bio-inspired nanomedicine strategies for synthetic blood ... · and blood donation was promoted as...

Advanced Review

Bio-inspired nanomedicine strategiesfor artificial blood componentsAnirban Sen Gupta*

Blood is a fluid connective tissue where living cells are suspended in noncellularliquid matrix. The cellular components of blood render gas exchange (RBCs),immune surveillance (WBCs) and hemostatic responses (platelets), and the non-cellular components (salts, proteins, etc.) provide nutrition to various tissues inthe body. Dysfunction and deficiencies in these blood components can lead tosignificant tissue morbidity and mortality. Consequently, transfusion of wholeblood or its components is a clinical mainstay in the management of trauma, sur-gery, myelosuppression, and congenital blood disorders. However, donor-derived blood products suffer from issues of shortage in supply, need for typematching, high risks of pathogenic contamination, limited portability and shelf-life, and a variety of side-effects. While robust research is being directed toresolve these issues, a parallel clinical interest has developed toward bioengi-neering of synthetic blood substitutes that can provide blood’s functions whilecircumventing the above problems. Nanotechnology has provided excitingapproaches to achieve this, using materials engineering strategies to create syn-thetic and semi-synthetic RBC substitutes for enabling oxygen transport, plateletsubstitutes for enabling hemostasis, and WBC substitutes for enabling cell-specific immune response. Some of these approaches have further extended theapplication of blood cell-inspired synthetic and semi-synthetic constructs for tar-geted drug delivery and nanomedicine. The current study provides a compre-hensive review of the various nanotechnology approaches to design syntheticblood cells, along with a critical discussion of successes and challenges of thecurrent state-of-art in this field. © 2017 Wiley Periodicals, Inc.

How to cite this article:WIREs Nanomed Nanobiotechnol 2017, 9:e1464. doi: 10.1002/wnan.1464

INTRODUCTION

The term ‘bio-inspired’ refers to mimicry or adap-tation of biological structures and properties in

designing technologies that can simulate or leveragethe function of the original biological entity. The fieldof nanotechnology and nanoengineering has pro-vided exciting opportunities to achieve this at the

atomic, molecular, and macromolecular scales, inboth medical and nonmedical areas. In the medicalfield, the impact of ‘bio-inspired design’ is realized inthe areas of cellular engineering, tissue engineering,regenerative medicine, drug delivery and nanomedi-cine, and a variety of materials engineering to simu-late extracellular matrix components.1–3 A uniquearea of bio-inspired design that has generated signifi-cant academic and clinical research interest duringpast 30 years is that of bio-inspired nanoscale andmicroscale engineering of synthetic (or semi-syn-thetic) blood cells using a variety of biomaterials-based systems. The central driving force for thisresearch is to develop technologies that can simulatethe structure and/or property of blood cells as well asplasma, to allow utilization of these technologies as‘blood substitute’ when donor-derived blood is either

*Correspondence to: [email protected]

Department of Biomedical Engineering, Case Western Reserve Uni-versity, Cleveland, OH, USA

Conflict of interest: The authors have declared no conflicts of inter-est for this article.

A. Sen Gupta is an inventor on patents US9107845 andUS9107963 in the area of ‘Synthetic Platelets’ and ‘Heteromultiva-lent Nanoparticle Compositions’.

Volume 9, November/December 2017 © 2017 Wiley Per iodica ls , Inc. 1 of 33

not readily available or poses significant contamina-tion risks.

Blood is a fluid connective tissue where livingcells are suspended in noncellular liquid matrix. Thecellular components of blood render gas exchange(RBCs), immune surveillance (WBCs) and hemostaticresponses (platelets), and the noncellular components(salts, proteins, etc.) provide nutrition to various tis-sues in the body. Figure 1(a) shows a schematic rep-resentation of blood circulatory path in the body,Figure 1(b) shows a representative histologic stain ofblood smear depicting RBC, WBC, and platelet, andFigure 1(c)–(e) shows cartoon depictions and repre-sentative scanning electron microscopy (SEM) images

of the three blood cell types. The research focus onpreserving and transporting donor-derived bloodstarted during World War I to treat wounded sol-diers, and by World War II blood transfusionsbecame widely available. During the 1950s, multipleblood banks were established in the United Statesand blood donation was promoted as a form of civicresponsibility. Subsequent development of many pro-cesses, e.g., freezing of RBCs, isolation of specificblood components via centrifugation, apheresis, andso on, have enhanced the utilization of whole bloodand its components. Currently, whole blood as wellas isolated components are clinically approved fortransfusion medicine applications in civilian and

Superior

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FIGURE 1 | (a) Arterial and venous circulation pathways in the body that carries RBCs, WBCs, and platelets for respective biological functions;(b) representative histology stain of blood smear showing RBC, WBC, and platelet; (c) schematic cartoon and representative SEM image of RBC;(d) schematic cartoon and representative SEM image of WBC; and (e) schematic cartoon and representative SEM images of resting and activeplatelets.

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battlefield trauma (e.g., in damage control resuscita-tion), surgical settings (e.g., transplants), chronic andacute anemia, and disease-associated, drug-induced,or congenital bleeding disorders.4–8 However,according to the Red Cross, only about 38% of USpopulation is eligible to donate blood at any giventime and only about 10% actually donate. In addi-tion, blood-based products have somewhat limitedshelf-life due to risks of pathogenic contamination.Also, RBCs and platelets upon storage develop stor-age lesions, which affect their stability, posttransfu-sion circulation lifetime, and relevantbioactivities.9,10 Currently, RBCs have a shelf-life of20–40 days, while platelet suspensions have a shelf-life of 3–5 days, at room temperature.11–14 Signifi-cant preclinical and clinical research has been direc-ted toward enhancing the shelf-life of blood productsby cold storage, freezing, lyophilization, and so forth,as well as, through development of pathogen reduc-tion technologies like psoralen-based or riboflavin-based UV irradiation, extensive serological testing ofdonor blood, leukoreduction, and specialized storageprotocols.15–25 These approaches have indeed par-tially improved the shelf-life and safety of blood pro-ducts, and even that partial improvement can lead tosignificant benefit in transfusion medicine. Blood pro-ducts may also present risks of refractoriness, graft-versus-host disease, transfusion-associated immuno-suppression, and acute lung injury. Also, portabilityof blood products, especially to remote civilian andbattlefield locations, continues to be a logistical chal-lenge. Altogether these various reasons continue toaffect widespread use of blood products.25,26 Thesechallenges can be potentially avoided by utilizingnanoscale and microscale engineering ofbiomaterials-based synthetic (and semi-synthetic)blood substitutes.27–34 In fact, the interest in syn-thetic blood substitutes developed during the HIVcrisis of the 1980s due to fear of contaminated bloodproducts.4 That, combined with issues of limited sup-ply, difficulty in storage and portability, limited shelf-life, high cost, and so on, have been driving theresearch on synthetic blood substitutes for past sev-eral decades, and several artificial blood productshave undergone preclinical and clinical stage studies.However, currently no product is clinically approvedby the FDA for human applications in the UnitedStates. A 2008 meta-analysis of 16 clinical trials offive different artificial blood products suggestedincreased health risks in patients treated with suchproducts, especially for RBC substitutes.35 Althoughsuch analyses have resulted in some apprehension inclinical utility of these products, it has also directedsignificant emphasis on understanding and resolving

the problems posed by these products at fundamentalmechanistic and physiological levels. The purpose ofthe current study is to provide a comprehensive reviewof current state-of-art in blood substitute technologies(Box 1), emphasizing the role of bio-inspired

BOX 1

ARTIFICIAL BLOOD COMPONENTS

Integration of biomaterials engineering, bloodbiology, and transfusion medicine areas has ledto development of bio-inspired technologiesthat simulate the functions of RBCs, platelets,WBCs, and plasma. For synthetic RBC substi-tutes, the most studied designs are HBOCswhere Hb molecules are directly polymerized,cross-linked, covalently modified with stability-enhancing polymers, or encapsulated withinmicro- or nanoparticles. As the O2-transportingmechanisms of Hb require enzyme-mediatedredox environment, newer HBOC designs havecoencapsulated suitable enzymes and redoxagents with Hb. Other promising approachesfor oxygen carriers include PFC emulsions andsynthetic porphyrin systems. For synthetic plate-let substitutes, the most studied designs areessentially ‘super-fibrinogen’ systems wheremicro- or nanoparticles are surface-coated withfibrinogen, fibrinogen-relevant peptides orfibrin-specific motifs to amplify the fibrinogen-mediated ‘aggregation’ mechanism of activeplatelets. Some approaches have also focusedon mimicking the ‘adhesion’ mechanisms of pla-telets by modifying nanoparticle surfaces withrecombinant GPIbα or peptides to bind vWFand recombinant GPIa-IIa to bind collagen.Newer designs have focused on combining theplatelet-inspired ‘aggregation’ and ‘adhesion’mechanisms via heteromultivalent modificationof nanoparticles with vWF-binding, collagen-binding and GPIIb-IIIa-binding peptides. Otherinteresting approaches include surface-decoration of nanoparticles with polypho-sphate (PolyP) moieties to modulate coagula-tion mechanisms and biointerfacing ofpolymeric nanoparticles with blood cell-derivedmembrane to impart specific bioactivities.Recent studies have also focused on incorporat-ing RBC and platelet-inspired shape, size, andmechanical flexibility aspects in the design ofsynthetic RBCs and platelets. As for plasma sub-stitutes, several crystalloid and colloid com-pounds are currently clinically approved.

WIREs Nanomedicine and Nanobiotechnology Bio-inspired artificial blood nanotechnology


nanomedicine approaches in their design, and criticallydiscussing the successes and potential challenges inthis area.

RBC SUBSTITUTES AND OXYGENCARRIER SYSTEMS

In blood, the primary function of red blood cells(RBCs) is the transport of oxygen and carbon diox-ide to and from tissues, by virtue of binding of thegases to hemoglobin (Hb) within the RBCs. Theaverage amount of Hb in adult human RBCs (alsoknown as mean corpuscular Hb or MCH) is 27–31picograms per cell (~250 million Hb molecules). Hbis a tetrameric protein of two α- and twoβ-polypeptide chains, each bound to an iron-containing heme group capable of binding one oxy-gen molecule. Figure 2(a) shows a multiscale sche-matic view of RBC, Hb inside RBC, and chemicalstructure of iron-containing ‘heme’ group insideHb. The binding of oxygen to Hb is positively coop-erative, such that a small change in oxygen partialpressure (pO2) can result in a large change in oxygenbound or released by Hb, resulting in a sigmoidalshape of the binding curve (Figure 2(b)).36 Theoxygen-carrying iron in Hb is in its reduced ‘ferrous’(Fe2+) state, which when oxidized to form methemo-globin (MetHb) with iron in the oxidized ‘ferric’ state

(Fe3+), is unable to bind oxygen.37,38 Due to this rea-son, in natural RBCs the oxygen transport mechan-ism of Hb is coupled to redox cycles (e.g., driven byenzyme NAD-cytochrome b5 reductase), such thatthe Fe(II)-containing Hb can be maintained in itsoxygen-binding state. Irreversible conversion of Hbto MetHb not only inhibits its oxygen-carryingcapacity, but also leads to dysregulated vascular toneand inflammatory reactions. Furthermore, Hb inRBCs has the capability to undergo conformationalchanges to allow saturation loading with O2 in thelungs (higher O2 affinity) and then off-load O2 in thetissue capillaries (lower O2 affinity). Such reversibleconformational control of Hb and its O2 affinity areaided by allosteric effector molecules like 2,3-diphosphoglycerate (2,3-DPG) formed inside RBCsas a glycolytic intermediate. Therefore, engineeringof RBC-inspired synthetic substitute poses a formida-ble challenge with regards to maintaining suchoxygen-carrying thermodynamic and kinetic charac-teristics of Hb, maintaining the redox environmentand minimizing irreversible MetHb formation.39 Thethree main types of RBC-inspired oxygen carrier sys-tems that have been designed using nanotechnologyapproaches and have undergone extensive preclinicaland some clinical research so far are, Hb-based oxy-gen carriers (HBOCs), perfluorocarbon (PFC)-basedemulsions, and iron (Fe2+)-containing porphyrins.

HBOC SystemsHBOCs are essentially semi-synthetic systems as theyutilize natural Hb as the oxygen-carrying component,either in chemically modified cell-free suspensionsor conjugated and cross-linked with polymers alongwith protective enzymes, or encapsulated withinbiomaterials-based nanocarrier and microcarrier vehi-cles.29,40 The Hb used in these systems is usuallyderived from outdated human or bovine RBCs or fromrecombinant technologies.40–46 When using outdatedhuman or bovine RBCs, the Hb is isolated via cell lysis,purified by sterile filtration and chromatographictechniques and sterilized (e.g., by low heat).47 Theoxygen-binding equilibrium kinetics of Hb is positivelycooperative in nature, where the Hb binds the first O2

slowly, which in turn increases the binding affinity forthe second, third, and fourth O2 molecule to Hb,resulting in the classic sigmoidal O2 equilibrium curve(OEC). Using such cell-free Hb suspensions presentsthe advantage of minimum antigenicity and the abilityto transport oxygen in plasma more efficiently becauseof the lack of interference by cell membrane. In fact, inthe early 20th century, suspension of cell-free Hb inlactated Ringer’s solution was used to intravenously

RBC(7-10 μ in diameter)

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FIGURE 2 | (a) Multiscale representation of RBC, the hemoglobin(Hb) protein structure inside RBC, and the ‘Heme’ porphyrin structureinside Hb; and (b) representative oxygen binding curve for Hb showingthe cooperative binding nature (sigmoidal curve).



treat fifteen patients, however, a large number of themwere reported to develop renal toxicity and cardiovas-cular complications.47 Similar results were also foundin the 1950s when US Navy treated several patientswith cell-free Hb.48 Cell-free Hb was also found tohave a very short circulation residence time because incell-free environment the Hb tetramer rapidly dissoci-ates into dimeric and monomeric forms that can bindto plasma immunoglobulins, and can undergo rapidclearance by the reticulo-endothelial system (RES) intospleen and liver, as well as renal clearance into kidneys,leading to Hb-based toxicities in these organs.49,50

Additionally, cell-free Hb and its dissociated deriva-tives can also extravasate into subendothelial regionand rapidly sequester the natural vasodilator nitricoxide (NO), resulting in conversion of NO into nitrate(dioxygenation reaction) and oxy-Hb to Met-Hb.51

This NO-scavenging results in vasoconstriction andcardiovascular complications. Furthermore, theabsence of the allosteric effector molecule 2,3-DPG incell-free Hb can lead to unnaturally high oxygen affin-ity, making oxygen off-loading problematic. Cell-freeHb can also change blood osmolarity, leading to alter-ation of blood volumes and associated side-effects.Altogether, these reasons have resulted in cell-freehuman Hb being deemed problematic for oxygen-carrying applications. Instead of human Hb, studieshave also been conducted with bovine Hb, but this alsopresents similar issues of stability, extravasation, NO-scavenging and renal clearance issues. One interestingway to address many of these issues is by developmentof recombinant Hb (e.g., in E. Coli) where specificmutations can enable decrease in dissociation andreduction in NO-binding capacities, but the correctcombination of mutations that can lead to an ideal Hbdesign is still not completely known.52–54 Recombinanttechnologies are also substantially expensive. There-fore, a significant volume of research has been focusedon molecular stabilization and functional modulationof Hb utilizing chemical modifications like cross-link-ing, polymerization, and macromeric surface conjuga-tions. The goals of these modifications are to reduceHb dissociation, extravasation and renal clearance,while maintaining reasonable circulation life-time andoxygen loading/off-loading capacities.

Chemically Modified HBOCsChemical cross-links in Hb can be created both intra-and intermolecularly. For example, Hb can be cross-linked intramolecularly between its two α-subunitsusing acylation with bis-(3,5 dibromosalicyl)-fumarate(also known as Diaspirin) and using this strategy onhuman Hb led to a product called HemAssist fromBaxter (Deerfield, Illinois, USA).40,55,56 This product

showed an increase in circulation time up to 12 hcompared to <6 h for unmodified Hb, but the cross-linked Hb unfortunately showed a 72% increase inmortality rates in human patients compared to saline,and clinical trials were discontinued.57 An analogouscross-linking approach between the α-subunits ofrecombinant Hb using Glycine as the cross-linkingagent led to a product called Optro from Somatogen(Boulder, Colorado, USA), which also resulted in risksof cardiac arrest and mortality.58–60 Instead of site-specific intramolecular cross-linking only, polymerizedHb was created using bifunctional cross-linkingreagents like glutaraldehyde (e.g., the products Hemo-pure from Biopure (Cambridge, Massachusetts, USA)and PolyHeme from Northfield Labs (Evanston, Illi-nois, USA) and o-raffinose (e.g., the product Hemo-Link from Hemosol (Toronto, Ontario, Canada)).61,62

One challenge in these approaches is to precisely con-trol polymer molecular weight and rigorous purifica-tion steps are necessary to ensure product quality.PolyHeme was reported to progress into Phase III clin-ical trials in the US in treating trauma-associatedblood loss and showed a decreased need of naturalblood transfusions.60 Clinical trials with HemoPurealso showed a reduced need of additional blood trans-fusions in cardiac surgery.63 Although HemoPure hasnot been approved by the FDA in the US, it has beenreported to have clinical approval in South Africa foracutely anemic patients. HemoLink also advanced tophase III clinical trials but was discontinued in 2003when patients receiving treatment experienced adversecardiac events. In fact, all of these products in theirclinical trials have shown high risks of transient hyper-tension, organ damage through micro- vascular con-striction and dysfunction, gastro-intestinal distress,nephrotoxicity, neurotoxicity, and increased mortal-ity.64,65 Instead of intramolecular cross-linking andintermolecular polymerization, modification of Hbhas also been carried out with macromeric bioconju-gation to increase stability and vascular residence timewhile reducing immune recognition.66–68 Importantexamples of this approach are found in polyethyleneglycol (PEG) modification of Hb (e.g., the productsHemospan from Sangart Inc. (San Diego, California,USA) and PEG-Hb from Enzon (Piscataway, New Jer-sey, USA) and poly(oxyethylene) modification of pyri-doxylated cross-linked Hb (e.g., the product PHPfrom Apex Bioscience Inc. (Chapel Hill, North Caro-lina, USA). PEG-ylated Hb products have undergoneextensive clinical trials and the studies showed risks ofbradycardia and elevation of hepatic pancreaticenzymes even at low doses.69 Nonetheless, the Phase Iand Phase II clinical trials showed that Hemospan waswell-tolerated in humans for efficient oxygen delivery,



and Phase III trials in orthopedic surgery patients werecarried out in Europe. The trials suggested that therisk of cardiovascular and renal dysfunctions still per-sisted with such chemically modified Hb products.70

Products like PHP have also indicated such risks ofcardiovascular and renal dysfunctions. During the pasttwo decades, it has been identified that cell-free Hb(including chemically modified versions) are potentscavengers of NO via rapid irreversible binding (rateconstant ~ 107 M−1s−1), which in turn affects systemicand pulmonary vascular tone, resulting in vasocon-striction, hypertension, and lowering of cardiac out-put.71,72 A resolution of this issue has been attemptedby modifying Hb molecule into becoming an NO car-rier through S-nitrosylation of cysteine residues in theβ-subunits of Hb or imparting the ability of enzymatictransformation of Hb into a source NO donor in pres-ence of nitrites, but with limited success in vivo.73

Natural RBCs contain enzymes like catalase (CAT)and superoxide dismutase (SOD) that help mitigatethe oxidative stresses stemming from superoxide moi-eties in injured and ischemic tissues. Based on thisrationale, in an interesting approach these enzymeshave been cross-linked to polymerized Hb to formPolyHb-SOD-CAT, which showed combined advan-tages of long circulation time and reduced oxidativedamage.74,75 Another interesting approach is to incor-porate regulatory molecules such as 2,3-DPG andMetHb reductase along with Hb in appropriateHBOC systems, to prevent Hb oxidation. In anotherrecent approach, a product named HemoTech hasbeen developed that uses purified bovine Hb cross-linked intramolecularly with ATP and intermolecu-larly with adenosine, and conjugated with reducedglutathione (GSH).76 The novelty of this design is theuse of pharmacologically active molecules (ATP, aden-osine, and GSH) as the chemical modifiers, whereATP regulates vascular tone through purinargic recep-tors, adenosine counteracts the vasoconstrictive prop-erties of Hb via stimulation of adenosine receptors,and GSH shields the ‘heme’ from NO and reactiveoxygen species. The preclinical and early phase clini-cal studies have shown that HemoTech works as aneffective oxygen carrier in treating blood loss, ane-mia, and ischemic vascular conditions, and furtherstudies are expected. In another recent nanotechnol-ogy approach, core-shell cluster structures wereformed by conjugating human serum albumin(HSA) on Hb using Hb surface lysines conjugated toHSA cysteine-34 using α-succinimidyl-ε-maleimidecross-linker.77 These Hb-HSA clusters are envisagedto have low risks of rapid clearance and extravasa-tion, and high circulation stability. A further modifi-cation of these Hb-HSA core-shell nanoclusters was

recently reported where antioxidant enzymes andplatinum nanoparticles were embedded in the HASpockets for protection of Hb.78 These nanoclusterdesigns have only been evaluated for their oxygen-binding capacity, redox properties, and stabilityin vitro, and rigorous in vivo studies would beneeded to establish their in vivo applicability.Figure 3 shows some of the prominent designs basedon chemical modification of cell-free Hb. In spite ofpromising preclinical and some clinical results, mostof the chemically modified Hb products have been

FIGURE 3 | Schematic representation of design and componentsof RBC-inspired chemically modified prominent HBOC nanosystems,along with their design/trade names.



withdrawn from clinical studies and discontinued inproduction, due to substantial clinical data indicat-ing more risks than benefit, owing to chemical heter-ogeneity of final product, variable stability,suboptimal vascular residence time, nonideal oxy-gen loading/off-loading capabilities, rapid irreversi-ble conversion to MetHb, and increasedcardiovascular and renal dysfunction issues. Whilesome of the newer products are refining their designand processing to address these issues, a paralleldirection of the research and development hasfocused on encapsulation of Hb within variousmicro- and nanocarrier vehicles.

Encapsulated HBOC SystemsThe advent of microparticulate and nanoparticulatetechnologies have revolutionized the delivery of phar-maceutical compounds, by encapsulating the com-pounds within such particulate vehicles that canprotect the compounds from plasma-induced effects,increase the circulation time of the compounds andallow sustained delivery to cells, tissues, and organs.Naturally, this concept was also adapted to createHBOCs that encapsulate Hb within suitable particu-late carrier vehicles. In fact, the concept and demon-stration of bio-inspired artificial cells was presentedas early as the 1950s and 1960s by Chang and col-leagues, by encapsulating Hb as well as other pro-teins and enzymes within microvesicles bound bypolymeric membranes. The membrane material origi-nally used was collodion (cellulose nitrate) and laterchanged to biodegradable PEG-polylactide (PEG-PLA).79,80 These ‘Hb corpuscles’ showed OECs simi-lar to RBCs and retained activity of RBC-relevantenzymes like 2,3-DPG, carbonic anhydrase, andCAT.79–81 However, a principal challenge was posedby the rapid macrophagic removal of these micron-sized polymeric vesicles from circulation, resulting invery short vascular residence time. Reducing thediameter to ~1 micron only marginally improved thecirculation, and a significant research effort wasdirected toward further improving the vascular resi-dence time by modifying the surface of the polymericvesicles with lipids and polysaccharides. In an analo-gous approach, Djordjevich et al. reported on encap-sulation of Hb in micron and submicron size lipidvesicles (liposome-encapsulated Hb or LEH), withmembrane made of phospholipids andcholesterol.31,82–84 This design was to essentiallymimic the physiological state of Hb in RBCs where itis encased within the cell membrane in a protectedenvironment that preserved the suitable redoxmechanisms for Hb function. A number of variationsof this design followed, e.g., ‘neohemocytes,’ ‘TRM-

645 Neo Red Cells’ and so on, where the primaryfocus was to maintain uniform Hb-encapsulationlevels and uniform size distribution of the vesicles,prevent vesicle destabilization or fusion over time,and thereby enhance vesicle stability upon storagewhile maintaining the RBC-mimetic oxygen transportproperties of the encapsulated Hb.85–87 During the1990s the ‘Stealth Liposome’ technology was estab-lished, where nanoscale lipid vesicles (100-200 nm indiameter) were surface-functionalized with PEG todecrease fusion and destabilization upon storage,reduce opsonization and prevent rapid macrophagicuptake, and this design significantly enhanced thecirculation residence time.88,89 Consequently, thistechnology was adapted to form Hb-encapsulatedPEG-ylated liposomal vesicles (HbV).90–92 For HbVpreparation, 1,2-dioctadecadienoyl-sn-glycero-3-phosphatidylcholine (DODPC) was used as the majormembrane phospholipid, such that γ-irradiationinduced radiolysis of water molecules in the vesiclesgenerated hydroxy (OH) radicals that promotedintermolecular polymerization of dienoyl groups inDODPC to produce remarkably stable liposomesthat could withstand freeze-thawing, freeze-drying,and rehydration processes. The incorporation of Hbin PEG-ylated submicron sized stealth liposome vesi-cles resulted in substantial improvement of circula-tion life-time (~60 h in some animal models) andmany refinements of this approach have beenreported during the past two decades regardingHbVs.93–96 Incorporation of Hb within lipidic vesi-cles are analogous to incorporation within cell mem-branes, and therefore the oxygen transportationability of HbVs were partly similar to natural RBCs,with comparable oxygen saturation and releasekinetics. Also, the liposomal encapsulation of Hbreduces its scavenging effect on NO and therebyreduces the subsequent negative effects on vascula-ture. The encapsulation also prevents glomerularclearance of Hb and thereby reduces nephrotoxicity.The current optimized HbV product contains about30,000 Hb molecules encapsulated within one PEG-ylated liposomal vesicle of ~250 nm in diameter.31,91

In comparison, a natural RBC is ~7 μm in diameterand ~2 μm in thickness, containing about 250 millionHb molecules. The HbVs have undergone extensiveresearch in preclinical animal models for potentialuse as a synthetic RBC substitute in transfusion andresuscitative approaches in perioperative settings,massive hemorrhagic shock and hemodilution inci-dents, and oxygenation of ischemic as well as trans-planted tissues and organs.31,91–95 These studiesshow significant promise of HbVs as semi-syntheticRBC mimics, however, these systems can still present



issues of broad size distribution of the vehicles, varia-tions in Hb-encapsulation efficiencies, variable phar-macokinetics, and complement-mediated immuneresponse in vivo. Further research is currently beingdirected toward resolving these issues for potentialclinical translation of HbV designs as a syntheticRBC substitutes.31,96–98 Interestingly, instead ofencapsulating Hb, some research approaches havealso attempted to encapsulate oxygen (O2) directlywithin phospholipid microvesicles (2–4 μm in diame-ter) to deliver O2 to deoxygenated RBCs in circula-tion.99,100 Although these oxygen-loadedmicrobubbles were found to be stable for a fewweeks in storage with only small extent of oxygenloss, in vivo they have a very short circulation life-time (<1 h). Therefore, treatment with these systemswould require multiple or repeated dosing, whichmay then lead to negative effects of dysregulated oxi-dative stress and associated immune response. There-fore, long-term safety profile of this technology needsto be rigorously evaluated. Encapsulation of Hb hasalso been studied in other microparticle and nano-particle systems besides using liposomal vesicles. Inpioneering work by Chang et al., Hb has also beenencapsulated within nanoscale polymeric particlesmade from PEG-PLA and analogous block copoly-mers.101,102 These polymeric nanoparticles, about80–200 nm in diameter, can allow oxygen transportkinetics of encapsulated Hb at ranges similar to natu-ral RBCs and the polymeric material is biocompatibleand biodegradable. Enzymes that maintain the redoxenvironment for stability and O2/CO2 transport ofHb (e.g., carbonic anhydrase, CAT, SOD, MetHbreductase, etc.) can also be encapsulated within thesame nanoparticles to further refine their functiontoward mimicking RBC action.103 This concept hasalso been adopted for other polymer systems likepoly(ε-caprolactone)/poly(L-lactic acid) (PCL/PLA)copolymers, poly(L-lysine) (PLL), poly(lactic-co-glycolic acid) (PLGA)/PEG copolymers, and soon.104,105 Amphiphilic block-copolymer systems alsoprovide the ideal building blocks for designing poly-mer vesicles, otherwise known as polymersomes,analogous to liposomes. These polymersome systemshave also been recently utilized to load Hb, to createpolymerosome-encapsulated Hb (PEH).106 The Hbloading in these PEH systems has been reported to be1–2 mg/mL, compared to human blood (i.e., withinRBC) concentration of ~150 mg/mL. Utilization ofhollow fiber membrane module based extrusion sys-tem can provide an interesting way to manufacturethese PEH systems.107 These PEH systems can encap-sulate both bovine and human Hb, and can showoxygen binding and release equilibrium kinetics and

other biophysical parameters similar to RBCs. Thisindicates considerable promise toward the applicationof PEH systems as a synthetic RBC substitute in trans-fusion medicine, but currently very limited in vivoevaluation data are available for these systems. Apotential issue with polymersome systems may betheir higher shell thickness compared to liposomes,which can lead to longer diffusion time for oxygen tosaturate the encapsulated Hb or to be released fromthe Hb to tissues. Modulation of polymer molecularweight of the shell and therefore of the shell-thicknesscan provide a way to influence oxygen transport fromPEH systems. The higher stability of polymersomescompared to liposomes, both in storage and in vivo,may provide additional advantages for its use as Hb-encapsulated artificial RBC systems. Hb has also beenencapsulated in microparticles by its coprecipitationwith calcium carbonate (CaCO3), followed by cross-linking with glutaraldehyde and selective dissolutionof CaCO3, with Hb quantity per microparticle closeto natural RBCs.108 These Hb-microparticles haveshown oxygen binding and release characteristics sim-ilar to free Hb, but much enhanced circulation life-time compared to free Hb. Analogous Hbmicroparticles carrying about 80% Hb content com-pared to natural RBCs, have been recently reportedwhere Hb and MnCO3 were coprecipitated, immedi-ately followed by HSA addition for encapsulation andstabilization of the particles.109 These particles haveshown limited NO scavenging and reduced effect onvasoconstriction. Recent reports have also demon-strated the capability of covalently conjugating Hbdirectly to the hydrophobic or hydrophilic domain ofblock-copolymers and then micellize the resultantconjugates to form Hb-loaded micelles.110,111 Inanother interesting nanotechnology approach,MnCO3 nanoparticles were used as templates todeposit layer-by-layer (L-B-L) assemblies of Hb anddialdehyde heparin (DHP), followed by cross-linkingto stabilize the layers and selective dissolution of thetemplate core.112 A similar approach was also used toform L-B-L coated nanotubes where alternate layersof Hb, DHP, and the enzyme CAT were deposited, tocreate systems for potential application in treatingoxidative stress.113 These complex nanoscale struc-tures have been characterized in vitro for their mor-phology, stability, cytotoxicity, and in some casesbiofunctionality, but no preclinical evaluation foroxygen-carrying efficacy in vivo, has been reportedyet. Figure 4 shows some representative designs andcomponents for encapsulated Hb systems that haveundergone and are currently still undergoing in vitroand in vivo evaluation for RBC-mimetic oxygen car-rier application.



Some Hb-encapsulation approaches havefocused on the physico-mechanical properties of nat-ural RBCs that significantly influence their biologicalfunctions. Natural healthy RBCs are biconcave dis-coid in morphology, with a diameter of ~8 μm and athickness of ~2 μm, and are highly flexible (Young’smodulus 0.1–0.2 kPa), that enables them to changetheir shape and morphology when passing throughmicrovascular circulation.114,115 The mechanicalintegrity and viscoelastic nature of RBCs during theircyclical deformation is maintained by a two-dimensional spectrin network attached to the cyto-solic side of their membrane. Oxygenated Hb rendersRBCs significantly more deformable than deoxyge-nated Hb, and this enables the deformation of RBCsto transport oxygen in the microvasculature. Thesize, shape, and flexibility of RBCs are also known toinfluence their hemodynamic distribution in the

blood flow field, where in mid to large vessels theRBCs flow in the center of the parabolic flow field,while in small vessels and capillaries RBCs canbecome distributed throughout for efficient oxygenexchange.116 These considerations have recently ledto biomaterials-based micro- and nanoscale mimicryof RBC-relevant size, shape, and flexibility attributesinto Hb-encapsulating synthetic constructs. Forexample, polyelectrolyte driven L-B-L assembly hasbeen used to create polymeric particles that mimicthe shape and deformability of natural RBCs.117 Inthis design, Hb and BSA were electrostatically depos-ited on the surface of RBC-shaped PLGA particles of~7 μm diameter and 400 nm shell thickness, andthen the PLGA core was selectively dissolved to yieldRBC-shaped Hb-loaded particles capable of highelastic deformation. Similar RBC-mimetic particleshave been fabricated using PEG hydrogel system in a

FIGURE 4 | Schematic representation of design and components of RBC-inspired Hb-encapsulated prominent HBOC nanosystems, along withtheir design/trade names.



stop-flow-lithography (SFL) approach where themechanical properties of resultant particles could becontrolled by modulating cross-linking density of thehydrogel systems.118 In a different approach, RBC-mimetic particles were fabricated from acrylatehydrogels using a ‘particle replication in nonwettingtemplates’ (PRINT) technology.119 These particleswere made in 2–3 μm molds, such that, upon hydra-tion, the particles swelled to disks with diametersbetween 5.2 and 5.9 μm, and heights between 1.22and 1.54 μm. Filling of the molds resulted in a menis-cus, leaving the particles thinner in the middle andthicker at the edges, resembling the biconcave natureof RBCs. All of these RBC-mimetic particle designshave demonstrated elastic deformation capabilitiesin vitro for transport through narrow channels, andin vivo they have shown variable circulation lifetimedepending on their elastic modulus. Althoughresearch approaches using these particles havereported Hb encapsulation via physical trapping orcovalent tethers (e.g., via ethyl(dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide (EDC/NHS)chemistry), detailed oxygen transport capabilities andin vivo transfusion medicine potential have not beenexplored or reported in detail. In another recentapproach, liposome-encapsulated actin-Hb(LEAcHb) synthetic RBCs with natural RBC-mimeticshape properties were prepared using a polymerizedactin core.120 Although these particles were muchsmaller (�136.8 nm) than RBCs, the biconcave shapealong with the mechanical support of the membraneimproved the half-life from �8 to >72 h. In naturalRBCs, the negative surface charge electrostaticallyprevents RBC aggregation over a distance of 20 nmand this rationale has led to some research in mim-icking RBC-relevant surface charge on Hb-encapsulating PEG–PLA nanoparticles (<200 nm indiameter) using cetyltrimethylammonium bromide(CTAB) or anionic sodium dodecyl sulfate (SDS) sur-factants.121 Cationized particles were found to havea half-life of ~11 h (eightfold higher than untreatedparticles), while the anionized particles were quicklyeliminated, giving a half-life of <1 h. In yet anotherrecent approach, a novel amphiphilic polymeric sys-tem was developed using polyethylene imine (PEI)modified with palmitic acid and was used to formtoroidal-shaped nanoparticles (termed nanobialys,~200 nm diameter) that can encapsulate Hb, as wellas, maintain redox enzymatic environment for Hbactivity by coencapsulation of 2,3-DPG and leukomethylene blue.122 These novel Hb-containing parti-cles, termed Erythromer, have shown some promiseof oxygen transport in vivo, and detailed biocompati-bility studies (e.g., for PEI which can pose

cytotoxicity issues), circulation lifetime and stability,Hb-loading capacity. oxygen transport capabilities,and so on would need to be further evaluated toestablish their clinical potential as synthetic RBC sub-stitute in transfusion medicine. It is interesting tonote that a hypothetical bio-inspired concept ofmicroparticulate ‘artificial RBCs’ termed Respirocytewas proposed almost two decades ago by RobertA. Freitas Jr in his paper ‘A Mechanical ArtificialRed Blood Cell: Exploratory Design in MedicalNanotechnology,’ but an ideal Hb-loaded syntheticRBC substitute for in vivo transfusion applicationsstill remains to be achieved and clinicallyapproved.123 A high volume of research continues tobe directed in this area in the United States and glob-ally, that may ultimately lead to a viable RBCsubstitute.

PFC-Based Oxygen Carrier SystemsDue to the issues of stability, circulation lifetime,potential NO-scavenging properties, and toxicity ofHb-based oxygen-carrying nanotechnologies, a paral-lel research interest has developed in non-Hb basedfully synthetic macromolecular (polymeric) systemscapable of transporting oxygen. To this end, the classof macromolecules that have undergone the mostrobust volume or research and translational activitiesis that of PFCs. PFCs are uniquely both hydrophobicand lipophobic which contributes to their biologicalinertness, and this property has resulted in utilizationof liquid PFCs in magnetic resonance imaging andgas-filled PFC microbubbles in ultrasound imagingcontrast agents in biomedical diagnosticapplications.124–127 PFCs have the ability to dissolveoxygen physically, where O2 is loosely bound by vander Waals type interaction between PFC macromers,and not chemically bonded as they usually are withHb. This dissolution equilibrium of O2 in PFCs fol-lows Henry’s law (i.e., oxygen partial pressure),resulting in a linear oxygen binding/transport profile,in contrast to the sigmoid cooperative binding profilefound for oxygen binding to Hb.128,129 Figure 5shows chemical structure of some representativePFCs that have been investigated extensively for oxy-gen transport applications, as well as, the linear pro-file of O2 binding to PFCs compared to the nonlinearexponential profile of O2 binding to Hb or HBOCs.PFCs are biologically inert liquids and gases, charac-terized by strong intramolecular and weak intermo-lecular C–F and C–C bonds. This essentially meansthat a much higher oxygen partial pressure isrequired to ensure sufficient dissolution and transportof oxygen by PFCs, compared to that by



Hb. However, due to the linear profile, oxygen off-loading capacity is better for PFCs compared toHb. Due to immiscibility of PFCs in water, intravas-cular application of PFCs for oxygen-carrying appli-cation requires rigorous emulsification of PFCs withsuitable surfactants (e.g., phospholipids) and the oxy-gen solubility in PFC emulsions is independent of thesurfactant used.130 Several PFC-based emulsions havebeen investigated extensively as oxygen carriers in pre-clinical and clinical stages.118–121 Fluosol-DA (GreenCross Corp., Osaka, Japan Green Cross Corp.,Japan), an emulsion of 10–20% perfluorodecalin inalbumin, was extensively tested in preclinical animalmodels and was the first PFC system to progress intoclinical trials and to receive FDA approval in 1989.However, this product was soon recalled because ofissues like low oxygen transport capacity (0.4 mLoxygen/100 mL compared to blood’s reported oxygentransport capacity of 20.1 mL/100 mL), prematureoxygen release, short shelf-life, temperature instability,slow excretion, and serious side-effects.131 Fluosol wasalso implicated in reducing platelet counts (thrombocy-topenia), febrile symptoms, and macrophage activation(inflammation trigger). The limitations of Fluosol PFCsystems were significantly resolved by newer generationPFC compounds like perfluoroctyl bromide (also

known as perflubron), perfluorodecyl bromide, andperfluorodichlorooctane.132–138 Using these compoundsin specific ratios along with egg-yolk phospholipids, aPFC-based oxygen carrier named Oxygent (AlliancePharmaceutical Corp, La Jolla, California, USA Alli-ance Pharmaceutical Corp, La Jolla, California, USA)was developed where the droplet size could be main-tained at 160–180 nm in diameter, that showedreduced macrophage activation.135,136 Intravascular cir-culation half-life for Oxygent droplets was found to be~10 h for a dose of 1.8 g/kg in humans.138,139 Oxygentwas found to render higher oxygen delivery comparedto Fluosol, but was still ~30% less efficient than naturalRBCs. Another analogous PFC product was developedusing perfluorodicholorooctane, egg yolk phospholipid,and triglyceride, named Oxyfluor (HemaGen, St. Louis,Missouri, USA), with droplet diameters in the range of220–250 nm.137 This product has also shown similardelay in macrophagic uptake and therefore similar cir-culation residence time as Oxygent. Due to nanoscalediameter, these PFC droplets can occupy small plasmavolumes and can circulate through microcapillaries,thereby providing significant ability of oxygen deliveryin microcirculation. In preclinical models in vivo, bothOxygent and Oxyfluor have shown promising benefitin oxygen delivery, although both products have also

(a)(d)

(e)

(f)

(b)

(c)

Perfluorodecalin (FLUOSOL)

Perfluorotripropylamine

Perfluoromethylcyclohexylpiperidin

Perfluorooctylbromide (PERFLUBRON)

Dichloroperfluorooctane (OXYFLUOR)

Whole blood, Hb = 140 g/L

Perflubron (90%)

Fluosol (20%)

HBOC, Hb = 70g/L

0 100 200 300

pO2 (mm Hg)

To

tal

O2 c

on

ten

t (m

L/d

L)

400 500

25

20

15

10

5

0

F2

F2

F2

F2

CCF

FC

C

C

C

CF2

CF2

F2C

F2C

C NC C

C

CF3F3C

CF2

CF3

F2C

F2

F2F2

F2

F2C

F2C

F2C

F2

F2 F2 F2 F2

C

C CC C

NF2C CF CF3FC

F2

CC

F2

F2

CF2

CBr

F2

C

F2

C

F2

C

F3C

F2

C

F2

C

F2

C

F2

CCF2

CF2

CF2

C Cl

Cl

F2

FIGURE 5 | (a–e) Chemical structure of various perfluorocarbon (PFC) compounds that have been studied for oxygen-carrying applications; (f )oxygen-binding curves of whole blood and HBOC systems (cooperative sigmoid binding characteristics) compared with that of PFC-based oxygencarriers (linear binding characteristics).



shown some platelet-depleting (thrombocytopenic)effects.140 In several large animal (e.g., canine) modelsof hemodilution and cardiopulmonary bypass, thesePFC systems have shown the ability to increase oxygentransport and metabolism.141 In fatal hemorrhagicmodels, these oxygen carriers could improve resuscita-tion and reduce mortality significantly. These promisingresults have led to clinical evaluation of such productsin human patients undergoing major noncardiac sur-gery, hemodilution, and hemorrhage.142–144 In suchscenarios, Oxygent was evaluated as a substitute forautologous blood transfusion and this product was ableto significantly transport oxygen and reverse transfu-sion triggers. These results indicated that such PFCbased oxygen carrier systems are at least as effective asautologous blood to reverse transfusion triggers andtherefore can act as RBC substitutes to some extent.However, advanced clinical trials of such products inpatients undergoing cardiopulmonary bypass were ter-minated in the United States because of increased riskof stroke and adverse events, although deeper analysisof the data has suggested that these adverse events wereassociated more with the study protocol rather than thePFC product itself.139 Oxycyte (Oxygen Biotherapeu-tics Inc., Morrisville, North Carolina, USA), is a third-generation PFC emulsion containing an aqueous 60%emulsion of perfluoro-tert-butylcyclohexane with puri-fied egg yolk phospholipids, that has undergone pre-clinical trials in several animal models and someclinical trials in patients with traumatic brain and spi-nal cord injury.145–147 Although some safety concernswere raised regarding Oxycyte’s effect on the immunesystem and possible hemorrhagic side effects, recentanimal studies have alleviated FDA concerns and stud-ies are under way. Perftoran (Russia) is another PFCemulsion of perfluorodecalin and perfluoromethylcyclo-piperidine in a nonionic surfactant that has been widelyinvestigated in Russia.148,149 Although this product hasshown adverse effects such as hypotension and pulmo-nary complications, randomized clinical trials with Perf-toran conducted in Mexico City on patientsundergoing cardiac valvuloplasty showed higher intrao-perative pO2 levels, and reduced need for allogenicRBCs. Further clinical studies are expected to establishthe benefit of this product.

Synthetic Porphyrin SystemsThe oxygen-transporting active site of natural Hb isthe ‘heme’ part, which consists of a porphyrin tetra-pyrrole ring surrounding an iron atom (Figure 2(a)).Based on this, researchers have developed Fe(II)-bearing porphyrin systems for oxygen trans-port.150,151 To this end, ‘picket fence’ Fe2+ porphyrin

molecules were created that demonstrated thatreversible oxygenation of myoglobin and Hb is possi-ble via immobilization of the five-coordinate ferrous‘heme’ in a sterically hindered hydrophobicmatrix.152 These systems showed cooperative oxygenbinding similar to natural Hb, but were prone to irre-versible oxidation in aqueous media. To resolve thisissue, a hydrophobic environment was created forthese molecules, for example, using liposomes,where amphiphilic Fe2+-porphyrin bearing four alkyl-phosphocholine groups were efficiently embeddedinto the bilayer of phospholipid vesicles (e.g., Lipid-Heme).153,154 The resultant vesicles demonstratedreversible binding and release of oxygen similar tonatural Hb. Instead of lipid vesicles, HSA particleshave also been used to incorporate these syntheticFe2+ porphyrin systems, and these have shownoxygen-transporting efficiency similar to RBCs. Sur-face modification of these particles with PEG haveresulted in increased circulation time and reducedoxidation. Another porphyrin-based interestingdesign is HemoCD, that is comprised of a 1:1 com-plex of 5,10,15,20-tetrakis (4-sulfonatophenyl)porphinatoiron(II) (Fe[II] TPPS) and a per-O-methylated β-cyclodextrin dimer having a pyridinelinker (termed HemoCD or Py3CD).151 This systemshowed oxygen affinities similar to natural Hb andwas found to be gradually autoxidized in aqueousenvironment via nucleophilic attack of water mole-cule to the O2–Fe bond. The circulation stability ofthese systems could be further enhanced by surfacedecoration with PEG-based dendrimers.155 Figure 6shows representative design schematic and nanoma-terials used for the various porphyrin-based oxygencarrier systems. The in vivo application and clinicalpromise of these nanoengineered porphyrin systemsas oxygen carriers need to be further evaluated inappropriate preclinical animal models.

PLATELET SUBSTITUTES, SYNTHETICHEMOSTATS, AND PLATELET-INSPIRED DRUG DELIVERY SYSTEMS

Platelets are anucleated blood cells primarily respon-sible for blood clotting and hemostasis. Platelets areproduced from mature megakaryocytes and underhemodynamic blood flow environment they have theability to undergo margination toward the blood ves-sel wall while the RBCs congregate more toward thecenter of the blood vessel, in a parabolic flowprofile.156–158 By virtue of this, platelets can stay inconstant surveillance of the vessel wall and can rap-idly provide hemostatic respond where needed. As



depicted in Figure 7, platelet’s mechanisms for hemo-static response at a vessel bleeding site is mediated by(1) rapid adhesion of platelets at the site by bindingto specific proteins like von Willebrand Factor (vWF)and collagen that are exposed at the site, (2) activa-tion and rapid aggregation of platelets at the site viainter-platelet bridging by blood protein fibrinogen(Fg) interacting with platelet surface integrin GPIIb-IIIa (primary hemostasis), and (3) facilitation ofcoagulation cascade mechanisms on the negativelycharged phosphoserine-rich membrane surface ofactive platelets aggregated at the site.159,160 Loss ofplatelets due to trauma or surgery, as well as, drug-induced or congenital defects in platelet number andfunction, can lead to a variety of bleeding complica-tions, and transfusion of natural platelets are rou-tinely used in the clinical treatment of suchconditions.161–164 These transfusions primarily use

allogeneic platelet concentrates (PCs) suspended inautologous plasma, stored at room temperature andthese PCs have a high risk of pathologic (mostly bac-terial) contamination, resulting in very short shelf-life(~3–5 days).164,165 Pathogen reduction technologies(e.g., psoralen-based UV treatment) can reduce con-tamination risks, but so far these have extended thePC shelf-life only marginally.165–168 Several otherplatelet-derived products are currently under investi-gation, e.g., frozen (−80�C) platelets, cold-stored (4�C)platelets, lyophilized platelets, platelet-derived micro-particles and infusible platelet membranes (IPM), andtrehalose-stabilized platelets (Thrombosomes; Cell-Phire, Rockville, Maryland, USA), with a vision toextend shelf-life of platelet-based hemostatic productswithout compromising biofunctional properties.169–172

PCs and natural platelet-derived products are usuallyobtained from pooled blood of multiple donors, and

FIGURE 6 | Schematic representation of design and components of some porphyrin-based oxygen-carrying nanosystems, along with theirdesign/trade names.



this presents significant risks of blood-borne infectionthat, in turn, necessitates rigorous serological testingand expensive leukoreduction techniques.23,173 Due tothese various issues with natural platelet-based sys-tems, a parallel significant interest has emerged in syn-thetic platelet substitutes that can render efficienthemostasis while allowing advantages of large-scalepreparation, minimum contamination risk via effectivesterilization, longer shelf-life, no need for blood typematching and reduced risks of biologic or pathologicside effects.30,34,35,174

To this end, a variety of platelet-inspired sys-tems have focused on mimicking platelet’s biochemi-cal mechanisms of injury site-specific adhesion,aggregation, and coagulation promotion via surfacemodification of microparticle and nanoparticle sys-tems with platelet surface-relevant glycoproteins andligands. It is to be noted here that platelets are micro-scale (2–3 μm diameter) discoid particles that have alarge variety of surface glycoproteins and receptorspresent at different densities. For example, plateletsurface integrin αIIbβ3 (GPIIbIIIa) that binds to theligand fibrinogen (Fg) is present at ~80,000 copiesper platelet, surface glycoprotein GPIb-IX-V that

participates in binding to vWF is present at ~50,000copies on the surface, and surface protein α2β1(GPIaIIa) that binds to collagen is present at ~3000copies per platelet.175 Besides these, there are multi-ple other surface proteins [e.g., cell adhesion mole-cules or CAMs, thrombin receptors, adenosinediphosphate (ADP) receptors, selectins, tetraspanins,α5β1 and α6β1 integrins, etc.] that are present at den-sities of tens to hundreds to thousands. In simulatingthe major hemostatic functions and activity of plate-lets on semi-synthetic or synthetic nanotechnologyplatforms, the focus has not necessarily been to simu-late the exact densities of interactions but rather thetype of interactions. For designs that have directlyused platelet-derived membranes for vesicle fabrica-tion or nanoparticle coating, the surface density ofinteractive motifs is dependent upon whatever bioac-tive surface proteins remain in the membrane postiso-lation and processing. In contrast, for fully syntheticdesigns the number of ligands per unit surface areaper particle can be theoretically estimated based ondimension of particles and concentration (e.g., mole%) of ligands reacted with particles, and that numbercan vary from hundreds to thousands per particle.

Vessel injury Vessel Haemostasis

Inactive

GPllb-llla

PSGL-1 GPlba

VWFP-selectin

ActiveGPllb-llla

Activeplatelet

Fg

Collagen

Primary hemostasis(active platelet adhesion

and aggregation)

Secondary hemostasis(coagulation cascade and

fibrin generation)

GPIbaVWF

Fg

FIX

FXIa

GPlalla or

GPVI

FIXa

FX

Prothrombin(FII)

FXa

FVa

Thrombin (FIIa) Fibrin

FVIIIa

Quiescentplatelet

FIGURE 7 | Molecular mechanisms of platelet-mediated hemostasis in bleeding vessel, showing respective components of primary hemostasisand secondary hemostasis.



Furthermore, since platelet’s physico-mechanicalproperties (e.g., shape, size, flexibility, etc.) can influ-ence their margination behavior under hemodynamicflow environment, some recent research approacheshave also been directed toward leveraging platelet’sbiophysical parameters in designing nanoparticle andmicroparticle based ‘synthetic platelet’ systems. Inanother interesting approach, conventional drugdelivery particles have been coated with platelet-derived membranes, to impart bio-inspired targetingproperties. Figure 8 shows some representativedesigns and components for platelet-inspired nano-medicine systems for hemostatic applications asplatelet substitutes and the following sectionsdescribe the approach and functional promise ofthese designs. It is to be noted that in parallel toleveraging and mimicking platelet’s hemostaticmechanisms, a subset of ‘synthetic hemostat’ researchhas also focused on development and utilization ofbody’s coagulation factor components and coagula-tion cascade outputs as injectable natural, semi-synthetic or synthetic molecules to augment hemosta-sis. Classic examples of this are the development ofrecombinant coagulation factors, a variety of naturaland synthetic biomaterials that induce activation ofplatelets and/or coagulation, peptide-modified syn-thetic polymers that mimic and amplify mechanismsof fibrin strengthening, and drug delivery platformsthat can deliver pro-coagulant molecules at bleedingsites.176–185 These systems are not necessarily ‘syn-thetic blood substitutes,’ but they form an importantarea of biomaterials and nanotechnology research interms of modulating blood clotting properties, withpotential applications in treating hemorrhagic com-plications and promoting wound healing. Detaileddiscussions of these systems are beyond the scope ofthe current review, but several informative reviewson many of these systems are availableelsewhere.178,186–188

Nanomedicine Systems Inspired by PlateletAdhesion MechanismsNatural platelets adhere to the injury site via severalspecific mechanisms. Upon vascular injury, theinjured endothelial cells lining the luminal wall ofblood vessels secrete vWF molecules from their Wei-bel Palade bodies.189 The injury site also has exposedsubendothelial collagen. The vWF molecule is usuallysecreted as a globular protein, which unravels underhemodynamic shear forces at the injury site and canmultimerize on injured endothelium and exposed col-lagen.190 The unraveled vWF exposes the A1-domain, which has binding specificity to platelet

surface receptor component GPIbα of the GPIb-IX-V complex. This vWF-to-platelet GPIbα interactionis shear-dependent and reversible, and leads toplatelet attachment and rolling at the injurysite.190,191 Concomitantly, platelet surface glycopro-teins GPIa-IIa and GPVI, with binding capability tocollagen, can anchor onto the exposed collagen atthe injury site and this synergistically strengthensthe adhesion of platelets at the site.191 The subse-quent platelet-mediated mechanisms of coagulationand hemostasis happen in tandem such adhesionevents. Therefore, inspired by these adhesionmechanisms, nanomedicine research has focused ondeveloping surface-engineered particulate systemsthat simulate platelet’s vWF-binding and collagen-binding capabilities. One of the earliest designs inthis aspect was ‘plateletosomes,’ that involved isola-tion of platelet membrane glycoproteins viadetergent-based extraction techniques and their sub-sequent incorporation within the lipid membrane ofliposomes.192 While this approach ensures retentionof platelet’s own biofunctional surface proteins, theextraction, purification, sterilization, and liposomemembrane incorporation make this strategy poten-tially complex and expensive to scale up. Thesecomplex issues could be avoided by utilizing recom-binant technologies, where recombinant GPIbα(rGPIbα that binds to vWF’s A1 domain) and GPIa-IIa (rGPIa-IIa, that binds to collagen) could be con-jugated on the surface of liposomes, latex beads, oralbumin-based particles to create synthetic systemsthat simulate platelet’s injury site-adhesive capabil-ities.193,194 In further advancement of this approach,both rGPIbα and rGPIa-IIa have been coconjugatedon the surface of liposomes and albumin particles,and this combination demonstrated higher bindingto collagen surfaces in presence of soluble vWF athigher shear rates, thereby closely mimicking naturalplatelet adhesion.195

Recombinant technology approaches to makeanalogous platelet surface protein can be quiteexpensive in the context of scaling up and clinicaltranslation. Also, the large size of these recombi-nant protein fragments can lead to issues of sterichindrance regarding their combinatorial decorationon the surface of nano- and microparticles. Theseissues can be potentially addressed by using smallmolecular weight peptides that have vWF-bindingand collagen-binding capabilities. An early exampleof this is found in reports focusing on the develop-ment of GPIbα-relevant 15-mer peptides that havebinding capability to vWF.196 These peptides weretethered onto liposomal surfaces stabilized viacross-linking, and the resultant liposomal



FIGURE 8 | Schematic representation of design and components of platelet-inspired intravenously injectable synthetic hemostat nanosystems,along with their design/trade names where applicable.



constructs, 150–200 nm in diameter, have beentested as ‘synthetic platelet substitutes.’197 Althoughthe biochemical characterizations of these con-structs have been reported, the actual hemostaticefficacy evaluation of these constructs in vitro andin vivo has not been demonstrated in any preclini-cal animal model. In a more recent approach,researchers have utilized a vWF-binding peptide(VBP) sequence TRYLRIHPQSWVHQI derivedfrom the C2 domain (residues 2303–2332) of thecoagulation factor FVIII and a collagen-bindingpeptide (CBP), which is a 7-mer repeat of theGlycine(G)–Proline(P)–Hydroxyproline(O) tri-peptide (i.e., -[GPO]7-) with helicogenic affinityto fibrillar collagen but minimal affinity to plate-let collagen receptors GPIa/IIa and GPVI(i.e., minimal risk of systemically activating plate-lets), to mimic platelet adhesion mechanisms onnanoparticles.198,199 Using liposomes as modelnanoparticles, VBPs and CBPs were conjugatedonto the particle surface. Liposomes decoratedwith only the VBP moieties exhibited enhancedadhesion onto vWF-coated surfaces or on colla-gen surfaces in presence of soluble vWF, underhigh shear flow. Studies also indicated that thisvWF-binding mechanism of VBP is possiblyoccurring at the D0–D3 domain of vWF and notthe platelet GPIbα-interactive A1 domain.199

These studies suggested that the VBP-decoratednanoparticles would be capable of binding tovWF without interfering with natural platelet’sbinding to the same vWF. Liposomes decoratedwith only CBP moieties exhibited significantbinding to collagen-coated surfaces under flow, atall shear conditions. With a vision for mimickingplatelet’s synergistic adhesion mechanisms, theVBP and CBP moieties were coconjugated on lip-osome surface, and the resultant heteromultiva-lently decorated particles showed significantlyhigher adhesion to ‘vWF + collagen’ surfaces atlow-to-high shear ranges, compared to liposomesbearing VBP or CBP moieties alone. Interestingly,when compared to liposomes surface-decoratedheteromultivalently with rGPIbα and CBP, lipo-somes surface-decorated with VBP and CBPshowed significantly higher levels of adhesion on‘vWF + collagen’ surface, suggesting that thelarge rGPIbα fragments may cause steric maskingof smaller CBP moieties that can be avoided byusing the VBP moieties.198 This emphasizes theutilization of small peptides when heteromultiva-lent functionalization is needed for synergisticbioactivity, e.g., in nanoparticle-based mimicry of thedual adhesion mechanisms of platelets.

Nanomedicine Systems Inspired by PlateletAggregation MechanismsFor the hemostatic action of natural platelets, adhe-sion to vWF and collagen at the injury site triggersseveral signal transduction pathways that ultimatelylead to platelet activation, resulting in change inplatelet morphology from biconvex discoid to stellarand spreading of platelets at the injury site.200,201

These activation mechanisms also result in stimula-tion of platelet surface integrin GPIIb-IIIa from aresting to a ligand-binding conformation, that canbind the blood protein fibrinogen (Fg) via peptidesRGD and HHLGGAKQAGDV (also known as H-12) in the α and γ chains on both termini ofFg.202,203 As a result, active platelets can undergo Fg-mediated aggregation at the vascular injury site. Theactivated platelets also secrete agonists like ADP, aswell as facilitate generation of thrombin, that canfurther activate neighboring platelets, adding to theaggregating population. Rationalizing from theseprocesses, a large volume of research has been car-ried out to mimic platelet’s aggregation mechanismsby decorating synthetic particle surfaces by Fg itself,Fg fragments, or Fg-based RGD and H-12 peptides.Some of the earliest designs of this approach involvedsurface engineering of polymeric beads and RBCswith Fg or Fg-derived RGD peptides.204–208 Thesedesigns were essentially ‘super-fibrinogen’ systemswhere the particle (or RBC) surface decoration withFg or Fg-derived RGD peptides were meant toamplify the aggregation of active platelets via multi-valent interaction with the stimulated GPIIb-IIIaintegrins on activated platelet surface. These designsdemonstrated hemostatic promise in vivo in severalpreclinical models, but have failed to progress intoclinical translation, possibly due to the limitations ofobtaining sufficient antigen-matched autologousRBCs (or from universal donors). To address suchlimitations, some research efforts have been directedtoward creating ‘universal donor-like RBCs’ bymasking RBC surface antigens with synthetic poly-mer coatings, or enzymatically converting the A andB antigens into O type, or stimulating stem cells intobecoming universal donor type RBCs.209 The scale-up efficacy and translational promise of suchapproaches are yet to be determined. Another Fg-based platelet-relevant nanotechnology approach thathas undergone preclinical and clinical evaluation inthe US and Europe is Fibrocaps (ProFibrix, Leiden,The Netherlands), where a solution of fibrinogen andthrombin are separately spray-dried and then com-bined to produce a mixture of suspendable micropar-ticles.210 Fibrocaps have been used in clinical trials



for treatment of bleeding during surgery and trauma-related injury, and have shown significant reductionin time-to-hemostasis after direct administration tosurgical wound within a Gelatin Sponge, comparedto the sponge alone. One limitation of this product isthat it needs to be directly applied to the wound(sprayed or applied in a sponge) and therefore canonly be applied to accessible wound sites and notintravenously. Several other Fg-based products thathave undergone extensive research as platelet-inspired semi-synthetic hemostats are Synthocytes,Thrombospheres, and Fibrinoplate, all of which areessentially made of human albumin microparticlessurface-coated with Fg.211,212 These products haveundergone preclinical in vivo studies and some earlyphase clinical trials, but more detailed clinical evalua-tions are needed to establish their in vivo safety pro-file and treatment efficacies. In an analogousapproach, Fg has also been coated on the surface ofliposomes and the resultant vesicles have shown theability to increase platelet recruitment and aggrega-tion on collagen-covered surfaces in vitro. Anotherinteresting nanomaterial reported in recent years isfibrin-targeted microgel and nanogel systems that canrespond to hemodynamic environment at an injurysite to potentially enhance platelet recruitment andaggregation, as well as, directly seal the bleeding site.This research has been demonstrated recently withpolyispopropyl acrylamide based low-cross-linkedmircogel particles surface-decorated with fibrin-specific recognition motifs, that can enable binding toinjury site by specific interaction with nascent fibrinprotofibrils.213 Mechanistically, the binding of theseconstructs would require prior presence of sufficientfibrin (i.e., sufficient propagation of coagulationmechanisms) at the injury site. Therefore, rationaliz-ing from the reported use of fibrin-specific constructsfor targeted drug delivery to thrombi, these gel parti-cles may find effective applications in clot-targetedtherapeutic delivery.214

Using Fg to surface-coat micro- and nanoparti-cles for mimicking and leveraging platelet’shemostasis (and thrombosis)-relevant aggregationmechanisms may present several issues of storage sta-bility, immunogenicity, and off-target activity. Arefined approach utilizing the same design rationaleis to use Fg-relevant small molecular weight peptidesfor particle surface decoration. The earliest examplesof this are found in designs utilizing RBCs or poly-meric particles with Fg-relevant RGD peptides, andsimilar approaches have also been reported in recentstudies.204,215 Most of these approaches have utilizedlinear small RGD peptides, e.g., CGRGD or GRGDS,

that have binding capability to platelet surface integ-rin GPIIb-IIIa. Two potential issues with using thesepeptides are (1) their ubiquitous nature to bind tomany different integrins on other cells (i.e., lack ofplatelet-specificity) and (2) their reported ability totrigger activation of resting platelets (i.e., systemicpro-thrombotic risk).216,217 Nonetheless, from a fea-sibility demonstration standpoint, decoration ofmicro- and nanoparticles with these peptides haveresulted in platelet-mimetic constructs that haveshown promising hemostatic ability in vitro andin vivo.215,218 The issue of specificity can be potentiallyresolved by utilizing peptides that have higher selectiv-ity to the active (Fg-binding) form of platelet GPIIb-IIIa. To this end, researchers have used Fg γ-chain rele-vant H-12 peptide (i.e., HHLGGAKQAGDV) or Fgfunction-mimicking GPIIb-IIIa-specific cyclic RGDpeptides (e.g., cyclo-CNPRGDY[-OEt]RC) to decoratealbumin, polymeric or liposomal particles to createsynthetic platelet mimics, that may provide higherfunctional specificity in hemostatic action.219–223 TheH-12 peptide decorated liposomal particles have beenfurther studied for targeted delivery of ADP (a plateletagonist) to augment hemostatic capability and the H-12 peptide has also been used to surface-decorate disc-shaped ‘nanosheets’ made of poly-lactate-co-glycolate(PLGA) polymer, for potential topical hemostaticapplications.224,225 All of these micro- and nanoparti-cle designs that utilize peptides to mimic Fg-mediatedplatelet aggregation mechanisms, have shown thecapability of inducing platelet aggregation in vitro, andsome have shown hemostatic promise in vivo in a vari-ety of preclinical animal models including tail veinbleeding, tail transection, femoral artery bleeding, liverresection and blunt trauma. Clinical translation ofthese designs and technologies will be dependent onestablishing large-scale manufacturing processes,demonstrating sterilizability and long shelf-life withoutcompromising bioactivity, and evaluating systemicsafety, circulation lifetime and hemostatic efficacy inlarger animals of chronic and acute bleeding scenarios.

Nanomedicine Systems That CombinePlatelet’s Adhesion and AggregationMechanismsPlatelet-mediated hemostasis is rendered by the cooper-ative action of the adhesive and aggregatory functional-ities of platelets.226,227 Therefore, in recent years therehas been significant interest in developing nanomedi-cine systems that can combine these two functions on asingle particle platform. To this end, latex beads weresurface-decorated simultaneously with rGPIbα proteinfragments and H-12 peptides and liposomes were



surface-decorated with rGPIbα protein fragments andcollagen-binding peptides.198,228 Studies with theseconstructs revealed that while combining two differentmotifs on a nanoparticle surface (i.e., heteromultivalentmodification), if there is a significant size differencebetween the motifs (e.g., large rGPIbα fragment com-pared to small peptides), the motifs need to be pre-sented at different canopy levels from the particlesurface by using different spacer lengths, in order tominimize steric interference (or masking) of the smallermotifs by the larger motifs. Such steric interferenceissues can be potentially resolved by using only smallpeptides to combine the adhesive and aggregatory func-tions of platelets. This was demonstrated recently bycombining the platelet-mimetic adhesion-promotingpeptides VBP and CBP with the platelet-relevantaggregation-promoting cyclic RGD-based Fg-mimeticpeptide (FMP) on liposomes or albumin-basedparticles.223,229–231 These ‘functionally integrated’platelet-mimetic systems showed higher hemostatic effi-cacy in vitro (in flow chamber set-up), as well as,in vivo (in mouse tail transection bleeding model), com-pared to systems that had adhesion functionality onlyor aggregation functionality only. The liposome-baseddesign was recently issued a patent (US 9107845) andregistered as ‘SynthoPlate’ and is being tested for treat-ing thrombocytopenia as well as trauma, in preclinicalanimal models. These studies suggest that mimickingplatelet-relevant multiple hemostatic functions via het-eromultivalent ligand modifications on biocompatiblenanoparticle platforms may lead to superior designsfor synthetic platelet substitutes (Box 2).

SYNTHETIC NANOMEDICINESYSTEMS USING RBC AND PLATELETMEMBRANE-BASED ‘CLOAKING’

A persistent challenge in nanomedicine design is theinteraction of nanoparticles with immune cellsin vivo, resulting in rapid uptake and clearance of theparticles by phagocytic processes of the mononuclearphagocytic system (MPS). One way to reduce suchclearance is the decoration of nanoparticle surfaceswith hydrophilic polymer brushes (e.g., with PEG)that reduces opsonization of particles in blood circu-lation and thereby reduces phagocytic recognitionand uptake. This approach has come to be known asthe ‘Stealth Nanoparticle’ technology.88,89,232 Inrecent years, researchers have focused on anotherway to reduce this clearance by utilizing particle sur-face presentation with ‘markers of self’ to evadeimmune recognition. To this end, a ‘marker of self’biomolecule that has garnered tremendous interest is

CD47, an integrin-associated cell surface glycopro-tein reportedly found on RBCs and polymorphonu-clear leukocytes.233 Surface modification ofnanoparticle systems by CD47 moieties has demon-strated a reduction in macrophagic uptake and

BOX 2

HETEROMULTIVALENT MODIFICATIONAND INTEGRATION OF BIOCHEMICALAND BIOPHYSICAL PARAMETERS

In many biological mechanisms, cell–cell interac-tions and cell–matrix interactions are mediatedby a concert of heterotypic ligand–receptormechanisms rather than a single type ofligand–receptor interactions. In case of bloodcells, this is found extensively in the intercellu-lar as well as cell–matrix interactions of plate-lets and WBCs. Recent advancement innanomedicine research has focused on elucidat-ing and exploiting these heteromultivalentinteractions in enhancing the bioactive perfor-mance of nanoparticle systems. For example,WBC interactions with inflamed endotheliumare mediated by a combination of integrin- andselectin-based binding and rolling mechanisms.Platelet interactions at the injury site aremediated by a combination of vWF-, collagen-,integrin-, and selectin-binding mechanisms.Such combinations can be efficiently simulatedon nanoparticle platforms by controlled code-coration of the particle surface with multipletypes of ligands. In carrying out such hetero-multivalent decorations, researchers shouldensure that the decorated motifs do not steri-cally interfere with each other and this can beachieved by controlling the size as well as thespacer lengths of motif conjugation. Anotheremerging area of cell-inspired nanomedicineresearch is the utilization of cell-mimetic bio-physical parameters, e.g., size, shape, mechani-cal modulus, surface charge, and so on,combined with the cell-inspired biochemical(e.g., ligand–receptor interactions) parameters.Bottom–up fabrication techniques like self-assembly and emulsion-directed precipitation,as well as, top–down fabrication techniques likecontrolled lithography, hydrodynamic jetting,nonwetting templates, thermostretching, andso forth can provide effective ways to incorpo-rate such morphological and physico-mechanical parameters in design refinement ofcell-inspired nanomedicine systems.



clearance.234 Rationalizing from this immune-evading promise of CD47 moieties and based on thefact that RBCs bear these moieties, researchers havedemonstrated an interesting approach of cloakingsynthetic nanoparticles (e.g., PLGA nanoparticles)with isolated RBC membranes to create biosyntheticnanomedicine systems for drug delivery.235–238 SuchRBC membrane based bio-interfacing of nanoparti-cles has demonstrated notable increase in circulationlifetime of the particles, as well as, ability of the par-ticles to allow passive targeting-based drug deliveryto tumors.239 In analogous approaches, variousresearch groups have also reported the developmentnanoparticles coated with isolated platelet mem-branes, and these biosynthetic particle systems haveshown the ability of delivering drugs to platelet-interactive disease states like metastatic tumors andbacterial infections.240–242 While bio-interfacing ofnanoparticles with isolated RBC and platelet-derivedmembranes represent an interesting approach to lev-erage biological complexity of native cell-surfacemoieties on synthetic particle platforms, this mayalso present significant challenges regarding clinicaltranslation. For example, utilization of RBC-derivedmembranes would necessitate either type matching ofRBCs or universal donor (type O) RBCs or utiliza-tion of patient’s own RBCs to create patient-specificsystems to minimize immunogenic risks. Similarly,utilization of platelet-derived systems would necessi-tate ensuring minimal immunogenicity and batch-to-batch reproducibility of membrane surface-proteincomposition to guarantee clinically translatable con-sistent bioactivity of the resultant nanoparticle tech-nologies.243 It has also been reported that extraction,isolation and purification processes applied to cellmembrane systems often result in significant loss ofmembrane protein functions and high heterogeneityin residual bioactivities.244 This can become a signifi-cant logistical barrier for quality control and func-tional uniformity of these membrane-derived bio-interfaced nanomedicine systems compared to fullysynthetic (e.g., function-specific synthetic peptide dec-orated) nanomedicine systems.

SYNTHETIC NANOMEDICINESTRATEGIES FOR WBC SUBSTITUTES

The primary function of circulating WBCs is to main-tain immune surveillance and protection against for-eign entities (e.g., bacteria) and diseased cells, andrender cytotoxic destruction and phagocytic clear-ance of these entities via complex concert of signaltransductions, intercellular interactions, and cellular

secretions. These complex multifactorial processeshave not been leveraged and mimicked effectively onsynthetic micro- or nanoparticle platforms. However,there has been some interesting synthetic approachesregarding functional mimicry of WBCs,e.g., (1) engineering of ‘Leukosomes’ where mem-brane glycoproteins from murine macrophages wereextracted and reconstituted into proteoliposomes thatcould deliver drugs (e.g., Dexamethasone) toinflamed vasculature, (2) engineering of ‘Leuko-poly-merosomes’ where polymeric vesicles were hetero-multivalently surface-decorated with selectin- andintegrin-binding motifs to render leukocyte-mimeticinteractions with ‘diseased’ cells for targeted releaseof hydrogen peroxide (H2O2) from these vesicles tokill the target cells, and (3) engineering of ‘Leuko-likeVectors (LLVs)’ where nanoporous silica particleswere surface-coated with cellular membranes isolatedfrom freshly harvested leukocytes to render proper-ties like avoidance of opsonization and macrophagicuptake, preferential binding to inflamed endothelium,and facilitated transport of therapeutics across theendothelium while avoiding the lysosomalentrapment.245–247 Figure 9 shows schematic repre-sentation of these various nanomedicine designapproaches based on leukocyte biology. Further rig-orous in vitro and in vivo evaluation of each of theseapproaches is needed to ensure manufacturing repro-ducibility, consistent composition, and bioactivity ofthe surface motifs, in vivo safety and therapeuticpromise.

NANOMEDICINE SYSTEMSMIMICKING PHYSICO-MECHANICALPROPERTIES OF BLOOD CELLS

The morphology and physico-mechanical propertiesof blood cells, especially RBCs and platelets, playimportant role in their collisional interactions underhemodynamic flow environment and thereby influ-ence their margination in flowing blood.158,248–252

RBCs are biconcave, discoid, highly flexible cells of8–10 μm in diameter, while circulating ‘resting’ plate-lets are biconvex, discoid, relatively stiffer cells,about 2–3 μm in diameter. Under hemodynamic flowenvironment, these physico-mechanical propertiesinfluence RBCs to congregate at the center of bloodvessels while platelets are pushed out to marginatetoward the vessel wall in a relatively cell-freezone.156,157 Based on these observations, someresearch approaches have tried to mimic the shape,size and flexibility of RBCs or platelets in nanomedi-cine design. Developing processes for the



manufacture of nonspherical, anisotrpic particles hasbecome an exciting area of nanomedicine research inrecent years, because the resultant particles showintriguing aspects regarding their hemodynamiclocalization, circulation life-time and interactionswith target cells and tissues.253 Some of these pro-cesses have been utilized to manufacture micro- andnanoparticle systems that mimic shape, size and flexi-bility aspects of RBCs and platelets. For example,using a process called particle replication in nonwet-ting templates (PRINT), researchers have manufac-tured RBC-mimetic discoid microparticles thatshowed higher circulation lifetime in vitro andin vivo.119,254,255 RBC- and platelet-mimetic particleshave also been developed utilizing techniques thatuse thermo-stretching of polymer templates followedby L-B-L assembly of polyelectrolytes on templatesand selective dissolution of templates, and these haveshown significant promise regarding in vivo biodistri-bution, microfluidic margination, wall adhesion, anddrug delivery.230,256–258 In a recent report, comparedto stiff spherical particles, platelet-mimetic softer dis-coid particles exhibited better margination and adhe-sion (lower koff) as measured by the duration ofparticle contact with the surface.259,260 In furtheradvancement of this research, platelet-mimetic softdiscoid particles were surface-decorated heteromulti-valently with VBP, CBP and RGD peptides, and thesefunctionally integrated ‘platelet-like nanoparticles’(PLNs) exhibited enhanced hemostatic efficaciesin vitro and in vivo, compared to stiff spherical parti-cles.230 Studies have also been carried out with

rGPIbα-decorated liposomes having various lipidmembrane fluidities that result in various levels ofmembrane flexibility.193 In these studies, it was foundthat ‘soft’ liposomes rolled slowly and showed betteradhesion on vWF-coated surfaces, compared to‘hard’ liposomes. Figure 10 shows a couple represen-tative schematics of anisotropic discoid particle fabri-cation technologies and corresponding fluorescenceor SEM images of the particles. These studies indicatethe promise of incorporating RBC- or platelet-inspired biophysical design parameters to furtherrefine the design of bio-inspired drug delivery parti-cles (Box 2).

CONCLUSION

Blood substitutes and artificial blood componentsremain highly sought after technologies that canpotentially resolve several limitations associated withthe widespread use of natural blood products,e.g., limited supply, expensive isolation and storage,limited portability, pathologic contamination risks,and biological side-effects. The use of milk, saline,Ringer’s solution, or animal-derived plasma to substi-tute for human blood, have been recorded in historyduring the 19th century.261 These materials couldpartly act as volume replacement systems to maintainosmotic balance (and possibly provide nutrition), butdid not have any component that essentiallysimulates the function of the various blood cells.Subsequent scientific research in this area ofvolume replacement therapies has led to the

FIGURE 9 | Schematic representation of design and components of some WBC (leukocyte)-inspired biosynthetic nanosystems, along with theirdesign/trade names.



development of plasma expander technologies undertwo main classes of materials, namely, crystalloids(e.g., sucrose, dextrose, etc.) and colloids(e.g., albumin, hydroxyethyl starch, etc.).262,263

While the identification of blood groups and theirsignificance in guiding blood transfusion happenedduring the late 1800s and early 1900s, the research issynthetic ‘functional’ substitutes of blood cells startedonly around the 1960s with the discovery of PFCsand this research intensified from the 1980s onwardbecause of blood transfusion-related HIV risk issues.Since then, nanotechnology, materials engineering,and deeper understanding of blood cell biology haveprovided unique avenues to design semi-syntheticand synthetic systems to mimic functions of RBCs,platelets, and leukocytes. To this end, bio-inspiredapproaches for mimicking RBC function havefocused mostly on capturing the oxygen transportingproperties of Hb, either by using cross-linked andpolymerized Hb directly or encapsulating Hb withina variety of microparticle and nanoparticle systems.The persistent challenges in these approaches are(1) limited availability of obtaining Hb from humanand bovine sources and infection risks associatedwith these sources, (2) limited mutation combinationsand high cost of obtaining Hb from recombinantsources, (3) maintaining consistent high loading effi-ciency of Hb, (4) presenting of the redox environ-ment that modulates Hb-mediated O2 and CO2

transport, (5) maintenance of reasonably long

circulation lifetime, (6) degradation or leakage of Hbwithin circulation, and (7) minimization of NO-scavenging by Hb leading to a variety of vascularand toxic side effects. Although some of the Hb-based synthetic RBC designs have partly addressedthese challenges, none has been able to completelyresolve these issues yet and that has remained as thebarrier to clinical translation. Newer RBC-inspirednanomedicine designs like Hb intermolecularly andintramolecularly cross-linked with pharmacologicallyactive agents, Hb polymerized with effector mole-cules and antioxidant enzymes, Hb encapsulatedalong with redox enzymes and effector moleculeswithin liposomes, polymersomes, or polymer-basedtoroidal nanoparticles, may provide refined strategiesto resolve these issues. PFC-based microparticle andnanoparticle systems have already undergone clinicalapproval for contrast agent applications in ultra-sound imaging and therefore leveraging this technol-ogy to clinically translate Hb-independent syntheticoxygen carriers, may be another promising option. Inthe field of bio-inspired approaches for mimickingplatelet function, majority of research has focused oncapturing the aggregation mechanism of active plate-lets by coating synthetic microparticle and nanoparti-cle platforms with Fg, Fg-fragments, Fg-relevantpeptides, or fibrin-binding motifs. While many ofthese designs have shown in vitro and in vivo hemo-static capabilities, they may also present systemicpro-thrombotic and pro-coagulant risks. Recent

Filling molds of controlled

geometry with polymers

and photo-curing

(a)

(b)

PRINT Technology

LBL assembly on templates

Heat

stretching

Rigid polymer sphere

(e.g. Polystyrene)Spheroidal

core Controlled coating on

spheroidal template

Discoid

particles

Core

dissolution

LBL assembly of

polyelectrolytes

Transferring cured

particles of moldedshape to receiver sheet

Transferred

particlesDiscoid particles

harvested by solvent

Particles 5-6 μ in diameter

Nanoscaleparticles

Microscaleparticles

Ⓡ

FIGURE 10 | Process scheme and representative particle images (fluorescence or SEM) for (a) the PRINT technology and (b) the template-mediated thermostretching and layer-by-layer assembly based technology to produce micro- and nanoparticles that mimic the size and shape ofblood cells (e.g., RBCs and platelets).



refinement of this design approach has focused onincorporating and combining platelet-mimetic injurysite-adhesive functions along with active plateletaggregatory functions and these nanomedicinedesigns have shown significant improvement in pro-moting hemostasis in a targeted fashion while mini-mizing systemic off-target activity. An importantaspect of this design refinement is to ensure that theparticle surface-decorating peptide ligands have spec-ificity of interactions with active platelets and injurysite-relevant exposed proteins (e.g., vWF and colla-gen), so that they do not interact with circulatingresting platelets to cause systemic thrombotic risks orpromote thrombin and fibrin generation in plasma tocause systemic coagulation risks. Another interestingresearch possibility in this area is the utilization ofnanoparticle system for targeted delivery ofcoagulation-modulating agents like PolyP, tranexa-mic acid (TXA), and various recombinant coagula-tion factors.264,265 Furthermore, an emerging areaof research in the field of blood cell-mimetic nano-medicine is the recent exploration of morphologicaland physico-mechanical parameters (e.g., size,shape, stiffness, flexibility, etc.) inspired by bloodcells (e.g., RBCs and platelets) that can enhance theperformance of synthetic microparticle and nanopar-ticle systems in the vascular compartment underhemodynamic flow environment. Integrating thesebiophysical aspects with the biochemical functional

aspects of RBCs and platelets, can lead to superiortechnologies for synthetic blood substitute applica-tions. As these technologies evolve, rigorousresearch should be performed to evaluate the biolog-ical performance of these technologies in standar-dized preclinical animal models, not only in thecontext of specific functions of the blood cells butalso regarding in vivo safety, especially off-targetside effects and immune response. Clinical transla-tion of these bio-inspired nanomedicine systems forsynthetic blood substitutes require a concerted effortfrom biomedical and materials engineers, bloodbiology experts, hematology, and trauma clinicians,as well as, appropriate regulatory agencies, to estab-lish standardized in vitro and in vivo evaluationmetrics and suitable clinical studies. As recentlyhighlighted in the ‘State of Science in TransfusionMedicine’ by the NIH, there is a significant clinicalinterest in donor-independent blood cells for trans-fusion applications.266 While one arm of theresearch is currently focusing on generation of bloodcells ex vivo, from precursor cells and stem cellsusing specialized bioreactors and cell culture tech-nologies, a parallel arm is continuing to work onsynthetic nanomedicine approaches for blood cellsubstitutes.267–271 Robust research and rigorous pre-clinical and clinical evaluation of these technologiescan lead to fully artificial blood substitutes fortransfusion applications in the future.

ACKNOWLEDGMENTS

ASG is supported by the National Institutes of Health (NIH) awards R01 HL121212 and R01 HL129179.The content and perspectives expressed in this publication are solely the responsibility of the author and do notnecessarily represent the official views of the National Institutes of Health.

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