Liposome-assisted synthesis of polymeric nanoparticles

Details Liposome-assisted synthesis of polymeric nanoparticles Liposome-assisted synthesis of polymeric nanoparticles

2000-05-25

2001-04-17

Foelak, Morton (Department: 1711)

Drug, bio-affecting and body treating compositions

Preparations characterized by special physical form

Liposomes

C427S213310

Reexamination Certificate

active

06217901

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to the creation of polymer nanoparticles capable of specifically binding to biological molecular targets and to the manufacture of such polymer nanoparticles, enabled through the use of liposomes.
BACKGROUND OF THE INVENTION
Molecular recognition events are ubiquitous in life. Example molecular recognition events include:
The recognition of foreign proteins or foreign biopolymers by soluble antibodies or T-cell receptors.
Pathogen surface molecules binding to cellular receptors (e.g. adhesion molecules) to gain access to the cytoplasm.
The binding of cytokines to cellular receptors. Cytokine binding triggers a wide range of signal transduction events in a cell. For instance in tumor metastasis, angiogenesis and tumor cell proliferation is promoted.
The binding of fibronectin by integrins to affect blood clotting.
Biologically active macromolecules are complex entities that have intricate three-dimensional structures. The exact spatial conformation of these macromolecules is directly related to their biological function. For example, proteins are composed of amino acids in well-defined sequences. Amino acids may have both polar and nonpolar side groups; the polar side groups may in turn be either charged or uncharged in aqueous buffers near neutral or physiological pH.
The three-dimensional structure of a particular polypeptide chain depends on the multiple spatially-specific interactions of its unique sequence, or primary structure. The chain folds into a three-dimensional structure in accordance with the multitude of specific interactions among the constituent amino acid units. For example, there are 10
26
possible primary structures for a 20-amino acid polypeptide. The secondary, tertiary, and quaternary structural possibilities increase the complexity.
Molecular imprinted polymers, or MIPs, have been developed for molecular recognition applications. Molecular imprinted polymers (MIPs) rely on a semi-directed method of forming recognition sites by using the intended target as a template around which monomers assemble. See, (a) Mosbach, K.,
Trends Biochem. Sci
., 19:9-14 (1994), (b) Wulff, G.,
Angew. Chem. Int Ed. Engl
., 34:1812-1832 (1995) (c) Mosbach, K. and Ramström, O;
Biotechnology
, 14:163-170 (1996) and (d) Takeuchi, T. and Matsui,
J. Acta Polymer
, 47:471-480 (1996). MIPs are fabricated by polymerizing monomers (e.g., acrylic acid) and cross-linking molecules (e.g., ethylene glycol diacrylate) in the presence of an “imprinting molecule” to produce a large, rigid, and insoluble polymer structure in which template molecules are embedded. Removal of the template molecules leaves imprints of the template molecule. These molecularly imprinted polymers are then capable of recognizing the molecules that were used as templates. A nonsolvent (e.g., chloroform) “porogen” is employed during polymerization to produce large pores in the bulk material. The porogen is required to allow target diffusion into the insoluble MIP's inner regions, where the large percentage of the imprints are located. MIP structures must be rigid to achieve target recognition. This rigidity is achieved by employing crosslinking molecules that have a high number of crosslinking moieties per molecular weight. Both bulk and suspension polymerization schemes have been employed. Bulk polymerization produces a rigid plastic product the size of the reaction container, while a polymeric sphere on the order of a micrometer in size (a microsphere) is formed through suspension polymerization.
The resulting MIP is a porous macroscopic solid, which is not soluble and, due to its size, settles out of solution. Each MIP has many imprint sites (10
8
-10
2
) with widely varying degrees of binding affinity and with only a small fraction of sites accessible to targets. The use of the porogen in the synthesis procedure results in large voids throughout the structure. The voids are intended to allow for diffusion of target molecules into and out of the imprinted sites. However, many sites are still inaccessible.
Unilamellar liposomes are stable microscopic spherical structures consisting of a lipid bilayer surrounding an aqueous core. The lipid bilayer acts as a permeability barrier, effectively separating aqueous solutes inside and outside the liposome. The stability of liposomes has made their use attractive as drug delivery vehicles.
Liposomes are capable of incorporating biomolecules into their lipid bilayer. These biomolecules include membrane-bound proteins (membrane-bound proteins are particularly important in molecular recognition). Such biomoiecules are involved in molecular recognition processes and are thus potential targets of therapeutic and diagnostic materials. Virus surface molecules have also been incorporated into liposomes. Such liposomes have been referred to as “virosomes”. Polymerization of water-soluble monomers in the interior of a liposome has been sparsely reported on in the literature. One successful polymerization method was reported by Torchilin, et.al.,
Makromol. Chem., Rapid Communication
, 8:457-460 (1987). Polymerization to form a crosslinked particle in the interior of a liposome may be desirable because the liposome can act as a vessel which limits the size of polymer particle.
Thus, the hollow cores of liposomes have been advantageously employed to encapsulate material for a wide variety of purposes, including cosmetic and drug delivery applications. Liposomes have also been used to present membrane-bound macromolecules for diagnostic, drug delivery and drug development applications. Another use of liposomes is to form polymer spheres of nanometer dimensions in the liposome interior. Here we propose to leverage the ability of liposomes to encapsulate molecules and to fix amphiphilic molecules and/or integral membrane proteins at the lipid bilayer to aid in the formation of polymer particles capable of molecular recognition for diagnostic and therapeutic applications.
The manufacture of diagnostic and therapeutic agents, including small molecules, peptides, and monoclonal antibodies that likewise recognize and bind to specific bio-molecular targets, is the basis of the pharmaceutical industry. A new class of materials capable of molecular binding to a broad range of therapeutically and diagnostically important targets could find widespread use.
SUMMARY OF THE INVENTION
The present invention is directed to a method for producing polymer particles, here called Synthetic Polymer Complements (“SPCs”) ranging in size from 20 to 1000 nm. The SPCs have surfaces that include functional groups that are complementary to surface sites of target molecules, resulting in the ability of the SPCs to selectively bind to molecular targets with high affinity. The molecular recognition capability of these particles enables their use in diagnostic, therapeutic, and separation applications.
The invention provides a method for the assembly of reactive monomers or building blocks, in a manner that at least partially encapsulates “template” molecules. Polymerization followed by template removal yields the SPC particle structure with molecular recognition sites on its surface. The SPC is then capable of selectively binding to “target” molecules when presented with the target molecule in a mixture of molecules. As used here, “templates”, “target templates”, and “targets” refer to the same molecular structure. The terms “template” and “target template” are used when discussing the SPC fabrication process; the term “target” is used for example when discussing the SPC in its end-use application.
Essentially, SPCs of the present invention are formed in the interior of liposomes. The SPC is formed by bringing an aqueous solution of building blocks (also referred to herein as a “building block set”) into contact with lipid constituents and with target template molecules. At least some of the building blocks have moieties that are complementary to moieties on the target templates, so that the building blocks self-assemble around the target template molecule. Liposom

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