Characterization of the interaction of nanobubble ultrasound contrast agents with human blood components.

Nanoscale ultrasound contrast agents, or nanobubbles, are being explored in preclinical applications ranging from vascular and cardiac imaging to targeted drug delivery in cancer. These sub-micron particles are approximately 10x smaller than clinically available microbubbles. This allows them to effectively traverse compromised physiological barriers and circulate for extended periods of time. While various aspects of nanobubble behavior have been previously examined, their behavior in human whole blood has not yet been explored. Accordingly, herein we examined, for the first time, the short and long-term effects of blood components on nanobubble acoustic response. We observed differences in the kinetics of backscatter from nanobubble suspensions in whole blood compared to bubbles in phosphate buffered saline (PBS), plasma, or red blood cell solutions (RBCs). Specifically, after introducing nanobubbles to fresh human whole blood, signal enhancement, or the magnitude of nonlinear ultrasound signal, gradually increased by 22.8 ± 13.1% throughout our experiment, with peak intensity reached within 145 s. In contrast, nanobubbles in PBS had a stable signal with negligible change in intensity (-1.7 ± 3.2%) over 8 min. Under the same conditions, microbubbles made with the same lipid formulation showed a -56.8 ± 6.1% decrease in enhancement in whole blood. Subsequent confocal, fluorescent, and scanning electron microscopy analysis revealed attachment of the nanobubbles to the surface of RBCs, suggesting that direct interactions, or hitchhiking, of nanobubbles on RBCs in the presence of plasma may be a possible mechanism for the observed effects. This phenomenon could be key to extending nanobubble circulation time and has broad implications in drug delivery, where RBC interaction with nanoparticles could be exploited to improve delivery efficiency.

Glioma stem cells activate platelets by plasma-independent thrombin production to promote glioblastoma tumorigenesis.

Background: The interaction between platelets and cancer cells has been underexplored in solid tumor models that do not metastasize, for example, glioblastoma (GBM) where metastasis is rare. Histologically, it is known that glioma stem cells (GSCs) are found in perivascular and pseudsopalisading regions of GBM, which are also areas of platelet localization. High platelet counts have been associated with poor clinical outcomes in many cancers. While platelets are known to promote the progression of other tumors, mechanisms by which platelets influence GBM oncogenesis are unknown. Here, we aimed to understand how the bidirectional interaction between platelets and GSCs drives GBM oncogenesis. Methods: Male and female NSG mice were transplanted with GSC lines and treated with antiplatelet and anti-thrombin inhibitors. Immunofluorescence, qPCR, and Western blots were used to determine expression of coagulation mechanism in GBM tissue and subsequent GSC lines. Results: We show that GSCs activate platelets by endogenous production of all the factors of the intrinsic and extrinsic coagulation cascades in a plasma-independent manner. Therefore, GSCs produce thrombin resulting in platelet activation. We further demonstrate that the endogenous coagulation cascades of these cancer stem cells are tumorigenic: they activate platelets to promote stemness and proliferation in vitro and pharmacological inhibition delays tumor growth in vivo. Conclusions: Our findings uncover a specific preferential relationship between platelets and GSCs that drive GBM malignancies and identify a therapeutically targetable novel interaction.

Platelet-Inspired Intravenous Nanomedicine for Injury-Targeted Direct Delivery of Thrombin to Augment Hemostasis in Coagulopathies.

Severe hemorrhage associated with trauma, surgery, and congenital or drug-induced coagulopathies can be life-threatening and requires rapid hemostatic management via topical, intracavitary, or intravenous routes. For injuries that are not easily accessible externally, intravenous hemostatic approaches are needed. The clinical gold standard for this is transfusion of blood products, but due to donor dependence, specialized storage requirements, high risk of contamination, and short shelf life, blood product use faces significant challenges. Consequently, recent research efforts are being focused on designing biosynthetic intravenous hemostats, using intravenous nanoparticles and polymer systems. Here we report on the design and evaluation of thrombin-loaded injury-site-targeted lipid nanoparticles (t-TLNPs) that can specifically localize at an injury site via platelet-mimetic anchorage to the von Willebrand factor (vWF) and collagen and directly release thrombin via diffusion and phospholipase-triggered particle destabilization, which can locally augment fibrin generation from fibrinogen for hemostatic action. We evaluated t-TLNPs in vitro in human blood and plasma, where hemostatic defects were created by platelet depletion and anticoagulation. Spectrophotometric studies of fibrin generation, rotational thromboelastometry (ROTEM)-based studies of clot viscoelasticity, and BioFlux-based real-time imaging of fibrin generation under simulated vascular flow conditions confirmed that t-TLNPs can restore fibrin in hemostatic dysfunction settings. Finally, the in vivo feasibility of t-TLNPs was tested by prophylactic administration in a tail-clip model and emergency administration in a liver-laceration model in mice with induced hemostatic defects. Treatment with t-TLNPs was able to significantly reduce bleeding in both models. Our studies demonstrate an intravenous nanomedicine approach for injury-site-targeted direct delivery of thrombin to augment hemostasis.

Beyond the thrombus: Platelet-inspired nanomedicine approaches in inflammation, immune response, and cancer.

The traditional role of platelets is in the formation of blood clots for physiologic (e.g., in hemostasis) or pathologic (e.g., in thrombosis) functions. The cellular and subcellular mechanisms and signaling in platelets involved in these functions have been extensively elucidated and new knowledge continues to emerge, resulting in various therapeutic developments in this area for the management of hemorrhagic or thrombotic events. Nanomedicine, a field involving design of nanoparticles with unique biointeractive surface modifications and payload encapsulation for disease-targeted drug delivery, has become an important component of such therapeutic development. Beyond their traditional role in blood clotting, platelets have been implicated to play crucial mechanistic roles in other diseases including inflammation, immune response, and cancer, via direct cellular interactions, as well as secretion of soluble factors that aid in the disease microenvironment. To date, the development of nanomedicine systems that leverage these broader roles of platelets has been limited. Additionally, another exciting area of research that has emerged in recent years is that of platelet-derived extracellular vesicles (PEVs) that can directly and indirectly influence physiological and pathological processes. This makes PEVs a unique paradigm for platelet-inspired therapeutic design. This review aims to provide mechanistic insight into the involvement of platelets and PEVs beyond hemostasis and thrombosis, and to discuss the current state of the art in the development of platelet-inspired therapeutic technologies in these areas, with an emphasis on future opportunities.

Hemoglobin-based Oxygen Carriers: Current State-of-the-art and Novel Molecules.

In blood, the primary role of red blood cells (RBCs) is to transport oxygen via highly regulated mechanisms involving hemoglobin (Hb). Hb is a tetrameric porphyrin protein comprising of two α- and two ß-polypeptide chains, each containing an iron-containing heme group capable of binding one oxygen molecule. In military as well as civilian traumatic exsanguinating hemorrhage, rapid loss of RBCs can lead to suboptimal tissue oxygenation and subsequent morbidity and mortality. In such cases, transfusion of whole blood or RBCs can significantly improve survival. However, blood products including RBCs present issues of limited availability and portability, need for type matching, pathogenic contamination risks, and short shelf-life, causing substantial logistical barriers to their prehospital use in austere battlefield and remote civilian conditions. While robust research is being directed to resolve these issues, parallel research efforts have emerged toward bioengineering of semisynthetic and synthetic surrogates of RBCs, using various cross-linked, polymeric, and encapsulated forms of Hb. These Hb-based oxygen carriers (HBOCs) can potentially provide therapeutic oxygenation when blood or RBCs are not available. Several of these HBOCs have undergone rigorous preclinical and clinical evaluation, but have not yet received clinical approval in the USA for human use. While these designs are being optimized for clinical translations, several new HBOC designs and molecules have been reported in recent years, with unique properties. The current article will provide a comprehensive review of such HBOC designs, including current state-of-the-art and novel molecules in development, along with a critical discussion of successes and challenges in this field.

Trauma-targeted delivery of tranexamic acid improves hemostasis and survival in rat liver hemorrhage model.

BACKGROUND: Trauma-associated hemorrhage and coagulopathy remain leading causes of mortality. Such coagulopathy often leads to a hyperfibrinolytic phenotype where hemostatic clots become unstable because of upregulated tissue plasminogen activator (tPA) activity. Tranexamic acid (TXA), a synthetic inhibitor of tPA, has emerged as a promising drug to mitigate fibrinolysis. TXA is US Food and Drug Administration-approved for treating heavy menstrual and postpartum bleeding, and has shown promise in trauma treatment. However, emerging reports also implicate TXA for off-target systemic coagulopathy, thromboembolic complications, and neuropathy. OBJECTIVE: We hypothesized that targeted delivery of TXA to traumatic injury site can enable its clot-stabilizing action site-selectively, to improve hemostasis and survival while avoiding off-target effects. To test this, we used liposomes as a model delivery vehicle, decorated their surface with a fibrinogen-mimetic peptide for anchorage to active platelets within trauma-associated clots, and encapsulated TXA within them. METHODS: The TXA-loaded trauma-targeted nanovesicles (T-tNVs) were evaluated in vitro in rat blood, and then in vivo in a liver trauma model in rats. TXA-loaded control (untargeted) nanovesicles (TNVs), free TXA, or saline were studied as comparison groups. RESULTS: Our studies show that in vitro, the T-tNVs could resist lysis in tPA-spiked rat blood. In vivo, T-tNVs maintained systemic safety, significantly reduced blood loss and improved survival in the rat liver hemorrhage model. Postmortem evaluation of excised tissue from euthanized rats confirmed systemic safety and trauma-targeted activity of the T-tNVs. CONCLUSION: Overall, the studies establish the potential of targeted TXA delivery for safe injury site-selective enhancement and stabilization of hemostatic clots to improve survival in trauma.

Intravenous administration of synthetic platelets (SynthoPlate) in a mouse liver injury model of uncontrolled hemorrhage improves hemostasis.

BACKGROUND: Clinical resuscitative treatment of traumatic hemorrhage involves transfusion of RBC, platelets and plasma in controlled ratios. However, use of such blood components, especially platelets, present many challenges including availability, portability, contamination risks, and short shelf-life, which limit the use of platelet transfusions outside of large trauma centers such as remote civilian hospitals and austere prehospital settings. This has prompted significant research in platelet substitutes that may resolve the above issues while providing platelet-mimetic hemostatic action. In this framework, we have developed a synthetic platelet surrogate, SynthoPlate, by integrative decoration of platelet function mimetic peptides on a biocompatible lipid nanovesicle platform. We have previously demonstrated hemostatic capability of SynthoPlate in correcting tail-bleeding time in thrombocytopenic mice. Building on this, we hypothesized that SynthoPlate transfusion would decrease bleeding in a murine model of acute hemorrhagic shock. METHODS: A validated model of uncontrolled intraperitoneal hemorrhage, via liver laceration was used to induce hemorrhagic shock in mice. SynthoPlate, control (unmodified) particles, and normal saline were administered as pretreatment and recue infusions to mice undergoing liver laceration and evaluated for hemostatic benefit by determining differences in blood loss and monitoring real-time hemodynamic data. RESULTS: Pretreatment SynthoPlate transfusion resulted in significant reduction of blood loss following hemorrhage, compared with control particles or normal saline treatment (0.86 ± 0.16 g control particles [CP] vs. 0.84 ± 0.13 g normal saline [NS] vs. 0.68 ± 0.09 g SynthoPlate, p < 0.005). SynthoPlate transfused mice demonstrated improved hemodynamics taking significantly longer to develop post-injury hypotension (168.3 ± 106.6 seconds CP vs. 137 ± 58 seconds NS vs. 546.7 ± 329.8 seconds SynthoPlate, p < 0.05). SynthoPlate infusion following liver laceration, that is, rescue transfusion, also resulted in a significant decrease in blood loss (0.89 ± 0.17 g CP vs. 0.92 ± 0.19 g NS vs. 0.69 ± 0.18 g SynthoPlate, p < 0.05). CONCLUSION: Transfusion of SynthoPlate particles reduces blood loss in a murine model of liver injury, and SynthoPlates may represent a viable transfusion product for the mitigation of blood loss in acute, severe hemorrhagic shock.

Factor XII and uPAR upregulate neutrophil functions to influence wound healing.

Coagulation factor XII (FXII) deficiency is associated with decreased neutrophil migration, but the mechanisms remain uncharacterized. Here, we examine how FXII contributes to the inflammatory response. In 2 models of sterile inflammation, FXII-deficient mice (F12-/-) had fewer neutrophils recruited than WT mice. We discovered that neutrophils produced a pool of FXII that is functionally distinct from hepatic-derived FXII and contributes to neutrophil trafficking at sites of inflammation. FXII signals in neutrophils through urokinase plasminogen activator receptor-mediated (uPAR-mediated) Akt2 phosphorylation at S474 (pAktS474). Downstream of pAkt2S474, FXII stimulation of neutrophils upregulated surface expression of αMß2 integrin, increased intracellular calcium, and promoted extracellular DNA release. The sum of these activities contributed to neutrophil cell adhesion, migration, and release of neutrophil extracellular traps in a process called NETosis. Decreased neutrophil signaling in F12-/- mice resulted in less inflammation and faster wound healing. Targeting hepatic F12 with siRNA did not affect neutrophil migration, whereas WT BM transplanted into F12-/- hosts was sufficient to correct the neutrophil migration defect in F12-/- mice and restore wound inflammation. Importantly, these activities were a zymogen FXII function and independent of FXIIa and contact activation, highlighting that FXII has a sophisticated role in vivo that has not been previously appreciated.

Platelet microparticle-inspired clot-responsive nanomedicine for targeted fibrinolysis.

Intravascular administration of plasminogen activators is a clinically important thrombolytic strategy to treat occlusive vascular conditions. A major issue with this strategy is the systemic off-target drug action, which affects hemostatic capabilities and causes substantial hemorrhagic risks. This issue can be potentially resolved by designing technologies that allow thrombus-targeted delivery and site-specific action of thrombolytic drugs. To this end, leveraging a liposomal platform, we have developed platelet microparticle (PMP)-inspired nanovesicles (PMINs), that can protect encapsulated thrombolytic drugs in circulation to prevent off-target uptake and action, anchor actively onto thrombus via PMP-relevant molecular mechanisms and allow drug release via thrombus-relevant enzymatic trigger. Specifically, the PMINs can anchor onto thrombus via heteromultivalent ligand-mediated binding to active platelet integrin GPIIb-IIIa and P-selectin, and release the thrombolytic payload due to vesicle destabilization triggered by clot-relevant enzyme phospholipase-A2. Here we report on the evaluation of clot-targeting efficacy, lipase-triggered drug release and resultant thrombolytic capability of the PMINs in vitro, and subsequently demonstrate that intravenous delivery of thrombolytic-loaded PMINs can render targeted fibrinolysis without affecting systemic hemostasis, in vivo, in a carotid artery thrombosis model in mice. Our studies establish significant promise of the PMIN technology for safe and site-targeted nanomedicine therapies in the vascular compartment.

Bio-inspired nanomedicine strategies for artificial blood components.

Blood is a fluid connective tissue where living cells are suspended in noncellular liquid matrix. The cellular components of blood render gas exchange (RBCs), immune surveillance (WBCs) and hemostatic responses (platelets), and the noncellular components (salts, proteins, etc.) provide nutrition to various tissues in the body. Dysfunction and deficiencies in these blood components can lead to significant tissue morbidity and mortality. Consequently, transfusion of whole blood or its components is a clinical mainstay in the management of trauma, surgery, myelosuppression, and congenital blood disorders. However, donor-derived blood products suffer from issues of shortage in supply, need for type matching, high risks of pathogenic contamination, limited portability and shelf-life, and a variety of side-effects. While robust research is being directed to resolve these issues, a parallel clinical interest has developed toward bioengineering of synthetic blood substitutes that can provide blood's functions while circumventing the above problems. Nanotechnology has provided exciting approaches to achieve this, using materials engineering strategies to create synthetic and semi-synthetic RBC substitutes for enabling oxygen transport, platelet substitutes for enabling hemostasis, and WBC substitutes for enabling cell-specific immune response. Some of these approaches have further extended the application of blood cell-inspired synthetic and semi-synthetic constructs for targeted drug delivery and nanomedicine. The current study provides a comprehensive review of the various nanotechnology approaches to design synthetic blood cells, along with a critical discussion of successes and challenges of the current state-of-art in this field. WIREs Nanomed Nanobiotechnol 2017, 9:e1464. doi: 10.1002/wnan.1464 For further resources related to this article, please visit the WIREs website.

Ferric Chloride-induced Murine Thrombosis Models.

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, diabetes, obesity, cancer and chronic autoimmune rheumatologic disorders. Thrombi are the cause of most heart attacks, strokes and extremity loss, making thrombosis an extremely important public health problem. Since these thrombi stem from inappropriate platelet activation and subsequent coagulation, targeting these systems therapeutically has important clinical significance for developing safer treatments. Due to the complexities of the hemostatic system, in vitro experiments cannot replicate the blood-to-vessel wall interactions; therefore, in vivo studies are critical to understand pathological mechanisms of thrombus formation. To this end, various thrombosis models have been developed in mice. Among them, ferric chloride (FeCl3) induced vascular injury is a widely used model of occlusive thrombosis that reports platelet activation and aggregation in the context of an aseptic closed vascular system. This model is based on redox-induced endothelial cell injury, which is simple and sensitive to both anticoagulant and anti-platelets drugs. The time required for the development of a thrombus that occludes blood flow gives a quantitative measure of vascular injury, platelet activation and aggregation that is relevant to thrombotic diseases. We have significantly refined this FeCl3-induced vascular thrombosis model, which makes the data highly reproducible with minimal variation. Here we describe the model and present representative data from several experimental set-ups that demonstrate the utility of this model in thrombosis research.

A factor VIII-derived peptide enables von Willebrand factor (VWF)-binding of artificial platelet nanoconstructs without interfering with VWF-adhesion of natural platelets.

There is substantial clinical interest in synthetic platelet analogs for potential application in transfusion medicine. To this end, our research is focused on self-assembled peptide-lipid nanoconstructs that can undergo injury site-selective adhesion and subsequently promote site-directed active platelet aggregation, thus mimicking platelet's primary hemostatic actions. For injury site-selective adhesion, we have utilized a coagulation factor FVIII-derived VWF-binding peptide (VBP). FVIII binds to VWF's D'-D3 domain while natural platelet GPIbα binds to VWF's A1 domain. Therefore, we hypothesized that the VBP-decorated nanoconstructs will adhere to VWF without mutual competition with natural platelets. We further hypothesized that the adherent VBP-decorated constructs can enhance platelet aggregation when co-decorated with a fibrinogen-mimetic peptide (FMP). To test these hypotheses, we used glycocalicin to selectively block VWF's A1 domain and, using fluorescence microscopy, studied the binding of fluorescently labeled VBP-decorated nanoconstructs versus platelets to ristocetin-treated VWF. Subsequently, we co-decorated the nanoconstructs with VBP and FMP and incubated them with human platelets to study construct-mediated enhancement of platelet aggregation. Decoration with VBP resulted in substantial construct adhesion to ristocetin-treated VWF even if the A1-domain was blocked by glycocalicin. In comparison, such A1-blocking resulted in significant reduction of platelet adhesion. Without A1-blocking, the VBP-decorated constructs and natural platelets could adhere to VWF concomitantly. Furthermore, the constructs co-decorated with VBP and FMP enhanced active platelet aggregation. The results indicate significant promise in utilizing the FVIII-derived VBP in developing synthetic platelet analogs that do not interfere with VWF-binding of natural platelets but allow site-directed enhancement of platelet aggregation when combined with FMP.

A platelet-inspired paradigm for nanomedicine targeted to multiple diseases.

Platelets are megakaryocyte-derived anucleated cells found in the blood. They are mainly responsible for rendering hemostasis or clotting to prevent bleeding complications. Decreased platelet numbers or deficiencies in platelet functions can lead to various acute or chronic bleeding conditions and hemorrhage. On the other hand, dysregulated hyperactivity of the clotting process can lead to thrombosis and vascular occlusion. There is significant evidence that beyond hemostasis and thrombosis, platelets play crucial mechanistic roles in other disease scenarios such as inflammation, immune response and cancer metastasis by mediating several cell-cell and cell-matrix interactions, as well as aiding the disease microenvironment via secretion of multiple soluble factors. Therefore, elucidating these mechanistic functions of platelets can provide unique avenues for developing platelet-inspired nanomedicine strategies targeted to these diseases. To this end, the current review provides detailed mechanistic insight into platelets' disease-relevant functions and discusses how these mechanisms can be utilized to engineer targeted nanomedicine systems.

Photodynamic nanomedicine in the treatment of solid tumors: perspectives and challenges.

Photodynamic therapy (PDT) is a promising treatment strategy where activation of photosensitizer drugs with specific wavelengths of light results in energy transfer cascades that ultimately yield cytotoxic reactive oxygen species which can render apoptotic and necrotic cell death. Without light the photosensitizer drugs are minimally toxic and the photoactivating light itself is non-ionizing. Therefore, harnessing this mechanism in tumors provides a safe and novel way to selectively eradicate tumor with reduced systemic toxicity and side effects on healthy tissues. For successful PDT of solid tumors, it is necessary to ensure tumor-selective delivery of the photosensitizers, as well as, the photoactivating light and to establish dosimetric correlation of light and drug parameters to PDT-induced tumor response. To this end, the nanomedicine approach provides a promising way towards enhanced control of photosensitizer biodistribution and tumor-selective delivery. In addition, refinement of nanoparticle designs can also allow incorporation of imaging agents, light delivery components and dosimetric components. This review aims at describing the current state-of-the-art regarding nanomedicine strategies in PDT, with a comprehensive narrative of the research that has been carried out in vitro and in vivo, with a discussion of the nanoformulation design aspects and a perspective on the promise and challenges of PDT regarding successful translation into clinical application.

A cell-targeted photodynamic nanomedicine strategy for head and neck cancers.

Photodynamic therapy (PDT) holds great promise for the treatment of head and neck (H&N) carcinomas where repeated loco-regional therapy often becomes necessary due to the highly aggressive and recurrent nature of the cancers. While interstitial light delivery technologies are being refined for PDT of H&N and other cancers, a parallel clinically relevant research area is the formulation of photosensitizers in nanovehicles that allow systemic administration yet preferential enhanced uptake in the tumor. This approach can render dual-selectivity of PDT, by harnessing both the drug and the light delivery within the tumor. To this end, we report on a cell-targeted nanomedicine approach for the photosensitizer silicon phthalocyanine-4 (Pc 4), by packaging it within polymeric micelles that are surface-decorated with GE11-peptides to promote enhanced cell-selective binding and receptor-mediated internalization in EGFR-overexpressing H&N cancer cells. Using fluorescence spectroscopy and confocal microscopy, we demonstrate in vitro that the EGFR-targeted Pc 4-nanoformulation undergoes faster and higher uptake in EGFR-overexpressing H&N SCC-15 cells. We further demonstrate that this enhanced Pc 4 uptake results in significant cell-killing and drastically reduced post-PDT clonogenicity. Building on this in vitro data, we demonstrate that the EGFR-targeted Pc 4-nanoformulation results in significant intratumoral drug uptake and subsequent enhanced PDT response, in vivo, in SCC-15 xenografts in mice. Altogether our results show significant promise toward a cell-targeted photodynamic nanomedicine for effective treatment of H&N carcinomas.

A platelet-mimetic paradigm for metastasis-targeted nanomedicine platforms.

There is compelling evidence that, beyond their traditional role in hemostasis and thrombosis, platelets play a significant role in mediating hematologic mechanisms of tumor metastasis by directly and indirectly interacting with pro-metastatic cancer cells. With this rationale, we hypothesized that platelets can be an effective paradigm to develop nanomedicine platforms that utilize platelet-mimetic interaction mechanisms for targeted diagnosis and therapy of metastatic cancer cells. Here we report on our investigation of the development of nanoconstructs that interact with metastatic cancer cells via platelet-mimetic heteromultivalent ligand-receptor pathways. For our studies, pro-metastatic human breast cancer cell line MDA-MB-231 was studied for its surface expression of platelet-interactive receptors, in comparison to another low-metastatic human breast cancer cell line, MCF-7. Certain platelet-interactive receptors were found to be significantly overexpressed on the MDA-MB-231 cells, and these cells showed significantly enhanced binding interactions with active platelets compared to MCF-7 cells. Based upon these observations, two specific receptor interactions were selected, and corresponding ligands were engineered onto the surface of liposomes as model nanoconstructs, to enable platelet-mimetic binding to the cancer cells. Our model platelet-mimetic liposomal constructs showed enhanced targeting and attachment of MDA-MB-231 cells compared to the MCF-7 cells. These results demonstrate the promise of utilizing platelet-mimetic constructs in modifying nanovehicle constructs for metastasis-targeted drug as well as modifying surfaces for ex-vivo cell enrichment diagnostic technologies.

Approaches to synthetic platelet analogs.

Platelet transfusion is routinely used for treating bleeding complications in patients with hematologic or oncologic clotting disorders, chemo/radiotherapy-induced myelosuppression, trauma and surgery. Currently, these transfusions mostly use allogeneic platelet concentrates, while products like lyophilized platelets, cold-stored platelets and infusible platelet membranes are under investigation. These natural platelet-based products pose considerable risks of contamination, resulting in short shelf-life (3-5 days). Recent advances in pathogen reduction technologies have increased shelf-life to ~7 days. Furthermore, natural platelets are short in supply and also cause several biological side effects. Hence, there is significant clinical interest in platelet-mimetic synthetic analogs that can allow long storage-life and minimum side effects. Accordingly, several designs have been studied which decorate synthetic particles with motifs that promote platelet-mimetic adhesion or aggregation. Recent refinement in this design involves combining the adhesion and aggregation functionalities on a single particle platform. Further refinement is being focused on constructing particles that also mimic natural platelet's shape, size and elasticity, to influence margination and wall-interaction. The optimum design of a synthetic platelet analog would require efficient integration of platelet's physico-mechanical properties and biological functionalities. We present a comprehensive review of these approaches and provide our opinion regarding the future directions of this research.

EGF receptor-targeted nanocarriers for enhanced cancer treatment.

The 'nanomedicine' approach has revolutionized cancer therapy by enabling the packaging of therapeutic agents within engineered nanovehicles that can specifically accumulate within the tumor stroma and then be internalized within cancer cells, to render site-selective action while minimizing nonspecific uptake and harmful side effects. While the specific accumulation within the tumor stroma is rendered by the ability of the nanovehicles to passively permeate through the tumor's leaky vasculature, the cellular internalization is often achieved by exploiting receptor-mediated active endocytotic mechanisms using receptor-specific ligand decoration on the vehicle surface. To this end, a highly important receptor found in several cancers is the EGF receptor, which has been implicated in tumor aggression and proliferation. In this context, we provide a comprehensive review of the various approaches of ligand decorations on nanovehicles for active targeting to EGF receptors, and discuss their pros and cons towards optimizing the design of EGF receptor-targeted nanomedicine systems.

Optimization of a nanomedicine-based silicon phthalocyanine 4 photodynamic therapy (Pc 4-PDT) strategy for targeted treatment of EGFR-overexpressing cancers.

The current clinical mainstays for cancer treatment, namely, surgical resection, chemotherapy, and radiotherapy, can cause significant trauma, systemic toxicity, and functional/cosmetic debilitation of tissue, especially if repetitive treatment becomes necessary due to tumor recurrence. Hence there is significant clinical interest in alternate treatment strategies like photodynamic therapy (PDT) which can effectively and selectively eradicate tumors and can be safely repeated if needed. We have previously demonstrated that the second-generation photosensitizer Pc 4 (silicon phthalocyanine 4) can be formulated within polymeric micelles, and these micelles can be specifically targeted to EGFR-overexpressing cancer cells using GE11 peptide ligands, to enhance cell-specific Pc 4 delivery and internalization. In the current study, we report on the in vitro optimization of the EGFR-targeting, Pc 4 loading of the micellar nanoformulation, along with optimization of the corresponding photoirradiation conditions to maximize Pc 4 delivery, internalization, and subsequent PDT-induced cytotoxicity in EGFR-overexpressing cells in vitro. In our studies, absorption and fluorescence spectroscopy were used to monitor the cell-specific uptake of the GE11-decorated Pc 4-loaded micelles and the cytotoxic singlet oxygen production from the micelle-encapsulated Pc 4, to determine the optimum ligand density and Pc 4 loading. It was found that the micelle formulations bearing 10 mol % of GE11-modified polymer component resulted in the highest cellular uptake in EGFR-overexpressing A431 cells within the shortest incubation periods. Also, the loading of ∼ 50 µg of Pc 4 per mg of polymer in these micellar formulations resulted in the highest levels of singlet oxygen production. When formulations bearing these optimized parameters were tested in vitro on A431 cells for PDT effect, a formulation dose containing 400 nM Pc 4 and photoirradiation duration of 400 s at a fluence of 200 mJ/cm(2) yielded close to 100% cell death.