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Localized delivery of DNA to the cells by viral collagen-loaded silica colloidal crystals
 
Harshini Sarojini, Krishnakiran Medepalli, Derek A. Terry, Bruce W. Alphenaar, and Eugenia Wang
University of Louisville School of Medicine, Louisville, KY, USA
BioTechniques, Vol. 43, No. 2, August 2007, pp. 213–221
Full Text (PDF)
Abstract

Low-molecular-weight colloidal crystals with enhanced biocompatibility and ordered porous structure are used in drug-delivery systems. The objective of our study is to demonstrate the use of silica nanoscale colloid particles for localized recombinant DNA release. The colloids were coated with collagen-containing viral vector constructs of lentiviral green fluorescent protein (GFP), and solidified at 37°C. The colloid-collagen-viral vector platform (CCP) was transferred to cell monolayer cultures of human lung fibroblasts. Results show specific infection of cells directly beneath the platform, as evidenced by positive GFP in their cytoplasm, while neighboring cells show no cytoplasmic GFP. The infection of specific cells is probably due to the gradual release of viral particles from the collagen matrix by cell-secreted collagenase, which avoids overdosing the cells with viral particles, resulting from the cytopathic effect often seen with high-titer viral infection. Cells infected with the lentiviral-GFP or lentivirus alone, not incorporated into the colloid-collagen device, show caspase 3-associated apoptotic cell death. This suggests that colloidal crystal-coated collagen may be used as a powerful platform to deliver genes of choice to localized subgroups of specific cells of interest. This specificity in the delivery mode is beneficial for functional studies of gene-directed impact on a particular cell population of interest in a heterogeneous cell culture.

Introduction

Recently, extensive interest has been devoted to the study of nanoparticles for use as biomedical agents for disease diagnosis and treatment (1). However, efficient intracellular delivery has not developed the ability to avoid nonspecific effects, such as toxicity. Therefore, a strategy of cell-specific delivery is urgently needed to obtain the beneficial effects of using dosage levels sufficient for predicted results with minimal systemic damage to detoxifying tissues, such as liver, with the final successful outcome accompanied by better patient acceptance. The recent development of innovative methods for specific drug delivery has received significant attention globally, primarily in cancer (2,3); promising strategies include liposomes (4,5), antibodies (6,7), and viral particles (8,9,10). Among these approaches, ionically cross-linked chitosan nanoparticles prove to be efficient vehicles for transporting peptides across the nasal mucosa (11). This approach has laid the foundation for the present studies with nanosized colloidal crystals, which we describe here.

So far the uses of nanocrystals were primarily focused on the area of drug delivery via cell membrane permeation. Gold microparticles coated with DNA are introduced into cells by high-velocity acceleration to penetrate the cell membrane (12). However, this method is problematic due to the obvious difficulties in controlling either the precise amount of DNA or the precise cell penetration modality. In this report, we demonstrate that colloidal crystals are promising candidate materials for this type of biological application. A colloidal crystal is created when a dispersion of uniformly sized spheres (or colloids) is allowed to sediment out into a closely packed crystalline structure. Known primarily as a template for making three-dimensional (3-D) photonic crystals, colloidal crystals have also received attention in bioengineering applications; inverted colloidal crystals can be used as 3-D cell culture scaffolds (13). Advances in intracellular drug delivery, coupled with unique capabilities of colloidal crystals, provide an essential technology needed to overcome the selective barrier of the cell membrane.

We report here the combined use of viral vectors embedded in a collagen matrix adhering to a nanocolloid platform for precision gene delivery to a subpopulation of cells in culture. The virus mixture, packed into the voids of the colloid crystal, is released into the cells by collagenase enzyme secreted from the cells into the proximal supernatant medium. Our delivery system avoids cell death, since the final infection of the specific subpopulation of cells is time-dependent on cell-secreted collagenase, rather than overdosing the entire monolayer culture with all the lentiviral particles, as traditionally done, often resulting in cell death. Our colloid-collagen-viral vector platform (CCP) offers promise for applications (8,14,15) to deliver a broad range of agents, such as bacteriophages (16), plant (17,18), insect (19), and animal viruses (20,21).

Materials and Methods

Silica Colloids

The silica colloids were obtained from Dr. A.A. Zakhidov, University of Texas at Dallas. Colloidal crystals were grown using a slow crystallization process of monodispersed aqueous silica colloids in a glass cylinder, typically for 10 months at ambient temperature (22). Resulting deposits were polycrystalline with rod-like single crystals, which were sintered for several hours at 700°–750°C. The crystals thus obtained are closely packed, interconnected silicon dioxide spheres arranged in a face-centered cubic (FCC) lattice structure (Figure 1A). The diameter of such spheres can range from 20 nm to 10 µm with interconnected voids between the spheres; the silica colloids used in this investigation had sphere diameters between 200 and 400 nm. The crystals were cut using a low-speed diamond saw and polished to a thickness of <1 mm to ensure that when used they would be suspended above the monolayer cultures; thicker colloidal crystals would not be able to float above the cell culture, and their weight would cause them to adhere to the cell cultures and destroy the cells beneath them. Inverse carbon replicas of the colloidal crystals were fabricated using the phenolic resin route (22), followed by dissolution of the silica spheres in 2% hydrofluoric acid (HF).



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