An efficient, non-viral dendritic vector for gene delivery in tissue engineering.

Recent developments within the field of tissue engineering (TE) have shown that biomaterial scaffold systems can be augmented via the incorporation of gene therapeutics. The objective of this study was to assess the potential of the activated polyamidoamine dendrimer (dPAMAM) transfection reagent (SuperfectTM) as a gene delivery system to mesenchymal stem cells (MSCs) in both monolayer and 3D culture on collagen based scaffolds. dPAMAM-pDNA polyplexes at a mass ratio (M:R) 10:1 (dPAMAM : pDNA) (1 ug pDNA) were capable of facilitating prolonged reporter gene expression in monolayer MSCs which was superior to that facilitated using polyethylenimine (PEI)-pDNA polyplexes (2 ug pDNA). When dPAMAM-pDNA polyplexes (1 ug pDNA) were soak loaded onto a collagen-chondroitin sulphate (CS) scaffold prolonged transgene expression was facilitated which was higher than that obtained for a PEI-pDNA polyplex (2 ug pDNA) loaded scaffold. Transgene expression was dependent on the composite nature of the collagen scaffold with varying expression profiles obtained from a suite of collagen constructs including a collagen alone, collagen-CS, collagen-hydroxyapatite, collagen-nanohydroxyapatite and collagen-hyaluronic acid scaffold. Therefore, the dPAMAM vector described herein represents a biocompatible, effective gene delivery vector for TE applications which, via matching with a particular composite scaffold type, can be tailored for regeneration of various tissue defects.

Early-stage development of novel cyclodextrin-siRNA nanocomplexes allows for successful postnebulization transfection of bronchial epithelial cells.

BACKGROUND: Successful delivery of small interfering RNA (siRNA) to the lungs remains hampered by poor intracellular delivery, vector-mediated cytotoxicity, and an inability to withstand nebulization. Recently, a novel cyclodextrin (CD), SC12CDClickpropylamine, consisting of distinct lipophilic and cationic subunits, has been shown to transfect a number of cell types. However, the suitability of this vector for pulmonary siRNA delivery has not been assessed to date. To address this, a series of high-content analysis (HCA) and postnebulization assays were devised to determine the potential for CD-siRNA delivery to the lungs. METHODS: SC12CDClickpropylamine-siRNA mass ratios (MRs) were examined for size and zeta potential. In-depth analysis of nanocomplex uptake and toxicity in Calu-3 bronchial epithelial cells was examined using IN Cell(®) HCA assays. Nebulized SC12CDClickpropylamine nanocomplexes were assessed for volumetric median diameter (VMD) and fine particle fraction (FPF) and compared with saline controls. Finally, postnebulization stability was determined by comparing luciferase knockdown elicited by SC12CDClickpropylamine nanocomplexes before and after nebulization. RESULTS: SC12CDClickpropylamine-siRNA complexation formed cationic nanocomplexes of ≤200 nm in size depending on the medium and led to significantly higher levels of siRNA associated with Calu-3 cells compared with RNAiFect-siRNA-treated cells at all MRs (p<0.001, n=3×4), with evidence of toxicity only at MRs 50-100. Nebulization of SC12CDClickpropylamine nanocomplexes using the Aeroneb(®) Pro resulted in VMDs of ∼4 µm and FPFs of ∼57% at all MRs. SC12CDClickpropylamine-siRNA-mediated luciferase knockdown was found to be 39.8±3.6% at MR=20 before and 35.6±4.55% after nebulization, comparable to results observed using unnebulized commercial transfection reagent, RNAiFect. CONCLUSIONS: SC12CDClickpropylamine nanocomplexes can be effectively nebulized for pulmonary delivery of siRNA using Aeroneb technology to mediate knockdown in airway cells. To the best of our knowledge, this is the first study examining the suitability of SC12CDClickpropylamine-siRNA nanocomplexes for pulmonary delivery. Furthermore, this work provides an integrated nanomedicine-device combination for future in vitro and in vivo preclinical and clinical studies of inhaled siRNA therapeutics.

Therapeutic aerosol bioengineering of targeted, inhalable microparticle formulations to treat Mycobacterium tuberculosis (MTb).

Therapeutic aerosol bioengineering (TAB) of Mycobacterium tuberculosis (MTb) therapies using inhalable microparticles offers a unique opportunity to target drugs to the site of infection in the alveolar macrophages, thereby increasing dosing in the lungs and limiting systemic exposure to often toxic drugs. Previous work by us used sophisticated, high content analysis to design the optimal poly(lactide-co-glycolic) acid (PLGA) microparticle for delivery of drugs to alveolar macrophages. Herein, we applied this technology to three different anti-MTb drugs. These formulations were then tested for encapsulation efficiency, drug-release, in vitro killing against MTb and aerosol performance. Methods for encapsulating each of the drugs in the PLGA microparticles were successfully developed and found to be capable of controlling the release of the drug for up to 4 days. The efficacy of each of the encapsulated anti-MTb drugs was maintained and in some cases enhanced post-encapsulation. A method of processing these drug-loaded microparticles for inhalation using standard dry powder inhaler devices was successfully developed that enabled a very high respirable dose of the drug to be delivered from a simple dry powder inhaler device. Overall, TAB offers unique opportunities to more effectively treat MTb with many potential clinical and economic benefits resulting.

Cellular targeting and trafficking of drug delivery systems for the prevention and treatment of MTb.

Targeted delivery of anti-tubercular therapeutics to alveolar macrophages via inhalation aims to achieve optimal concentration of the therapeutic in the mycobacteria's niche environment. However, several challenges need to be overcome when designing a system to achieve this targeted, intracellular delivery. The first objective is to design a system that is suitable for inhalation, i.e. it must be capable of deposition in the alveolar region of the lungs. The theme of this commentary will be on the biological barriers for intracellular targeting to alveolar macrophages once particles are deposited in the lungs with emphasis on the delivery of anti-tubercular therapy and implications for novel vaccine formulations. The commentary focuses on four key features: 1) How Mycobacterium tuberculosis enters and is trafficked through macrophages, 2) the mechanism by which current drug delivery systems (DDS) enter and are trafficked through cells and 3) How an ideal DDS for anti-tubercular therapy would be trafficked through the macrophage and 4) the potential for using DDS for novel anti-tubercular therapy and vaccine development. These four features of targeted DDS shall be discussed in relation to some new findings from our own research.