A cost-effective, scalable technique for producing microspheres loaded with biologically active solid proteins is provided. The microspheres degrade over time and release biologically active VEGF, as demonstrated by the proliferation of HUVECs in vitro compared to negative controls. A defined concentration of microspheres can deliver a quantifiable level of VEGF with known release kinetics. The invention can be used with other growth factors and applied to tissue engineering applications such as the regeneration of peripheral nerve, bone, adipose tissue, and solid organs. The method of the invention includes the steps of dissolving a polymer with an organic solvent to produce a polymer solution; adding a biologically effective amount of a bioactive substance to the solution to produce a mixture of the polymer and the bioactive substance; vibrating the mixture to produce a bioactive substance-polymer complex; emulsifying the mixture to produce an emulsion comprising the bioactive substance-polymer complex; and extracting the organic solvent from the emulsion to produce microspheres comprising the polymer-bioactive substance complex, wherein the bioactivity of the bioactive substance is usefully preserved.

SUMMARY OF THE INVENTION

The present invention solves the problems in the prior art just described by providing a solid-encapsulation/single-emulsion/solvent extraction technique to encapsulate solid proteins and other bioactive substances into biodegradable microspheres. The present invention provides a cost-effective, scalable technique for producing microspheres loaded with biologically active proteins, and in particular solid proteins, together with compositions of such microspheres.

One method for making microspheres of the present invention that encapsulate and effect the controlled release of a bioactive substance includes the steps of dissolving a polymer with an organic solvent to produce a polymer solution, adding a biologically effective amount of a bioactive substance to the solution to produce a mixture of the polymer and the bioactive substance, vibrating the mixture to produce a bioactive substance-polymer complex, emulsifying the mixture to produce an emulsion of the bioactive substance-polymer complex and the organic solvent, and extracting the organic solvent from the emulsion to produce microspheres comprising the polymer-bioactive substance complex, so that the bioactivity of the bioactive substance is usefully preserved.

The present invention provides a system for delivering a therapeutic agent to tissue. The system includes biodegradable microspheres made as described above and encapsulating a therapeutic agent as the bioactive substance in a dispenser for administration of the microspheres to the tissue so that the microspheres release the therapeutic agent from the microspheres to the tissue.

The present invention further provides a drug delivery system. The drug delivery system includes biodegradable microspheres incorporating a drug made as previously described and a dispenser for administration of the microspheres. The release of the therapeutic agent or the drug from the microspheres occurs in two phases: an initial burst phase and a later steady-state phase.

In addition to methods and systems, the present invention provides microsphere compositions. Microspheres of the present invention contain bioactive substances such as polypeptides, therapeutic agents or drugs. A particular feature of the present compositions is that bioactive substances in their solid state, not dissolved in solution, may be contained in the microspheres with the bioactivity of the substance preserved. Another feature of the present invention is that solid bioactive substances are incorporated into microspheres without using the atomization-freeze process familiar to those skilled in the art. Further, the present invention does not make use of a tissue homogenizer or an ultrasonic atomizer.

DETAILED DESCRIPTION OF THE INVENTION

While the making and using of the various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts which can be embodied in a wide variety of specific contexts. The specific embodiments described herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention nor the scope of the claims appended hereto.

A bioactive substance, as defined herein, is an agent, or its pharmaceutically acceptable salt, which possesses therapeutic, prophylactic or diagnostic properties in vivo or which influences the biological structure, function, or activity of a cell or tissue of a living organism. Examples of suitable therapeutic and/or prophylactic biologically active agents include, for instance, proteins such as immunoglobulin-like proteins, antibodies, cytokines (e.g., lymphokines, monokines, chemokines), interleukins, interferons, erythropoietin, nucleases, tumor necrosis factor, colony stimulating factors, immunoadjuvants, insulin, enzymes, tumor suppressors, hormones (e.g., growth hormone and adrenocorticotrophic hormone), antigens (e.g., bacterial and viral antigens), growth factors; peptides; polypeptides; proteins, nucleic acids such as antisense molecules; and small molecules such as antibiotics, steroids, decongestants, neuroactive agents, anesthetics, sedatives, anti-tumor agents, cardiovascular agents, antineoplastics, antihistamines, hormones and vitamins. Examples of suitable diagnostic and/or therapeutic bioactive agents include radioactive isotopes and radiopaque agents.

A microsphere, as defined herein, includes a particle of a biocompatible solid-phase material having a diameter of about one millimeter to about one micrometer, or less, wherein the particle may contain a biologically active agent and, wherein the solid-phase material sustains the in vivo release of the biologically active agent from the microsphere. A microsphere can have a spherical, non-spherical or irregular shape. The typical microsphere shape is generally spherical.

A biocompatible material, as defined herein, means that the material, and any degradation products of the material, is non-toxic to the recipient and also presents no significant deleterious or untoward effects on the recipient's body, such as an immunological reaction at the injection site.

Particles of a biologically active agent include, for example, crystalline particles, non-crystalline particles, freeze dried particles and lyophilized particles. The particles may contain only the biologically active agent or may also contain one or more stabilizing agents and/or other excipients.

Typically, the solid-phase material of the microsphere is a biocompatible polymer that may be either a biodegradable polymer, a non-biodegradable polymer, blends thereof or copolymers thereof.

Biodegradable, as defined herein, means the polymer will degrade or erode in vivo to form smaller chemical species. Degradation can result, for example, by enzymatic, chemical and/or physical processes. Suitable biocompatible, biodegradable polymers include, for example, poly(lactide)s, poly(glycolide)s, poly(lactide-co-glycolide)s, poly(lactic acid)s, poly(glycolic acid)s, poly(lactic acid-co-glycolic acid)s, polycaprolactone, polycarbonates, polyesteramides, polyanhydrides, poly(amino acids), polyorthoesters, polyacetyls, polycyanoacrylates, polyetheresters, poly(dioxanone)s, poly(alkylene alkylate)s, copolymers of polyethylene glycol and polyorthoester, biodegradable polyurethanes, hydrogels, blends and copolymers thereof.

Biocompatible, non-biodegradable polymers suitable for the methods and compositions of the present invention include non-biodegradable polymers selected from the group consisting of polyacrylates, polymers of ethylene-vinyl acetates and other acyl substituted cellulose acetates, non-degradable polyurethanes, polystyrenes, polyvinyl chloride, polyvinyl fluoride, poly(vinyl imidazole), chlorosulphonate polyolefins, polyethylene oxide, hydrogels, blends and copolymers thereof.

Further, the terminal functionalities of a polymer can be modified. For example, polyesters may be blocked, unblocked or a blend of blocked and unblocked polymers. A blocked polyester is as classically defined in the art, specifically having blocked carboxyl end groups. Generally, the blocking group is derived from the initiator of the polymerization and is typically an alkyl group. An unblocked polyester is as classically defined in the art, specifically having free carboxyl endgroups.

Acceptable molecular weights for polymers used in the present invention may be determined by a person of ordinary skill in the art accounting for factors such as the desired polymer degradation rate, physical properties such as mechanical strength and rate of dissolution of polymer in solvent. Typically, an acceptable range of molecular weights is of about 2,000 Daltons to about 2,000,000 Daltons. In one embodiment, the polymer is a biodegradable polymer or copolymer. In another embodiment, the polymer is a poly(lactide-co-glycolide) (hereinafter "PLGA") with a lactide:glycolide ratio of about 1:1 and a molecular weight of about 5,000 Daltons to about 70,000 Daltons. In yet another embodiment, the PLGA used in the present invention has a molecular weight of about 5,000 Daltons to about 42,000 Daltons.

The invention is now described in an example demonstrating the successful microencapsulation of a solid polypeptide growth factor and the controlled released of the factor to stimulate cell proliferation. Reference is made to King T. W., and Patrick, C. W., Jr., "Development and in Vitro Characterization of Vascular Endothelial Growth Factor (VEGF)-loaded Poly(dl-lactic-co-glycolic Acid)/poly(ethylene Glycol) Microspheres Using a Solid Encapsulation/single Emulsion/solvent Extraction Technique," J. Biomed. Mater. Res., September 2000; 51(3):383-390, the disclosure of which is incorporated herein by reference. Reference is also made to King, Timothy W., "Enhanced in Vivo Angiogenesis Within a Model Tissue Engineered Construct Using Biodegradable Microspheres Containing Encapsulated VEGF," Ph.D. Dissertation, The University of Texas M.D. Anderson Cancer Center and The University of Texas Graduate School of Biomedical Sciences, Houston, Tex. May, 2000, the disclosure of which is also incorporated herein by reference.

The field of tissue engineering is one application that may benefit from the development of a therapeutic delivery system. Tissue engineering techniques offer a potential means to develop autologous tissues for repair of primary tissue defects, regardless of whether the defect is a solid organ, soft tissue, or bony tissue. The appropriate regulation of angiogenesis, however, is critical to the incorporation of a vascular network into a viable engineered tissue. Vascular endothelial growth factor (VEGF) is a potent stimulator of angiogenesis. Several investigators have demonstrated the ability of VEGF to stimulate therapeutic angiogenesis in vivo. One embodiment of the present invention is a controlled-release VEGF delivery system that regulates angiogenesis within tissue-engineered constructs. To illustrate the invention, the manufacture, characterization, in vitro degradation, and bioactivity of VEGF microspheres over a 28-day course are described.

One modality of controlled delivery of biologically active molecules to a tissue is microspheres of Poly(DL-lactide-co-glycolide) (PLGA)/polyethylene glycol (PEG). Tissues to which a bioactive substance may be delivered include, but are not limited to, engineered tissue for use in plastic and reconstructive surgery. Engineered tissue requires a blood supply for the engineered tissue to successfully integrate with natural tissue and to achieve the pliability of natural tissue. Angiogenesis is the process of the growth of blood vessels into tissue to supply the tissue with blood. To stimulate angiogenesis within an engineered tissue, vascular endothelial growth factor (VEGF) and bovine serum albumin (BSA) were coencapsulated into microspheres fabricated from PEG and 50/50 PLGA using a solid-encapsulation/single-emulsion/solvent extraction technique of the present invention.

Two VEGF/BSA ratios were studied: 1:2000 and 1:10,000. The studies include analysis of the loading efficiency and particle size distribution, bright-field microscopy, scanning electron microscopy, release kinetics, and an in vitro human umbilical vein endothelial cell (HUVEC) proliferation assay to assess biological activity of the released VEGF.

The microspheres were manufactured, stored, and degraded over 28 days. The burst release rates for 1:2000 and 1:10,000 VEGF/BSA microspheres were 71.87.+-.8.11 and 27.91.+-.1.71 ng/mL (mean.+-.standard error of the mean), respectively; steady-state release rates were 6.56.+-.1.10 and 2.21.+-.0.47 ng/mL, respectively. The microspheres released biologically active VEGF, and the VEGF increased the proliferation of HUVECs in culture (p.ltoreq.0.05). The data obtained from these studies demonstrate that a defined concentration of microspheres of the invention will deliver a quantifiable level of VEGF at a known release rate.

Microsphere production

To illustrate the solid-encapsulation/single-emulsion/solvent extraction technique of the present invention, 500 mg of 50/50 poly(DL-lactide-co-glycolide) (PLGA, inherent viscosity 0.61 dL/g in HFIP at 30.degree. C.) (Birmingham Polymers, Birmingham, Ala.) and 5 mg of polyethylene glycol (PEG; Mw: 4600) (Aldrich, Milwaukee, Wis.) were dissolved in 2 mL of methylene chloride (Fisher, Fair Lawn, N.J.) creating a 25% (w/v) solution. PEG was added to increase the degradation rate of the microspheres.

After allowing the polymers to dissolve, 100 mg of bovine serum albumin (BSA) powder (Sigma, St. Louis, Mo.) were added to the polymer solution and vortexed vigorously (Fisher, Fair Lawn, N.J.) at a medium-high setting for 30 seconds. Immediately, 10 mL of 0.3% (w/v) polyvinyl alcohol (PVA) were added to the protein--polymer solution and vortexed vigorously at a medium-high setting for an additional 30 seconds. The emulsion was added to a beaker containing an additional 90 mL of 0.3% PVA and 100 mL of 2% isopropyl alcohol and continuously stirred for 90 minutes at room temperature, allowing the organic solvent to be extracted from the polymer--protein complex. The resultant microspheres were centrifuged and washed in distilled water multiple times, frozen to -80.degree. C., and lyophilized. For the degradation study, control microspheres containing no protein were fabricated using the same methodology. Microspheres containing rhVEGF165 (R&D Systems, Minneapolis, Minn.) were manufactured by coencapsulating the VEGF with BSA. Two different ratios of VEGF--BSA (w/w) were manufactured and characterized: 1:2000 and 1:10,000 (i.e., 50 and 10 mg of VEGF were added to the 100 mg of BSA, respectively).

Claim 1 of 6 Claims

1. A system for delivering a therapeutic agent to a subject, the system comprising: biodegradable microspheres comprising a biodegradable polymer and a biologically effective amount of a polypeptide therapeutic agent having usefully preserved bioactivity, wherein the microspheres are obtained from a single emulsion of the biodegradable polymer, the polypeptide therapeutic agent, and an organic solvent, the method comprising: dissolving the biodegradable polymer with an organic solvent to produce a polymer solution; adding the polypeptide therapeutic agent to the polymer solution to produce a mixture of polymer and polypeptide therapeutic agent, wherein no additional processing medium is required; vibrating the mixture to produce a polypeptide therapeutic agent-polymer complex; emulsifying the mixture to produce an emulsion comprising the polypeptide therapeutic agent-polymer complex; and extracting the organic solvent from the emulsion to produce microspheres comprising the polypeptide therapeutic agent-polymer complex, wherein no additional emulsification or mechanical agitation is performed; and a means for delivering the microspheres to the subject in need thereof.
 

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