Letter Cite This: Nano Lett. XXXX, XXX, XXX−XXX
pubs.acs.org/NanoLett
Macrophage-Membrane-Coated Nanoparticles for Tumor-Targeted Chemotherapy Yu Zhang,† Kaimin Cai,‡ Chao Li,† Qin Guo,† Qinjun Chen,† Xi He,† Lisha Liu,† Yujie Zhang,† Yifei Lu,† Xinli Chen,† Tao Sun,† Yongzhuo Huang,§ Jianjun Cheng,*,‡ and Chen Jiang*,† †
Key Laboratory of Smart Drug Delivery, Ministry of Education, State Key Laboratory of Medical Neurobiology, Department of Pharmaceutics, School of Pharmacy, Fudan University, Shanghai 201203, China ‡ Department of Materials Science and Engineering, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United States § Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Shanghai 201203, China S Supporting Information *
ABSTRACT: Various delivery vectors have been integrated within biologically derived membrane systems to extend their residential time and reduce their reticuloendothelial system (RES) clearance during systemic circulation. However, rational design is still needed to further improve the in situ penetration efficiency of chemo-drug-loaded membrane delivery-system formulations and their release profiles at the tumor site. Here, a macrophagemembrane-coated nanoparticle is developed for tumor-targeted chemotherapy delivery with a controlled release profile in response to tumor microenvironment stimuli. Upon fulfilling its mission of tumor homing and RES evasion, the macrophage-membrane coating can be shed via morphological changes driven by extracellular microenvironment stimuli. The nanoparticles discharged from the outer membrane coating show penetration efficiency enhanced by their size advantage and surface modifications. After internalization by the tumor cells, the loaded drug is quickly released from the nanoparticles in response to the endosome pH. The designed macrophage-membrane-coated nanoparticle (cskc-PPiP/PTX@ Ma) exhibits an enhanced therapeutic effect inherited from both membrane-derived tumor homing and step-by-step controlled drug release. Thus, the combination of a biomimetic cell membrane and a cascade-responsive polymeric nanoparticle embodies an effective drug delivery system tailored to the tumor microenvironment. KEYWORDS: tumor microenvironment, macrophage-membrane coating, cascade-responsiveness, biomimetic delivery system, breast-cancer targeting
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characteristics or behaviors such as immunological response and off-target protein adsorption during systemic circulation.5,6 Nanoparticles camouflaged by biologically derived cell membranes have recently attracted attention for their prolonged circulation in vivo.7,8 Their innate self-recognition features optimize their dynamic properties by cloaking the contents to evade RES elimination and immunological surveillance. The membrane camouflage strategy can achieve a superior half-life in systemic circulation and improve tumor adhesion ability without loss of drug-loading capacity or the nanosize advantage.9,10 Various delivery vectors including goldbased nanoplatforms, upconversion nanoparticles, and mesoporous silica nanoparticles have been integrated into macrophage-membrane systems with good stability during systemic circulation.11,12 The innate inflammation-directed chemotactic
anoparticles have been explored as a promising delivery vector for cancer therapeutic agents with the potential for great impact on future public health.1,2 To achieve favorable antineoplastic effects, the formulations should cross multiple physiological barriers by responding appropriately to different intracorporal milieus. A nanoparticle requires long residence in systemic circulation with considerable stability against plasma dilution, opsonization, and reticuloendothelial system (RES) clearance.3,4 Upon passive or active accumulation at a cancerous site, the tumor uptake amount and drug-release efficiency will be the key factors in its general curative effect. Therefore, the rational design of drug delivery systems must incorporate both stabilizing strategies and on-demand drugrelease mechanisms. Miscellaneous approaches involving particle size, surface charge, morphology, and terminal modification have been investigated to extend circulation halflife and improve targeting ability. Nevertheless, synthetic nanomaterials have still been reported to possess dangerous © XXXX American Chemical Society
Received: December 14, 2017 Revised: February 21, 2018
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DOI: 10.1021/acs.nanolett.7b05263 Nano Lett. XXXX, XXX, XXX−XXX
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Nano Letters ability of macrophages could drive the vector to accumulate in chronic inflammatory tumor tissue, offering promise for orthotopic therapies such as photothermic and photodynamic therapy.13,14 However, barriers to chemo-drug delivery by macrophage-membrane-camouflaged systems remain. In the classical mononuclear phagocytic system, macrophages are inherently driven to eliminate malignant cells and deplete fibrosis.15,16 However, contrary to its phagocytotic nature, the macrophage-derived vesicle must instead be engulfed by tumor cells to achieve drug delivery. After accumulation at tumor sites, the membrane coating becomes an obstacle impeding drug release. Therefore, an appropriate membrane escape tactic is an additional prerequisite for chemo-drug delivery platforms with biologically derived membrane coatings. Meanwhile, we noted that a similar membrane escape phenomenon had been discovered in the intracellular release process in endosomes. Cationic polymeric drug carriers, such as H+-capturing sponges, can induce an excessive influx of electrolyte and water into the acidic late endosome to achieve an internal equilibrium of electric neutrality and ion strength, thus impelling the endosome membrane to burst and causing extravasation of the nanoparticle contents.17,18 This phenomenon is termed the proton sponge effect.19 Our previous studies on poly(amino acid)-based vectors also showed favorable endosome escape and intracellular drug-release abilities.20−22 Like the endosomal environment, the tumor microenvironment features reduced pH owing to oncogenic transformation and abnormal metabolism.23,24 Thus, the acidic tumor-tissue microenvironment may provide H+-rich conditions enabling nanoparticle escape from the membrane enclosure, similar to that occurring in the late endosome via the proton sponge effect. Herein, inspired by the proton sponge effect, a biomimetic macrophage-membrane-coated nanoparticle (cskc-PPiP/PTX@ Ma) was engineered to exhibit step-by-step release behavior in response to the differences in pH in the tumor microenvironment. Natural macrophage membranes with their associated membrane proteins were reconstructed into vesicles without loss of their inflammatory tumor-homing ability. During systemic circulation, the vesicle membrane was expected to serve as a concealing cloak against opsonization and RES clearance and as a tumor-homing navigator to enhance tumor accumulation. In the first release stage, once the tumortargeting task of the membrane cloak was complete, the interstitial pH would cause the membrane-coated formulation to undergo expansion and eruption, removing the coat. The discharged nanoparticles could then be further taken up by tumor cells, assisted by their surface modification with a targeting peptide. In the second release stage, the encapsulated chemo-drug would finally be released from the nanoparticles in response to the intracellular pH of the tumor cells. This stepby-step release strategy should optimize the drug-release kinetics in the tumor microenvironment while maintaining the tumor-targeting ability of the membrane-coating system in systemic circulation. Paclitaxel (PTX), a classic hydrophobic anticancer drug, was chosen as the model drug to test the formulation’s delivery capacity and therapeutic effect in an orthotopic breast-cancer-bearing mouse model. The biodistribution, pharmacodynamics, and general tissue toxicity suggested that the resulting formulation, cskc-PPiP/PTX@ Ma, offered promise for breast-cancer therapy. The preparation of the membrane-coated nanoparticles is illustrated in Figure 1A. For the construction of the internal
Figure 1. A. Scheme of the preparation of membrane-coated nanoparticles. B. Microscope images (63× oil lens, crop) of cskcPPiP/PTX@Ma (a) and PPC8/PTX@Ma (b) in buffers at pH 7.4 and 6.5.
nanoparticle, amphiphilic bola-pattern polymers with selected side chains were synthesized by the dual-end PEGylation of poly(β-amino ester) for hydrophobic drug loading.25,26 The pH-sensitive polymer was functionalized with a cationic 2aminoethyldiisopropyl group (PPiP) to tune its buffer capacity to the extracellular pH of the tumor region. The pH-insensitive polymer with neutral octyl group side chains (PPC8) shared the same carbon number but lacked the branched structure and protonation capacity of the pH-sensitive polymer (Scheme S1, Table S1). The molar ratio of backbone to side chain monomer was tuned to 1.05:1 to produce a symmetric structure with both ends terminating in acrylate groups for further terminal PEGylation. To further facilitate tumor cell uptake after tumor homing and membrane exuviation, a synthetic D-form oligopeptide with the sequence cskc was chosen as the targeting ligand for nanoparticle surface modification. This oligopeptide was reported to show high affinity to the insulin-like growth B
DOI: 10.1021/acs.nanolett.7b05263 Nano Lett. XXXX, XXX, XXX−XXX
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Figure 2. (A) Illustration of membrane escape and drug-release mechanism. Cumulative drug-release profile of PPiP/PTX@Ma (B) and PPC8/ PTX@Ma (C) at various pH values. DLS (D, E) and TEM (F, G) images of PPiP/PTX in buffer at pH 7.4 and 6.5. Changes in the ζ potential (H) and CMC value (I) of the polymers PPiP and PPC8 at various pH values.
prepared nanoparticle with freshly extracted macrophage membrane and were proven to possess sufficient drug-loading capacity for in vivo drug delivery (Table S2). The morphology of cskc-PPiP/PTX@Ma consisted of uniform spheres at pH 7.4 but cracked at pH 6.5 to give sickle-shaped particles (Figure 1Ba). Meanwhile, PPC8/PTX@Ma maintained its globular shape intact with changing pH (Figure 1Bb). DLS data further
factor 1 receptor (IGF1R), which is aberrantly highly expressed on tumor cells because of metabolic disturbance.27−29 The targeting polymer (cskc-PPiP) was optimized by terminal conjugation of the IGF1R-targeting peptide on the hydrophilic PEG end of PPiP. All the polymers were characterized by 1H NMR spectroscopy (Figures S1−S6). The membrane-coated nanoparticles were prepared by repeated extrusion of the asC
DOI: 10.1021/acs.nanolett.7b05263 Nano Lett. XXXX, XXX, XXX−XXX
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Figure 3. (A) Penetration efficiency of PPC8@Ma and cskc-PPiP@Ma into tumor spheroids with 2 h of incubation. Upper panels show results in pH 7.4 medium and lower panels in pH 6.5, scale bar 100 μm, z-axis depth 20 μm. (B) Tumor sections of mice 24 h after injection with cskc-PPiP, PPC8@Ma, PPiP@Ma, and cskc-PPiP@Ma, scale bar 100 μm. DAPI (blue), CD34 (green), and Probe (red) stained the cell nuclei, blood vessels, and formulation trace, respectively.
confirmed the change in the size of membrane-coated nanoparticles with hydration status (Figure S7A,B). Meanwhile, both formulations exhibited a small negative charge with the membrane coating in comparison with that of the nude particles (Figure S7C). This interesting phenomenon suggested that the change in morphology might provide a way to achieve membrane decortication and discharge of the interior nanoparticle in response to the acidity of tumor tissue. The release kinetics of both formulations were then investigated in milieus
simulating tumor tissue and intracellular acidity (Figure 2A). When the pH was decreased from 7.4 to 6.5, PPiP/PTX@Ma showed only a slight rise in the accumulative release plateau (Figure 2B), although the external membrane was cracked (Figure 1B). Meanwhile, the internal drug-loading nanoparticles (PPiP/PTX) maintained their nanoscale shape intact despite morphological expansion from pH 7.4 (Figure 2D,F) to pH 6.5 (Figure 2E,G). When the pH approached 5.0, simulating the endosomal pH environment, the drug release D
DOI: 10.1021/acs.nanolett.7b05263 Nano Lett. XXXX, XXX, XXX−XXX
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Figure 4. Biodistribution and in vivo antitumor efficacy. (A) IVIS images of mice injected with near-infrared probe-loaded cskc-PPiP and cskc-PPiP@ Ma at designated time points. (B) 3D reconstruction of fluorescence signal in cskc-PPiP@Ma-treated mouse at 48 h. (C) Heart (H), liver (Li), spleen (S), lung (Lu), kidney (K), and tumor (T) excised from the above-mentioned mouse. (D) Quantification of PTX concentration in organs and tumor tissue excised from mice treated with Taxol, cskc-PPiP/PTX, PPC8/PTX@Ma, and cskc-PPiP/PTX@Ma (n = 4). Body weight (E) and tumor volume (F) data were recorded during the 3 week treatment course. (G) Tumor tissue apoptosis in mice treated with saline, Taxol, PPC8/ PTX@Ma, PPiP/PTX@Ma, and cskc-PPiP/PTX@Ma. Green signals indicate apoptotic cells in tumor section. Scale bar, 100 μm.
of PPiP/PTX@Ma was rapidly enhanced by the additional H+. The release behavior of PPiP/PTX (Figure S8A) was consistent with that of PPiP/PTX@Ma, which confirmed that the drug
unpacking relied on particle disassembly rather than the membrane escape process. In contrast, PPC8/PTX@Ma showed consistent size and appearance at pH 7.4 and 6.5 E
DOI: 10.1021/acs.nanolett.7b05263 Nano Lett. XXXX, XXX, XXX−XXX
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mouse models bearing orthotopic tumors. Tumor sections were stained with DAPI (blue) to locate tumor cells and CD34 (green) to show intratumor vessels (Figure 3B). Another fluorescence probe (red) allowed tracing of the internal nanoparticles in the tumor tissue. Significantly greater tumor accumulation and distal penetration from the vessel toward the interior of the tumor were found in the PPiP@Ma- and cskcPPiP@Ma-treated groups than in the PPC8@Ma-treated group. Moreover, cskc-PPiP@Ma displayed generally higher fluorescence intensity than PPiP@Ma, indicating greater cellular uptake, which was deduced to be related to the IGF1R-mediated uptake pathway. Furthermore, general biodistribution was investigated by the injection of near-infrared fluorescence-probe-loaded cskcPPiP@Ma and cskc-PPiP followed by observation at designated time points (2, 4, 12, 24, and 48 h; Figure 4A). The cskcPPiP@Ma group showed rapid tumor homing within 2 h and long residence at the cancerous site with only slight signal attenuation until 48 h, whereas the cskc-PPiP group displayed significant liver and kidney accumulation in the first 2 h and gradual clearance in 24 h. The 3D reconstructed fluorescent photograph of the cskc-PPiP@Ma-treated mouse also demonstrated the favorable tumor accumulation of PPiP@Ma (Figure 4B). The major organs (heart, liver, spleen, lung, and kidney) and whole tumor tissue showed similar distribution tendencies (Figure 4C, left cskc-PPiP, right cskc-PPiP@Ma). The distribution profiles of various drug-loading formulations were further evaluated by the quantitative determination of the PTX concentrations in perfused and saline-washed organs and tumor tissues 24 h postinjection (Figure 4D and Figure S13). Because of its tumor-tissue pH-responsive escape mechanism and active targeting, cskc-PPiP/PTX@Ma showed greater tumor accumulation than PPC8/PTX@Ma. In addition, despite its lack of membrane coating, cskc-PPiP/PTX showed greater tumor accumulation than PTX in 50% cremophor−ethanol solution due to its active targeting ability. Since slight fluorescent signals and drug accumulation were observed in other organs, we assessed the systemic toxicity of various formulations. The polymers (PPiP, PPC8) and biologically derived membrane materials (Ma) were found to be nontoxic to 293 cells, which indicated their good biocompatibility and low toxicity (Figure S14A). The uptake experiment in the 293 cell line also showed little PTX internalization, as the intact membrane coating protected the internal drug-loading nanoparticles (Figure S14B). H&Estained sections showed inflammation in the portal area of the liver treated with Taxol, while no significant organic injury was observed in organs from the mice treated with membranecoated formulations (Figure S14C). The inflammation may have been caused by the cosolvents of Taxol, ethanol, and cremophor, which could induce liver damage with repeated injection.31,32 In the next pharmacodynamics experiments, MTT and apoptosis tests were conducted to evaluate the in vitro cytotoxicity of the naked drug-loaded nanoparticles to MDAMB-231 cells. The cell viability and IC50 values demonstrated that all the naked particle formulations exhibited valid antitumor efficacy in vitro (Figure S15A). To further elucidate the antitumor efficacy of those membrane-coated nanoparticles, we evaluated the apoptosis of MDA-MB-231 cells treated with Taxol, PPC8/PTX@Ma, PPiP/PTX@Ma, and cskc-PPiP/ PTX@Ma, all in PBS pH 6.5 before incubation. The annexinV (green) signal indicated everted phosphatidylserine during
(Figure S9), which was consistent with its slow release at all pH values (Figure 2C), even slower than that of PPC8/PTX (Figure S8B). This result indicated that the membrane-coating strategy contributed to the integrity and stability of the formulation. Changes in the ζ potential and critical micelle concentration (CMC) values of different polymeric nanoparticles indicated that the stepwise protonation of the PPiP polymer might contribute to the morphological expansion and gradual loss of drug-loading capacity of PPiP/PTX@Ma (Figure 2H,I). A titration test showed good buffering capacity of PPiP in the pH range 6.0−7.0, while it became too cationic to complete micellization at pH 5.0 (Figure S10). In the acidic extracellular microenvironment, the interior PPiP/PTX nanoparticle performed as a H+-absorbing proton sponge and could escape from the ruptured coating after equilibrium disruption of the capsular membrane structure. After uptake by the tumor cells, the even lower intracellular pH finally caused nanoparticle disassembly and drug release. The buffering capacity derived from the chemical structure of the polymeric materials led to different drug-release behavior under different biomimetic conditions. The cellular internalization behavior of cskc-PPiP/PTX was then tested in the MDA-MB-231 cell line. A synthetic oligopeptide targeting IGF1R was chosen to facilitate uptake by breast-cancer cells.29,30 The ligand modification rate was optimized to 20%, as increased modification did not further improve the uptake efficiency (Figure S11A,B). The internalization mechanism of cskc-PPiP/PTX was then studied by individually inhibiting different endocytosis pathways by treating the cells with filipin complex (caveolae-mediated pathway), phenylarsine oxide (PhAsO and clathrin-dependent pathways), colchicine (macropinocytosis), and ice incubation (ATP-dependent pathway). The results showed that the uptake of coumarin-loaded particles (cskc-PPiP/coumarin) mainly relied on ATP-facilitated caveolae-mediated and clathrindependent pathways (Figure S11C,D). Confocal images of cskc-PPiP/coumarin traces in the cytoplasm showed obvious overlap with the acidic late endosome after 0.5 h of incubation, suggesting that cskc-PPiP/PTX might follow the same internalization process and undergo second-stage intracellular drug release (Figure S11E). Based on the in vitro uptake results in monolayer tumor cells, we further explored the tumor-penetrating ability of cskcPPiP@Ma and the control PPC8@Ma both in vitro and in vivo by constructing red BODIPY-loaded formulations (Figure 3A). We conducted an in vitro experiment on tumor spheroids at two different pH values to evaluate the correlation with internal nanoparticle escape. In the blood-mimicking milieu (pH 7.4), both cskc-PPiP@Ma and PPC8@Ma showed only surface adsorption on tumor spheroids because their size limited infiltration into tight intercellular junctions without additional assistance. In the tumor-tissue microenvironment-mimicking milieu (pH 6.5), cskc-PPiP@Ma displayed considerable penetration efficiency since the internal nanoparticles underwent membrane escape. Meanwhile, PPC8@Ma maintained membrane integration and adsorption on the spheroid surface. In addition to the size and flexibility advantages resulting from membrane escape, the targeting ligand was also found to contribute to the general penetration efficiency. The penetration data of PPiP@Ma and cskc-PPiP@Ma demonstrated that cskc modification facilitated faster penetration than that of plain PPiP after membrane escape (Figure S12, pH 6.5, 0.75 h of incubation). The penetration study was then repeated in F
DOI: 10.1021/acs.nanolett.7b05263 Nano Lett. XXXX, XXX, XXX−XXX
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ACKNOWLEDGMENTS We acknowledge the support from National Science Fund for Distinguished Young Scholars (Grant 81425023), National Natural Science Foundation of China (Grant 81373355), and Fudan-SIMM Joint Research Fund (Grant FU-SIMM 20174009). Jianjun Cheng acknowledges support from the NSF (DMR-1309525) and NIH (R01 1R01CA207584 and R21 CA198684). Kaimin Cai acknowledges Beckman Institute Graduate Fellowship support at the University of Illinois at Urbana−Champaign. Yu Zhang acknowledges China Scholarship Council (CSC).
early apoptosis, while PI (red) marked membrane-permeable cells in late apoptosis (Figure S15B). The MTT results showed that PPC8/PTX@Ma, because the drug-loaded nanoparticles were sealed in intact membrane coatings, had lower cytotoxicity than PPiP/PTX@Ma and cskc-PPiP/PTX@Ma, as the enclosed drug-loading nanoparticles escaped from the membrane vesicles during PBS incubation via the proton sponge effect, unleashing their antitumor efficacy. The cskc-PPiP/ PTX@Ma demonstrated particularly enhanced antitumor ability because of its facilitated cellular internalization. The in vivo antitumor effects of various PTX formulations were further investigated in an orthotopic breast-cancer tumor model by intravenous administration every 4 days for 3 weeks. The mice’s body weight and tumor volume were recorded every 2 days to evaluate the general toxicity and antitumor efficacy (Figure 4E,F and Figure S16). The cskc-PPiP/PTX@Ma-treated group showed significant control of the tumor burden while maintaining a healthy body weight. The mice in the Taxol group suffered from continuous weight loss, mainly due to nonselective biodistribution and excipient toxicity (Figure S14C). Tumor sections from mice in each group were stained for tissue-level apoptosis detection (Figure 4G). The samples from the cskc-PPiP/PTX@Ma group displayed the most extensive cell apoptosis, indicating the remarkable in vivo antitumor effect of cskc-PPiP/PTX@Ma. In summary, we have developed a macrophage-membranecoated nanoparticle delivery system with a step-by-step release profile in response to the pH differences in the tumor microenvironment. The resulting formulation (cskc-PPiP/ PTX@Ma) exhibited favorable tumor-homing ability in systemic circulation and high biocompatibility resulting from its membrane coating. The unique buffering property of the PPiP materials for the interior nanoparticle further endowed the formulation with tuned drug-release kinetics responsive to extracellular and intracellular tumor microenvironment stimuli. The combination of biomimetic cell membranes and responsive polymeric nanoparticles could inspire the rational design of membrane-coating systems for tailored chemo-drug delivery to tumors.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.7b05263. Detailed methods, synthesis and characterization of polymers, characterization and pharmacodynamics data of control formulations, and internalization mechanism study (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. ORCID
Kaimin Cai: 0000-0001-9442-8312 Yongzhuo Huang: 0000-0001-7067-8915 Jianjun Cheng: 0000-0003-2561-9291 Chen Jiang: 0000-0002-4833-9121 Notes
The authors declare no competing financial interest. G
DOI: 10.1021/acs.nanolett.7b05263 Nano Lett. XXXX, XXX, XXX−XXX
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