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.

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 Approaches for Enabling RNAi Therapeutics.

RNA interference (RNAi) is a primitive evolutionary mechanism developed to escape incorporation of foreign genetic material. siRNA has been instrumental in achieving the therapeutic potential of RNAi by theoretically silencing any gene of interest in a reversible and sequence-specific manner. Extrinsically administered siRNA generally needs a delivery vehicle to span across different physiological barriers and load into the RISC complex in the cytoplasm in its functional form to show its efficacy. This review discusses the designing principles and examples of different classes of delivery vehicles that have proved to be efficient in RNAi therapeutics. We also briefly discuss the role of RNAi therapeutics in genetic and rare diseases, epigenetic modifications, immunomodulation and combination modality to inch closer in creating a personalized therapy for metastatic cancer. At the end, we present, strategies and look into the opportunities to develop efficient delivery vehicles for RNAi which can be translated into clinics.

Engineered Bio-inspired Multifunctional Peptide- and Protein-based Therapeutic Biomolecules for Better Wound Care.

Developing non-immunogenic therapeutic biomolecules for facilitating blood clotting followed by wound healing via therapeutic angiogenesis, still remains a formidable challenge. Excessive blood loss of accident victims and battalions cause a huge number of deaths worldwide. Patients with inherited bleeding disorders face acute complications during injury and post-surgery. Biologically-inspired peptide-based hemostat can act as a potential therapeutic for handling coagulopathy. Additionally, non-healing wounds for patients having ischemic diseases can cause severe clinical complications. Advancement in stabilized growth-factor-based proangiogenic therapy may offer effective possibilities for the treatment of ischemic pathology. This review will discuss nature-inspired biocompatible stabilized peptide- and protein-based molecular medicines to serve unmet medical challenges for handling traumatic coagulopathy and impaired wound healing.

Bioinspired Non-Immunogenic Multifunctional Sealant for Efficient Blood Clotting and Suture-Free Wound Closure.

Engineering bioinspired peptide-based molecular medicine is an emerging paradigm for the management of traumatic coagulopathies and inherent bleeding disorder. A hemostat-based strategy in managing uncontrolled bleeding is limited due to the lack of adequate efficacy and clinical noncompliance. In this study, we report an engineered adhesive peptide-based hybrid regenerative medicine, sealant 5, which is designed integrating the structural and functional features of fibrin and mussel foot-pad protein. AFM studies have revealed that sealant 5 (55.8 ± 6.8 nN adhesive force) has higher adhesive force than fibrin (46.4 ± 7.3 nN adhesive force). SEM data confirms that sealant 5 retains its network-like morphology both at 37 and 60 °C, inferring its thermal stability. Both sealant 5 and fibrin exhibit biodegradability in the presence of trypsin, and sealant 5 also showed biocompatibility in the presence of fibroblast cells. Engineered sealant 5 efficiently promotes hemostasis with enhanced adhesiveness and less blood-loss than fibrin. In vivo data suggests that in heparinized conditions, sealant 5 ceases bleeding at 212.3 ± 15.1 s, whereas fibrin halts bleeding at 294.3 ± 21.4 s and blood-loss is ∼4-fold less in sealant 5 than in fibrin. In a heparinized system, sealant 5 facilitates faster blood-clotting than fibrin (∼82 s faster) and RADA-16, a reported peptide-based sealant (∼113 s faster). Additionally, in the case of sealant 5, the process of clotting mimicry-like fibrin is independent of the body's own coagulation system. Sealant 5 efficiently halts bleeding for both external and internal wounds, even for a heparinized system overcoming the bacterial infection. ELISA data and PMBC cell proliferation data support the non-immunogenic feature of sealant 5. Though fibrin and sealant 5 have exhibited comparable efficacy in suture-free wound closure, in vivo H&E staining images have revealed infiltration of very few immune cells as well as the presence of abundant collagen formation in the case of sealant 5-treated wound. Such nature-inspired non-immunogenic sealants offer exciting possibilities for the treatment of uncontrolled bleeding vis-à-vis wound closure.

Peptide-based topical agents and intravenous hemostat for rapid hemostasis.

Traumatic coagulopathy due to severe external injury and internal hemorrhage is life-threatening to accident victims and soldiers on the battlefield, causing considerable number of deaths worldwide. Patients with inherited bleeding disorders (such as haemophilia, von Willebrand disease, inherited qualitative platelet defects, and afibrinogenemia) also contribute to the vast number of deaths due to abnormal bleeding, and these patients are difficult to handle during surgery. Platelets and different plasma proteins play an essential role in blood coagulation and in the maintenance of the body's hemostatic balance. The improper function or deficiency of these factors cause abnormal bleeding. To address such bleeding disorders, external clotting agents (such as extracellular protein-inspired natural and synthetic peptide-based sealants and peptide-functionalized polymer/liposome-based sealants) have been developed by different groups of researchers. The primary focus of this review is to provide molecular insights into the existing biologically inspired peptide-based sealants, highlighting the advantages and limitations of such reported designed sealants to handle blood clotting, and also provide insights into the design of improved next-generation surgical sealants.

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 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.

Coupling growth-factor engineering with nanotechnology for therapeutic angiogenesis.

Therapeutic angiogenesis is an emerging paradigm for the management of ischemic pathologies. Proangiogenic Therapy is limited, however, by the current inability to deliver angiogenic factors in a sustained manner at the site of pathology. In this study, we investigated a unique nonglycosylated active fragment of hepatocyte growth factor/scatter factor, 1K1, which acts as a potent angiogenic agent in vitro and in a zebrafish embryo and a murine matrigel implant model. Furthermore, we demonstrate that nanoformulating 1K1 for sustained release temporally alters downstream signaling through the mitogen activated protein kinase pathway, and amplifies the angiogenic outcome. Merging protein engineering and nanotechnology offers exciting possibilities for the treatment of ischemic disease, and furthermore allows the selective targeting of downstream signaling pathways, which translates into discrete phenotypes.