Synthetic approaches to RBC mimicry and oxygen carrier systems.

Whole blood or red blood cell (RBC) transfusions are highly significant, clinically, for blood replacement therapies in traumatic injuries, presurgical conditions, and anemias. However, natural RBC-based products suffer from limited shelf life due to pathological contamination and also present risks of refractoriness, graft-versus-host disease, immunosuppression, and acute lung injury. These issues can be only partially resolved by pathogen reduction technologies, serological blood testing, leukoreduction, and specialized storage; hence, they severely affect the efficacy and safety of the blood products. Consequently, there is a significant interest in synthetic RBC analogues that can mimic its oxygen-transport properties while allowing convenient manufacture, reproducibility, long shelf life, and reduced biological risks. To this end, the current Review provides a comprehensive description and discussion of the various research approaches and current state-of-the-art in synthetically mimicking RBC's oxygen-carrying biochemical properties, as well as the biophysical parameters (shape, size and mechanical modulus) that influence RBCs' hemodynamic transport properties in blood flow.

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.

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.

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.

In vitro and in vivo hemostatic capabilities of a functionally integrated platelet-mimetic liposomal nanoconstruct.

There is significant clinical interest in synthetic platelet substitutes that can mimic platelet's hemostastic functionalities while allowing scale-up, minimal biological contamination, and long shelf-life. To this end, mimicking active platelet's hemostatically relevant matrix-adhesion properties and aggregation properties independently and then integrating them via heteromultivalent ligand decoration on a single synthetic particle can lead to an efficient platelet substitute design. We have recently reported on the feasibility of this approach in vitro, using liposomes as model particles. Building on these studies, here we demonstrate the capability of optimizing the platelet-mimetic properties of our liposomal constructs in vitro via modulating the ligand-decoration densities and ligand ratios. In addition, we demonstrate the enhanced hemostatic efficacy of the functionally-integrated platelet-mimetic constructs in vivo. Liposomes were surface-decorated with collagen- and VWF-binding peptides (CBP and VBP) to mimic platelet adhesion and a fibrinogen-mimetic peptide (FMP) to promote platelet aggregation. Modulation of VBP- and CBP-densities and relative ratios enabled optimizing construct adhesion under varying shear-flow conditions. Modulation of FMP-density enabled enhancement of construct-promoted platelet aggregation. The VBP-, CBP- and FMP-decorations were integrated on a single liposome, and these functionally-integrated constructs showed significantly higher hemostatic efficacy in vivo in a mouse tail-transection model compared to 'adhesion-only' or 'aggregation-only' constructs.

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.