Christa L. Modery-Pawlowski

Platelet-like Nanoparticles: Mimicking Shape, Flexibility, and Surface Biology of Platelets To Target Vascular Injuries

Targeted delivery of therapeutic and imaging agents in the vascular compartment represents a significant hurdle in using nanomedicine for treating hemorrhage, thrombosis, and atherosclerosis. While several types of nanoparticles have been developed to meet this goal, their utility is limited by poor circulation, limited margination, and minimal targeting. Platelets have an innate ability to marginate to the vascular wall and specifically interact with vascular injury sites. These platelet functions are mediated by their shape, flexibility, and complex surface interactions. Inspired by this, we report the design and evaluation of nanoparticles that exhibit platelet-like functions including vascular injury site-directed margination, site-specific adhesion, and amplification of injury site-specific aggregation. Our nanoparticles mimic four key attributes of platelets, (i) discoidal morphology, (ii) mechanical flexibility, (iii) biophysically and biochemically mediated aggregation, and (iv) heteromultivalent presentation of ligands that mediate adhesion to both von Willebrand Factor and collagen, as well as specific clustering to activated platelets. Platelet-like nanoparticles (PLNs) exhibit enhanced surface-binding compared to spherical and rigid discoidal counterparts and site-selective adhesive and platelet-aggregatory properties under physiological flow conditions in vitro. In vivo studies in a mouse model demonstrated that PLNs accumulate at the wound site and induce ∼65% reduction in bleeding time, effectively mimicking and improving the hemostatic functions of natural platelets. We show that both the biochemical and biophysical design parameters of PLNs are essential in mimicking platelets and their hemostatic functions. PLNs offer a nanoscale technology that integrates platelet-mimetic biophysical and biochemical properties for potential applications in injectable synthetic hemostats and vascularly targeted payload delivery.

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 platelets shape, size and elasticity, to influence margination and wall-interaction. The optimum design of a synthetic platelet analog would require efficient integration of platelets 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.

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 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.

Targeted killing of metastatic cells using a platelet- inspired drug delivery system

The ‘targeted nanomedicine’ approach has revolutionized cancer therapy by packaging drugs within nanovehicles that can accumulate preferentially within the tumor due to the ‘enhanced permeation and retention’ (EPR) mechanism (passive targeting) and further bind to cancer cells via specific ligand–receptor interactions (active targeting). While these approaches have shown promise in well-vascularized primary tumors, their use for metastatic sites faces challenges due to high heterogeneity in the expression levels of tumor-associated targetable receptors. Therefore, alternative strategies for metastatic cell-specific targeting are of great clinical interest. To this end, we are exploring binding interactions of metastatic cells with host cells in the vascular compartment and whether such interactions can be exploited for metastasis-targeted delivery of drug-loaded nanovehicles. For ‘proof-of-concept’ of this approach, using high-metastatic MDA-MB-231 versus low-metastatic MCF-7 human breast cancer cells, we demonstrate that high-metastatic cells have enhanced binding interactions with active platelets via several cell-surface proteins. Utilizing this information and using liposomes as a model nanovehicle, we have created delivery systems that can actively bind to the high-metastatic cells via platelet-inspired heteromultivalent interactions. Using a mono-culture of MCF-7 versus MDA-MB-231, we demonstrate that these systems enhance the delivery of Doxorubicin (DOX) to the high-metastatic cells to cause significant cell-killing. Furthermore, incubating co-cultures of MCF-7 and MDA-MB-231 cells with the DOX-loaded platelet-inspired nanovehicles, we demonstrate that the targeted binding, DOX delivery and cell-killing are quite selective towards the high-metastatic cells. Our results suggest that this platelet-inspired targeting can lead to unique strategies for metastasis-selective nanomedicine.