Biofunctionalized nanoparticles: an emerging drug delivery platform for various disease treatments

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Biofunctionalized nanoparticles: an emerging drug delivery platform for various disease treatments Rajendran J.C. Bose1,2, Soo-Hong Lee2 and Hansoo Park1 1 2

School of Integrative Engineering, Chung-Ang University, Seoul, Republic of Korea Department of Biomedical Science, College of Life Science, CHA University, Republic of Korea

Biological barriers, such as phagocytosis and nonspecific distribution, are major factors limiting the clinical translation of nanomedicine. Biomimetic and bioengineering strategies have been used to overcome these challenges. In particular, natural cell membrane-based biofunctionalized nanoparticles (CMFNPs) have gained widespread attention owing to their cell surface mimetic characteristics and tailored nanomaterial features. These hybrid nanocarriers show strong potential for the delivery of myriad therapeutic agents. Herein, we highlight the most recent advances in CMFNP-based drug delivery systems and address the challenges and opportunities in the field.

Introduction Nanoparticles (NPs) have unique properties, such as flexible physicochemistry and a high surface area:volume ratio, which enable targeted functionalization with different molecules by surface coating [1–3]. Although this property is particularly relevant for surface interactions, it also facilitates the multifunctionality and functionalization of NPs for specific applications [4–6]. NP-mediated drug delivery has several advantages, including enhancing drug pharmacokinetics and enabling the therapeutic use of drugs; however, there are also few drawbacks, such as lack of specificity and adverse effects [7–9]. Nevertheless, these collective features are encouraging for the clinical development of nanopharmaceuticals. Although significant efforts have been devoted to the translation of NPs from the bench to market, numerous clinical trials have failed at the late stages because of severe toxicity, which is critically dictated by the surface properties of NPs [10,11]. To overcome these challenges, several new NP engineering approaches have been explored, including bioengineering and biomimetic strategies [12]. In particular, natural CMFNPs have become increasingly important in drug delivery applications [13,14], mainly because natural cell membrane components have complex structures that are responsible for vital cellular functions that are difficult to mimic Corresponding author: Lee, S.-H. ([email protected]), Park, H. ([email protected]) 1359-6446/ß 2016 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.drudis.2016.06.005

using synthetic materials [15]. The translocation of a natural cell membrane to an NP surface offers the combined advantages of a biomimetic cell membrane surface and the tailored flexibility of material chemistry [13,16]. For example, cell membrane-mediated biofunctionalization to an NP surface resulted in immuneevading properties by reducing the absorption of opsonic protein and ‘self’ signals through the surface proteins [17–19]. This biofunctionalization process also provides synthetic NPs with a natural homing ability, and can be used as a template for active targeting functionalization [20]. The natural cell membranebased lipid components on the NP surface also provided greater colloidal stability and slow down the drug release (acting as a ‘molecular fence’) [2,21–23]. Recently, a variety of CMFNPs have been developed for different drug delivery applications using various cell sources, such as red blood cells (RBCs), white blood cells (WBCs), platelet cells (PCs), mesenchymal stem cells (MSCs), cancer cells (CCs), and bacteria [18,24–27]. Currently, CMFNPs are in preclinical development, and collective information on their method of preparation and characterization is required to achieve bench-to-bed translation [13]. Therefore, here we provide updated information on the different methods of preparation for the characterization and application of CMFNPs. We also address the major challenges and opportunities related to the application of CMFNPs in the field of nanomedicine (Box 1).

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BOX 1

Challenges to, and opportunities for, the use of CMFNPs in nanomedicine

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Challenges Complex fabrication process  Reproducibility issues: lack of precise control over the fabrication process.  The experimental variables (type of cell source, material properties, and use of different cell membrane isolation techniques and particle fabrication methods) can influence the clinical outcome. Practical difficulties  Significant challenges include the identification and selection of the most appropriate cell type and material core for the preparation of biofunctionalized NPs.  RBCs, WBCs, SCs, and CCs have been extensively tested as biomimetic membrane sources. However, most of these cell types have been evaluated in preclinical trials for their safety and efficacy, and the results show significant heterogeneity in terms of their efficacy. Lack of standard characterization methods  Lack of physicochemical and preclinical characterization methods.  Insufficient knowledge on the toxicity, immunogenicity, and biodistribution profiles of CMFNPs. Scalability complexities  Current fabrication methods were tested on a laboratory scale.  Pilot scale studies with optimized results are a prerequisite for clinical translations. Instability issues  Preserving cell membrane components (proteins) of the native structure poses practical difficulties.  Stability of physically or chemically fused cell membrane on the material surface is questionable. Regulatory uncertainty  Cell membrane particles are comparatively new to the field of nanomedicine. Therefore, a range of preclinical tests is required.  The lack of clear regulatory and/or safety guidelines for the development of nanomedicine-related products is an additional barrier to the clinical translation process. Opportunities  Artificial cell-based therapy.  Personalized medications (tailored nanomedicines to the needs of an individual patient).  Therapeutic and prophylactic vaccines for the treatment of cancer and autoimmune diseases (immunotherapy).  Targeted drug delivery (treatment for cancer, cardiovascular, and antibiotic-resistant microbes)  Non-invasive therapy and drug delivery (on-demand or stimuliresponsive drug or therapeutic agent delivery and improvement of PTT).  Detoxification (bioscavengers, such as cell-mimetic micro- and nanomotors).  Imaging and diagnostics (biosensors).

Biofunctionalization methods and characterizations of CMFNPs A top-down fabrication process has been commonly used for the preparation of CMFNPs owing to the simplicity and efficiency of this process (Fig. 1 and Table 1) [20,28]. The major steps of CMFNP formulation include preparation of cell membrane-derived vesicles, fabrication of a material core, and fusion of the material core with cell-derived membrane vesicles by either physical extrusion 1304

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Drug Discovery Today  Volume 21, Number 8  August 2016

or electrostatic interactions [18,25,29]. The cell membranes are separated by hypotonic lysis with mild homogenization and are purified by serial centrifugation steps [29]. The drug-encapsulated or drug-conjugated NP core is formulated by a suitable method, such as nanoprecipitation, double-emulsion solvent evaporation, or a self-assembly process [21,25,30]. Finally, a physical extrusion or electrostatic attraction method is used [28]. The lipid and protein compositions of a natural cell membrane vary substantially among cell types, which is an important property for performing complex cellular functions. Therefore, to exploit the unique advantages of various cell surfaces effectively, researchers have explored different cell sources, including RBCs, WBCs, PCs, SCs, and CSs, for the biofunctionalization of NPs [18,20,21,25,28]. Among these various cell types, RBCs have been used as the primary source for the biofunctionalization of NPs owing to the variety of surface proteins responsible for the immune-evading properties of RBCs, which lead to extended circulation [17]. For example, CD47 was identified on the RBC surface as a self-marker that actively signals the presence of macrophages and prevents their uptake [19]. Similarly, other surface proteins, such as C8-binding protein, homologous restriction protein, decay accelerating factor, membrane cofactor protein, complement receptor 1, and CD59, show resistance to complement actions [19]. Zhang et al. prepared RBC membrane-functionalized NPs (RBCNPs) using a top-down fabrication method. The RBC membrane-derived vesicles were prepared by hypotonic lysis followed by mild sonication and the intracellular components were removed by a series of centrifugation steps [28]. Finally, the purified RBC membrane-derived vesicles were fused with preformulated poly (lactic-co-glycolic acid) (PLGA) NPs using the extrusion method. The translocation of intact RBC membranes and their associated surface proteins onto the NPs endowed the latter with immuneevasion properties [28]. It was demonstrated that the prepared RBCNPs had a PLGA polymeric core approximately 70 nm in diameter and an RBC membrane-based lipid layer 7–8 nm thick, as determined with dynamic light scattering and transmission electron microscopy (TEM). Furthermore, surface protein analysis by gel electrophoresis revealed that the RBC membrane proteins were efficiently transferred to the surface of the RBCNPs. The results showed that the RBCNPs had a superior circulation half-life compared with polyethylene glycol (PEG)-coated PLGA NPs [28]. Several features of both preformulated NPs and natural cell sources have been shown to influence the efficiency of the biofunctionalization process [31]. Various interfacial aspects of the cell membrane and NPs, including size, membrane coverage, orientation, effects of the surface charge of the NP, and surface curvature on the RBC membrane, have been shown to affect the functionalization process [31]. The membrane cloaking process was shown to be valid for particles within the 65–340-nm size range [31]. The surface glycans of the source cell membranes and the surface charge of polymeric NPs also have a crucial role in processing efficiency [31]. In addition, the cell membrane:particle core ratio is another important factor that determines the membrane coverage on the surface of the NP [31]. The amount of protein or protein density has been used to optimize the biofunctionalization process [17,20]. Recently, various researchers extended this RBC membrane-based biofunctionalization procedure to different materials, including silica, gelatin, and gold NPs [13,14].

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Cell source

Nanoparticle fabrication

Separation of cells

Extrusion or Electrostatic interaction In vitro cell culture

Cell lysis

Cell membranederived vesicle preparation

Nanoparticle core preparation

Cell membranefunctionalized NPs Drug Discovery Today

FIGURE 1

General method for the fabrication of cell membrane-functionalized nanoparticles.

PCs have numerous advantages, including natural adhesion to an inflamed vascular subendothelium and circulating pathogens [21]. Hu et al. prepared natural platelet membrane-functionalized PLGA-NPs (PNPs) using a slightly modified method of the RBCNP procedure [21]. The PC membrane was separated by repeated freeze–thaw cycles, mild sonication, and centrifugation. Then, the separated PC membranes were coated onto the surface of the PLGA NPs by electrostatic attraction. The authors took advantage of the charge distribution between the outer and inner lipid layers of the PC membranes to achieve an efficient functionalization process. They manipulated the negatively charged surface of PLGA-NPs so that there was electrostatic charge repulsion between the outer lipid layer of the platelet membrane and the PLGA-NP surface, resulting in a ‘right-side-out’ assembly of the PC membranes [21]. Physicochemical characterizations revealed that the PNPs were slightly larger in size (115 nm) than bare PLGA-NPs (100 nm) and showed an equivalent surface charge to that of PCs ( 30 mV). The core–shell structure of PNPs was further revealed by TEM. Western blot analysis was applied to confirm the protein content of the PNPs, which showed that most of the platelet surface proteins comprised immunomodulatory proteins (CD47, CD55, and CD59), integrin components (aIIb, a2, a5, a6, b1, and b3), and transmembrane proteins (GPIba, GPIV, GPV, GPVI, GPIX, and CLEC-2), thus enabling the PNPs to function like natural PCs [21]. The therapeutic potential of PNPs was demonstrated by assessing their selective adherence to damaged vasculatures and Staphylococcus aureus [21].

WBCs (macrophages and monocytes) have numerous advantages, such as natural tumor tropic, adhesive, and trans-endothelial migration properties, which can be used to target CCs or diseased endothelium for drug delivery [18,29,32]. WBCs have unique surface proteins, such as LFA-1, CD45, and CD3z, which have numerous roles in tumor adhesion and transmigration via interactions with inflamed endothelial cells [18]. For the preparation of WBCs or leukocytes, a relatively similar process to that described for RBCNPs has been used with slight modifications to the purification (discontinuous sucrose density gradient centrifugation) and functionalization processes [18]. Leukocyte membrane-functionalized silica microparticles (SMPs), known as leuko-like vectors (LLVs), were prepared by self-assembly, which was driven by electrostatic and hydrophobic interactions between the negatively charged leukocyte membranes ( 31.16 mV) and positively charged SMPs (+7.4 mV). The efficiency of the biofunctionalization process was confirmed by a charge shift of the LLV ( 26 mV) from +7.4 mV (SMPs). The authors used scanning electron microscopy (SEM), TEM, DOT blotting, and Fourier transform infrared spectroscopy to further characterize the LLV structure and protein content. In addition, they demonstrated the therapeutic potential of LLVs using in vitro experiments (phagocytosis and trans-endothelial penetration assays) and in vivo experiments (tumor tropic accumulation) [18]. Recently, this WBC membranebased biofunctionalization process was extended to silica NPs and PLGA-NPs [29]. MSCs have several advantages, such as in vitro expansion and an inherent homing property to the CCs and site of tissue injury [33]. Bose et al. demonstrated a method of preparation of MSC www.drugdiscoverytoday.com

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TABLE 1

Summary of CMFNPs and their applications Biofunctionalized NPs

Source cells

Material core

Therapeutic agent

Functions

Refs

RBC-mimicking NPs

RBC membranes

PLGA-NPs PLGA-NPs Au-NPs –

– DOX – STRAIL

[28] [22] [4] [33]

PLGA-NPs

–

PLGA-NPs

NP–toxin complexes

PLGA-NPs

Hydrogel–NP–toxin complexes Vanc

Long circulating nanocarrier Long circulating nanocarrier Long circulating nanocarrier Natural tumor-targeting system Long circulation nanocarrier; active targeting functionalization Nanosponges as toxin decoys; nanotoxoid vaccine Nanosponges as toxin decoys; nanotoxoid vaccine On-demand antibiotic delivery; toxin decoys Photodynamic anticancer therapy; targeting functionalized NPs Remotely triggered anticancer drug delivery platform; long circulating carrier Detoxification of organophosphate poisoning Cancer immunotherapy; targeted nanovaccine delivery system

[49]

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Bone marrow-derived SC nanoghosts (NGs) Folate and AS1411 ligand-inserted RBC membranes RBC membranes

Gelatin-NPs Dual-targeting ligand (FA and TPP) functionalized RBC membranes CPT-loaded RBC membranes

Upconversion NPs (UCNPs)

MC450

C5F12 microbubbles

CPT

RBC membranes

PLGA NPs

–

Mannose-inserted RBC membranes

Redox-sensitive PLGA-NPs

MPLA Hgp-100(25–33) peptide

Biomimetic nanomotor sponge Cell-mimicking Janus micro motor

RBC membranes

Gold nanowires

–

Au and Albumin-coated magnesium micromotors

–

LLVs

WBC membranes

Silica-NPs

DOX

Macrophage cell membrane camouflaged NPs (MPCM) Monocyte-derived nanoghost camouflaged NPs Platelet-NPs

PC membranes

[1]

[16] [45] [30] [48]

[51 [36]

Biomimetic nanomotors for detoxification Biomimetic Janus micromotors for detoxification

[44]

Biomimetic LLV to deliver drugs; extravasations through inflamed endothelium; natural cancer-targeting system Targeted cancer therapy

[46]

[43]

[32]

PLGA-NPs

DOX

Natural cancer-targeting system

[29]

PLGA-NPs

Docetaxel

Disease-specific targeted drug delivery; (restenosis therapy) Pathogen-targeted antibiotic delivery; toxin decoys

[21]

Vanc PC-mimicking nanovehicles (PM-NV)

sTRAIL-decorated PCs (sTRAIL-PCs)

Acryl amide nanogels

DOX

Circulating CC-targeting system; sequential drug delivery system; combinatorial anticancer therapy (TRAIL and DOX)

[26]

CC NPs

Mouse melanoma cells

PLGA-NPs

–

Cancer nanovaccine platform; natural cancer-targeting system; (homotypic targeting)

[20]

Bacterial membrane-NPs

Escherichia coli outer membrane vesicles (OMVs)

Au-NPs

–

Nanovaccine for antibacterial infection

[27]

MSC membrane-NPs

Genetically modified AdMSCs expressing CXCR4 receptor

PLGA-NPs

–

MSC-mimetic nanocarriers; targeting functionalization through CXCR4-SDF-1a

[25]

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membrane-coated hybrid NPs (SHNPs) using genetically modified adipose-derived MSCs (AdMSCs) as an alternative cell source [25]. CCs have inherent homotypic binding properties, and CC membrane components can be used as potent tumor antigens for the treatment of cancer by immunotherapy. Fang et al. used a similar procedure to that described for RBCNPs to prepare CC membrane-functionalized NPs (CCNPs) from B16–F10 mouse melanoma CCs [20]. With respect to drug delivery applications, properties of the material core significantly influence the drug-loading and releasing capabilities of CMFNPs. It was shown that the method of material core preparation, drug chemistry, drug solubility, and method of drug incorporation with the material core also influenced the carrier efficacy of CMFNPs [12]. For instance, Aryal et al. reported that chemically conjugated doxorubicin (DOX)-RBCNPs resulted in improved efficiency in terms of drug incorporation and sustained release [22]. CMFNPs are structurally sophisticated systems requiring robust characterization methods. Currently, the Nanotechnology Characterization Laboratory (NCL) and Frederick National Laboratory for Cancer Research serve as a ‘national resource and knowledge base’ for the characterization of nanomedicine [11,34]. Numerous physicochemical, in vitro, and in vivo characterization methods developed at the NCL have been used to predict the biofunctionalization process efficiency and clinical efficacy of CMFNPs [14,34]. Tables 2 and 3 provide a list of characterization methods that have been applied for the characterization of CMFNPs in recent studies, along with their advantages and disadvantages.

Biofunctionalized NPs for drug delivery applications Biofunctionalized NPs represent a promising nano drug delivery platform owing to their superior biomimicking ability with tailored targeting features [35]. Drug-loaded CMFNP formulations can be tailored for sustained or ‘on-demand’ drug release by tuning their physicochemical properties [30]. The selection of cell membranes allows for the design of nanocarriers with desired biological features for extended circulation and site-specific localization [35]. Moreover, CMFNPs can also be used for detoxification and cancer immunotherapeutics [36,37]. Here, we highlight the potential applications of CMFNP-based drug delivery systems (Fig. 2 and Table 1).

Biofunctionalized NPs as long-circulating drug carriers Systemic administration of nanomedicines often fails to meet clinical expectations [38]. The interface between the synthetic material surface and a biological system is responsible for the activation of the immune system, resulting in rapid clearance of the nanomedicine from the circulation [39]. Therefore, a long circulation half-life of a nanocarrier was found to be important for targeted drug delivery and sustained release [40]. PEGylation has been considered as an efficient strategy to extend the circulation half-life of a nanocarrier [41]. Despite the clinical success of PEGylated nanomedicine, unexpected immunogenic responses, known as the ‘accelerated blood clearance (ABC) phenomenon’, have been observed [19,41]. To overcome these challenges, the use of RBCNPs has been proposed owing to their longer circulation half-life (39.6 h) compared with PEGylated NPs (15.8 h) [19]. The researchers explained that the reason behind this effect was the

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presence of a self-signaling protein (CD47), which restricted the phagocytosis process through interactions with the signal regulatory protein alpha expressed by macrophages [19,42]. This potential advantage of RBC membranes has also been extended to the construction of biomimetic micro- and nanomotors, which show advanced mobility and biological functions [43–45]. In different experiments, WBC membrane-coated NPs also demonstrated biomimetic long circulation capabilities compared with silica and PLGA-NPs, resulting in leukocyte-like biological functions, including extended blood circulation, transmigration through the inflamed endothelium, and natural tumor tropic properties [18,29,46].

Biofunctionalized nanocarriers for photodynamic cancer therapy Photothermal therapy (PTT) and photodynamic therapy (PDT) have emerged as new strategies for the treatment of various cancers. PTT is a minimally invasive method in which photon energy is transformed into heat to destroy the CCs [47]. By contrast, PDT uses photosensitizers (PS) that become cytotoxic upon irradiation with laser light. Overall, the clinical efficacy of phototherapy-mediated cancer treatments critically relies on the precise delivery of PS agents to CCs [47]. Recently, long-circulating CMFNPs were demonstrated to achieve significant therapeutic efficacy of PTT and PDT. The clinical efficacy of PDT is dependent on the precise delivery of PS to the CCs and the efficient generation and release of singlet oxygen species. Ding et al. improved the targeting ability of a biofunctionalized PDT nanovector by inserting active targeting ligands, leading to an extended circulation time with increased tumor localization of the PDT nanovector [48]. Gold nanocages (AuNCs) have also been investigated for PTT. However, their short circulation half-life has so far limited their clinical efficacy. Piao et al. recently showed the combined advantages of RBC membranes and AuNCs to improve the efficacy of PTT. RBC membrane functionalization to the AuNCs enables a prolonged circulation time and significantly improved cancer localization, resulting in enhanced therapeutic efficacy of PTT [39].

Biofunctionalized NPs for sustained drug delivery applications The functionalization of RBC membranes has been used to control drug release kinetics from a polymeric matrix. A recent experiment conducted by Aryal et al. demonstrated that the RBC membranefunctionalized polymeric NP surfaces can act as a diffusion barrier, which could slow the release of DOX 1.2 times compared with bare particles, thereby improving the therapeutic efficacy of DOX in acute myeloid leukemia cells [22]. Li et al. developed a biomimetic, enzyme-responsive antibiotic delivery system using RBC membrane-functionalized supramolecular gelatin NPs (SGNPs) loaded with vancomycin (vanc). The antibiotic release was achieved by the degradation of polymeric NPs with gelatinase, which is present at the bacterial infection site. In addition, RBC membranes on SGNPs provided versatile functions, including detoxification, sustained drug release, and an extended circulation half-life [30]. Hsieh et al. reported a biomimetic ‘acoustically responsive anticancer drug delivery system’ based on a camptothecin (CPT)loaded RBC membrane functionalized with perfluro-n pentene (C5F12), termed ‘RBC membrane droplets’ (RBCMDs). RBCMDs undergo a transition from liquid droplets to a gas state upon highwww.drugdiscoverytoday.com

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TABLE 2

Examples of CMFNPs and their characteristics Biofunctionalized NPs Characterization methods (drug-polymeric core) Size Surface Morphology Surface Drug-loading and (hydrodynamic charge release studies (shape) protein diameter) (Zeta potential) characterization

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RBCNPs (Vanc-Gelatin)

RBCMB RBCmicrobubbles-CPT

LLV-DOX

Macrophage cell membrane camouflaged mesoporous silica NPs (MPCM-DOX)

90–130 nm

1.7 mm

1–2 mm

47.8–65.1 nm

Vanc-loading and release efficiency accessed by dialysis and quantified by RP-HPLC; drug-loading efficiency (DLE) 63.7%; drug-loading content (DLC) 11.4%

In vitro phagocytosis assay; assessment of enzyme (gelatinase)-responsive drug release; toxin removal capability of RBCNPs

[30]

Light microscopy

SDS-PAGE analysis

Anticancer drug (CPT-loading efficiency calculated by fluorescence intensities; DLC 2–3%; DLE 62–97%

In vitro phagocytosis assay; In vitro and in vivo anticancer activity for ultrasoundtriggered drug release

[49]

26 mV

TEM and SEM

Dot blot analysis

–

In vitro assay for endothelial cross penetration and DOX release; in vitro phagocytosis assay; in vivo tumor-tropic efficiency of LLVs

[46]

16.9 mV

TEM

–

DOX encapsulation efficiency evaluated by UV–vis spectroscopy; DLE 35.7%.

In vitro phagocytosis [32] assay; in vivo pharmacokinetic analysis and tumor-tropic efficiency of MPCM-camouflaged MSNCs

SDS PAGE and Western blot; TEM-immune gold labeling for further confirmation

Docetaxel loading and release quantified by HPLC; DLC 2.1  0.04 Vanc loading and release quantified by HPLC; DLC 4%

Assessment of pharmacokinetic, biodistribution, and immunocompatibility properties of PCNPs; assessment of natural targeting ability of PCNPs of damaged vasculature and pathogens

[21]

–

DOX loading and release quantified by spectrophotometer

In vitro phagocytosis assay; in vitro assay for site-specific sequential drug delivery; in vivo pharmacokinetics analysis; in vivo antitumor efficacy and elimination of circulating tumor cells

[26]

SDS PAGE and Western blot analysis

–

Assessment of natural cancer-targeting ability of CCNPs

[20]

8.7 mV TEM

–

113.4  1.2 nm

29.5  1.2 mV TEM

PC–PLGA-NPs-vanc (PCNPs)

200.3  3.1 nm

30.2  1.0 mV

Platelet mimicking nanovehicles; PMNV-sTRAIL and DOX

120.9 nm

21.3 mV

110 nm

35 to

TEM

40 mV TEM

intensity focused ultrasound ionization, which triggers drug release, and have shown great potential in anticancer therapy [49].

Biofunctionalized NPs for targeted drug delivery applications Targeted nanocarriers have improved the therapeutic efficacy of drugs with minimal adverse effects [40]. Currently, disease 1308

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Preclinical functional characterizations

SDS-PAGE analysis

26 to

PC-PLGA-docetaxel NPs (PCNPs-DOX)

CCNPs

Refs

targeting has been made possible by NP functionalization with ligands that target specific sites. Various conjugation chemistries have been used to improve the target specificity of NPs [40]. However, these chemical methods have a few drawbacks, such as high cost and complex chemistry [40]. Alternatively, the natural cell membrane-based biofunctionalization process has proven to

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Effective targeting of pathogens

Overcoming biological barriers

Disease-specific targeting system

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Transendothelial migration

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Targeting vascular disease

Immunotherapy

Evading immune systems

Long circulating carrier Circulating tumor cell (CTC)targeting carrier

Camouflaged drug carrier Natural tumor-homing ability

Antigen-presenting cell (APC)targeting carrier Drug Discovery Today

FIGURE 2

Applications of cell membrane-functionalized nanoparticles.

confer NPs with robust targeting functions because of their surface proteins [20]. In addition, active targeting ability can be achieved by inserting targeting ligands, such as the aptamer AS1411. For instance, Fang et al. demonstrated the active targeting ability of RBCNPs by inserting folate and the nucleolin-targeting aptamer AS1411 [1]. Despite these advantages, the natural targeting functionalization of NPs has considerable limitations, such as limited cell sources and their heterogeneous safety and efficacy profiles. Additionally, preserving the native structure of cell membrane surface components, such as proteins, and the instability of physically or chemically fused natural cell membranes on NPs also pose practical difficulties. Cancer immunotherapeutics are recent therapeutic modalities encompassing therapeutic cancer vaccines, which are designed to stimulate the immune system against cancer antigens [36,50]. Targeting antigen-presenting cells (APCs) is a crucial factor determining the clinical efficacy. Guo et al. prepared mannose-modified RBCNPs to target the mannose receptor on APCs, resulting in increased antigen-induced anticancer immune responses [36]. Alternatively, biofunctionalization of NPs using a natural cell source with intrinsic targeting abilities has shown significant advantages for natural targeting to the diseased site [21,51]. Fang et al. demonstrated the natural homotypic targeting abilities of a

CCNP platform prepared from the human melanoma CC line MDA-MB-435, which was demonstrated to have homotypic binding properties [20]. The CC membrane preferentially increased the affinity of the drug-loaded NPs to the source CCs because of the presence of cell adhesion molecules, resulting in improved particle uptake (40-fold) compared with control NPs [20]. The CCNP platform also demonstrated good potential as a cancer vaccine vehicle. The monophosphoryl lipid A (MPLA) adjuvant-encapsulated CCNP system has numerous tumor membranes, resulting in robust anticancer immune responses [20]. Similarly, PCs have inherent natural adherence properties to the injured vascular subendothelium and circulating pathogens, including staphylococci and streptococci [21]. The transference of the PC membrane and its associated adhesion proteins to PLGANPs (PNPs) provides multiple PC-like functions, such as reduction of the immune response, and PC-like binding properties, enabling selective adhesion to the diseased subendothelium and superior adhesion to specific pathogens [21]. This specific targeting feature of the PNP platform has been demonstrated for versatile targeted drug delivery applications. For instance, the antimicrobial efficacy of vanc-loaded PNPs, vanc-RBCNPs, and free vanc were compared for their antimicrobial efficacy in vitro and in vivo [21]. The vancPNPs showed significant improvement in MRSA252 (a strain of www.drugdiscoverytoday.com

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TABLE 3

Analytical modalities for the evaluation of the characteristics of biofunctionalized NPs

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Characterization methods

Properties can be analysed

Advantages

Limitations

Dynamic light scattering (DLS)

Size; size distribution (poly dispersity); stability

Nondestructive method; assured accuracy and reproducibility; compatible with multiple solvent; economic

Assumption of spherical shape of sample; limited resolution; insensitive to large particles; difficulties in measuring polydispersed samples

Electrophoretic light scattering

Zeta potential (surface charge); stability

Minimum sample requirement; sensitive method; user friendly; economic

Electro-osmotic effect; lack of reproducibility

SEM; environmental SEM (ESEM); TEM

Size and size distribution; shape (morphology); aggregation; surface protein analysis (immune gold labeling)

High resolution (subnanometer accuracy); TEM has higher spatial resolution than SEM; direct measurement and visualization

Conductive material coating required; specific nature of sample required (dry for SEM, liquid for TEM); insensitivity to size or size distribution measurement for heterogeneous samples; sample damage/alternation; expensive

Atomic force microscopy (AFM)

Surface properties; shape; size and size distribution; structure; aggregation

Direct measurement of surface topography; 3D sample surface mapping; subnanoscale resolution

Sophisticated method; application limited to exterior of material surface

Nuclear magnetic resonance (NMR)

Chemical composition, structure, and conformational changes

Nondestructive method; inexpensive

Low sensitivity; time-consuming process

Infrared spectroscopy (IR); Fourier transform infrared (FTIR)

Surface properties of bioconjugates; structural conformation

Inexpensive; assured accuracy and reproducibility

Interference and strong absorbance of H2O (IR); relatively less sensitivity for nanoscale analysis

SDS-PAGE; DOT blot (slot blotting); Western blotting

Protein analysis

Gold standard method for protein analysis (SDS-PAGE) and identification (Western blotting); Dot blotting is simplest method of western blotting suitable for identification of most of cell membrane proteins

Greater variability; skilled personnel required

Mass spectroscopy (MS)

Molecular weight, composition, and structure, including proteins and peptides

Assured accuracy and reproducibility; high sensitivity method

Sophisticated method; skilled personnel required

Circular dichroism (CD)

Thermal stability; structure and conformational changes of proteins/DNA

Nondestructive method; gold standard method for accelerated stability analysis of proteins

Less sensitive than absorption methods; weak CD signal for nonchiral chromophores

Surface enhanced Raman (SERS); Tip-enhanced Raman spectroscopy (TERS)

Conformational changes of protein–metallic NP conjugates and their structural and chemical properties

Increased spatial resolution; topological information of nanomaterials

Limited spatial resolution (only to micrometers); lack of reproducibility

methicillin-resistant S. aureus) reduction compared with vancRBCNPs, which was attributed to the targeting effect of the PNPs. In addition, the selective PNP adhesion to the diseased vasculatures was used for targeted antiproliferative drug (DOX) delivery for the treatment of coronary restenosis. These results demonstrate the benefit of PNP-directed delivery for improving drug localization to diseased vasculatures [21]. Circulating tumor cells (CTCs) are responsible for cancer metastases in distant organs via hematogenous dissemination. Fundamental studies have revealed that CTCs can locally induce thrombosis, including platelet activation and fibrin deposition, to form a protective cloak called tumor cell-induced PC aggregation (TCIPA), which in turn protects CTCs from immune cells, allowing them to spread to distant tissues [52,53]. The key mechanisms contributing to the effects of TCIPA include biomolecular binding, such as via P-selectin and CD44 receptors, and structurebased capture. Biomimetic functionalization of synthetic NPs with 1310

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PC membranes along with surface conjugation of tumor-specific apoptosis-inducing ligand cytokine (TRAIL) show a potential advantage of CTC adhesion for targeted drug delivery [54]. Such PCmimetic ‘Trojan horses’ were incorporated into CTC-induced thrombosis in the vasculature to deliver cancer-killing drugs within the CTC thrombi at a high local concentration, and were shown to be efficient in CTC neutralization to attenuate metastasis [54]. In another study, Hu et al. used the potential advantage of PC mimicry to develop a sequential and site-specific anticancer drug delivery system. This PC membrane-coated nanovehicle enabled the sequential delivery of TRAIL and DOX, which activate the extrinsic apoptosis and intrinsic apoptosis pathways, respectively [26].

Concluding remarks and future perspectives Although constrains remain, the advancement of nanomedicine is in full swing as it proceeds to fuel both academic and industrial

research [55]. To mitigate the challenges associated with conventional nanomedicines, the applications of CMFNPs have been widely explored [14]. CMFNPs have been prepared by using a topdown fabrication method, in which the natural cellular membranes can be efficiently functionalized on the NPs [35]. Combining the benefits of both synthetic and natural platforms, CMFNPs show significant therapeutic potential, including selective targeting and prolonged circulation by evasion of the immune system, and a precise and strong biological response with ensured safety [24,49]. In particular, most CMFNPs showed an extended circulation half-life, increasing the likelihood that they will accumulate at the desired sites specifically within CCs [19,25]. In addition to their long circulation time, much attention has been paid to the use of CMFNPs to enhance the efficacy of therapeutic molecules [13,14]. With the increasing interest in natural target functionality, research in this area is also likely to expand. The presence of physiological barriers is a principal factor limiting drug accumulation at diseased sites, and remains the greatest challenge to drug developers [56]. Site-specific delivery of nanotherapeutics is a promising strategy to overcome this challenge, which will likely realize a paradigm shift in nanopharmaceuticals [55]. Cell membrane-based biofunctionalization of materials conferred site-specific natural targeting abilities to NPs [21]. Moreover, additional targeting capabilities were achieved by the insertion of targeting ligands [48]. Engineering the flexibility of a material core in the CMFNP platform can result in versatile drug carrier features, in which multiple drugs or therapeutic agents can be incorporated and released in either a sustained or controlled manner [26,30]. Combinatorial drug delivery is a potential strategy that provides a synergistic pharmacological effect for efficient disease treatments [26]. The rational design of CMFNPs resulted in the release of combined therapeutic agents either simultaneously or in a sequential manner, which could improve the efficacy of drugs several fold [26]. Furthermore, the cell membrane coating can be selected to have desired therapeutic benefits. For instance, a RBC membrane can be used to extend the nanocarrier circulation, and other cell membranes, such as those of WBCs, PCs, or SCs, can be used for natural targeting functionalities [18,24,35,39]. Currently, RBC-, PC-, WBC-, SC-, and CC-derived membrane vesicles have garnered

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the most attention owing to their unique therapeutic and natural targeting potentials [13]. In particular, the use of CMFNPs based on these cell sources in drug delivery applications has been investigated at different preclinical stages [14]. CC membranebased natural targeting functionalization is a novel method that could be applied for advanced cancer treatments, such as cancer targeting and immunotherapeutics [20]. Furthermore, WBC membrane-mediated functionalization enables the materials to cross biological barriers, including the endothelium and tumor membranes [29,32]. Similarly, PC membrane-functionalized NPs have multifaceted therapeutic abilities, including vascular-targeted drug delivery and pathogen-specific antibiotic delivery [15,21]. The potential advantages of AdMSC-functionalized NPs for various disease treatments are also under investigation [25]. The above-mentioned therapeutic capabilities of CMFNPs can be readily translated to the clinical stage or efficiently applied for the development of small-molecule ‘nanoversions’. Despite these advances in the development and application of CMFNPs, the field is still in its infancy and there are several challenges that need to be overcome to meet clinical expectations. We have listed the key challenges and opportunities in Box 1. This situation is likely to change in the near future. Overall, biofunctionalized nanocarriers hold great potential for the treatment of various diseases. Looking forward, the cell membrane-mediated biomimetic functionalization process holds tremendous promise to revolutionize nanomedicine across various disciplines and specialties. We expect that CMFNPs could also eventually replace current cellular therapies and be used for the development of artificial cells.

Acknowledgments This research was supported by National Research Foundation of Korea funded by the Ministry of Science, ICT & Future Planning (NRF-2016R1A2A1A05004987), the Bio & Medical Technology Development Program of the NRF funded by the Korean government, MSIP (NRF-2014M3A9D3033887) and a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (Grant Number: HI15C1744).

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