Sunny Kumar

Platelet-like Nanoparticles: Mimicking Shape, Flexibility, and Surface Biology of Platelets To Target Vascular Injuries

Targeted delivery of therapeutic and imaging agents in the vascular compartment represents a significant hurdle in using nanomedicine for treating hemorrhage, thrombosis, and atherosclerosis. While several types of nanoparticles have been developed to meet this goal, their utility is limited by poor circulation, limited margination, and minimal targeting. Platelets have an innate ability to marginate to the vascular wall and specifically interact with vascular injury sites. These platelet functions are mediated by their shape, flexibility, and complex surface interactions. Inspired by this, we report the design and evaluation of nanoparticles that exhibit platelet-like functions including vascular injury site-directed margination, site-specific adhesion, and amplification of injury site-specific aggregation. Our nanoparticles mimic four key attributes of platelets, (i) discoidal morphology, (ii) mechanical flexibility, (iii) biophysically and biochemically mediated aggregation, and (iv) heteromultivalent presentation of ligands that mediate adhesion to both von Willebrand Factor and collagen, as well as specific clustering to activated platelets. Platelet-like nanoparticles (PLNs) exhibit enhanced surface-binding compared to spherical and rigid discoidal counterparts and site-selective adhesive and platelet-aggregatory properties under physiological flow conditions in vitro. In vivo studies in a mouse model demonstrated that PLNs accumulate at the wound site and induce ∼65% reduction in bleeding time, effectively mimicking and improving the hemostatic functions of natural platelets. We show that both the biochemical and biophysical design parameters of PLNs are essential in mimicking platelets and their hemostatic functions. PLNs offer a nanoscale technology that integrates platelet-mimetic biophysical and biochemical properties for potential applications in injectable synthetic hemostats and vascularly targeted payload delivery.

Shape and size-dependent immune response to antigen-carrying nanoparticles.

The immune system has evolved to recognize and respond to a wide variety of pathogens and produce distinct immune responses against diverse pathogenic structures. Despite remarkable advances, the general mechanisms by which the immune system differentiates the structure of antigen presenting particulates have yet to be elucidated. Using particles of various sizes and shapes, we assessed the role of morphological features of particles in antigen presentation and subsequent processing by the immune cells. Ovalbumin was used as a model antigen. Spherical polystyrene particles of 193 nm and 521 nm diameters were successfully stretched to form rod-shaped particles of 376 nm and 1530 nm in length, respectively. Ovalbumin conjugation to these different particle types was optimized to achieve ~50 μg of ovalbumin conjugation per mg of particle. In vivo immunization study results revealed that small spherical particles (193 nm in diameter) produced a Th1-biased response whereas rod-shaped particles (1530 nm in length) produced a Th2-biased response against ovalbumin. Among different particle types, smaller spherical (193 nm) particles generated stronger Th1 and Th2 immune responses compared to the other particle types. In vitro studies with dendritic cells indicated that spherical (193 nm) and rod (1530 nm) shaped particles were internalized by dendritic cells and delivered ovalbumin. These results provide evidence for size- and shape-dependent modulation of immune responses and this knowledge can be leveraged to rationally design and develop next generation vaccines against a wide range of pathogens.

Peptides as skin penetration enhancers: mechanisms of action.

Skin penetrating peptides (SPPs) have garnered wide attention in recent years and emerged as a simple and effective noninvasive strategy for macromolecule delivery into the skin. Although SPPs have demonstrated their potential in enhancing skin delivery, they are still evolving as a new class of skin penetration enhancers. Detailed studies elucidating their mechanisms of action are still lacking. Using five SPPs (SPACE peptide, TD-1, polyarginine, a dermis-localizing peptide and a skin penetrating linear peptide) and a model hydrophobic macromolecule (Cyclosporine A, CsA), herein we provide a mechanistic understanding of SPPs. To evaluate the mechanism and safety of SPPs, their effects on skin lipids, proteins and keratinocyte cells were evaluated. Three SPPs (SPACE, Polyarginine and TD-1) significantly enhanced CsA penetration into the skin. SPPs did not alter the skin lipid barrier as measured by skin resistance, transepidermal water loss (TEWL) and Fourier transform infrared (FTIR) spectroscopic analysis. In contrast, SPPs interacted with skin proteins and induced changes in skin protein secondary structures (α-helices, β-sheet, random coils and turns), as evaluated by FTIR analysis and confirmed by in-silico docking. SPPs enhanced CsA skin penetration, via a transcellular pathway, enhancing its partitioning into keratin-rich corneocytes through concurrent binding of SPP with keratin and CsA. Interaction between SPP and keratin best correlated with measured CsA skin transport. Many SPPs appeared to be safe as shown by negligible effect on skin integrity, nominal skin irritation potential and cytotoxicity. Among the peptides tested, SPACE peptide was found to be least toxic to keratinocytes, and among the most effective at delivering CsA into the skin.

Synergistic antitumor activity of camptothecin–doxorubicin combinations and their conjugates with hyaluronic acid

Combinations of topoisomerase inhibitors I and II have been found to synergistically inhibit cancer cell growth in vitro, yet clinical studies of these types of combinations have not progressed beyond phase II trials. The results of clinical combinations of topoisomerase (top) I and II inhibitors typically fall within one of two categories: little to no improvement in therapeutic efficacy, or augmented toxicity compared to the single drug counterparts. Hence, despite the promising activity of top I and II inhibitor combinations in vitro, their clinical applicability has not been realized. Here, we report the use of polymer-drug conjugates as a means to co-deliver synergistic doses of top I and II inhibitors camptothecin (CPT) and doxorubicin (DOX) to tumors in vivo in a 4T1 breast cancer model. At specific molar ratios, DOX and CPT were found to be among the most synergistic combinations reported to date, with combination indices between 0.01 and 0.1. The identified optimal ratios were controllably conjugated to hyaluronic acid, and elicited significant tumor reduction of murine 4T1 breast cancer model when administered intravenously. This study elucidates a method to identify synergistic drug combinations and translate them to in vivo by preserving the synergistic ratio via conjugation to a carrier polymer, thus opening a promising approach to translate drug combinations to clinically viable treatment regimens.

Topical delivery of Cyclosporine A into the skin using SPACE-peptide.

Cyclosporine A (CsA) is used for the treatment of psoriasis; however systemic administration of CsA is potentially life threatening and there are long-term side effects. Topical application of CsA has the potential to overcome this hurdle; however, its use is limited by poor water solubility and low permeability. Here, we report the use of a physical mixture of SPACE-peptide and CsA in an aqueous ethanol solution to enhance the dermal absorption of the drug. The aqueous ethanol solution (hydroethanolic solution) containing 5mg/mL CsA and 50mg/mL of free SPACE-peptide (SP50) delivered about 30% of topically applied CsA into the porcine skin in vitro and led to an approximately 9-fold (p<0.01) increase in accumulation in viable epidermis compared to the hydroethanolic solution without SPACE-peptide (control group). In vivo biodistribution and pharmacokinetic studies performed using SKH1 hairless mice also confirmed the efficacy of SP50 in dermal delivery of CsA and also demonstrated its advantages over other routes in terms of minimizing its systemic absorption. Topical application of SP50 significantly increased the localization of CsA in the target skin (113.1±13.6(μg/g)/mg) compared to all other groups (p<0.01). In addition, SP50 led to significantly higher skin/blood ratio (443.4±181.5) and skin/liver ratio (1059.5±110.8) of CsA compared to all other groups (p<0.01). The SP50 formulation reported here offers a promising approach for the dermal delivery of CsA.

De Novo Design of Skin-Penetrating Peptides for Enhanced Transdermal Delivery of Peptide Drugs

Skin-penetrating peptides (SPPs) are attracting increasing attention as a non-invasive strategy for transdermal delivery of therapeutics. The identification of SPP sequences, however, currently performed by experimental screening of peptide libraries, is very laborious. Recent studies have shown that, to be effective enhancers, SPPs must possess affinity for both skin keratin and the drug of interest. We therefore developed a computational process for generating and screening virtual libraries of disulfide-cyclic peptides against keratin and cyclosporine A (CsA) to identify SPPs capable of enhancing transdermal CsA delivery. The selected sequences were experimentally tested and found to bind both CsA and keratin, as determined by mass spectrometry and affinity chromatography, and enhance transdermal permeation of CsA. Four heptameric sequences that emerged as leading candidates (ACSATLQHSCG, ACSLTVNWNCG, ACTSTGRNACG, and ACSASTNHNCG) were tested and yielded CsA permeation on par with previously identified SPP SPACE (TM) . An octameric peptide (ACNAHQARSTCG) yielded significantly higher delivery of CsA compared to heptameric SPPs. The safety profile of the selected sequences was also validated by incubation with skin keratinocytes. This method thus represents an effective procedure for the de novo design of skin-penetrating peptides for the delivery of desired therapeutic or cosmetic agents.

Nucleic acid delivery into skin for the treatment of skin disease: Proofs-of-concept, potential impact, and remaining challenges

Nucleic acids (NAs) hold significant potential for the treatment of several diseases. Topical delivery of NAs for the treatment of skin diseases is especially advantageous since it bypasses the challenges associated with systemic administration which suffers from enzymatic degradation, systemic toxicity and lack of targeting to skin. However, the skins protective barrier function limits the delivery of NAs into skin after topical application. Here, we highlight strategies for enhancing delivery of NAs into skin, and provide evidence that translation of topical NA therapies could have a transformative impact on the treatment of skin diseases.

Enhanced epidermal localization of topically applied steroids using SPACE™ peptide

The balance of efficacy and safety of topical corticosteroids (TCs) depends on their ability to penetrate into and be retained within the skin. Here, we evaluated the ability of SPACE™ peptide to enhance epidermal delivery and localization of three model TCs. In vitro and in vivo skin penetration studies were performed to evaluate penetration of TCs into and across the skin in the presence of various formulations of SPACE™ peptide. Topical formulations of corticosterone containing free SPACE™ peptide produced significantly enhanced epidermal penetration and localization. Ratio of drug deposition in the skin and receiver (efficacy/safety, indicative of ratio of local to systemic uptake) exhibited higher values for SPACE™ peptide-based formulation as compared to aqueous and hydroethanolic solutions and Cortizone™ cream. Mass spectrometry analysis showed that SPACE™ peptide associates with corticosterone, which may explain its enhanced retention effect. SPACE™ peptide also enhanced dermal retention of two more TCs (hydrocortisone and triamcinolone acetonide) compared to the vehicle control. An in vivo study in mice further established the ability of SPACE™ peptide to enhance skin retention of hydrocortisone without producing elevated blood concentrations. These results show that SPACE™ peptide is an effective additive to the formulation for enhanced skin localization of topical steroids.