Microspheres containing condensed polyanionic bioactive agents and methods for their production

• US 6,395,253 B2
• Filed: 04/23/1998
• Issued: 05/28/2002
• Est. Priority Date: 04/23/1998
• Status: Expired due to Fees

• Current Assignee: University of Michigan
• Original Assignee: Board of Regents of the University of Michigan (University of Michigan)
• Inventors: Levy, Robert J., Cohen, Hagit, Labhasetwar, Vinod
• Primary Examiner(s): Ketter, James
• Assistant Examiner(s): SCHNIZER, RICHARD A
• Application Number:
  US09/065,892
• Publication Number:
  US 20020012652A1
• Time in Patent Office: 1,496 Days
• Field of Search: 427/2.3,213 424/85.2,426,493,1.25,497 435/69.1,320.1 514/12,44 525/54.2 604/28,96,509 536/23.5
• US Class Current: 424/1.25
• CPC Class Codes: A61K 9/5153 Polyesters, e.g. poly(lactide-co-glycolide)
• CPC Class Codes: A61K 9/5192 Processes

Abstract

The invention describes improved methods for incorporating nucleic acids into polymeric microspheres and/or nanospheres through the use of a condensing agent.

Claims

1. A method for making microspheres comprising a condensed nucleic acid, said method comprising the steps of:

(a) dissolving at least one biocompatible biodegradable polymer in a water-immiscible organic solvent to yield an organic phase;

(b) dissolving a nucleic acid in water to yield a first aqueous phase;

(c) emulsifying the organic and first aqueous phases to yield a first emulsion;

(d) dissolving a condensing agent in water to yield a second aqueous phase;

(e) emulsifying the first emulsion and the second aqueous phase to yield a second emulsion; and

(f) removing the organic solvent from the second emulsion to yield microspheres comprising a condensed nucleic acid.

2. The method according to claim 1, wherein the nucleic acid is DNA.

3. The method according to claim 2, wherein the DNA comprises a promoter operably linked to a sequence encoding a protein.

4. The method according to claim 3, wherein the encoded protein is a therapeutic protein.

5. The method according to claim 4, wherein the therapeutic protein is a growth factor.

6. The method according to claim 5, wherein the growth factor is selected from the group consisting of transforming growth factor-beta (TGF-β

), fibroblast growth factor (FGF), platelet derived growth factor (PDGF), insulin like growth factor (IGF), and bone morphogenic factor (BMP).

7. The method according to claim 4, wherein the therapeutic protein is a hormone.

8. The method according to claim 7, wherein the hormone is selected from the group consisting of growth hormone (GH) and human parathyroid hormone (PTH).

9. The method according to claim 2, wherein the DNA comprises more than one DNA molecule.

10. The method according to claim 2, wherein the DNA is viral DNA.

11. The method according to claim 10, wherein the viral DNA is adenoviral DNA.

12. The method according to claim 2, wherein the condensing agent is a polycation.

13. The method according to claim 12, wherein the polycation is polylysine.

14. The method according to claim 12, wherein the polycation is a polypeptide.

15. The method according to claim 12, wherein the biocompatible biodegradable polymer is polylactic polyglycolic acid (PLGA) and the water-immiscible organic solvent is chloroform.

16. The method according to claim 15, wherein the second aqueous phase further includes an emulsifying agent.

17. The method according to claim 16, wherein the emulsifying agent is polyvinyl alcohol (PVA).

18. A method for making microspheres comprising condensed DNA, said method comprising the steps of:

(a) dissolving polyglycolic polylactic acid in chloroform to yield an organic phase;

(b) dissolving DNA in water to yield a first aqueous phase;

(c) emulsifying the organic and first aqueous phases to yield a first emulsion;

(d) dissolving poly-L-lysine and polyvinyl alcohol in water to yield a second aqueous phase;

(e) emulsifying the first emulsion and the second aqueous phase to yield a second emulsion; and

(f) evaporating the organic solvent from the second emulsion to yield microspheres comprising a condensed DNA.