Self-Assembly of Extracellular Vesicle-like Metal–Organic Framework

Article Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

pubs.acs.org/JACS

Self-Assembly of Extracellular Vesicle-like Metal−Organic Framework Nanoparticles for Protection and Intracellular Delivery of Biofunctional Proteins Gong Cheng,†,‡ Wenqing Li,†,‡ Laura Ha,† Xiaohui Han,† Sijie Hao,† Yuan Wan,†,‡ Zhigang Wang,†,‡ Fengping Dong,§ Xin Zou,† Yingwei Mao,§ and Si-Yang Zheng*,†,‡,∥ †

Department of Biomedical Engineering, ‡Penn State Materials Research Institute, §Department of Biology, and ∥Penn State Hershey Cancer Institute, The Pennsylvania State University, University Park, Pennsylvania 16802, United States S Supporting Information *

ABSTRACT: The intracellular delivery of biofunctional enzymes or therapeutic proteins through systemic administration is of great importance in therapeutic intervention of various diseases. However, current strategies face substantial challenges owing to various biological barriers, including susceptibility to protein degradation and denaturation, poor cellular uptake, and low transduction efficiency into the cytosol. Here, we developed a biomimetic nanoparticle platform for systemic and intracellular delivery of proteins. Through a biocompatible strategy, guest proteins are caged in the matrix of metal−organic frameworks (MOFs) with high efficiency (up to ∼94%) and high loading content up to ∼50 times those achieved by surface conjunction, and the nanoparticles were further decorated with the extracellular vesicle (EV) membrane with an efficiency as high as ∼97%. In vitro and in vivo study manifests that the EV-like nanoparticles can not only protect proteins against protease digestion and evade the immune system clearance but also selectively target homotypic tumor sites and promote tumor cell uptake and autonomous release of the guest protein after internalization. Assisted by biomimetic nanoparticles, intracellular delivery of the bioactive therapeutic protein gelonin significantly inhibits the tumor growth in vivo and increased 14-fold the therapeutic efficacy. Together, our work not only proposes a new concept to construct a biomimetic nanoplatform but also provides a new solution for systemic and intracellular delivery of protein.

■

INTRODUCTION Proteins play dynamic and diverse roles in various cellular processes, including metabolism, signaling, energetics, gene regulation, and immune response. Many diseases arise from functional alteration or deficiency of intracellular proteins.1 Therefore, protein therapy has great potential in cell manipulation and disease treatment.2−4 Protein therapeutics is often realized indirectly through transfection of DNA or mRNA encoding the effector protein.5,6 Intracellular delivery of proteins offers high specificity, low toxicity, transient modulation of cell function, and no risk of genomic alteration for medical treatment, which distinguishes it from conventional gene therapy.7,8 Nonetheless, the formulation and delivery of proteins are fundamentally challenging because proteins have poor cell membrane permeability and high susceptibility to degradation and denaturation.9,10 To address these issues, integration of proteins with cell-penetrating molecules or functional nanocarriers to assist the internalization is one of the pioneer explorations.11−15 Especially, polymeric capsules and cages can partly protect proteins to alleviate the tendency of proteins to degrade,16,17 while low protein loading efficiency © XXXX American Chemical Society

and capacity would limit the therapeutic effectiveness. Moreover, implementation of synthetic carriers in systemic delivery of protein often suffers from rapid clearance by the phagocyte system18,19 and low delivery efficiency to the targeted tissue,20 due to their exogenous nature and the lack of specific targeting domains. An ideal delivery system should encapsulate cargo proteins with high efficiency and capacity in a compatible way, protect the proteins from protease attack and mononuclear phagocyte system clearance in the physiological environment, selectively deliver the protein cargo to targeted tissues, and release proteins after internalization. To develop such a biocompatible nanosystem, herein, we designed a biomimetic nanosystem that assembles cargo proteins in pH-responsive metal−organic framework (MOF) nanoparticles and camouflages the nanoparticles with an extracellular vesicle membrane (EVM) (Figure 1). Proteins are caged in the MOF (ZIF-8)21,22 matrix by selfassembly of blocks of metal nodes (Zn2+) and organic ligands Received: April 2, 2018

A

DOI: 10.1021/jacs.8b03584 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Article

Journal of the American Chemical Society

delivery,29 which was realized by adjusting the concentration of the organic ligands (Figure S1) and proteins (Figure S2). Various proteins with different isoelectric points or molecular weight, including bovine serum albumin (BSA), cytochrome c (Cyt c), and gelonin, could be encapsulated in MP nanoparticles without altering the morphology or crystal structure of the MOF matrix (Figures 2A and S3 and Table S1). Successful incorporation of protein cargos in MP nanoparticles is evidenced by the presence of characteristic protein absorption peaks around 1667 cm−1 (CO)30 in Fourier transform infrared (FTIR) spectroscopy (Figures 2A, S3F). Importantly, the protein loading efficiency (percentage of MP-encapsulated proteins over total proteins) is as high as ∼94% at low initial protein concentration. Over ∼41% protein loading capacity (weight percentage of proteins in MP nanoparticles) can be achieved at high initial protein concentration (Figure S4). It is 3−50 times those achieved by surface conjunction or adsorption,31,32 which would greatly reduce the therapeutic dosage. Notably, MOFs have a characteristic pore aperture size of ∼1 nm (Figure S5), and MP nanoparticles also inherit the unique porous structure (Figure 2B). Thus, the MP nanoparticles could prevent the proteases (size: 2−10 nm),33 a major threat for proteins in the biological environment, from accessing the caged proteins, thereby protecting the caged proteins from digestion by protease (Figure S6). Moreover, the MP nanoparticles are pH-responsive for stimulus−release of guest cargos (Figure 2C). Although the MP nanoparticles are stable at physiological pH (Figure S7), they degraded in a few hours under acidic conditions (Figure S8), as metal−ligand bonds in MOFs are easily hydrolyzed to yield the protonated ligand under acidic condition.34 EVs were collected by ultracentrifugation from the culture medium of human breast adenocarcinoma MDA-MB-231 cells (Figure 2E).26 EVM (Figure 2F) was extracted through a hypotonic treatment and followed by ultracentrifugation to reconstitute the proteolipid patches (Figure S9). Self-assembly of the EVM on the MP nanoparticle surface was driven by ultrasonication and repeated extrusion. Notably, some specific interactions (e.g., electrostatic, hydrophilic, and coordination)35−37 between the EV membrane and MOF matrix particles could contribute to the successful preparation of the EMP nanoparticles (Figure S10). The ratio of EVM to MP nanoparticles was optimized to modulate the uniformity, membrane thickness, and surface stability of the coating (Figure S11). As a result, EVM patches could completely cover the MP nanoparticles and form a distinctive corona of the EMP nanoparticles (Figure 2G). In comparison to MP nanoparticles, the surface morphology is slightly changed due to the presence of a biofunctional membrane on the nanoparticles (Figure S12). Our strategy could deplete most of the tumorigenic contents (∼75% EV proteins and ∼70% EV RNA) inside the EVs (Figure S13), reducing the potential risk of tumor metastasis and growth resulting by EVs in downstream applications. Over 97% of the MP nanoparticles are successfully decorated with the EVM (Figure 2H). Biomimetic nanoparticles without loading cargo proteins have a similar protein molecular weight distribution pattern to that of the EVM (Figure 2I), while it is different from that of EVs, implying they inherit surface receptors and membrane proteins of the EVM. Physicochemical characterizations show that the final EMP nanoparticles have a slightly larger size than the bare MP nanoparticles (Figure 2J,K and Table S2) and possess a nearly equivalent surface charge to that of EV and EVM (Figure

Figure 1. Schematic illustration of the procedure of preparing biomimetic EMP nanoparticles for homotypic targeting and intracellular delivery of guest proteins. I: Caging protein cargo by selfassembly of blocks of inorganic nodes and organic ligands to synthesize MOF-protein (MP) nanoparticles. II: Extraction of extracellular vesicle membrane (EVM) through a hypotonic treatment of extracellular vesicle (EV). III: Self-assembly of EVM on MP nanoparticle surface by ultrasonication and extrusion to form EVMOF-protein (EMP) nanoparticles. IV: Systemic and intracellular delivery of the guest proteins by EMP nanoparticles.

(2-methylimidazole) to form the MOF-protein (MP) nanoparticles. The MP nanoparticles possess a large internal surface area and noncovalent affinity,23 enabling a remarkably high protein loading efficiency (∼94%) and loading capacity (∼41%). After internalization by cells, the pH-responsive metal−ligand bonds enable a release of proteins in acidic endosomes and lysosomes.24 In previous studies, we and others documented that extracellular vesicles (EVs) could contain integrin-associated transmembrane protein CD47, which mediates a “do not eat me” signal to evade phagocytosis and membrane-anchored proteins to enhance specific endocytosis by homotypic cells.25−28 Thus, it is promising that camouflage of the nanoparticles with EVM to mimic the EVs would assist the nanoparticles in escaping from phagocytosis, target specific tissue, and penetrate the membrane barriers to achieve systemic and intracellular protein delivery. To demonstrate the overall approach, we caged gelonin, an rRNA disruption N-glycosidase, as the cancer therapeutic protein to form MP nanoparticles of ∼80 nm in diameter and enveloped the MP nanoparticles with EVM derived from MDA-MB-231 tumor cells. In vitro and in vivo experiments demonstrate that EV-MOF-protein (EMP) nanoparticles can protect the cargo protein, reduce uptake by the mononuclear phagocyte system, enhance blood circulating time, preferentially be delivered to the homotypic tumor from which the EVs derived, and achieve significant therapeutic efficacy in cancer treatment.

■

RESULTS AND DISCUSSION Assembly, Optimization, and Characterization of MP and EMP Nanoparticles. Protein cargos are encapsulated in MOF (ZIF-8) nanoparticles by self-assembly in an aqueous phase. By taking advantage of the flexible design of metal− organic nanoparticles, the morphology and diameter of MP nanoparticles were tuned to the optimal size range for B

DOI: 10.1021/jacs.8b03584 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Article

Journal of the American Chemical Society

Figure 2. Assembly and characterization of biomimetic nanoparticles. (A) FTIR characterization of the assembly of the model proteins (BSA) in MP nanoparticles. (B) N2 adsorption−desorption isotherms reveal that the MP nanoparticles have a similar porous structure to that of the MOF nanoparticles. The decrease of cavity volume of the MP nanoparticles is due to the encapsulation of proteins in the frame structure. (C) pHresponsive release kinetics of the guest proteins from the MP nanoparticles. The protein cargos are more efficiently released at pH = 5.0 in comparison to pH = 7.4. (D−G) TEM images of MP, EVs, EVM, and EMP nanoparticles, respectively. Scale bars: 100 nm. (H) Confocal microscopy image of EMP nanoparticles camouflaged with fluorescence-labeled EVM: (1) The green fluorescence is from encapsulated protein (fluorescein isothiocyanate tagged BSA, fluoBSA) in MP nanoparticles, (2) the red fluorescence is from the EVM, and (3) the merged fluorescence image. Scale bar: 2 μm. (I) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protein patterns of (1) markers, (2) EVs, (3) EVM, and (4) biomimetic nanoparticles without loading cargo proteins. (J, K) Size distribution of (J) MP and (K) EMP nanoparticles measured by DLS. (L) Zeta potential of MP nanoparticles, EVs, EVM, and EMP nanoparticles.

compared with the bare MP nanoparticles. In comparison to the liposome-enveloped MP nanoparticles (LMPs), the EMP nanoparticles also reduced protein adsorption by 4-fold (Figure 3A and B), indicating the critical role of the EVM for reducing opsonization. Macrophages are major components of the immune defense system and play a critical role in clearing foreign objects from the circulation. Therefore, the ability of the biomimetic EMP nanoparticles to inhibit internalization by macrophages was studied using a murine macrophage-like cell line, RAW264.7. The MP, EMP, and LMP nanoparticles encapsulated with equivalent fluorescence proteins were incubated with RAW264.7 cells for 2 h. Laser scanning microscopy (Figure 3C) clearly shows the apparent decrease of internalization of nanoparticles by the camouflage of the EV membrane. Flow cytometry analysis further confirms that uptake of the EMP

2L). The stability of the EMP nanoparticles was tested over time through dynamic light scattering (DLS) and transmission electron microscopy (TEM) analysis (Figure S14). Even after 3 days, the size and morphology of the EMP nanoparticles do not significantly change in comparison to the freshly prepared ones, which can be attributed to the stabilizing effect by the biological membrane’s hydrophilic surface glycans.38 Reduced Particle Opsonization and Phagocytosis. Nanoparticles adsorb proteins from physiological fluids, which affects their biological identity, results in the recognition and clearance by phagocytes, and ultimately determines the biological fate of nanoparticles.19,39,40 To study if the biomimetic EMP nanoparticles can reduce the adsorption of highly abundant serum proteins, we incubated the EMP nanoparticles with fluorescent IgG. Indeed, the EMP nanoparticles reduced protein adsorption by approximately 6-fold C

DOI: 10.1021/jacs.8b03584 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Article

Journal of the American Chemical Society

preferentially target certain distant cells and tissues.43 Homotypic self-seeding has been proposed as an important mechanism for tumor growth.44 Therefore, we hypothesize that EMP nanoparticles enveloped with specific tumor-cell-derived EVM could target tumors from which the EVs are derived. To confirm the homotypic targeting ability of EMP nanoparticles, the biomimetic nanoparticles with EVM derived from MDAMB-231 tumor cells were incubated with different cells, including MDA-MB-231, 293T human embryonic kidney cells, 3T3 mouse embryo fibroblasts, CAD mouse central nervous system-derived cells, MCF7 human breast adenocarcinoma cells, and SH-SY5Y human neuroblastoma cells. As revealed by laser scanning microscopy and flow cytometry (Figure 4A and B), MDA-MB-231 cells exhibit the highest uptake efficiency of the EMP nanoparticles, about 2−8-fold higher than other cells in the group (Figure 4C). These results reveal that a highly homotypic targeting interaction can be achieved between the EMP nanoparticles and the source tumor cells. Furthermore, the influence of nanoparticles and EVM camouflage on the intracellular transduction of the protein cargo into MDA-MB-231 tumor cells was studied (Figures 4D, S15, and S16). In comparison to the low efficiency of direct protein transduction (∼1.27%), the MP nanoparticles dramatically promote the intracellular delivery of protein cargos with ∼43.7% transduction efficiency (percentage of cells transduced). More importantly, the EMP nanoparticles further enhance the transduction efficiency to ∼77.5%, while the lipiddecorated LMP nanoparticles have a transduction efficiency of only ∼28.2% (Figure S17). Thus, EVM camouflage of the MP nanoparticles enables specific targeting to the homotypic cells and significantly enhances the protein transduction efficiency. Internalization and Intracellular Release Mechanism. To elucidate the internalization mechanism of the EMP nanoparticles, we incubated the cells with various endocytosis inhibitors, including chlorpromazine (CHL, inhibitor of clathrin-mediated endocytosis), amiloride (AMI, inhibitor of Na+/H+ pump related macropinocytosis), dynasore (DYN, inhibitor of dynamin), methyl-beta-cyclodextrin (MBD, inhibitor of cholesterol-dependent endocytosis), and nystatin (NYS, inhibitor of lipid raft-caveolae endocytosis).45,46 As indicated by flow cytometry analysis (Figure 5A), inhibitors DYN and MBD resulted in significant reduction of intracellular delivery of proteins, implying EMP nanoparticle uptake is mainly mediated by pathways of dynamin- and cholesterol-dependent endocytosis.46,47 We further analyzed the intracellular trafficking of EMP nanoparticles in MDA-MB-231 cells using a red fluorescent endosome marker (FM4-64) to track the endocytosis (Figure 5B). EMP nanoparticles rapidly attached to the cell membrane within 30 min. Colocalization of green fluorescent from the protein with red fluorescence from the endosome was observed in 2 h, suggesting the EMP nanoparticles entered the endosomes. At 4 h, we observed considerable green fluorescence depleted out of the endosomes. The green fluorescence gradually diffused to the whole cytoplasm as time progressed (Figures S18 and S19 and Movie S1), indicating the successful intracellular delivery of proteins. We then further studied the mechanism of protein release to the cytosol. As shown in Figure S20, the intracellular release of proteins could be ascribed to the “proton-sponge” effect.48 After internalization by cells, the EMP nanoparticles go through the endocytosis pathway, and the intravesicular pH drops along the endocytic pathway, from pH 6.0−6.5 in early

Figure 3. Biomimetic EMP nanoparticles reduce surface opsonization and prevent phagocytosis. (A) Fluorescence spectra of fluorescent IgG absorbed on MP, biomimetic EMP, and LMP nanoparticles. (B) Quantification of fluorescent IgG (n = 5) absorbed on MP, biomimetic EMP, and LMP nanoparticles. (C) Laser scanning fluorescence microscopy images of RAW264.7 cells incubated with MP, EMP, and LMP nanoparticles for 2 h. The cell nuclei were stained with Hoechst (blue). Nanoparticles with encapsulated fluoBSA are green fluorescent. Scale bar: 50 μm. (D) Flow cytometry analysis of RAW264.7 cells incubated with MP, biomimetic EMP, and liposome-enveloped MP (LMP) nanoparticles for 2 h. Negative control (NC) represents the RAW264.7 cells without any treatment. (E) Quantification of the mean fluorescence intensity (MFI) of the RAW264.7 cell uptake. ***P < 0.001.

nanoparticles is only ∼30% of that of the original MP nanoparticles (Figure 3D and E). More intriguingly, the stealth effect of EVM cannot be simply replaced by the lipid membrane (Figure 3E), although the envelope of the lipid membrane also reduced the absorption of serum proteins (Figure 3A and B). Notably, differing from the pure LMP nanoparticles, the EMP nanoparticles also contain certain EV membrane proteins. In previous work, we and others also demonstrated the presence of CD47 antigen on MDA-MB-231 cells derived EVs,26,41 which could mediate a signal to resist the phagocyte-dependent clearance.27,42 Homotypic Targeting of MDA-MB-231 Tumor Cells and Assisted Intracellular Protein Delivery Using EMP Nanoparticles. EVs can recognize specific cell types through their surface receptors, and tumor-cell-derived EVs can D

DOI: 10.1021/jacs.8b03584 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Article

Journal of the American Chemical Society

Figure 4. EMP nanoparticles assist tumor cell targeting and promote the intracellular transduction of protein cargos. (A) Laser scanning fluorescence microscopy images of MDA-MB-231, 293T, 3T3, CAD, MCF-7, and SY5Y cells incubated with EMP nanoparticles enveloped with EMV derived from MDA-MB-231 cells. The cell nuclei were stained with Hoechst (blue), and nanoparticles with encapsulated fluoBSA are green fluorescent. Scale bar: 50 μm. (B, C) Flow cytometry analysis of different cells incubated with EMP nanoparticles for 2 h compared with cells without EMP nanoparticle treatment, respectively. ***P < 0.001. (D) Flow cytometry analysis of MDA-MB-231 cells after protein transduction with bare protein, MP, EMP, and LMP nanoparticles, respectively.

endosomes to pH 4.5−5.5 in late endosomes and lysosomes.24 Notably, due to the acid-labile noncovalent bond, the designed pH-responsive MOF matrix of the EMP nanoparticles can release the organic ligand of the imidazole derivative, which shows a buffering effect upon protonation of the imidazole ring.49,50 Using a ratiometric dye (Lysosensor Yellow/Blue DND-160), we determined the lysosomal pH (Figure 5C), and the evolution of lysosomal pH was observed by laser scanning microscopy (Figure 5D). We observed the intracellular vesicle pH dramatically increased in the first 4 h upon incubation with the EMP nanoparticles, due to the buffering effect of the release ligands. Furthermore, we also observed rapid diffusion of zinc ions in the whole cytosol in a few hours (Figure S21), indicating the interference with endosome pH would lead to the disruption of the endosomal membrane and release of guest molecules through the “proton-sponge” effect. EMP Nanoparticles Suppress Tumor Growth in a Mouse Xenograft Model. Before application of the EMP nanoparticles in the intracellular delivery of therapeutic proteins, we studied their intracellular evolution of the components and the biocompatibility. Figure S21 reveals the intracellular evolution of the zinc ions upon internalization of EMP nanoparticles. Apparently, the released zinc ions were secreted out of the cells in several hours, because cells have a complex and important zinc homeostasis system, and there are many proteins that are dedicated to Zn2+ transport and buffering.51 Interestingly, differing from rapid clearance of the zinc ions from the intracellular environment, the EVM

component from the internalized EMP decayed relatively slowly. As shown in Figure S22, after internalization for 12 h, EVM was mainly located in the perinuclear region and cell peripheries. Furthermore, we also observed the colocalization of green and red fluorescence, which may be due to the fusion of EVM with the cellular membrane.52 After internalization for 24 h, a large amount of green fluorescence located in the cell peripheries disappeared, implying that part of the EVM was expelled from the cell. In addition, considerable green fluorescence was still located in the perinuclear region and colocalized with the red fluorescence, implying the EVM with abundant sphingomyelin may recycle back to the plasma membrane.53 With the extension of time, diffused and decreased green fluorescence from EVM was observed, indicating the gradual clearance or transformation of EVM. We also checked the cell toxicity of the biomimetic nanoparticles and their components. As shown in Figure S23, the nanoparticles have no significant cytotoxicity at the reasonably low dosage used in our subsequent experiments, despite the potential toxicity of zinc ions.54 To further evaluate the effectiveness of MP and EMP nanoparticles for protein transduction and their therapeutic potential, encapsulation and delivery of bioactive therapeutic proteins in vitro and in vivo were studied. Gelonin, an Nglycosidase protein with a molecular weight of 28 kDa, triggers cell apoptosis by cleaving a specific glycosidic bond in rRNA and thereby disrupting protein synthesis.55 However, besides the susceptibility and the fragile nature of the protein, unlike E

DOI: 10.1021/jacs.8b03584 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Article

Journal of the American Chemical Society

Figure 5. Internalization of EMP nanoparticles goes through the endocytosis pathway. (A) Efficiency of EMP nanoparticles uptake by MDA-MB-231 cells in the presence of different endocytosis inhibitors. ***P < 0.001. (B) Laser scanning fluorescence microscopy analysis of time-dependent intracellular traffic of EMP nanoparticles in MDA-MB-231 cells. Scale bar: 10 μm. (C) Mean vesicle pH in MDA-MB-231 cells at different times. ***P < 0.001. (D) Laser scanning microscopy analysis of MDA-MB-231 cells after incubation with EMP nanoparticles for the indicated periods of time. Intracellular pH was measured using a ratiometric sensor (LysoSensor Yellow/Blue DND-160). The yellow lines are plotted to indicate the cell borders. Scale bar: 20 μm.

was observed in the mice treated with empty MOF nanoparticles or gelonin alone (Figure S24C), while intratumoral injection of gelonin-loaded MP nanoparticles shows a remarkable inhibition effect toward tumor growth, indicating the successful intracellular delivery of the bioactive proteins in vivo. Besides, no significant change in mice body weight was observed during the treatment (Figure S24D), implying the safety of the nanoparticles. After camouflaging with EV membrane, systemic delivery of therapeutic proteins by the EMP nanoparticles was achieved. To evaluate the tumor-targeting capability of EMP nanoparticles, EMP nanoparticles loaded with indocyanine green (ICG)-labeled proteins (gelonin) were administered to the MDA-MB-231 tumor-bearing mice intravenously via the tail vein. Mice injected with EMP or MP nanoparticles exhibited apparent fluorescence signal at the tumor sites after 4 h postinjection (Figure 6A), indicating the accumulation of nanoparticles at the tumor sites. Moreover, the mice group treated with EMP nanoparticles camouflaged with MDA-MB231 EVM showed much higher fluorescence intensity than the groups of mice treated with protein and MP nanoparticles, indicating the notable targeting ability of EMP nanoparticles. As the time was extended, EMP nanoparticles continuously accumulated at the tumor site in the first 24 h. Relatively strong fluorescence could be observed even after 72 h, while fluorescent signals from other parts of the body were no longer detectable (Figure 6A and B). Mice were euthanized after 3 days, and tumor and major organs were retrieved for ex

other ribosomal toxins (e.g., ricin and abrin), gelonin lacks a translocation domain, and thus its intracellular delivery is a great challenge.56 We caged gelonin proteins into MP nanoparticles and further camouflaged them with EVM derived from MDA-MB-231 cells as the EMP nanoparticles. The therapeutic potential of the MP and EMP nanoparticles was first examined by assessing the in vitro cytotoxicity of nanoparticles against MDA-MB-231 cells using the CCK-8 assay. Without loading the therapeutic proteins, the MOF nanoparticles and EVM-enveloped EMOF nanoparticles have no significant cytotoxicity to MDA-MB-231 cells under 80 μg mL−1 (Figure S23B). However, a live−dead cell assay clearly reveals that massive cell apoptosis happened within 6 h after incubation with the EMP nanoparticles (Figure S24). MP and EMP nanoparticles with loaded gelonin induce significant cell apoptosis, and the EMP nanoparticles have a significantly lower half-maximal inhibitory concentration (IC50: ∼0.025 μM) than that (IC50: ∼0.05 μM) of MP nanoparticles (Figure S24B), while the dose is about 5−10 times lower than that in previous studies.55,57 In contrast, native gelonin has no apparent cytotoxicity against MDA-MB-231 cells due to their poor membrane permeability. We further checked the effectiveness of intracellular delivery of gelonin by nanoparticles in vivo. To evaluate the in vivo toxicity, phosphate-buffered saline (PBS), MOF nanoparticles, gelonin, and gelonin-loaded MP nanoparticles were injected directly into mouse orthotopic xenograft tumors formed with MDA-MB-231 cells, respectively. Similar to the in vitro results, a negligible therapeutic effect (P > 0.1) F

DOI: 10.1021/jacs.8b03584 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Article

Journal of the American Chemical Society

Figure 6. Biomimetic EMP nanoparticles increase accumulation of therapeutic proteins in tumor through systemic administration. (A) In vivo fluorescence imaging of the MDA-MB-231 tumor-bearing mice at different times after tail vein intravenous injection of ICG-labeled protein (gelonin) (I), EMP (II), and MP (III) nanoparticles. (B) Time-dependent variation of fluorescence intensity at tumor sites from the mice (n = 5). **P < 0.01, ***P < 0.001. (C) Ex vivo fluorescence imaging of tumor and major organs collected from representative mice after euthanasia at day 3 postinjection. (D) ROI analysis of the fluorescent signals from the tumors and major host organs (n = 5). *P < 0.05, ***P < 0.001. (E) Laser scanning microscopy images of mouse tumor sections. Mice were euthanized at day 3 postinjection. The nuclei were stained with Hoechst (blue). Scale bar: 50 μm.

vivo imaging. The retained fluorescence intensity of the EMP nanoparticles at tumor sites is significantly higher than those of other organs and those of the protein alone and the MP nanoparticle groups (Figure 6C). Quantitative region-ofinterest (ROI) analysis shows that fluorescence intensity at the tumor sites of mice treated with the EMP nanoparticles is about 11- and 4-fold higher than those treated with protein alone and MP nanoparticles (Figure 6D), respectively. Additionally, the promoted protein transduction efficiency was confirmed by laser scanning microscopy analysis of cryosections of the tumors (Figure 6E). Apparently, much brighter red fluorescence from ICG labels was observed in the mouse tumor cryosection samples treated with the EMP nanoparticles than those treated with native proteins or the MP nanoparticles. Taken together, the above results demonstrate that the EVM camouflage can alleviate the rapid clearance of nanoparticles, selectively target their homotypic tumors, and promote the proteins’ intracellular transduction. The potential of systemic delivery, homotypic targeting, and intracellular transduction of therapeutic proteins by the EMP nanoparticles to inhibit tumor growth in vivo was then studied using the orthotopic MDA-MB-231 tumor-bearing mice. Apparently, in comparison to the PBS control group, no significant difference was observed on the tumor growth after intravenous injection of native gelonin (Figure 7A). However, the EMP and MP nanoparticles can effectively inhibit the tumor growth. More importantly, tumor size in mice treated

with the EMP nanoparticles is significantly smaller than those in all other groups, revealing that camouflage of EVM remarkably improves the therapeutic efficacy. In addition, during the treatment, no significant difference in the body weight of mice was observed among different groups (Figure 7B). Tumors were also collected and weighed after euthanasia of mice (Figure 7C and D), which further confirms the therapeutic effect of EMP nanoparticles. Histologic analysis of the tumor sections stained with hematoxylin and eosin (H&E) indicates an apparent decrease of tumor cell density in tumor tissue after systemic administration of the EMP nanoparticles (Figure 7E). Besides, the in situ TUNEL assay clearly demonstrates massive cell apoptosis in the tumor tissue after treatment by the EMP nanoparticles (Figure 7F), which is about 4-fold that of the group treated with the MP nanoparticles (Figure 7G). On the contrary, PBS and gelonin alone treatments resulted in negligible cell apoptosis. Histological examination of the main organs including heart, liver, lung, spleen, and kidney finds no pathological abnormalities in the mice treated with native gelonin, MP nanoparticles, or EMP nanoparticles (Figures 7H and S25), indicating negligible systemic toxicity of the gelonin proteins and the MP and EMP nanoparticles. Collectively, the EVMcamouflaged EMP nanoparticles can apparently target homotypic tumor tissue, assist intracellular transduction of therapeutic protein in systemic delivery, and show high potency G

DOI: 10.1021/jacs.8b03584 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Article

Journal of the American Chemical Society

Figure 7. Systemic transduction of therapeutic proteins (gelonin) for inhibition of tumor growth. (A) MDA-MB-231 tumor growth curves after tail vein intravenous injection of PBS, gelonin, MP, and EMP nanoparticles (n = 5). Arrows represent the injection time points. *P < 0.05, **P < 0.01. (B) Body weight variation of MDA-MB-231 tumor-bearing mice during treatment (n = 5). (C) Representative images of MDA-MB-231 xenograft tumors of the mice after different treatments. (D) Weight of the tumors collected from mice euthanized at day 22 with different treatments (n = 5). (E) Representative images of histology analysis (H&E) of MDA-MB-231 xenograft tumors from the mice after different treatments. (F) Representative TUNEL analysis of apoptosis in the tumor tissues after different treatments. The tumor sections were stained with fluorescein-dUTP (green) for apoptosis and Hoechst for the nucleus (blue). (G) Quantification results of the Tunnel assay (n = 5). (H) Histological (H&E) images of the major host organs collected on day 22 show no evident systemic toxicity of the biomimetic EMP nanoparticles. Scale bars in E, F, and H are all 100 μm.

■

for inhibition of tumor growth with minimal toxicity to other major organs.

■

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.8b03584. Detailed experimental procedures; characterization methods; and additional figures and tables (PDF) Movie showing the successful intracellular delivery of proteins (AVI)

CONCLUSION

To sum up, we developed a biomimetic metal−organic framework nanosystem by all-in-one self-assembly and EV camouflage for systemic and intracellular delivery of therapeutic proteins. The biomimetic EMP nanoparticles have high loading efficiency (up to ∼94%) and loading capacity (up to ∼41%) without compromising protein activity. We demonstrate that the biomimetic EMP nanoparticles not only protect the protein cargos but also alleviate the phagocyte-dependent clearance, selectively target the tumor site, and promote the systemic and intracellular delivery of protein cargos. Cellular internalization of EMP nanoparticles undergoes the endocytosis pathway, and the release of guest molecules exploits a “proton-sponge” effect. Using the therapeutic protein gelonin as a model, we applied the biomimetic nanosystem for tumor therapeutics in vitro and in vivo, and it showed high anticancer efficacy and low toxicity. It is important to note that the overall camouflage strategy with tumor-cell-derived EVs is potentially translational. We envision that the EMP nanosystem would provide a new tool for systemic and intracellular protein delivery.

■

AUTHOR INFORMATION

Corresponding Author

*[email protected] ORCID

Gong Cheng: 0000-0002-2217-6408 Si-Yang Zheng: 0000-0002-0616-030X Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank the Penn State Materials Research Institute and Huck Institute of Life Sciences for their support. This work is supported by the Pennsylvania State University start-up fund and the National Institutes of Health under Award Number DP2CA174508. H

DOI: 10.1021/jacs.8b03584 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Article

Journal of the American Chemical Society

■

(29) Wilhelm, S.; Tavares, A. J.; Dai, Q.; Ohta, S.; Audet, J.; Dvorak, H. F.; Chan, W. C. W. Nat. Rev. Mater. 2016, 1, 16014. (30) Barth, A. Biochim. Biophys. Acta, Bioenerg. 2007, 1767, 1073− 1101. (31) Zhou, L.; Chen, Z.; Dong, K.; Yin, M.; Ren, J.; Qu, X. Adv. Mater. 2014, 26, 2424−2430. (32) Xiang, J.; Xu, L.; Gong, H.; Zhu, W.; Wang, C.; Xu, J.; Feng, L.; Cheng, L.; Peng, R.; Liu, Z. ACS Nano 2015, 9, 6401−6411. (33) Smith, D. M.; Simon, J. K.; Baker, J. R., Jr Nat. Rev. Immunol. 2013, 13, 592−605. (34) Howarth, A. J.; Liu, Y.; Li, P.; Li, Z.; Wang, T. C.; Hupp, J. T.; Farha, O. K. Nat. Rev. Mater. 2016, 1, 15018. (35) Ojida, A.; Inoue, M.-a.; Mito-oka, Y.; Tsutsumi, H.; Sada, K.; Hamachi, I. J. Am. Chem. Soc. 2006, 128, 2052−2058. (36) Parodi, A.; Quattrocchi, N.; van de Ven, A. L.; Chiappini, C.; Evangelopoulos, M.; Martinez, J. O.; Brown, B. S.; Khaled, S. Z.; Yazdi, I. K.; Enzo, M. V.; Isenhart, L.; Ferrari, M.; Tasciotti, E. Nat. Nanotechnol. 2013, 8, 61−68. (37) Yang, J.; Chen, X.; Li, Y.; Zhuang, Q.; Liu, P.; Gu, J. Chem. Mater. 2017, 29, 4580−4589. (38) Hu, C.-M. J.; Fang, R. H.; Wang, K.-C.; Luk, B. T.; Thamphiwatana, S.; Dehaini, D.; Nguyen, P.; Angsantikul, P.; Wen, C. H.; Kroll, A. V.; Carpenter, C.; Ramesh, M.; Qu, V.; Patel, S. H.; Zhu, J.; Shi, W.; Hofman, F. M.; Chen, T. C.; Gao, W.; Zhang, K.; Chien, S.; Zhang, L. Nature 2015, 526, 118−121. (39) Tenzer, S.; Docter, D.; Kuharev, J.; Musyanovych, A.; Fetz, V.; Hecht, R.; Schlenk, F.; Fischer, D.; Kiouptsi, K.; Reinhardt, C.; Landfester, K.; Schild, H.; Maskos, M.; Knauer, S. K.; Stauber, R. H. Nat. Nanotechnol. 2013, 8, 772−781. (40) Schöttler, S.; Becker, G.; Winzen, S.; Steinbach, T.; Mohr, K.; Landfester, K.; Mailänder, V.; Wurm, F. R. Nat. Nanotechnol. 2016, 11, 372−377. (41) Kowal, J.; Arras, G.; Colombo, M.; Jouve, M.; Morath, J. P.; Primdal-Bengtson, B.; Dingli, F.; Loew, D.; Tkach, M.; Théry, C. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, E968−E977. (42) Liu, X.; Pu, Y.; Cron, K.; Deng, L.; Kline, J.; Frazier, W. A.; Xu, H.; Peng, H.; Fu, Y.-X.; Xu, M. M. Nat. Med. 2015, 21, 1209−1215. (43) Gay, L. J.; Felding-Habermann, B. Nat. Rev. Cancer 2011, 11, 123−134. (44) Kim, M.-Y.; Oskarsson, T.; Acharyya, S.; Nguyen, D. X.; Zhang, X. H. F.; Norton, L.; Massagué, J. Cell 2009, 139, 1315−1326. (45) Iversen, T.-G.; Skotland, T.; Sandvig, K. Nano Today 2011, 6, 176−185. (46) Gilleron, J.; Querbes, W.; Zeigerer, A.; Borodovsky, A.; Marsico, G.; Schubert, U.; Manygoats, K.; Seifert, S.; Andree, C.; Stoter, M.; Epstein-Barash, H.; Zhang, L.; Koteliansky, V.; Fitzgerald, K.; Fava, E.; Bickle, M.; Kalaidzidis, Y.; Akinc, A.; Maier, M.; Zerial, M. Nat. Biotechnol. 2013, 31, 638−646. (47) Macia, E.; Ehrlich, M.; Massol, R.; Boucrot, E.; Brunner, C.; Kirchhausen, T. Dev. Cell 2006, 10, 839−850. (48) Varkouhi, A. K.; Scholte, M.; Storm, G.; Haisma, H. J. J. Controlled Release 2011, 151, 220−228. (49) Pack, D. W.; Putnam, D.; Langer, R. Biotechnol. Bioeng. 2000, 67, 217−223. (50) Moreira, C.; Oliveira, H.; Pires, L. R.; Simões, S.; Barbosa, M. A.; Pêgo, A. P. Acta Biomater. 2009, 5, 2995−3006. (51) Eide, D. J. Biochim. Biophys. Acta, Mol. Cell Res. 2006, 1763, 711−722. (52) Parolini, I.; Federici, C.; Raggi, C.; Lugini, L.; Palleschi, S.; De Milito, A.; Coscia, C.; Iessi, E.; Logozzi, M.; Molinari, A.; Colone, M.; Tatti, M.; Sargiacomo, M.; Fais, S. J. Biol. Chem. 2009, 284, 34211− 34222. (53) Koivusalo, M.; Jansen, M.; Somerharju, P.; Ikonen, E. Mol. Biol. Cell 2007, 18, 5113−5123. (54) Ye, Z.; Wu, S.; Zheng, C.; Yang, L.; Zhang, P.; Zhang, Z. Langmuir 2017, 33, 12952−12959. (55) Veenendaal, L. M.; Jin, H.; Ran, S.; Cheung, L.; Navone, N.; Marks, J. W.; Waltenberger, J.; Thorpe, P.; Rosenblum, M. G. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 7866−7871.

REFERENCES

(1) Leader, B.; Baca, Q. J.; Golan, D. E. Nat. Rev. Drug Discovery 2008, 7, 21−39. (2) Lu, Y.; Sun, W. J.; Gu, Z. J. Controlled Release 2014, 194, 1−19. (3) Schwarze, S. R.; Ho, A.; Vocero-Akbani, A.; Dowdy, S. F. Science 1999, 285, 1569−1572. (4) Wang, M.; Zuris, J. A.; Meng, F.; Rees, H.; Sun, S.; Deng, P.; Han, Y.; Gao, X.; Pouli, D.; Wu, Q.; Georgakoudi, I.; Liu, D. R.; Xu, Q. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, 2868−2873. (5) Jensen, S. A.; Day, E. S.; Ko, C. H.; Hurley, L. A.; Luciano, J. P.; Kouri, F. M.; Merkel, T. J.; Luthi, A. J.; Patel, P. C.; Cutler, J. I.; Daniel, W. L.; Scott, A. W.; Rotz, M. W.; Meade, T. J.; Giljohann, D. A.; Mirkin, C. A.; Stegh, A. H. Sci. Transl. Med. 2013, 5, 209ra152. (6) Yin, H.; Kanasty, R. L.; Eltoukhy, A. A.; Vegas, A. J.; Dorkin, J. R.; Anderson, D. G. Nat. Rev. Genet. 2014, 15, 541−555. (7) Yin, H.; Song, C.-Q.; Dorkin, J. R.; Zhu, L. J.; Li, Y.; Wu, Q.; Park, A.; Yang, J.; Suresh, S.; Bizhanova, A.; Gupta, A.; Bolukbasi, M. F.; Walsh, S.; Bogorad, R. L.; Gao, G.; Weng, Z.; Dong, Y.; Koteliansky, V.; Wolfe, S. A.; Langer, R.; Xue, W.; Anderson, D. G. Nat. Biotechnol. 2016, 34, 328−333. (8) Yu, M.; Wu, J.; Shi, J.; Farokhzad, O. C. J. Controlled Release 2016, 240, 24−37. (9) D’Astolfo, D. S.; Pagliero, R. J.; Pras, A.; Karthaus, W. R.; Clevers, H.; Prasad, V.; Lebbink, R. J.; Rehmann, H.; Geijsen, N. Cell 2015, 161, 674−690. (10) Pakulska, M. M.; Miersch, S.; Shoichet, M. S. Science 2016, 351, aac4750. (11) Nischan, N.; Herce, H. D.; Natale, F.; Bohlke, N.; Budisa, N.; Cardoso, M. C.; Hackenberger, C. P. R. Angew. Chem., Int. Ed. 2015, 54, 1950−1953. (12) Gu, Z.; Biswas, A.; Zhao, M.; Tang, Y. Chem. Soc. Rev. 2011, 40, 3638−3655. (13) Ghosh, P.; Yang, X.; Arvizo, R.; Zhu, Z.-J.; Agasti, S. S.; Mo, Z.; Rotello, V. M. J. Am. Chem. Soc. 2010, 132, 2642−2645. (14) Brodin, J. D.; Sprangers, A. J.; McMillan, J. R.; Mirkin, C. A. J. Am. Chem. Soc. 2015, 137, 14838−14841. (15) Sun, W.; Ji, W.; Hall, J. M.; Hu, Q.; Wang, C.; Beisel, C. L.; Gu, Z. Angew. Chem., Int. Ed. 2015, 54, 12029−12033. (16) Zhao, M. X.; Liu, Y. R.; Hsieh, R. S.; Wang, N.; Tai, W. Y.; Joo, K. I.; Wang, P.; Gu, Z.; Tang, Y. J. Am. Chem. Soc. 2014, 136, 15319− 15325. (17) Liang, K.; Ricco, R.; Doherty, C. M.; Styles, M. J.; Bell, S.; Kirby, N.; Mudie, S.; Haylock, D.; Hill, A. J.; Doonan, C. J.; Falcaro, P. Nat. Commun. 2015, 6, 7240. (18) Butcher, N. J.; Mortimer, G. M.; Minchin, R. F. Nat. Nanotechnol. 2016, 11, 310−311. (19) Monopoli, M. P.; Aberg, C.; Salvati, A.; Dawson, K. A. Nat. Nanotechnol. 2012, 7, 779−786. (20) Mitragotri, S.; Burke, P. A.; Langer, R. Nat. Rev. Drug Discovery 2014, 13, 655−672. (21) Huang, X.-C.; Lin, Y.-Y.; Zhang, J.-P.; Chen, X.-M. Angew. Chem., Int. Ed. 2006, 45, 1557−1559. (22) Banerjee, R.; Phan, A.; Wang, B.; Knobler, C.; Furukawa, H.; O’Keeffe, M.; Yaghi, O. M. Science 2008, 319, 939−943. (23) Furukawa, H.; Cordova, K. E.; O’Keeffe, M.; Yaghi, O. M. Science 2013, 341, 1230444. (24) Sorkin, A.; von Zastrow, M. Nat. Rev. Mol. Cell Biol. 2002, 3, 600−614. (25) Kamerkar, S.; LeBleu, V. S.; Sugimoto, H.; Yang, S.; Ruivo, C. F.; Melo, S. A.; Lee, J. J.; Kalluri, R. Nature 2017, 546, 498−503. (26) Wan, Y.; Cheng, G.; Liu, X.; Hao, S.-J.; Nisic, M.; Zhu, C.-D.; Xia, Y.-Q.; Li, W.-Q.; Wang, Z.-G.; Zhang, W.-L.; Rice, S. J.; Sebastian, A.; Albert, I.; Belani, C. P.; Zheng, S.-Y. Nat. Biomed. Eng. 2017, 1, 0058. (27) Jaiswal, S.; Jamieson, C. H. M.; Pang, W. W.; Park, C. Y.; Chao, M. P.; Majeti, R.; Traver, D.; van Rooijen, N.; Weissman, I. L. Cell 2009, 138, 271−285. (28) El Andaloussi, S.; Mager, I.; Breakefield, X. O.; Wood, M. J. A. Nat. Rev. Drug Discovery 2013, 12, 347−357. I

DOI: 10.1021/jacs.8b03584 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Article

Journal of the American Chemical Society (56) Chu, T. C.; Marks, J. W.; Lavery, L. A.; Faulkner, S.; Rosenblum, M. G.; Ellington, A. D.; Levy, M. Cancer Res. 2006, 66, 5989−5992. (57) Ellington, A. D.; Marks, J. W.; Chitai Chu, T.; Lavery, L.; Faulkner, S.; Levy, M.; Rosenblum, M. G. Cancer Res. 2005, 65, 1455.

J

DOI: 10.1021/jacs.8b03584 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX