Microencapsulated materials and method of making same

Abstract

A method of forming microspheres of a bioactive material, such as a protein polymer or drug by nebulizing a solubilized form of a material to be encapsulated and an encapsulating material, such as albumin, in a stirred chilled solvent system comprising a vegetable oil, mineral oil and/or a lower alcohol such that the formed microspheres demonstrate intracellular bioactivity when taken up by macrophages.

1 . A method of encapsulating antisense oligonucleotides (oligomers) to NF-kB, comprising the steps of:
 a) dissolving albumin in water;   b) solubilizing said antisense oligonucleotides (oligomers) to NF-kB in an aqueous phase selected from the group consisting of phosphate buffered saline (PBS), saline, water, and water polyoxyethylene sorbitan monooleate;   c) mixing said dissolved albumin and said solubilized antisense oligonucleotides (oligomers) to NF-kB together;   d) cooling said mixture formed in step c);   e) cooling a nonaqueous first solvent comprising either a lower carbon alcohol selected from the group consisting of methanol, ethanol, propanol, and butanol or a bioinert oil;   f) emulsifying the mixture of step d) with cooled nonaqueous first solvent while said nonaqueous first solvent is stirred so as to form microspheres containing antisense oligonucleotides (oligomers) to NF-kB;   g) crosslinking said microspheres with glutaraldehyde while stirring;   h) washing said microspheres of step g) with a second solvent;   i) sizing said microspheres of step h); and,   j) freeze drying said microspheres of step i),   whereby said antisense oligonucleotides (oligomers) to NF-kB are encapsulated in said microspheres.   
   
   
       2 . The method of  claim 1 , wherein said bioinert oil is either a vegetable or mineral oil. 
   
   
       3 . The method of  claim 3 , wherein said bioinert inert vegetable or mineral oil is selected from the group consisting of olive oil, canola oil, cottonseed oil, mineral oil, mixtures of the foregoing oils and subcomponents of the foregoing oils. 
   
   
       4 . A method of encapsulating a material selected from the group consisting of antisense oligonucleotides (oligomers) to NF-kB, a tumor vaccine containing antigens derived from B 16 murine melanoma cells, (N,N′-bis[3,5-diacetylphenyl]decanediamide amidinohydrazone tetrahydrochloride (“CNI-1493”), clodronate, comprising the steps of:
 a) dissolving albumin in water;   b) solubilizing said material in an aqueous phase selected from the group consisting of phosphate buffered saline (PBS), saline, water, and water polyoxyethylene sorbitan monooleate;   c) mixing said dissolved albumin and said solubilized material together;   d) cooling said mixture formed in step c);   e) cooling a nonaqueous first solvent comprising either a lower carbon alcohol selected from the group consisting of methanol, ethanol, propanol, and butanol or a bioinert oil;   f) emulsifying the mixture of step d) into with cooled nonaqueous first solvent while said nonaqueous first solvent is stirred so as to form microspheres containing said material;   g) crosslinking said microspheres with glutaraldehyde while stirring;   h) washing said microspheres of step g) with a second solvent;   i) sizing said microspheres of step h); and,   j) freeze drying said microspheres of step i),   whereby said material is encapsulated in said microspheres.   
   
   
       5 . A method for microenencapsulating a bioactive material, comprising the steps of:
 a) preparing a mixture by combining a dissolved biodegradable polymer and said bioactive material solubilized in an aqueous solution selected from the group consisting of phosphate buffered saline (PBS), saline, water and water with polyoxyethylene sorbitan monooleate;   b) cooling said mixture;   c) emulsifying said mixture of step b) with a cooled nonaqueous first solvent comprising either a lower carbon alcohol selected from the group consisting of methanol, ethanol, propanol, and butanol or an inert oil while stirring said nonaqueous first solvent so as to form microspheres;   d) crosslinking said microspheres; and,   e) washing said crosslinked microspheres with a second solvent.   
   
   
       6 . The method of  claim 5 , wherein said bioinert oil is either a vegetable or mineral oil. 
   
   
       7 . The method of  claim 5 , wherein said bioinert inert vegetable or mineral oil is selected from the group consisting of olive oil, canola oil, cottonseed oil, mineral oil, mixtures of the foregoing oils and subcomponents of the foregoing oils. 
   
   
       8 . The method as claimed in  claim 5 , wherein said bioactive material is antisense oligonucleotides (oligomers) to NF-kB. 
   
   
       9 . The method as claimed in  claim 5 , wherein said bioactive material is pyrrolidine dithiocarbamate. 
   
   
       10 . The method as claimed in  claim 5 , wherein the first solvent is an inert oil. 
   
   
       11 . The method as claimed in  claim 5 , wherein the bioactive material contains oligonucleotide. 
   
   
       12 . The method as claimed in  claim 5 , wherein the bioactive material is a therapeutic agent. 
   
   
       13 . The method as claimed in  claim 5 , wherein the bioactive material is a drug selected from group consisting of drugs, synthetic drugs, and bioactive proteins. 
   
   
       14 . The method as claimed in  claim 5 , wherein the bioactive material is an aqueous solubilizable drug. 
   
   
       15 . The method as claimed in  claim 5 , wherein the bioactive material is Clodronate. 
   
   
       16 . The method as claimed in  claim 5 , wherein the bioactive material is a guanylhydrazone which can inhibit p38 MAP kinase. 
   
   
       17 . The method as claimed in  claim 6 , further comprising a step f): drying the microspheres. 
   
   
       18 . A bioactive microencapsulated material selected from the group consisting of antisense oligonucleotides (oligomers) to NF-kB, a tumor vaccine containing antigens derived from B16 murine melanoma cells, (N,N′-bis[3,5-diacetylphenyl]decanediamide amidinohydrazone tetrahydrochloride (“CNI-1493”), and clodronate, said bioactive microencapsulated material formed by a method, comprising the steps of:
 a) dissolving albumin in water;   b) solubilizing said material in an aqueous phase selected from the group consisting of phosphate buffered saline (PBS), saline, water, and water polyoxyethylene sorbitan monooleate;   c) mixing said dissolved albumin and said solubilized material together;   d) cooling said mixture formed in step c);   e) cooling a nonaqueous first solvent comprising either a lower carbon alcohol selected from the group consisting of methanol, ethanol, propanol, and butanol or a bioinert oil;   f) emulsifying the mixture of step d) into with cooled nonaqueous first solvent while said nonaqueous first solvent is stirred so as to form microspheres containing said material;   g) crosslinking said microspheres with glutaraldehyde while stirring;   h) washing said microspheres of step g) with a second solvent;   i) sizing said microspheres of step h); and,   j) freeze drying said microspheres of step i),   
   
   
       19 . A bioactive microencapsulated material which retains substantial bioactivity after uptake by macrophages, said material being selected from the group consisting of antisense oligonucleotides (oligomers) to NF-kB, a tumor vaccine containing antigens derived from B16 murine melanoma cells, (N,N′-bis[3,5-diacetylphenyl]decanediamide amidinohydrazone tetrahydrochloride (“CNI-1493”), and clodronate.