The clinical utility of siRNA therapy has been hampered due to poor cell penetration, nonspecific effects, rapid degradation, and short half-life. We herewith proposed the formulation development of STAT6 siRNA (S6S) nanotherapeutic agent by encapsulating them within gelatin nanocarriers (GNC). The prepared nanoformulation was characterized for size, charge, loading efficiency, release kinetics, stability, cytotoxicity, and gene silencing assay. The stability of S6S-GNC was also assessed under conditions of varying pH, serum level, and using electrophoretic assays. In vitro cytotoxicity performance was evaluated in human adenocarcinoma A549 cells following MTT assay. The developed formulation resulted in an average particle size, surface charge, and encapsulation efficiency as 70 ± 6.5 nm, +10 ± 1.5 mV, and 85 ± 4.0%, respectively. S6S-GNC showed an insignificant (P < 0.05) change in the size and charge in the presence of buffer solutions (pH 6.4 to 8.4) and FBS (10% v/v). A549 cells were treated with native S6S, S6S-lipofectamine, placebo-GNC, and S6S-GNC using untreated cells as a control. It was observed that cell viability was decreased significantly with S6S-GNC by 55 ± 4.1% (P < 0.001) compared to native S6S (2.0 ± 0.55%) and S6S-lipofectamine complex (40 ± 3.1%). This investigation infers that gelatin polymer-based nanocarriers are a robust, stable, and biocompatible strategy for the delivery of siRNA.

Abstract. The aim of this investigation was to develop and evaluate freeze-dried mannosylated liposomes for the targeted delivery of selenium. Dipalmitoylphosphatidylcholine, distearoylphosphatidylglycerol, and cholesterol were dissolved in a chloroform and methanol mixture and allowed to form a thin film within a rotatory evaporator. This thin film was hydrated with a sodium selenite (5.8 μM) solution to form multilamellar vesicles and homogenized under high pressure to yield unilamellar nanoliposomes. Seloaded nanoliposomes were mannosylated by 0.1%w/v mannosamine (Man-Lip-Se) prior to being lyophilized. Mannosamine concentration was optimized with cellular uptake studies in M receptor expressing cells. Non-lyophilized and lyophilized Man-Lip-Se were characterized for size, zeta potential, and entrapment efficiency. The influence of liposomal composition on the characteristics of Man-Lip-Se were evaluated using acidic and basic medium for 24 h. Thermal analysis and powder X-ray diffraction were used to determine the interaction of components within the Man-Lip-Se. The size, zeta potential and entrapment efficiency of the optimum Man-Lip-Se were observed to be 158±28.9 nm, 33.21±0.89 mV, and 77.27±2.34%, respectively. An in vitro Se release of 70-75% up to 24 h in PBS pH 6.8 and <8% Se release in acidic media (0.1 N HCl) in 1 h was observed. The Man-Lip-Se were found to withstand gastric-like environments and showed sustained release. Stable freeze-dried Man-Lip-Se were successfully formulated with a size of <200 nm, ∼75% entrapment, and achieved controlled release of Se with stability under acidic media, which may be of importance in the targeted delivery of Se to the immune system.

Nutraceuticals are important due to their inherent health benefits. However, utilization and consumption are limited by their poor water solubility and instability at normal processing and storage conditions. Herein, we propose an elegant and novel approach for the delivery of nutraceuticals in their active form using hydrocolloid matrices that are inexpensive and non-toxic with GRAS status. Iota-carrageen and curcumin have been chosen as models of hydrocolloid and nutraceutical compound, respectively. The iota-carrageenan network maintains a stable organization after encapsulating curcumin molecules, protects them from melting and then releases in a sustained manner. These findings lay a strong foundation for developing value-added functional foods and medicinal foods.

Signal transducer and activator of transcription 6 (STAT-6) is a transcription factors that is highly expressed in various types of cancers including lung cancer and its expression was reported to be significantly correlated with tumor progression. Several studies showed that down-regulation of STAT-6 using short interfering RNA (siRNA) leads to the induction of apoptosis and inhibition of tumor growth. The use of siRNA for cancer therapy represents a promising approach for STAT-6 silencing due to its efficient knockdown of targeted genes compared to other conventional anti-cancer therapies. Despite this huge therapeutic potential, the clinical utility of siRNA therapy has been greatly hampered due to its poor cell penetration, rapid degradation, short half-life and non-specific effects. To overcome these limitations, we herewith propose to formulate STAT-6 siRNA (S6S) as a nanotherapeutic by encapsulating them within the gelatin nanocarrier (GNC) to achieve its successful intratumoral delivery. The nanoformulation was prepared by two step desolvation technique and characterized exhaustively for loading efficiency, size, charge, release kinetics, stability and cytotoxicity performance. The stability of S6S-GNC was assessed under conditions of varying pH and serum level to determine the stability of developed formulation. The S6S-GNC was incubated in a phosphate buffer (pH 6.4, 7.4 and 8.4) to assess the influence of pH on charge and size of the GNC. Additionally, the stability was also accessed using an electrophoretic assay. In vitro cytotoxicity was evaluated against A549 lung cancer cells following MTT cytotoxicity assay. The developed formulation contained ≈10,000 GNC/ml, and the average particle size and surface charge was observed to be 69.6±6.5 nm and +10±1.5 mV, respectively. The optimized S6S-GNC formulations showed an encapsulation efficiency of 85±4%. S6S-GNC showed an insignificant (p&gt;0.05) change in the size in the presence of buffer solutions (pH 6.4 to 8.4) and FBS (10% v/v). A549 cells were treated with native S6S, S6S-lipofectamine, placebo GNC and S6S-GNC using untreated cells as a control. It was observed that cell viability was decreased significantly with S6S-GNC by 55±4 % (p&lt;0.001) compared to native S6S (2±0.5%) and S6S-lipofectamine (40±3 %). Placebo GNC treatment showed &gt;97% viability of cells demonstrating non-toxicity and safety of GNC formulation The increased cytotoxicity with S6S-GNC can be attributed to the accumulation of positively charged GNCs through electrochemical diffusion. Overall, these results suggest FDA approved gelatin polymer based nanocarriers may be a more robust, stable and biocompatible strategy to deliver S6S via parenteral route with the potential to develop them as cancer targeted nanocarrier by subsequently anchoring them to cancer specific ligands (such as RGD, Folate etc) via freely available amine and carboxy terminals of gelatin. Citation Format: Susanne R. Youngren, Rakesh K. Tekade, Peter R. Hoffmann, Mahavir B. Chougule. Biocompatible nanocarrier mediated delivery of STAT-6 siRNA to cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3313. doi:10.1158/1538-7445.AM2013-3313