Development and Evaluation of siRNA Loaded Gelatin Nanocarriers for the Treatment of Asthma

dc.contributor.advisor Chougule, Mahavir B.
dc.contributor.advisor Morris, Kenneth R. Youngren-Ortiz, Susanne R.
dc.contributor.department Pharmaceutical Sciences 2016-06-28T21:05:43Z 2016-05
dc.description.abstract Asthma is a chronic inflammatory disease that impacts about 300 million people worldwide and affects over 8% of Americans. Existing asthma treatments, such as anti-histamines or steroids, do not address the underlying cellular processes responsible for asthma pathogenesis and pathophysiology and therefore, more targeted approaches are needed for mitigating the disease. There is a continued need for drug or oligonucleotide delivery systems which could selectively deliver anti-asthmatic therapeutics directly to their site of action. T helper 2 (Th2) cell-mediated asthma progression plays a major role in inflammatory mediator production and airway mucus hyper-secretion associated with chronic atopic (allergic) asthma. The signal transducer and activator of transcription 6 (STAT6) is an essential transcription factor in asthma pathogenesis and progression via Th2 cells. RNAi is a naturally occurring gene silencing process that has a high degree of specificity and the potential to silence genes of interest. However, the clinical utility of RNAi therapy has been hampered due to poor cell penetration, nonspecific effects, rapid degradation, and short half-life. The central hypothesis of this work is that receptor-guided STAT6 siRNA loaded gelatin nanocarriers will selectively deliver STAT6 siRNA to T cells and silence STAT6 mRNA and protein expression thereby inhibiting the transcription of master gene GATA3 involved in the initiation and progression of asthma and attenuate allergic asthma. The overall objective of this research work was to formulate and characterize gelatin nanocarriers for the targeted delivery of STAT6 siRNA to T cells and to evaluate their effects in vitro. The development and evaluation of receptor-targeted STAT6 siRNA loaded gelatin nanocarriers represents the first critical step in the pre-clinical development of a novel nanotherapeutic for asthma therapy. To establish the effectiveness of gelatin nanocarriers of STAT6 siRNA for the targeted cellular delivery of siRNA for the treatment of asthma, a proof-of-concept formulation was evaluated. The objective was that to develop a gelatin based STAT6 siRNA loaded nanocarrier (S6S-GNC) and to evaluate if it provided biostability and delivery of STAT6 siRNA to cancer cells to exert anti-cancer activity through STAT6 mediated anti-proliferation and pro-apoptotic effects in a human lung cancer cell line. The prepared nanoformulation was characterized for size, charge, loading efficiency, release kinetics, stability, as well as in vitro cytotoxicity and protein downregulation in A549 cells treated with S6S-GNC. This investigation inferred gelatin polymer-based nanocarriers to be a robust, stable and biocompatible strategy for the delivery of STAT6 siRNA. The main objective of this study was to prepare, optimize, and characterize the interleukin receptor targeting peptide conjugated STAT6 siRNA loaded gelatin nanocarriers (S6S-GNC-P) and to evaluate their in vitro efficacy under cell binding, cell internalization, antiproliferative, and STAT6 protein and mRNA downregulation assays following treatment in a mouse Th2 cell line. The formulation was optimized using Taguchi orthogonal array design of experiments, regression analysis, and desirability index. The S6S-GNC were PEGylated using a carbodiimide crosslinking of maleimide terminated PEG5000. The PEGylated S6S-GNC were peptide conjugated through a stable thioether bond between the maleimide functionalized PEG and the cystamide containing interleukin targeting peptide. The optimized S6S-GNC-P formulation contained approximately 4800 PEG5000 in a brush-border configuration and 720 peptide molecules per gelatin nanocarrier and had a particle size of 98 ± 3.6 nm, PDI of 0.66 ± 0.02, zeta potential of +6.3 ± 0.54, and STAT6 siRNA loading efficiency of 19%. The S6S-GNC-P underwent a sustained diffusion-based STAT6 siRNA release through a matrix-based spherical system, as confirmed with kinetic modeling. The formulation was found to be stable in the presence of simulated physiological media and under electrophoretic mobility assays in the presence of RNAse. The S6S-GNC dose dependency studies found that 100 nM STAT6 siRNA equivalent doses were superior than other concentrations in the range of 25-200 nM siRNA for maximal STAT6 protein downregulation compared to the scrambled siRNA loaded GNC control. The S6S-GNC-P did not impart any significant cell cytotoxicity in Th2 cells in the concentration range of 25-250 nM STAT6 siRNA, but did significantly (p<0.001) reduce STAT6 protein expression in Th2 treated cells compared to the medium, the STAT6 siRNA positive, and scrambled siRNA-GNC-P controls, as well as the PEGylated S6S-GNC formulation. The S6S-GNC-P treated Th2 cells had a significant decrease in STAT6 mRNA expression levels when compared to the medium control (p<0.01) and the PEGylated S6S-GNC (p<0.001). Controlled release Gem-GNC with particle size of approximately 180 nm, EE% >90%, and LE% of ~9% were successfully developed for pulmonary delivery by the inhalation route of administration. The target particle size value was 150 nm, so the employed predicted equation generated via linear regression modeling for particle size led to the volume ratio of 7:10 of 90% v/v ethanol, which was selected by fixing the gelatin and genipin concentrations to 1% w/v and 0.02% w/w, respectively. The SEM and TEM images had shown that the developed Gem-GNC were uniform in particle size and were of a smooth spherical morphology with particle size of approximately 200 nm. The Taguchi analysis determined that genipin concentration was the most influential parameter on particle size since it had the largest S/N ratio range. DSC of lyophilized Gem-GNC indicated that the Gem and excipients where molecularly dispersed. PXRD analysis of the lyophilized Gem-GNC found that Gem within the Gem-GNC had an amorphous configuration. The formulation was found to be stable in the presence of pH 6.4-8.4 DPBS solutions. The release mechanisms of Gem from the Gem-GNC were found to be non-Fickian diffusion and erosion from a matrix-based nanocarrier. The Gem-GNC exhibited a controlled release of Gem, which is highly desirable for the long term constant delivery of the formulation with reduced dosing intervals. The nebulized Gem-GNC exhibited a mass median aerodynamic diameter (MMAD) of 2.0 ± 0.16 µm, geometric standard deviation (GSD) of 2.7 ± 0.16, and fine particle fraction (FPF) of 75.2 ± 2.4%. A549 cells treated with Gem-GNC obtained IC50 of 0.023 μM, however the Gem solution obtained an IC50 of 0.013 µM at 72 hr. The H460 cells treated with Gem-GNC obtained 5–fold lower IC50 at 48 hr when compared to the Gem solution, whereas at 72 hr the IC50 for Gem-GNC was 10-fold lower than the IC50 of Gem solution (** p<0.01). The developed Gem-GNC was found to be effective in protecting Gem from degradation and was able to delivery Gem within the tumor cells to exert anticancer activity. The development and evaluation of the Gem-GNC provides evidence that an aerosolized GNC approach may be useful for the delivery of therapeutics to the lungs, possibly for lung cancer treatment. More studies are warranted to fully illustrate the safety profile in order to form risk and benefit comparisons. The novel formulation of S6S-GNC-P was intended to be delivered by the intravenous route of administration. However the third objective, which was focused on the development and evaluation of aerosolized gemcitabine-loaded gelatin nanocarriers (Gem-GNC), depicted the potential for inhalation delivery of S6S-GNC-P by nebulization. A gemcitabine loaded gelatin nanocarrier (Gem-GNC) formulation was optimized and evaluated for their aerodynamic properties following nebulization. Future studies are necessary to evaluate the effects of administered gelatin nanocarriers, because they have potential for antigenic or immunogenic effects in the highly sensitive lungs of asthmatic patients. Ph.D.
dc.description.institution University of Hawaii at Hilo
dc.embargo.liftdate 2017-06-27
dc.format.extent 462 pages
dc.language.iso en
dc.subject Pharmaceutical sciences
dc.subject Asthma
dc.subject Gelatin
dc.subject Inhalation
dc.subject Nanoparticle
dc.subject Parenteral
dc.subject siRNA
dc.title Development and Evaluation of siRNA Loaded Gelatin Nanocarriers for the Treatment of Asthma
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