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Red Blood Cells for Drug Delivery

Junjie Yan, Jicheng Yu, Chao Wang, and Zhen Gu*

Dr. J. Yan, J. Yu, Dr. C. Wang, Prof. Z. GuJoint Department of Biomedical EngineeringUniversity of North Carolina at Chapel Hill and North Carolina State UniversityRaleigh, NC 27695, USAE-mail: [email protected]. Z. GuCenter for Nanotechnology in Drug Delivery and Division of Molecular PharmaceuticsUNC Eshelman School of PharmacyUniversity of North Carolina at Chapel HillChapel Hill, NC 27599, USA

The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/smtd.201700270.

DOI: 10.1002/smtd.201700270

Meanwhile, RBCs present complete biodegradability without any toxic by-products, and simultaneously keep the organisms off the toxic effects from the loaded drugs.[7] In addition to a bicon-cave shape and high surface to volume ratio (A/V), RBCs are lacking of nucleus and many organelles. These unique traits provide sufficient space for inner car-goes loading and surface modification.[8] Recently, RBCs were also demonstrated to reduce uptake of nanoparticles (NPs) by many mononuclear phagocytic system

(MPS) organs such as the liver and spleen, except the lung.[9] More importantly, the ample natural markers (such as CD47, proteins, sialic acid, and glycan) on the membrane of RBCs contribute to absence of immunogenic clearance of RBC-asso-ciated formulations.[10]

Due to the long period of research exploration and opti-mization, RBC-based carriers have become one of the most crucial nature-derived DDSs in nanomedicine, ranging from loading approaches,[5c,8,11,12] cargoes,[6a,7,13] RBC-derived categories [11c,d,14] to applications in imaging,[15] transfusion medicine,[16] detoxification, and vaccination.[17] However, some challenging issues, such as integrity preservation, storage, contamination, restricted targeting, and penetrating capability of RBC-based nanocarriers need further perfection for clinical translation. Particularly, various bioresponsive RBC-based platforms have been recently explored to facilitate targeted drug release, presenting reduction of dosing frequency and enhanced therapeutic efficacy (see Section 5).

Herein, we summarize the advances in the research of RBCs for drug delivery, primarily including loading approaches, cat-egories of RBCs, and loading cargoes, especially focusing on the latest progress in RBC membrane-cloaked NPs and RBC-mimetic NPs. Moreover, the state-of-the-art stimuli-responsive RBC-based DDSs, the drug release from which can be triggered by magnetic field, light, ultrasound, enzymes, pH, temperature, and glucose, are also introduced (Figure 1). An outlook and prospects of this field are also discussed in the end.

2. Categories of RBC Carriers

As the increasing demand of diversity, accuracy, and control-lability of multiscale in modern medicine, it is largely insuffi-cient to conquer all the issues of DDSs by natural RBCs alone. For instance, the micrometer size of RBCs confines their use to vascular drug delivery, which promotes the birth of RBC membrane-cloaked NPs and provides various functions when different core materials are introduced. On the other hand, although approaches of RBC loading without the need for

Red blood cells (RBCs), or erythrocytes, have been promising endogenous candidates for drug delivery for more than four decades. Armed with a better understanding of the diverse mechanisms and paths for drug delivery, researchers have achieved great progress in manufacturing RBC-based carriers, from both the scientific and clinical viewpoints. Here, the loading methods and loading therapeutics by RBC carriers are surveyed, and recent advances in the design of stimuli-triggered RBC carriers highlighted. Key devel-oping trends and challenges, are also discussed, and an outlook is presented.

Nanomedicine

1. Introduction

With the rapid progress in materials and chemistry, consid-erable improved achievement has been exploited in drug delivery systems (DDSs).[1] Despite this, many of DDSs are exogenous based and far beyond the acceptable demands in clinics, because they often fail to match the complexity nor-mally behaved from the natural biological entities, exhibiting poor performance in treatment efficacy, pharmacokinetics, and biocompatibility.[2] For example, PEGylation is the most widely applicable strategy to shield from the reticuloendothelial system (RES) and to improve in vivo circulation lifespan and stability.[3] However, the immune system can be potentially activated upon PEGylation and lose effect when multiple administrations are required.[4] By contrast, endogenous carriers possess inherent biological properties from the natural evolution and keep them-selves away from these issues.[5] Among them, red blood cells (RBCs), also known as erythrocytes, are one of the most his-torical and popular natural carriers serving for drug delivery since 1970s because of their excellent accessibility (the most abundant cell in the human body), excellent biocompatibility, tunable loading capacity, and long blood circulation half-life (≈120 d in humans).[6]

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ex vivo manipulations with RBC have been designed and showed excellent efficacy and safety in preclinical studies,[11b,18] many modified RBCs still have such limitations upon being uti-lized in vivo due to the possible damage during ex vivo manipu-lation, such as the destruction of plasma membrane integrity and reduction in circulatory half-life of the modified RBCs.

2.1. Pristine RBCs

Generally, pristine RBC-decorated nanomaterials present longer circulation time, but the clearance of decorated-RBCs is much faster than RBCs themselves.[11d] Also, RBC carriers may otherwise alter the activity of cargoes. For example, coupling to RBC protects cargoes from plasma inhibitors and modulates the interactions between NPs and membranes, particularly via masking cargoes by RBC glycocalyx.[19] Additionally, RBC car-riers can alter the distribution of nanocarriers within the vas-cular lumen, redistributing them from the marginal layer to the mainstream.[11d] Based on these variations, RBCs not only are utilized to load drugs, but also are leveraged for carriage of nanovectors. As the approaches mentioned above, molecules can be loaded on/into RBCs either by encapsulation or surface binding, but the encapsulation methods with ex vivo loading process will inevitably be confronted with the challenges of loss of RBCs’ biocompatibility,[20] and the sizes of encapsulated molecules must be small enough to enter the membrane pores of RBCs during the hypotonic procedure. By contrast, surface

binding approaches provide access for molecules with more controllable sizes, surface potential and functions (imaging, diagnosis, and therapy). Recently, Wang et al. constructed an RBC-based microcarrier for orthogonal near-infrared (NIR) upconversion coregulated site-specific oxygen delivery and photo dynamic treatment of hypoxia tumor.[21] Moreover, this microplatform was also prominent to avoid biological uptakes by the inflamed endothelium, MPS, and RES.

Pristine RBCs take up ≈50% of blood volume and are well-known as RBC carriers for drug delivery for over 35 years.[22] RBCs are characterized with an average diameter of ≈7 µm, a thickness of ≈2 µm, and a large volume of 90 µm3, whereas the average diameters of capillaries are typically ≈2–3 µm, the high quantity and conflicting dimension of RBCs will both influence the way of travel of RBCs in blood vessels.[23] Despite of the high flexibility and elasticity, which supply RBCs with the capabilities to squeeze through narrow capillaries, it is still difficult for RBC carriers to deliver molecules to targeted sites

Junjie Yan received his Ph.D. degree from University of Science and Technology of China in 2014, in the Department of Polymer Science and Engineering. Then, he joined Jiangsu Institute of Nuclear Medicine (Wuxi) as a research asso-ciate. Currently, he is a visiting scholar in the joint Department of Biomedical

Engineering at University of North Carolina (UNC) at Chapel Hill and North Carolina State University, under the guidance of Prof. Zhen Gu. His research interests focus on synthetic methodologies and applications of functional materials in molecular imaging, detection, and theranos-tics of cancer and other diseases.

Zhen Gu obtained his Ph.D. degree at the University of California, Los Angeles, under the guidance of Prof. Yi Tang in the Department of Chemical and Biomolecular Engineering. He was a post-doctoral associate working with Prof. Robert Langer and Prof. Daniel Anderson at MIT and Harvard Medical School. He is currently an

Associate Professor in the Joint Department of Biomedical Engineering at University of North Carolina (UNC) at Chapel Hill and North Carolina State University. He also holds a joint position in the Eshelman School of Pharmacy and Department of Medicine at UNC. His group studies controlled drug delivery, bioinspired materials, and nanobi-otechnology, especially for cancer and diabetes treatment.


Figure 1. Schematic of RBC carriers for drug delivery applications, sum-marized from different perspectives. Loading methods typically include surface binding, encapsulation, and mechanical extrusion. Loading car-goes mainly contain therapeutic drugs, enzymes, and nanocarriers. RBC categories primarily include pristine RBCs, RBC membrane-cloaked nano-particles, RBC mimetics, and nanoerythrosomes. Responsive RBC-based carriers can be triggered under stimuli, such as magnet, light, ultrasound, pH, enzyme, heat, and glucose.



in the absence of any alteration or modification to the composi-tion and the structure of RBCs owing to their restricted space of activity. However, this limitation could be advantageous for spe-cific delivery of thrombolytic agents. For example, tissue-type plasminogen activator (tPA) is a protein used in the medical treatment of thrombolysis, but it is not suitable for prophy-laxis because of its short circulation and unspecific dissolu-tion of preexisting and nascent clots. When tPA was coupled to the surface of RBCs, the fibrinolytic activity of RBC-tPA in the blood increased more than tenfold compared to that of free tPA.[24] More significantly, RBC-tPA only dissolved nas-cent clots, while causing no side effects on preexisting clots or extravascular tissues.

To address the micrometer-sized limitations of RBCs in drug delivery, Lejeune et al. prepared pristine RBC-membrane derived nanoerythrosomes via sonication and physical extrusion respectively, with a diameter ranging from 100 to 200 nm.[25] Generally, one RBC can be converted into 4000–5000 nano-erythrosomes via either a sonication dismembrator or probes or electrical break-down (Figure 2).[11c,26] In the beginning, this kind of natural derivative from RBC membranes pre-sented promise in vitro experiments; however, the formulation of nanoerythrosomes sacrificed the biocompatibility of RBC membranes, which rendered fast clearance from the blood and limited its wide exploration in vivo.[27] Recently, the artesunate-conjugated nanoerythrosomes were found to lose their integri-ties after only 9 h, which was much shorter than the circulation time of natural RBCs.[28]

2.2. RBC Membrane-Cloaked Nanoparticles

The desirable requirements for nanocarriers contributing to drug delivery mainly include long circulation period in blood, stable and persistent drug release, as well as highly efficient and specific targeting ability without rapid clearance by immune cells. Generally, PEGyation has been valued as a gold standard to improve the circulation time by creating a stealthy hydration shell, preventing nanocarriers, such as liposomes and micelles, from opsonization and offering better tumor accumulation via enhanced permeability and retention (EPR) effect.[1a,29] Up to now, PEGylation technique has given birth to several clin-ical products involving PEGylated liposomes (Doxil, Caelyx) and PEGylated proteins (Oncospar, PEG-Intron, PEGASYS, Neulasta).[30] However, recent study revealed the unexpected anti-PEG immunological response and inspired the investiga-tion of zwitterionic polymers such as poly(sulfobetaine) and poly(carboxybetaine), whose strong hydration was shown to keep off the nonspecific protein absorption.[31]

On the other hand, natural materials get along well with bio-logical surroundings and have aroused extensive interests in biomedical applications. For example, RBCs have been viewed as a significant reference to design and synthesize novel delivery platforms because of their unique structures and various sur-face proteins. However, the use of pristine RBCs is largely restricted owing to the micrometer size and limited space of activity. In 2011, Zhang and co-workers developed a biomimetic approach to decorate biodegradable poly(lactic-co-glycolic acid)

(PLGA) with RBC membranes (Figure 3).[32] Remarkably, this top-down method efficiently reduced the overall diameter of the resulting NPs. Moreover, the bioactivity of RBC mem-branes was greatly retained with long circu-lation time, and no denaturation of proteins was observed as that occurred via a chemical bioconjugation strategy.[33] Additionally, the more controllability and tunability of syn-thetic NPs can also supply RBC membrane-coated hybrid nanomaterials with more func-tions for advanced drug delivery. Generally, the fabrication process of RBC membrane-cloaked NPs basically follows a two-step routine.[11c,32] First, RBC membranes vesi-cles with various sizes are easily prepared by mechanical extrusion through polycarbonate track etch membrane filters (0.1–10 µm) after initial hypotonic treatment. Then, RBC membranes and NPs are extruded together for several times to fuse each other.

The most important aspects for successful RBC membranes coating are membrane/NPs ratios and surface charges.[34] Taking PLGA NPs (diameter around 100 nm) as an example, a recommended ratio was 100 µL blood per mg polymer to ensure the complete shielding of NPs. High sialic acid content renders RBC membranes negatively charged, and the strong electrostatic interaction with positively charged NPs could possibly


Figure 2. Fabrication of nanoerythrosomes using extrusion and sonication methods. Reproduced with permission.[11c] Copyright 2016, The Royal Society of Chemistry.



collapse the fluidic lipid bilayer and lead to severe aggregation. Therefore, the cloaked NPs are usually negatively charged, with efficient camouflaging as characterized by transmission elec-tron microscopy (TEM). The integrities of RBC membranes were greatly retained with CD47 markers at the same density as natural RBCs, and the surface of cloaked NPs was mostly with right-side-out membrane orientation (≈84%) according to the fluorescence quenching performance. On this basis, Gao et al. recently encapsulated perfluorocarbon (PFC) within PLGA NPs and subsequent cloaking with RBC membranes led to the forma-tion of PFC@PLGA-RBC membranes NPs (Figure 4).[35] Com-bining the high oxygen solubility of PFC and long blood circu-lation of RBCs, this nanoscaffold remarkably circumvented the issue of tumor hypoxia and greatly improved the responses of the tumors to radiotherapy treatment. Yang and co-workers also

cloaked melanin NPs with RBC membranes, simultaneously obtaining excellent photothermal property, enhanced blood retention and improved tumor accumulation.[36] Upon light irradiation (808 nm), this nanoplatform presented higher photo thermal therapy efficacy than that of bare melanin NPs. In another latest work, Zhang et al. described a RBC membrane-cloaked nanogel scaffold with combinatorial antivirulence and bioreductive antibiotic delivery against Staphylococcus aureus infection.[37] The disulfide bond crosslinkers in the network facilitated the accelerated drug delivery from RBC-nanogels in the intracellular reductive environment and therefore realized an improved antibacterial performance. More importantly, this powerful strategy was not only suitable for organic NPs, but also versatile to inorganic ones such as gold NPs, gold nanoc-ages, and magnetic iron oxide nanoparticles/nanoclusters.[38]


Figure 3. Synthesis and characterization of RBC membrane-cloaked NPs. A) Schematics of the preparation process of the RBC membrane-cloaked NPs. B) TEM images of RBC membrane-cloaked NPs, negative stained with uranyl acetate. C) RBC membrane-cloaked NPs exhibit enhanced circulation as compared to both PLGA NPs and corresponding PEGylated NPs. Reproduced with permission.[32] Copyright 2011, National Academy of Science.



Recently, Zhang and co-workers expanded this approach to hybrid membrane cloaking by introducing additional functionality for specific application.[39] They applied 1:1 protein weight ratio of RBC membrane to platelet membrane to cloak PLGA NPs, and each membrane was labeled with a dye for further Förster resonance energy transfer analysis. The reten-tion of the two membranes on the surface of PLGA NPs were nearly the same to the 1:1 input by the confocal laser micros-copy, and the core–shell structure with negative surface charge were verified via both TEM and dynamic light scattering. More significantly, proteins of RBC membrane (CD235a, CD47) and platelet membrane (CD41, CD61, CD47) were both largely pre-served on the surface of final [RBC-P]Ps, retaining similar cir-culation and distribution profiles of individually cloaked NPs.

2.3. RBC-Mimetic Nanoparticles

The properties of materials are not only related to their com-positions, but also dependent on their chemical structures.[40] It is usually shown that particles with deformable and non-spherical shapes exhibit enhanced circulation time,[41] thus several methods aiming to building RBC-mimetic NPs have been established. The strategy of layer-by-layer (LbL) assembly of polyelectrolytes on a template was developed by Doshi et al. (Figure 5A).[42] Typically, the biconcave NPs core templates were generated utilizing hollow polystyrene (PS) or PLGA spheres upon heat or solvent-induced fluidization. Afterward, cationic

poly(4-styrene sulfonate), anionic polymers poly(allylamine hydrochloride) (PAH) and hemoglobin (Hb) were stepwise deposited onto the templates via electrostatic interactions, followed by the layer crosslinking via glutaraldehyde and sub-sequent removal of templates, resulting in the formation of RBC-mimetic polymeric NPs. The elastic module of RBC-mimetic NPs can be readily tuned by template/shell materials, shell thickness and the density of crosslinking towards the same order of magnitude as that of natural RBCs. In addition, these RBC-mimetic NPs (7 ± 2 µm) can flexibly flow through narrow capillaries (5 µm in diameter) and restore their original shapes when getting out of the channels, presented comparable features as natural RBCs. Shi and Li groups created a facile templated framework pyrolysis approach for the preparation of RBC-shaped, hydrophilic, and hollow mesoporous carbon nanocapsules (HMCNs) (Figure 5B).[43] However, this strategy was not based on a mechanically deformed template, it resulted from the physical collapse of the remaining carbonaceous framework shell after the removal of silica cores, which was not strong enough to support the spherical hollow structures.

Photolithography presents advantages in synthesizing non-spherical well-defined particles, but its bench nature of pro-cess restricts the particle throughput and photoresist materials during manufacturing are not biocompatible.[44] The stop-flow lithography technique in microfluidic devices well resolves these problems, achieving improved resolution, increased throughout and multifunctional particles with sharp inter-faces.[45] DeSimone and co-workers initiated the approach of


Figure 4. A) Scheme of the preparation of PFC@PLGA-RBCM. PFC solutions were encapsulated inside the PLGA shell, which was then coated with RBCM. B) Dynamic PA monitoring of 4T1 solid tumors to determine the tumor oxygenation status by measuring the ratios of oxygenated hemoglobin (λ = 850 nm) and deoxygenated hemoglobin (λ = 750 nm). C) Quantification of tumor hypoxia densities at different time intervals. Reproduced with permission.[35] Copyright 2017, Wiley-VCH.



particle replication in nonwetting templates (PRINT), which was dedicated to the rapid synthesis of nonspherical parti-cles with high throughput and precisely controlled parameter such as shape, size, and elasticity (Figure 6).[46] For example, hydrogel-based RBC mimetics were synthesized to be equipped with the shape and deformability of RBCs via PRINT approach, utilizing 2-hydroxyethyl acrylate and poly(ethylene glycol) di acrylate (MW = 4000 g mol−1) as monomer and crosslinker, respectively. The negative-charged surfaces of RBC mimetics were realized by the addition of 2-carboxyethyl acrylate (10%, by weight), and the module of these RBC mimetics could be widely tuned by the ratio of crosslinker. For instance, by increasing the amount of crosslinker from 1% to 10%, the elastic moduli increased from 7.8 to 63.9 kPa, which was comparable to the same magnitude of elastic module of natural RBCs (26 ± 7 kPa). Finally, the deformability of RBC mimetics was similar to that of natural RBCs, namely, 1% crosslinked RBC mimetics stretched within the narrow channels with 239% increase in length and recovered original morphology when the flow was absent. Recently, Kozlovskaya et al. produced RBC-mimicking hydrogel capsules by consecutive photolithography and LbL

assembly approaches, which presented pH-triggered shape responses by the utilization of poly(methacrylic acid).[47]

Except from the physical geometry-mimetic manner, the introduction of chemical RBC components has been used to mimic the characteristics of natural RBCs as well. Different from whole RBC membranes coating strategy, conjugating pro-teins of RBC membranes to the surface of NPs is an alternative to mimic the properties of natural RBCs. For instance, CD47 is a glycoprotein that has ability to protect RBCs from the uptake by macrophages, and it has been used to cover PS NPs through biotin–streptavidin chemistry. As expected, the phagocytic uptake of PS NPs was greatly decreased after CD47 coating, and the antiphagocytosis effect could be impeded when PS NPs were conjugated with anti-CD47 antibody, confirming it was a CD-47-mediated blocking mechanism.[48] Subsequently, Discher and co-workers demonstrated that minimal “self” pep-tides designed computationally from human CD47 obviously increased the circulation period of PS NPs by the delay of mac-rophage-mediated uptake, which facilitated persistent circula-tion of paclitaxel (PTX) in tumor cites and the tumors shrunk more than the NPs without self-peptides.[49] As CD47 binding


Figure 5. A) LbL strategy to synthesize synthesis RBC-mimetic NPs from hollow PS template and PLGA template (electrohydrodynamic jetting). (a,b) SEM images of RBC-mimetic NPs. (a) RBC mimetics prepared from PS template by LbL deposition of PAH/BSA with crosslinked layers. (b) RBC-mimetic NPs prepared from PLGA template by LbL deposition of PAH/BSA and subsequent dissolution of the template. (c) Comparison of elastic mod-ulus of RBC-mimetic NPs with pristine RBCs and PLGA particles. (d) RBC-mimetic NPs (7 ± 2 µm) flowing through glass capillary (5 µm in diameter) (*, P < 0.001, n = 5; scale bars, 5 µm; insets, 2 µm). Reproduced with permission.[42] Copyright 2009, National Academy of Science. B) Schematic of the synthesis for HMCNs and the corresponding framework transformations. TEM images of SiO2@SiO2/C (a) and corresponding element mapping (b: silica, c: oxygen, d: carbon). (e,f) TEM images of HMCNs after oxidation. Reproduced with permission.[43] Copyright 2014, Wiley-VCH.



to signal-regulatory protein alpha leads to the inhibition of inflammatory cell attachment, Discher and co-workers verified that coating of polyvinyl chloride or polyurethane surfaces with CD47 obviously reduced human neutrophil and human mono-cyte derived macrophages, indicating CD47 coated surfaces could stand up to inflammatory cell interactions.[50]

3. Loading Methods

To develop effective RBC-based carriers and meet different purposes, a plenty of innovative techniques have been accom-plished. Primarily, there are two methods to load cargoes by either encapsulation within RBCs inner space or conjugated to the surface of RBCs.[11a]

3.1. Encapsulation

The membranes of RBC are composed of lipids and proteins in nearly mass proportion,[51] and these proteins act as channels for transportation of hydrophilic or hydrophobic cargoes.[52] Although molecules can reversibly pass through the protein channels of RBCs, the encapsulation efficiency of this method dependent on the concentration gradient is usually very low.[53] While in a hypotonic condition, it is an irreversible process consuming energy, under which RBC membranes swell and the pores on the membranes can open when the osmotic pres-sure exceeds the required energy for penetration through RBC membranes, accumulating cargoes in the cytosol.[5c,54] With

further ex vivo hypertonic/isotonic treatment, the membrane pores of RBCs can be sealed and molecules can be encapsulated within the inner space efficiently, preserving biophysical and immunological characteristics.[5c,52] Depending on the different procedures, osmotic lysis-based approaches mainly include hypotonic hemolysis, hypotonic dialysis, hypotonic dilution, and hypotonic preswelling, followed by the development of automated loading device (red cell loader).[55] In a recent study, Hamidi et al. described a coencapsulation method to simultane-ously load bovine serum albumin (BSA) protein and drug (phe-nytoin), obtaining prominently improved loading parameters and more controllable drug release behavior.[56] Other methods, such as chemical perturbation and electroporation are also used to encapsulate molecules into RBCs. However, these methods are sometimes not favored since their destructive damage to the membranes of RBCs is irreversible.[57] Besides the method mentioned above, endocytosis, electric cell fusion, lipid fusion, and novel cell penetrating peptides (CPPs)-mediated strategy are alternatively employed to fulfill RBCs encapsulation.[58]

3.2. Surface Binding

Apart from the physical encapsulation, cargoes can also be covalently or noncovalently coupled to the surface of RBCs. Coupling to the surface of RBCs can not only change the phar-macokinetics of cargoes, but also prolong their blood circulation and alter tissue distribution in vivo. By contrast, attachment of cargoes to the surface of RBCs does not obviously influ-ence the organ distribution of RBCs.[9] Initially proposed by


Figure 6. PRINT approach of fabricating RBC-mimetic NPs. A) Graphical depiction of the PRINT process used to fabricate RBC-mimetic NPs. Briefly (top to bottom), an elastomeric fluoropolymer mold (green) with disc-shaped wells was covered by an aliquot of the prepolymer mixture (red). The mold was passed through a pressured nip (black) covered by a high-energy sheet (gray), wicking away excess liquid from the mold surface while filling the wells of the mold. The filled mold was cured photochemically, yielding crosslinked hydrogel particles, which were harvested from the mold by freezing onto a thin film of 1% poly(vinyl alcohol) in water (blue) and peeling away the mold. Melting of this layer resulted in a suspension of hydrogel particles. Fluorescent images of hydrated RBC-mimetic NPs with varying percent crosslinker: B) 10% crosslinked, C) 5% crosslinked, D) 2% crosslinked, and E) 1% crosslinked. Scale bars are 20 µm. Reproduced with permission.[46a] Copyright 2011, National Academy of Science.



Muzykantov and co-workers, avidin–biotin interaction is an often used strategy to bind various kinds of bioactive agents.[59] Biotinylation of RBCs can be achieved either by N-hydroxysuc-cinimido biotin (NHS-biotin) or biotin hydrazide, Magnani et al. reported that NHS-biotin provided a cell recovery over 90%, about 1000 biotin molecules were successfully attached onto each RBC cell (mouse) and the RBC’s 24 h survival in circula-tion was not reduced.[60] Another covalent method is based on a positive targeting strategy, which is typically realized by firstly anchoring affinity ligands (peptides, antibodies, and aptamers) to the membranes of RBCs, followed by site-specific cargoes conjugation.[61] The extent of conjugation could be calculated according to the surface densities of membrane proteins via this approach, and it maximally decreases the risk of sacrificing the surface properties of RBCs.

Noncovalent coupling to surface of RBCs is well-known as a “hitchhiking” strategy to increase the in vivo circula-tion of cargoes (Figure 7).[9,62] The driving force of this tac-tics possibly results from the nonspecific physical inter-actions such as electrostatic, hydrogen bonding, van der Waals, and hydrophobic force.[11c] Although it is efficient in improving circulation lifetime as encapsulation approach, the loaded cargoes via “hitchhiking” strategy can finally detach from RBCs surface because of the shear forces and physical interactions with vascular walls.[62b] Recently, Simberg and co-workers made use of the membrane printing method to attach ligands to the surface of RBCs.[63] In their study, a

model ligand immunoglobulin (IgG) was chemically conju-gated with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol)-3400 and then integrated with RBC membranes via lipid transfer. Unlike the instability encoun-tered in glycosylphosphatidylinositol anchored proteins, IgG was retained in the membrane of RBCs with a prolonged terminal half-life (≈73 h). In another latest work, Moghimi and co-workers demonstrated that the adverse cardiopulmo-nary reactions in sensitive human subjects from intravenous injections of nanopharmaceuticals could be dampened or cir-cumvented by RBCs “hitchhiking” strategy, which improved the circulation times of nanopharmaceuticals and prevented macrophage recognition (Figure 8).[6b] Upon intravenous injection, RBC-bound carboxylated PS NPs did not increase pulmonary arterial pressure (PAP) significantly, while unbound NPs led to a considerable rise in PAP.

4. Loading Cargoes by RBCs

Although the loading capacity of RBCs is limited, many kinds of molecules with different sizes have been encapsulated.[6a,7,64] Also surface binding strategy provides direct covalent or nonco-valent coupling of cargoes to RBCs. In light of the different ulti-mate purposes, the loading objects dedicating to drug delivery primarily contain drugs, enzyme therapeutics, and nanocar-riers (Table 1).


Figure 7. Schematic of the preparation of RBCs adsorbed crosslinked NPs (RBC-cNPs) via a “hitchhiking” strategy (electrostatic interactions) and subsequent shear-induced release. cNPs were coassembled using heparin and thiolated poly-l-lysine (PLL-SH), followed by disulfide bond formation from thiol groups under oxidative conditions. Reproduced with permission.[62d] Copyright 2016, Wiley-VCH.



4.1. Small-Molecule Drugs

Up to now, various kinds of pharmaceuticals and biopharma-ceuticals have been combined with RBCs, including antiinflam-matory, antiviral, anticancer, and antiinfectious agents.[7,65]

4.1.1. Antiinflammatory Drugs

Corticosteroids and nonsteroidal antiinflammatory drugs are the two most antiinflammatory agents. As an outstanding rep-resentative, dexamethasone (DEX) is extensively used to treat acute inflammation, allergy, and immunology. However, long-term use of DEX could possibly lead to diverse side effects such as Kurshin syndrome, skin atrophy, pathological fracture, diabetes, peptic ulcer, and glaucoma,[66] which restrict their use in clinics. The perfect release behavior of DEX should be slow and persistent with satisfactory minimum dose, however, repeated administration at intervals of a few hours are required to achieve goal.[67] By contrast, when prodrug DEX-phosphate was loaded into RBCs, one administration could keep the effec-tive concentration of DEX for several days.[68] In cystic fibrosis

patients’ model, monthly infusions of RBCs loaded with DEX maintained the effective DEX level in the blood for 4 weeks and significantly reduced the inflammatory reactions.[69] Similar results were also observed in patients with inflammatory bowel disease (IBD), both in adults and pediatrics.[70] More impor-tantly, compared with free drug, treatment with DEX-loaded RBCs greatly decreased the possibilities of side effects from 80% to 25%.[71]

4.1.2. Anticancer Drugs

Up to now, chemotherapy is still the most commonly used therapy towards cancers, and plenty of carriers such as liposomes, micelles, polymeric NPs, and inorganic NPs have been constructed for anticancer drugs delivery, either by active targeting (decorated with specific ligands) or passive targeting (EPR effect).[72] Although PEGylation has been regularly adopted to increase the half-life circulation of drugs, it still needs frequent dosing and subsequently results in drug resist-ance.[73] With the aid of RBCs shielding, the circulation half-life of dequalinium increased from 4 h to around 5–6 d.[74] Initially,


Figure 8. Overcoming adverse reactions to NPs through RBCs “hitch-hiking” strategy. A) Differential interference contrast/fluorescence microscopy images of carboxylated PS NPs (750 nm) binding to human and pig (inset) RBCs. Scale bars, 10 µm. B) SEM image of a human RBC binding to PS NPs. Scale bar, 1 µm. C,D) Quantitative characterization of RBC-NPs interaction by fluorescence activated cell sorter (FACS). The results show the fraction of bound NPs and cells for humans and pigs (C) and how NPs are distributed on cells (D). E) Complement responses (sC5b-9 measurements) to RBC-bound and unbound NPs in human and pig whole blood. F) Hemodynamic changes in pigs measured by PAP variation. Reproduced with permission.[6b] Copyright 2017, Nature Publishing Group.



doxorubicin (DOX) was loaded into RBCs with RBC membrane crosslinking by glutaraldehyde,[75] which obviously enhanced RBCs uptake by macrophages and obtained improved thera-peutic effect in the treatment of lymphoid tumor.[76] Compared with free daunorubicins, the RBC membrane-cloaked counter-parts facilitated patients with acute leukemia a sustainable drug level in plasma, significantly decreasing the administration fre-quency and reducing side effects. These advantages were attri-buted to the long circulation of RBCs and the corresponding slower drug release from RBC-based carriers. Similar effica-cies were also available in utilization of other anticancer drugs including PTX, camptothecin (CPT), etc.[77]

4.1.3. Antiinfectious Drugs

RBC carriers have also presented potential in the specific delivery of antiinfectious drugs in macrophages cells located in the RES.[13b] For examples, Magnani et al. found that RBCs loaded with antiinfectious drug azidothymidine (AZT) increased the recognition and phagocytosis by macrophages and exhibited enhanced antiviral effect in comparison with free drugs for the treatment of human immunodeficiency virus (HIV) infection.[78] Moreover, they also used RBCs to encapsulate other antiinfectious drugs in the therapies for both HIV and acquired immune deficiency syndrome (AIDS),[79]

which paved the way for the further systematic trials in both animal models and human studies. Lanao and co-workers studied pharmacokinetics and biodistribution of amikacin encapsulated in RBCs carrier and showed that RBC-encapsu-lated amikacin preferred to accumulate in RES organs such as liver and spleen.[80] Hepatitis C virus (HCV) infection was typically treated with interferon-α (IFN-α) and ribavirin (RIBA), but accompanying high toxicity and complication under high dosage. Sulkowski enhanced the delivery of IFN-α and RIBA to the liver by RBCs shielding and attained improved therapeutic efficacy.[81] However, this strategy was not suitable for some antiinfectious drugs, Lisovskaya and co-workers demonstrated that antimalarial agent clotrimazole enhanced lysis to RBCs and this kind of oxidative damage subjected RBCs to rapid cleavage by hepatic RES macrophages.[82]

4.2. Enzyme

Aside from chemical drugs, therapeutic proteins, especially enzymes, have also been encapsulated in RBCs by the inspi-ration of enzyme replacement therapy and detoxification, with sustainable therapeutic levels in plasma and reduction of the frequency of dosage.[7,83] More importantly, the absence of chemical modification maximally preserves the bioactivity of enzymes and biocompatibility and pharmacokinetics of


Table 1. Some representative examples of RBC-based DDSs described in this article.

Platform Cargo Method Application Study stage Ref.

Pristine RBCs Small-molecule drugs

Dexamethasone Encapsulation Cystic fibrosis, IBD, chronic obstructive pulmonary disease, ulcerative Colitis

In humans [68–71]

Doxorubicin Surface binding Lymphoma In humans [75,76]

AZT Encapsulation HIV infection, AIDS In mice [78,79]

IFN-α and RIBA Encapsulation HCV infection In humans [81]

Enzymes or proteins l-Asparaginase Encapsulation Acute lymphoblastic leukemia In humans [85–89]

Glucocerebrosidase Encapsulation Gaucher’s disease In humans [90]

Thymidine phosphorylase Encapsulation Genetic mitochondrial deficiency In humans [91]

Adenosine deaminase

(Pegademase)

Encapsulation Severe combined immunodeficiency associated

with deficiency of adenosine deaminase

In humans [92–94]

Factor IX Encapsulation Hemophilia B In humans [95]

Tissue-type plasminogen

activator

Surface binding Thrombolysis In rats [24]

Nanocarriers Upconversion NPs Surface binding Oxygen delivery and photodynamic therapy In vitro [21]

RBC membrane-

cloaked NPs

PLGA NPs Mechanical extrusion Theranostics and in vivo imaging In mice [32–35,37]

Gold nanocages Mechanical extrusion Photothermal therapy In mice [38b]

Magnetic iron oxide NPs/nanoclusters Mechanical extrusion MRI-guided photothermal therapy In mice [38c,d]

Nanoerythrosomes Daunorubicin Surface binding Leukemia In vitro [25]

Fasudil Encapsulation Pulmonary arterial hypertension In rats [26]

Artesunate Surface binding Antimalarial therapy In rats [28]

RBC-mimetic NPs Iron NPs, heparin (PLGA, PS as templates) Encapsulation New preparation method In vitro [42]

Doxorubicin (mesoporous carbon

nanocapsules as templates)

Encapsulation Cancer therapy In mice [43]

Paclitaxel Encapsulation Cancer therapy In mice [49]



RBCs,[84] achieving remarkable reduction immunogenic reac-tions and improvement of therapeutic index. So far, more than 20 therapeutic enzymes have been encapsulated into carrier RBCs.[6a,7]

The most well-known therapeutic enzyme encapsulated in RBCs is l-asparaginase, which has been used for the treatment of the acute lymphoblastic leukemia (ALL) since 1967.[85] Typi-cally, l-asparaginase converts plasmatic l-asparagine (l-Asn) into l-aspartate plus ammonia, and malignant cells are defi-cient in asparagine synthetase thus they are unable to synthe-size l-Asn to satisfy metabolic demands, leading to the death of tumor cells.[86] However, use of l-asparaginase free drug is usu-ally encountered with side effects including short half-life in plasma, anaphylaxis, and blood coagulations. These problems were partially circumvented by PEGylation until RBC carriers were employed to encapsulate l-asparaginase, which elimi-nated plasma asparagine more efficiently and was confirmed in diverse animal models.[87] In human studies, a Phase I–II trial testing GRASPA on 24 patients in relapsed ALL also showed a reduction in both allergic reactions and coagulation disor-ders.[88] More significantly, the enhanced half-life in plasma rendered one single injection of GRASPA (150 IU kg−1) to get same effect as eight intravenous injections of free l-aspar-aginase.[88] In an updated Phase III trial, GRASPA showed enhanced asparaginase activity and significantly reduced the incidence of hypersensitivity in the nonallergic patients.[89]

Among these RBC-loaded enzymes, many of them are inten-tionally leveraged to treat the congenital diseases and some are even being tested in humans such as glucocerebrosidase, thymidine phosphorylase, and adenosine deaminase (ADA). Glucocerebrosidase is an enzyme used in replacement therapy for Gaucer’s disease, and it is one of the oldest enzymes stabi-lized via the encapsulation by RBCs.[90] Mutations in the gene encoding for the thymidine phosphorylase cause mitochon-drial neurogastroinstestinal encephalomyopathy. Upon using RBC-loaded thymidine phosphorylase, the urinary excretions of thymidine and deoxyuridine were 6% and 13% of the pre-therapy values respectively, and the plasma concentrations also decreased.[91] ADA deficiency leads to elevated cellular levels of deoxyadenosine triphosphate and systemic accumulation of its precursor. PEG-conjugated adenosine deaminase (Pegademase) was originally used for enzyme replacement therapy, but was limited by short half-life in plasma, frequent administrations, and high cost.[92] Bax et al. entrapped Pegademas in human RBCs and obtained a higher ADA concentration compared with free ADA.[93] Later, they subjected the ADA-loaded RBCs to an

adult-type ADA deficient patient, with ADA protection from antigenic responses and retention of therapeutic activities.[94] During the 9 years of therapy, 2–3 weekly administration was both metabolically and clinically effective.

Hemophilia B is a disorder of blood coagulation caused by deficiency of factor IX, and usually the conventional treatment is recurrent infusions of deficient factor IX. The typical 2 or 3 infusions per week increase the risk of complications and provoke immune response. Sinauridze et al. utilized RBCs to encapsulate factor IX and achieved five- to tenfold increase of elimination half-life from 8.8 ± 5.6 to 73.9 ± 16 h, with suffi-cient plasma concentration of factor IX for more than 15 d.[95]

5. Responsive RBC Carriers

Many drug agents have difficulties in penetrating intact RBC membranes and are generally released from RBCs via slow diffusion through the RBC membranes and the gradual deg-radation of RBCs. On the other hand, the treatment efficacy of therapeutics is closely related to the administration method,[96] which requires the development of intelligent DDSs to achieve precision drug release. As is known, functional natural and synthetic carriers, such as stimuli-responsive carriers and long-circulating carriers, are playing a predominant role for the effective release of drugs at the site of interest,[97] and some promising candidates have been demonstrated in RBC-based DDSs (Table 2).

5.1. Magnetic-Responsive DDSs

Magnetically controlled specific localization of targeted region with the utilization of an external magnetic field was first pro-posed by Zimmermann and Pilwat.[98] Sprandel et al. have entrapped ferromagnetic particles by the hypotonic treatment, before which sonication was demanded to avoid further aggre-gation of magnetic NPs.[99] Compared with other entrapped molecules, the higher cytotoxicity of ferrofluids led to great loss of cells and low entrapment. It was found that only 20–30% of RBC ghosts retained their biconcave shapes and the dam-aged cells were highly leaky to ferrofluids. Although this freshly prepared magnetic RBC ghosts were utilized to fulfill a mag-netic-responsive circulation, the magnetic field only acceler-ated the efflux without targeting ability. Later, Jain and Vyas coated magnetite with silicone oil, obviously decreasing the


Table 2. Summary of representative responsive RBC carriers summarized in this article.

Stimuli RBC components Loaded cargoes Ref.

Magnet Pristine RBCs Fe3O4 NPs, diclofenac sodium, 5-Aza-2-deoxycytidine [38c,d,98–103]

Light Pristine RBCs, RBC membranes, RBC mimetics Au NPs, MSNPs, ICG, DOX, PTX, Ce6 [77a,b,104–106]

Ultrasound Pristine RBCs, RBC membranes Nanodroplets, ICG, CPT [77c,109]

Enzyme Pristine RBCs, RBC membranes Vancomycin [110]

pH Pristine RBCs Anionic polymers [113]

Temperature RBC membranes ICG, DOX [111]

Glucose Pristine RBCs, RBC membranes Glc-insulin [114]



cytotoxic effect of magnetic particles towards the cytoderms of RBCs and increasing the amount of magnetite-loaded RBCs with retaining biconcave discoid shape to 40–50%.[100] The existence of magnetite rendered the cytoderms of RBCs more porous, resulting in a faster drug release profile. These mag-netic RBCs were responsive to an external magnetic field, but the magnetic responsiveness could be kept only for a limited period due to the accelerating efflux rate caused by the cyto-toxic effect of magnetite particles towards the cytoderms of RBCs. For example, the percentage of the magnetic RBCs decreased remarkably from 50% to 2% when the entrapment extended from 3 to 12 h. Cinti et al. developed a magnetic-con-trolled RBC-based DDS with attachment of a viral spike fusion hemagglutinin glycoprotein on the surface of RBCs, with both superparamagnetic NPs and anticancer drug 5-Aza-2-deoxycyti-dineen capsulated inside.[101] These smart magnetic-responsive RBCs behaved similar to virus with excellent fusogenicity with target cells, and they can be readily delivered to targeted sites through an external magnetic field. More importantly, these engineered magnetic RBCs considerably improved the circula-tion half-life of 5-Aza-2-deoxycytidineen (15–25 min in human), efficiently escaping from the physical degradation before being delivered to the targeted tissues. Due to the high fusogenicity and improved biostability, an increased bioavailability of the drug was obtained with only tenth dose of the standard therapy.

Considering the possible damage to the RBC membranes and undesirable encapsulation of unwanted compounds, the opposite manner of coating RBCs with magnetic NPs were lately reported.[102] Typically, the magnetic NPs were first covered with crosslinked silica shells, and then RBCs were physically absorbed with the core–shell magnetic nanoparti-cles (CSMNs) through electrostatic attraction. Hybrid RBCs–CSMNs were very stable within the initial 2 h, but the distinct decrease of zeta potential after 5 h indicated detachment of CSMNs from RBCs, which could be overcome when RBCs–CSMNs were stored in medium with excessive CSMNs. Fur-thermore, the density of surface positive charges should be accurately controlled by tuning the ratio of condensation mono-mers since higher amino density would induce the rupture of RBC membranes and cause hemolysis. Aside from the poten-tial as a DDS, magnetic RBCs are also promising candidates for contrast agents[103] and imaging-guided therapy because of the intrinsic magnetic and photothermal properties of the mag-netic particles,[38c,d] the employment of an external magnetic field achieved small increase in tumor temperature (≈1.9 °C) but without obvious increase of the tumor inhibition rates.

5.2. Light Responsive DDSs

Recently, NIR light has been utilized as an emerging stimulus in drug release for combined theranostics due to its noninva-sive manner, deep penetration and easy manipulation with remote and precise control. Generally, the power of the con-tinuous wave used in NIR is not strong enough to destroy RBC membranes and induce subsequent drug release, so laser light absorption centers are usually necessary. Delcea et al. leveraged highly concentrated gold NPs to absorb onto the surface of RBC membranes, generating aggregates of gold NPs due to the

electrostatic interactions between glycocalyx (negative charged) and gold NPs (positive charged).[104] Upon NIR laser was illumi-nated, gold NPs could convert electromagnetic waves into heat, and the local temperature increase led to the pore formations on the RBC membranes, which resulted in the improved drug release from the interior volumes of RBCs. Alternatively, Liu’s group designed a NIR light-responsive DDS by coencapsu-lating a photothermal agent, an indocyanine green–BSA nano-complex (ICG–BSA) and an anticancer drug DOX in RBCs, which were then decorated with RGD (Arg–Gly–Asp) peptide targeting αvβ3-integrin.[105] Under an 808 nm NIR laser illumi-nation (0.5 W cm−2) for 5 min, the temperature of RBCs sus-pension increased from 34 to 50 °C, and the integrate structure of RBCs was totally destroyed with a burst DOX release (≈80%). Later, they developed another light-responsive DDS based on photodynamic effect, which could cause serious hemolysis under laser irradiation due to the generation of singlet oxygen and reactive oxygen species.[106] Chlorin e6 (Ce6) photosensi-tizer was directly absorbed into RBCs, along with the encapsu-lation of DOX. This hybrid DDS was highly stable under dark, however, the structures of RBC membranes were significantly destroyed under 660 nm LED light illumination (5 mW cm−2, 10 min), accompanying rapid release of DOX. Compared to the model based on photothermal mechanism, this photosensi-tizer-decorated RBC scaffold tremendously increased the sensi-tivity of light-responsive drug delivery. Moreover, this DDS was also applicable to entrap large proteins such as horse radish peroxidase with subsequent light-controlled release.[106]

As mentioned above, pristine RBCs are not good candidates for drug delivery because of their large size. Therefore, Li and co-workers constructed a nanosized NIR light-responsive RBC-mimetic DDS.[77b] The interior hybrid poly(caprolactone)-ester terminated polymeric NPs (≈150 nm) were prepared by solvent evaporation methods with simultaneous loading of an anti-cancer drug PTX, followed by coating with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine to offer a melting transition around 41.5 °C. Next, hypotonically derived RBC membranes with 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide were cloaked onto the polymeric cores to endow RBC mimetics with long circulation. Upon 808 nm laser irradiation (3 W cm−2, 5 min), the temperature of RBCs increased to ≈55 °C and about 44.8% of PTX was released, giving rise to enhanced synergetic chemophotothermal therapy. In another work, mesoporous silica nanoparticles (MSNPs, ≈90 nm) was alternatively used as inner core materials for the coloading of Ce6 and DOX, fol-lowed by the same strategy of RBC membranes cloaking.[77a] With a laser stimulus (2 W cm−2, 5 min), the DOX concentra-tion in tumor increased twofold and the combined therapy effi-cacy was improved. Especially, this RBC-mimetic significantly inhibited the tumor growth and metastasis in a metastatic 4T1 breast cancer mouse model.

5.3. Acoustic Responsive DDSs

Ultrasound has been one of the most widely used approaches for biomedical and clinical applications because of its nonin-vasiveness, high penetration depth, safety, and low cost.[107] Generally, microbubbles or nanodroplets containing low




boiling points compounds (i.e., perfluorocarbon) need to be stabilized by synthetic lipids.[108] Recently, Hsieh et al. devel-oped anticancer drugs CPT loaded biomimetic RBC droplets, cloaking the liquid cores (perfluoropentane loading) with RBC membranes.[77c] This DDS presented excellent in vivo biocompatibility and improved circulation time. Under high intensity focused ultrasound irradiation for 3 min, droplet vaporization facilitated efficient CPT release and simultane-ously caused remarkable damages to cancer cells, obtaining a synergistic effect of chemotherapy and ultrasound therapy. Moreover, acoustic droplet vaporization considerably increased the signal to noise ratio of the ultrasound echo signal from 15 to 40 dB (droplet concentration 64 × 106 droplets mL−1), which could be potentially used for ultrasound imaging. Due to the low acoustic contrast, RBCs cannot be monitored via conven-tional ultrasound. To solve this issue, Klibanov and co-workers coencapsulated an optical absorber, ICG, into RBCs to offer additional photoacoustic (PA) signals, which can be detected via existed ultrasound technique with high resolution.[109] ICG-RBCs achieved a penetration depth of 18 mm and gener-ated a 17 dB PA signal increase above the background, exhibi-ting great promise in imaging-guided therapy.

5.4. Other Stimuli-Responsive Systems

Other stimuli such as enzyme, pH, temperature, and glucose have also been employed in RBC-based carriers to trigger targeted drug release. For example, Li et al. built an enzyme-responsive antibiotic delivery system, which was composed of a supramolecular gelatin nanoparticle core and a RBC membrane shell, with encapsulation of vancomycin (Van) antibiotics.[110] This hybrid nanocarrier effectively accumulated at the infec-tion site via EPR effect, typically accompanying with overexpres-sion of gelatinase, which resulted in degradation of the gelatin core and subsequent release of Van. This enzymatic respon-sive manner not only significantly improved the bioavailability of antibiotics and reduced demanding dose, but also greatly excluded from potential antibiotic resistance. Through photo-thermal effect, the trigger can be either light or temperature, thus Hui et al. fabricated an upper critical solution temperature poly(N-acryloylglycinamide-co-butyl acrylate) with preload of ICG and DOX in micelle state, acting as a photothermal agent and an anticancer drug respectively.[111] Upon laser irradiation at a target site, the local immediate temperature increase transformed the polymer core from hydrophobic to hydrophilic. A 5 min illumination (808 nm, 0.8 W cm−2) achieved a twofold DOX release with triggered cytotoxicity specifically to irradiated tumor cells, and the release response followed an “on-and-off” profile.

On the other hand, pH gradient exists in cellular, tissue, and organ levels and it has been widely used as a stimulus for designing responsive DDSs.[112] Recently, Wang and Chen syn-thesized anionic hyperbranched poly(l-lysine iso-phthalamide)s (HPLPs) with CPPs mimicking properties.[113] Namely, HPLPs are hydrophilic at physiological pH and cannot penetrate cell membranes. However, at late endosomal pH, HPLPs turned into hydrophobic conformation and presented hemolytic activity due to the pore formation on RBCs instead of membranes dissolution. Additionally, HPLPs had no obvious cytotoxicity

against HeLa cells up to concentration of 5 mg mL−1 and could selectively release preloaded drugs from late endosome/lyso-some to cytoplasm.

Moreover, Gu’s group recently constructed a glucose-respon-sive smart insulin delivery platform by the integration of glu-cose derivative-modified insulin (Glc-Insulin) with RBCs, which have abundant membrane proteins of glucose transporters (GLUTs) (Figure 9).[114] This binding is reversible and thus the insulin can be readily released from RBCs once hyperglycemia occurs, which is controlled by the competitive binding between the glucose and GLUT. The utilization of RBCs controlled the glucose level at ≈200 mg dL−1 at least 24 h and increased gradu-ally over 4 d, which was possibly attributed to the biostability and long circulation of RBCs. Besides employment of RBC ghosts, the strategy of cloaking NPs with RBC membranes also accomplished comparable insulin delivery efficacy.

6. Conclusion and Outlook

These years, RBC-mediated DDSs have received considerable attentions for various biomedical applications due to their superior biocompatibility and long circulation. Additionally, the integration of new strategies for formulation preparation offers technical support to promise in translational medicine.

However, there are some critical issues that should not be ignored for further development and translation of RBC-based DDSs.[115] First, both integrities of RBC components (pris-tine RBCs, RBC membranes) and loading molecules should be maximally retained after the loading process. For example, RBC membranes can be possibly damaged by osmotic stress and crosslinking. Excess coupling of therapeutic molecules to RBCs surface can also reduce their elasticity and deformability, which may subsequently engender high risk of activating com-plement and trigger the undesirable clearance of RBCs by RES and decrease its circulation period in the blood.[116] The major complement protective proteins on human RBCs are decay accelerating factor and CD59, while the low express of comple-ment receptor 1 CR1 is insufficient to affect lysis.[117] There-fore, the approaches of activation of the alternative pathway of complement by RBC-based DDSs have been employed to cir-cumvent these issues.[118] In contrast, the hitchhiking strategy of loading molecules onto RBCs are preferred due to their minimal hemolysis effect towards RBCs. On the other hand, not only the loading methods, but also the loading molecules (on RBCs surface or in RBCs inner space) can potentially elicit RBCs leakage and cause side effects. Also, it is more difficult to control the parameters of natural materials than that of arti-ficial ones, a standard protocol is therefore highly demanded to direct the cargo loading by RBC-based carriers.

Second, RBC components are usually performed ex vivo, so the storage and latent contamination of RBCs during the whole operation should be extremely concerned.[64] Storage lesion of RBCs can result in enhanced clearance, nitric oxide scavenging and immunoregulatory reactions, such as acute lung injury or higher mortality rate. Moreover, RBC damage can probably lead to consequences including vascular occlusion by adhesive RBCs and kidney failure caused by released Hb.[119] For example, pigs transfused with RBCs with a storage period of 28 d had much




higher levels of plasma heme compared to that transfused with RBCs stored for 2 d, and extensive endothelial damage to the vasculature and kidney were observed in the former model.[120] Typically, cryopreservation, anaerobic condition, and the glu-cose-6-phosphate dehydrogenase paradigm are employed to preserve the qualities of RBCs.[17a] Recently, Pan et al. devised high-throughput in vitro assays to determine the sensitivity of loaded RBCs to potential damaging insults including osmotic, mechanical, oxidative, and complement stress, as well as RBCs agglomeration, and testing the biocompatibility of RBC-based DDSs deserves more attention though.[121] Meanwhile, synge-neic blood sources are demanded to ensure the safety and pre-vent the immune responses. When RBCs are further used as carriers for drug delivery in blood transfusion at preclinical and clinical levels, RBCs should be collected from a blood bank and obey with strict blood types match.[16b]

Last but not the least, RBCs are incapable of penetrating and crossing tissue barriers except within the vascular space.[122] Ideally, the expected platform based on RBCs could be readily manipulated with controllable targeting ability to any sites in vivo, but current research of RBC-based nanocar-riers across microscopic barriers such as the blood–brain bar-rier and endothelial barriers are scarce. Thanks to the up-to-date flourish in RBC membrane-cloaked NPs, more and more responsive RBC-derived DDSs have been constructed and remarkably facilitated the on demand drug release at targeted regions, simultaneously inheriting outstanding biocompat-ibility and superior circulation of RBCs. Appropriate selection

of triggers, including physical and physiological triggers should be taken into account from translational perspective, based on targeted positions, release kinetics, and therapeutic dosage. For example, deep penetration often involves ultrasound and magnetic field-mediated approaches. Closed-loop controlled release usually relies on physiological signal-based stimuli.[123] In addition, innovations accumulated for further RBC-based DDSs could also guide the design of other cellular particulate-mediated DDSs, including stem cells,[124] white blood cells,[125] platelet,[126] cancer cells,[127] bacteria,[128] and leukocytes.[129]

AcknowledgementsThis work was supported by the grants from the Alfred P. Sloan Foundation, American Diabetes Association (ADA) (1-15-ACE-21), and JDRF (2-SRA-2016-269-A-N).

Conflict of InterestThe authors declare no conflict of interest.

Keywordsdrug delivery, erythrocytes, nanomedicine, nanoparticles, red blood cells

Received: August 4, 2017Revised: September 8, 2017

Published online:


Figure 9. Schematic of the glucose-responsive insulin delivery system based on RBCs. Glucosamine-modified derivative was attached to RBCs by interacting with glucose receptor/transporter (GLUT) on plasma membranes. Reproduced with permission.[114] Copyright 2017, Wiley-VCH.




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