Nanostructured lipopeptide-based membranomimetics for stabilizing bacteriorhodopsin.

Nanostructured 7-9-residue cyclic and unstructured lipopeptide-based facial detergents have been engineered to stabilize the model integral membrane protein, bacteriorhodopsin. Formation of a cylindrical-type micelle assembly induced by facial amphipathic lipopeptides resembles a biological membrane more effectively than conventional micelles. The hydrophobic face of this cylindrical-type micelle provides extended stability to the membrane protein and the hydrophilic surface interacts with an aqueous environment. In our present study, we have demonstrated experimentally and computationally that lipopeptide-based facial detergents having an unstructured or ß-turn conformation can stabilize membrane proteins. However, constrained peptide detergents can provide enhanced stability to bacteriorhodopsin. In this study, we have computationally examined the structural stability of bacteriorhodopsin in the presence of helical, beta-strand, and cyclic unstructured peptide detergents, and conventional detergent-like peptides. Our study demonstrates that optimal membranomimetics (detergents) for stabilizing a specific membrane protein can be screened based on the following criteria: (i) hydrodynamic radii of the self-assembled peptide detergents, (ii) stability assay of detergent-encased membrane proteins, (iii) percentage covered area of detergent-encased membrane proteins obtained computationally and (iv) protein-detergent interaction energy.

Engineered vitamin E-tethered non-immunogenic facial lipopeptide for developing improved siRNA based combination therapy against metastatic breast cancer.

RNA interference based therapeutic gene silencing is an emerging platform for managing highly metastatic breast cancer. Cytosolic delivery of functional siRNA remains the key obstacle for efficient RNAi therapy. To overcome the challenges of siRNA delivery, we have engineered a vitamin E-tethered, short, optimum protease stabilized facial lipopeptide based non-immunogenic, biocompatible siRNA transporter to facilitate the clinical translation in future. Our designed lipopeptide has an Arginine-Sarcosine-Arginine segment for providing optimum protease-stability, minimizing adjacent arginine-arginine repulsion and reducing intermolecular aggregation and α-tocopherol as the lipidic moiety for facilitating cellular permeabilization. Interestingly, our designed non-immunogenic siRNA transporter has exhibited significantly better long term transfection efficiency than HiPerFect and can transfect hard to transfect primary cell line, HUVEC. Our engineered siRNA therapeutics demonstrated high efficacy in managing metastasis against triple negative breast cancer by disrupting the crosstalk of endothelial cells and MDA-MB-231 and reduced stemness and metastatic markers, as evidenced by downregulating critical oncogenic pathways. Our study aimed at silencing Notch1 signalling to achieve "multi-targeted" therapy with a single putative molecular medicine. We have further developed mechanistically rational combination therapy combining Notch1 silencing with a repurposed drug m-TOR inhibitor, metformin, which demonstrated synergistic interaction and enhanced antitumor efficacy against cancer metastasis.

Harnessing Peptide-Functionalized Multivalent Gold Nanorods for Promoting Enhanced Gene Silencing and Managing Breast Cancer Metastasis.

Small interfering RNA (siRNA) has become the cornerstone against undruggable targets and for managing metastatic breast cancer. However, an effective gene silencing approach is faced with a major challenge due to the delivery problem. In our present study, we have demonstrated efficient siRNA delivery, superior gene silencing, and inhibition of metastasis in triple-negative breast cancer cells (MDA-MB-231) using rod-shaped (aspect ratio: 4) multivalent peptide-functionalized gold nanoparticles and compared them to monovalent free peptide doses. Multivalency is a new concept in biology, and tuning the physical parameters of multivalent nanoparticles can enhance gene silencing and antitumor efficacy. We explored the effect of the multivalency of shape- and size-dependent peptide-functionalized gold nanoparticles in siRNA delivery. Our study demonstrates that peptide functionalization leads to reduced toxicity of the nanoparticles. Such designed peptide-functionalized nanorods also demonstrate antimetastatic efficacy in Notch1-silenced cells by preventing EMT progression in vitro. We have shown siRNA delivery in the hard-to-transfect primary cell line HUVEC and also demonstrated that the Notch1-silenced MDA-MB-231 cell line has failed to form nanobridge-mediated foci with the HUVEC in the co-culture of HUVEC and MDA-MB-231, which promote metastasis. This antimetastatic effect is further checked in a xenotransplant in vivo zebrafish model. In vivo studies also suggest that our designed nanoparticles mediated inhibition of micrometastasis due to silencing of the Notch1 gene. The outcome of our study highlights that the structure-activity relationship of multifunctional nanoparticles can be harnessed to modulate their biological activity.

Emerging concepts in designing next-generation multifunctional nanomedicine for cancer treatment.

Nanotherapy has emerged as an improved anticancer therapeutic strategy to circumvent the harmful side effects of chemotherapy. It has been proven to be beneficial to offer multiple advantages, including their capacity to carry different therapeutic agents, longer circulation time and increased therapeutic index with reduced toxicity. Over time, nanotherapy evolved in terms of their designing strategies like geometry, size, composition or chemistry to circumvent the biological barriers. Multifunctional nanoscale materials are widely used as molecular transporter for delivering therapeutics and imaging agents. Nanomedicine involving multi-component chemotherapeutic drug-based combination therapy has been found to be an improved promising approach to increase the efficacy of cancer treatment. Next-generation nanomedicine has also utilized and combined immunotherapy to increase its therapeutic efficacy. It helps in targeting tumor immune response sparing the healthy systemic immune function. In this review, we have summarized the progress of nanotechnology in terms of nanoparticle designing and targeting cancer. We have also discussed its further applications in combination therapy and cancer immunotherapy. Integrating patient-specific proteomics and biomarker based information and harnessing clinically safe nanotechnology, the development of precision nanomedicine could revolutionize the effective cancer therapy.

Engineered Histidine-Enriched Facial Lipopeptides for Enhanced Intracellular Delivery of Functional siRNA to Triple Negative Breast Cancer Cells.

Cytosolic delivery of functional siRNA remains the major challenge to develop siRNA-based therapeutics. Designing clinically safe and effective siRNA transporter to deliver functional siRNA across the plasma and endosomal membrane remains a key hurdle. With the aim of improving endosomal release, we have designed cyclic and linear peptide-based transporters having an Arg-DHis-Arg template. Computational studies show that the Arg-DHis-Arg template is also stabilized by the Arg-His side-chain hydrogen bonding interaction at physiological pH, which dissociates at lower pH. The overall atomistic interactions were examined by molecular dynamics simulations, which indicate that the extent of peptide_siRNA assembly formation depends greatly on physicochemical properties of the peptides. Our designed peptides having the Arg-DHis-Arg template and two lipidic moieties facilitate high yield of intracellular delivery of siRNA. Additionally, unsaturated lipid, linoleic acid moieties were introduced to promote fusogenicity and facilitate endosomal release and cytosolic delivery. Interestingly, such protease-resistant peptides provide serum stability to siRNA and exhibit high efficacy of erk1 and erk2 gene silencing in the triple negative breast cancer (TNBC) cell line. The peptide having two linoleyl moieties demonstrated comparable efficacy with commercial transfection reagent HiPerFect, as evidenced by the erk1 and erk2 gene knockdown experiment. Additionally, our study shows that ERK1/2 silencing siRNA and doxorubicin-loaded gramicidin-mediated combination therapy is more effective than siRNA-mediated gene silencing-based monotherapy for TNBC treatment.

Engineered isopeptide bond stabilized fibrin inspired nanoscale peptide based sealants for efficient blood clotting.

Designing biologically inspired nanoscale molecular assembly with desired functionality is a challenging endeavour. Here we report the designing of fibrin-inspired nanostructured peptide based sealants which facilitate remarkably fast entrapping of blood corpuscles (~28 seconds) in contrast to fibrin (~56 seconds). Our engineered sealants are stabilized by lysine-aspartate ionic interactions and also by Nε(γ-glutamyl) lysine isopeptide bond mediated covalent interaction. Each sealant is formed by two peptides having complementary charges to promote lysine-aspartate ionic interactions and designed isopeptide bond mediated interactions. Computational analysis reveals the isopeptide bond mediated energetically favourable peptide assemblies in sealants 1-3. Our designed sealants 2 and 3 mimic fibrin-mediated clot formation mechanism in presence of transglutaminase enzyme and blood corpuscles. These fibrin-inspired peptides assemble to form sealants having superior hemostatic activities than fibrin. Designed sealants feature mechanical properties, biocompatibility, biodegradability and high adhesive strength. Such nature-inspired robust sealants might be potentially translated into clinics for facilitating efficient blood clotting to handle traumatic coagulopathy and impaired blood clotting.

Engineered Nanostructured Facial Lipopeptide as Highly Efficient Molecular Transporter.

Designing an effective peptide based molecular transporter for the intracellular delivery of hydrophilic therapeutic biomacromolecules remains a considerable challenge. Highly basic oligoarginine and lipidated arginine rich cell penetrating peptides have been reported in the literature as molecular transporters, which were extensively used for cellular internalization of significantly large biopharmaceuticals. However, oligoarginine based molecular transporters with l-arginine residues pose significant challenges due to proteolytic instability and limited stability of noncovalent peptide-cargo nanocomplexes. Exploiting the rational peptide designing strategy, we have engineered protease-resistant facial lipopeptide based molecular transporter having arginine-sarcosine-arginine moiety to minimize adjacent arginine-arginine pair repulsion. N-Methylated amino acid sarcosine was incorporated as a spacer between two adjacent arginine residues, which provides proteolytic stability to the designed peptide and minimizes intermolecular aggregation of peptides. Two stearyl moieties were incorporated to facilitate cellular internalization. Interestingly, our designed lipopeptide exhibits significantly enhanced cellular internalization with only six l-arginine residues compared to stearylated oligo-nona-arginine. Additionally, enhanced proteolytic stability of such class of molecular transporter enables increased cargo internalization, and we anticipate that our engineered multifunctional, proteolytically stable, nanostructured facial lipopeptide based molecular transporter can have major impact in advancing drug delivery technologies.