|Year : 2014 | Volume
| Issue : 1 | Page : 1-5
Nanotechnology-based artificial platelets
Feroz Alam, Mohammed Naim, Suhail-ur Rahman, Mariam Shadan
Department of Pathology, J.N. Medical College, Aligarh Muslim University, Aligarh, Uttar Pradesh, India
|Date of Submission||25-Oct-2013|
|Date of Acceptance||21-Nov-2013|
|Date of Web Publication||29-Jan-2014|
Department of Pathology, J.N. Medical College, Aligarh Muslim University, Aligarh 202002, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Platelets are essential components of blood. Because of drawbacks in collection and storage of platelets, marked shortage is felt especially in the areas endemic for dengue fever and malaria. To meet the shortage, various synthetic and semisynthetic compounds are being produced and tested worldwide, which either enhance platelet function or are a substitute to it. In this study, we discuss these semiartificial and artificial substitutes of platelets along with other compounds that enhance the activity of platelets, with special emphasis on their mechanism of action and biofeasibility.
Keywords: artificial platelets, nanosheets, nanotechnology
|How to cite this article:|
Alam F, Naim M, Rahman Su, Shadan M. Nanotechnology-based artificial platelets. Egypt J Haematol 2014;39:1-5
| Introduction|| |
Platelets are small cell fragments (1-2 μm) originating from megakaryocytes in the bone marrow. There is a marked shortage of platelets especially in the areas endemic for dengue fever and malaria. Collection and storage of platelets have their own drawbacks , . The platelet membrane has integral glycoproteins (GPs) essential in the initial events of adhesion and aggregation, leading to formation of the platelet plug during hemostasis. Platelet GP receptors react with aggregating agents such as collagen on the damaged vascular endothelial surface, fibrinogen, and von Willebrand factor to facilitate platelet-platelet and platelet-endothelial cell adhesion. The major GPs are the Ib-IX complex, whose main binding protein is von Willebrand factor, and IIb/IIIa, which specifically binds fibrinogen. Storage organelles within the platelet include the 'dense' granules, which contain nucleotides, calcium, and serotonin, and α granules containing fibrinogen, von Willebrand factor, platelet-derived growth factor, and many other clotting factors. Following adhesion, the platelets are stimulated to release the contents of their granules essential for platelet aggregation. The GPIIb/IIIa complex is a heterodimeric receptor of the integrin family and it is present on the membrane of platelets. Upon activation by an agonist, that is adenosine diphosphate (ADP) or thrombin receptor-associated protein, GPIIb/IIIa receptors stored in internal pools move to the platelet surface  . Furthermore, a conformational change of the GPIIb/IIIa receptor (both the newly arrived and the receptors already present) occurs, making the receptor accessible for interaction  . The GPIIb/IIIa receptor interacts with at least three adhesive proteins: fibronectin, vWF, and fibrinogen. These interactions are critical for platelet adhesion, spreading, and aggregation  . The platelets also provide an extensive phospholipid surface for the interaction and activation of clotting factors in the coagulation pathway. This aggregate formed in the primary hemostasis is a hemostatic plug that seals the vessel wall, and then secondary hemostasis can take place.
| Fibrinogen and its segments|| |
The first (semi)artificial platelets made were coated with fibrinogen; hence, platelets were able to bind them with their GPIIb/IIIa receptor and the substitutes can enhance platelet aggregation. The use of complete fibrinogen molecule to coat platelet substitutes has some disadvantages. The fibrinogen purification process is complicated because fibrinogen from human blood is not stable, and its activity in solution is extremely low. It also raises the potential for transmitting infectious diseases  .
The H12 fibrinogen sequence is a dodecapeptide 'HHLGGAKQAGDV-H12' present on the fibrinogen γ-chain carboxy-terminal (γ 400-411) and exists only in the fibrinogen domain. Hence, it is a highly specific fibrinogen marker binding to the platelet receptor GPIIb/IIIa. One more peptide sequence, the RGD (arginine-glycine-aspartine) sequence, also binds with the GPIIb/IIIa platelet receptor, but the RGD sequence is the cell attachment site of a large number of other adhesive extracellular matrix, blood, and cell surface proteins, and nearly half of the over 20 known integrins recognize this sequence in their adhesion protein ligands  . Okamura et al.  coated their platelet substitutes for the first time with the H12 fibrinogen sequence because the H12 sequence interacts very specifically with the GPIIb/IIIa receptor on the platelets. As H12 binds specifically and only to GPIIb/IIIa, this sequence was preferred over the RGD sequence.
Wong and Chang cross-linked fibrinogen to polymerized hemoglobin, creating a product with both platelet-like properties and oxygen-carrying abilities. Polyhemoglobin has been tested in clinical settings and in trauma situation where large amounts are infused; hemodilution can occur, necessitating the administration of platelets and/or clotting factors. Tested in a hemodiluted setting, polyhemoglobin-fibrinogen blood clotted faster, and clots adhered better than blood containing polyhemoglobin alone  . Fibrinogen could also be conjugated to other hemoglobin-based oxygen carriers to yield other oxygen-carrying platelet substitutes.
| Coated erythrocytes|| |
Coller  discovered that inert beads coated with fibrinogen cause platelets in rest to agglutinate spontaneously; this interaction, however, needed to be enhanced by the addition of an agonist ADP. Later in 1983, it was found that formaldehyde-fixed platelets with fibrinogen covalently bound on their surface also cause the platelets to agglutinate  . These findings started a series of experiments with a wide range of platelet substitutes, starting with coated erythrocytes. Agam and Livne were the first to report fibrinogen-coated erythrocytes; they covalently cross-linked fibrinogen molecules to the red cells membrane by incubation with formaldehyde. These erythrocytes had the capability to enhance agonist-induced platelet aggregation in vitro. The platelet-dependent aggregation induced by ADP or thrombin was proportional to the fibrinogen density on the red cell membrane; the fibrinogen density on the erythrocytes could vary between 58 and 1400 molecules per cell  . Coller et al.  produced arginine-glycine-aspartine (RGD)-coated erythrocytes; these platelet substitutes were called thromboerythrocytes because these are the erythrocytes that gained a thrombocytic function. These thromboerythrocytes bind to the platelet with their conjugated RGD sequence.
| Coated albumin microcapsules/microspheres|| |
Yen et al.  produced fibrinogen-coated albumin microspheres (FAM) that were shown to be hemostatically active in thrombocytopenic rabbits, these were named thrombospheres. Later, Levi and colleagues produced fibrinogen-coated albumin microcapsules called synthocytes. The administration of synthocytes in rabbits that were made thrombocytopenic (either by antiplatelet antibodies or chemotherapy) resulted in a significant reduction in enhanced bleeding. The enhancement of primary hemostasis seemed to be because of the facilitation of adhesion of remaining platelets in circulation by the synthocytes. The mechanism of action was discovered by a perfusion experiment in which the fibrinogen-coated microcapsules were added to human whole blood and perfused over an endothelial matrix. Scanning electron microscopic images showed aggregates formed on the endothelial matrix, composed of microcapsules, platelets, and connecting fibrin fibers  . Both the products, FAMs and synthocytes, were nonthrombogenic, and they acted by facilitating adhesion of the remaining platelets to the endothelium. A major disadvantage of FAMs and synthocytes was that they relied essentially on the presence of natural platelets.
Takeoka and colleagues started with a novel platelet substitute consisting of latex beads with a diameter of 1 μm. Human serum albumin was adsorbed onto the surface of the latex beads and then either the H12 sequence or the RGD peptide was conjugated on the surface by a disulfide link. They concluded that the H12-conjugated latex beads preferentially interacted with an activated platelet surface through GPIIb/IIIa receptors and facilitated platelet accumulation at the sites of hemostasis. The adhesion of H12 latex beads was suppressed in the presence of free H12 as an inhibitor of GPIIb/IIIa binding, showing that the adhesion was specific. The RGD-coated beads caused agglutination of nonactivated platelets, which makes it a nonusable product  . Agglutination of nonactivated platelets would lead to nonselective enhanced thrombosis in the human body.
These studies concluded that particles coated with H12 sequence could be suitable as an alternative to human platelets. Okamura and colleagues in 2005 produced biocompatible and biodegradable particles by conjugating the H12 sequence to polymerized albumin particles (polyAlb). H12-polyAlb was shown to specifically interact with an activated platelet surface through GPIIb/IIIa receptors and facilitate platelet accumulation at the sites of hemostasis  . The problem with H12-polyAlb was its short half-life in circulation (~10 min). Polyethylene glycol (PEG) was attached to the H12-conjugated polyAlb forming an H12-PEG-polyAlb complex. This complex not only maintained the property of specific binding with activated platelets only, but also increased its half-life to about 6 h  . The mechanism of action of the H12-PEG-polyAlb complex is that platelets flowing in the circulation adhere to the collagen that is exposed at the site of the vascular injury. The platelets are then activated and bind to the H12-PEG-polyAlb particles present in the circulation, and the activated platelet-bound H12-PEG-polyAlb provides a lot of H12 sequences, which are available for binding with other platelets. H12-PEG-polyAlb particles thereby promote thrombus formation by accelerating and enhancing the aggregation of the flowing platelets. H12-PEG-polyAlb particles even contribute for the thrombus by their own mass effect, and hence can also work in patients with severe thrombocytopenia  .
| Coated liposomes|| |
Nishiya et al.  developed several liposome-based platelet substitutes, bearing recombinant fragments of GPIa/IIa (rGPIa/IIa) and Ib α (rGPIb α). The first study with significant hemostatic effects was conducted by Nishiya in 2004 in which liposomes with H12 conjugated on their surface were prepared. These liposomes interacted with activated platelets by binding of the fibrinogen H12 sequence to the GPIIb/IIIa platelet receptor. Triggered by this interaction, the liposome released encapsulated material that was within them. The rate of content release was dose dependent, depending upon the surface density of H12 on the liposome. In addition, other modes of interaction between the liposome and platelets may occur, such as internalization of the liposomes by the platelets upon interaction. When the liposomes are conjugated with octa-arginine instead of H12, the liposomes are internalized, thereby releasing their contents inside the activated platelets  .
Use of liposome as artificial platelets has the advantage of strengthening the hemostatic ability by utilization of their well-known targeted drug delivery function. Okamura and colleagues encapsulated potent platelet agonist, ADP, as a drug to be carried by the liposomes. ADP is normally stored in dense platelet granules and released after cellular activation; it then functions to reinforce or maintain platelet aggregation. They formed a H12-ADP-liposome complex  and confirmed that H12-conjugated liposomes bind specifically to GPIIb/IIIa of activated platelets. Okamura et al.  visualized the specific accumulation of the liposomes at the sites of vascular injury by encapsulating the contrast dye iopamidol into the H12-conjugated liposomes (H12-iopamidol-liposome complex), which provided the first visual evidence that H12-conjugated particles specifically interact with activated platelets.
In another study in 2010, Okamura and colleagues created different kind of liposome vesicles, all bearing the H12 sequence and containing ADP (H12-ADP vesicles). These vesicles varied in membrane flexibility and lamellarity, which regulated the release of the liposomal content. They claimed to have obtained a recipe to regulate the hemostatic ability of their vesicles by controlling the ADP release with the aid of different membrane properties. They succeeded by correcting the bleeding time in severely thrombocytopenic rabbits and rats and even by varying the hemostatic ability. The mechanism of the ADP release and its regulation depends on the fact that vesicles incorporated in the platelet aggregates are strongly bound to neighboring platelets and are subjected to physical forces, which pull on the liposome and continuously change its shape. This deformability depends on the surface flexibilities and lamellarities, which are in turn dependent on the composition of the liposomal membrane. The deformability (including a possible disruption) is correlated with the amount of ADP released from the liposome vesicle. Less flexibility and higher lamellarity will release more ADP  . In short, liposome-based substitutes are able to selectively bind activated platelets at the site of vascular injury, and liposomes can also encapsulate agonist agents that can be delivered to the sites of vascular injury leading to an enhanced hemostatic effect.
| Nanosheets|| |
Okamura and colleagues in 2010 prepared a new artificial platelet substitute, disk-shaped biodegradable nanosheets. These nanosheets were made of biodegradable poly(d, l-lactide-co-glycolide) (PLGA). These PLGA nanosheets were coated with the H12 sequence on their surface along with spherical microparticles made of PLGA, which were coated with the same density of H12 on their surface; both these nanosheets and spherical microparticles had an exactly equal volume. A flow experiment was conducted in which thrombocytopenic blood flowed over a collagen surface with addition of either the nanosheets or spherical microparticles (both conjugated with a fluorescent marker). This experiment showed that the nanosheets adhered to the collagen surface at twice the rate of the spherical microparticles and concluded that ellipsoidal nanosheets adhered more effectively than classical spherical microparticles of same volume. Because of the larger contact area of the ellipsoidal nanosheets, there were more binding sites available, supporting and increasing the adhesive strength  . This experiment confirmed earlier findings, which showed that the adhesive strength was significantly increased by increasing the aspect ratio of the minor and major axes of the particle from 1 to 10  . Hence, the greatest advantage in using nanosheets is having a large contact area for the target site.
Furthermore, by changing the aspect ratio, the nanosheets tend to align in the more near-wall region of the blood vessel  . Flowing near the vessel wall makes it more likely for the nanosheets to bind at the site of injury. Okamura et al.  also produced rectangular nanosheets with a very high aspect ratio of 60, which did not adhere to the collagen surface. These nanosheets did not flow in the near-wall region because of interference with the erythrocytes present in the blood. These experiments showed that the adhesive rate of the carriers on a collagen surface can be controlled by the change in their shape and that the best adherence is obtained with ellipsoidal nanosheets with a medium aspect ratio, as these will flow in the near-wall region of the vessel. The nanosheets also induced a two-dimensional spreading of platelet thrombi, which was in contrast with spherical microparticles three-dimensional piling  . The spreading of the thrombi might be because of the ultrathin structure of the nanosheets, as platelets bind to either side of the sheet, with a broad scaffold. Three-dimensional piled up thrombi may lead to vessel occlusion and can have severe consequences. Therefore, disk-shaped biodegradable nanosheets can be a useful artificial platelet substitute.
Synthetic platelets by Bertram et al. were synthesized by binding the polypeptide poly(lactic-co-glycolic acid)-poly-l-lysine to PEG arms, terminated with RGD functionalities to form a nanoparticle of ~170 nm in core diameter  . These synthetic platelets are administered intravenously; they work by adhering to activated platelets and increasing the number of platelets at the site of injury. The polyester core of the nanoparticle induces clot formation by activating the coagulation cascade  . It is thought that the surface charge of the particles and their aggregation in water is the mechanism by which they activate platelets and induce thrombi formation. Synthetic platelets are composed of the same polymer as used in dissolvable sutures, surrounded by a water soluble polymer; the molecules surrounding these polymers help the synthetic platelets bind to the activated platelets only, avoiding uncontrollable/unwanted clots that could lead to embolisms, making them theoretically safe for use in the human body  .
Clottocytes are a theoretical design by Robert A. Freitas Jr, which are artificial, mechanical nanobots to simulate platelets, able to achieve complete hemostasis in 1 s. The nanobots - powered by serum oxyglucose - would contain a fiber mesh able to unfold near the site of injury and adhere to the blood vessel. The clottocytes would be ~2 μm in diameter, roughly the same size as the body's own platelets. The mesh itself would be biodegradable, and, upon release, a soluble film coating the appropriate parts of the mesh would dissolve in contact with the plasma to expose sticky mesh. This 'stickiness' would be blood group specific to trap the antigen carrying the red blood cells. Each mesh would overlap with a neighboring mesh and attract enough blood cells to immediately stop bleeding, requiring no additional or increased concentration of cells  . These clottocytes may be considered as a part of the future-induced hemostasis. Their size relative to natural platelets means that they can operate at the cellular level with other blood components and move safely around the vascular system without blocking any vessel. As already mentioned, clottocytes could induce complete hemostasis in ~1 s, as this is the amount of time it has been estimated for the clottocyte mesh to unfurl and adhere to the damaged vessel wall. Because of the predicted density of the clottocytes, each nanobot would be no more than 370 μm away from each other; hence, if the vessel wall is severely damaged, enough devices could work together, each mesh overlaying with its neighbor to ensure that the bleeding is stopped  .
| Vasculoid|| |
Vasculoid is a complex idea originated because of the ability of nanotechnology to convert the dreams of yesterday into today's reality. The idea of the vasculoid originated in the minds of Freitas and Phoenix  ; they desired a complex artificial robot that was expected to perform all the functions of natural human blood, with the same immaticulate articulation and precision.
| Infusible platelet membranes|| |
Platelet microparticles are microvesicles of the platelet membranes, which are formed spontaneously during storage. The microparticles have similar hemostatic properties as the intact platelets. They can be produced from outdated platelets, with a shelf life of only 5 days; a considerable amount of outdated platelets are available. These microparticles are produced by lysing the platelets (by freeze-thawing), heating the product to inactivate the viruses, and lyophilization  . The product has a shelf life of over 2 years when stored at 4°C. Phase I and II trials of infusible platelet membranes have been conducted, and the product has been shown to be safe and effective so far.
| Conclusion|| |
Platelets are an essential component of blood but face scarcity in amount and drawbacks in the storage procedures. Hence, production of alternatives to meet the crisis is necessary now. Nanotechnology has rejuvenated the idea of an artificial platelet substitute. The approach is being used to develop artificial platelets and/or to increase the activity of the pre-existing ones. In future, it is possible that a complex system can be developed, which can replace the platelets and obliviate all the scarcity in terms of number and shelf life.
| Acknowledgements|| |
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