Development and Characterization of Modified Chitosan Lipopolyplex for an Effective siRNA Delivery.

Cytotoxicity, speedy degradation, and limited cellular absorption are the foremost features influencing the successful delivery of RNAs. Chitosan (Cs) is a polymer that offers an advantage due to its bio-compatibility and biodegradable nature, making it an ideal polycationic vector for delivering siRNA. In this study, chitosan has been modified with arginine in order to increase its encapsulation of siRNA and improve cellular absorption. It was discovered that arginine and guanidino moieties could transport through membranes of cells and play an important part in membrane permeability. FTIR and 13C NMR were used to characterize the complex. These chitosan-arginine (CsAr) siRNA complexes are further encapsulated in anionic DPPC/cholesterol liposomes to combine the effects of liposome-chitosan-arginine complexes called lipopolyplexes (LCAr). Formed LCAr were investigated for their lipid/CsAr-siRNA ratios, size, zeta-potential, heparin, and serum RNase stability by agarose gel retardation, and cell uptake efficiency compared to their "parent" polyplexes. Results revealed complete lipidation of CsAr-siRNA polyplexes at lipid mass ratio 10 resulting in lipopolyplexes in the 120 to 230nm range. Polyplex entrapped ~70% of siRNA, whereas lipidation increases siRNA encapsulation to ~95%. Developed LCAr showed ~4 times less hemolytic potential as compared to the parent polyplexes at the highest siRNA dose. The CsAr-siRNA and its lipid-coated form showed enhanced cellular association as compared to the marketed Lipofectamine 2000 proving its effectiveness in siRNA delivery. CsAr-liposome conjugation is simple and safe, and serves as a robust carrier for gene transport in physiological situations without compromising transfection efficacy.

Analysis of RNA Interference Targeted Against Human Antigen R (HuR) to Reduce Vascular Endothelial Growth Factor (VEGF) Protein Expression in Human Retinal Pigment Epithelial Cells.

VEGF-A or vascular endothelial growth factor-A is an important factor in enabling neovascularization and angiogenesis. VEGF-A is regulated transcriptionally as well as post transcriptionally. Human antigen R (HuR) belonging to the embryonic lethal abnormal vision (ELAV) family is a key regulator promoting stabilization of VEGF-A mRNA. In this research we investigate, whether HuR targeted RNA interference would enable the reduction of the VEGF-A protein in human retinal pigment epithelial cells (ARPE-19) in-vitro, in normoxic conditions. Three siRNA molecules with sequences complementary to three regions of the HuR mRNA were designed. The three designed siRNA molecules were individually transfected in ARPE-19 cells using Lipofectamine™2000 reagent. Post-transfection (24 h, 48 h, 72 h), downregulation of HuR mRNA was estimated by real-time polymerase reaction, while HuR protein and VEGF-A protein levels were semi-quantitatively determined by western blotting techniques. VEGF-A protein levels were additionally quantified using ELISA techniques. All experiments were done in triplicate. The designed siRNA could successfully downregulate HuR mRNA with concomitant decreases in HuR and VEGF-A protein. The study reveals that HuR downregulation can prominently downregulate VEGF-A, making the protein a target for therapy against pathological angiogenesis conditions such as diabetic retinopathy.

Liposome-polyethylenimine complexes for the effective delivery of HuR siRNA in the treatment of diabetic retinopathy.

Diabetic retinopathy (DR) is a vision-impairing complication of diabetes, damaging the retinal microcirculatory system. Overexpression of VEGF (vascular endothelial growth factor) is implicated in the pathogenesis of DR. Human antigen R (HuR) is an RNA-binding protein that favorably regulates VEGF protein expression by binding to VEGF-encoding mRNA. Downregulating HuR via RNA interference strategies using small interfering RNAs (siRNAs) may constitute a novel therapeutic method for preventing VEGF protein overexpression in DR. Delivery of siRNAs to the cellular cytoplasm can be facilitated by cationic peptides or polymers and lipids. In this study, a cationic polymer (polyethylenimine (PEI)) and lipid nanoparticles (liposomes) were co-formulated with siRNA to form lipopolyplexes (LPPs) for the delivery of HuR siRNA. LPPs-siRNA were analyzed for size, zeta potential, serum stability, RNase stability, heparin stability, toxicity, and siRNA encapsulation efficiency. Cellular uptake, downregulation of the target HuR (mRNA and protein), and associated VEGF protein were used to demonstrate the biological efficacy of the LPPs-HuR siRNA, in vitro (human ARPE-19 cells), and in vivo (Wistar rats). In vivo efficacy study was performed by injecting LPPs-HuR siRNA formulations into the eye of streptozotocin (STZ)-induced diabetic rats after the development of retinopathy. Our findings demonstrated that high retinal HuR and VEGF levels observed in the eyes of untreated STZ rats were lowered after LPPs-HuR siRNA administration. Our observations indicate that intravitreal treatment with HuR siRNA is a promising option for DR using LPPs as delivery agents.

Formulation development and evaluation of therapeutic contact lens loaded with ganciclovir.

PURPOSE: In the present investigation ganciclovir (GAN) loaded microparticles dispersed in hydrogel-based contact lenses were fabricated, characterized and evaluated for eye irritation. METHODS: GAN-Hydroxy Propyl Methyl Cellulose (HPMC) microparticles were prepared by solvent evaporation method and evaluated for entrapment efficiency, drug content and drug release. The Polyhydroxyethylmethacrylate (pHEMA) contact lenses were synthesized by free radical polymerization reaction using crosslinkers like ethylene glycoldimethacrylate and photoinitiator such as IRGACURE 1173®, in UVB light, λ 365 nm. The GAN-HPMC microparticles when incorporated into the premonomer mixture and polymerized together give rise to a particle dispersion system in the hydrogel contact lenses. The contact lenses were studied for surface morphology, transmittance, swelling, drug release, Na+ion permeability and hens egg test chorioallantoic membrane assay (HETCAM). RESULTS: Hydrogel contact lens exhibited satisfactory surface morphology, transmittance, swelling, Na+ion permeability (3.72 × 106 mm2/min) and a release of 48 h suggesting a potential for prolonged ocular drug delivery. Furthermore, HETCAM exhibited no signs of ocular irritation. CONCLUSION: The developed delivery platform is a promising alternative to conventional dosage forms like eye drops, suspensions and ointments due to its increase in the residence time attributed to its prolonged release profile.

Role of small interfering RNA (siRNA) in targeting ocular neovascularization: A review.

Ocular neovascularization (NV) plays a central role in the pathogenesis of various ocular diseases including diabetic retinopathy, age-related macular degeneration, retinoblastoma, retinitis pigmentosa and may lead to loss of vision if not controlled in time. Several clinical trials elucidate the central role of vascular endothelial growth factor (VEGF) in the pathogenesis of the ocular neovascularization. The advent and extensive use of ocular anti-VEGF therapy heralded a new age in the treatment of retinal vascular and exudative diseases. RNA interference (RNAi) can be used to inhibit the in-vitro and in-vivo expression of specific genes and thus provides an extremely useful method for investigating gene activity with minimal toxicity. siRNA targeting VEGF overcomes many drawbacks associated with the conventional treatment available for the treatment of ocular neovascularization. However, delivery methods that protect the siRNA against degradation and are appropriate for long-term care will help increase the effectiveness of RNAi-based anti-VEGF ocular therapies. Several nanotechnology approaches have been explored by formulation scientists for delivery of siRNA to the eye; targeting particularly VEGF for the treatment of NV. This review mainly focuses on current updates in various pre-clinical and clinical siRNA strategies for targeting VEGF involved in the development of ocular neovascularization.

Methods for evaluating penetration of drug into the skin: A review.

BACKGROUND: Skin being the largest organ of the human body plays a very important role in the permeation and penetration of the drug. In addition, the transdermal drug delivery system (TDDS) plays a major role in managing dermal infections and attaining sustained plasma drug concentration. Thus, evaluation of percutaneous penetration of the drug through the skin is important in developing TDDS for human use. MATERIAL AND METHODS: Various techniques are used for getting the desired drug penetration, permeation, and absorption through the skin in managing these dermal disorders. The development of novel pharmaceutical dosage forms for dermal use is much explored in the current era. However, it is very important to evaluate these methods to determine the bioequivalence and risk of these topically applied drugs, which ultimately penetrate and are absorbed through the skin. RESULTS: Currently, numerous skin permeation models are being developed and persuasively used in studying dermatopharmacokinetic (DPK) profile and various models have been developed, to evaluate the TDD which include ex vivo human skin, ex vivo animal skin, and artificial or reconstructed skin models. CONCLUSION: This review discusses the general physiology of the skin, the physiochemical characteristics affecting particle penetration, understand the models used for human skin permeation studies and understanding their advantages, and disadvantages.