Poly(l-lysine) nanostructured particles for gene delivery and hormone stimulation
Introduction
Modern drug delivery relies on the use of drug carrier vehicles that facilitate the delivery of cargo to the target cells, tissues and organs. Examples of carriers include polymers [1], micelles [2], liposomes [3], solid nano- and microparticles [4], polymer capsules [5], and other macromolecular and supramolecular assemblies. Advantages of carrier vehicles include shielding the drug from degradation by the body and reducing potential toxic effects of the drug. Additionally, for hydrophobic water insoluble therapeutics carrier vehicles markedly increase the deliverable payload. Targeted delivery of the drug is also made possible by the attachment of ligands that interact with specific receptors. Recently, we reported the preparation of colloidal drug carriers via infiltration of polymers into sacrificial porous template particles [6], [7], [8]. Crosslinking of the adsorbed polymer chains and template removal yields polymer hydrogel replica particles. This approach has been shown to be versatile in terms of polymer types and crosslinking strategies, and accommodates the use of biodegradable polypeptides and linkages to facilitate biomedical applications of the replica particles.
During the past decade, gene therapy has become a worldwide research focus and has been advanced considerably. The main objective in gene therapy is constructing an efficient gene delivery system able to transfer the therapeutic DNA to the targeted tissues and cells. However, despite extensive research, the development of an efficient and safe gene delivery system remains a main challenge for gene therapy. Poly (l-lysine) (PLL) polymers are one of the first cationic polymers used for gene transfer [9]. Currently, PLL has been widely used as a non-viral gene vector. They are polypeptides with amino-acid lysine as a repeat unit and are biodegradable. This property is very useful for in vivo applications [10]. Nevertheless, PLL shows a low level of transfection efficiency, primarily owing to the lack of rapid release of PLL/DNA complexes from endosomes [11]. Moreover, PLL suffers from immunogenicity and toxicity caused by its amino-acid backbone [12].
Polymer replica particles are well suited for the adsorption and coadsorption of therapeutics for their concurrent delivery. Specifically, in this work we designed replica particles based on PLL crosslinked via a homobifunctional linker to support coadsorption of a plasmid DNA and a peptide hormone for concurrent transfection and induction of a cellular function. We outline the preparation of the PLL replica particles (PLLRP), verify their cytotoxicity, and demonstrate their successful use as colloidal carriers for the concurrent gene and drug delivery.
To investigate the gene expression and hormone simulation, we chose SPT7pTL, a vector expressing the human SPT7 nuclear transcription factor, and alpha-melanocyte-stimulating hormone (α-MSH) as a reporter hormone. B16-F1 cells were used because these cells respond specifically to α-MSH. α-MSH is a potent stimulator of melanogenesis in mammalian melanocytes and in melanoma cells which binds to a specific receptor on the cell surface, melanocortin-1 receptor (MC1-P), and induces activation of tyrosinase, the key enzyme for melanin formation, through stimulation of adenylate cyclase and protein kinase A [13]. α-MSH is of interest as a model for signal transduction effects from cyclic AMP up to the final product, in this case the quantifiable pigment melanin. It is already known that α-MSH can be covalently coupled to molecules using well-established procedures [14]. From an earlier study [14], it became clear that α-MSH coupled to PLL can be incorporated into multilayer films and that certain biological properties of such α-MSH derivatives remain active in this formulation. Recently, we have also shown that it is possible to use poly (l-glutamic acid) (PGA) as a carrier [15]. Furthermore, based on this PLLRP gene delivery system, we simultaneously evaluated the melanin stimulation and gene expression in these cells by fluorescence microscopy. To further understand the bi-functionality, we labeled the SPT7pTL and PGA-α-MSH with YOYO-1 and Rhodamine, respectively, to follow its intracellular pathway by confocal microscopy.
Section snippets
Materials
Poly (l-lysine) hydrobromide (PLL, MW = 30 kDa) and poly-l-glutamic acid (PGA, 54 kDa) were purchased from Sigma. The SPT7pTL plasmid (5.3 kb) was grown in E. coli and purified by a Giagen kit (Qiagen, US). The purity and integrity of the plasmid were assessed by absorption spectroscopy (A260/A280 ratio) and electrophoresis on a 1% agarose gel. The DNA concentration was determined by UV absorbance at 260 nm using a Cary 400 spectrophotometer.
PLL replica particle (PLLRP) preparation and ζ-potentials characterization
PLL (or FITC-PLL) was infiltrated into porous silica
Characterization of PLL replica particles
The preparation of PLLRP involved polymer infiltration into mesoporous silica (MS) particles, crosslinking of the adsorbed chains using a homobifunctional crosslinker and finally removal of the template particles [6]. This linker reacts with the substrate amine groups to form an amidine linkage which retains the positive charge (Scheme 1). The morphology of PLLRP was analyzed by TEM (Fig. 1A) and visualized by fluorescence microscopy (Fig. 1B). The diameter of the PLLRP measured by fluorescence
Conclusions
Our data suggests that the PLLRP is a promising vector for gene therapy and hormone stimulation. PLLRP is not cytotoxic. Moreover, PLLRP could lead to efficient gene delivery and hormone stimulation followed by melanin production, simultaneously. The pathway of the DNA molecules was also visualized by confocal microscopy, and we found that SPT7pTL could enter into the nucleus. The entry of hormone into the cytoplasm and nuclei of cells induces melanin secretion.
Acknowledgments
This work was supported by the project ANR06-BLAN-0197-01/CartilSpray, from the “Agence Nationale de la Recherche”, the “Fondation Avenir”, the “Ligue contre le Cancer, du haut Rhin, Région Alsace” and “Cancéropôle du Grand Est”. This work was also supported by the Australian Research Council under the Discovery Project and Federation Fellowship schemes, and by FAST. X.Z and C.M thanks the Faculté de Chirurgie Dentaire of Strasbourg for financial support. N.J is indebted to CHU de Nancy
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Both authors contributed equally to this work.