Surface modification of lamellar particles

Details Surface modification of lamellar particles Surface modification of lamellar particles

2001-09-06

2003-10-21

Page, Thurman K. (Department: 1615)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

C424S184100, C424S489000, C424S499000, C424S501000

Reexamination Certificate

active

06635254

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention relates generally to positively charged lamellar particles and to methods for preparing such particles. More specifically, the present invention relates to lamellar particles formed from a biodegradable and biocompatible polymer and which carry a cationic charge on their surface, to compositions containing these particles, and to methods for preparing such particles and compositions.
Lamellar particles prepared by the controlled precipitation of crystalline polymers such as poly-L-lactide (PLA) and polybetahydroxybutyrate (PHB) have been described in PCT International Publication No. WO 96/01695. These particles have been termed polymeric lamellar substrate particles (PLSP). Due to a large surface area and a relatively hydrophobic surface these particles can be used to adsorb antigenic materials, such as influenza vaccine and tetanus toxoid, and have been shown to have utility as vaccine adjuvants. The term “adjuvant” refers to a material that can be added to a vaccine formulation in order to improve the immune response. The lamellar particles can be prepared in different sizes (length and thickness). The surfaces of the lamellar particles can be modified by conditioning the particles, for example by storing them in a buffer solution for a period of weeks. The surface of the particle undergoes polymer degradation resulting in altered antigenic adsorption and release properties. The adsorption properties of the lamellar particles can be characterized by the measurement of an adsorption isotherm. When prepared from polymers such as poly-L-lactide the lamellar particles carry a net negative charge as measured, for example using the method of particle electrophoresis using a Malvern Zeta Sizer 4 (laser doppler anemometry). While such a negative charge can be advantageous for the sorption of certain antigens (particularly when the antigen carries a net positive charge under the conditions adopted for antigen loading), in other situations the net negative charge can be disadvantageous.
A particular problem arises when it is desired to use the lamellar particles for the delivery of polynucleotides (antisense agents and DNA). Polynucleotides carry a net negative charge due to the presence of phosphate groupings. The adsorption of polynucleotides to lamellar particles is very poor due to electrostatic charge repulsion. It would be advantageous to have lamellar particles that carry a net positive charge. It will be appreciated by the person skilled in the art that lamellar particles carrying a net positive charge could also be useful for the delivery of vaccine antigens that were negatively charged, i.e., acidic proteins where adsorption could be effected at pH conditions below the isoelectric point of the antigen.
We have found that it has not previously been possible to produce lamellar particles with a strong positive charge that remains on the particles. We have coated negatively charged lamellar particles produced from poly-L-lactide with an adsorbed cationic material, for example a cationic surfactant, such as cetyltrimethyl ammonium bromide, or a cationic polymer, such as chitosan (polyglucosamine), diethylaminoethyl dextran (DEAE-dextran), or polyethyleneimine. It is possible thereby to change the negatively charged lamellar particles into positive particles, as measured by particle electrophoresis. Unfortunately, the positively charged material is not strongly adsorbed to the particle surface and has a poor stability. The adsorbed cationic material is sensitive to centrifugation, dialysis and high concentrations of electrolyte. With the effect that much of the positively charged material is desorbed or washed off the surface of the particle. This presents a significant problem in further processing, such as particle clean-up and polynucleotide adsorption.
BRIEF SUMMARY OF THE INVENTION
It is therefore an object of the present invention to provide lamellar particles with a positive charge which remains on the particles and a method for producing such particles. The present applicant has developed a novel method of producing positively charged lamellar particles.
The method of the invention may provide positively charged lamellar particles wherein the loss of surface attached material during subsequent processing (e.g., during clean-up and polynucleolide adsorption) is substantially reduced as compared to particles that are surface modified by a simple coating (physisorption process).
According to the present invention, there are also provided compositions comprising lamellar particles which carry a cationic charge on their surface.
In a preferred embodiment the present invention provides compositions comprising lamellar particles which carry a cationic charge on their surfaces and a material, for example genetic material.
DETAILED DESCRIPTION OF THE INVENTION
The cationic charge on the lamellar particles typically arises from a cationic material attached to or incorporated into the particles. The positively charged lamellar particles are typically produced by co-precipitation of the particles in the presence of a cationic material. The cationic polymer is preferably adsorbed to the surface of the lamellar particle.
The particles of the present invention carry a net positive charge, and the loss of surface-attached material during subsequent processing, such as clean-up and polynucleolide adsorption, is substantially reduced as compared to particles that are surface-modified by a simple coating (physisorption process).
The amount of surface attached-material that is lost during processing can be evaluated in a quantitative fashion. The surface charge (zeta potential, measured in mV) on the lamellar particles suspended in a buffer of low ionic strength is measured using the technique of particle electrophoresis.
Preferably, cleaning of the particles, such as by the addition of aqueous buffer or water and recovery of the ‘washed’ particles by centrifugation or filtration should not result in a greater than 80% loss of surface charge as measured by particle electrophoresis in the same buffer of low ionic strength. More preferably, such loss of charge should be less than 50% of that for the unwashed particles and, most preferably, less than 35% of the value for unwashed particles.
The lamellar particles of the invention may be prepared from any biodegradable and biocompatible polymer. Suitable polymers are preferably crystalline.
We use the term “biodegradable polymer” to include polymeric systems at least a part of which can degrade into low molecular weight compounds which are known to be involved normally in metabolic pathways. We also use the term to include polymer systems which can be attacked in the biological milieu, so that the integrity of the system, and in some cases of the macromolecules themselves, is affected and gives fragments or other degradation by-products which can move away from their site of action, but not necessarily from the body.
The biodegradable polymer used is preferably at least 5 percent by weight crystallizable.
The biodegradable polymer in the particles is preferably at least 5 percent by weight crystalline, more preferably at least 30%, more preferably at least 50%, still more preferably at least 70%, and most preferably at least 90% crystalline.
Whether or not a polymer is crystalline, and the degree of crystallinity, can be determined by methods well known in the art, for example X-ray diffraction methods as applied to polymers or by differential scanning calorimetry.
Suitable polymers for use in the particles of the present invention include poly-L-lactide (PLA) and polyalkanoic acids, such as polyhydroxybutyrate (PHB) and polyhydroxyvalerate (PHV).
The polymer may be a mixture of PLA with another biodegradable polymer or with a biocompatible but non-degradable polymer, either as a copolymer or as a blend of polymers. In either case, the resulting mixture should still be at least in part crystalline and preferably at least 5% by weight crystalline. The content of a non-crystallizable or non-cryst

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