A platelet-inspired paradigm for nanomedicine ... - Future Medicine

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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. KEYWORDS: drug delivery n hemostasis n immune response n inflammation n metastasis n platelet n platelet-inspired nanomedicine n thrombosis

Platelets are anucleated cells produced from mature megakaryocytes [1]. Their most wellestablished role is in the regulation of hemostasis [2]. Dysregulation of platelets’ hemostatic mechanisms leads to occlusive thrombosis, which is evident in myocardial infarction, stroke and peripheral vascular disease [3]. Beyond hemostasis and thrombosis, there is growing evidence that platelets have multiple mechanistic roles in other pathologies such as inflammation, immune response and cancer metastasis [4,5] via various ligand-mediated interactions, as well as secretion of molecules that influence the disease microenvironment. F igure 1 shows scanning electron micrographs of human platelet morphology changes from the resting to activated state (Figure 1A), selected platelet surface molecules involved in physiologic and pathologic functions (Figure 1B) and various molecules secreted from platelet granules (Figure 1C). Understanding platelets’ physiological role in hemostasis and pathological role in thrombosis, inflammation, immune response and cancer metastasis can provide unique avenues for developing nanomedicine strategies in these disease areas. This review is organized into five focus areas, for each of which we provide a brief description of platelets’ mechanistic involvement. We then review and discuss how these mechanisms are utilized for nanomedicine development.

Platelets in hemostasis Platelet involvement in hemostasis The mechanism of injury site adhesion, activation and aggregation of platelets to form the hemostatic plug is known as primary hemostasis. Natural quiescent platelets are biconvex discoid cells approximately 2–5 µm in diameter with an elastic modulus of 10–50 kPa [6,7]. Theoretical modeling and experimental analyses have indicated that these key biophysical properties of platelets play significant roles in their interactions with the bigger (~8 µm diameter) biconcave disc-shaped and more flexible red blood cells (RBCs) in blood flow, which leads to their expulsion and margination from the bulk RBC flow in the center of the vessel towards the vessel wall [8]. This margination enhances the platelets’ adhesion probability at a vascular injury site when needed. The principal adhesion mechanisms of platelets include the binding of platelet surface glycoprotein component GPIba of the receptor GPIb-IX-V to von Willebrand factor (vWF) secreted by the injured endothelial cells, in addition to the binding of platelet surface glycoproteins GPIa‑IIa and GPVI to subendothelial collagen at the injury site [9]. These adhesion processes, as well as the action of agonists such as ADP and thrombin, lead to activation of platelets, which change shape from discoid biconvex to stellate [10]. Platelet activation also triggers multiple signaling pathways,

10.2217/NNM.13.113 © 2013 Future Medicine Ltd

Nanomedicine (2013) 8(10), 1709–1727

Christa L Modery‑Pawlowski1, Hsiao-Hsuan Kuo2, William M Baldwin 3rd2 & Anirban Sen Gupta*1 Department of Biomedical Engineering, Case Western Reserve University, 2071 Martin Luther King Jr Drive, Cleveland, OH 44106, USA 2 Department of Immunology, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195, USA *Author for correspondence: [email protected] 1

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Figure 1. Morphological changes in platelets. Important biomolecules expressed on the surface of active platelets and a representative list of granule contents secreted by platelets upon activation. (A) Platelets change from discoid to pseudopodal stellate morphology upon activation, and fully activated platelets have a spread morphology with stable adhesion to the substrate surface. Scale bars: 2 µm. (B) Activated platelets express several receptors on the surface that mediate platelet adhesion (e.g., GPVI and GPIa‑IIa, which bind collagen, and GPIba of GPIb-IX-V, which binds vWF), platelet activation (e.g., receptors for ADP, thrombin and thromboxane A2) and platelet aggregation (e.g., stimulated GPIIb‑IIIa, which binds Fg). (C) Activated platelets also secrete a variety of agonists, chemokines, cytokines, proteins, enzymes and adhesion molecules from their granules (a and dense) and lysosomes. CAM: Cell adhesion molecule; Fg: Fibrinogen; MMP: Matrix metalloprotease; TP: Thromboxane A2; vWF: von Willebrand factor.

resulting in further production of ADP, thrombin and thromboxane to enhance platelet activation and aggregation, and induce conformational changes in the platelet surface integrin GPIIb-IIIa into a stimulated form that can bind fibrinogen (Fg) [11]. Stimulated GPIIb-IIIa binds Fg via the RGD sequence within Fg’s a-chains and the HHLGGAKQAGDV sequence (H12 peptide) within the g-chains [12]. Fg can bridge 1710

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active platelets via such multivalent interactions, leading to platelet aggregation at the injury site. Furthermore, a regulated flip-flop mechanism on the membrane of these aggregated active platelets causes phosphoserines from the membrane inner leaflet to become exposed on the outer leaflet, and this leads to colocalization and activation of multiple coagulation factors on the platelet membrane [13]. These procoagulant processes lead to the formation of thrombin, which breaks down Fg to fibrin, arresting the platelet plug and other blood components to form the final clot [14]. This coagulation mechanism is called secondary hemostasis. Figure 2 shows platelets’ involvement in these hemostatic mechanisms, and mimicking this with synthetic platforms provides a way to develop artificial platelet analogs for hemostatic therapies. Platelet-inspired hemostatic nanomedicine Based on platelets’ primary hemostatic mechanisms, several past approaches have involved mimicking either the mechanisms of platelet adhesion by modifying synthetic particle surfaces with recombinant GPIba and GPIa-IIa, or amplifying the platelet aggregation function by decorating synthetic particle surfaces with Fg or Fg-derived peptides [15]. The resultant products have shown promising preclinical data, but have either failed to progress into clinical trials or have shown very limited efficacy in clinical studies [15]. One possible drawback of these approaches may be the utilization of only adhesion or only aggregation as the platelet-mimetic function, but not a combination of them. It is well established that both adhesion and aggregation functionalities in tandem are critical for primary hemostasis [16]. This was further corroborated in a recent study in which synthetic particles surface-modified with platelet aggregation-promoting Fg-derived H12 peptides and those surface-modified by vWF adhesion-promoting recombinant GPIba motifs, when used in combination, showed significantly higher hemostatic function compared with either of these particles alone [17]. Injecting two different types of surface-modified particles to achieve a cumulative hemostatic effect may be difficult in translation. Hence, it can be rationalized that integrating both functions on a single synthetic platform may result in a more efficacious platelet-mimetic design with superior hemostatic abilities. Such integration can pose significant challenges regarding simultaneous conjugation of various large protein fragments (e.g., Fg or Fg fragments, recombinant GPIba future science group

A platelet-inspired paradigm for nanomedicine targeted to multiple diseases

and GPIa-IIa) onto one particle owing to mutual steric interference [18–20]. To resolve this issue, Ravikumar et al. utilized heteromultivalent decoration of particle platforms with small peptide ligands that can allow cooperative mechanisms of platelet-mimetic adhesion and aggregation promotion. Specifically, they conjugated vWFbinding peptides, collagen-binding peptides and Fg-mimetic GPIIb-IIIa-binding peptides to lipids (e.g., distearylphosphatidylethanolamine) via PEG spacers to form distearylphosphatidylethanolamine–PEG peptide systems, and have subsequently assembled them into heteromultivalently decorated unilamellar liposomes [19,20]. This approach allows for modulation of the peptide decoration densities to optimize the bioactivity of the corresponding ligands for hemostatic function [21]. This functionally integrated design has demonstrated significantly enhanced hemostatic activity, both in vitro and in vivo, compared with designs that only bear adhesion or aggregation functionality [21]. Since physical parameters such as shape, size and mechanical modulus also play significant

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roles in platelets’ hemodynamic margination properties [8], such parameters have become attractive in platelet-inspired particle design. Several researchers have demonstrated that anisotropic particles (e.g., spheroids, ellipsoids, discs and rods) have a higher capability of margination and adhesion compared with spherical particles [22,23]. In addition, particles in a 1–5 µm diameter range have been shown to have a higher margination propensity, compared with particles in a submicron range [24]. It has also been demonstrated that shear gradients in a hemodynamic flow environment substantially influence the margination and localization of discoid platelets [25] . Combination of these observations suggests that biconvex discoid particles, a few micrometers in diameter, may provide an improved platform for platelet mimicry in a hemodynamic environment. Regarding platelet-mimetic mechanical modulus, a notable example is that of albumin-based soft discoid particles, which have shown platelet-mimetic vWF adhesion [26]. Platelets’ physicomechanical properties are highly dynamic as platelets go

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Figure 2. Cell–cell and cell–matrix interactions of platelets in primary hemostasis (platelet margination, adhesion and aggregation at vascular injury site) and platelet-mediated stimulation of secondary hemostasis (coagulatory mechanisms triggered on the activated platelet surface leading to crosslinked fibrin formation). The margination is influenced by platelet shape, size and modulus. The adhesion is principally mediated by GPIba binding to vWF and GPVI and GPIa‑IIa binding to collagen. The aggregation is principally mediated by GPIIb‑IIIa binding to Fg and the coagulatory pathways are facilitated by phospholipids on the activated platelet surface. Fg: Fibrinogen; RBC: Red blood cell; vWF: von Willebrand factor; WBC: White blood cell.

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from a quiescent to active state in response to shear or adhesion (Figure 1A), and biomaterials that can undergo such environmentally responsive changes can provide additional functions in platelet-inspired particle design. As active platelet membranes also help coagulation, some research approaches have been directed towards adapting these mechanisms on synthetic platforms. For example, phosphoserine-rich liposomes have been engineered to modulate coagulatory capabilities [27]. Platelets also secrete various biomolecules (e.g., ADP, thrombin and vWF) in modulating primary and secondary hemostasis. Hence, platelet-mimetic approaches can leverage drug delivery concepts to render artificial hemostatic designs that can locally secrete hemostasis-modulating factors. Examples of this can be seen in designs that incorporate platelet interaction-mimetic ligand motifs on the surface of particles loaded with agonists (e.g., ADP) and drugs (e.g., dexamethasone) [28,29]. Figure 3 shows selected research results in the use of artificial platelet analogs for hemostatic therapy and more details are available elsewhere [15].

Platelets in atherosclerosis thrombosis & restenosis Platelet involvement in the pathology of atherosclerosis, thrombosis & restenosis When the hemostasis mechanisms discussed in the ‘Platelet involvement in hemostasis’ section, are dysregulated, this leads to hyperactivity of platelet-mediated clot formation, which is the main characteristic of thrombosis. Anatomically, thrombosis can be venous (RBC-rich red clot) or arterial (platelet-rich white clot); however, irrespective of this distinction, platelet-relevant mechanisms play a pivotal role in thrombosis [30,31]. Restenosis is a phenomenon by which thrombosis and inflammatory processes can reoccur to occlude the vessel after a thrombosed artery has been cleared via interventional procedures (e.g., angioplasty and stenting). Atherosclerosis is an even more complex multifactorial disease of blood vessels that is initiated by inflammatory events partly mediated by platelets, and can then propagate into subendothelial plaque formation, erosion and rupture, leading to acute occlusive thrombosis. Platelets play a significant role in mediating these inflammatory and thrombotic events in atherosclerosis and restenosis [32–34]. The role of platelets in thrombosis stems from their previously described ability to undergo rapid adhesion, activation and aggregation at the site of endothelial injury, and 1712


to concomitantly propagate coagulation mechanisms on active platelet membrane surfaces. As previously described, the initial injury site adhesion is mediated by vWF–platelet GPIba and collagen–platelet GPIa-IIa and GPVI interactions, and the aggregation is mediated principally by Fg interaction with active platelet GPIIb-IIIa. The GPIIb-IIIa integrins can also bind to vWF domains and fibronectin, which provide additional mechanisms of thrombus stabilization. Besides GPIIb-IIIa integrin, another major surface marker for activated platelets is P-selectin, which is present within cytoplasmic a-granules in quiescent platelets and becomes upregulated on the platelet surface as the granules fuse with the cell membrane upon platelet activation. P-selectin mediates recruitment of inflammatory cells by interaction with PSGL-1 on leukocytes, and also interacts with other platelets via sulfatides, as well as with endothelial cells via GlyCAM-1, CD34 and MadCAM-1 [35,36]. In normal hemostasis, these clot-forming mechanisms are precisely balanced by innate mechanisms of clot retraction, dissolution and healing. For example, plasminogen and tissue-type plasminogen activator interact to generate plasmin, which, in turn, digests fibrin to dissolve the clot. In addition, endothelium-secreted thromboregulators such as nitric oxide and prostacyclin reduce the prothrombotic activity of platelets [37]. In pathological arterial thrombosis, this fine balance is disrupted, resulting in excessive platelet activity and clotting. In venous thrombosis, recent research findings suggest that platelet-mediated adhesion and aggregation mechanisms overlap with blood hypercoagulability, blood flow variations and damaged endothelium (together known as Virchow’s triad of contributing factors), which act in concert [38,39]. The platelet-relevant mechanisms described above also have a significant spatiotemporal involvement in inflammatory processes for atherosclerosis and restenosis. As mentioned previously, P-selectin expressed on active platelets can directly interact with leukocytes via PSGL-1, as well as via other receptor–ligand pairs, such as CD40–CD40L (CD154) and CD11b/CD18–b2 integrins [40]. Activated platelets also secrete proinflammatory biomolecules such as IL-1b, RANTES, MCP-1 and macrophage colony-stimulating factor, which promote inflammatory cell recruitment and transformations that facilitate atherosclerosis [34]. Activated platelets also secrete PDGF and TGF-b, which stimulate smooth muscle cell (SMC) migration, proliferation and collagen synthesis. In addition, future science group


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Figure 3. Representative results of various aspects in platelet-mimetic and platelet-inspired synthetic hemostat research. (A) Fluorescent micrographs of: liposomes surface-decorated with rGPIba or VBP undergoing adhesion on vWF surfaces under flow; liposomes surface-decorated with CBP undergoing adhesion on collagen surfaces under flow; liposomes bearing both rGPIba and CBP showing not much enhanced adhesion on a vWF–collagen mixed surface due to steric interference between the surface-decorated motifs; and liposomes bearing both VBP and CBP showing significantly enhanced adhesion on a vWF–collagen surface due to the cumulative effect of vWF and collagen binding without mutual steric interference between the motifs. (B) Enhancement of primary hemostasis by liposomes bearing adhesive functionalities (VBP and CBP decorations) and platelet-aggregatory functionalities (fibrinogen-mimetic cRGD peptides) and corresponding results of this design regarding liposome adhesion (green), platelet aggregation on liposomes (red) and resultant colocalization (yellow). Scale bars: 50 µm. (C) Enhanced hemostatic function of a mixed population of H12- and rGPIba-decorated latex beads, compared with H12 latex beads only and unmodified latex beads. (D) Enhanced hemostatic effect of ADP-releasing H12-decorated liposomal vesicles compared with those without ADP. (E) Significant enhancement of hemostatic efficacy in vivo in reducing bleeding time in a mouse tail transection model when adhesion functionalities (VBP and CBP) and aggregation functionalities (FMP) are integrated on the same liposomal vehicle compared with adhesion- or aggregation-only designs. (F) Particles with a 2–5‑µm diameter marginate and bind more compared with submicron-diameter particles. (G) Particles of anisotropic shapes (e.g., disk and rod) have a higher adhesion probability to a substrate under flow conditions compared with spherical particles. (H) Representative results and scanning electron micrograph of platelet shape- and size-mimetic albumin-based particles decorated with GPIba fragment for vWF adhesion. Scale bar: 2 µm. *p < 0.05; **p < 0.002. CBP: Collagen-binding peptide; FMP: Fibrinogen-mimetic peptide; H: Height; R: Radius; rGPIba: Recombinant GPIba; VBP: von Willebrand factor-binding peptide; vWF: von Willebrand factor. (A) Adapted with permission from [19]; (B) adapted with permission from [20]; (C) adapted with permission from [17]; (D) adapted with permission from [28]; (E) adapted with permission from [21]; (F) adapted with permission from [24]; (G) adapted with permission from [23]; (H) adapted with permission from [26].



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activated platelets and macrophages produce matrix metalloproteases, which aid further platelet activation and aggregation, as well as vascular matrix degradation for infiltration of SMCs and inflammatory cells [41,42]. Figure 4 depicts platelet-relevant interactions in thrombosis, and demonstrates how utilizing these can provide unique methods of targeted nanomedicine for these diseases. Platelet-inspired nanomedicine targeted to thrombotic disease sites Several research strategies have been directed towards nanovehicles that actively target atherothrombotic sites. For example, echogenic liposomes that actively target Fg, fibrin or ICAM-1 using surface-conjugated antibodies were shown to enhance ultrasound imaging of atheroma in pigs [43,44]. Such echogenic liposomes have also provided a way for using ultrasound-induced cavitation for targeted delivery of thrombolytic drugs (e.g., tissue-type plasminogen activator and streptokinase) [45,46]. A PEG-lipid-based micelle system surface modified with a fibrin-targeted pentapeptide has been reported for targeted delivery of a fluorescent probe and a therapeutic agent (hirulog) to atherosclerotic sites in ApoE-/- mice [47]. Fibrin-targeted nanovehicles have also been Leukocyte

reported for the clot-targeted delivery of imaging agents and atherosclerosis-targeted delivery of antiproliferative agents (e.g., paclitaxel and fumagillin) [48–50]. Gas-filled microbubbles surface modified with platelet integrin GPIIb-IIIaspecific antibody abciximab (marketed as ReoPro®; Eli Lilly, IN, USA) were shown to enhance targeted molecular imaging of thrombus [51]. The Sen Gupta group has developed liposomes surface modified with peptide ligands that have high affinity to activated integrin GPIIb-IIIa and P-selectin on the active platelet surface [52–54]. In vascularly targeted applications, the nanovehicles need to not only actively bind to the disease site, but also ensure that binding is stable under hemodynamic flow. To this end, they have demonstrated that vehicles bearing heteromultivalent ligand decorations provide a unique way to enhance binding selectivity, as well as stable retention under flow [55]. Thrombus targeting has also been suggested for albumin-based particles surface-decorated with GPIba fragments for binding vWF on diseased endothelium [26]. Similar vWF-binding was also recently reported for nanoparticles made of gelatin (denatured collagen) [56]. In another study, a lipid–polymer nanoparticle surface decorated with peptides that target basement membrane collagen IV, was able

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Figure 4. Cell–cell and cell–matrix interactions of platelets in thrombosis and possible restenotic and inflammatory processes. Platelet hyperactivity and aggregation is mediated by GPIIb‑IIIa–Fg interaction and P-selectin–PSGL-1 interaction; P-selectin-based interactions also lead to recruitment and adhesion of leukocytes. Fg: Fibrinogen; vWF: von Willebrand factor.

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to undergo enhanced binding to vascular injury sites in vivo and deliver paclitaxel to reduce SMC activity [57]. In another interesting study, the nanomedicine design utilized the presence of a high shear environment near thrombotic sites to allow polymeric nanoparticle microaggregates to undergo shear-induced disintegration and release thrombolytic drugs [58]. Figure 5 shows selected research results in the area of nanomedicine systems targeted to thrombotic diseases. Besides targeting active platelets or matrix proteins, nanomedicine research has also been directed towards targeting other cell types in athero sclerosis, thrombosis and restenosis; for example, macrophages, neutrophils and SMCs [59].

Platelets in inflammation Platelet involvement in inflammation pathology As indicated previously, platelets also play a major role in facilitating acute and chronic inflammatory responses. In any inflammatory response, the process of leukocyte homing to the inflamed tissue involves leukocyte recognition of injury site and migration towards the endothelium, initial tethering and rolling of leukocytes along the endothelium, activation of leukocytes causing expression of various cell adhesion molecules, firm adhesion followed by diapedesis through the endothelium, and migration through the tissue to the source of injury/infection along with transformation into the macrophage phenotype. Platelets have been shown to orchestrate many of these steps through direct interaction with inflammatory cells and release of soluble factors. Platelets are one of the first cellular responders to distress signals and can accumulate rapidly at the site of injury or pathogen invasion [60]. Upon activation at this site, platelets release a variety of chemokines, cytokines and growth factors, which further facilitate the inflammatory response. For example, CXCL4 (or PF-4), which is released from active platelet a-granules, is a chemoattractant for neutrophils, monocytes and fibroblasts, and is also a stimulant for monocyte differentiation into macrophages [61]. CXCL4 and CCL3 stimulate the release of histamine from basophils and stimulate eosinophil adhesion to the endothelium [62], while CXCL8 (or IL-8) recruits neutrophils to the site of inflammation [63]. Additionally, CXCL7 promotes neutrophil chemotaxis and adhesion to the endothelium [64]. RANTES (or CCL5), which is secreted by activated platelets, also enhances recruitment of monocytes and shear-resistant monocyte arrest [65]. Platelet-secreted cytokines such as IL-1b future science group

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and b-thromboglobulin can activate endothelial cells, causing the release of IL-6, CXCL8 and monocyte chemoattractant protein-1 (or CCL2), as well as increasing the expression of adhesion molecules such as E-selectin, VCAM-1, ICAM-1 and avb3 for leukocyte binding [66,67]. Growth factors released from platelet a-granules, such as PDGF and TGF-b, are potent chemotactic agents for neutrophils and monocytes [68], and also influence fibroblast and SMC proliferation [69]. Besides releasing proinflammatory soluble factors, direct interactions between active platelets and inflammatory cells are also important in inflammation. Activated platelets can directly bind to circulating leukocytes [70] via the interaction of platelet P-selectin with leukocyte PSGL-1 [40]. Activated leukocytes express b2 -integrins (e.g., aMb2 and aLb2), which are involved in stronger cell–cell adhesions. For example, LFA-1 and MAC-1 (also called aMb2 or CD11b/ CD18) bind to platelet ICAM-2 and GPIba, respectively, and enable strong platelet–leukocyte binding [71,72]. Another important ligand– receptor pair implicated in platelet–leukocyte– endothelium interactions is CD40–CD40L (CD154) [73]. CD40 is expressed on activated leukocytes and endothelial cells, and CD40L is expressed on active platelets and stimulated leukocytes, and hence these interactions facilitate the platelet–endothelium–leukocyte triad. These various interactions, along with plateletsecreted proinflammatory factors, have been implicated in several inflammatory conditions including atherosclerosis (described previously), allergic inflammation (e.g., asthma, rhinitis and eczema), inflammatory bowel disease (IBD) and rheumatoid arthritis (RA) [74–77]. Figure 6 shows a schematic of platelet-mediated cell–cell and cell–matrix interactions in inflammation. Owing to platelets’ direct and mediatory roles in these inflammatory processes, platelet-inspired nanomedicine may provide unique avenues of inflammation-targeted drug delivery, by inhibiting/reducing platelets’ mechanistic involvement at cellular and molecular levels, as well as by targeting the delivery of anti-inflammatory and antiproliferative agents to the inflammation site using platelet-mimetic delivery vehicles. Platelet-inspired nanomedicine targeted to inflammatory diseases Agents that inhibit platelets’ interactions with inflammatory cells have been investigated for their therapeutic effect in inflammatory conditions. For example, selectin antagonists have been investigated in allergic asthma, showing www.futuremedicine.com

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Figure 5. Representative results of platelet-relevant and platelet-inspired therapeutic approaches in the diagnosis and treatment of thrombotic and restenotic events. (A) Data regarding liposomes that target activated platelets, showing one-to-one correspondence between (Ai) liposome fluorescence and (Aii) phase-contrast images of active platelets in vitro, (Aiii) enhanced binding of ligand-decorated liposomes to activated platelets compared with quiescent platelets depicted by flow cytometry and (Aiv) active binding of fluorescently labeled platelet-targeted liposomes to a carotid artery injury site in vivo in a rat model (scale bar: 10 µm). (Av) Dual-targeted liposomes directed to active platelets (simultaneous targeting of GPIIb-IIIa and P-selectin), (Avi) fluorescence micrographs of the (Avia) dual targeted liposomes showing enhanced binding to activated platelets under flow in vitro compared with (Avib) only GPIIb‑IIIa- or (Avic) only P-selectin-targeted liposomes, with (Avid) albumin surface and (Avie) unmodified liposomes as control (scale bar: 50 µm), (Avii) which are also depicted via quantitative fluorescence intensity measurements. (Bi) Fabrication of von Willebrand factor-binding gelatin NPs loaded with tPA and (Bii) efficacy of enhanced TIMI with the tPA-loaded gelatin NPs along with ultrasound-triggered release (circles), compared with tPA alone (triangles) or tPA-plus-ultrasound (squares). (Ci) CREKA peptide‑modified micelles for targeting to fibrin-rich clot sites (scale bar: 200 µm) and (Cii) enhanced in vivo targeting of the micelles to a vascular clot site indicated by green fluorescence labeling of the micelles (scale bar: 20 µm). (Di) The design of a lipid–polymer complex NP surface decorated with peptides that target subendothelial matrix at the injury site and (Dii) efficacy of the targeted NP to target an Angio site in vivo. (E) Design of (Ei) PLGA-based NPs and (Eii) microscale conglomerate NPs loaded with thrombolytic drug, which can undergo shear-induced disintegration and drug release when injected into (Eiii) a vascular occlusion, resulting in (Eiv) the removal of occlusion. (E) Scale bar: 100 µm. *p < 0.05 vs tPA plus transthoracic ultrasound. **p < 0.01 vs tPA alone.

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Figure 5. Representative results of platelet-relevant and platelet-inspired therapeutic approaches in the diagnosis and treatment of thrombotic and restenotic events (cont.). Angio: Angioplastic injury site; NP: Nanoparticle; PLGA: Poly(lactic-co-glycolic acid); TIMI: Thrombolysis in myocardial infarction; tPA: Tissue-type plasminogen activator. (Ai–iv) Adapted with permission from [54] ; (Av–vii) adapted with permission from [55] ; (B) adapted with permission from [56] ; (C) adapted with permission from [47] ; (D) adapted with permission from [57] ; (E) adapted with permission from [58] .

significant reductions in airway hyperactivity [78]. NSAIDs, particularly aspirin, that inhibit platelet activation, have shown therapeutic benefit in inflammatory diseases such as RA [79,80]. P-selectin antagonists, such as quinolone salicylic acids, have shown therapeutic promise in rat models of arthritis [81]. Additionally, a recombinant PSGL-1 antibody reduced immune cell accumulation and joint severity in a mouse model of RA [82]. Similarly, a humanized monoclonal antibody to PSGL-1 is in development by Selexys Pharmaceuticals (OK, USA) for the treatment of IBD [201]. Although these therapies themselves are not ‘nanomedicine’ approaches, they demonstrate the efficacy of platelet-relevant therapies in inflammation. Hence, delivering these agents in an inflammation-targeted fashion packaged within platelet-inspired delivery vehicles may provide a method for better therapeutic efficacy. Regarding nanoparticle-based approaches for delivery of anti-inflammatory agents, Richards et al. demonstrated that liposomes loaded with the anti-inflammatory drug clodronate can be passively taken up by macrophages at the site of arthritic inflammation [83]. Similarly, passive Mac1 (αMβ2 or CD11b/CD18)

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uptake of PEGylated nanoparticles into inflamed synovium has been shown to increase drug accumulation due to enhanced vascular permeability in a rat RA model [84]. In a rat model of IBD, Lamprecht et al. demonstrated that drugs encapsulated in nanoparticles accumulated at the site of inflamed tissue better than a solution of free drug, as a result of enhanced vascular permeability [85]. Nanoparticle surface charge has also been shown to be an important factor, as negatively charged particles showed enhanced adherence to inflamed colonic mucosa in colitis-induced rats [86]. Beyond these studies of passive accumulation of nanoparticles, surface modification of particles with targeting ligands has been demonstrated to enhance the therapeutic effect. To this end, most targeting approaches have involved targeting the inflamed endothelium. For example, an RGD peptide that binds to avb3 on inflamed endothelial cells was attached to dexamethasone phosphate-loaded liposomes to demonstrate enhanced targeting and therapy in a rat model of arthritis [87]. Minaguchi et al. reported on the use of sialyl Lewis X-modified glycoliposomes for effective targeting of inflamed endothelium in a mouse arthritis

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Figure 7. Representative results of platelet-relevant therapeutic approaches in the treatment of inflammatory diseases such as arthritis and colitis. (A) Therapeutic effect of a P-selectin antagonist (rPSGL-Ig) compared with just saline in an arthritis model. (B) Anti-inflammatory drug FK506 (tacrolimus) loaded within polymeric nanoparticles exhibited higher therapeutic efficacy than free drug in an animal model of inflammatory bowel disease. (C) Enhanced binding of E-selectintargeted (a-E) leukocyte–endothelial cell adhesive particles on TNF-a-induced inflamed endothelium compared with normal endothelium in vivo in a mouse model. (D) Higher therapeutic efficacy (lower arthritis score) of dexamethasone phosphate-containing RGD-decorated liposomes (squares) compared with nontargeted drug-loaded liposomes (circles), RGD-decorated liposomes without drug (triangles) and buffer control (diamonds) in an adjuvant-induced arthritis model in rats. (E) Enhanced treatment effect (reduced macrophage index) of MLVc or SLVc liposomes in reducing inflammatory effect in a synovial joint arthritis model in rats. (F) Enhanced accumulation over time of selectin-targeting Sialyl Lewis X-modified liposomes in an arthritis model in mice, compared with nonarthritic (control) mice. For color image see online at www.futuremedicine.com/doi/full/10.2217/NNM.13.113. *p < 0.05 compared with FK506 solution; **p < 0.05 compared with FK506 + amio and FK506 + tro; ***p < 0.05 compared with the right two histogram bars; ****p < 0.001 compared with saline‑treated rats; *****p < 0.05. CAIA: Collagen antibody-induced arthritis; MLVc: Clondronate encapsulated multilamellar; SUVc: Clondronate encapsulated unilamellar.

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Figure 7. Representative results of platelet-relevant therapeutic approaches in the treatment of inflammatory diseases such as arthritis and colitis (cont.). (A) Adapted with permission from [81] ; (B) adapted with permission from [85]; (C) adapted with permission from [89]; (D) adapted with permission from [87]; (E) adapted with permission from [83]; (F) adapted with permission from [88] .

model [88]. Sakhalkar et al. reported on polylactic acid/PEG block copolymer nanoparticles that specifically target inflamed endothelium via surface decoration of antibodies against E-selectin, ICAM-1 and VCAM-1 [89]. Figure 7 shows selected examples of research regarding inflammationtargeted therapy. Although these approaches do not explicitly claim ‘platelet-inspired’ design, it is to be noted that the approaches use several ligand–receptor mechanisms that overlap and mediate the triad interactions between active platelets, stimulated endothelium and inflammatory cells. Therefore, platelet-inspired delivery vehicles can target multiple cellular participants in inflammation and provide unique methods for inflammation-localized drug delivery.

Platelets in immune responses Platelet involvement in immune response modulation It is now appreciated that many of the platelet interactions described in the context of hemostasis, thrombosis and inflammation also empower platelets to modulate innate and adaptive immune responses. Activated platelets can undergo multireceptor-mediated interactions with stimulated endothelial cells and various leukocytes (Figure 6), that are highly relevant in immune responses. In addition, platelet-released soluble factors also facilitate inflammatory immune responses. As representative examples of the immunomodulatory roles of platelets, there are two notable platelet-mediated events in the host’s response to organ transplants and microorganisms. In the initial phases of organ transplantation, when the organ is recovering from ischemia following explantation from the donor and reperfusion in the recipient, significant inflammation is driven by innate immune responses [90]. In addition, incompatible antigens coded by the MHC are the principal targets of adaptive immune responses elicited by genetically mismatched transplants [90]. In clinical studies, intravascular aggregates of activated platelets have been observed in transplanted organs by immunohistological staining at the time of reperfusion and later during rejection [91–93]. In renal ischemia-reperfusion injury, Ley et al. found that expression of P-selectin on platelets directed neutrophil infiltration into the kidneys [94]. Similarly, platelets have been implicated in the increase of perivascular leukocyte infiltration in transplants future science group

during lymphocyte- and antibody-mediated rejection [95,96]. RANTES, which is secreted by activated platelets, can instigate monocyte arrest and promote immune response on transplantassociated inflamed vasculature [65]. In transplant rejection, antibodies and lymphocytes can bind by antigen-specific receptors to allogeneic endothelial cells, and can subsequently initiate attachment of platelets through several mechanisms. For example, antibodies can activate the complement cascade to lyse endothelial cells and expose subendothelial collagen, which then leads to platelet adhesion and aggregation, resulting in graft infarction or microthrombi formation [97]. Studies have also shown that transplant-associated stimulated endothelial cells can exocytose vWF and P-selectin, which can then lead to platelet recruitment [98,99]. Using a model of skin allografts, Morrell et al. directly visualized the rolling and arrest of platelets on capillary endothelium that secreted vWF due to stimulation by antibodies reacting to MHC antigens [95,100]. Baldwin et al. found that antibodies to MHC antigens can cause platelet adhesion to capillary endothelium in renal allografts via P-selectin-mediated interactions [101]. In addition to P-selectin, platelet-derived CD40L (CD154) has also been found to induce lymphocytic infiltration and rejection of cardiac allografts in mice [102]. Serotonin, released by stimulated platelets, has also been reported to have an important mechanistic role in allograft rejection [103]. Platelets can also contribute to fibrosis that is associated with chronic graft failure, especially in lung and kidney transplants. A recent study regarding platelet-derived serotonin in systemic sclerosis suggests that platelets might contribute to fibrosis of chronic rejection via the induction of extracellular matrix synthesis through TGF-b-dependent pathways [104]. Platelets are also known to mediate immune responses to microorganisms [105,106]. For example, bacteria and other microorganisms have been reported to have the capability to stimulate platelet activation, resulting in the expression of Toll-like receptors, which allow the active platelets to directly bind and capture bacteria, kill them by thrombocidins and allow subsequent clearance by phagocytes [105,106]. Platelet-mediated recruitment of neutrophils and T cells provides further immune support against microorganisms. All of these observations suggest the prominent role of platelets in immune responses, whether beneficial www.futuremedicine.com

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Figure 8. Representative results of platelet adhesion- and active platelet-relevant biomolecule expression and platelet-associated treatment implications in immune mechanisms of transplant rejection. (A) Enhanced von Willebrand factor expression and staining on vascular endothelium of skin transplants in (Aii) antibody-injected mice versus (Ai) control mice. The arrow in (Aii) indicates von Willebrand factor staining and platelet aggregates. (Bi) Platelet‑mediated enhanced leukocyte recruitment (dark myeloperoxidase staining) at a skin graft site in antibody‑injected mice compared with antibody-injected, platelet-depleted mice. (C) Serial sections of kidney transplant in mice, exhibiting (Ci) high presence of leukocytes via H&E staining and (Cii) concomitant presence of platelets via P-selectin staining. (D) The presence of active platelets (brown von Willebrand factor staining) and leukocytes in a cardiac allograft inflamed section (bottom half) compared with the noninflamed section (top half). (E) Evidence of platelet recruitment and activation being an early mechanistic factor of graft rejection in a skin graft model in mice, indicating that plasma levels of PF4 (secreted from a-granules of activated platelets) are much higher over time in graft-bearing mice compared with mice with no grafts. (F) Platelet-depletion resulted in significantly higher graft survival in a skin graft model in mice. For color image see online at www.futuremedicine.com/doi/full/10.2217/NNM.13.113. *p < 0.03. H&E: Hematoxylin and eosin. (A) Adapted with permission from [95] ; (B) adapted with permission from [95] ; (C) adapted with permission from [90] ; (D) adapted with permission from [100] ; (E) adapted with permission from [96] ; (F) adapted with permission from [96] .

(e.g., protection against microorganisms) or harmful (e.g., organ transplant rejection). Therefore, platelet-inspired vehicles can potentially be used for immunomodulatory strategies. 1720


Platelet-inspired nanomedicine targeted to immune responses Depleting platelets or blocking platelet function with antibodies has demonstrated the potential of future science group


platelet-relevant therapy in experimental immunemediated inflammatory diseases. Figure 8 shows some recent examples of research establishing the importance of platelets in the immune response to transplants and demonstrating the effect of platelet depletion on transplant survival. Systemic depletion or blockage of platelet function has limited clinical potential because of adverse systemic side effects (e.g., hemorrhage). Instead, localized delivery of anti-inflammatory drugs or immunomodulatory agents can be achieved by nanovehicles that exploit platelet-mimetic binding mechanisms at the transplant site. To this end, antibodyconjugated nanoparticles have been explored in models of immune-mediated inflammation; for example, immunoliposomes conjugated with antiselectin antibodies to deliver dexamethasone in antibody-induced glomerulonephritis [107]. Selective binding to immunological mediators of inflammation, such as complements, may also offer additional targets for binding of plateletinspired vehicles [108]. Radiolabeled platelets have been used to diagnose early stages of transplant rejection in humans [109]. A recent review by Garraud et al. also emphasizes the potential use of transfused natural platelets as therapeutic immune cells [106]. Localized delivery of immunosuppressive drugs using platelet-inspired vehicles may make it possible to avoid many undesirable effects associated with systemic overimmunosuppression of these drugs, including cancer, infections and renal dysfunction. Therefore, plateletinspired drug delivery systems can usher in a new era of targeted immunomodulatory therapies in various applications.

Platelets in cancer metastasis Platelet involvement in the pathology of metastasis Tumor metastasis is the phenomenon of cancer spreading from a primary tumor site to secondary distant sites in the body, and is the main reason behind cancer-related morbidity and mortality. The metastatic process is highly complicated, involving cancer cell detachment from the primary tumor, epithelial-to-mesenchymal transformation, migratory intravasation within neighboring blood and lymph vessels, and hematologic transport via the circulatory system. This occurs while avoiding immune surveillance, arrest and adhesion at secondary sites, and results in extravasation from the circulatory system followed by angiogenetic progression of the metastatic microenvironment, and finally colonization and growth at the secondary sites [110]. There is a compelling experimental and clinical evidence that suggests future science group

Review

a significant role of platelets in these processes [111–113]. In 1865, Trousseau had made the observation that migratory thrombophlebitis could be an indicator of occult malignancy [114], which is now known as Trousseau’s syndrome. Similar observations were made by Billroth, who postulated that tumor platelet microemboli play a critical role in metastasis [115]. Gasic et al. demonstrated that platelet number influences cancer metastastatic potential [116]. Pinedo et al. put forward the role of a platelet-derived secretome in tumor angiogenesis, a major component of tumor metastasis [117]. The interactions between activated platelets and tumor cells have been reported as a functional component of metastasis by other research groups [118]. Modery-Pawlowski et al. have recently demonstrated that prometastatic breast cancer cell lines express high levels of platelet-interactive receptors [119]. The potential involvement of active platelets in metastasis is further corroborated by the reports that active platelets promote epithelial-to-mesenchymal transition in cancer cells [120], secrete proangiogenic cytokines (e.g., VEGF-A and PDGF) and proteases (e.g., MMP-2 and MMP‑9) to influence tumor angiogenesis, matrix degradation and cancer cell migration [121], activate and aggregate around circulating tumor cells to form a ‘platelet cloak’ that renders avoidance of immune surveillance in circulation [122], facilitate the attachment of circulating tumor cells onto the vascular wall at secondary sites [123], and mediate metastatic microenvironment development via various secreted molecules [118]. These possible mechanisms of platelet involvement are shown schematically in Figure 9. Therefore, platelet-inspired therapeutic approaches may provide unique pathways for metastasis-targeted detection and treatment. Platelet-inspired nanomedicine targeted to metastasis Several preclinical and clinical studies have been carried out regarding antiplatelet and anticoagulant agents for therapeutic effect in malignancy [112,124]. These studies are not essentially ‘nanomedicine’ strategies, but involve systemic administration of antiplatelet and anticoagulant agents such as dipyridamole (blocks ADP-induced platelet activation), aspirin (blocks thromboxane pathway of platelet activation), integrin GPIIb-IIIa-directed antibody (blocks platelet aggregation and leads to platelet fragmentation) and heparin (inactivates coagulation factors). Although these studies have shown some therapeutic benefit in metastatic malignancy, the results have been fraught with systemic risks of www.futuremedicine.com

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hemostatic dysregulation and hemorrhage [112]. In such cases, platelet-mimetic nanoconstructs that have enhanced binding ability to metastatic cells, can lead to technologies that allow metastasis-targeted drug delivery or can be used in diagnostic ex vivo cell capture methods for metastasis detection. The potential of this approach is demonstrated in selectin-coated or anti-CD34 antibody-coated microfluidic devices for preferential capture or killing of circulating tumor cells, and in nanoconstructs targeted to sites of upregulated avb3 expression as a surrogate indicator of metastasis-associated angiogenesis [125,126]. Modery-Pawlowski et al. have recently demonstrated the potential of actively targeting metastatic cells using the multireceptor-mediated interactions between active platelets and metastatic cells. In these studies, it has been established that the prometastatic breast cancer line MDA-MB-231 had enhanced expression of platelet-interactive receptors compared with the low-metastatic breast cancer line MCF-7, and subsequently demonstrated that liposomes heteromultivalently surface decorated with ligands, which can simultaneously bind to these multiple receptors, can target the prometastatic cells at a significantly enhanced level compared with the low-metastatic cells [119]. Figure 10 shows

some examples of platelet involvement in metastasis and the feasibility of some platelet-inspired nanotechnologies for metastasis-targeted diagnosis and therapy. We envisage that nanomedicine research on platelet mimicry can extend its clinical repertoire by customization of plateletmimetic constructs for metastasis-targeted delivery of imaging and therapeutic agents.

Conclusion We have provided mechanistic descriptions of the involvement of platelets in hemostasis, thrombosis, inflammation, immune response and metastasis, and have reviewed available experimental demonstrations, as well as provided perspectives on how such mechanisms are utilized to develop nanomedicine strategies targeted to these areas. Platelet-mediated interactions in these five scenarios have multiple overlapping ligand–receptor mechanisms, and deconvoluting the precise spatiotemporal distribution and role of these mechanisms can provide unique insights into disease-specific targeting strategies. Beyond the ligand–receptor-mediated interactions, the secretome from platelets, endothelium, macrophages and SMCs has critical roles in modulating various physiological and pathological mechanisms. Therefore, understanding the Active platelets

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Figure 9. Platelet involvement in hematologic pathways of cancer aggression and metastasis. Platelets have been reported to play important roles in facilitating cancer cell intravasation, protection of CTCs from immune surveillance, adhesion and extravasation of cancer cell at secondary and distal sites, and development of a metastatic microenvironment. CAM: Cell adhesion molecule; CTC: Circulating tumor cell; ECM: Extracellular matrix; Fg: Fibrinogen; vWF: von Willebrand factor.

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Figure 10. Representative results of involvement of platelets in metastasis and development of platelet-inspired approaches in metastasis-targeted diagnosis and therapy. (A) Treatment of Ep5 breast cancer cells with platelets induces an epithelial–mesenchymal transition-relevant invasive morphology, compared with when treated with just a buffer (scale bar: 50 µm). (B) There is a high correlation between active platelet number and number of metastases in a lung metastasis model in mice. (Ci) P-selectin-coated tubes were capable of arresting HL60 cancer cells under flow and (Cii) P-selectin nanoparticles were capable of delivering Cy3‑siRNA (red color) to the HL60 cells. (Di) Surfaces coated with active platelets were capable of enhanced attachment of metastatic MDA-MB-231 cells (scale bar: 50 µm), (Dii) calcein-stained activated platelets (green) were capable of enhanced binding to the MDA‑MB-231 cells (blue 4´,6-diamidino-2-phenylindole and orange–red phalloidin stain) in culture under flow (scale bar: 20 µm), and (Diii) platelet-inspired liposomes simultaneously targeting to b3 integrins and P-selectin were capable of binding to MDA-MB-231 cells to deliver a hydrophobic rhodamine (red fluorescence) payload (scale bar: 20 µm). (A) Adapted with permission from [120] ; (B) adapted with permission from [116] ; (C) adapted with permission from [127] ; (D) adapted with permission from [119] .

role of such a secretome can provide additional avenues of utilizing platelet-inspired delivery vehicles as depots for the controlled release of secreted molecules that aid or inhibit mechanistic pathways as needed. Such nanomedicine approaches should also utilize the role of platelets’ physical attributes, as well as biophysical parameters of platelets’ microenvironment in rendering platelet-mimetic functions.

Future perspective Platelet’s multifunctional mechanistic roles provide a novel paradigm for nanomedicine strategies targeted to various diseases. The design of such vehicles should integrate the biochemical interactions as well as the biophysical attributes of natural platelet function in physiological and pathological scenarios. Platelet-mimetic construct future science group

design should involve biomaterial systems that are biocompatible, can be produced in bulk with high quality and can be easily sterilized to ensure extended storage life. Platelets have unique abilities to respond to their biochemical and biophysical microenvironment. Biomaterials that allow such environmentally responsive properties may provide unique refinement to platelet-inspired construct designs. Since many of platelets’ biointeractive capabilities are mediated by multiple ligand–receptor processes acting in concert, mimicking this via suitable heteromultivalent ligand decorations of nanomedicine platforms may provide enhanced disease selectivity. Special attention should also be given to ensure that the constructs themselves do not induce pathologic thrombotic and inflammatory events. Furthermore, natural platelet functions are substantially www.futuremedicine.com

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regulated by a variety of molecules secreted from the platelets themselves, stimulated endothelium and leukocytes. Therefore, by leveraging drug delivery technologies in the platelet-inspired nanomedicine constructs, one can envisage mimicry or inhibition of these regulatory pathways as required. Utilizing a platelet-inspired nanomedicine paradigm based on physiologic and pathologic roles of platelets can lead to a multitude of targeted therapy applications in various diseases.

Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

Executive summary Platelets are principally involved in maintaining hemostasis, but they can also mediate pathologic conditions of thrombosis, inflammation, immune reaction and cancer metastasis. The physiologic and pathologic roles of platelets are rendered by multiple ligand–receptor interactions, for example, fibrinogen–GPIIb‑IIIa, PSGL1–P-selectin, von Willebrand factor–GPIba, collagen–GPVI, collagen–GPIa‑IIa and CD40L(CD154) –CD40. In addition to ligand–receptor-mediated surface interactions, platelets also produce a variety of cytokines, chemokines and growth factors in various physiologic and pathologic events. Exploiting platelets’ cell–cell and cell–matrix interactions via heteromultivalent modification of synthetic vehicles can provide a unique platelet-mimetic paradigm for nanomedicines targeted to various conditions where platelets play a critical mechanistic role. Development of platelet-inspired construct design should ensure that the constructs can be produced conveniently with consistent quality, the necessary heteromultivalent ligand modifications can be easily achieved, constructs can be sterilized easily for large-scale applications, constructs with a long storage life and portability, and do not cause unwanted systemic thrombotic and inflammatory incidents in vivo. Ongoing and future research regarding platelet-inspired nanomedicine platforms should leverage the heterotypic biochemical interactions of platelets, as well as the unique physical parameters (e.g., shape, size and modulus) and environmentally responsive properties (e.g., flow- or shear-sensitive properties) that influence the hemodynamic behavior and biointeractive functions of natural platelets.

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