Lipid-coated microgels for the triggered release of doxorubicin

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Abstract

We have systematically engineered a polymeric, multi-component drug delivery system composed of a lipid-coated hydrogel microparticle (microgel). The design of this delivery system was motivated by the recent elucidation of the mechanism of regulated secretion from the secretory granule and the compositional and structural features that underlie its ability to store and release endogenous drug-like compounds. The present work describes the assembly and response of a prototype construct which displays several important features of the secretory granule, including its high drug loading capacity, and triggered microgel swelling, resulting in the burst release of drug. To achieve this, ionic microgels were synthesized, and loaded with doxorubicin via ion exchange. These microgels were then coated with a lipid bilayer, and the release of doxorubicin was triggered from the gels using either lipid-solubilizing surfactants or electroporation. The use of a microanalytical technique is featured utilizing micropipette manipulation that allows the study of the behavior of individual microparticles. The lipid-coated microgels were electroporated in saline solution; they swelled and disrupted their bilayer coating over a period of several seconds and exchanged doxorubicin with the external plasma saline over a period of several minutes. It is envisioned that this system will ultimately find utility in drug delivery systems that are designed to release chemotherapeutic agents and peptides by the application of a triggering signal.

Introduction

The emerging aim in controlled release drug delivery is to achieve the correct dose of drug at only the disease site with the most effective pharmacodynamic profile [1]. The first goal is to solubilize drug or bind drug to a matrix that changes the biodistribution in an efficacious way. The second is to induce release by a local or externally applied trigger involving a change in drug binding mediated by a chemical transformation or a phase transition in the matrix. Interestingly, triggered and local release is a ubiquitous feature of biological systems involved in regulated secretion, where extracellular regulators (such as histamine) acting on specialized secretory cells control the secretion of hormones [2]. These secretory cells contain an organelle, commonly known as the secretory granule, the lumen of which is composed of a cross-linked poly-anionic condensed polymer network encapsulated within a lipid membrane [3], [4], [5]. The condensed network functions as an osmotically inert particulate storage depot for the stable encapsulation of high concentrations of small molecules and proteins [6]. The secretory granule is coated with a lipid membrane and, in a process controlled by extracellular regulators, the granule membrane fuses with the cell’s plasma membrane. This results in the formation of an aqueous channel (fusion pore) between the external physiological medium and granule lumen. Exchange of ions and water across this channel induces the polymer network to undergo a phase transition where the network rapidly swells and so triggers the release of its contents in a burst profile into the extracellular medium [4], [5], [7]. A multi-component drug delivery system which closely mimics these properties of the secretory granule will likely have utility for triggered release of drugs [8].

Motivated by the initial work of Prof. Julio M. Fernandez [4], [9] and the fact that a wide variety of natural [10] and synthetic poly-electrolytes have already been used to form condensed phase nano- or microparticles [11], [12], [13], we have begun the systematic engineering of synthetic systems that reconstruct several of the compositional, structural and materials property features of the natural secretory granule in order to provide for similar functional performance [8], [9]. The features described herein which have been designed to mimic the secretory granule are: (i) a carboxylated network polymer particle [14]; (ii) ionic binding of a small molecule to the network [5]; (iii) pH-induced condensation of the network [15]; (iv) coating of the network with an ion-impermeable lipid bilayer to stabilize the condensed phase of the gel in a solution which would otherwise swell the gel [16]; (v) electromechanical rupture of the bilayer coating induced by gel network swelling; and, finally, (vi) release of the bound ion via ion exchange with monovalent cations [17]. The construction of a prototype system displaying these behaviors has been achieved (Fig. 1, Fig. 2). The prototype utilizes a lipid-coated condensed hydrogel microparticle (microgel) which was loaded with the potent chemotherapeutic weak base cation doxorubicin, also known as adriamycin.

This paper describes both the assembly and response of such an environmentally responsive microgel, and includes the use of a microanalytical technique which combines micropipette manipulation with electroporation. This allows us to trigger membrane breakdown, microgel swelling and subsequent drug release for a single microgel particle. Our ultimate aim is to develop a novel drug delivery system for the site-specific triggered release of a variety of therapeutic molecules.

Section snippets

Materials

4-Nitrophenyl methacrylate (NPMA) was synthesized from the corresponding acid chloride and 4-nitrophenol. Methacryoyl chloride (8.13 g, 80.1 mmol, 90% tech.) was added dropwise in 30 ml of ethyl acetate to a 4°C solution of 4-nitrophenol (10 g, 71 mmol) in 150 ml of dry ethyl acetate and suspended sodium carbonate (9.01 g, 85 mmol) under a N2 atmosphere. The reaction was allowed to warm to room temperature (3 h). The suspended sodium salt was filtered (fritted glass funnel) and the reaction was

pH response and microgel volume change

For the production of this prototype, a modification of the precipitation polymerization developed by Kawaguchi was chosen, because of the control this method affords over the size, polydispersity and composition of the resultant microgels. After hydrolysis in hydroxide, the resultant microgels were between 6 and 7 microns in diameter in phosphate buffered saline (PBS) at pH 7.4. To check the pH response of these microgels, the micropipette manipulation technique [26] was used to transfer a

Summary and conclusions

We can now review the whole mechanism of swelling and release for a drug-loaded, lipid bilayer-coated microgel. The sequence of events for the swelling and release of drug occurs in three stages. Initially, the permeability of the membrane must be sufficiently compromised (e.g., by electroporation or membrane dissolution or other permeabilizing species), but only to an extent that it allows proton efflux from the microgel and a sodium ion influx into the gel particle (Fig. 1), i.e. large-scale

Acknowledgements

We thank Drs. J. Fernandez and P. Verdugo for helpful discussions, Dr. P. Marszalek for guidance on microelectroporation, and Dr. John Rice for reviewing the manuscript. We thank Access Pharmaceuticals Inc., Dallas, TX, for support of this work.

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    Present address: Chem Codes Inc., Commercial Park West, Durham, NC 27713, USA.

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