Lyophilized platelets inhibit platelet aggregation with simultaneous paradoxical promotion of platelet adhesion.

Lyophilized platelets have been explored as a potential hemostatic agent due to their long-term ambient storage capabilities that make them readily available in various scenarios. Additionally, their high biocompatibility and the key role of platelet interactions in various clinical conditions make them a promising platform for drug delivery. To explore these applications and for wider clinical deployment, the interactions between lyophilized platelets and fresh platelets must be examined. This project characterized receptor expression on the lyophilized platelet surface and their ability to bind fibrinogen using flow cytometry. The effect of lyophilized platelets on aggregation of unaltered platelets was assessed using light transmission aggregometry while the effect on adhesion was evaluated using static and microfluidic assays. Lyophilized platelets maintained significant levels of GPIIb and GPVI receptors on their surface, though the expression was reduced from fresh platelets. Additionally, lyophilized platelets maintained GPIb expression similar to fresh platelets. Furthermore, 15.8% of the lyophilized platelets exhibited the active conformation of the GPIIb/IIIa receptor, indicating a significant increase over fresh platelets. Lyophilized platelets also exhibited an increase in exposed phosphatidylserine and fibrinogen binding. Despite the effect of lyophilized platelets in promoting the adhesion of fresh platelets on a collagen-coated surface, their net effect was inhibitory on platelet aggregation. This study demonstrates that lyophilized platelets can have paradoxical effects on platelet adhesion and aggregation, which could have an impact for clinical applications. Detailed characterization and engineering of these effects will be important for their continued development as a drug delivery platform.

Platelet cancer cell interplay as a new therapeutic target.

The interaction between circulating tumor cells and platelets is a key factor in cancer metastasis. These interactions, driven by a variety of receptors, support circulating tumor cells by protecting them from immune detection, cushioning them from shear stress, and promoting their arrest at the endothelium. Additionally, platelets have been shown to accumulate in the primary tumors, promoting tumor growth and angiogenesis by releasing growth factors. Furthermore, tumor cells can interact with platelets by inducing aggregation, which further protects cancer cells. However, the platelet cancer cell interplay also offers new approaches to develop targeted therapies. The accumulation of platelets in tumors has successfully been leveraged to deliver chemotherapeutics and imaging agents. Likewise, these platelet-based interactions have been utilized to target cancer cells in circulation. Although these current systems have limitations including drug loading and storage, leveraging platelet-cancer cell interactions to effectively target circulating tumor cells and tumors shows great promise for future cancer treatments.

Radiation nanosensitizers in cancer therapy-From preclinical discoveries to the outcomes of early clinical trials.

Improving the efficacy and spatial targeting of radiation therapy while sparing surrounding normal tissues has been a guiding principle for its use in cancer therapy. Nanotechnologies have shown considerable growth in terms of innovation and the development of new therapeutic approaches, particularly as radiosensitizers. The aim of this study was to systematically review how nanoparticles (NPs) are used to enhance the radiotherapeutic effect, including preclinical and clinical studies. Clinicaltrials.gov was used to perform the search using the following terms: radiation, cancer, and NPs. In this review, we describe the various designs of nano-radioenhancers, the rationale for using such technology, as well as their chemical and biological effects. Human trials are then discussed with an emphasis on their design and detailed clinical outcomes.

Anti-Platelet Effect Induced by Iron Oxide Nanoparticles: Correlation with Conformational Change in Fibrinogen.

Iron oxide nanoparticles are developed for various biomedical applications, however, there is limited understanding regarding their effects and toxicity on blood components. The particles traveling in circulation inevitably interact with blood cells and plasma proteins and may interfere with hemostasis. Specifically, this study focuses on the influence of superparamagnetic iron oxide nanoparticles (SPIONs) coated with a biocompatible polymer, polyvinyl alcohol (PVA), on platelet function. Here, engineered SPIONs that are functionalized with various PVA coatings to provide these particles with different surface charges and polymer packing are described. These formulations are assessed for any interference with human platelet functions and coagulation, ex vivo. Positively charged SPIONs induce a significant change in platelet GPIIb-IIIa conformation, indicative of platelet activation at the dose of 500 µg mL-1 . Remarkably, engineered PVA(polyvinyl alcohol)-SPIONs all display a robust dose-dependent anti-platelet effect on platelet aggregation, regardless of the PVA charge and molecular weight. After assessing hypotheses involving SPION-induced steric hindrance in platelet-platelet bridging, as well as protein corona involvement in the antiplatelet effect, the study concludes that the presence of PVA-SPIONs induces fibrinogen conformational change, which correlates with the observed dose-dependent anti-platelet effect.

Platelet decoys inhibit thrombosis and prevent metastatic tumor formation in preclinical models.

Platelets are crucial for normal hemostasis; however, their hyperactivation also contributes to many potentially lethal pathologies including myocardial infarction, stroke, and cancer. We hypothesized that modified platelets lacking their aggregation and activation capacity could act as reversible inhibitors of platelet activation cascades. Here, we describe the development of detergent-extracted human modified platelets (platelet decoys) that retained platelet binding functions but were incapable of functional activation and aggregation. Platelet decoys inhibited aggregation and adhesion of platelets on thrombogenic surfaces in vitro, which could be immediately reversed by the addition of normal platelets; in vivo in a rabbit model, pretreatment with platelet decoys inhibited arterial injury-induced thromboembolism. Decoys also interfered with platelet-mediated human breast cancer cell aggregation, and their presence decreased cancer cell arrest and extravasation in a microfluidic human microvasculature on a chip. In a mouse model of metastasis, simultaneous injection of the platelet decoys with tumor cells inhibited metastatic tumor growth. Thus, our results suggest that platelet decoys might represent an effective strategy for obtaining antithrombotic and antimetastatic effects.

Ultrasound-sensitive nanoparticle aggregates for targeted drug delivery.

Here we describe injectable, ultrasound (US)-responsive, nanoparticle aggregates (NPAs) that disintegrate into slow-release, nanoscale, drug delivery systems, which can be targeted to selective sites by applying low-energy US locally. We show that, unlike microbubble based drug carriers which may suffer from stability problems, the properties of mechanical activated NPAs, composed of polymer nanoparticles, can be tuned by properly adjusting the polymer molecular weight, the size of the nanoparticle precursors as well as the percentage of excipient utilized to hold the NPA together. We then apply this concept to practice by fabricating NPAs composed of nanoparticles loaded with Doxorubicin (Dox) and tested their ability to treat tumors via ultrasound activation. Mouse studies demonstrated significantly increased efficiency of tumor targeting of the US-activated NPAs compared to PLGA nanoparticle controls (with or without US applied) or intact NPAs. Importantly, when the Dox-loaded NPAs were injected and exposed to US energy locally, this increased ability to concentrate nanoparticles at the tumor site resulted in a significantly greater reduction in tumor volume compared to tumors treated with a 20-fold higher dose of the free drug.

Assessment of whole blood thrombosis in a microfluidic device lined by fixed human endothelium.

The vascular endothelium and shear stress are critical determinants of physiological hemostasis and platelet function in vivo, yet current diagnostic and monitoring devices do not fully incorporate endothelial function under flow in their assessment and, therefore, they can be unreliable and inaccurate. It is challenging to include the endothelium in assays for clinical laboratories or point-of-care settings because living cell cultures are not sufficiently robust. Here, we describe a microfluidic device that is lined by a human endothelium that is chemically fixed, but still retains its ability to modulate hemostasis under continuous flow in vitro even after few days of storage. This device lined with a fixed endothelium supports formation of platelet-rich thrombi in the presence of physiological shear, similar to a living arterial vessel. We demonstrate the potential clinical value of this device by showing that thrombus formation and platelet function can be measured within minutes using a small volume (0.5 mL) of whole blood taken from subjects receiving antiplatelet medications. The inclusion of a fixed endothelial microvessel will lead to biomimetic analytical devices that can potentially be used for diagnostics and point-of-care applications.

Shear-Activated Nanoparticle Aggregates Combined With Temporary Endovascular Bypass to Treat Large Vessel Occlusion.

BACKGROUND AND PURPOSE: The goal of this study is to combine temporary endovascular bypass (TEB) with a novel shear-activated nanotherapeutic (SA-NT) that releases recombinant tissue-type plasminogen activator (r-tPA) when exposed to high levels of hemodynamic stress and to determine if this approach can be used to concentrate r-tPA at occlusion sites based on high shear stresses created by stent placement. METHODS: A rabbit model of carotid vessel occlusion was used to test the hypothesis that SA-NT treatment coupled with TEB provides high recanalization rates while reducing vascular injury. We evaluated angiographic recanalization with TEB alone, intra-arterial delivery of soluble r-tPA alone, or TEB combined with 2 doses of intra-arterial infusion of either the SA-NT or soluble r-tPA. Vascular injury was compared against stent-retriever thrombectomy. RESULTS: Shear-targeted delivery of r-tPA using the SA-NT resulted in the highest rate of complete recanalization when compared with controls (P=0.0011). SA-NT (20 mg) had a higher likelihood of obtaining complete recanalization as compared with TEB alone (odds ratio 65.019, 95% confidence interval 1.77, >1000; P=0.0231), intra-arterial r-tPA alone (odds ratio 65.019, 95% confidence interval 1.77, >1000; P=0.0231), or TEB with soluble r-tPA (2 mg; odds ratio 18.78, 95% confidence interval 1.28, 275.05; P=0.0322). Histological analysis showed circumferential loss of endothelium restricted to the area where the TEB was deployed; however, there was significantly less vascular injury using a TEB as compared with stent-retriever procedure (odds ratio 12.97, 95% confidence interval 8.01, 21.02; P<0.0001). CONCLUSIONS: A novel intra-arterial, nanoparticle-based thrombolytic therapy combined with TEB achieves high rates of complete recanalization. Moreover, this approach reduces vascular trauma as compared with stent-retriever thrombectomy.

Influence of Surface Charge and Polymer Coating on Internalization and Biodistribution of Polyethylene Glycol-Modified Iron Oxide Nanoparticles.

The aim of this study was to investigate the influence of the surface charge and coating of Superparamagnetic Iron Oxide Nanoparticles (SPIONs) on their in vitro and in vivo behaviors. Neutral and negatively-charged PEG-based SPIONs were synthesized and compared to Resovist, a carboxydextran-based SPION currently used in clinics. Their cytotoxicity, cell internalization, and potential as contrast agents for magnetic resonance imaging were assessed. Neutral pegylated SPIONs were internalized less readily by the reticuloendothelial system and showed a lower uptake by the liver, compared to negatively-charged SPIONs (with carboxydextran and PEG). These results suggested that the charge of functionalized SPIONs was more relevant for their biological interactions than the nature of their coating.

Dispersion of titanate nanotubes for nanomedicine: comparison of PEI and PEG nanohybrids.

In the present study, we report the dispersion of titanate nanotubes (TiONts) via polymer grafting (PolyEthylene Glycol, PEG) or polymer adsorption (polyethylene imine, PEI) where different TiONts/polymer ratios have been investigated. The TiONts/PEI and TiONts/PEG nanohybrids were characterized by scanning and transmission electron microscopy as well as by zeta potential measurements in order to determine both their dispersion state and stability in water (at different pH for zetametry). The nature of the chemical bonds at the surface of these nanohybrids was investigated by Fourier-transformed infrared (FTIR) spectroscopy while the grafting densities of PEG on the nanotubes were quantified by thermogravimetric analyses (TGA). The nanohybrids reported here are promising tools for biotechnology applications due to their tubular morphology, their very good dispersion in water and the reactivity of their surface.

Anti-platelet agents augment cisplatin nanoparticle cytotoxicity by enhancing tumor vasculature permeability and drug delivery.

Tumor vasculature is critically dependent on platelet mediated hemostasis and disruption of the same can augment delivery of nano-formulation based chemotherapeutic agents which depend on enhanced permeability and retention for tumor penetration. Here, we evaluated the role of Clopidogrel, a well-known inhibitor of platelet aggregation, in potentiating the tumor cytotoxicity of cisplatin nano-formulation in a murine breast cancer model. In vivo studies in murine syngeneic 4T1 breast cancer model showed a significant greater penetration of macromolecular fluorescent nanoparticles after clopidogrel pretreatment. Compared to self-assembling cisplatin nanoparticles (SACNs), combination therapy with clopidogrel and SACN was associated with a 4 fold greater delivery of cisplatin to tumor tissue and a greater reduction in tumor growth as well as higher survival rate. Clopidogrel enhances therapeutic efficiency of novel cisplatin based nano-formulations agents by increasing tumor drug delivery and can be used as a potential targeting agent for novel nano-formulation based chemotherapeutics.

PEGylated liposomal Gemcitabine: insights into a potential breast cancer therapeutic.

PURPOSE: Nanoencapsulation of chemotherapeutics is an established method to target breast tumors and has been shown to enhance the efficacy of therapy in various animal models. During the past two decades, the nucleoside analog Gemcitabine has been under investigation to treat both recalcitrant and localized breast cancer, often in combination with other chemotherapeutics. In this study, we investigated the chemotherapeutic efficacy of a novel Gemcitabine-encapsulated liposome previously formulated by our group, GemPo, on both sensitive (4T1) and recalcitrant (MDA-MB-231) breast cancer cell lines. METHODS: Gemcitabine free drug and liposomal Gemcitabine were compared both in vitro and in vivo using breast cancer models. RESULTS: We demonstrated that GemPo differently hindered the growth, survival and migration of breast cancer cells, according to their drug sensitivities. Specifically, whereas GemPo was a more potent cytotoxic and apoptotic agent in sensitive breast cancer cells, it more potently inhibited cell migration in the resistant cell line. However, GemPo still acted as a more potent inhibitor of migration, in comparison with free Gemcitabine, irrespective of cell sensitivity. Administration of GemPo in a 4T1-bearing mouse model inhibited tumor growth while increasing mice survival, as compared with free Gemcitabine and a vehicle control. Interestingly, the inclusion of a mitotic inhibitor, Paclitaxel, synergized only with free Gemcitabine in this model, yet was as effective as GemPo alone. However, inclusion of Paclitaxel with GemPo significantly improved mouse survival. CONCLUSIONS: Our study is the first to demonstrate the pleiotropic effects of Gemcitabine and Gemcitabine-loaded nanoparticles in breast cancer, and opens the door for a novel treatment for breast cancer patients.

P2Y12 receptor inhibition augments cytotoxic effects of cisplatin in breast cancer.

Expression of P2Y12 receptors has been documented in some cancer cell lines like C6 glioma, renal carcinoma and colon carcinoma. However, its direct role in altering response to chemotherapeutics has not been studied. In this study, we characterize the expression of P2Y12 receptor in breast cancer cell lines and evaluate its role in enhancing the cytotoxic effects of cisplatin. We observed a significant upregulation in P2Y12 expression in 4T1 breast cancer cell line with cisplatin treatment. Co-administration of P2Y12 inhibitor with cisplatin resulted in significantly higher cytotoxic response in 4T1 cancer cell line. This was mediated by HIF1α-dependent upregulation of cellular apoptotic pathways. These findings identify P2Y12 receptor as a potential target to enhance antitumor efficacy of chemotherapeutic agents like cisplatin.

Titanate nanotubes: towards a novel and safer nanovector for cardiomyocytes.

Actively contractile cardiomyocyte (CM) monolayer represents an interesting tool to study both cardiac diseases and injuries. However, this model is poorly transfectable with conventional agents. Consequently, there is a need to develop new carriers that could overcome this problem. Titanate nanotubes (TiONts) could be a potential candidate due to possibly higher cell uptake as a direct consequence of their shape. On the basis of this rationale, TiONts were assessed for their cytotoxicity and internalization pathways. Cytotoxicity was assessed for TiONts either functionalized with PEI or unfunctionalized and its spherical counterpart P25 TiO2. No cytotoxic effect was observed under TiONts, TiONts-PEI1800 and P25 TiO2 exposed conditions. The tubular morphology was found to be an important parameter promoting internalization while reversing the charge was assessed as non-additional. Internalization was found to occur by endocytosis and diffusion through the membrane. A preliminary transfection study indicated the potential of TiONts as a nanocarrier.

Mechanistic studies of Gemcitabine-loaded nanoplatforms in resistant pancreatic cancer cells.

BACKGROUND: Pancreatic cancer remains the deadliest of all cancers, with a mortality rate of 91%. Gemcitabine is considered the gold chemotherapeutic standard, but only marginally improves life-span due to its chemical instability and low cell penetrance. A new paradigm to improve Gemcitabine's therapeutic index is to administer it in nanoparticles, which favour its delivery to cells when under 500 nm in diameter. Although promising, this approach still suffers from major limitations, as the choice of nanovector used as well as its effects on Gemcitabine intracellular trafficking inside pancreatic cancer cells remain unknown. A proper elucidation of these mechanisms would allow for the elaboration of better strategies to engineer more potent Gemcitabine nanotherapeutics against pancreatic cancer. METHODS: Gemcitabine was encapsulated in two types of commonly used nanovectors, namely poly(lactic-co-glycolic acid) (PLGA) and cholesterol-based liposomes, and their physico-chemical parameters assessed in vitro. Their mechanisms of action in human pancreatic cells were compared with those of the free drug, and with each others, using cytotoxity, apoptosis and ultrastructural analyses. RESULTS: Physico-chemical analyses of both drugs showed high loading efficiencies and sizes of less than 200 nm, as assessed by dynamic light scattering (DLS) and transmission electron microscopy (TEM), with a drug release profile of at least one week. These profiles translated to significant cytotoxicity and apoptosis, as well as distinct intracellular trafficking mechanisms, which were most pronounced in the case of PLGem showing significant mitochondrial, cytosolic and endoplasmic reticulum stresses. CONCLUSIONS: Our study demonstrates how the choice of nanovector affects the mechanisms of drug action and is a crucial determinant of Gemcitabine intracellular trafficking and potency in pancreatic cancer settings.

Rationally designed oxaliplatin-nanoparticle for enhanced antitumor efficacy.

Nanoscale drug delivery vehicles have been extensively studied as carriers for cancer chemotherapeutics. However, the formulation of platinum chemotherapeutics in nanoparticles has been a challenge arising from their physicochemical properties. There are only a few reports describing oxaliplatin nanoparticles. In this study, we derivatized the monomeric units of a polyisobutylene maleic acid copolymer with glucosamine, which chelates trans-1,2-diaminocyclohexane (DACH) platinum (II) through a novel monocarboxylato and O --> Pt coordination linkage. At a specific polymer to platinum ratio, the complex self-assembled into a nanoparticle, where the polymeric units act as the leaving group, releasing DACH-platinum in a sustained pH-dependent manner. Sizing was done using dynamic light scatter and electron microscopy. The nanoparticles were evaluated for efficacy in vitro and in vivo. Biodistribution was quantified using inductively coupled plasma atomic absorption spectroscopy (ICP-AAS). The PIMA-GA-DACH-platinum nanoparticle was found to be more active than free oxaliplatin in vitro. In vivo, the nanoparticles resulted in greater tumor inhibition than oxaliplatin (equivalent to 5 mg kg⁻¹ platinum dose) with minimal nephrotoxicity or body weight loss. ICP-AAS revealed significant preferential tumor accumulation of platinum with reduced biodistribution to the kidney or liver following PIMA-GA-DACH-platinum nanoparticle administration as compared with free oxaliplatin. These results indicate that the rational engineering of a novel polymeric nanoparticle inspired by the bioactivation of oxaliplatin results in increased antitumor potency with reduced systemic toxicity compared with the parent cytotoxic. Rational design can emerge as an exciting strategy in the synthesis of nanomedicines for cancer chemotherapy.