Method for high supercoiled DNA content microspheres

Details Method for high supercoiled DNA content microspheres Method for high supercoiled DNA content microspheres

1998-12-10

2001-03-06

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, C264S004400, C264S004600

Reexamination Certificate

active

06197229

ABSTRACT:

BACKGROUND OF THE INVENTION
The importance of DNA based therapeutics, in particular in gene therapy, has led to increased research and development in this area (see, for example, Friedmann, T.
Science,
1989, 244, 1275; Miller, A. D.
Nature,
1992, 260, 455; Mulligan, R. C.
Science,
1993, 260, 926; Wilson, J. M.
Nature,
1993, 365, 691; Crystal, R. G.
Nature Med.,
1995, 1, 15). The use of these therapeutics can be problematic, however, because during drug delivery the DNA is subject to degradation. To maximize the power of these agents, it would be desirable to develop a mode of delivery in which the DNA-based therapeutic is protected from degradation.
Towards this end, several nano- or micro-encapsulation techniques have been developed and have been described in the literature (see, for example, Langer, R. S.
Science
1990, 249, 1527; Kato et al.
J Biol. Chem.
1991, 266, 3361; Jong et al.,
J. Controlled Release
1997, 47, 123; Mathiowitz et al.,
Nature
1997, 386, 410; Smith et al.
Adv. Drug Del. Rev.
1997, 26, 135). Additionally, U.S. Pat. No. 5,407,609 by Tice et al. describes a method of microencapsulating biological or immunological agents to form a microencapsulated product. More recently, Ciftci et al. have developed a method to introduce DNA into mammalian cells using a polymer based gene delivery system. (Ciftci et al.,
Pharmaceutical Res.
1997, 14, s-639) This method, however only results in an encapsulation efficiency of 33-49%.
In particular, one of the most common techniques for preparation of biodegradable polymer microspheres encapsulating hydrophilic molecules is the double-emulsion solvent evaporation method. Using this technique, the molecule to be encapsulated is placed in aqueous solution while the polymer is dissolved in an organic phase commonly consisting of methylene chloride or ethyl acetate. The two phases (volume organic/volume aqueous=3-20) are emulsified, typically by sonication or homogenization. This primary emulsion is then added to a second aqueous phase and again mixed by homogenization to form the (water-in-oil)-in-water double emulsion. Upon evaporation of the partially water-miscible solvent, the polymer-containing droplets harden to form microspheres which can then be isolated by filtration or centrifugation. Lyophilization removes water from the interior aqueous phase resulting in a dry suspension of the encapsulated material within the polymer matrix. Unfortunately, however, the encapsulation efficiency of DNA into the hydrophobic matrix of PLGA was low (~20%) using this method. Additionally, the use of this method leads to a tendency of plasmid DNA to be converted from its supercoiled state to a nicked or linear state. The preservation of the supercoiled DNA is important because it is known that supercoiled DNA retains the highest level of bioactivity (Xu et al.,
Biochem.
1996, 35, 5616; Yamaizumi et al.,
Mol. Cell Biol.
1983, 3, 511).
Clearly, many of the methods described above still present a problem for DNA therapeutics because of the tendency of DNA therapeutics to degrade during and after the encapsulation process. Specifically, DNA stress induced degradation is encountered during homogenization and lyophilization. Furthermore, the DNA is susceptible to diffusing out of the aqueous phase, thus decreasing the encapsulation efficiency. Therefore, a method of encapsulating DNA based therapeutics that retains the integrity of the DNA (maximizes the supercoiled-DNA content) and increases the encapsulation efficiency would be desirable.
SUMMARY OF THE INVENTION
Recognizing the need to develop improved delivery systems, the present invention provides methods for the formulation of high supercoiled-DNA content systems and microspheres. In one aspect, the present invention provides a method for the formulation of a high supercoiled DNA content system including formulating an emulsion having a polymer dissolved in organic solvent surrounding an aqueous inner phase containing DNA, and lowering the temperature of the emulsion below the freezing point of the aqueous inner phase. In another aspect, the method includes the step of removing the organic solvent and removing water from the aqueous inner phase to form the system. The system may include microspheres or another implantable structure.
In yet another aspect the invention provides a method for the formulation of high supercoiled DNA content microspheres which increases the encapsulation efficiency of DNA in microspheres and also prevents the degradation of supercoiled DNA during and after formulation, specifically during the homogenization and lyophilization processes. This method includes the formulation of a primary emulsion, and subsequently lowering of the temperature of the primary emulsion below the freezing point of the aqueous inner phase. Finally, the primary emulsion is transferred to a water-based surfactant solution and subjected to homogenization to form a secondary microsphere emulsion. Stirring of the secondary emulsion allows the removal of the organic phase and hardening of the microspheres, which are then isolated, frozen and lyophilized.
In yet another aspect of the invention, a primary emulsion is formed which contains a DNA nicking inhibitor in addition to DNA and buffer. The presence of the DNA nicking inhibitor ensures that the integrity of the DNA is retained. The primary emulsion thus formed with the DNA nicking inhibitor can be utilized in each of the methods described above to provide systems and microspheres with increased encapsulation efficiency and DNA integrity.
In still another aspect, the present invention provides a method for the cryopreparation of water soluble low molecular weight compounds to increase their encapsulation efficiency.


REFERENCES:
patent: 3885011 (1975-05-01), Renoux et al.
patent: 5075109 (1991-12-01), Tice et al.
patent: 5407609 (1995-04-01), Tice et al.
patent: 266119 B1 (1994-07-01), None
patent: WO95/24929 (1995-09-01), None
patent: WO97/03702 (1997-02-01), None
patent: WO97/17063 (1997-05-01), None
patent: WO98/31398 (1998-07-01), None
patent: WO98/51279 (1998-11-01), None
Jong Y.S. et al. “Controlled Release of Plasmid DNA”Journal of Controlled Release, 1997, 47, 123-124.
Crystal, R.G., “The Gene as the Drug”,Nature Medicine, 1995, 1, 15.
Jones et al., “Poly(DL-lactide-co-glycolide)-Encapsulated Plasmid DNA Elicits Systemic and Muscosal Antibody Responses to Encoded Protein after Oral Administration”,Vaccine, 1997, 15, 814.
Langer, R., “New Methods of Drug Delivery”,Science, 1990, 249, 1527.
Mathiowitz et al., “Biologically Erodable Microspheres as Potential Oral Drug Delivery Systems”,Nature, 1997, 386, 410.
Miller, A.D., “Human Gene Therapy Comes of Age”,Nature, 1992, 357, 455.
Mulligen, R.C., “The Basic Science of Gene Therapy”,Science, 1993, 260, 926.

Affiliated with

Also associated with

Rating

Method for high supercoiled DNA content microspheres does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Method for high supercoiled DNA content microspheres, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for high supercoiled DNA content microspheres will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2475190