules may have limited solubility, prohibiting this method altogether.
  • the encapsulation method should be less expensive to carry out than conventional methods, where cost may be a principal factor in the slow development of controlled release injectable depots. For example, passive loading of polymer particles may waste a considerable amount of macromolecule, leaving a majority of the macromolecule in the solution following pore closure.
  • the present technology includes systems, methods, articles, and compositions that relate to sorbing and/or encapsulating an agent, such as a biomolecule, with a solid polymer, such as a porous self-healing polymer, where the polymer may be in the form of particles or microspheres.
  • the delivery system may also take on various forms, may be a portion of other forms, or may be coated onto other forms, including various shapes or devices such as drug eluting stents, sutures, screws, tissue engineering scaffolds, and blood circulating nanoparticles, among others.
  • a delivery system comprises a solid polymer matrix comprising an ionic affinity trap, the ionic affinity trap is operable to sorb an agent from an aqueous solution.
  • An agent can be sorbed to the ionic affinity trap.
  • the solid polymer matrix can be a self-healing polymer.
  • the self-healing polymer can comprise one or more pores that comprise the ionic affinity trap, where at least a portion of the pores can be interconnected.
  • the delivery system can further include an agent sorbed to the ionic affinity trap and wherein the pore partially or fully encapsulates the agent and prevents the agent from exiting the pore.
  • the solid polymer matrix can comprises a biodegradable polymer and can comprise a copolymer of lactic acid and glycolic acid.
  • the solid polymer matrix can take the form of a microparticle or microsphere.
  • the ionic affinity trap can comprise a metal salt, such as aluminum hydroxide, aluminum phosphate, potassium phosphate, magnesium carbonate, calcium phosphate, or combinations thereof, and can comprise an ionomer gel.
  • the ionic affinity trap can comprise ionized end groups of the polymer, where the ionized end groups can comprise carboxylate groups.
  • the agent can comprise a biomolecule, drug, or antigen, where the biomolecule can comprise a protein, peptide, proteoglycan, lipoprotein, or nucleic acid.
  • the agent can also comprise an immunocontraceptive.
  • the solid polymer matrix can further comprise a plasticizer.
  • a method of making a delivery system comprises sorbing an agent to an ionic affinity trap, wherein a solid polymer matrix comprises the ionic affinity trap and an aqueous solution comprises the agent.
  • the method can further comprise partially or fully encapsulating an agent in a pore by increasing the temperature of the self-healing polymer to about its T g or above, where the solid polymer matrix includes a self-healing polymer comprising the pore.
  • the aqueous solution in the sorbing step can comprise less than about 1 mg/mL of the agent. At least 90% of the agent in the sorbing step can be sorbed to the ionic affinity trap.
  • a delivery system can include a self-healing polymer comprising one or more pores, where the pore or pores include an ionic affinity trap and an agent, such as a biomolecule, associated with the ionic affinity trap.
  • the delivery system can be made by providing the self-healing polymer having one or more pores, where the pore(s) includes an ionic affinity trap.
  • the self-healing polymer is contacted with an agent having affinity for the ionic affinity trap so that the agent associates with the ionic affinity trap.
  • the self-healing polymer can then partially or fully encapsulate the agent, preventing the agent from exiting the pore(s).
  • the delivery system can also include a self-healing polymer where a portion of the self-healing polymer comprises ionized end groups and an agent sorbed to the ionized end groups.
  • a delivery system can be made by contacting the self-healing polymer with an agent having affinity for the ionized end groups and optionally
  • FIG. 1 Self-healing microencapsulation of large molecules in PLGA microsphere injectable controlled-release depots. Scanning electron micrographs of self-microencapsulating microspheres (SM-1, Tables 1 and 2) before (A) and after (B) healing of polymer pores in the presence of 230 mg/mL lysozyme at 42.5° C. Laser confocal fluorescent micrographs (C) of the cross-sectional distribution of BSA-Coumarin (in white domains) after self-healing encapsulation of the protein in microspheres (20-63 ⁇ m in diameter) (SM-2, Tables 1 and 2).
  • SM-microsphere porosity (E)
  • E SM-microsphere porosity
  • FIG. 2 Assessment of lysozyme aggregation and leuprolide controlled release after self-healing microencapsulation (SM) without organic solvent.
  • Insoluble aggregation recorded after microencapsulation of encapsulation-labile lysozyme (A) according to self-healing (Formulations A-D without organic solvent) and standard solvent-evaporation (Formulations E and F with organic solvent) processes.
  • SM microspheres were prepared from 11 (Formulations A, and B) and 51 (Formulations C-F) kDa M w , PLGA 50/50, and in the presence (B, D, F) and absence (A, C, E) of 0.45 M sucrose in the aqueous lysozyme solution.
  • Formulations A and B, C and D, and E and F, respectively, correspond to SM-4, SM-3, and TM-1 in Tables 1 and 2.
  • SM-5 controlled-release leuprolide from SM-microspheres
  • C serum testosterone suppression in rats
  • FIG. 3 Active self-microencapsulation of ovalbumin (OVA) and tetanus toxoid (TT) in Al(OH) 3 adjuvant-containing SM-microspheres (ASM, Tables 1 and 2).
  • Scanning electron micrographs (A, B) depict the SM-microsphere morphology after loading entire OVA mass from 0.5 mg/mL OVA solution and self-healing at 37° C.
  • a and B formulations respectively correspond to ASM-1 and ASM-3 in Tables 1-4.
  • FIG. 4 Goodness of self-encapsulation in PLGA microspheres as indicated by elevated lysozyme loading (open bar chart) and minimal initial burst (closed bar chart) of enzyme employing diversity of pore-forming excipients/initial water phase volume to create the PLGA 50/50 pore network.
  • A Effect of MgCO 3 (porosigen/stabilizer) loading (3 ( ⁇ ) and 4.5 ( ⁇ ) % w/w) and addition of sucrose (0 ( ⁇ ) and 0.45 ( ⁇ ) M) in protein loading solution on cumulative release of BSA from self-microencapsulated microspheres.
  • Actual BSA loading in ⁇ , ⁇ , ⁇ , and ⁇ SM formulations was 4.25 ⁇ 0.05, 5.65 ⁇ 0.06, 7.26 ⁇ 0.09, and 5.54 ⁇ 0.04%, respectively.
  • ASM-1, ASM-2, and ASM-3 PLGA microsphere formulations consists of 3.2% w/w Al(OH) 3 and 3.5% w/w trehalose as porosigen and Al(OH) 3 lyophilization stabilizer.
  • ASM-2 and ASM-3 contain 2.5 and 5% w/w DEP, respectively.
  • Blank and ASM-1 PLGA microspheres were incubated at 25 and 43° C. for 48 h.
  • ASM-2 and ASM-3 PLGA microspheres were incubated for 48, 24, and 30 h at 10, 25, and 37° C., respectively.
  • Preparation process of blank ASM PLGA microspheres is given in Table 1.
  • FIG. 9 Photomicrographs of 3.2 wt % alhydrogel/3.8 wt % trehalose/PLGA microparticles before (A) and after (B) self-healing at 25° C. for 24 h and 43° C. for 48 h.
  • FIG. 10 Photomicrographs of 3.2 wt % alhydrogel/3.8 wt % trehalose/2.5 wt % DEP/PLGA microparticles before (A) and after (B) self-healing at 10° C. for 48 h, 25° C. for 24 h, and 37° C. for 30 h.
  • FIG. 11 Photomicrographs of 3.2 wt % Alhydrogel/3.8 wt % Trehalose/5 wt % DEP/PLGA microparticles before (A) and after (B) self-healing.
  • FIG. 13 Prolonged serum testosterone suppression by leuprolide acetate (LA)-PLGA particles in male Sprague-Dawley rats. Effect of dosing interval (2 ⁇ , 3 ⁇ , and 4 ⁇ ) of LA-PLGA particles on serum testosterone suppression.
  • LA leuprolide acetate
  • Biomolecules such as peptides, proteins, or polysaccharides dissolved in aqueous solution can be self-microencapsulated in poly(lactic-co-glycolic acid) (PLGA) by placing the biomacromolecule solution in contact with solid PLGA, preformed with an interconnected pore-network, at below the polymer glass transition temperature (T g ), and then healing the pores at a temperature at or above the T g .
  • PLGA poly(lactic-co-glycolic acid)
  • Healed polymers can then slowly release the biomacromolecules under physiological conditions for over 1 month.
  • Benefits of the present approach include improved compatibility with biotechnology-derived drugs, reduced manufacturing cost and residual organic solvent, the ability to create new biomaterial architectures, and facile use among non-formulation scientists and clinicians.
  • Modern synthetic polymeric biomaterials are widely used to slowly release medicines over days to years after administration to the body. These polymers are configured in numerous biomedical and pharmaceutical three-dimensional forms (e.g., spheres, rods, coatings, porous matrices) including micro- to millimeter scale injectable depots, drug-eluting stents, scaffolds for engineering tissues, and blood-circulating nanometer scale particles and can be made biodegradable or nondegradable.
  • drugs particularly peptides and proteins, are most commonly microencapsulated by first combining drug with a polymer dissolved in organic solvent.
  • the drug is either micronized (e.g., by homogenization, sonication, or grinding) or molecularly dissolved in the solvent, to yield drug domains, which later become dispersed in the final polymer matrix. Both steps can compromise stability of encapsulated proteins and other biomolecules.
  • the organic solvent is removed to clinically acceptable levels and the polymer is dried before use. Described here is a new self-microencapsulation paradigm based on the polymer's own spontaneous “self-healing” capacity in aqueous media.
  • features of this new approach include a simple mixing process (e.g., as mixing naked DNA to lipofectin gene delivery vector), the absence of exposure of drug to organic solvent during encapsulation (e.g., as supercritical fluid polymer processing), and mild processing conditions (e.g., as spray-congealing for commercial manufacture of PLGA-encapsulated growth hormone).
  • a simple mixing process e.g., as mixing naked DNA to lipofectin gene delivery vector
  • the absence of exposure of drug to organic solvent during encapsulation e.g., as supercritical fluid polymer processing
  • mild processing conditions e.g., as spray-congealing for commercial manufacture of PLGA-encapsulated growth hormone
  • SM-1 injectable self-microencapsulating microsphere
  • PLGA lactic and glycolic acids
  • FIG. 1A After leaching the sugar, pores on the scale of 250 to 2500 nm were easily viewed by electron microscopy ( FIG. 1A ).
  • the dry microspheres were incubated at 4° C. ( ⁇ hydrated T g ⁇ 30° C.) in concentrated aqueous lysozyme solution at 230 mg/mL for 48 h to allow the protein to enter the open polymer pores.
  • SM microspheres prepared by several different conditions were loaded with protein and incubated under physiological conditions (in PBS+0.02% Tween 80, pH 7.4 at 37° C.) for 48 h to observe the “initial burst release” of protein ( FIG. 4 ), which is often too high with poorly encapsulated material.
  • optimal porosigen loading e.g. 1.5-4.5% (w/w magnesium carbonate/polymer matrix), 5 measurements typically exhibited an optimal initial burst release of protein ( ⁇ 20% release).
  • the measured loading and initial burst values were within the desirable range as established by clinically used PLGA depots and required loading times were on the order of 12 hours ( FIG. 1F ).
  • SM microspheres As expected by the mild encapsulation conditions (37-43° C. temperature exposure) by the new approach—no harsh mixing or organic solvent exposure—protein stability was also improved with SM microspheres relative to microspheres prepared by traditional emulsion-based microencapsulation techniques (e.g., water-in-oil-in-water—solvent evaporation, w/o/w, as used for the Lupron Depot).
  • traditional emulsion-based microencapsulation techniques e.g., water-in-oil-in-water—solvent evaporation, w/o/w, as used for the Lupron Depot.
  • the model enzyme, lysozyme well-established to undergo aggregation during solvent evaporation, the potential stability improvement of the enzyme in SM microspheres was evaluated relative to solvent evaporation control groups.
  • Desirable polypeptides were also successfully self-microencapsulated by PLGAs and released slowly and continuously over a period of >1 month.
  • the most commonly delivered peptide from PLGA depots, leuprolide acetate, used to suppress testosterone in prostate cancer patients to inhibit growth of the hormone-dependent cancer was loaded in SM PLGA microspheres employing an ionic affinity trap, ZnCO 3 , to create pores for self-encapsulation and to facilitate continuous release of peptide.
  • the resulting SM microspheres ( FIG. 5 ) encapsulated 3.0 ⁇ 0.2% (w/w peptide/polymer matrix) leuprolide acetate (5 measurements) and released the peptide in vitro slowly and continuously for 2 months ( FIG. 2B ).
  • OVA or TT protein antigens were loaded into SM PLGA containing, as an ionic affinity trap, lyophilization-stabilized Al(OH) 3 adjuvant (ASM, Table 1), which absorbed the antigen into the polymer matrix from surrounding 0.5 or 0.8 or 1 mg/mL antigen solution, with 98 ⁇ 1% EE (6 measurements) and 1.0 ⁇ 0.05% OVA loading (6 measurements) (ASM-3, tables S3 and S4) or 87 ⁇ 0.4% EE (3 measurements) and 1.6 ⁇ 0.03% TT loading (Table 5).
  • ASM lyophilization-stabilized Al(OH) 3 adjuvant
  • Spontaneous self-healing in homogenous polymer systems has been described in nano-scale cracks of solid rocket propellants, following bullet holes in plastic plates, film formation from latex particles, and across lap joints of polymer films.
  • the process mechanism which has found to be ubiquitous to polymers in the vicinity of their T g or above has been analyzed in detail to involve multiple elements such as: a) polymer chain interdiffusion, b) minimization of energetically unfavorable interfacial area, and c) transfer of energy stored in a defect.
  • a clinician can mix sterile SM microspheres with an injectable solution of vaccine (e.g., tetanus toxoid) before injecting into women of child-bearing age, providing improved immunity for their unborn children against neonatal infection.
  • an injectable solution of vaccine e.g., tetanus toxoid
  • New biomaterial architectures e.g., drug-eluting stent coatings
  • process-sensitive large molecules as previously unchartered formulation conditions e.g., high temperature, reactive molecules, organic solvent
  • SM microsphere formulations For manufacturing, a mixture of several different SM microsphere formulations, each having distinct design characteristics (release kinetics, size, surface biofunctional groups) can be combined for a drug of interest in a single sterile mixing step. With the absence of aseptic processing of organic solvents, this paradigm can have significant cost savings, which was a critical factor in halting production of the Nutropin Depot, the first and only FDA approved injectable protein depot. The simplicity of self-microencapsulation can also significantly facilitate various controlled release approaches, providing more rapid advancement of controlled release products and technology.
  • Tetrahydrofuron was purchased from Fisher-Scientific (Pittsburgh, Pa., USA). Tetramethyl-rhodamine (TMR)-dextran was purchased from Invitrogen Corporation (Carlsbad, Calif., USA). Tetanus toxoid (TT) (3120 Lf/mL) and equine tetanus antitoxin were received as gift samples respectively from Serum Institute of India Ltd. (Pune, India) and U.S. Food and Drug Administration (Silver Spring, USA). Human tetanus immune globulin (HyperTETTM S/D, 250 units) was purchased from Talecris Biotherapeutics, Inc. (Research Triangle Park, N.C., USA).
  • Leuprolide acetate (Batch #091203) was purchased from Shanghai Shjnj Modern Pharmaceutical Technology Co., Ltd (Shanghai, China). Male Sprague-Dawley rats were procured from Charles River Laboratories. Isoflurane was purchased from Baxter Healthcare Corporation (Deerfield, Ill., USA). B-D MicrotainerTM blood collection and serum separation tubes were purchased from Becton, Dickinson and Company (Franklin Lakes, N.J., USA). Goat anti-human IgG-alkaline phosphatase was purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, Pa., USA).
  • BSA Conjugating BSA to a pH-insensitive fluorescent coumarin: About 1.2 g BSA was dissolved in 40 mL of 0.2 M sodium bicarbonate (pH 4.5). To this, 2 mL of 10 mg/mL 7-methoxycoumarin-3-carbonyl azide in dimethyl sulfoxide (DMSO) was added while stirring. The solution was stirred continuously at room temperature in darkness for 90 min. To quench the reaction, 4 mL of 1.5 M hydroxylamine hydrochloride was added and then the solution was extensively dialyzed using a 25,000 Da M w cut-off membrane against degassed distilled water at 4° C.
  • DMSO dimethyl sulfoxide
  • SM self-microencapsulating
  • CH 2 Cl 2 methylene chloride
  • Suitable initial water phase was added to the corresponding polymer solution and immediately homogenized as per the first homogenization conditions specified in Table 1 using a Tempest IQ 2 homogenizer (The VirTis Company, Gardiner, N.Y., USA) equipped with a 10 mm shaft in an ice water bath to create the respective first emulsion.
  • a Tempest IQ 2 homogenizer The VirTis Company, Gardiner, N.Y., USA
  • PLGA 50:50 Conc.(mg polymer in 1 WP Formulation Initial water phase M w mL CH 2 Cl 2 ) + volume First Second homog./ code (WP) composition (kDa) excipient ( ⁇ L) homog. vortexing SM-1 500 mg trehalose in 51 320 175 20,000 rpm Homog.
  • microspheres were then transferred on a rigged rotator (Glas-Col, Terre Haute, Ind., USA) to prevent interparticle healing in an incubator maintained at specified temperature for specified duration (self-healing conditions), as listed in Table 2. Microspheres were then removed, washed 10 times with distilled and deionized water (ddH 2 O), centrifuged at 3000-3800 rpm for 5-10 min, and freeze-dried.
  • ddH 2 O distilled and deionized water
  • TE encapsulation
  • Coomassie (modified Bradford) protein assay A modified Bradford assay was used to determine protein concentrations. Briefly, appropriate volume of standard or sample was mixed with Coomassie PlusTM reagent (Thermo Fisher Scientific, Rockford, Ill., USA) in a 96-well plate (Nalge Nunc International, Rochester, N.Y., USA). Then, the absorbance was read at 595 nm within 30 min using a Dynex II MRX microplate reader (Dynex Technology Inc., Chantilly, Va., USA).
  • UV detection at 215 and 280 nm and fluorescence detection with excitation and emission wavelengths of 278 nm and 350 nm for BSA, OVA and lysozyme, and 384 nm and 480 nm for BSA-Coumarin were used.
  • High performance liquid chromatography (HPLC) analysis of leuprolide acetate Analysis of leuprolide acetate was accomplished by HPLC, with a gradient of acetonitrile (Solvent A) and 0.05 M sodium phosphate buffer, pH 7.0 (Solvent B) on a Nova-Pak C18 column (4 ⁇ m, 3.9 ⁇ 150 mm, Waters Corporation, Milford, Mass., USA). The gradient method was 0 min (20% A), 6 min (30% A), 9.5 min (37% A), 11.5 min (37% A), 16.5 min (50% A), and 19 min (20% A), followed by a 2 min recovery. UV detection was measured at 215 nm and 280 nm, and fluorescence detection was performed at excitation and emission wavelengths of 278 and 350 nm, respectively.
  • microspheres were dissolved in ethyl acetate and centrifuged at 6,000 rpm for 5 min. The supernatant polymer solution was removed and the aluminum hydroxide-OVA sediment was washed twice with ethyl acetate. The sediment was then dried at 30° C. using the VacufugeTM to remove ethyl acetate. One milliliter of 190 mM sodium citrate solution was added to the dried aluminum hydroxide-OVA pellet, mixed thoroughly, and incubated at 37° C. for 3 days with constant agitation. In control studies, this duration was determined to be sufficient for complete elution of OVA from the aluminum hydroxide gel.
  • Enzyme-linked immunosorbent assay Antigenically active TT was determined by the ELISA. Except for final incubation step with p-nitrophenyl phosphate liquid substrate, all initial ELISA steps were performed at room temperature. Briefly, 100 ⁇ L of 2-3 international units (IU)/mL of equine tetanus antitoxin in PBS (pH 7.2) was added to 96-well microtitration plates (Nalge Nunc International, Rochester, N.Y., USA) and incubated overnight. The plates were washed 3-5 times between all steps with PBS containing 0.05% TweenTM 20 (pH 7.2).
  • Phosphate blocking buffer PBB, PBS/0.5% BSA/0.05% BrijTM 35, pH 7.4 was used as a diluent for all TT samples and antibodies (except equine tetanus antitoxin above). Standard TT with known concentration and test samples were diluted at 2-fold steps in coated plates using PBB as a diluent. The plates were held for 2 h and washed. Then, 100 ⁇ L of human anti-TT IgG (HyperTETTM S/D, 1:5000 dilution) was added and allowed to react for 2 h followed by 100 ⁇ L of goat anti-human IgG-alkaline phosphatase diluted 1:20000 in PBB for another 2 h.
  • PBB Phosphate blocking buffer
  • the plates were washed and 100 ⁇ l of p-nitrophenyl phosphate liquid substrate was added. After 30 min incubation at 37° C., the absorbance was read at 405 nm on a Dynex II MRX microplate reader (Dynex Technology Inc., Chantilly, Va., USA) equipped with Revelation 4.21 Software. Log/Logit curve fitting model was used to plot the standard curve and calculate unknown concentration of TT in test samples.
  • ⁇ 4-10 passive BSA/lysozyme/leuprolide acetate SM PLGA microspheres
  • 20 active OVA or TT self-encapsulated Al(OH) 3 —PLGA microspheres
  • PBST phosphate buffered saline
  • BSA active TT self-encapsulated Al(OH) 3 —PLGA microspheres
  • AAA amino acid analysis
  • SEM Scanning electron microscopy
  • Determination of polymer matrix porosity of SM microencapsulating microspheres Measurement of polymer matrix porosity of blank SM microencapsulating PLGA microspheres was done by Porous Materials, Inc. (Ithaca, N.Y., USA) using an AMP-60K-A-1 mercury porosimeter, generating pore volume versus pressure data. The pore volume was reported as volume per gram microspheres (cc/g). Total microsphere volume was calculated as the sum of the pore volume and the polymer volume, where the polymer density (1.25 g/cc for 51 kDa PLGA 50:50, provided by manufacturer) and weight of the porosimetry sample were used to calculate the pore volume.
  • Percent porosity was calculated as the pore volume per total microsphere volume. Pressure associated with microspheres' packing and surface wetting, before mercury intrusion into the pores had taken place, was not calculated into the final pore volume as has been reported previously (S4). The method of determination of porosity utilized large amount of microspheres sample ( ⁇ 250 mg) and there was no significant difference (p>0.05) among the different measurements (three measurements) of the same formulation. Hence, only one test was run for the measurement of polymer matrix porosity of various SM microspheres formulations.
  • the dextran-FITC was extracted using acetone to dissolve the PLGA and concentrating the insoluble dextran-FITC using centrifugation (10,000 rpm at 10 min), and repeating 3-fold. Dextran-FITC was dissolved in PBS, pH 7.4, and loading was determined via HPLC with fluorescence (without column separation) using 20 or 40 ⁇ L injection volume and a 1 mL/min PBS, pH 7.4 mobile phase. The fluorescence of the dextran-FITC was measured respectively at an excitation and emission wavelength of 490 and 520 nm.
  • leuprolide acetate self-encapsulated PLGA microspheres (1 ⁇ 2-month dose), leuprolide acetate solution (1 ⁇ 1-month dose), and blank SM PLGA microspheres without drug (1 ⁇ dose) in a liquid vehicle (1% w/v carboxymethylcellulose and 2% w/v mannitol), and commercial 1-month Lupron Depot (Abbott Laboratories, North Chicago, Ill., USA) (2 ⁇ dosing at days 0 and 28) were subcutaneously injected at the back (lower neck portion) of rats (6 animals/study group). Total dose of leuprolide acetate was based on 100 ⁇ g/kg/day.
  • Animal body weight considered for dosing leuprolide acetate was 425 g which is projected body weight of male Sprague Dawley rat at midpoint (day 28) of the study (as per the weight (g)/age (weeks) curve given by Charles River Laboratories).
  • Blood samples were collected via jugular vein stick before (day ⁇ 7 and 0 for baseline testosterone level) and after (1, 7, 14, 21, 28, 35, 42, 49, and 56 days) injection of preparations. The collected blood samples were immediately transferred to B-D MicrotainerTM blood collection and serum separation tubes previously incubated in ice, centrifuged at 8,000 rpm for 10 min, and then the serum was removed and stored in microcentrifuge tubes at ⁇ 20° C. until further use.
  • Serum testosterone levels were assayed by radioimmunoassay using a TESTOSTERONE Double Antibody-125I RIA Kit (MP Biomedicals LLC., Solon, Ohio, USA) at the University of Pennsylvania Diabetes Center (Philadelphia, Pa., USA). Lowest detection limit of testosterone was 0.1 ng/mL. In case of samples which exhibited testosterone level below the detection limit, a 0.1 ng/mL value was used for statistical evaluation and plotting the curve.
  • Active SM PLGA microspheres were irradiated by using 60 Co as irradiation source (Michigan Memorial Phoenix Project, University of Michigan) at 2.5 MRad dose and 0.37 MRad/h dose rate. Briefly, about 250 mg active SM PLGA microspheres were freeze-dried, placed in 5-mL ampoules and then ampoules were sealed under vacuum. All the samples were irradiated at room temperature.
  • An unpaired Student's t-test was used to assess statistical significance between numerous SM PLGA microsphere formulations with respect to polymer porosity, protein and peptide loading, stability, and in vitro release, and in vivo testosterone level. Results were considered statistically significant if p ⁇ 0.05.
  • both dyes were successfully encapsulated at very similar levels as indicated by the following data determined experimentally (five measurements): SM PLGA microspheres self-encapsulated 0.30 ⁇ 0.01 and 0.22 ⁇ 0.01% w/w TMR-dextran and FITC-dextran 4 kDa, respectively.
  • lysozyme is a common model protein for testing protein damage during microencapsulation, we self-microencapsulated this enzyme and monitored its loading in terms of monomeric, total, and enzymatically active protein content.
  • the stability of lysozyme encapsulated via self-healing microencapsulation was compared with the enzyme encapsulated via a traditional w/o/w process (TM-1, Tables 1 and 2).
  • TM-1 traditional w/o/w process
  • sucrose as a differentially soluble material
  • TE method double emulsion
  • loading solution self-microencapsulation method
  • reduced lysozyme loading % w/w lysozyme/polymer matrix
  • the percentage (% w/w) of loaded lysozyme that exists as intact soluble monomer was slightly higher for the self-microencapsulated microsphere (SM-3, Tables 1 and 2) formulations than the TM-1 formulations.
  • the fraction of total lysozyme loaded as insoluble aggregates was significantly less (p ⁇ 0.05) for self-microencapsulation technique when compared to TE method.
  • lysozyme loaded via TE method (TM-1, Tables 1 and 2) underwent 40 ⁇ 4% and 12 ⁇ 2% insoluble aggregation (three measurements) with or without sucrose in the inner water phase, respectively ( FIGS. 2F and E).
  • the amount of lysozyme self-encapsulated in 0, 1.5, 4.3, 11.0% w/w MgCO 3 loaded blank PLGA 50:50 microspheres was 4.5 ⁇ 0.2, 6.4 ⁇ 0.1, 9.8 ⁇ 0.3, and 8.7 ⁇ 0.4% w/w (lysozyme/polymer matrix), respectively (three measurements).
  • the release rate of lysozyme from above mentioned formulations was directly related to the amount of ionic affinity trap (MgCO 3 ) loaded into the blank PLGA 50:50 microspheres ( FIG. 6B ).
  • MgCO 3 ionic affinity trap
  • the cumulative amount of lysozyme released from self-microencapsulated microspheres formulations was 30 ⁇ 1, 41 ⁇ 1, 51 ⁇ 4, and 57 ⁇ 4% respectively for 0, 1.5, 4.3, and 11.0% w/w MgCO 3 loaded blank PLGA 50:50 microspheres.
  • the amount of protein remaining in the microspheres after 28 days of release, including soluble monomer, soluble aggregates, and insoluble aggregates, was similarly quantified and >90% mass balance (total recovery) was achieved for all four formulations.
  • total amount of lysozyme recovered from self-microencapsulated microspheres formulations was 92 ⁇ 2, 91 ⁇ 1, 99 ⁇ 4, and 110 ⁇ 4% (three measurements) respectively for 0, 1.5, 4.3, and 11.0% w/w MgCO 3 loaded PLGA 50:50 microspheres.
  • the total amount of soluble protein, both released over 28 days and recovered as residual soluble monomer was significantly higher (p ⁇ 0.05) for 4.3 and 11.0% w/w MgCO 3 -based self-encapsulated microspheres formulations.
  • total (released+residual) amount of soluble protein recovered for 0, 1.5, 4.3, and 11.0% w/w MgCO 3 loaded PLGA 50:50 microspheres was 83 ⁇ 2, 85.0 ⁇ 1, 95 ⁇ 4, and 107 ⁇ 4%, respectively.
  • specific activity of the residual lysozyme remaining in the self-encapsulated microspheres after 28 days of release was analyzed. The specific activity was calculated based upon the total amount of soluble protein analyzed, both monomer and aggregated.
  • the specific activity given as the percentage of the specific activity of the native, standard lysozyme was 102 ⁇ 6, 116 ⁇ 19, 100 ⁇ 5, and 97 ⁇ 5% respectively for 0, 1.5, 4.3, and 11.0% w/w MgCO 3 loaded PLGA 50:50 microspheres.
  • the soluble lysozyme retained in the self-microencapsulated microspheres after 28 days of release was still completely active within experimental error for all formulations.
  • SM PLGA microspheres (SM-3, Table 1) were loaded with FITC-dextran (SM-3, Table 2) and the loaded biomacromolecular dye was analyzed at various times before and after initiating self-encapsulation by increasing temperature from 4° C. (T ⁇ T g ) to 42.5° C. (T>T g ). At each time point, microspheres were washed extensively with ddH 2 O to remove any unencapsulated dye. As shown in FIG. 1F , only background levels of dextran were loaded when at the low temperature for 20 h. However, after increasing temperature to 42° C.
  • PLGA self-healing microencapsulation provides long-term delivery of bioactive large molecules in vivo—Long-term testosterone suppression in rats after single injection of leuprolide acetate self-encapsulated PLGA microspheres:
  • Commercially available injectable PLGA microsphere-based formulation of leuprolide (Lupron DepotTM) is prepared by the traditional (water-in-oil-water) encapsulation method.
  • the ability of PLGA self-healing microencapsulation to provide long-term in vivo release was evaluated by assessing long-term testosterone suppression in male Sprague-Dawley rats (six rats/study group) in comparison with commercial Lupron DepotTM and negative controls ( FIG. 2C ).
  • the initial serum testosterone level (1-2 ng/mL) increased to ⁇ 5 ng/mL after one day of subcutaneous injection of Lupron DepotTM, leuprolide self-encapsulated PLGA microspheres and leuprolide solution.
  • LHRH luteinizing hormone-releasing hormone
  • LHRH luteinizing hormone-releasing hormone
  • the testosterone levels fell below the castration level (0.5 ng/mL) within a week and remained under that level for 6-7 weeks following a single injection of leuprolide acetate self-encapsulated PLGA microspheres and 8 weeks after two injections (day 0 and 28) of Lupron DepotTM.
  • ASM-1 3.2% w/w Al(OH) 3 /3.5% w/w trehalose/0% w/w diethyl phthalate (DEP)/PLGA microspheres
  • ASM-2 3.2% w/w Al(OH) 3 /3.5% w/w trehalose/2.5% w/w DEP/PLGA microspheres
  • ASM-3 3.2% w/w Al(OH) 3 /3.5% w/w trehalose/5% w/w DEP/PLGA microspheres
  • active SM PLGA microspheres to actively self-microencapsulate very sensitive vaccine antigen (TT) after sterilization of blank microspheres with gamma radiations was investigated and compared with the results obtained prior to irradiation (Table 5). There was no significant difference in active loading of TT before and after sterilization of active SM PLGA microspheres, indicating the effectiveness of this novel strategy to self-encapsulate vaccine antigens after terminal sterilization of microspheres. For example, with 400 ⁇ g initial incubation mass of TT from 0.8 mg/mL loading solution, all the active SM PLGA microsphere formulations self-encapsulated TT equivalent to about 1.6% w/w polymer loading and 87% encapsulation efficiency (3 measurements).
  • SM PLGA microspheres with respectively 3.2, 3.5, and 5% w/w of Al(OH) 3 , trehalose and DEP were found to be an optimal formulation for active self-healing microencapsulation of protein at physiological temperature (37° C.) with high encapsulation efficiency.
  • DEP successful employment of DEP to reduce the required temperature for self-healing opens-up the door to self-microencapsulate temperature-sensitive molecules in higher M w PLGA at or below physiological temperature.
  • ASM-3 PLGA microspheres (3.2% w/w Al(OH) 3 /3.5% w/w trehalose/5% w/w DEP/PLGA microspheres) largely retained OVA (i.e., 60-73%) after 1-day of exposure to the citrate buffer, whereas unencapsulated Al(OH) 3 released all the protein (97 ⁇ 0.8% release).
  • ASM-3 PLGA microspheres released OVA slowly in a controlled manner over a period of 10 days (48 ⁇ 4.4% release (three measurements) after 10 days), indicating an effective active self-encapsulation of protein in Al(OH) 3 —PLGA microspheres.
  • OVA monomer or antigenic TT from ASM-3 PLGA microspheres (3.2% w/w Al(OH) 3 /3.5% w/w trehalose/5% w/w DEP/PLGA microspheres) was also significantly different (p ⁇ 0.05) than unencapsulated Al(OH) 3 ( FIGS. 3C and D).
  • OVA-Al(OH) 3 control gel exhibited 76, 90, and 99% OVA monomer release and TT-Al(OH) 3 control gel exhibited 87, 95, and 98% antigenic TT release respectively after 1, 3, and 7 days.
  • ASM-3 PLGA microspheres exhibited very less initial burst (17% OVA monomer or 32% antigenic TT release after 1 day) and provided slow and continuous release of OVA monomer or antigenic TT over a period of 28 days (49 and 68% OVA monomer or 83 and 99% antigenic TT release respectively after 14 and 28 days). After 28 days of release, 19 ⁇ 3% soluble OVA monomer and 10 ⁇ 2% insoluble OVA aggregate (covalent and non-covalent) was recovered from ASM-3 PLGA microspheres with a total recovery of 98 ⁇ 3% (three measurements).
  • injectable PLGA microspheres represent an approach to control the release of vaccine antigens to reduce the number of doses in the immunization schedule and optimize the desired immune response via selective targeting of antigen to antigen presenting cells.
  • the present technology provides delivery systems having high encapsulation efficiencies for one or more various agents.
  • the delivery system can include an ionic affinity trap, such as a metal salt, in combination with a solid polymer matrix to enable a large portion or substantially the entire amount of an agent outside the polymer to enter pores in the polymer, for example, where the agent is subsequently encapsulated following self-healing of the polymer.
  • an ionic affinity trap such as a metal salt
  • the delivery system includes a solid polymer matrix.
  • the solid polymer matrix can include a polymer, such as a self-healing polymer, and can include one or more pores, an ionic affinity trap, an agent, and anything else associated with the delivery system.
  • the solid polymer matrix can include a self-healing polymer that includes one or more pores including an ionic affinity trap that can be used to sorb an agent, where the pores are then partially or completely closed to encapsulate the agent and prevent it from leaving the pore(s).
  • the polymer can include a porous self-healing polymer that is able to alter its shape following a treatment.
  • the delivery system comprises a self-healing polymer, where a portion of the self-healing polymer comprises ionized end groups and an agent sorbed to the ionized end groups.
  • the self-healing polymer can be biodegradable and can degrade or erode over time.
  • the ionic affinity trap may also act as an adjuvant in some cases.
  • the delivery system can further include the use of a plasticizer to plasticize the polymeric material and manipulate the temperature at which self-healing begins to occur. Control of these properties can be important for encapsulation of certain agents that may be damaged at elevated temperatures (e.g., 43° C. or higher) commonly used with moderate molecular weight self-healing polymers.
  • the delivery system can include a porous self-healing polymer, optionally a differentially soluble material such as a saccharide or disaccharide, and one or more ionic affinity traps and plasticizers.
  • the differentially soluble material can be employed to obtain a porous self-healing polymer network and/or to stabilize the polymeric material.
  • the ionic affinity trap and plasticizer can improve the encapsulation efficiency and the self-healing property of the polymer, respectively.
  • a self-healing polymer having a porous network, such as PLGA microspheres, can be prepared using established methods, such as those described in U.S. Pat. Appl. Pub.
  • a solution comprising an agent is placed in contact with a self-healing polymer having pores or one that can form pores when in contact with the solution.
  • the self-healing polymer experiences a condition that causes spontaneous polymer chain rearrangement, which in turn causes the accessible pores (pores having access to the polymer surface) to close.
  • the agent becomes entrapped, encapsulated, or absorbed within the self-healing polymer when these pores close.
  • the self-healing polymer can be: poly(dicyclopentadiene); poly(dimethyl siloxane); poly(diethoxy siloxane); furan-maleimide-based polymers; dicyclopentadiene-based polymers; anthracene-maleimide based polymers; 1,1,1-tris-(cinnamoyloxymethyl)ethane (TCE)-based polymers; poly(ethylene-co-butylene); methyl methacrylate (MMA) embedded polypropylene fibers; epoxy with a urea-formaldehyde microcapsule; ionomers including hydrocarbon polymers bearing pendant carboxylic acid groups that are either partially or completely neutralized with metal or quaternary ammonium ions (e.g., Surlyn 8920, Surlyn 8940, Nucrel 960, and Nucrel 925); epoxy resins-diglycidyl ether of bisphenol A, diglycidyl ether of bisphenol F;
  • self-healing polymeric materials having reactive furfuryl functionality including poly(furfuryl methacrylate) and poly(furfuryl methacrylate)-co-poly(methyl methacrylate), as described by Kavitha et al., Applied Materials & Interfaces, vol. 1, no. 7, 1427-1436 (2009).
  • self-healing polymeric materials based on furan-functionalized, alternating thermosetting poly ketones and bis-maleimide as described by Zhang et al., Macromolecules 2009, 42, 1906-1912.
  • the polymeric material can also be a biodegradable material.
  • the polymeric material can be in several forms, including particles such as particles, including microspheres, and various shapes of tissue engineering scaffolds.
  • the self-healing polymer can be copolymers of lactic acid and glycolic acid (PLGA) and related copolymers, including any polymer containing a polyester with lactic and/or glycolic acid repeat units.
  • the polymers may be made using any method, and may be linear, star, branched, cross-linked, or any configuration so long as the polymer has lactic and/or glycolic repeat units, which may be liberated by hydrolysis.
  • the pore-containing polymers may be preformed prior to the encapsulation step; i.e., the microparticles, microspheres, tissue engineering scaffold, nanoparticles, drug-eluting stent, suture, or screw may be formed according to known methods prior to contact with the agent to be encapsulated.
  • PLGA is a polyester composed of one or more of three different hydroxy acid monomers, d-lactic, l-lactic, and/or glycolic acids.
  • the polymer can be made to be highly crystalline (e.g., poly(l-lactic acid)), or completely amorphous (e.g., poly(d,l-lactic-co-glycolic acid)), can be processed into most any shape and size (e.g., down to ⁇ 200 nm), and can encapsulate molecules of virtually any size.
  • PLGA microspheres and other injectable implants have an established safety record and are used in several different marketed products from various companies worldwide. For example, these controlled-release products are capable of controlling the release of peptides and proteins slowly and continuously from about 1 to 6 months, or even longer.
  • PLGA microparticles In addition to the depot effect, smaller PLGA microparticles (e.g., less than 10 ⁇ m) have demonstrated adjuvant activity via their uptake by macrophages and dendritic cells (DCs), and their localization in lymph nodes, and to induce cytotoxic T lymphocyte (CTL) responses.
  • DCs macrophages and dendritic cells
  • CTL cytotoxic T lymphocyte
  • the mild inflammatory response produced by PLGA microspheres is hypothesized as being involved in their adjuvant characteristics. Most significant are reports of long-lasting antibody responses, many neutralizing above protective levels, in numerous animal models following a single dose of PLGA microparticle encapsulated antigens including displays of immunological memory after 1 year of immunization and protection against challenge.
  • PLGA can be in the form of particles, such as microspheres, which can be prepared using a double emulsion-solvent evaporation microencapsulation method.
  • PLGA along with a saccharide or disaccharide, such as trehalose, and CH 2 Cl 2 can be homogenized at 10,000 rpm using a Tempest IQ2 homogenizer (The VirTis Company, Gardiner, N.Y.) equipped with a 10 mm shaft in an ice/water bath for 1 min to prepare the first emulsion.
  • a Tempest IQ2 homogenizer The VirTis Company, Gardiner, N.Y.
  • Two milliliters of 5% (w/v) PVA solution can then be immediately added to the first emulsion and the mixture vortexed (Genie 2, Fisher Scientific Industries, Inc., Bohemia, N.Y.) for 15 sec. to produce the w/o/w double emulsion.
  • the resulting emulsion can be poured into 100 mL of 0.5% (w/v) PVA solution under rapid stirring and hardened at room temperature for about 3 hours. Hardened microspheres can be collected by centrifugation, washed three times with purified water, and freeze-dried.
  • samples can be flash-frozen in liquid nitrogen and placed on a Freezone 6 freeze-drying system (Labcono, Kansas City, Mo.) at 133 ⁇ 10 ⁇ 3 mbar or less vacuum at a condenser temperature of ⁇ 46° C. for 48 hours.
  • Three percent MgCO 3 powder (w/w of polymer) can suspended in the polymer solution before encapsulation when ionic affinity trap-containing PLGA microspheres are desired.
  • microspheres can be first coated with gold for 200 sec. by a vacuum coater (Desk II, Denton Vacuum, Inc., Hill, N.J.). Microsphere morphology can then be observed using a scanning electron microscope (S3200N Variable Pressure SEM, Hitachi) with a voltage of 15 keV. For size distribution analysis, the size of more than 200 particles can be measured from SEM micrographs and the weight-averaged mean radius of the microspheres can be calculated. To observe the microsphere cross section, polymer specimens can be pre-cut by a razor blade on a glass slide before coating with gold.
  • the delivery system can be prepared as follows.
  • PLGA microparticles loaded with an ionic affinity trap e.g., Alhydrogel
  • an ionic affinity trap e.g., Alhydrogel
  • alhydrogel can be concentrated to the desired concentration by removing water.
  • Trehalose solutions double the required concentration
  • Alhydrogel and trehalose can then be mixed at 1:1 (v/v) ratio.
  • Polymer solution (1 mL) can be prepared by dissolving the required amount of PLGA (250 or 350 mg/mL) in methylene chloride.
  • the self-healing material comprising the self-healing polymer can be acceptably terminally sterilized (e.g., by gamma irradiation) with little or no loss in polymer molecular weight.
  • a sterile solution of agent and sterile microspheres of polymeric material can then be combined to effect encapsulation.
  • a sterile agent solution can be added to sterile and dry microspheres.
  • the polymer can be subjected to numerous stresses (e.g., excess heat, mixing, etc.) that normally cannot be used after loading the agent because certain agents (e.g., peptide/protein/DNA) may degrade under such conditions.
  • stresses e.g., excess heat, mixing, etc.
  • certain agents e.g., peptide/protein/DNA
  • the element of control over the ultimate polymeric material morphology and the kind of microsphere prepared can be vastly increased if encapsulation is performed after microsphere (scaffold) preparation.
  • the self-healing polymer can be in various configurations, and is not limited to particles or microspheres, for example.
  • the self-healing polymer can be formed in various shapes and articles of various sizes.
  • the self-healing polymer can be used as a polymer coating on drug-eluting stents, prepared as nanopartices, and formed into tissue engineering scaffolds, including shapes that replace portions of tissue or shapes that conform to various tissues.
  • the materials and methods provided herein are also applicable to self-healing materials used as tissue engineering scaffolds, as well as any type of biomaterial or any other polymer encapsulation system (e.g., agricultural) that requires the need to encapsulate molecules that do not strongly partition into the polymer phase, but instead are encapsulated within the pores of a self-healing polymer.
  • This is particularly useful for aqueous-based materials such as biological materials present in their native state in aqueous solution.
  • the microspheres can be incubated in an aqueous solution comprising an agent, where the agent can be at a fairly low concentration (e.g., about 0.5 to 1 mg/mL).
  • the aqueous mixture of polymeric material and agent can then be incubated between about 10° C. to about 43° C. over a period of time, for example, ranging from hours to days.
  • the porous network of the polymeric material heals thereby encapsulating the agent with a high encapsulation efficiency (e.g., >99%).
  • Incubation temperature for loading the agent into the delivery system varies as the composition of the polymeric material varies; e.g., with or without an plasticizer.
  • the incubating temperature can be tuned from about 10° C. to about 43° C. to achieve a very high encapsulation efficiency of the agent and to improve the self-healing property of the polymer.
  • plasticizer for example, self-healing of a moderate molecular weight polymer can occur readily at about 37° C. instead of about 43° C.
  • the self-healing polymer can be combined with a solution comprising an agent at a relatively low concentration, where nearly the entire amount of agent is taken up into the polymer pores, and the agent is then encapsulated within the polymer following self-healing of the polymer, where each step includes applying an appropriate temperature adjustment.
  • the encapsulation efficiency (weight of agent encapsulated/weight agent in solution exposed to polymer) is greater than 50%. In some embodiments, the encapsulation efficiency is greater than 60%. In some embodiments, the encapsulation efficiency is greater than 70%. In some embodiments, the encapsulation efficiency is greater than 80%. In some embodiments, the encapsulation efficiency is greater than 90%. In some embodiments, the encapsulation efficiency is greater than 95%. And in some embodiments, the encapsulation efficiency is greater than 99%.
  • a differentially soluble material such as a saccharide or similar material, can be used in forming the pores in the self-healing polymeric material.
  • a saccharide or similar material can be used in forming the pores in the self-healing polymeric material.
  • the saccharide portion can be dissolved without dissolving the polymer to leave empty pores in the polymer.
  • Useful saccharides include disaccharides such as trehalose, sucrose, and lactose.
  • Other saccharides that can be used include polysaccharides, such as dextran, and glycosaminoglycans, such as heparin.
  • the saccharide can be used to stabilize an ionomer gel used as the ionic affinity trap, for example, as described by A L Clausi, S A Merkley, J F Carpenter, T W Randolph, J Pharm Sci 97, 2049 (2008).
  • Other pore-forming saccharides that can be used include mannose and mannitol.
  • the differentially soluble material can include a water-soluble osmotic material in order to create the pores in a porous self-healing polymer.
  • Mg and Al salts can be used to create a percolating pore network; i.e., pores that interconnect with the surface of the polymer.
  • Other basic salts are described by Zhu et al. in Pharmaceutical Research, Vol. 17, No. 3, 2000.
  • useful components for making pores and/or stabilizing proteins include those described in S. E. Bondos, A. Bicknell, Analytical Biochemistry 316 (2003) 223-231.
  • Examples of such materials that may promote protein solubility include: kosmotropes including MgSO 4 at 0-0.4 M, NH 4 SO 4 at 0-0.3 M, Na 2 SO 4 at 0-0.2 M, Cs 2 SO 4 at 0-0.2 M; weak kosmotropes including NaCl 0-1 M, KCl 0-1M; Chaotropes including CaCl 2 0-0.2 M, MgCl2 0-0.2 M, LiCl 0-0.8 M, RbCl 0-0.8 M, NaSCN 0-0.2 M, NaI 0-0.4 M, NaClO4 0-0.4 M, NaBr 0-0.4 M, Urea 0-1.5 M; Amino acids including glycine 0.5-2%, L-arginine 0-5 M; sugars and polyhydric alcohols including Sucrose 0-1M, Glucose 0-2 M, Lactose 0.1-0.5 M, Ethylene glycol 0-60% v/v, Xylitol 0-30%
  • Ionic affinity traps used to sorb an agent in the present delivery systems include bases such as metal salts and metal hydroxides.
  • the ionic affinity trap may also be in the form of a gel and can include various ionomers.
  • aluminum hydroxide and calcium phosphate gels are known as ionomers.
  • aluminum hydroxide and aluminum phosphate are two such ionic affinity traps that are particularly useful.
  • Some ionic affinity traps may further act as an adjuvant to stimulate the immune system and increase the response to a vaccine.
  • the agent is a macromolecule such as a protein antigen
  • the ionic affinity trap may also perform as an adjuvant when the delivery system is used for vaccination.
  • the ionic affinity trap can include aluminum hydroxide and/or aluminum phosphate.
  • Aluminum hydroxide AlhydrogelTM, 2%) and aluminum phosphate (Adju-PhosTM) are available from Accurate Chemical and Scientific Corporation (Westbury, N.Y.). These aluminum materials can also act as adjuvants when the present delivery system is used with or as a vaccine.
  • Aluminum hydroxide and aluminum phosphate can be prepared by exposing aqueous solutions of aluminum ions to alkaline conditions under very controlled circumstances, which in the case of aluminum phosphate takes place in the presence of phosphate ions.
  • ionic affinity trap Various soluble aluminum salts can be used for the production of the ionic affinity trap, but the experimental conditions—temperature, concentration and even rate of addition of reagents—can strongly influence the results.
  • Other metal salts that can be used as an ionic affinity trap include those disclosed in Zhu et al. in Pharmaceutical Research, Vol. 17, No. 3, 2000.
  • Colloidal or sub-colloidal suspensions of aluminum hydroxide can be characterized by particle size distribution, electrical charge, and the hydrated colloid nature of the precipitate formed. Alterations of the preparation recipe can give rise to various forms of aluminum hydroxide which differ with respect to their physico-chemical characteristics, stability and protein adsorption.
  • One model for the structure of aluminum hydroxide takes form in a ring-structure of six members, each member consisting of an Al 3+ surrounded by six coordinated water molecules in an octohedral shape.
  • the coordinated water molecules are oriented with the oxygen toward the aluminum ion.
  • the high charge of the Al 3+ is believed to weaken the bond between oxygen and hydrogen thus facilitating the removal of protons, especially under alkaline conditions.
  • Deprotonization thus facilitated by the alkalinity, is believed to lead to the initial formation of dimers by dihydroxyl bridges between octohedras and later to the formation of the six-membered ring-structure and even larger structures.
  • the ratio of aluminium to hydroxide approaches 1:3.
  • Al(OH) 3 is misleadingly simple.
  • the model thus described is a generalized model that does not consider crystalline forms or inclusion of other ions.
  • aluminium hydroxide precipitated from aluminium chloride can be described as Al(OH) 2.55 (Cl) 0.45 , existing as a polymer of ten fused six-membered rings and if precipitated from sulphate as Al(OH) 2.30 (SO4) 0.35 and based on three fused such rings.
  • the ionic affinity trap can bind agents such as proteins, including protein antigens, where the ionic affinity trap can be an aluminum salt adjuvant. Without the use of such adjuvants, proteins may be only weakly immunogenic.
  • Aluminum salts are currently the only adjuvants generally approved for use in vaccines for humans. Despite their approved use, the mechanism of action is still poorly understood. Among a variety of nonmutually exclusive proposed mechanisms, roles as depots for antigen induction of inflammatory responses and delivery of antigen into antigen presenting cells are proposed.
  • the two most common aluminum salts employed as adjuvants are the phosphate and hydroxide forms.
  • the salts themselves have been well characterized with aluminum hydroxide (AlhydrogelTM) can usually be found in a crystalline state, whereas aluminum phosphate can exist in an amorphous form.
  • AlhydrogelTM aluminum hydroxide
  • the points of zero charge are 4.0-5.5 and about 11 for the phosphate and hydroxide salts, respectively.
  • proteins seem to better adsorb to the oppositely charged salts through simple electrostatic effects, although apolar and ion displacement interactions may play a role as well.
  • the ionic affinity trap can be calcium phosphate.
  • Calcium phosphate can function as an adjuvant and can be used to potentiate the immune response of vaccines and to prepare adsorbed allergen extracts. It can be well tolerated and readily resorbed by the body and it is believed to potentiate the immune response by the depot mechanism whereby the antigen is adsorbed during the preparation of the vaccine and slowly released following administration. Calcium phosphate is also believed to act by presenting the adsorbed antigen to antigen presenting cells as a particulate antigen.
  • Calcium phosphate has a molecular composition close to Ca 3 (PO 4 ) 2 , where the calcium/phosphorus molar ratio (Ca/P) can vary from 1.35 to 1.83 depending on the rate of mixing during the precipitation reaction.
  • the properties of the precipitate are strongly dependent on the precipitation conditions. For example, calcium phosphate precipitated by rapid mixing can adsorb about 100% of diphtheria toxoid while calcium phosphate precipitated by slow mixing can adsorb about 58% of the same dose of diphtheria toxoid.
  • Another useful ionic affinity trap is aluminum phosphate.
  • the ionic affinity trap can be alum, which includes aluminum and potassium.
  • ionic affinity traps include extracellular matrix-like materials, including dextran sulfate, chitosan, and hyaluronic acid.
  • Layer-by-layer assembly based on charge. For example, start with a negatively charged agent, such as heparin, inside the polymer pores and then bind heparin-binding growth factors; e.g., fibroblast growth factors and vascular endothelial growth factors. Alternating incubations of growth factor and heparin create a network of growth factor stabilized in between heparin layers; i.e., heparin-growth factor-heparin-growth factor and so on.
  • a negatively charged agent such as heparin
  • heparin-binding growth factors e.g., fibroblast growth factors and vascular endothelial growth factors.
  • Alternating incubations of growth factor and heparin create a network of growth factor stabilized in between heparin layers; i.e., heparin-growth factor-heparin-growth factor and so on.
  • nucleating agent for crystallization inside the polymer at a concentration above saturation; e.g., using features as described in U.S. Pat. No. 5,869,604. This includes using exogenous nucleating agents such as minerals, transition metal ions such as copper and lead, highly absorbent structures such as zeolites, preformed crystal seeds of amino acids, and preformed crystal seeds of polypeptides other than the agent being loaded into the pores.
  • Creating a Donnan equilibrium e.g., charged species that can not escape the pores
  • a pH gradient precipitating the protein at its isoelectric pH.
  • a plasticizer can be used to plasticize the polymeric material and manipulate the temperature at which self-healing begins to occur.
  • Useful plasticizers include diethyl phthalate, tributyl acetylcitrate, and similar compounds.
  • Other useful plasticizers include those that (1) cause a pore network to form (e.g., by osmotic pressure), (2) stabilize the encapsulated molecule, and (3) cause the encapsulated molecule to preferentially distribute inside the polymer pores (e.g., either in solution, in solid state, or sorbed to a structure of some kind) relative to the outside solution.
  • the agent to be encapsulated may be any material, compound, or biomolecule of interest that can associate with the ionic affinity trap.
  • the methods provided herein are particularly useful for agents that would be subject to degradation when exposed to conditions used in preparing pore-containing polymers.
  • agents include biomolecules such as proteins, peptides, proteoglycans, lipoproteins, and nucleic acids, such as RNA and DNA.
  • the agent can be a small molecule or a large colloidal particle (e.g., virus), or any bioactive substance, such as a biomacromolecule.
  • the agent to be encapsulated should have an affinity for the ionic affinity trap.
  • the agent can be present at a low concentration (e.g., 1 mg/mL or lower) so that the ionic affinity trap acts to bind and effectively load the porous polymeric material with the agent. Loading of the porous polymeric material is therefore not dependent on passive diffusion, which typically requires a high concentration of agent to be loaded in order to obtain the desired loading level.
  • the present technology may also be used for small molecules, e.g., drugs used in drug-eluting stents or in nanoparticle delivery. Hydrophilic molecules can be problematic to encapsulate in drug-eluting stents by conventional methods.
  • the present technology may also be used to encapsulate nanoparticulate materials (e.g., viruses) without drying the polymer. For example, some materials may be denatured or degraded in whole or part by a drying process and hence the present methods can avoid drying is such instances.
  • PLGA microspheres loaded with ovalbumin can be prepared as follows to demonstrate an embodiment of a delivery system constructed in accordance with the present technology.
  • the effects of formulation and incubation parameters on agent loading were ascertained indicated in Tables 6-13.
  • Ovalbumin is used as a model to demonstrate loading of a protein antigen, for example. The following parameter effects were determined.
  • FIGS. 9-11 Self-healing of microparticles loaded with ovalbumin is further illustrated in FIGS. 9-11 .
  • FIG. 9 depicts 3.2 wt % Alhydrogel/3.8 wt % Trehalose/PLGA microparticles before (A) and after (B) self-healing by incubating at about 25° C. for about 24 hours and about 43° C. for about 48 hours.
  • FIG. 10 depicts 3.2 wt % Alhydrogel/3.8 wt % Trehalose/2.5 wt % DEP/PLGA microparticles before (A) and after (B) self-healing by incubating at about 10° C. for about 48 hours, at about 25° C. for about 24 hours, and at about 37° C. for about 30 hours.
  • FIG. 11 depicts 3.2 wt % Alhydrogel/3.8 wt % Trehalose/5 wt % DEP/PLGA microparticles before (A) and after (B) self
  • a delivery system is used to microencapsulate Leuprolide, a potent agonistic analogue of luteinizing hormone-releasing hormone, inhibits the secretion of pituitary gonadotropin when administered chronically in therapeutic doses.
  • Microsphere depot formulations of leuprolide can be developed for long-term testosterone suppression.
  • the present technology can employ “sorption loading.” Basically, this involves taking ground PLGA of relatively low MW (from the manufacturer), which has ionized end group (in this case carboxylate, but could be made anything), and then incubating low peptide solution concentrations. The ionic interaction causes the peptide to “sorb” to the polymer. The full nature of this sorption is not fully understood, other than it requires the ionized end group and a high enough temperature for polymer chain mobility. It could be mostly at the surface, i.e., adsorption, or mostly in the bulk, i.e., absorption.
  • the sorption group in FIG. 13 is generated with injections every two, three, or four weeks at the same dosage as the Lupron Depot.
  • the polymer is of low MW and takes up more water than the polymers generated for most self-healing microencapsulation methods.
  • we expect the peptide sorbs by penetration directly into the polymer phase and combines with carboxylic end groups (the ionic affinity trap), as the acid end-group PLGA takes up sufficient water for peptide permeation.
  • the efficacy of LA-PLGA particles to provide long-term in vivo LA release was evaluated by assessing long-term testosterone suppression ability of LA-PLGA particles in male Sprague-Dawley rats.
  • the treatment of experimental animals was in accordance with University committee on use and care of animals (University of Michigan UCUCA), and all NIH guidelines for the care and use of laboratory animals.
  • Male Sprague-Dawley rats of 6 weeks old were housed in cages and given free access to standard laboratory food and water, and allowed one week to acclimate prior to study initiation. Animals were anesthetized with 2-4% isoflurane administered by a calibrated vaporizer (Midmark, Orchard Park, N.Y., USA).
  • the leuprolide acetate (1 ⁇ ) and LA-PLGA particles (2 ⁇ (day 0, 14, 28, and 42), 3 ⁇ (day 0, 21, and 42), and 4 ⁇ (day 0 and 28) in a liquid vehicle (1% w/v carboxymethylcellulose and 2% w/v mannitol), and commercial Lupron Depot (2 ⁇ (day 0 and 28)) were subcutaneously injected at the back (lower neck portion) of rats (6 animals/study group).
  • the dose of leuprolide acetate was 100 ⁇ g/kg/day.
  • Animal body weight considered for dosing leuprolide acetate was 425 g which is projected body weight of male Sprague Dawley rat at midpoint (day 28) of the study (as per the weight (g)/age (weeks) curve given by Charles River Laboratories).
  • Blood samples were collected via jugular vein stick before (day ⁇ 7 and 0 for baseline testosterone level) and after (1, 7, 14, 21, 28, 35, 42, 49, and 56 days) injection of preparations. The collected blood samples were immediately transferred to B-D MicrotainerTM blood collection and serum separation tubes previously incubated in ice, centrifuged at 8,000 rpm for 10 min, and then the serum was removed and stored in microcentrifuge tubes at ⁇ 20° C. until further use.
  • Serum testosterone levels were assayed by Radioimmunoassay using a TESTOSTERONE Double Antibody-125I RIA Kit (MP Biomedicals LLC., Solon, OH, USA) at the University of Pennsylvania Diabetes Center (Philadelphia, Pa., USA). Lowest detection limit of testosterone was 0.1 ng/mL.
  • LA leuprolide acetate
  • the residual particles were washed three times with deionized water (1 mL water/10 mg particles) and then freeze-dried.
  • LA absorbed PLGA particles were passed through sieves to obtain 20-63 ⁇ m and stored at ⁇ 20° C. until further use.
  • LA leuprolide acetate
  • Benefits of the current technology include: a) the ability to prepare agent encapsulated products in a straightforward manner, b) reduction in the cost of manufacturing as loss of expensive agents during encapsulation is reduced and a smaller quantity of agent (about 0.5 to 1 mg/mL) can be used to achieve very high encapsulation efficiency (99% or more), c) can be used with multiple agents, d) can be used at point-of-care, and e) allows terminal sterilization of the delivery system prior to agent loading (i.e., no aseptic manufacturing with organic solvents is required).
  • Example embodiments are provided so that this disclosure will be thorough, and will fully convey the scope to those who are skilled in the art. Numerous specific details are set forth such as examples of specific components, devices, and methods, to provide a thorough understanding of embodiments of the present disclosure. It will be apparent to those skilled in the art that specific details need not be employed, that example embodiments may be embodied in many different forms, and that neither should be construed to limit the scope of the disclosure. In some example embodiments, well-known processes, well-known device structures, and well-known technologies are not described in detail. Equivalent changes, modifications and variations of some embodiments, materials, compositions and methods can be made within the scope of the present technology, with substantially similar results.
  • the words “desire” or “desirable” refer to embodiments of the technology that afford certain benefits, under certain circumstances. However, other embodiments may also be desirable, under the same or other circumstances. Furthermore, the recitation of one or more desired embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the technology.
  • the word “include,” and its variants, is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the materials, compositions, devices, and methods of this technology.
  • the terms “can” and “may” and their variants are intended to be non-limiting, such that recitation that an embodiment can or may comprise certain elements or features does not exclude other embodiments of the present technology that do not contain those elements or features.
  • compositions or processes specifically envisions embodiments consisting of, and consisting essentially of, A, B and C, excluding an element D that may be recited in the art, even though element D is not explicitly described as being excluded herein.
  • compositional percentages are by weight of the total composition, unless otherwise specified. Disclosures of ranges are, unless specified otherwise, inclusive of endpoints and include all distinct values and further divided ranges within the entire range. Thus, for example, a range of “from A to B” or “from about A to about B” is inclusive of A and of B. Disclosure of values and ranges of values for specific parameters (such as temperatures, molecular weights, weight percentages, etc.) are not exclusive of other values and ranges of values useful herein. It is envisioned that two or more specific exemplified values for a given parameter may define endpoints for a range of values that may be claimed for the parameter.
  • Parameter X is exemplified herein to have value A and also exemplified to have value Z, it is envisioned that Parameter X may have a range of values from about A to about Z.
  • disclosure of two or more ranges of values for a parameter (whether such ranges are nested, overlapping or distinct) subsume all possible combination of ranges for the value that might be claimed using endpoints of the disclosed ranges.
  • Parameter X is exemplified herein to have values in the range of 1-10, or 2-9, or 3-8, it is also envisioned that Parameter X may have other ranges of values including 1-9, 1-8, 1-3, 1-2, 2-10, 2-8, 2-3, 3-10, and 3-9.
  • “A” and “an” as used herein indicate “at least one” of the item is present; a plurality of such items may be present, when possible. “About” when applied to values indicates that the calculation or the measurement allows some slight imprecision in the value (with some approach to exactness in the value; approximately or reasonably close to the value; nearly). If, for some reason, the imprecision provided by “about” is not otherwise understood in the art with this ordinary meaning, then “about” as used herein indicates at least variations that may arise from ordinary methods of measuring or using such parameters.

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WO2021016211A1 (fr) 2019-07-19 2021-01-28 The Regents Of The University Of Michigan Matrice polymère poreuse autocicatrisante pour l'encapsulation de macromolécules actives et procédés
WO2024054879A1 (fr) 2022-09-06 2024-03-14 Regents Of The University Of Michigan Particules polymères biodégradables pour l'administration d'agents thérapeutiques chargés positivement

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WO2011088229A3 (fr) 2012-01-05

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