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.

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.

Zika virus: Molecular responses and tissue tropism in the mammalian host.

Zika virus (ZIKV) outbreaks have raised alarm because of reports of congenital Zika virus syndrome in infants. The virus is also known to cause the debilitating Guillain-Barré syndrome in adults. As a result, extensive research has been carried out on the virus over the past few years. To study the molecular responses of viral infectivity in mammals, in vitro two-dimensional and three-dimensional cellular models have been employed. The in vivo models of mouse, pig, chicken, and nonhuman primates are primarily used to investigate the teratogenicity of the virus, to study effects of the virus on specific tissues, and to study the systemic effects of a proposed antiviral agent. The virus exhibits wide tissue tropism in the mammalian host. The major host tissues of viral persistence and propagation are neural tissue, ocular tissue, testicular tissue and placental tissue. An understanding of the function of viral components, viral replication cycle, and the molecular responses elicited in the host tissues is imperative for designing antiviral treatment strategies and for development of vaccines. This review provides an update on ZIKV research models and mammalian host responses with respect to ZIKV tissue infection.

Hydrophobic and electrostatic interactions between cell penetrating peptides and plasmid DNA are important for stable non-covalent complexation and intracellular delivery.

Cell penetrating peptides are useful tools for intracellular delivery of nucleic acids. Delivery of plasmid DNA, a large nucleic acid, poses a challenge for peptide mediated transport. The paper investigates and compares efficacy of five novel peptide designs for complexation of plasmid DNA and subsequent delivery into cells. The peptides were designed to contain reported DNA condensing agents and basic cell penetrating sequences, octa-arginine (R8 ) and CHK6 HC coupled to cell penetration accelerating peptides such as Bax inhibitory mutant peptide (KLPVM) and a peptide derived from the Kaposi fibroblast growth factor (kFGF) membrane translocating sequence. A tryptophan rich peptide, an analogue of Pep-3, flanked with CH3 on either ends was also a part of the study. The peptides were analysed for plasmid DNA complexation, protection of peptide-plasmid DNA complexes against DNase I, serum components and competitive ligands by simple agarose gel electrophoresis techniques. Hemolysis of rat red blood corpuscles (RBCs) in the presence of the peptides was used as a measure of peptide cytotoxicity. Plasmid DNA delivery through the designed peptides was evaluated in two cell lines, human cervical cancer cell line (HeLa) and (NIH/3 T3) mouse embryonic fibroblasts via expression of the secreted alkaline phosphatase (SEAP) reporter gene. The importance of hydrophobic sequences in addition to cationic sequences in peptides for non-covalent plasmid DNA complexation and delivery has been illustrated. An alternative to the employment of fatty acid moieties for enhanced gene transfer has been proposed. Comparison of peptides for plasmid DNA complexation and delivery of peptide-plasmid DNA complexes to cells estimated by expression of a reporter gene, SEAP. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

N-glycans and metastasis in galectin-3 transgenic mice.

Poly-N-acetyl-lactosamine (polyLacNAc) on N-glycans facilitate lung specific metastasis of melanoma cells by serving as high affinity ligands for galectin-3, expressed in highest amounts in the lungs, on almost all its tissue compartments including on the surface of vascular endothelium. PolyLacNAc not only aids in initial arrest on the organ endothelium but in all the events of extravasation. Inhibition of polyLacNAc synthesis, or competitive inhibition of its interaction with galectin-3 all inhibited these processes and experimental metastasis. Transgenic galectin-3 mice, viz., gal-3(+/+) (wild type), gal-3(+/-) (hemizygous) and gal-3(-/-) (null) have been used to prove that galectin-3/polyLacNAc interactions are indeed critical for lung specific metastasis. Gal-3(+/-) mice which showed <50% expression of galectin-3 on the lungs also showed proportionate decrease in the number of B16F10 melanoma metastatic colonies affirming that galectin-3 and polyLacNAc interactions are indeed key determinants of lung metastasis. However, surprisingly, the number and size of metastatic colonies in gal-3(-/-) mice was very similar as that seen in gal-3(+/+) mice. The levels of lactose binding lectins on the lungs and the transcripts of other galectins (galectin-1, -8 and -9) which are expressed on lungs and have similar sugar binding specificities as galectins-3, remain unchanged in gal-3(+/+) and gal-3(-/-) mice. Further, inhibition of N-glycosylation with Swainsonine (SW) which drastically reduces metastasis of B16F10 cells in gal-3(+/+) mice, did not affect lung metastasis when assessed in gal-3(-/-) mice. Together, these results rule out the possibility of some other galectin taking over the function of galectin-3 in gal-3(-/-) mice. Chimeric mice generated to assess if absence of any effect on metastasis is due to compromised tumor immunity by replacing bone marrow of gal-3(-/-) mice with that from gal-3(+/+) mice, also failed to impact melanoma metastasis. As galectin-3 regulates several immune functions including maturation of different immune cells, compromised tumor immunity could be the major determinant of melanoma metastasis in gal-3(-/-) mice and warrants thorough investigation.

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.

The Brilliance of the Zebrafish Model: Perception on Behavior and Alzheimer's Disease.

Alzheimer's disease (AD) has become increasingly prevalent in the elderly population across the world. It's pathophysiological markers such as overproduction along with the accumulation of amyloid beta (Aß) plaques and neurofibrillary tangles (NFT) are posing a serious challenge to novel drug development processes. A model which simulates the human neurodegenerative mechanism will be beneficial for rapid screening of potential drug candidates. Due to the comparable neurological network with humans, zebrafish has emerged as a promising AD model. This model has been thoroughly validated through research in aspects of neuronal pathways analogous to the human brain. The cholinergic, glutamatergic, and GABAergic pathways, which play a role in the manifested behavior of the zebrafish, are well defined. There are several behavioral models in both adult zebrafish and larvae to establish various aspects of cognitive impairment including spatial memory, associative memory, anxiety, and other such features that are manifested in AD. The zebrafish model eliminates the shortcomings of previously recognized mammalian models, in terms of expense, extensive assessment durations, and the complexity of imaging the brain to test the efficacy of therapeutic interventions. This review highlights the various models that analyze the changes in the normal behavioral patterns of the zebrafish when exposed to AD inducing agents. The mechanistic pathway adopted by drugs and novel therapeutic strategies can be explored via these behavioral models and their efficacy to slow the progression of AD can be evaluated.

Neurosensory Prosthetics: An Integral Neuromodulation Part of Bioelectronic Device.

Bioelectronic medicines (BEMs) constitute a branch of bioelectronic devices (BEDs), which are a class of therapeutics that combine neuroscience with molecular biology, immunology, and engineering technologies. Thus, BEMs are the culmination of thought processes of scientists of varied fields and herald a new era in the treatment of chronic diseases. BEMs work on the principle of neuromodulation of nerve stimulation. Examples of BEMs based on neuromodulation are those that modify neural circuits through deep brain stimulation, vagal nerve stimulation, spinal nerve stimulation, and retinal and auditory implants. BEDs may also serve as diagnostic tools by mimicking human sensory systems. Two examples of in vitro BEDs used as diagnostic agents in biomedical applications based on in vivo neurosensory circuits are the bioelectronic nose and bioelectronic tongue. The review discusses the ever-growing application of BEDs to a wide variety of health conditions and practices to improve the quality of life.

Targeted drug therapy in nonsmall cell lung cancer: clinical significance and possible solutions-part II (role of nanocarriers).

INTRODUCTION: Nonsmall cell lung cancer (NSCLC) accounts for 80-85% of the cases of lung cancer. The conventional therapeutic effective dosage forms used to treat NSCLC are associated with rigid administration schedules, adverse effects, and may be associated with acquired resistance to therapy. Nanocarriers may provide a suitable alternative to regular formulations to overcome inherent drawbacks and provide better treatment modalities for the patient. AREAS COVERED: The article explores the application of drug loaded nanocarriers for lung cancer treatment. Drug-loaded nanocarriers can be modified to achieve controlled delivery at the desired tumor infested site. The type of nanocarriers employed are diverse based on polymers, liposomes, metals and a combination of two or more different base materials (hybrids). These may be designed for systemic delivery or local delivery to the lung compartment (via inhalation). EXPERT OPINION: Nanocarriers can improve pharmacokinetics of the drug payload by improving its delivery to the desired location and can reduce associated systemic toxicities. Through nanocarriers, a wide variety of therapeutics can be administered and targeted to the cancerous site. Some examples of the utilities of nanocarriers are codelivery of drugs, gene delivery, and delivery of other biologics. Overall, the nanocarriers have promising potential in improving therapeutic efficacy of drugs used in NSCLC.

Targeted drug therapy in non-small cell lung cancer: Clinical significance and possible solutions-Part I.

INTRODUCTION: Non-small cell lung cancer (NSCLC) comprises of 84% of all lung cancer cases. The treatment options for NSCLC at advanced stages are chemotherapy and radiotherapy. Chemotherapy involves conventional nonspecific chemotherapeutics, and targeted-protein/receptor-specific small molecule inhibitors. Biologically targeted therapies such as an antibody-based immunotherapy have been approved in combination with conventional therapeutics. Approved targeted chemotherapy is directed against the kinase domains of mutated cellular receptors such as epidermal growth factor receptor (EGFR), anaplastic lymphoma kinases (ALK), neurotrophic receptor kinases (NTRK) and against downstream signaling molecules such as BRAF (v-raf murine sarcoma viral oncogene homolog B1). Approved biologically targeted therapy involves the use of anti-angiogenesis antibodies and antibodies against immune checkpoints. AREAS COVERED: The rationale for the employment of targeted therapeutics and the resistance that may develop to therapy are discussed. Novel targeted therapeutics in clinical trials are also included. EXPERT OPINION: Molecular and histological profiling of a given tumor specimen to determine the aberrant onco-driver is a must before deciding a targeted therapeutic regimen for the patient. Periodic monitoring of the patients response to a given therapeutic regimen is also mandatory so that any semblance of resistance to therapy can be deciphered and the regimen may be accordingly altered.

Galectin-3 expressed on different lung compartments promotes organ specific metastasis by facilitating arrest, extravasation and organ colonization via high affinity ligands on melanoma cells.

Interactions between molecules on the surface of tumor cells and those on the target organ endothelium play an important role in their arrest in an organ. Galectin-3 on the lung endothelium and high affinity ligands poly-N-acetyllactosamine (polyLacNAc) on N-oligosaccharides on melanoma cells facilitate such interactions. However, to extravasate and colonize an organ the cells must stabilize these interactions by spreading to retract endothelium, degrade exposed basement membrane (BM) and move into parenchyma and proliferate. Here, we show that galectin-3 is expressed on all the major compartments of the lungs and participates in not just promoting adhesion but also in spreading. We for the first time demonstrate that both soluble and immobilized galectin-3 induce secretion of MMP-9 required to breach vascular BM. Further, we show that immobilized galectin-3 is used as traction for the movement of cells. Downregulation of galactosyltransferases-I and -V resulted in significant loss in expression of polyLacNAc and thus reduced binding of galectin-3. This was accompanied with a loss in adhesion, spreading, MMP-9 secretion and motility of the cells on galectin-3 and thus their metastasis to lungs. Metastasis could also be inhibited by blocking surface polyLacNAc by pre-incubating cells with truncated galectin-3 (which lacked oligomerization domain) or by feeding mice with modified citrus pectin in drinking water. Overall, these results unequivocally show that polyLacNAc on melanoma cells and galectin-3 on the lungs play a critical role in arrest and extravasation of cells in the lungs and strategies that target these interactions inhibit lung metastasis.