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Red Blood Cells as Smart Delivery Systems Xiao Han, Chao Wang, and Zhuang Liu Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00758 • Publication Date (Web): 03 Jan 2018 Downloaded from http://pubs.acs.org on January 3, 2018

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Bioconjugate Chemistry

Red Blood Cells as Smart Delivery Systems

Xiao Han1, Chao Wang*2, and Zhuang Liu*1

1, Institute of Functional Nano & Soft Materials (FUNSOM), Jiangsu Key Laboratory for Carbon-based Functional Materials and Devices, Soochow University, Suzhou, Jiangsu 215123, China 2, Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Raleigh, NC 27695, USA.

* Corresponding author: E-mail: [email protected], [email protected]

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Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Red blood cells (RBCs) also called erythrocytes are the most abundant type of blood cells. Recently, RBCs have been extensively studied as drug delivery systems for their remarkable properties, including inherent bio-compatibility, low immunogenicity, flexibility and long systemic circulation. Over the years, a number of different RBC-based drug delivery systems including genetically engineered RBCs, non-genetically engineered RBCs, as well as RBC membrane coated nanoparticles have been explored, aiming at diverse biomedical applications. These techniques may address many challenging issues faced by traditional drug delivery systems, as demonstrated by many successful preclinical results. Novel techniques dedicated to produce drug-carrying RBCs are currently undergoing transition from preclinical research to clinical realm. In this topical review, we will summarize the latest progresses in the development of RBC-based smart delivery systems for various biomedical applications.

TOC


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Introduction Red blood cells (RBCs) (also called erythrocytes), the most abundant type of blood cells in the human body, are mainly used for oxygen transportation. Hemoglobin inside the RBCs permits them to transport oxygen from the lungs. Normal human RBCs have a diameter of 7–8 µm and an average volume of 90 fluid ounce.1 Unlike many other cells in mammals, red blood cells are lack of a nucleus and can change their shape to fit through the blood vessels in the body.

2

As the most

abundant type of blood cells, RBCs serve as an ideal candidate for in vivo drug delivery with long circulation time, high cargo loading capacity, excellent biocompatibility and low immunogenicity.3-5 The crucial self-markers covered on the surface allow RBCs to circulate in vivo for a long period of time (~120 d in human and ~50 d in mice) without being cleared by macrophages.6,7 RBCs are completely biodegradable without generating toxic bi-products. Meanwhile the semipermeable membrane of RBC can prevent encapsulated cargoes from rapid clearance and therefore achieve sustained release.8 More importantly, mature RBCs do not contain any genetic materials, enabling them fewer safety risks than other gene and cell therapies. 9

Based on these intrinsic properties of RBCs, multiple strategies have been developed for RBC engineering, which can be divided into genetic engineering and non-genetic engineering approaches, so as to fabricate various RBC-based delivery systems. In addition, many studies have designed nano-sized particles coated with membranes of erythrocytes to mimic RBCs as smart drug carriers.10 This Topical Review highlights recently developed representative RBC-based system for promoting drug delivery performance, which could inspire further approaches to better utilize the RBCs as platforms to treat various diseases.

Genetic engineering of RBCs Genetic engineering for the cell therapy is an approach that inserts or replaces new DNA in the host genome to induce the gene express for the therapeutic purposes.11 Genetic engineering approach that makes RBCs express therapeutic proteins has been wildly developed and commercialized for treatment of different diseases. The lack of a nucleus of RBCs limits their genetically modification at the mature stage. Therefore, an alternative method would be engineering erythroid precursors in 3 ACS Paragon Plus Environment


order to express proteins on mature RBCs. For example, Rubius Therapeutics is a commercialized RBC-based technical platform founded and launched by Flagship VentureLabs®. By genetically modifying hematopoietic stem cells, their final RBCs (Red-Cell Therapeutics™) can display a variety of therapeutic proteins including receptor agonists, antigens and binders, enzymes, and combinations thereof on the cell surface or in the cytosol to treat various diseases including cancer, autoimmune disorders and other rare diseases.12 In another work reported by Shi et al., by genetically engineering erythroid precursors, RBCs expressing sortase-modifiable proteins on their membrane were generated. These engineered RBCs can be further labeled with a variety of functional probes through sortase-catalyzed reaction without damaging the cell in vivo.13

A recent research reported a retroviral-based strategy to genetically modify RBCs from hematopoietic stem and progenitor cells (HSPCs) with single domain antibodies (VHHs) to detoxify botulinum neurotoxin serotype A (BoNT/A), a neuron toxin that leads to flaccid paralysis and death. Glycophorin A (GPA) and Kell are expressed on both HSPCs and matured RBCs, which make them suitable targets for modification. Type A VHHs-based neutralizing agents (VNA/A) recognizes and neutralizes BoNT/A specifically. By fusing virus vectors encoding chimeric proteins with VNA at the N’-terminus of murine glycophorin A (GPA) and at the C’-terminus of Kell, the generated RBCs were able to express both GPA-VNA and Kell-VNA proteins on the surface. Note that after bone marrow reconstitution, both GPA-VNA/A- and Kell-VNA/A-producing mice survived the infection of BoNT/A of increased dose at weekly intervals. Remarkably, both group even survived after challenging with 10,000 times the lethal dose (LD50) of BoNT/A. It was found that the transfusion of these engineered-RBCs into naive mice afforded antitoxin-capacity for up to 28 days.14

The ultimate fate of erythrocytes is being cleared after apoptosis-like programmed cell death in the spleen and by the reticuloendothelial system (RES).3 Therefore, the natural route of RBC removal in body could be exploited to induce antigen-specific T-cell deletion for induction of tolerance. Pishesha et al. used genetically engineered RBCs as substrates for sortase to covalently attach peptides from autoantigens, such as myelin oligodendrocyte glycoprotein and insulin-derived 4 ACS Paragon Plus Environment

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peptide. In order to generate enough blood sourse for modification, CRISPR/Cas9 was used to introduce sortasable LPETGG motif at the C terminus of the Kell protein into murine germ line and Kell-LPETGG mice were served as blood donors. Transfusion of the modified RBCs with related autoantigens relieved the sign of disease in experimental autoimmune encephalomyelitis (EAE), as well as maintained normoglycemia in a type 1 diabetes (T1D) mouse model.15 (Fig.1)

Figure 1. a) Schematic for Kell C-terminal sortase labeling with GGG-carrying antigens peptides. b) Evaluation and quantification of mature Kell-LPETGG RBCs for sortase labeling by incubation of RBCs with biotin-containing probes in the presence or absence Sortase A. Cytofluorimetry was performed with anti-TER119, a RBC surface marker, and antibiotin antibodies. (n = 3; **P < 0.01, unpaired t test with Holm–Sidak adjustment). c) Schematic for prophylactic Type-1 diabetic treatment. Blood glucose levels were measured to monitor T1D progression in NOD mice, considered diabetic when glucose levels were >250 mg/dL, **P < 0.01 (log-rank test). d) Individual 5 ACS Paragon Plus Environment


blood glucose level measurement in mice treated with RBC or RBC-InsB9–23.15 Copyright 2017 National Academy of Sciences

For the genetic engineering, in vitro production of transferable RBCs should be generated from immature HSPCs. Genetically engineering of HSPCs enables minimal change of the biogenesis of generated RBCs compared to normal matured RBCs, as judged by cell size, surface protein expression and hemoglobin content. These approaches were able to provide a safe supply of RBCs and have been proved to be clinically applicable.16 In addition, as all human red cells do not have their nuclei and without any genetic materials, the final gene-free RBCs may be safer and more easily controlled than other gene and cell therapies (e.g. CAR-T cell therapy or stem cell therapy).

Non-genetically engineered RBCs Besides genetically engineering of erythroid precursors, many ex-vivo methods including encapsulation, absorption and bioconjugation are applied to engineer RBCs for the delivery of therapeutics without genetic modification, rendering ‘carrier RBCs’. Over the years, efforts have been made to design carrier RBCs for delivery of cargoes in models ranging from small animals to human patients in clinical studies.17 Compared with traditional liposomal drug delivery systems, carrier RBCs are possessed of a longer circulation half-life due to the self-markers on the membrane. For example, CD47 is a surface protein that regulate phagocytic uptake by macrophages through interactions with SIRPα.18 For human erythrocytes, it was found that complement receptor 1 (CR1) and decay-accelerating factor (DAF) can prevent inappropriate self-recognitoin by alternative complement pathway through inhibiting C3 convertases.19 Also, CD59 and C8 binding protein (C8bp) on the erythrocyte membrane are able to prevent full assembly of the membrane attack complex.20, 21 As for mouse erythrocytes, besides CD47 and DAF, complement receptor 1 (CR1)– related gene y protein (Crry) protects erythrocytes from spontaneous complement attack by inhibiting C3 convertase. 22

This cellular loading strategy may also prolong the release of cargoes. As reported by Rossi et al., dexamethasone 21-phosphate, a therapeutic agent for chronic obstructive pulmonary disease 6 ACS Paragon Plus Environment

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(COPD), was loaded inside erythrocytes. A single administration of drug-load erythrocytes to patients with COPD was able to maintain dexamethasone concentration in blood for up to 7 days.23 Multiple strategies have been developed to load RBCs with cargoes, including electric pulses24 and hypotonic dialysis25. As reported by Muthuvel in 2005, formate dehydrogenase-loaded RBCs along with carbicarb, an alkalinizing agent which can increase the intracellular pH, facilitated the removal of formate in a rat model.26 According to a previous study by our group, a model drug, doxorubicin (DOX) and a photothermal agent, Indocyanine Green (ICG) were first capsulated with BSA and followed by dialysis against hypotonic buffer at 4 ℃ for 30 min. The swollen RBCs were then transferred to a hypertonic buffer and dialyzed at 37 ℃ to reconstitute osmotic pressure and reseal those molecules inside RBCs. During these processes, adenosine triphosphate (ATP) and glutathione (GSH) were added to maintain the structure of RBCs and protect them from oxidative damage.25 The methods discussed above require disruption of the cell membrane to create pores for drugs to diffuse in. A new method that encapsulates protein into RBCs using cell penetrating peptide (CPP) was reported by Yang’s group.27 L-asparaginase, a clinically used drug for the treatment of acute lymphoblastic leukemia (ALL), was conjugated to CPP via a disulfide linkage. CPP-conjugated L-asparaginase was capable of transporting across the membrane and L-asparaginase would be disassociated with CPP via the break of disulfide linkage due to the presence of GSH and other reductase activities within the RBC. Through this method, a loading efficiency of 8% of L-asparaginase was observed.

Beside the interior space of the RBC, the large surface-to-volume ratio of RBC is beneficial for cell hitchhiking or surface reengineering. Through electrostatic interactions, van der Waals and/or hydrophobic interactions, nanoparticles can attach to the surface of RBCs.3 According to Samir, by attaching to the surface of RBCs, the vascular circulation time of 220-nm polystyrene nanoparticles was dramatically enhanced to 100-fold longer retention time than their free counterparts. This strategy was effective for nanoparticles ranging in diameter from 100 nm to 1.1 µm.18 According to Anselmo et al, a longer retention time and a ~7-fold higher accumulation in lungs could be achieved by hitchhiking polystyrene nanoparticles onto the surface of RBCs.2 This cellular hitchhiking strategy has provided a new method to solve two major issues in nanoparticle-based 7 ACS Paragon Plus Environment


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therapeutic strategies: the avoidance of metabolic organs such as liver and spleen and the accumulation of nanoparticles to sites that is difficult to reach such as lungs and brain. This strategy has also exhibit great application in the design of controlled release drug loading system. Recently, different groups including ours have attempted to fabricate novel RBC-based drug delivery systems. As reported by Delcea, gold nanoparticles anchored on the surface of RBCs could achieve controlled release of drug from the inner cavities under the irradiation of a NIR laser.28 Our group reported that chlorin e6 (Ce6) could be anchored into the membrane of RBCs by simple mixing without altering the membrane stability. Upon radiation of laser, the membrane breakdown will trigger the release of DOX inside the RBCs.29

In addition to the physical attachment, biological molecules can be linked to the exterior of the RBC through covalent conjugation. Biotin-avidin bridge is a common surface engineering strategy to load cargoes. In a previous work, Tat antigen, a HIV regulatory protein, can be effectively bind to erythrocytes through biotin-avidin bridge. The obtaining RBC-Tat was proved to be a good approach to deliver antigen to DC, thereby generating Tat-specific CD4+ and CD8+ T cells.30 In another

work,

our

group

has

successfully

conjugate

antigen

peptide

(OVA257–264,

SIINFEKL)-loaded major histocompatibility complex-I (pMHC-I) and anti-CD28 antibody onto the surface of RBCs to obtain artificial antigen presenting cells by biotin-avidin bridge. The obtained artificial antigen presenting cells (aAPC) were further tethered with interleukin 2 (IL-2) as a T cell proliferation signal by DSPE-PEG-anti-His antibody complex to mimic the paracrine activation pathway. When incubated with splenocytes in vitro, such aAPC-IL2 showed favorable CD8+ T cell activation functions, triggering significantly enhanced T cell proliferation and increased inflammatory cytokine secretion. These activated splenocytes could effectively damage cancer cells via antigen-specific manner, indicating that this RBC-based aAPC system is promising for T cell re-education and activation. This strategy might be a potential promising method for in vitro adoptive T cell transfer.31 (Fig.2)


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Figure 2. a) Schematic illustration to show the fabrication of RBC-based aAPCs modified with pMHC-I, aCD28, and IL2, as well as the mechanism of RBC-based aAPCs to activate CD8+ T cells. b) Schematic illustration of in vitro T cell re-education and cancer-cell-specific killing. c) Efficacy of cancer-cell-specific lysis by naive splenocytes and R-aAPC-IL2-activated splenocytes from C57 mice. d) Dimer staining to show the T cell receptor specificity of the CD8+ T cell populations in naïve splenocytes and aAPC-IL2 treated splenocytes. 31 Copyright 2017 John Wiley & Sons, Inc.



In another example, Wang et al. loaded magnetic nanoparticles (e.g. iron oxide nanoparticles (IONPs)) onto the surface of RBCs to develop a “magnetic RBC”. These magnetic RBCs could be used for tumor targeting.32 IONPs were firstly modified with dopamine and further coated with Ce6-grafted poly(allylamine hydrochloride) (PAH). The IONPs were conjugated onto the surface of RBCs through biotin-avidin bridge. Before loading, DOX was loaded inside RBCs via hypotonic osmosis. In order to enhance the stability of engineered RBCs, the surface of the IONPs was further modified with PEG. The obtaining DOX@RBC-IONP-Ce6-PEG exhibited a greatly prolonged blood-circulation time and a significantly enhanced tumor-homing ability in response to magnetic targeting. The in vivo therapy results further demonstrate that the combination therapy has a remarkable synergistic effect which could inhibit tumor growth with low dose of therapeutic agents. (Fig.3)

Figure 3. a) A scheme showing the preparation steps of theranostic RBCs modifi ed with IONPs, Ce6, DOX, and PEG. b) A schematic illustrating in vivo magnetic tumor targeting and fluorescence imaging (Ce6) of mice bearing two subcutaneous 4T1 tumors on opposite flanks after i.v. injection of RBC-IONP-Ce6-PEG or an equivalent dose of free IONP-Ce6-PEG nanoparticles. White and green arrows point to tumors with and without a magnet attached, respectively.32 Copyright 2014 John Wiley & Sons, Inc

Some biological molecules including peptides and antibodies can serve as bridges on the surface of RBCs. As reported by Wang et al. glucose derivative-modified insulin can effectively bind to the membrane of RBCs probably via glucose transporter molecule, an abundant membrane protein. In a hyperglycemia environment, the binding is reversible, resulting in the releasing of insulin due to the competitive interaction of free glucose. 33 10 ACS Paragon Plus Environment

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Possessed with these ideal properties mentioned above, RBCs could serve as intriguing cells as drug delivery systems. Cargoes can be loaded either inside RBCs or on the outside membrane through different ex vivo approaches. These carrier RBCs are safe carriers for a wide array of cargoes, as documented in several excellent reviews. 1, 3, 17, 34, 35 Also, as a large number of RBCs are cleared by RES each day, carrier RBCs have also been designed as natural drug delivery systems targeting the RES. 3 Based on that, several preclinical studies have been carried out to treat RES-related diseases using RBCs as drug delivery system.36 Non-RES targeting and controlled release of encapsulated cargoes can be achieved through further surface engineering of carrier RBC, for example magnetic nanoparticles loading33 or RGD-peptide modification25. Besides intriguing drug delivery systems, RBCs can also be designed as artificial antigen presenting cells via surface modification. 30, 32

RBC Membrane coated particles based delivery system Inspired by those remarkable properties of RBCs, the researchers have designed drug carriers with similar chemical characteristics and biological functions that mimic RBCs in the bloodstream. One strategy is to make nano-sized vesicle with RBC membrane, which can be obtained by series of membrane extrusion. In 1994, Lejeuen et.al first prepared nano-sized erythrocyte vesicle through membrane extrusion. According to their results, extrusion of erythrocyte membrane through pores of 0.4 µm and 1 µm both yielded the nano-sized erythrocyte vesicle with mean diameter of 0.1 µm. This is probably due to the spontaneous formation of RBC membrane vesicle during the extrusion.37 As discussed above, the micrometer size of RBCs greatly limit their ability to extravascular diffusion.33 The small size enables the erythrocyte vesicles to penetrate certain tissue and finally achieve intracellular drug delivery. As reported by Guo et al., Celecoxib (CB), a cyclooxygenase-2 specific inhibitor used to treat Alzheimer's disease, was encapsulated into erythrocyte membrane through extrusion. The therapeutic efficacy of CB alone is limited by its poor solubility and low blood-brain barrier permeability. These limitations can be avoided by intranasal delivery of erythrocyte encapsulated CB (CB-RBCMs) probably because of the high flexibility of red blood cell membrane. Of note, compared to phospholipid membrane encapsulated CB liposomes (CB-PSPD-LPs), CB-RBCMs were able to release CB in a slower and more constant rate in vitro 11 ACS Paragon Plus Environment


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and achieved better therapeutic effects against Alzheimer’s disease on elevating neurogenesis and inhibiting the apoptosis of neurons.38

The other strategy is to cover the nanoparticles with RBC membrane. This process is achieved through 2 steps: the derivation of membrane vesicles from RBCs and the fusion of the vesicle to the core through extrusion. (Fig.4) A lipid bilayer coverage of 5-10 nm was observed out of the core according to transmission electron microscopy (TEM) image, which is in line with the thickness of the natural RBC membrane. Meanwhile the zeta potential of RBC membrane coated nanoparticles was close to the value of RBC membrane vesicle. 39, 40 Both experimental results confirmed the successful coating of nanoparticles by RBC membrane. This extrusion method can coat a variety of negatively charged cores including polymers (e.g. poly(lactic-co-glycolic acid) (PLGA)39,

40

,

melanin41 ), inorganic nanoparticles42, 43, QDs44 with RBC membrane. The positively charged core will cause aggregation with the membrane due to the electrostatic interactions, resulting in the disorder and collapse of fluidic lipid bilayer.3


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Figure 4. a) Schematic of the preparation process of RBC membrane-camouflaged nanoparticles (RBC-NPs). RBC membrane-derived vesicles are extruded together with polymeric nanoparticle cores to form the final RBC-NPs. b) Proteins in emptied RBCs, RBC-membrane-derived vesicles, and purified RBC-membrane-coated PLGA nanoparticles were solubilized and resolved on a polyacrylamide gel. c) RBCmembrane-coated PLGA nanoparticles, PEG-coated lipid-PLGA hybrid nanoparticles, and bare PLGA nanoparticles were incubated in 100% fetal bovine serum and monitored for absorbance at 560 nm for 4 h. d) DiD-loaded nanoparticles were injected intravenously through the tail vein of mice. At various time points blood was withdrawn intraorbitally and measured for fluorescence at 670 nm to evaluate the systemic circulation lifetime of the nanoparticles (n = 6 per group).40 Copyright 2011, National Academy of Sciences.



Owing to the self-marker proteins, RBC membrane serves as perfect stealth coating camouflage with high biocompability and immune-evasion ability.6 According to a previous study, equivalent density of CD47 on the membrane of RBC membrane coated nanoparticle to natural RBCs was identified. The CD47 on the membrane camouflage are in right-side-out orientations, exposing their extracellular domains for molecular interactions probably due to the asymmetric charge of the membrane, which cause the electrostatic repulsion with the PLGA core.7 Piao et al. successfully fused natural RBC membranes onto surfaces of the gold nanocage. The yielding RBC-AuNCs exhibit high stability and a longer circulation time in a mouse model compared to poly(vinylpyrrolidone) (PVP) covered AuNCs.43 Similar results were observed by Hu et al, in which the RBC membrane camouflaged poly(lactic-co-glycolic acid (PLGA) nanoparticles had an in vivo circulation half-life of nearly 40 hours, a significant improvement over the 16 hours of the PEGylated nanoparticles using a mouse model.45 Recently, our group has reported a nanoscale RBC-mimic system by encapsulating perfluorocarbon (PFC), an artificial blood substitute with extremely high oxygen solubility, within PLGA. These particles were further camouflaged with RBC membrane, obtaining PFC@PLGA-RBCM nanoparticles. The PFC@PLGA core showed excellent capability of dissolving oxygen and the RBC membrane enabled a much longer retention upon intravascular injection. After i.v. injection for 24 h, mice were treated with radiotherapy at the dose of 8 Gy. According to the result, mice receiving PFC@PLGA-RBCM and X-ray radiation showed remarkable inhibition to tumor growth, which is more effective than X-ray group at the same dose. This is probably due to the relieved hypoxia in tumor microenvironment by oxygen-dissolved PFC, which greatly enhanced the treatment efficacy during RT.46 (Fig.5)


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Figure 5. a) Scheme of the preparation of PFC@PLGA-RBCM. PFC solutions were encapsulated inside the PLGA shell, which was then coated with RBCM. b) Schematic illustration of PFC@PLGA-RBCM nanoparticles penetrating inside solid tumors through blood vessels. c) Photo-acoustic images 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) before PFC@PLGA-RBCM injection and at different time points after PFC@PLGA-RBCM injection. Tumor-bearing mice injected with naked PFC@PLGA nanoparticles were used as the control. d) Quantification of the time-dependent oxyhemoglobin saturation levels in the tumors based on imaging data in (c). e) Tumor growth curves of different groups of tumors after various treatments indicated. Five mice with one subcutaneous 4T1-tumor per mouse were used in each group.46 Copyright 2017 John Wiley & Sons, Inc.

According to Zhang et al., a cell membrane-templated polymerization method was used to prepare RBC membrane-coated nanogels for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infection. In brief, RBC ghosts were mixed with acrylamide and a redox-sensitive crosslinker

cystine

dimethacrylate

and

a

photoinitiator

lithium

phenyl-2,4,6-Trimethylbenzoylphosphinate, followed by sonication, extrusion and gelation. The antibiotic vancomycin was added into the solution during gelation process.47 The redox-responsive hydrogel core is designed to deliver antibiotics while the RBC membrane is used for absorbing and 15 ACS Paragon Plus Environment


neutralizing pore-forming toxin secreted by bacteria. Compared to free antibiotics and non-responsive nanogels, RBC coated nanogels exhibited remarkable antibacterial activity in vitro. Similar strategies were reported by Zhang’s Group, in which the RBC-NPs serve as nanosponges to absorb and neutralize membrane-damaging toxin, therefore diverting the toxin away from its cellular targets.48

Despite the long circulation time of RBC-membrane coated nanoparticles, it was worth noting that target-selectivity is an important feature in applying RBC membrane coated nanoparticle for disease treatment, especially in cancer treatment. According to Fang et al, a lipid-insertion technique was employed to prepare RBC membrane coated nanoparticles with targeting ability, in which a folate-PEG-lipid conjugate or a nucleolin-targeting aptamer AS1411 was inserted into RBC membrane ghost before membrane coating on the cores. These lipid insertion techniques exhibit excellent targeting ability in vitro.49 Guo et al. proposed an antigen peptide delivery system with erythrocyte-membrane enveloped PLGA nanoparticles. In their study, mannose was inserted into the membrane of RBCs through DSPE-PEG to target antigen-presenting cells (APCs) in the lymphatic organ through interaction with mannose receptor. An antigen peptide, hgp 10025-33, was covalently linked to PLGA core through NHS and 2-(pyridyldithio)-ethylamine (PDA) reaction. MPLA, an FDA-approved lipid-like derivative of lipopolysaccharide (LPS) which binds to toll-like receptor 4 (TLR-4), was inserted into the membrane through gentle mixing. This formulation of peptide and adjuvant combines the intrinsic property of RBC membrane-camouflaged particles and APC-targeting ability. Compared to non-targeted RBC-PLGA-hgp formulations, mannose inserted RBC membrane coated PLGA-hgp nanoparticles exhibited a superiority of DC uptake and activation both in vitro and in vivo. This vaccine formulation shows a great tumor prevention ability and inhibition of both tumor growth and metastasis. In the similar way, other targeting ligand such as folic acid can be linked to the surface of RBC vesicles before extrusion to possess tumor targeting ability.50

Prospective The ultimate objective of engineered RBCs or RBC membrane-derived vesicles and particles is to 16 ACS Paragon Plus Environment

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effectively deliver cargoes while maintaining their surface characteristics and causing minimum unwanted immune response.35 Therefore, it is necessary to test the in vivo performance before clinical use. Most preclinical studies in this field were tested in small animals and further experiments needs to be done in larger animals before testing in humans. Another issue might be the blood-supply. In small animal experiments, whole blood collected form syngeneic animal models was used to avoid unwanted immunogenic responses. For further clinical use, engineered RBCs or RBC-derived carrier system should match the blood type and Rh compatibility of patients.51 The usage of blood collected form the patient himself or type O; Rh negative blood might be a solution. However, the modification of foreign molecules on the membrane of RBCs should more or less induce alloimmunization, which is a potential risk in clinical use especially after repeated administrations. It is believed that the extent of alloimmunization is related with the density of cargoes on the cell surface. Systemic administration of foreign proteins also increases significant risk of inducing a strong antibody response. For example, when it comes to biotin-avidin bridge, which is the common surface modification strategy, the use of avidin or streptavidin can induce immunogenicity and streptavidin is considered to be even highly immunogenic.52

In addition, although the RBC membrane-coated nanoparticles exhibit longer retention time compared with bare nanoparticles, the retention time of such nanoparticles is still much shorter than RBCs themselves. Therefore it is necessary to test the properties of membrane after the cores have been coated.53 Meanwhile, a variety of strategies have been successfully developed to load drugs inside RBCs. These methods, however, will cause membrane damage and therefore result in a loss of structural integrity and change of osmosis pressure, rendering them prone to recognition and clearance by the host immune system.27 Meanwhile, compared with other cell or cell membrane based therapeutic strategies, it would be hard for RBC-based drug delivery system to fulfill local release except for RES targeting, limiting their potential applications.3

While most RBC-based drug delivery systems are still in proof-of-concept stage, some have been proved to be effective and safe in preclinical studies and will likely be promising clinical treatments. The first clinical trial of carrier RBCs was reported by Beutler in 1977 when a patient with 17 ACS Paragon Plus Environment


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Gaucher’s disease was intravenously administrated with glucocerebrosidase coated with human erythrocytes. The patient was considerably improved and no evident side effect was observed.54 Several technologies using ex vivo devices to encapsulate drugs into the cavity of erythrocytes have entered clinical trials (Table 1). Other commercialized RBC-based drug delivery systems are under development. For example, erythrocyte-encapsulated thymidine phosphorylase for the treatment of mityochondrial neurogastrointestinal encephalomyopathy on Balb/c mice model and beagle dog model is currently under development by Orphan Technologies.55 Rubius Therapeutics is another commercialized RBC-based technical platform for red blood cell-base therapy. As we can imagine, RBC-based cell therapy will be as a new class of medicines to address a wide array of indications in the foreseeable future.

Sponsor

Intervention

ERYtech Pharma

native L asparaginase loaded RBCs

Erydel

Dexamethasone sodium phosphate loaded RBCs (EryDex)

Applications

Phase

NCT number

Acute Lymphoblastic Leukemia

Phase 2

Acute myeloblastic leukemia

Phase 2

NCT0072 3346 NCT0151 8517 NCT0181 0705

Recovery and survival of EryDex in non-patient volunteers

Phase 1

NCT0238 0924

Table 1. List of RBC-based drug delivery system undergoing clinical trials. (From www.clinicaltrials.gov)


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Author Information Corresponding Authors [email protected], [email protected]

Notes The authors declare no competing financial interest.

Acknowledgment This article was partially supported by the National Basic Research Programs of China (973 Program) (2016YFA0201200), the National Natural Science Foundation of China (51525203), Joint International Research Laboratory of Carbon-Based Functional Materials and Devices, Collaborative Innovation Center of Suzhou Nano Science and Technology, and a Project Funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions.

References (1) (2) (3) (4) (5) (6) (7)

(8)

(9) (10)

Pierige, F., Serafini, S., Rossi, L., and Magnani, M. (2008) Cell-based drug delivery. Adv. Drug Delivery Rev. 60, 286-295. Anselmo, A. C., and Mitragotri, S. (2014) Cell-mediated delivery of nanoparticles: taking advantage of circulatory cells to target nanoparticles. J. Controlled Release 190, 531-541. Tan, S., Wu, T., Zhang, D., and Zhang, Z. (2015) Cell or cell membrane-based drug delivery systems. Theranostics 5, 863-881. Yoo, J.-W., Irvine, D. J., Discher, D. E., and Mitragotri, S. (2011) Bio-inspired, bioengineered and biomimetic drug delivery carriers. Nat. Rev. Drug Discovery 10, 521-535. Fang, R. H., Hu, C. M., and Zhang, L. (2012) Nanoparticles disguised as red blood cells to evade the immune system. Expert Opin. Biol. Ther. 12, 385-389. Oldenborg, P. A. (2000) Role of CD47 as a Marker of Self on Red Blood Cells. Science 288, 2051-2054. Hu, C. M., Fang, R. H., Luk, B. T., Chen, K. N., Carpenter, C., Gao, W., Zhang, K., and Zhang, L. (2013) 'Marker-of-self' functionalization of nanoscale particles through a top-down cellular membrane coating approach. Nanoscale 5, 2664-2668. Rossi, L., Pierige, F., Antonelli, A., Bigini, N., Gabucci, C., Peiretti, E., and Magnani, M. (2016) Engineering erythrocytes for the modulation of drugs' and contrasting agents' pharmacokinetics and biodistribution. Adv. Drug Delivery Rev. 106, 73-87. Hamidi, M., Zarrin, A., Foroozesh, M., and Mohammadi-Samani, S. (2007) Applications of carrier erythrocytes in delivery of biopharmaceuticals. J. Controlled Release 118, 145-160. Luk, B. T., and Zhang, L. (2015) Cell membrane-camouflaged nanoparticles for drug delivery. J. Controlled Release 220, 600-607. 19 ACS Paragon Plus Environment


(11) (12) (13)

(14)

(15)

(16)

(17)

(18) (19)

(20)

(21)

(22)

(23)

(24) (25)

(26)

Maeder, M. L., and Gersbach, C. A. (2016) Genome-editing technologies for gene and cell therapy. Mol. Ther. 24, 430-446. http://www.rubiustx.com/ Shi, J., Kundrat, L., Pishesha, N., Bilate, A., Theile, C., Maruyama, T., Dougan, S. K., Ploegh, H. L., and Lodish, H. F. (2014) Engineered red blood cells as carriers for systemic delivery of a wide array of functional probes. Proc Natl Acad Sci U S A 111, 10131-10136. Huang, N. J., Pishesha, N., Mukherjee, J., Zhang, S., Deshycka, R., Sudaryo, V., Dong, M., Shoemaker, C. B., and Lodish, H. F. (2017) Genetically engineered red cells expressing single domain camelid antibodies confer long-term protection against botulinum neurotoxin. Nat Commun 8, 423. Pishesha, N., Bilate, A. M., Wibowo, M. C., Huang, N. J., Li, Z., Dhesycka, R., Bousbaine, D., Li, H., Patterson, H. C., Dougan, S. K. et al. (2017) Engineered erythrocytes covalently linked to antigenic peptides can protect against autoimmune disease. Proc. Natl. Acad. Sci. U.S.A. 114, 3157-3162. Giarratana, M. C., Kobari, L., Lapillonne, H., Chalmers, D., Kiger, L., Cynober, T., Marden, M. C., Wajcman, H., and Douay, L. (2005) Ex vivo generation of fully mature human red blood cells from hematopoietic stem cells. Nat. Biotechnol. 23, 69-74. Villa, C. H., Anselmo, A. C., Mitragotri, S., and Muzykantov, V. (2016) Red blood cells: Supercarriers for drugs, biologicals, and nanoparticles and inspiration for advanced delivery systems. Adv. Drug Delivery Rev. 106, 88-103. Chambers, E., and Mitragotri, S. (2007) Long circulating nanoparticles via adhesion on red blood cells: mechanism and extended circulation. Exp. Biol. Med. 232, 958-966. Fearon, D. T. (1979) Regulation of the amplification C3 convertase of human complement by an inhibitory protein isolated from human erythrocyte membrane. Proc. Natl. Acad. Sci. U.S.A. 76, 5867-5871. Schönermark, S., Rauterberg, E., Shin, M., Löke, S., Roelcke, D., and Hänsch, G. (1986) Homologous species restriction in lysis of human erythrocytes: a membrane-derived protein with C8-binding capacity functions as an inhibitor. J. Immunol. 136, 1772-1776. Davies, A., Simmons, D. L., Hale, G., Harrison, R. A., Tighe, H., Lachmann, P., and Waldmann, H. (1989) CD59, an LY-6-like protein expressed in human lymphoid cells, regulates the action of the complement membrane attack complex on homologous cells. J. Exp. Med. 170, 637-654. Miwa, T., Zhou, L., Hilliard, B., Molina, H., and Song, W.-C. (2002) Crry, but not CD59 and DAF, is indispensable for murine erythrocyte protection in vivo from spontaneous complement attack. Blood 99, 3707-3716. Rossi, L., Serafini, S., Cenerini, L., Picardi, F., Bigi, L., Panzani, I., and Magnani, M. (2001) Erythrocyte ‐ mediated delivery of dexamethasone in patients with chronic obstructive pulmonary disease. Biotechnol. Appl. Biochem. 33, 85-89. Tsong, T. Y. (1991) Electroporation of cell membranes. Biophys. J. 60, 297-306. Sun, X., Wang, C., Gao, M., Hu, A., and Liu, Z. (2015) Remotely Controlled Red Blood Cell Carriers for Cancer Targeting and Near-Infrared Light-Triggered Drug Release in Combined Photothermal-Chemotherapy. Adv. Funct. Mater. 25, 2386-2394. Muthuvel, A., Rajamani, R., Manikandan, S., and Sheeladevi, R. (2006) Detoxification of formate by formate dehydrogenase-loaded erythrocytes and carbicarb in folate-deficient 20 ACS Paragon Plus Environment

Page 20 of 23

Page 21 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(27)

(28)

(29)

(30)

(31)

(32)

(33) (34) (35) (36)

(37)

(38)

(39)

(40)

(41)

methanol-intoxicated rats. Clin. Chim. Acta. 367, 162-169. He, H., Ye, J., Wang, Y., Liu, Q., Chung, H. S., Kwon, Y. M., Shin, M. C., Lee, K., and Yang, V. C. (2014) Cell-penetrating peptides meditated encapsulation of protein therapeutics into intact red blood cells and its application. J. Controlled Release 176, 123-132. Delcea, M., Sternberg, N., Yashchenok, A. M., Georgieva, R., Bäumler, H., Möhwald, H., and Skirtach, A. G. (2012) Nanoplasmonics for dual-molecule release through nanopores in the membrane of red blood cells. ACS Nano 6, 4169-4180. Gao, M., Hu, A., Sun, X., Wang, C., Dong, Z., Feng, L., and Liu, Z. (2017) Photosensitizer Decorated Red Blood Cells as an Ultrasensitive Light-Responsive Drug Delivery System. ACS Appl. Mater. Interfaces 9, 5855-5863. Corinti, S., Chiarantini, L., Dominici, S., Laguardia, M. E., Magnani, M., and Girolomoni, G. (2002) Erythrocytes deliver Tat to interferon-γ-treated human dendritic cells for efficient initiation of specific type 1 immune responses in vitro. J. Leukocyte Biol. 71, 652-658. Sun, X., Han, X., Xu, L., Gao, M., Xu, J., Yang, R., and Liu, Z. (2017) Surface-Engineering of Red Blood Cells as Artificial Antigen Presenting Cells Promising for Cancer Immunotherapy. Small 13. Wang, C., Sun, X., Cheng, L., Yin, S., Yang, G., Li, Y., and Liu, Z. (2014) Multifunctional theranostic red blood cells for magnetic-field-enhanced in vivo combination therapy of cancer. Adv. Mater. 26, 4794-4802. Wang, C., Ye, Y., Sun, W., Yu, J., Wang, J., Lawrence, D. S., Buse, J. B., and Gu, Z. (2017) Red Blood Cells for Glucose-Responsive Insulin Delivery. Adv. Mater. 29 Hu, C. M., Fang, R. H., and Zhang, L. (2012) Erythrocyte-inspired delivery systems. Adv. Healthcare Mater. 1, 537-547. Pierige, F., Bigini, N., Rossi, L., and Magnani, M. (2017) Reengineering red blood cells for cellular therapeutics and diagnostics. Wiley Interdiscip. Rev.: Nanomed. Nanobiotechnol 9. Kruse, C., James, G., Freehauf, C., and Williams, C. (1987) Methotrexate loaded erythrocytes carriers: optimization their formation, their characterization, and their pharmacological efficiency in treating hepatoma 129 ascites tumors in mice. Adv. Biosci.(Ser.) 67, 137-144. LEJEUNE, A., MOORJANI, M., GICQUAUD, C., and LACROIX, J. (1994) Nanoerythrosome, a New Derivative of Erythrocyte Ghost: Preparation and Antineoplastic Potential as Drug Carrier for. Anticancer Res. 14, 915-920. Guo, J. W., Guan, P. P., Ding, W. Y., Wang, S. L., Huang, X. S., Wang, Z. Y., and Wang, P. (2017) Erythrocyte membrane-encapsulated celecoxib improves the cognitive decline of Alzheimer's disease by concurrently inducing neurogenesis and reducing apoptosis in APP/PS1 transgenic mice. Biomaterials 145, 106-127. Luk, B. T., Hu, C. M., Fang, R. H., Dehaini, D., Carpenter, C., Gao, W., and Zhang, L. (2014) Interfacial interactions between natural RBC membranes and synthetic polymeric nanoparticles. Nanoscale 6, 2730-2737. Hu, C.-M. J., Zhang, L., Aryal, S., Cheung, C., Fang, R. H., and Zhang, L. (2011) Erythrocyte membrane-camouflaged polymeric nanoparticles as a biomimetic delivery platform. Proc. Natl. Acad. Sci. U.S.A. 108, 10980-10985. Jiang, Q., Luo, Z., Men, Y., Yang, P., Peng, H., Guo, R., Tian, Y., Pang, Z., and Yang, W. (2017) Red blood cell membrane-camouflaged melanin nanoparticles for enhanced 21 ACS Paragon Plus Environment


(42)

(43)

(44)

(45)

(46)

(47)

(48) (49)

(50)

(51) (52)

(53) (54)

(55)

photothermal therapy. Biomaterials 143, 29-45. Ren, X., Zheng, R., Fang, X., Wang, X., Zhang, X., Yang, W., and Sha, X. (2016) Red blood cell membrane camouflaged magnetic nanoclusters for imaging-guided photothermal therapy. Biomaterials 92, 13-24. Piao, J.-G., Wang, L., Gao, F., You, Y.-Z., Xiong, Y., and Yang, L. (2014) Erythrocyte membrane is an alternative coating to polyethylene glycol for prolonging the circulation lifetime of gold nanocages for photothermal therapy. ACS Nano 8, 10414-10425. Guo, X., Zhang, Y., Liu, J., Yang, X., Huang, J., Li, L., Wan, L., and Wang, K. (2016) Red blood cell membrane-mediated fusion of hydrophobic quantum dots with living cell membranes for cell imaging. J. Mater. Chem. B 4, 4191-4197. Hu, C. M., Fang, R. H., Luk, B. T., and Zhang, L. (2014) Polymeric nanotherapeutics: clinical development and advances in stealth functionalization strategies. Nanoscale 6, 65-75. Gao, M., Liang, C., Song, X., Chen, Q., Jin, Q., Wang, C., and Liu, Z. (2017) Erythrocyte-Membrane-Enveloped Perfluorocarbon as Nanoscale Artificial Red Blood Cells to Relieve Tumor Hypoxia and Enhance Cancer Radiotherapy. Adv. Mater. 29. Zhang, Y., Zhang, J., Chen, W., Angsantikul, P., Spiekermann, K. A., Fang, R. H., Gao, W., and Zhang, L. (2017) Erythrocyte membrane-coated nanogel for combinatorial antivirulence and responsive antimicrobial delivery against Staphylococcus aureus infection. J. Controlled Release 263, 185-191. Hu, C.-M. J., Fang, R. H., Copp, J., Luk, B. T., and Zhang, L. (2013) A biomimetic nanosponge that absorbs pore-forming toxins. Nat. Nanotechnol. 8, 336-340. Fang, R. H., Hu, C. M., Chen, K. N., Luk, B. T., Carpenter, C. W., Gao, W., Li, S., Zhang, D. E., Lu, W., and Zhang, L. (2013) Lipid-insertion enables targeting functionalization of erythrocyte membrane-cloaked nanoparticles. Nanoscale 5, 8884-8888. Rao, L., Meng, Q. F., Bu, L. L., Cai, B., Huang, Q., Sun, Z. J., Zhang, W. F., Li, A., Guo, S. S., Liu, W., Wang, T. H., et al. (2017) Erythrocyte Membrane-Coated Upconversion Nanoparticles with Minimal Protein Adsorption for Enhanced Tumor Imaging. ACS Appl. Mater. Interfaces 9, 2159-2168. Gao, W., and Zhang, L. (2015) Engineering red-blood-cell-membrane-coated nanoparticles for broad biomedical applications. AIChE J. 61, 738-746. Chinol, M., Casalini, P., Maggiolo, M., Canevari, S., Omodeo, E., Caliceti, P., Veronese, F., Cremonesi, M., Chiolerio, F., and Nardone, E. (1998) Biochemical modifications of avidin improve pharmacokinetics and biodistribution, and reduce immunogenicity. Br. J. Cancer 78, 189-197. Shi, J., Kantoff, P. W., Wooster, R., and Farokhzad, O. C. (2017) Cancer nanomedicine: progress, challenges and opportunities. Nat. Rev. Cancer 17, 20-37. Beutler, E., Dale, G., Guinto, D., and Kuhl, W. (1977) Enzyme replacement therapy in Gaucher's disease: Preliminary clinical trial of a new enzyme preparation. Proc. Natl. Acad. Sci. U.S.A. 74, 4620-4623. Levene, M., Coleman, D. G., Kilpatrick, H. C., Fairbanks, L. D., Gangadharan, B., Gasson, C., and Bax, B. E. (2012) Preclinical toxicity evaluation of erythrocyte-encapsulated thymidine phosphorylase in BALB/c mice and beagle dogs: an enzyme-replacement therapy for mitochondrial neurogastrointestinal encephalomyopathy. Toxicol. Sci. 131, 311-324. 22 ACS Paragon Plus Environment

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Red Blood Cells as Smart Delivery Systems - ACS Publications

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