| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | September 15, 1998 |
| PATENT TITLE |
Preparation of ionically cross-linked polyphosphazene microspheresy by coacervation |
| PATENT ABSTRACT |
A method is provided for preparing polyphosphazene microspheres wherein the polyphosphazene microspheres are produced by coacervation. A solution containing a polyphosphazene is admixed with a solution containing a salt of a monovalent ion such as a salt of a Group I element (for example, NaCl) to form a dispersion containing polyphosphazene coacervate microdroplets. The dispersion then is admixed with a solution containing a salt of a multivalent ion, such as a salt of a Group II element (for example, CaCl.sub.2) to form a suspension of polyphosphazene microspheres. The polyphosphazene microspheres then are recovered from the suspension. Such method enables one to obtain high yields of microspheres having a controlled size distribution. Polyphosphazene microspheres containing biological material can be produced by providing a biological material in the polyphosphazene solution that is mixed with the solution containing a salt of a monovalent ion. The biological material may be an antigen or other biological material selected from proteins, nucleic acids, polysaccharides and synthetic compounds having biological activity. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | July 2, 1996 |
| PATENT REFERENCES CITED |
Deasy, Drugs and the Pharmaceutical Sciences, vol. 20, Microencapsulation and Related Drug Processes, Marcel Dekker, Inc., New York, pp. 61-95 (1984). Allcock, et al., Macromolecules, vol. 22, pp. 75-79 (1989). Bano, et al., Biotechnology, vol. 9, pp. 468-471 (May 1991). Axelos, et al., Macromolecules, vol. 27, pp. 6594-6602 (1994). Payne, et al., Advances in Mucosal Immunology, Mestecky, et al., eds., Plenum Press, New York, pp. 1475-1480 (1995). |
| PATENT CLAIMS |
What is claimed is: 1. A method of producing ionically cross-linked polyphosphazene microspheres comprising: (a) admixing an aqueous solution containing a water-soluble polyphosphazene polyelectrolyte with a solution containing a salt of a monovalent ion to form a dispersion containing polyphosphazene coacervate microdroplets; (b) admixing said dispersion with a solution containing a salt of multivalent ion to form a suspension of polyphosphazene microspheres. 2. The method of claim 1, and further comprising: (c) recovering said ionically cross-linked polyphosphazene microspheres from said suspension. 3. The method of claim 1 wherein said salt of a monovalent ion is a salt of a Group I element. 4. The method of claim 3 wherein said Group I element is sodium. 5. The method of claim 4 wherein said salt of a monovalent ion is NaCl. 6. The method of claim 1 wherein said salt of a multivalent ion is a calcium salt. 7. The method of claim 6 wherein said salt of a multivalent ion is CaCl.sub.2. 8. The method of claim 1 wherein said polyphosphazene polyelectrolyte is poly›di(carboxylatophenoxy)phosphazene!. 9. The method of claim 1 wherein said microspheres have diameter of from about 1 to about 10 .mu.m. 10. A method of producing ionically cross-linked polyphosphazene microspheres comprising: (a) admixing an aqueous solution containing a water-soluble polyphosphazene polyelectrolyte with a solution containing water soluble polymer to form a water-soluble interpolymer complex of said polyphosphazene polyelectrolyte and said water-soluble polymer; (b) admixing said interpolymer complex with a solution containing a salt of a monovalent ion to form a dispersion containing polyphosphazene coacervate microdroplets of said interpolymer complex; (c) admixing said dispersion with a solution containing a salt of a multivalent ion to form a suspension of microspheres of said interpolymer complex. 11. The method of claim 10, and further comprising: (d) recovering said microspheres from said suspension. 12. The method of claim 10 wherein said water-soluble polymer is poly(ethylene oxide-propylene oxide). 13. A method of producing ionically cross-linked polyphosphazene microspheres containing biological material comprising: (a) admixing an aqueous solution containing a water-soluble polyphosphazene polyelectrolyte and biological material with a solution containing a salt of a monovalent ion to form a dispersion containing coacervate microdroplets; (b) admixing said dispersion with a solution containing a salt of a multivalent ion to form a suspension of said ionically cross-linked polyphosphazene microsphere containing biological material. 14. The method of claim 13, and further comprising: (c) recovering said microspheres from said suspension. 15. The method of claim 13 wherein said salt of a monovalent ion is a salt of a Group I element. 16. The method of claim 15 wherein said Group I element is sodium. 17. The method of claim 16 wherein said salt of a monovalent ion is NaCl. 18. The method of claim 13 wherein said salt of a multivalent ion is a calcium salt. 19. The method of claim 18 wherein said salt of a multivalent ion is CaCl.sub.2. 20. The method of claim 13 wherein said polyphosphazene polyelectrolyte is poly›di(carboxylatophenoxy)phosphazene!. 21. The method of claim 13 wherein said microspheres have diameter of from about 1 to about 10 .mu.m. 22. The method of claim 13 wherein said biological material is selected from the group consisting of proteins, biologically active synthetic compounds, nucleic acids, and polysaccharides. 23. The method of claim 13 wherein said biological material is an antigen. 24. The method of claim 23 wherein said antigen is derived from the group consisting of rotavirus, measles virus, mumps virus, rubella virus, polio virus, hepatitis A virus, hepatitis B virus, Herpes virus, human immunodeficiency virus, influenza virus, Haemophilus influenza, Clostridium tetani, Corynebacterium diphtheria, and Neisseria gonorrhoae. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
BACKGROUND OF THE INVENTION This invention relates to the preparation of polyphosphazene microspheres. More particularly, this invention relates to the preparation of polyphosphazene microspheres by coacervation of the polyphosphazene. Water soluble polymers and polymeric hydrogels, such as polyphosphazene hydrogels, may be used to microencapsulate antigens for delivery to mucosal surfaces and for the controlled release of antigen at the mucosal surface, or for injection. Such encapsulated antigen also may be administered orally or intranasally. Polyphosphazene microspheres may be employed as pharmaceutical carriers for a variety of prophylactic and/or therapeutic agents. For example, polyphosphazene microspheres may serve as immunoadjuvants, whereby such microspheres may contain any of a variety of antigens, such as antigens which may be derived from cells, bacteria, virus particles, or portions thereof, the antigen may be a protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, or combination thereof, which elicits an immunogenic response in an animal, for example, a mammal, bird, or fish. In general, the encapsulated antigen may be mixed with a polyphosphazene solution, microparticles of the polyphosphazene and antigen are formed, and the polyphosphazene is crosslinked ionically or covalently to form a stable biodegradable microparticle. The microparticles adhere to mucosal surfaces such as the mucosal lining of the gastrointestinal tract, increasing takeup by the reticuloendothelium of antigen as it is released over time. The polyphosphazene may be cross-linked ionically with a polyion or divalent cation, such as calcium chloride. Methods for preparation of synthetic polymer microspheres including polyelectrolyte hydrogel microspheres have been described; however, these methods require the use of organic solvents and surfactants. Examples of such methods are described in U.S. Pat. No. 4,948,586, issued to Bohm, et al. Hydrogel microspheres or nanoparticles can be prepared in aqueous solutions by a simple coacervation process using natural polyelectrolytes, such as gelatin and some synthetic polyelectrolytes. High concentrations of salt or elevated temperatures were used to induce coacervate formation. Microsphere preparation according to these methods, however, requires the use of a water-insoluble "core" material in a liquid or solid form, such as oils, parafin or water-insoluble drugs. (Deasy, et al., Microencapsulation and Related Drug Processes, Marcel Dekker, Inc., New York, pgs. 61-95 (1984); U.S. Pat. No. 4,622,244, issued to Lapka, et al.) The polymer-rich coacervate droplets deposit on the surface of dispersed water-insoluble core material and then coalesce to form the coating which is usually then cross-linked by chemical means. U.S. Pat. No. 5,149,543, issued to Cohen, et al., teachaes that ionically cross-linked polyphosphazene hydrogel microspheres can be prepared by spraying an aqueous polyphosphazene solution into a solution containing multivalent cations. This method then was modified to produce microspheres in a micron size range. (Payne, et al., Advances in Mucosal Immunology, Mestecky, et al., eds. Plenum Press, New York, pgs. 1475-1480 (1995).) This process, however, requires complicated spraying equipment, and it is difficult to control the microsphere size distribution, especially to achieve high yield and narrow size distribution of microspheres having sizes in the micron range. The Cohen, et al. patent also teaches that ionically cross-linked polyphosphazene hydrogel microspheres are unstable in the presence of monovalent ions and their treatment with excess aqueous potassium chloride at pH 7.5 results in cleavage of the ionic cross-links. Disintegration of microspheres in phosphate buffered saline (pH 7.4) was also observed. (Bano, et al., Bio Technology, Vol. 9, pgs. 468-471 (1991).) Allcock, et al., U.S. Pat. No. 5,053,451, also teach cleavage of ionic cross-links in the presence of KC1. Axelos, et al., Macromolecules, Vol. 27, No. 22, pgs. 6594-6602 (1995) demonstrate that a monovalent salt, such as sodium chloride, dissolves or precipitates hydrogels formed by ionic cross-linking of polyelectrolyte with divalent metals. It is therefore an object of the present invention to provide a method of producing ionically cross-linked polyphosphazene hydrogel microspheres with a controlled microsphere size distribution without the use of elevated temperatures, organic solvents, water-insoluble core materials, or complex manufacturing equipment. In such a method, a solution containing a polyphosphazene is subjected to aqueous coacervation using a salt of a monovalent ion. It is a further object of the present invention to provide a method for encapsulating biological materials by mixing biological material with either polyphosphazene solution before microsphere preparation, or with prepared polyphosphazene microspheres. In accordance with an aspect of the present invention, there is provided a method of producing polyphosphazene microspheres. In such method, a solution containing a polyphosphazene is subjected to coacervation. The term "coacervation" as used herein means the separation of a macromolecular solution into two immiscible liquid phases. One phase is a dense coacervate phase, concentrated in the macromolecules, and the other phase is a polymer deficient phase. Coacervation is a result of a molecular dehydration of the polymer and may be induced by a temperature change, the addition of a non-solvent, or the addition of a micro-salt (simple coacervation), or may be induced by the addition of another polymer to form an interpolymer complex (complex coacervation). Coacervates may be described as liquid crystals and mesaphases and are more fluid than other systems with higher structural order, such as micelles. Such systems are in dynamic equilibrium and changes in the conditions may result in either the reformation of a one-phase system or the formation of a flocculate or precipitate. (Burgess, Macromolecular Complexes in Chemistry and Biology, Dubin, et al., eds., Springer-Verlag, Berlin, pgs. 285-300 (1994).) The advantages of the method for making microspheres using coacervation are that it avoids the use of organic solvents, heat, complicated manufacturing equipment, such as spray equipment and eliminates generation of the aerosol. The method is highly reproducible and generates microspheres with an improved, more narrow microsphere size distribution, compared to the spray technique. Contrary to the microspheres obtained by spray method, coacervation microspheres do not contain significant amount of larger size aggregates or amorphous precipitate. This result is important for the preparation of microspheres for vaccine delivery since the uptake of these microspheres by M-cells is limited to the particles having diameter of 10 .mu.m or less (Payne, et al., 1995). A further advantage of the coacervation process that it enables the efficient control of the microsphere size by simply varying the concentration of the components. SUMMARY OF THE INVENTION In one embodiment, the polyphosphazene microspheres are produced by admixing a solution containing polyphosphazene polyelectrolyte with a solution containing a salt of a monovalent ion to form coacervate droplets. The dispersion then is admixed with a solution containing a salt of a multivalent ion, whereby the microspheres are stabilized. If desired, the polyphosphazene microspheres then are recovered from the dispersion. Polyphosphazenes are polymers with backbones consisting of alternating phosphorus and nitrogen, separated by alternating single and double bonds. Each phosphorous atom is covalently bonded to two pendant groups ("R"). The repeat unit in polyphosphazenes has the following general formula: ##STR1## wherein n is an integer. The substituent ("R") can be any of a wide variety of moieties that can vary within the polymer, including but not limited to aliphatic, aryl, aralkyl, alkaryl, carboxylic acid, heteroaromatic, carbohydrates, including glucose, heteroalkyl, halogen, (aliphatic)amino, including alkylamino-, heteroaralkyl, di (aliphatic) amino- including dialkylamino-, arylamino-, diarylamino-, alkylarylamino-, -oxyaryl including but not limited to -oxyphenylCO.sub.2 H, -oxyphenylSO.sub.3 H, -oxyphenylhydroxyl and -oxyphenylPO3H; -oxyaliphatic including -oxyalkyl, -oxy(aliphatic)CO.sub.2 H, -oxy(aliphatic)SO.sub.3 H, -oxy(aliphatic)PO.sub.3 H, and -oxy(aliphatic)hydroxyl, including -oxy(alkyl)hydroxyl; -oxyalkaryl, -oxyaralkyl, -thioaryl, -thioaliphatic including -thioalkyl, -thioalkaryl, -thioaralkyl, --NHC(O)O-- (aryl or aliphatic), --O--›(CH.sub.2).sub.x O!.sub.y --CH.sub.2)--O--›(CH.sub.2).sub.x O!.sub.y (CH.sub.2).sub.x NH(CH.sub.2).sub.x SO.sub.3 H, and --O--›(CH.sub.2).sub.x O!.sub.y --(aryl or aliphatic), wherein x is 1-8 and y is an integer of 1 to 20. The groups can be bonded to the phosphorous atom through, for example, an oxygen, sulfur, nitrogen, or carbon atom. In general, when the polyphosphazene has more than one type of pendant group, the groups will vary randomly throughout the polymer, and the polyphosphazene is thus a random copolymer. Phosphorous can be bound to two like groups, or two different groups. Polyphosphazenes with two or more types of pendant groups can be produced by reacting poly(dichlorophosphazene) with the desired nucleophile or nucleophiles in a desired ratio. The resulting ratio of pendant groups in the polyphosphazene will be determined by a number of factors, including the ratio of starting materials used to produce the polymer, the temperature at which the nucleophilic substitution reaction is carried out, and the solvent system used. While it is very difficult to determine the exact substitution pattern of the groups in the resulting polymer, the ratio of groups in the polymer can be easily determined by one skilled in the art. Phosphazene polyelectrolytes are defined herein as polyphosphazenes that contain ionized or ionizable pendant groups that render the polyphosphazene anionic, cationic or amphiphilic. The ionic groups can be in the form of a salt, or, alternatively, an acid or base that is or can be at least partially dissociated. Any pharmaceutically acceptable monovalent cation can be used as counterion of the salt, including but not limited to sodium, potassium, and ammonium. The phosphazene polyelectrolytes can also contain non-ionic side groups. The phosphazene polyelectrolyte can be biodegradable or nonbiodegradable under the conditions of use. The ionized or ionizable pendant groups are preferably not susceptible to hydrolysis under the conditions of use. A preferred phosphazene polyelectrolyte is a polyanion and contains pendant groups that include carboxylic acid, sulfonic acid, hydroxyl, or phosphate moieties. While the acidic groups are usually on nonhydrolyzable pendant groups, they can alternatively, or in combination, also be positioned on hydrolyzable groups. An example of a phosphazene polyelectrolyte having carboxylic acid groups as side chains is shown in the following formula: ##STR2## wherein n is an integer, preferably an integer between 10 and 300,000, and preferably between 1,000 to 300,000. This polymer has the chemical name poly ›di (carboxylatophenoxy) phosphazene! or, alternatively, poly›bis(carboxylatophenoxy)phosphazene! (PCPP). The phosphazene polyelectrolyte is preferably biodegradable to prevent eventual deposition and accumulation of polymer molecules at distant sites in the body, such as the spleen. The term biodegradable, as used herein, means a polymer that degrades within a period that is acceptable in the desired application, typically less than about five years and most preferably less than about one year, once exposed to a physiological solution of pH 6-8 at a temperature of approximately 25.degree. C.-37.degree. C. Polyphosphazenes, including phosphazene polyelectrolytes, can be prepared by a macromolecular nucleophilic substitution reaction of poly(dichloro phosphazene) with a wide range of chemical reagents or mixture of reagents in accordance with methods known to those skilled in the art. Preferably, the phosphazene polyelectrolytes are made by reacting the poly(dichloro phosphazene) with an appropriate nucleophile or nucleophiles that displace chlorine. Desired proportions of hydrolyzable to non-hydrolyzable side chains in the polymer can be obtained by adjusting the quantity of the corresponding nucleophiles that are reacted with poly(dichlorophosphazene) and the reaction conditions as necessary. Preferred polyphosphazenes have a molecular weight of over 1,000, more preferably from about 500,000 to about 1,500,000. The polyphosphazene may be contained in an appropriate solution, such as, for example, phosphate buffered saline (PBS), inorganic or organic buffer solutions, or aqueous solutions of biological materials, such as proteins, antigens, or mixtures thereof. The polyphosphazene may be present in the solution at any concentration, pH, and ionic strength, preferably at a concentration of from about 0.01% to about 1.5%, and a pH from 7 to 8. The polyphosphazene solution is admixed with a solution containing at least one salt of a monovalent ion, such as a salt of a Group I element, such as a sodium or lithium salt. Other salts of monovalent ions which may be employed include, but are not limited to, ammonium salts. In one embodiment, the salt of a monovalent ion is a salt of a Group I element. Preferably, the salt of a Group I element is a sodium salt, such as sodium chloride, sodium sulfate, or sodium phosphate. Preferably, the sodium salt is sodium chloride, or NaC1l. The salt of the monovalent ion may be present in the solution at any concentration and pH, preferably at a concentration of from about 0.1% to about 40% and a pH from 7 to 8. The resulting mixture of the polyphosphazene solution and the solution including a salt of a monovalent ion is allowed to stand for a period of time which is sufficient to allow the formation of a coacervate phase; i.e., coacervate microdroplets of polyphosphazene are formed in the mixture. After a significant amount of microdroplets have been formed in the mixture, the mixture is added to a solution including at least one salt of a multivalent ion, such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, or cadmium. In one embodiment, the salt of the monovalent ion is a calcium salt, such as calcium chloride, calcium bromide or calcium acetate. Preferably, the calcium salt is calcium chloride, or CaC1.sub.2. The salt of the multivalent ion may be present in the solution at any concentration and pH, preferably from about 1% to about 25% and a pH from 7 to 8. The Ca.sup.2+ ions in the solution serve as a cross-linker, whereby the microspheres are stabilized when they contact the solution containing the calcium salt. The suspension of the polyphosphazene microspheres in the resulting solution which includes a salt of a monovalent ion and a salt of a multivalent ion is stirred for a period of time sufficient to form a suspension of stabilized microspheres. If desired, the microspheres then may be recovered from the suspension by means known to those skilled in the art, such as, for example, by centrifugation. In another embodiment, the microspheres may be formed by preparing a water soluble interpolymer complex of a polyphosphazene and another water soluble polymer that can form a water-soluble interpolymer complex by means of electrostatic, hydrogen, or hydrophobic interactions. In one embodiment such a polymer is a poly (ethylene oxide-propylene oxide). The interpolymer complex can be formed at any molecular ratio which does not cause precipitation, any pH, any ionic strength, and any temperature, preferably at a pH from 7 to 8 and at room temperature. Induction of coacervation then is effected by the addition of a solution of a salt of a monovalent ion, such as hereinabove described to form interpolymer complex coacervate droplets. The microdroplets then may be stabilized by adding the dispersion containing the microspheres to a solution containing a salt of a multivalent ion as hereinabove described. The preparation of polyphosphazene microspheres by coacervation enables one to recover an increased yield of polyphosphazene microspheres having a size in the micron range (up to 90 differential percent by volume and 95 differential percent by number), and produce microspheres of other sizes if needed without the use of elaborate equipment. The microspheres, formed by coacervation, as hereinabove described, may be employed as carriers for a variety of prophylactic or therapeutic agents. In one embodiment, the microspheres may be employed as carriers of a biological material such as an antigen, which is capable of eliciting an immune response in an animal. The antigen may be derived from a cell, bacterium, virus particle, or a portion thereof. The antigen may be a protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, or a combination thereof, which elicits an immune response in an animal, including mammals, birds, and fish. The immune response may be a humoral immune response or a cell-mediated immune response. In the event the material to which the immune response is to be directed is poorly antigenic, it may be conjugated to a carrier such as albumin or to a hapten, using standard covalent binding techniques, for example, with one of the several commercially available reagent kits. In one embodiment, the microsphere is employed to deliver a nucleic acid sequence which encodes an antigen to a mucosal surface where the nucleic acid is expressed. Particular examples of antigens which may be contained in the polyphosphazene microspheres include, but are not limited to, viral proteins such as influenza proteins, human immunodeficiency virus (HIV) proteins, Herpes virus proteins, and hepatitis A and B proteins, and antigens derived from rotavirus, measles, mumps, rubella, and polio; and bacterial proteins and lipopolysaccharides such as Gram-negative bacterial cell walls, and antigens derived from organisms such as Haemophilus influenza, Clostridium tetani, Corynebacterium diphtheria, and Neisseria gonhorrhoae. In general, the antigen is mixed with the polymer solution prior to coacervation to insure dispersion of the antigen throughout the microsphere. The microspheres, which contain an antigen, can be administered as a vaccine by any method known to those skilled in the art that elicits an immune response, including parenterally (intravenously, intramuscularly, subcutaneously, intraperitoneally, etc.), orally, or by transmembrane or transmucosal administration. Preferably, the vaccine is administered transmucosally. Nonlimiting examples of routes of delivery to mucosal surfaces are intranasal (or generally, the nasal associated lymphoid tissue), respiratory, vaginal, and rectal. The dosage is determined by the antigen loading and by standard techniques for determining dosage and schedules for administration for each antigen, based on titer of antibody elicited by the microsphere antigen administration. BRIEF DESCRIPTION OF THE DRAWINGS The invention now will be described with respect to the drawings, wherein: FIG. 1 is a phase diagram for a coacervation system formed by mixing solutions of PCPP and sodium chloride, wherein the concentration of NaCl is plotted against polymer concentration; FIG. 2 is a schematic of the preparation of polyphosphazene microspheres; FIG. 3 is a graph of the differential percentage of microsphere diameters by volume and by number, as prepared by coacervation and spray methods; FIG. 4 is an electron micrograph of ionically cross-linked PCPP microspheres prepared by a coacervation method; FIG. 5 is a graph of the mean particle size diameter of the ionically cross-linked PCPP microspheres over time of coacervate droplet incubation in NaCl for different salt concentrations; FIG. 6 is a graph of the differential percentages of microspheres by volume and by number for ionically cross-linked PCPP microspheres prepared with different concentrations of CaCl.sub.2 ; and FIG. 7 is a graph of the differential percentages of microsphere diameters by volume and by number for the ionically cross-linked PCPP microspheres prepared with different concentrations of PCPP. DETAILED DESCRIPTION OF THE INVENTION |
| PATENT EXAMPLES | available on request |
| PATENT PHOTOCOPY | available on request |
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