Topical Synthetic Platelets Loaded With Gentamicin Decrease Bacteria in Deep Partial-Thickness Burns.

INTRODUCTION: Prolonged inflammation and infection in burns may cause inadequate healing. Platelet granules contain anti-inflammatory mediators that impact wound healing. Synthetic platelets (SPs) avoid portability and storage difficulties of natural platelets and can be loaded with bioactive agents. We evaluated wound healing outcomes in deep partial-thickness (DPT) burns treated topically with SP loaded with antibiotics. MATERIALS AND METHODS: Thirty DPT burns were created on the dorsum of two Red Duroc hybrid pigs. Six wounds were randomized into five groups: SP alone, SP loaded with gentamicin vesicles, SP with gentamicin mixture, vehicle control (saline), or dry gauze. Wounds were assessed from postburn days 3-90. Primary outcome was re-epithelialization percentage at postburn day 28. Secondary outcomes included wound contraction percentage, superficial blood flow relative to normal skin controls, and bacterial load score. RESULTS: Results showed that re-epithelialization with the standard of care (SOC) was 98%, SP alone measured 100%, SP loaded with gentamicin vesicles was 100%, and SP with gentamicin mixture was 100%. Wound contraction was 5.7% in the SOC and was ∼10% in both the SP loaded with gentamicin vesicles and SP with gentamicin mixture groups. Superficial blood flow in the SOC was 102.5%, SP alone was 170%, the SP loaded was 155%, and gentamicin mixture 162.5%. Bacterial load score in the SOC was 2.2/5.0 and was significantly less at 0.8/5.0 in SP loaded with gentamicin vesicles (P > 0.05). SP and gentamicin mixture scored 2.7 and 2.3/5.0. CONCLUSIONS: Topical SP treatment did not significantly improve outcomes. However, SP loaded with gentamicin-infused vesicles decreased bacterial load.

Targeting Thymidine Phosphorylase With Tipiracil Hydrochloride Attenuates Thrombosis Without Increasing Risk of Bleeding in Mice.

OBJECTIVE: Current antiplatelet medications increase the risk of bleeding, which leads to a clear clinical need in developing novel mechanism-based antiplatelet drugs. TYMP (Thymidine phosphorylase), a cytoplasm protein that is highly expressed in platelets, facilitates multiple agonist-induced platelet activation, and enhances thrombosis. Tipiracil hydrochloride (TPI), a selective TYMP inhibitor, has been approved by the Food and Drug Administration for clinical use. We tested the hypothesis that TPI is a safe antithrombotic medication. Approach and Results: By coexpression of TYMP and Lyn, GST (glutathione S-transferase) tagged Lyn-SH3 domain or Lyn-SH2 domain, we showed the direct evidence that TYMP binds to Lyn through both SH3 and SH2 domains, and TPI diminished the binding. TYMP deficiency significantly inhibits thrombosis in vivo in both sexes. Pretreatment of platelets with TPI rapidly inhibited collagen- and ADP-induced platelet aggregation. Under either normal or hyperlipidemic conditions, treating wild-type mice with TPI via intraperitoneal injection, intravenous injection, or gavage feeding dramatically inhibited thrombosis without inducing significant bleeding. Even at high doses, TPI has a lower bleeding side effect compared with aspirin and clopidogrel. Intravenous delivery of TPI alone or combined with tissue plasminogen activator dramatically inhibited thrombosis. Dual administration of a very low dose of aspirin and TPI, which had no antithrombotic effects when used alone, significantly inhibited thrombosis without disturbing hemostasis. CONCLUSIONS: This study demonstrated that inhibition of TYMP, a cytoplasmic protein, attenuated multiple signaling pathways that mediate platelet activation, aggregation, and thrombosis. TPI can be used as a novel antithrombotic medication without the increase in risk of bleeding.

Platelet-inspired synthetic nanoparticles improve hemostasis and hemodynamics in a rabbit model of abdominal hemorrhage.

BACKGROUND: Early platelet transfusion is associated with reduced mortality in traumatic hemorrhage. However, platelet usage is severely limited because of the challenges of donor availability, platelet portability, and storage. Here, we report on a bioinspired synthetic platelet (SP) nanoconstruct that utilizes liposome surface-decoration with peptides that mimic injury site-specific platelet adhesion to von Willebrand Factor and collagen, and fibrinogen-mediated platelet aggregation. Synthetic platelet has previously shown promising hemostatic outcomes in vitro and in vivo. Here, we evaluated hemostasis and hemodynamic effects of SP in a rabbit model of abdominal hemorrhage. METHODS: Twenty-three adult male New Zealand white rabbits (2.5-3.5 kg) were treated with either buffer, control particles (CPs), or SP. Under general anesthesia with invasive monitoring, rabbits underwent laparotomy with combined splenic and hepatic injury. Hemodynamics were monitored for 30 minutes and blood loss was quantified. Blood counts, aggregometry, catecholamine and platelet factor 4 (PF4) assays were performed at multiple timepoints. Analysis used analysis of variance and post hoc Tukey testing with α = 0.05. RESULTS: Rabbits in the SP (n = 7) group had significantly lower weight-normalized blood loss compared with both buffer (n = 8) and CP (n = 8) animals (21.1 vs. 33.2 vs. 40.4 g/kg, p < 0.001). Synthetic platelet-treated animals had higher systolic blood pressure area under curve compared with buffer- and CP-treated animals (1567 vs. 1281 vs. 1109 mm Hg*min, p = 0.006), although post hoc differences were only significant for the SP/CP comparison ( p = 0.005). Platelet counts, catecholamine levels, PF4, and aggregometry were similar between groups. CONCLUSION: Synthetic platelet treatment significantly reduced blood loss and improved hemodynamics in a rabbit abdominal hemorrhage model. Synthetic platelet has potential as an intravenous hemostatic platelet surrogate with donor-independent availability and scalable manufacture.

A Multichannel Portable Platform With Embedded Thermal Management for Miniaturized Dielectric Blood Coagulometry.

This article presents a standalone, multichannel, miniaturized impedance analyzer (MIA) system for dielectric blood coagulometry measurements with a microfluidic sensor termed ClotChip. The system incorporates a front-end interface board for 4-channel impedance measurements at an excitation frequency of 1 MHz, an integrated resistive heater formed by a pair of printed-circuit board (PCB) traces to keep the blood sample near a physiologic temperature of 37 °C, a software-defined instrument module for signal generation and data acquisition, and a Raspberry Pi-based embedded computer with 7-inch touchscreen display for signal processing and user interface. When measuring fixed test impedances across all four channels, the MIA system exhibits an excellent agreement with a benchtop impedance analyzer, with rms errors of ≤0.30% over a capacitance range of 47-330 pF and ≤0.35% over a conductance range of 2.13-10 mS. Using in vitro-modified human whole blood samples, the two ClotChip output parameters, namely, the time to reach a permittivity peak (Tpeak) and maximum change in permittivity after the peak (Δϵr,max) are assessed by the MIA system and benchmarked against the corresponding parameters of a rotational thromboelastometry (ROTEM) assay. Tpeak exhibits a very strong positive correlation (r = 0.98, p < 10-6, n = 20) with the ROTEM clotting time (CT) parameter, while Δϵr,max exhibits a very strong positive correlation (r = 0.92, p < 10-6, n = 20) with the ROTEM maximum clot firmness (MCF) parameter. This work shows the potential of the MIA system as a standalone, multichannel, portable platform for comprehensive assessment of hemostasis at the point-of-care/point-of-injury (POC/POI).

Platelets and hemostatic proteins are co-localized with chronic neuroinflammation surrounding implanted intracortical microelectrodes.

Intracortical microelectrodes induce vascular injury upon insertion into the cortex. As blood vessels rupture, blood proteins and blood-derived cells (including platelets) are introduced into the 'immune privileged' brain tissues at higher-than-normal levels, passing through the damaged blood-brain barrier. Blood proteins adhere to implant surfaces, increasing the likelihood of cellular recognition leading to activation of immune and inflammatory cells. Persistent neuroinflammation is a major contributing factor to declining microelectrode recording performance. We investigated the spatial and temporal relationship of blood proteins fibrinogen and von Willebrand Factor (vWF), platelets, and type IV collagen, in relation to glial scarring markers for microglia and astrocytes following implantation of non-functional multi-shank silicon microelectrode probes into rats. Together with type IV collagen, fibrinogen and vWF augment platelet recruitment, activation, and aggregation. Our main results indicate blood proteins participating in hemostasis (fibrinogen and vWF) persisted at the microelectrode interface for up to 8-weeks after implantation. Further, type IV collagen and platelets surrounded the probe interface with similar spatial and temporal trends as vWF and fibrinogen. In addition to prolonged blood-brain barrier instability, specific blood and extracellular matrix proteins may play a role in promoting the inflammatory activation of platelets and recruitment to the microelectrode interface. STATEMENT OF SIGNIFICANCE: Implanted microelectrodes have substantial potential for restoring function to people with paralysis and amputation by providing signals that feed into natural control algorithms that drive prosthetic devices. Unfortunately, these microelectrodes do not display robust performance over time. Persistent neuroinflammation is widely thought to be a primary contributor to the devices' progressive decline in performance. Our manuscript reports on the highly local and persistent accumulation of platelets and hemostatic blood proteins around the microelectrode interface of brain implants. To our knowledge neuroinflammation driven by cellular and non-cellular responses associated with hemostasis and coagulation has not been rigorously quantified elsewhere. Our findings identify potential targets for therapeutic intervention and a better understanding of the driving mechanisms to neuroinflammation in the brain.

Military Medicine and Medical Research as a Source of Inspiration and Innovation to Solve National Security and Health Challenges in the 21st Century.

The history of military medicine and research is rife with examples of novel treatments and new approaches to heal and cure soldiers and others impacted by war's devastation. In the 21st century, new threats, like climate change, are combined with traditional threats, like geopolitical conflict, to create novel challenges for our strategic interests. Extreme and inaccessible environments provide heightened risks for warfighter exposure to dangerous bacteria, viruses, and fungi, as well as exposure to toxic substances and extremes of temperature, pressure, or both providing threats to performance and eroding resilience. Back home, caring for our veterans is also a health-care priority, and the diseases of veterans increasingly overlap with the health needs of an aging society. These trends of climate change, politics, and demographics suggest performance evaluation and resilience planning and response are critical to assuring both warfighter performance and societal health. The Cleveland ecosystem, comprising several hospitals, a leading University, and one of the nation's larger Veteran's Health Administration systems, is ideal for incubating and understanding the response to these challenges. In this review, we explore the interconnections of collaborations between Defense agencies, particularly Air Force and Army and academic medical center-based investigators to drive responses to the national health security challenges facing the United States and the world.

Peptide-decorated liposomes promote arrest and aggregation of activated platelets under flow on vascular injury relevant protein surfaces in vitro.

Platelet-mimetic synthetic hemostats are highly attractive in transfusion medicine. To this end, past research reports have described particles that either amplify platelet aggregation or mimic platelet adhesion. However, a construct design that effectively combines both functionalities has not been reported. Here we describe the design of a liposomal construct simultaneously surface-decorated with three peptides (a vWF-binding peptide (VBP), a collagen-binding peptide (CBP), and an active platelet clustering cyclic-RGD (cRGD) peptide), that can integrate platelet-mimetic dual hemostatic activities of adhesion and aggregation. We first demonstrate that surface-immobilized cRGD-liposomes are capable of aggregating activated platelets onto themselves. Subsequently, we demonstrate that hetero-multivalent liposomes bearing VBP, CBP, and cRGD, when introduced in flow with ≈ 20,000 activated platelets per microliter, are capable of adhering to vWF/collagen surfaces and promoting the recruitment/aggregation of platelets onto themselves. We envision that optimizing this construct can lead to a highly refined synthetic hemostat design for potential application in transfusion medicine.

EGFR-mediated intracellular delivery of Pc 4 nanoformulation for targeted photodynamic therapy of cancer: in vitro studies.

In photodynamic therapy (PDT), the light activation of a photosensitizer leads to the generation of reactive oxygen species that can trigger various mechanisms of cell death. Harnessing this process within cancer cells enables minimally invasive yet targeted cancer treatment. With this rationale, here we demonstrate tumor-targeted delivery of a highly hydrophobic photosensitizer Pc 4 loaded within biocompatible poly(ethylene glycol)-poly(ɛ-caprolactone) block co-polymer micelles. The micelles were surface-modified with epidermal growth factor receptor (EGFR)-targeting GE11 peptides for active targeting of EGFR-overexpressing cancer cells, in vitro. Pc 4-loaded EGFR-targeted micelles were incubated with EGFR-overexpressing A431 epidermoid carcinoma cells for various time periods, to determine Pc 4 uptake by epifluorescence microscopy. The cells were subsequently photoirradiated, and PDT-induced cell death for various incubation periods was determined by MTT assay and fluorescence Live/Dead assay. Our results indicate that active EGFR targeting of the Pc 4-loaded micelles accelerates intracellular uptake of the drug. Consequently, this enhances the PDT-induced cytotoxicity within shorter time periods. FROM THE CLINICAL EDITOR: Photodynamic cancer therapy using Pc 4, a light activated and highly hydrophobic photosensitizer is demonstrated in this paper in vitro. Pc 4 was delivered in block-copolymer micelles surface-modified with GE11 peptides targeting EGFR-overexpressing cancer cells.

Nanomedicine approaches in vascular disease: a review.

Nanomedicine approaches have revolutionized the treatment of cancer and vascular diseases, where the limitations of rapid nonspecific clearance, poor biodistribution and harmful side effects associated with direct systemic drug administration can be overcome by packaging the agents within sterically stabilized, long-circulating nanovehicles that can be further surface-modified with ligands to actively target cellular/molecular components of the disease. With significant advancements in genetics, proteomics, cellular and molecular biology and biomaterials engineering, the nanomedicine strategies have become progressively refined regarding the modulation of surface and bulk chemistry of the nanovehicles, control of drug release kinetics, manipulation of nanoconstruct geometry and integration of multiple functionalities on single nanoplatforms. The current review aims to capture the various nanomedicine approaches directed specifically toward vascular diseases during the past two decades. Analysis of the promises and limitations of these approaches will help identify and optimize vascular nanomedicine systems to enhance their efficacy and clinical translation in the future. FROM THE CLINICAL EDITOR: Nanomedicine-based approaches have had a major impact on the treatment and diagnosis of malignancies and vascular diseases. This review discusses various nanomedicine approaches directed specifically toward vascular diseases during the past two decades, highlighting their advantages, limitations and offering new perspectives on future applications.

A polymer-based systemic hemostatic agent.

Uncontrolled noncompressible hemorrhage is a major cause of mortality following traumatic injuries in civilian and military populations. An injectable hemostat for point-of-care treatment of noncompressible hemorrhage represents an urgent medical need. Here, we describe an injectable hemostatic agent via polymer peptide interfusion (HAPPI), a hyaluronic acid conjugate with a collagen-binding peptide and a von Willebrand factor-binding peptide. HAPPI exhibited selective binding to activated platelets and promoted their accumulation at the wound site in vitro. In vivo studies in mouse tail vein laceration model demonstrated a reduction of >97% in both bleeding time and blood loss. A 284% improvement in the survival time was observed in the rat inferior vena cava traumatic model. Lyophilized HAPPI could be stably stored at room temperature for several months and reconstituted during therapeutic intervention. HAPPI provides a potentially clinically translatable intravenous hemostat.

Biomaterials and Advanced Technologies for Hemostatic Management of Bleeding.

Bleeding complications arising from trauma, surgery, and as congenital, disease-associated, or drug-induced blood disorders can cause significant morbidities and mortalities in civilian and military populations. Therefore, stoppage of bleeding (hemostasis) is of paramount clinical significance in prophylactic, surgical, and emergency scenarios. For externally accessible injuries, a variety of natural and synthetic biomaterials have undergone robust research, leading to hemostatic technologies including glues, bandages, tamponades, tourniquets, dressings, and procoagulant powders. In contrast, treatment of internal noncompressible hemorrhage still heavily depends on transfusion of whole blood or blood's hemostatic components (platelets, fibrinogen, and coagulation factors). Transfusion of platelets poses significant challenges of limited availability, high cost, contamination risks, short shelf-life, low portability, performance variability, and immunological side effects, while use of fibrinogen or coagulation factors provides only partial mechanisms for hemostasis. With such considerations, significant interdisciplinary research endeavors have been focused on developing materials and technologies that can be manufactured conveniently, sterilized to minimize contamination and enhance shelf-life, and administered intravenously to mimic, leverage, and amplify physiological hemostatic mechanisms. Here, a comprehensive review regarding the various topical, intracavitary, and intravenous hemostatic technologies in terms of materials, mechanisms, and state-of-art is provided, and challenges and opportunities to help advancement of the field are discussed.

ClotChip: A Microfluidic Dielectric Sensor for Point-of-Care Assessment of Hemostasis.

This paper describes the design, fabrication, and testing of a microfluidic sensor for dielectric spectroscopy of human whole blood during coagulation. The sensor, termed ClotChip, employs a three-dimensional, parallel-plate, capacitive sensing structure with a floating electrode integrated into a microfluidic channel. Interfaced with an impedance analyzer, the ClotChip measures the complex relative dielectric permittivity, ϵr , of human whole blood in the frequency range of 40 Hz to 100 MHz. The temporal variation in the real part of the blood dielectric permittivity at 1 MHz features a time to reach a permittivity peak, , as well as a maximum change in permittivity after the peak, , as two distinct parameters of ClotChip readout. The ClotChip performance was benchmarked against rotational thromboelastometry (ROTEM) to evaluate the clinical utility of its readout parameters in capturing the clotting dynamics arising from coagulation factors and platelet activity. exhibited a very strong positive correlation ( r = 0.99, p < 0.0001) with the ROTEM clotting time parameter, whereas exhibited a strong positive correlation (r = 0.85,  p < 0.001) with the ROTEM maximum clot firmness parameter. This paper demonstrates the ClotChip potential as a point-of-care platform to assess the complete hemostatic process using <10 µL of human whole blood.

RGD-modified liposomes targeted to activated platelets as a potential vascular drug delivery system.

Local drug delivery has become an important treatment modality for the prevention of thrombotic events following coronary angioplasty. In this study, we investigate the ability of liposomes bearing surface conjugated linear Arg-Gly-Asp (RGD) peptide (GSSSGRGDSPA) moieties to target and bind activated platelets, and the effect of such RGD-modified liposomes on platelet activation and aggregation. The binding of RGD-liposomes to human platelets was assessed by fluorescence microscopy, phase contrast microscopy and flow cytometry. The effect of RGD-modified liposomes on platelet activation and aggregation was investigated in vitro, with and without platelet agonists. RGD-liposomes were found to bind activated platelets at levels significantly greater than the control RGE-liposomes. The RGD-liposomes did not exhibit any statistically significant effect on platelet activation or aggregation. The results demonstrate the ability of the RGD-modified liposomes to target and bind activated platelets without causing significant platelet aggregation and suggests a feasible way for the development of a platelet-targeted anti-thrombogenic drug delivery system. Furthermore, the approach can be extended to the development of liposomes for other vascular targets, for application in drug delivery or gene therapy.

Polyphosphates and other phosphorus-containing polymers for drug delivery applications.

Poly(phosphate ester)s, polyphosphonates, and polyphosphazenes are three classes of phosphorus-containing polymers that have received wide attention over the past decade for their utility in biomedicine and tissue engineering. These three families of polymers can lead to a number of subclasses of polymers with varied properties. Significant research in this area has led to niche polymers with morphologies ranging from viscous gels to amorphous microparticles for utility in drug delivery. Furthermore, the pentavalency of phosphorus offers the potential for covalent linking of the drug. The classes of polymers discussed in this review are being explored in human clinical trials for vaccine delivery as well as delivery of oncolytic and CNS therapeutics. More applications in the areas of DNA delivery and tissue engineering are also being explored.

Nanotechnology applications in diagnosis and treatment of metastasis.

The lethality of solid tumors is in large part dependent on their ability to metastasize through hematologic and lymphatic transport pathways. The dissemination of cancer cells from the primary tumor to undergo transport, their ability to survive in transit and then to subsequently form metastatic colonies, is facilitated by a complex concert of signaling pathways and cell-cell and cell-matrix interactions. Elucidating these mechanistic components is highly valuable to guide the development of technologies for efficiently detecting and treating metastasis. To this end, in recent years nanotechnology approaches have provided several unique detection, characterization and treatment strategies. The current article will review these approaches to discuss their promise and challenges, specifically in metastatic cancer, above and beyond the usual nanomedicine applications in cancer therapy.

Heteromultivalent ligand-decoration for actively targeted nanomedicine.

Active targeting has become an important component of nanomedicine design where nanovehicles are surface-decorated with cell receptor-specific or disease matrix-specific ligands to enable site-selective binding, retention and delivery of theranostic cargo. In this context, there have been numerous reports regarding surface-modification of nanovehicles with antibodies, antibody fragments, carbohydrates, aptamers and peptides as targeting ligands. However, majority of these reports have focused on using a single type of targeting moiety on the vehicle surface. In any disease development and progression, multiple receptors and proteins are often spatio-temporally upregulated simultaneously and heterogeneously. Rationalizing from this, a significant advantage can be envisioned in targeting multiple entities simultaneously using vehicle co-decoration with multiple types of ligands, to enhance binding activity and targeting specificity. To this end, we present a comprehensive up-to-date review on research endeavors in heteromultivalent ligand-modification of nanovehicles and provide a mechanistic rationale as well as an insightful discussion of this promising area, including findings from our own research.

The effects of PEG-based surface modification of PDMS microchannels on long-term hemocompatibility.

The current study demonstrates the first surface modification for poly(dimethylsiloxane) (PDMS) microfluidic networks that displays a long shelf life as well as extended hemocompatibility. Uncoated PDMS microchannel networks rapidly adsorb high levels of fibrinogen in blood contacting applications. Fibrinogen adsorption initiates platelet activation, and causes a rapid increase in pressure across microchannel networks, rendering them useless for long term applications. Here, we describe the modification of sealed PDMS microchannels using an oxygen plasma pretreatment and poly(ethylene glycol) grafting approach. We present results regarding the testing of the coated microchannels after extended periods of aging and blood exposure. Our PEG-grafted channels showed significantly reduced fibrinogen adsorption and platelet adhesion up to 28 days after application, highlighting the stability and functionality of the coating over time. Our coated microchannel networks also displayed a significant reduction in the coagulation response under whole blood flow. Further, pressure across coated microchannel networks took over 16 times longer to double than the uncoated controls. Collectively, our data implies the potential for a coating platform for microfluidic devices in many blood-contacting applications.

Streptokinase loading in liposomes for vascular targeted nanomedicine applications: encapsulation efficiency and effects of processing.

Vascular diseases leading to thrombo-occlusion are the leading cause of morbidity and mortality worldwide. Revascularization and restoration of antegrade blood flow is critical for tissue survival and patient health. In this aspect, systemic administration of thrombolytics (e.g., streptokinase) to dissolve occlusive thrombi is a clinically established strategy. However, this strategy typically necessitates the administration of large doses, leading to a high incidence of hemorrhagic complications due to systemic side effects. To minimize this risk, liposomes specifically targeted to the site of thrombo-occlusion have been bioengineered by exploiting ligand-receptor relationships pertinent to thrombus-associated cell phenotypes. This study focuses on encapsulating streptokinase within these liposomes, specifically regarding the effect of liposome processing conditions on streptokinase encapsulation and activity. Theoretical calculations of encapsulation capacity agreed well with that reported in the literature. The experimental encapsulation efficiency values are 45.9 ± 34.0% (n = 9 ± SD) and 21.6 ± 30.0% (n = 6 ± SD), using two different methods. The liposome processing conditions are found to decrease streptokinase activity; however, over 30% remain active after processing, maintaining enough activity to be therapeutic especially when protected inside a vehicle targeted to the site of thrombo-occlusion. The insight gained from the research reported here would enable refining the design and the processing conditions of liposomal formulations of fibrinolytics to yield reduced variability in encapsulation efficiency and streptokinase activity. The design of a thrombus-targeted 'stealth' liposome reported earlier and the current findings of fibrinolytics' encapsulation and activity in such liposomes can be efficiently integrated to develop an efficient strategy for vascular nanomedicine.