Encapsulation of bioactive agents

Details Encapsulation of bioactive agents Encapsulation of bioactive agents

2002-05-29

2003-05-20

Nutter, Nathan M. (Department: 1711)

Plastic and nonmetallic article shaping or treating: processes

Encapsulating normally liquid material

Liquid encapsulation utilizing an emulsion or dispersion to...

C264S004300, C264S004330, C264S004600, C427S213300, C427S213310, C427S213360

Reexamination Certificate

active

06565777

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to improvements in encapsulation of bioactive agents, such as antigens, drugs and DNA for vaccination and gene therapy. In particular, the present invention relates to methods for encapsulating antigen and/or DNA, in aqueous solution, in a polymer microparticle, so that the microparticles when administered to a recipient deliver antigen to antigen presenting cells of the recipient, and/or induce expression of the DNA in the recipient in the antigen presenting cells. The present invention relates also to microparticles and compositions comprising microparticles.
BACKGROUND OF THE INVENTION
The present technology of microencapsulation is currently at least 10 years old but does not seem to have yielded any commercially successful products, despite many announced breakthroughs. Indeed, apart from WO-A-97/17063 (by the same inventors as the present invention), published methods have been found to be inefficient and unreliable.
Many published patents and applications are in the name of the Southern Research Institute (SRI). In particular, U.S. Pat. No. 5,407,609 purports to describe in example 7, an emulsion based method for the manufacture of hollow particles. Another emulsion based method for making particles that contain a protein, specifically BSA, is described in Sah et al (J. Microencapsulation, 1995, vol. 12, no. 1, pp 59-69). If these particles are to be used as a means of administering an encapsulated bioactive agent to antigen-presenting cells in the gut epithelium, it is of great importance that their size be below 10 microns in diameter. Larger particles are not endocytosed by the targeted gut cells and pass through the gut without effect.
However, the methods detailed in U.S. Pat. No. 5,407,609 and by Sah et al succeed in making relatively large particles, or at least particles over a wide range of sizes, where a significant portion of particles are larger than the biological activity cut off point of 10 microns. A large spread of particle sizes, such as that seen in U.S. Pat. No. 5,407,609 inevitably leads to much of the encapsulated agent being incorporated in particles of a size that are not appropriate for phagocytosis. Worse still, Sah et al obtained a mixture of particle sizes with an apparent minimum diameter of around 10 microns. Both art methods are, therefore, not specifically designed to produce encapsulated product available for uptake by antigen-presenting cells. The encapsulation method used by Sah also requires very high shear rates during encapsulation, shear rates that would have a detrimental effect upon, and are thus unsuitable for, encapsulation of agents such as DNA.
U.S. Pat. No. 5,407,609 makes reference to the construction of microbubble-like particles but emphasises that water soluble agents are not easily encapsulated in such particles due to a tendency for the encapsulated agent to migrate out of that part of the emulsion that will eventually constitute the internal content of the particles. To overcome this problem, U.S. Pat. No. 5,407,609 requires that as soon as the emulsion has formed it be immediately added to a large volume of extraction medium i.e. water. However, in doing so control over particle size may be lost.
The internal architecture of microparticles made using the Sah method is generally a honeycombed matrix, illustrated in the photographs that accompany the Sah paper, rather than a simple, hollow microbubble-like structure. Incomplete removal of solvent and/or water from the insides of these matrices during the solvent extraction step and subsequent lyophilisation can result in the premature degradation of the polymer of the particle and a corresponding fall in the pH of any encapsulated aqueous solution, leading to damage of an encapsulated agent.
The bioactive agent release profile of administered microparticles has been previously altered by changing the relative amounts of lactide and glycolide present in PLG polymer. Much of this data is however based on “microsphere” technology where the encapsulated agent is distributed throughout a polymer matrix and there is no known prior art that relates to control of the release profile of microbubble particles. Altering the relative amounts of lactide and glycolide present in the PLG polymer has shown limited success in the short term but it would be desirable to provide alternative means of controlling the release profile.
WO-A-97/17063 describes methods for obtaining significant levels of incorporation of DNA into microparticles however, the authors of the present invention would like to improve upon this incorporation efficiency.
The present invention thus seeks to overcome, or at least ameliorate, the problems observed in the art. In particular, preferred embodiments of the invention aim to provide improved technology for the encapsulation of bioactive agents such as antigen and/or nucleic acids in polymer microparticles for the delivery of antigen and/or gene based prophylactics and therapies.
SUMMARY OF THE INVENTION
Accordingly, the present invention provides a method of encapsulating DNA in a polymer microparticle, comprising:
dissolving polymer in a solvent to form a polymer solution;
preparing an aqueous solution of DNA;
combining the polymer and DNA solutions with agitation to form a water-in-oil emulsion;
adding the water-in-oil emulsion to a further aqueous phase containing a stabiliser or surfactant with agitation to form a (water-in-oil)-in-water emulsion;
adding the (water-in-oil)-in-water emulsion to excess of an aqueous phase to extract the solvent, thereby forming polymer microparticles of a size up to 10 microns in diameter, said microparticles containing DNA;
wherein the DNA comprises a coding sequence and induces expression of the coding sequence in a recipient following administration, and wherein the solvent comprises ethyl acetate.


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