Ultrasoft platelet-like particles stop bleeding in rodent and porcine models of trauma.

Uncontrolled bleeding after trauma represents a substantial clinical problem. The current standard of care to treat bleeding after trauma is transfusion of blood products including platelets; however, donated platelets have a short shelf life, are in limited supply, and carry immunogenicity and contamination risks. Consequently, there is a critical need to develop hemostatic platelet alternatives. To this end, we developed synthetic platelet-like particles (PLPs), formulated by functionalizing highly deformable microgel particles composed of ultralow cross-linked poly (N-isopropylacrylamide) with fibrin-binding ligands. The fibrin-binding ligand was designed to target to wound sites, and the cross-linking of fibrin polymers was designed to enhance clot formation. The ultralow cross-linking of the microgels allows the particles to undergo large shape changes that mimic platelet shape change after activation; when coupled to fibrin-binding ligands, this shape change facilitates clot retraction, which in turn can enhance clot stability and contribute to healing. Given these features, we hypothesized that synthetic PLPs could enhance clotting in trauma models and promote healing after clotting. We first assessed PLP activity in vitro and found that PLPs selectively bound fibrin and enhanced clot formation. In murine and porcine models of traumatic injury, PLPs reduced bleeding and facilitated healing of injured tissue in both prophylactic and immediate treatment settings. We determined through biodistribution experiments that PLPs were renally cleared, possibly enabled by ultrasoft particle properties. The performance of synthetic PLPs in the preclinical studies shown here supports future translational investigation of these hemostatic therapeutics in a trauma setting.

Wound-triggered shape change microgels for the development of enhanced biomimetic function platelet-like particles.

Platelets play a pivotal role in hemostasis and wound healing and conditional shape change is an important component of platelet functionality. In normal circumstances, platelets travel through the circulatory system in an inactive rounded state, which enables platelets to easily move to vessel walls for attachment. When an injury occurs, platelets are prompted by molecules, such as thrombin, to shift into a stellate shape and increase exposure of fibrin-binding receptors. When active, platelets promote hemostasis and clot retraction, which enhances clot stability and promotes healing. However, in conditions where platelets are depleted or hyporeactive, these functions are diminished and lead to inhibited hemostasis and healing. To treat platelet depletion, our group developed platelet-like particles (PLPs) which consist of highly deformable microgels coupled to fibrin binding motif. However, first generation PLPs do not exhibit wound-triggered shape change like native platelets. Thus, the objective of these studies was to develop a PLP formulation that changes shape when prompted by thrombin. To create thrombin-sensitive PLPs (TS-PLPs), we incorporated a thrombin-cleavable peptide into the microgel body and then evaluated PLP properties before and after exposure to thrombin including morphology, size, and in vitro clot retraction. Once thrombin-prompted shape change ability was confirmed, the TS-PLPs were tested in vivo for hemostatic ability and subsequent wound healing outcomes in a murine liver trauma model. We found that TS-PLPs exhibit a wound-triggered shape change, induce significant clot retraction following exposure to thrombin and promote hemostasis and healing in vivo after trauma.

Fibrin-specific poly(N-isopropylacrylamide) nanogels for targeted delivery of tissue-type plasminogen activator to treat thrombotic complications are well tolerated in vivo.

Targeted drug delivery for maintaining blood fluidity can reduce the risks associated with systemic anticoagulants that can lead to off-target bleeding. Recently, there has been much interest in targeted delivery of tissue-type plasminogen activator (tPA) for treating thrombotic complications. The work presented here characterizes a fibrin-specific nanogel (FSN) design for targeted delivery of tPA to treat thrombotic complications. Fibrin binding and clot degradation were characterized in vitro, and animal models of thrombosis were used to examine nanogel effects on coagulation parameters. In vitro assays showed tPA-FSNs attach to fibrin in a dose-dependent manner independent of tPA loading. In animal models of thrombosis, including an electrolytic injury to monitor clot properties in real time, and a lipopolysaccharide-induced disseminated intravascular coagulation (DIC) animal model, tPA-FSNs modulated fibrin/fibrinogen and platelet incorporation into clots and at optimized dosing could recover consumptive coagulopathy in DIC. Distribution of unloaded and tPA-loaded FSNs showed potential clearance of tPA-FSNs after 24 h, although unloaded FSNs may be retained at sites of fibrin deposits. Maximum tolerated dose studies showed tPA-FSNs have minimal toxicity up to 20 times the optimized therapeutic dose. Overall, these studies demonstrate the therapeutic efficacy of targeted fibrinolysis for systemic microthrombi and begin to evaluate key translational parameters for tPA-FSN therapeutics, including optimal tPA-FSN dosage in a DIC rodent model and safety of intravenous tPA-FSN therapeutics.

Nanosilver composite pNIPAm microgels for the development of antimicrobial platelet-like particles.

Platelets crucially facilitate wound healing but can become depleted in traumatic injury or chronic wounds. Previously, our group developed injectable platelet-like particles (PLPs) comprised of highly deformable, ultralow crosslinked pNIPAm microgels (ULCs) coupled to fibrin binding antibodies to treat post-trauma bleeding. PLP fibrin-binding facilitates homing to sites of injury, promotes clot formation, and, due to high particle deformability, induces clot retraction. Clot retraction augments healing by increasing clot stability, enhancing clot stiffness, and promoting cell migration into the wound bed. Because post-traumatic healing is often complicated by infection, the objective of these studies was to develop antimicrobial nanosilver microgel composite PLPs to augment hemostasis, fight infection, and promote healing post-trauma. A key goal was to maintain particle deformability following silver incorporation to preserve PLP-mediated clot retraction. Clot retraction, antimicrobial activity, hemostasis after trauma, and healing after injury were evaluated via confocal microscopy, colony-forming unit assays, a murine liver trauma model, and a murine full-thickness injury model in the absence or presence of infection, respectively. We found that nanosilver incorporation does not affect base PLP performance while bestowing significant antimicrobial activity and enhancing infected wound healing outcomes. Therefore, Ag-PLPs have great promise for treating hemorrhage and improving healing following trauma.

Extracellular-Matrix-Anchored Click Motifs for Specific Tissue Targeting.

Local presentation of cancer drugs by injectable drug-eluting depots reduces systemic side effects and improves efficacy. However, local depots deplete their drug stores and are difficult to introduce into stiff tissues, or organs, such as the brain, that cannot accommodate increased pressure. We present a method for introducing targetable depots through injection of activated ester molecules into target tissues that react with and anchor themselves to the local extracellular matrix (ECM) and subsequently capture systemically administered small molecules through bioorthogonal click chemistry. A computational model of tissue-anchoring depot formation and distribution was verified by histological analysis and confocal imaging of cleared tissues. ECM-anchored click groups do not elicit any noticeable local or systemic toxicity or immune response and specifically capture systemically circulating molecules at intradermal, intratumoral, and intracranial sites for multiple months. Taken together, ECM anchoring of click chemistry motifs is a promising approach to specific targeting of both small and large therapeutics, enabling repeated local presentation for cancer therapy and other diseases.

Selective Osmotic Shock for Islet Isolation in the Cadaveric Canine Pancreas.

Currently, islet isolation is performed using harsh collagenases that cause nonspecific injury to both islets and exocrine tissue, negatively affecting the outcome of cell transplantation. We evaluated a novel islet isolation protocol utilizing high concentrations of glucose to cause selective osmotic shock (SOS). Islets have a membrane glucose transporter that allows adaptation to changes in glucose concentrations while exocrine tissue can be selectively destroyed by these osmolar shifts. Canine pancreata were obtained within 15 min after euthanasia from animals ( n = 6) euthanized for reasons unrelated to this study. Each pancreas was divided into 4 segments that were randomized to receive 300 mOsm glucose for 20 min (group 1), 600 mOsm for 20 min (group 2), 300 mOsm for 40 min (group 3), or 600 mOsm for 40 min (group 4). Islet yield, purity, and viability were compared between groups. Mean ± standard error of the mean islet yield for groups 1 to 4 was 428 ± 159, 560 ± 257, 878 ± 443, and 990 ± 394 islet equivalents per gram, respectively. Purity ranged from 37% to 45% without the use of density gradient centrifugation and was not significantly different between groups. Islet cell viability was excellent overall (89%) and did not differ between treatment protocol. Islet function was best in groups treated with 300 mOsm of glucose (stimulation index [SI] = 3.3), suggesting that the lower concentration of glucose may be preferred for use in canine islet isolation. SOS provides a widely available means for researchers to isolate canine islets for use in islet transplantation or in studies of canine islet physiology.