Title:  Biocompatible polymeric delivery systems having functional groups attached to the surface thereof

United States Patent:  6,326,021

Assignee:  The Ohio State University Research Foundation (Columbus, OH)

Filed:  June 16, 2000

A method for making biocompatible polymeric matrices, particularly polymeric particles, that have functional groups on the surface thereof are provided. The method comprises: providing a biocompatible base polymer; providing a surface-active, functional polymer, hereinafter referred to as an "SAFP"; entangling chains of the base polymer with chains of the SAFP, both of which are in a mobile state; and then demobilizing the base polymer chains to form a polymeric particle or matrix having a specific geometry. Polymeric particles having functional groups on the surface thereof are also provided. The particles comprise a biocompatible base polymer and an SAFP. The SAFP comprises one or more interactive regions for physically cross-linking with the base polymer, and one or more hydrophilic regions. The particles have a core region and an outer region having a an outer surface. The core region contains a plurality of biocompatible base polymer chains. The outer region of the particle contains a plurality of biocompatible base polymer chains and the interactive regions of the SAFP. The hydrophilic functional region or regions of the SAFP chains extend from the surface of the particle when the particle is placed in an aqueous solution.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for making a polymeric matrix having functional groups on the surface thereof. The method comprises providing a base polymer; providing an SAFP; physically cross-linking chains of the SAFP with chains of the base polymer, and then de-mobilizing the base polymer chains to form a solid polymeric particle or porous or nonporous sheet of any moldable shape.

The base polymer is a biocompatible polymer. As used herein the term biocompatible refers to a polymer that is approved for use in the body by the Food and Drug Administration. Examples of biocompatible polymers are poly(ethylene-covinyl acetate), and silicone rubber cross linked to poly (dimethyl siloxan sulfoxide) and derivatives thereof. Preferably, the base polymer is biodegradeable and bioresorbable. As used herein the term biodegradeable refers to a base polymer that breaks down into oligomeric and/or monomeric units over a period of time, typically hours to months, when implanted or injected into the body of a mammal. As used herein, a bioresorbable polymer is one whose degradative products are metabolized in vivo or excreted from the body via natural pathways. Such polymers include, for example, a polymer from the linear polyester family, such as polylactic acid, polyglycolic acid or polycaprolactone and their associated copolymers, e.g. poly (lactide-co-glycolide) at all lactide to glycolide ratios, and both L-lactide or D,L lactide. Polymers such as polyorthoester, polyanhydride, polydioxanone and polyhyroxybutyrate may also be employed. Preferably, the base polymer is amorphous, i.e., it is not crystalline. It is also preferred that the base polymer not generate crystalline residues upon degradation in vivo. Preferably, the weight-averaged molecular weight of the base polymer is above 10,000 daltons. The polydispersity, I=Mw/Mn, is preferably less than 2.5.

The surface active, functional polymer (SAFP)is composed of a polymeric-backbone having an interactive region for physically cross-linking with the base polymer. Preferably, the backbone of the SAFP comprises a plurality of interactive regions. The interactive regions of the SAFP penetrate into the dispersed phase of an oil in water emulsion. The SAFP also comprises a hydrophilic region carrying one or more functional groups. Preferably the SAFP comprises a plurality of hydrophilic regions for extending from the surface of the base polymer when the final particle is placed into an aqueous solution. The hydrophilic regions are components of the backbone or are pendant groups which are attached to the backbone. The SAFP is a homopolymer or, preferably, a random co-polymer or a triblock co-polymer. Preferably, the weight-averaged molecular weight of the SAFP is from 4000 to 25,000 daltons. Preferably the functional groups are from 1-50% by weight of the SAFP. The SAFP is soluble at the interphase.

The SAFP further comprises functional groups which are covalently bonded to the backbone of the SAFP or pendant groups which are attached to the hydrophilic region of the SAFP backbone. The functional groups encompass conjugatable groups such as for example amines, hydroxyls, carbonyls, thiols, and carboxylic acids for covalently bonding of other bioactive molecules to the surface of the particle. The linkages formed following conjugation of the bioactive molecules to the conjugatable groups include amides, esters, and thioethers. Examples of SAFP which have conjugatable functional groups include (poly) lysine, acetylated poly (lysine); poly (glutanic acid, and poly(oxyethylene)-poly (oxyproplene) copolymers. The functional groups also encompass bioactive molecules such as for example, ligands, antibodies, peptides, nucleic acids and compounds that allow the particles to avoid the RES. The ligands are employed to target the particles to cells having receptors which interact with the ligand. One example of a ligand which may be attached to the backbone of the SAFP is the folate ligand which is used to target molecules to cancer cells. The antibodies target the particles to cells which overexpress antigens that are immunospecific for the antibodies. Examples of nucleic acids that are suitable functional groups are cDNA and RNA molecules that encode a gene product and antisense DNA. Examples of peptides are synthetic peptide vaccines. One example of a bioactive molecule that allows the particles to avoid the RES is polyethylene glycol (PEG).

The base polymer chains are interacted with the SAFP chains under conditions that mobilize both the base polymer and the SAFP chains and that allow the SAFP chains to become physically entangled with the base polymer chains. Thereafter the base polymer chains are de-mobilized to provide a polymeric particle or matrix of specific geometry, e.g., a sheet.

Preferably, an oil in water/emulsion and evaporation method is used to form particles. Alternatively, an oil in oil emulsion may be used. In the oil in water emulsion method the base polymer is mobilized by dissolving in a organic solvent, such as for example methylene chloride, to provide a solution of the base polymer. An aqueous solution containing the SAFP is added to a solution of the base polymer and the resulting mixture vigorously agitated for a time sufficient to form an oil in water emulsion. Preferably, the amount of SAFP added to the mixture is less than 0.5% (w/w) compared to the base polymer so the bulk properties of the polymer in the final polymeric particle will be minimally effected. More preferably, the amount of SAFP added is less than 0.05% (w/w) compared to the base polymer. The temperature of the reaction is from 5-40^oC. When an acetylated poly (lysine) is used as the SAFP, the preferred pH is about 8.0. When a non-acetylated poly (lysine) is used as the SAFP, the preferred pH is from about 10 to about 12.

The base polymer is then de-mobilized by evaporating the organic solvent. The resulting particles are microspheres or nanospheres, both of which are spherical receptacles comprising a polymeric matrix. Preferably, the microspheres have a diameter in the range of 200x10^-6 m to 0.5x10^-6 m range, more preferably 1-100 .mu.m. Preferably the nanospheres have a diameter in the range of 20x10^-9 m to 1000x10^-9 m, more preferably 50 to 200 nm.

Optionally, a drug is added to the polymer solution for encapsulation or incorporation into the particle. Suitable drugs which may be incorporated into the particle are a cytotoxic drug, such as for example doxorubicin; an anti-coagulant agent, such as heparin; an anti-oxidant such as vitamin E; compounds that regulate cellular proliferation, and anti-inflammatory drugs, such as corticosteroids. During formation of the particle, the drugs are incorporated into the bulk base polymer matrix.

The bioactive molecules, such as the ligands, the antibodies, and the peptides may be attached to the SAFP backbone prior to the time the SAFP is interacted with the base polymer. Alternatively, the bioactive molecules are attached to the particle via the conjugatable groups after formation of the particle.

Good results have been obtained using PLGA as the base polymer, an acetylated poly(L-lysine) as the SAPFP and an oil-in-water emulsion-solvent evaporation method.

Preferably, the SAFP is synthesized by exposing poly(L-lysine) to acetic anhydride.

Preferably, the molecular weight distribution of the poly(L-lysine) is 4,000 to 25,000. 30 Preferably, acetic anhydride is added in 300-fold to 700-fold excess compared to the lysine residues present (mol/mol) to obtain a partially acetylated poly(L-lysine) which has both hydrophilic and lipophilic characteristics. During microsphere formation, the lipophilic portion of the acetylated poly(L-lysine) is non-covalently connected to the microsphere through partial entanglement with the base polymer chains. The hydrophilic portion of each acetylated poly(L-lysine) molecule extends from the surface of the microsphere. The .epsilon.-amino groups of the unacetylated lysines in the hydrophilic portion of the acetylated poly(L-lysine) are available for conjugation.

Good results have also been obtained using PLGA as the base polymer and poly-(L lysine) as the SAFP. PLGA (0.20 dl/g, 50/50) was dissolved in 750 mg of CH₂ Cl₂ (1 ml). Poly-1-lysine was dissolved in 1.0 ml buffer (pH 10, 0.1 M Borax/NaOH) at a concentration of 0.5%. The polylysine solution was then added to the PLGA/CH₂ Cl₂ solution and vortexed for 15-20 seconds. The resulting emulsion was then quickly poured into 100 ml hardening buffer (pH 10, 0.1M Borax/NaOH) and stirred for 2 hours. The mixture was then filtered through an 0.45 um membrane filter and washed three times with deionized and distilled water. The resulting microparticles are resuspended in a small amount of water and lypholized. The resulting microparticles have a diameter of 20-40 .mu.m and contain 2-8 .mu.mol of .epsilon.-amino groups per gram of microsphere.

Polymeric sheets having functional groups on the surface thereof are made by swelling a sheet made of the base polymer using conventional techniques, such as heat or solvating in a solvent such as ethanol, and then contacting the swollen surface of the sheet with an aqueous solution containing the SAFP for preferably from 1 to 24 hours. Thereafter, the sheet is deswollen and, preferably, rinsed and dried to provide a polymeric sheet having functional groups on the surface thereof. The sheet can be formed into particles using standard methods.

Particles prepared as described above are useful for delivering or targeting drugs, diagnostic agents, vaccines and genes to the circulation or specific sites of a mammalian body.

Claim 1 of 20 Claims

What is claimed is:

1. A method for preparing a biocompatible polymeric matrix having functional groups on the surface thereof, comprising:

a) providing a first solution comprising a poly lactide-co-glycolide base polymer and an organic solvent;

b) providing a second solution comprising a surface-active functional polymer and a solvent;

c) mixing said first solution with said second solution to provide an emulsion in which chains of the base polymer become physically entangled with chains of the surface-active polymer; and

d) evaporating said organic solvent to provide a polymeric matrix comprising chains of the base polymer which are physically entangled with chains of the surface-active polymer.

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