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1The aim of this study was to compare protein-loaded inhalable microparticles 2 manufactured using a range of biocompatible polymers including hydroxypropyl cellulose 3 (HPC), chitosan, hyaluronic acid, alginate, gelatin, ovalbumin and poly (lactide-co-4 glycolide)(PLGA). Spray drying was used to prepare microparticles containing bovine 5 serum albumin labeled with flourescein isothiocyanate (BSA-FITC). Particles of respirable 6 size and high protein loading were obtained. No evidence of BSA degradation was seen 7 from PAGE analysis. The microparticles were mixed with mannitol as a carrier and 8 powder aerosolization was assessed with a multi-dose dry powder inhaler (DPI) using a 9 multi-stage cascade impactor. The mass median aerodynamic diameter (MMAD) ranged 10 between 2.9-4.7m. Potential polymer toxicity in the lungs was compared by impinging 11 the particles on Calu-3 monolayers and assessing the cytotoxicity, induction of cytokine 12 release, changes in transepithelial permeability and electrical resistance. No toxic effects 13were observed with most of the polymers though some evidence of compromised cell 14 monolayer integrity was seen for PLGA and ovalbumin. PLGA and gelatin microparticles 15 caused a significant increase in IL-8 release. Of the polymers studied, PLGA showed the 16 greatest toxicity. Certain polymers showed particular promise for specific protein delivery 17 needs in the lungs, such as HPC to improve flow properties, sodium hyaluronate for 18 controlled release, and chitosan and ovalbumin for systemic delivery. 19

The development of small interfering RNA (siRNA) to silence specific genes offers a new means of understanding and treating a range of respiratory diseases, including inflammatory lung disease. The alveolar macrophage (AM) is a key component of the inflammatory process in the lungs, associated with high levels of gene expression in inflammatory lung disease and therefore an attractive target for therapeutic siRNA. Delivery of siRNA to macrophages presents a significant delivery challenge, as fully differentiated alveolar macrophages are difficult to access and transfect. In this study we engineered particles suitable for inhalation that would efficiently transfect macrophages postinhalation. The process for encapsulation of siRNA in poly(lactic-co-glycolic acid) microparticles (MPs) was optimized using a double emulsion technique, and the resulting particles were characterized for size, shape, aerosol characteristics, encapsulation efficiency, and integrity of encapsulated siRNA. The cell uptake of the siRNA-loaded microparticles was determined by flow cytometry, confocal laser scanning microscopy (CLSM), and high-content analysis (HCA) with MPs capable of transfecting up to 55% of cells. Anti-TNFα siRNA-MPs were then prepared to study the functional activity of encapsulated siRNA in LPS-stimulated macrophages as a model of inflammation. The anti-TNFα siRNA-MPs were able to decrease TNFα expression by 45% over 48 h in the differentiated human monocytic cell line THP-1 compared to negligible knockdown using commercial transfection reagents and offered significant, sustained siRNA knockdown of TNFα in primary monocytes for up to 72 h.

Abstract. Pulmonary delivery of therapeutic peptides and proteins has many advantages including high relative bioavailability, rapid systemic absorption and onset of action and a non-invasive mode of administration which improves patient compliance. In this study, we investigated the effect of spraydrying (SD) and spray freeze-drying processes on the stability and aerosol performance of parathyroid hormone (PTH) (1-34) microparticles. In this study, the stabilisation effect of trehalose (a non-reducing sugar) and Brij 97 (a non-ionic surfactant) on spray-dried PTH particles was assessed using analytical techniques including circular dichroism (CD), fluorescence spectroscopy, modulated differential scanning calorimetry and an in vitro bioactivity assay. Physical characterisation also included electron microscopy, tap density measurement and laser light diffraction. The aerosol aerodynamic performance of the formulations was assessed using the Andersen cascade impactor. Based on these studies, a formulation for spray freeze-drying was selected and the effects of the two particle engineering techniques on the biophysical stability and aerosol performance of the resulting powders was determined. CD, fluorescence spectroscopy and bioactivity data suggest that trehalose when used alone as a stabilising excipient produces a superior stabilising effect than when used in combination with a non-ionic surfactant. This highlights the utility of CD and fluorescence spectroscopy studies for the prediction of protein bioactivity post-processing. Therefore, a method and formulation suitable for the preparation of PTH as a dry powder was developed based on spray-drying PTH with trehalose as a stabiliser with the bioactivity of SD PTH containing trehalose being equivalent to that of unprocessed PTH.

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.

SummaryThe emergence of RNA interference (RNAi) offers a potentially exciting new therapeutic paradigm for respiratory diseases. However effective delivery remains a key requirement for their translation into the clinic and has been a major factor in the limited clinical success seen to-date. Inhalation offers tissue-specific targeting of the RNAi to treat respiratory diseases and a diminished risk of off-target effects. In order to deliver RNAi directly to the respiratory tract via inhalation "smart" non-viral carriers are required to protect the RNAi during delivery/aerosolisation and enhance cell-specific uptake to target cells. Herein, we review the state of the art in therapeutic aerosol bioengineering (TAB) and specifically non-viral siRNA delivery platforms for delivery via inhalation. This includes developments in inhaler device engineering and particle engineering including manufacturing methods and excipients used in TAB that underpin the development of "smart" cell-type specific delivery systems to target siRNA to respiratory epithelial cells and/or alveolar macrophages.

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