Inhalable, bioresponsive microparticles for targeted drug delivery in the lungs.

OBJECTIVE: There is a growing interest in developing bioresponsive drug delivery systems to achieve greater control over drug release than can be achieved with the conventional diffusion controlled polymeric delivery systems. While a number of such systems have been studied for oral or parenteral delivery, little or no work has been done on bioresponsive delivery systems for inhalation. Using the raised elastase levels present at sites of lung inflammation as a proof-of-concept model, we endeavoured to develop a prototype of inhalable elastase sensitive microparticles (ESMs). METHODS: Microparticles degradable by the enzyme elastase were formed by crosslinking the polymer alginate in the presence of an elastase substrate, elastin, using Ca(+2) ions and subsequent spray drying. KEY FINDINGS: The bioresponsive release of a protein cargo in the presence of elastase demonstrated the enzyme-specific degradability of the particles. The microparticles showed favorable properties such as high drug encapsulation and good powder dispersibility. Potential polymer toxicity in the lungs was assessed by impinging the microparticles on Calu-3 cell monolayers and assessing changes in transepithelial permeability and induction of cytokine release. The microparticles displayed no toxic or immunogenic effects. CONCLUSIONS: With a manufacturing method that is amenable to scale-up, the ability to be aerosolised efficiently from a first-generation inhaler device, enzyme-specific degradability and lack of toxicity, the ESMs show significant promise as pulmonary drug carriers

Targeted Liposomal Drug Delivery to Monocytes and Macrophages

As the role of monocytes and macrophages in a range of diseases is better understood, strategies to target these cell types are of growing importance both scientifically and therapeutically. As particulate carriers, liposomes naturally target cells of the mononuclear phagocytic system (MPS), particularly macrophages. Loading drugs into liposomes can therefore offer an efficient means of drug targeting to MPS cells. Physicochemical properties including size, charge and lipid composition can have a very significant effect on the efficiency with which liposomes target MPS cells. MPS cells express a range of receptors including scavenger receptors, integrins, mannose receptors and Fc-receptors that can be targeted by the addition of ligands to liposome surfaces. These ligands include peptides, antibodies and lectins and have the advantages of increasing target specificity and avoiding the need for cationic lipids to trigger intracellular delivery. The goal for targeting monocytes/macrophages using liposomes includes not only drug delivery but also potentially a role in cell ablation and cell activation for the treatment of conditions including cancer, atherosclerosis, HIV, and chronic inflammation

In vivo animal models for drug delivery across the lung mucosal barrier.

Over recent years the research focus within the field of respiratory drug delivery has broadened to include a wide range of potential applications for inhalation by delivering drugs not just onto the lung mucosa but across it. The range of drugs being assessed is broad and includes both current and novel therapies and there are a growing number of additives that appear capable of enhancing systemic absorption. Comprehensive characterisation of drug delivery to the lungs is a complex task involving the determination of delivered, deposited and (for systemically-targeted drugs) absorbed dose. As it is difficult to simulate in vitro, in vivo whole animal models are still key to inhaled drug development. Because of the anatomical complexities and interspecies differences in the lungs, the appropriate choice of species and drug delivery method is vital during study design. New delivery devices designed specifically for animal studies as well as more sophisticated methods to determine drug deposition and absorption after inhalation are improving the information derived from these studies

Long noncoding RNA are aberrantly expressed in vivo in the cystic fibrosis bronchial epithelium.

Long non-coding RNAs (lncRNAs) have emerged recently as key regulatory molecules with diverse roles at almost every level of the regulation of gene expression. The roles of these RNAs in the pathogenesis of cystic fibrosis (CF); a lethal multisystem, autosomal recessive disorder have yet to be explored. Our aim was to examine the expression profile of lncRNA, in the airway epithelium of people with CF. We examined the expression of 30,586 lncRNAs by microarray (Human LncRNA Array v3.0, Arraystar, Inc.), in vivo in bronchial cells isolated from endobronchial brushings obtained from CF and non-CF individuals. In total, we identified 1,063 lncRNAs with differential expression between CF and non-CF individuals (fold change ≥3, p≤0.001). The microarray also contained probes for ∼26,109 protein coding transcripts, of which 720 were differentially expressed between CF and non-CF brush samples (fold change ≥3, p≤0.001). Confirmation of a selection of differentially expressed coding mRNA and lncRNA transcripts such as XIST and TLR8 was achieved using qRT-PCR. Gene ontology bioinformatics analysis highlighted that many processes over-represented in the CF bronchial epithelium are related to inflammation. These data show a significantly altered lncRNA and mRNA expression profile in CF bronchial cells in vivo. Dysregulation of some of these lncRNAs may play important roles in the chronic infection and inflammation that exists in the lungs of people with CF

A Novel Methodology for Bio-electrospraying Mesenchymal Stem Cells that Maintains Differentiation, Immunomodulatory and Pro-reparative Functions

Mesenchymal stem cells (MSCs) are an important cell source for tissue engineering (TE) and cell therapies for several reasons including ease of isolation from multiple tissues, uncomplicated ex vivo culture, ability to self-renew and differentiate into numerous cell types, MSC/immune cell interactions and pro-reparative properties. Current MSC therapies involve administration via intravenous (I.V.) injection. However, this can result in MSC entrapment and failure to target injured site. In TE, artificial 3D constructs are being investigated as strategies for direct delivery of MSCs to a target area. However, these constructs have numerous limitations including lack of cell infiltration, poor cell functionality and limited diffusion of nutrients and oxygen through the scaffolds. We are investigating the jetting methodology bio-electrospraying (BES) as an alternative strategy for MSCs delivery in vivo that may overcome obstacles associated with I.V. injections and scaffold transplantation. For BES in vivo, low voltages, stable jetting and a single needle configuration are highly desirable. A commercially available electrospray apparatus Spraybase was used to electrospray mouse bone marrowderived MSCs (mBMSCs) at low voltages (* 3–6 kV) in vitro. Stable jetting conditions with a single needle at these low voltages were established by employing a ringshape electrode for potential difference, specific culture medium and the use of high mBMSCs numbers to overcome viscosity difficulties. The viability and functionality of the mBMSCs following BES was determined by analysing expression of specific surface markers, multilineage differentiation, suppression of T- cell activation and proreparative capabilities. We show that mBMSCs post-BES functioned similarly to non-bio-electrospray (non-BES) control mBMSCs for all parameters examined. This methodology may subsequently enable targeted delivery of MSCs to an injury site in vivo and potentially avoid the complications associated with MSCs entrapment and the limitations associated with artificial scaffolds

Respiratory Tissue Engineering: Current Status and Opportunities for the Future.

Currently, lung disease and major airway trauma constitute a major global healthcare burden with limited treatment options. Airway diseases such as chronic obstructive pulmonary disease and cystic fibrosis have been identified as the fifth highest cause of mortality worldwide and are estimated to rise to fourth place by 2030. Alternate approaches and therapeutic modalities are urgently needed to improve clinical outcomes for chronic lung disease. This can be achieved through tissue engineering of the respiratory tract. Interest is growing in the use of airway tissue-engineered constructs as both a research tool, to further our understanding of airway pathology, validate new drugs, and pave the way for novel drug therapies, and also as regenerative medical devices or as an alternative to transplant tissue. This review provides a concise summary of the field of respiratory tissue engineering to date. An initial overview of airway anatomy and physiology is given, followed by a description of the stem cell populations and signaling processes involved in parenchymal healing and tissue repair. We then focus on the different biomaterials and tissue-engineered systems employed in upper and lower respiratory tract engineering and give a final perspective of the opportunities and challenges facing the field of respiratory tissue engineering

Development of a gene-activated scaffold platform for tissue engineering applications using chitosan-pDNA nanoparticles on collagen-based scaffolds.

Biomaterial scaffolds that support cell infiltration and tissue formation can also function as platforms for the delivery of therapeutics such as drugs, proteins, and genes. As burst release of supraphysiological quantities of recombinant proteins can result in adverse side effects, the objective of this study was to explore the potential of a series of collagen-based scaffolds, developed in our laboratory, as gene-activated scaffold platforms with potential in a range of tissue engineering applications. The potential of chitosan, a biocompatible material derived from the shells of crustaceans, as a gene delivery vector was assessed using mesenchymal stem cells (MSCs). A transfection efficiency of \u3e45% is reported which is similar to what is achieved with polyethyleneimine (PEI), a non-viral gold standard vector, without causing cytotoxic side effects. When the optimised chitosan nanoparticles were incorporated into a series of collagen-based scaffolds, sustained transgene expression from MSCs seeded on the scaffolds was maintained for up to 28days and interestingly the composition of the scaffold had an effect on transfection efficiency. These results demonstrate that by simply varying the scaffold composition and the gene (or combinations thereof) chosen; the system has potential for a myriad of therapeutic applications

Screening of siRNA Nanoparticles for Delivery to Airway Epithelial Cells Using High Content Analysis

Background: Delivery of siRNA to the lungs via inhalation offers a unique opportunity to develop novel methods of treating a range of poorly treated respiratory conditions. However progress has been greatly hindered by safety and delivery issues. This study developed a high-throughput method for screening novel nanotechnologies for pulmonary siRNA delivery Methodology: Following physico-chemical analysis, the ability of PEI-PEG/siRNA nanoparticles to facilitate siRNA delivery was determined using high content analysis (HCA) in Calu-3 cells. Results obtained from HCA were validated using confocal microscopy. Finally, cytotoxicity of the PEI-PEG/siRNA particles was analysed by HCA using the Cellomics® multiparamter cytotoxicity assay. Conclusions: PEI-PEG/siRNA nanoparticles facilitated increased siRNA uptake and luciferase knockdown in Calu-3 cells compared to PEI/siRNA

Controlled release of vascular endothelial growth factor from spray-dried alginate microparticles in collagen-hydroxyapatite scaffolds for promoting vascularization and bone repair.

A major limitation with current tissue-engineering approaches is creating functionally vascularized constructs that can successfully integrate with the host; this often leads to implant failure, due to avascular necrosis. In order to overcome this, the objective of the present work was to develop a method to incorporate growth factor-eluting alginate microparticles (MPs) into freeze-dried, collagen-based scaffolds. A collagen-hydroxyapatite (CHA) scaffold, previously optimized for bone regeneration, was functionalized for the sustained delivery of an angiogenic growth factor, vascular endothelial growth factor (VEGF), with the aim of facilitating angiogenesis and enhancing bone regeneration. VEGF was initially encapsulated in alginate MPs by spray-drying, producing particles of \u3c 10 µm in diameter. This process was found to effectively encapsulate and control VEGF release while maintaining its stability and bioactivity post-processing. These VEGF-MPs were then incorporated into CHA scaffolds, leading to homogeneous distribution throughout the interconnected scaffold pore structure. The scaffolds were capable of sustained release of bioactive VEGF for up to 35 days, which was proficient at increasing tubule formation by endothelial cells in vitro. When implanted in vivo in a rat calvarial defect model, this scaffold enhanced vessel formation, resulting in increased bone regeneration compared to empty-defect and VEGF-free scaffolds. This biologically functionalized scaffold, composed entirely of natural-based materials, may offer an ideal platform to promote angiogenesis and tissue regeneration. Copyright © 2015 John Wiley \u26 Sons, Ltd