Krishnakumar Subramanian

Translational siRNA Therapeutics Using Liposomal Carriers: Prospects & Challenges

Gene silencing has emerged as a promising strategy for molecular therapy of various malignant, viral, hereditary and inflammatory disorders. However, its translation from lab to clinic is yet to gain momentum due to the numerous problems that plague its development. A multi-functional siRNA delivery system with desired properties such as enhanced immune compatibility, target specificity, high cell uptake and excellent silencing efficiency is required to understand the challenges involved in the selection and modification of small interfering RNA (siRNA), factors influencing the complexation process and the response of the biological system to the formulation. Liposomes have been used as delivery systems due to its versatility in handling different types of drugs, tunable size, charge and surface functionalities that improve its effectiveness in vivo. This review highlights the challenges involved in gene silencing and describes the progression of liposomal systems used in gene silencing. The rationale in introducing chemical modifications in siRNA, synthesizing designer cationic lipids and evolution of hybrid liposomal systems has been elaborated, emphasizing their merits and short-comings. Finally, a description of the current state of clinical trials involving liposomal formulations has been included to provide an unbiased perspective of the future of liposomal systems and gene silencing tools as therapeutic tools.

Management of retinoblastoma: opportunities and challenges

Abstract Nano-delivery systems have significantly evolved over the last decade for the treatment of cancer by enabling site-specific delivery and improved bioavailability. The widely investigated nanoparticle systems are biodegradable polyesters, dendrimers, liposomes, mesoporous silica and gold nanoparticles. These particles when conjugated with different targeting motifs enhance the therapeutic efficiency of the drug molecules and biocompatibility. However, the application of such systems towards the treatment of retinoblastoma (RB), a rapidly spreading childhood eye cancer, still remains in its infancy. Nanoparticle-based systems that have been investigated for RB therapy have displayed improved drug delivery to the most restricted posterior segment of the eyes and have increased intra-vitreal half-life of the chemotherapy agents highlighting its potential in treatment of this form of cancer. This review focuses on the challenges involved in the treatment of RB and highlights the attempts made to develop nano-dimensional systems for the treatment of RB.

Molecular pathology of retinoblastoma

Retinoblastoma (RB) is an embryonic neoplasm of retinal origin. For many years, scientists have sought the fundamental origins of tumorigenesis, with the ultimate hope of discovering a cure. Indeed, these efforts have led to a significant understanding that multiple molecular and genetic aberrations, such as uncontrolled proliferation and the inhibition of apoptosis that contribute to the canonical characteristics of tumor biology. Despite these advances, a thorough understanding, such as the precise cells, which are the targets of neoplastic transformation, especially in solid tumors, is currently lacking. The focus of this review is to emphasize the molecular defects involved in the RB tumor progression and mechanisms associated with inhibition of tumor cell apoptotic processes. This review also discusses the importance of target molecules characterization and their potential therapeutic or prognostic use in RB disease.

Comparative docking of dual conformations in human fatty acid synthase thioesterase domain reveals potential binding cavity for virtual screening of ligands

Human fatty acid synthase (hFASN), a homo dimeric lipogenic enzyme with seven catalytic domains, is an important clinical target in cancer, metabolic syndrome and infections. Here, molecular modelling and docking methods were implemented to examine the inter-molecular interactions of thioesterase (TE) domain in hFASN with its physiological substrate, and to identify potential chemical inhibitors. TE catalyses the hydrolysis of thioester bond between palmitate and the 4’ phosphopantetheine of acyl carrier protein, releasing 16-carbon palmitate. The crystal structure of hFASN TE in two inhibitory conformations (A and B) were geometry-optimized and used for molecular docking with palmitate, orlistat (a known FASN inhibitor) and virtual screening against compounds from National Cancer Institute (NCI) database. Relatively, low binding affinity was observed during the complex formation of palmitate with A (−.164 kcal/mol) and B (−.332 kcal/mol) forms of TE, when compared with orlistat-docked TE (A form: −5.872 kcal/mol and B form: −5.484 kcal/mol), clearly indicating that the native inhibited conformation (crystal structure) was unfavourable for substrate binding. We used these orlistat dual binding modes as positive controls for prioritizing the ligands during virtual screening. From 2, 31,617 molecules in the NCI database, 916 high-scoring compounds (hit ligands) were obtained for A-form and 4582 for B-form of the TE-domain, which were then ranked according to glide docking score, XP H bond score, absorption, distribution, metabolism and excretion and binding free energy (Prime/MM-GBSA). Consequently, two top scoring ligands (NSC: 319661 and NSC: 153166) emerged as promising drug candidates that may be tested in FASN-over-expressing diseases.

'Nano-in-Nano' Hybrid Liposomes Increase Target Specificity and Gene Silencing Efficiency in Breast Cancer induced SCID Mice.

Graphical abstract Figure. No Caption available. Abstract Gene silencing has immense potential in the treatment of cancer. However, enhancement of its efficiency requires the development of specifically targeted and safe carrier systems. Cationic carriers are generally limited by their immunogenicity. Hence, in this study, we report hybrid liposomes encapsulating Poly (L‐lysine)‐siRNA complex to silence epithelial cell adhesion molecule (EpCAM), highly expressed in epithelial cancers. The hybrid liposomes LL1 (Egg PC:DSPE‐PEG, 10:0) and hybrid immunoliposomes LL2 (Egg PC:DSPE‐PEG, 8:2) linked with EpCAM antibody as the targeting ligand showed an encapsulation efficiency of 70% and 86%, respectively. LL2 liposomes with a zeta potential of −26 mV exhibited good colloidal stability in phosphate buffered saline containing bovine serum albumin and fetal bovine serum at 37 °C. Cell uptake studies showed increased uptake of the LL2 when compared to LL1 liposomes. Finally, the hybrid immunoliposomes were evaluated for their efficacy in regressing the tumor volume in SCID mice. Eight doses each of 0.15 mg/kg, which is among the lowest reported siRNA concentrations, were administered to the animals. About 45% reduction in tumor volume was achieved after 28 days in the mice treated with LL2 when compared with the positive control and LL1 treated groups. Thus, our results demonstrate that the ‘nano–in–nano’ concept of encapsulating poly (l–Lysine) complexed EpCAM siRNA in immunoliposomes may be a promising strategy to treat EpCAM‐positive epithelial cancers, especially as an adjuvant therapy.

EpCAM-targeted liposomal si-RNA delivery for treatment of epithelial cancer

Abstract Background: RNA interference (RNAi) technology using short interfering RNA (si-RNA) has shown immense potential in the treatment of cancers through silencing of specific genes. Cationic non-viral vectors employed for gene delivery exhibit toxic effects in normal cells limiting their widespread use, therefore, site-specific delivery using benign carriers could address this issue. Objective: Design of a non-toxic carrier that enables site-specific delivery of si-RNA into the cancer cells is of prime importance to realize the promise of gene silencing. Methods: In the present study, non-cationic liposomes encapsulating si-RNA against epithelial cell adhesion molecule (EpCAM) were developed and characterized for encapsulation efficiency, colloidal stability, in vitro and in vivo gene silencing efficacy. Results: PEGylated liposomes containing phosphatidyl choline and phosphatidyl ethanolamine exhibited maximum si-RNA encapsulation efficiency of 47%, zeta potential of -21 mV, phase transition temperature of 51 °C and good colloidal stability in phosphate-buffered saline (PBS) containing bovine serum albumin (BSA) and plasma protein (PP) at 37 °C. Conjugation of epithelial cell adhesion molecule (EpCAM) antibody to the liposomes resulted in enhanced cell internalization and superior down-regulation of EpCAM gene in MCF-7 cell lines when compared with free si-RNA and the non-targeted liposomes. In vivo evaluation of immunoliposomes for their efficacy in regressing the tumor volume in Balb/c SCID mice showed about 35% reduction of tumor volume in comparison with the positive control when administered with an extremely low dose of 0.15 mg/kg twice a week for 4 weeks. Conclusion: Our results exhibit that the nanocarrier-mediated silencing of EpCAM gene is a promising strategy to treat epithelial cancers.

A rare case of giant multicystic solitary fibrous tumor of the orbit

ABSTRACT Solitary fibrous tumor (SFT) is a rare spindle cell tumor of the orbit of mesenchymal origin. Though these tumors are mostly solid, partial or complete cystic changes can rarely occur. Only six such previous cases of cystic fibrous tumor of the orbit have been mentioned in the literature. We report a case of an elderly male who presented with a huge left sided medial orbital mass. Magnetic resonance imaging showed a predominant cystic orbital mass separated by septae and suggested a diagnosis of Hydatid cyst. The patient underwent complete excision of the mass and histopathology and immunohistochemistry were suggestive of cystic SFT. Cystic degeneration in SFT is extremely rare and can be a harbinger of malignancy, and pose risk of recurrence. Close follow up and monitoring is required for all such cases.

Orbital angioleiomyoma: A rare orbital neoplasm

ABSTRACT A 44-year-old male patient presented with painless progressive proptosis of left eye for the last 20 years. Examination revealed a purplish vascular mass extending from the medial orbital region to the surface of the globe. He underwent complete excision of the mass via an anterior orbitotomy approach. Histopathology and immunohistochemistry revealed a diagnosis of angioleiomyoma. No recurrence was noted at 1 year of follow-up. Angioleiomyomas are benign smooth muscle tumors with an additional vascular component. Their occurrence in the orbit is extremely rare with only three cases reported in literature till date. We report a fourth case of angioleiomyoma of the orbit with the longest duration of presentation of 20 years.

Protein Adsorption Characteristics and Inflammatory Response of Lipoplexes of DNA and si-RNA

Objective: A major limitation in gene delivery applications employing nanocarriers is the inflammatory response elicited when administered in vivo. The mode of complexation of the oligonucleotide with the carrier can alter its interactions with biomolecules, a fact that has not been explored for lipoplexes hitherto. Materials: Liposomes prepared by thin film hydration were used to form lipoplexes of si-RNA and DNA, which exhibited a smaller size and a shift toward negative zeta potential when compared with blank liposomes. Results: The oligonucleotides wrap over the liposome surface and the surface coverage depends on the number of base pairs. The colloidal stability, protein resistance and cell uptake of lipoplexes were found to be dependent on surface charge and PEG. The lipoplexes with si-RNA did not induce cytokine production in BALB/c mice. Conclusion: The results highlight the importance of PEGylation for achieving good protein resistance without compromising cell uptake and therapeutic efficiency.

Membrane Proteome of Invasive Retinoblastoma: Differential Proteins and Biomarkers

Purpose: Retinoblastoma (RB) is a pediatric ocular cancer which is caused due to the aberrations in the RB1 gene. The changes in the membrane proteomics would help in understanding the development of the retinoblastoma and could identify candidates for biomarkers and therapy.