(T1130-03-15) Development and In Vitro Characterization of STAT6 siRNA Loaded PEGylated Gelatin Nanocarriers as an Anti-Asthmatic Therapy

Purpose: Asthma is a chronic inflammatory disease affecting 300 million people worldwide, among 25 million are in USA. Current asthma treatments, such as antihistamines or steroids, do not address the underlying cellular process responsible for asthma pathogenesis and therefore, targeted approaches are needed for combating the disease. STAT6 is an essential transcription factor in asthma pathogenesis and progression. Currently, there are no FDA approved STAT6 inhibitors available. Nanocarrier (NC) based targeted approaches help in increasing the stability and site-specific delivery of siRNA and reduce the risk of adverse effects. The work hypothesized that the formulation of STAT6 siRNA loaded PEGylated gelatin nanocarriers (GNC) would exhibit stability in different physiologically relevant media, protect the siRNA from degradation, and promote STAT6 protein downregulation in Th2 cells. The main purpose of this study was to development, optimization and in vitro characterization of PEGylated STAT6 siRNA loaded gelatin nanocarriers (S6S-GNC).

Methods: Type A cationic gelatin nanocarriers were prepared using the 2-step desolvation technique with some modifications. The S6S-GNC were developed using Taguchi orthogonal array design of experiments of acetaminophen (APAP) loaded gelatin nanoparticles, which underwent regression analysis to predict full factorial data. The independent factors were gelatin concentration, volume ratio of desolvating agent added to the gelatin solution, and APAP concentration. Gelatin concentration was selected as the orthogonally distributed parameter. The orthogonal array (L₉, 3³) was utilized to determine the optimal parameters and to analyze the effect of these parameters. Nine combinations were formed (F1-F9). Regression analysis of the Taguchi dataset was performed to predict full factorial data (F10-F36) to better design a formulation with desired properties. S6S-GNC were prepared and PEGylated using the established 2-step desolvation technique with s modifications as illustrated schematically in figure 1. The particle size of S6S-GNC and PEGylated S6S-GNC were determined by dynamic light scattering using a NICOMP ZLS 380 analyzer (PSS-NICOMP, Santa Barbara, USA). The zeta potential of the formulations was measured and calculated by Smoluchowski’s equation. For entrapment efficiency (EE%) evaluation, 100 µl of formulation and 400 µl distilled deionized water was placed on the top of the Vivaspin filter membrane and centrifuged at 16,200×g for 20 min. The aqueous filtrate was analyzed to determine the free STAT6 siRNA by UV-Vis Spectrophotometer. Loading efficiency was analyzed from EE data. For In vitro release studies 2 ml of S6S-GNC formulation was placed inside a dialysis bag (MWCO 100 kDa) and were placed in 50 ml of PBS pH 7.4 under constant agitation condition (300 rpm) at 37°C. At predetermined time intervals 0.5 ml of dissolution medium was collected and equal volume of fresh dissolution medium was added. The samples were analyzed for S6S from the developed formulations. For stability studies under serum and pH medium, 200 µl of S6S-GNC was incubated in 400 µl PBS pH 6.4, 7.4 and 8.4 for 1 hr (n=3) to assess the influence of pH on the surface charge and size of the nano-formulation. Stability studies were also performed under the presence of 10%v/v FBS at pH 7.4.

Results: It was observed that, non-PEGylated S6S-GNC showed particle size of 93 ± 3.9 nm and PEGylated S6S-GNC showed 97 ± 3.6 nm. There was a non-significant (p >0.05) increase in particle size. The PDI of the non-PEGylated (0.58 ± 0.02) and the PEGylated S6S-GNC (0.55 ± 0.01) did not show significant difference (p >0.05). The zeta potential of the PEGylated S6S-GNC (+6.8 ± 0.74mV) showed a significant reduction (p < 0.001) compared to zeta potential of non-PEGylated S6S-GNC (+17.1 ± 0.48 mV). Both Non and PEGylated S6S-GNC formulations showed high entrapment efficiencies of approximately 99% and loading efficiencies of approximately 0.27%.

The PEGylated S6S-GNC showed < 20% cumulative percent siRNA within 1 hr. After 24 hr, it released 77 ± 15% siRNA, at 48 hr approximately 92% cumulative siRNA release and by 72 hr had complete siRNA release. This represents a sustained release compared to the free siRNA solution, which had complete siRNA release within 1 hr. Difference in particle size and PDI of the formulation following incubations in DPBS containing 10% FBS at 1, 24, 48, 72, and 96 hr. were non-significant (p >0.05), which shows that S6S-GNC is stable.

Conclusion: Developed and optimized the PEGylated S6S-GNC formulation which is stable and showed sustained release with average particle size of 97 ± 3.6 nm, PDI of 0.55, zeta potential of +6.8 ± 0.74 mV. In the future experiments, formulation and in vitro and in vivo evaluation of IL4R targeting peptide conjugated PEGylated S6S-GNC will be performed.

References: Youngren SR, Tekade RK, Gustilo B, Hoffmann PR, Chougule MB. STAT6 siRNA matrix-loaded gelatin nanocarriers: formulation, characterization, and ex vivo proof of concept using adenocarcinoma cells. Biomed Res Int. 2013;2013:858946. doi:10.1155/2013/858946


Figure 1: Formulation of PEGylated S6S-GNC (S6S-GNC).