Preferential Cancer Cell Self-Recognition and Tumor Self-Targeting

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Preferential Cancer Cell Self-Recognition and Tumor Self-Targeting by Coating Nanoparticles with Homotypic Cancer Cell Membranes Jing-Yi Zhu, Di-Wei Zheng, Mingkang Zhang, Wuyang Yu, Wen-Xiu Qiu, Jing-Jing Hu, Jun Feng, and Xian-Zheng Zhang Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.6b02786 • Publication Date (Web): 11 Aug 2016 Downloaded from http://pubs.acs.org on August 13, 2016

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Preferential Cancer Cell Self-Recognition and Tumor Self-Targeting by Coating Nanoparticles with Homotypic Cancer Cell Membranes Jing-Yi Zhu,† Di-Wei Zheng,† Ming-Kang Zhang,† Wu-Yang Yu,† Wen-Xiu Qiu,† JingJing Hu,† Jun Feng,*,† Xian-Zheng Zhang† †

Key Laboratory of Biomedical Polymers of Ministry of Education & Department of

Chemistry, Wuhan University, Wuhan 430072, P. R. China

ABSTRACT: The ultimate goal in cancer therapy and diagnosis is to achieve highly specific targeting to cancer cells. Coated with the source cancer cell membrane specifically derived from the homologous tumors, the nanoparticles are identified with the self-recognition internalization by the source cancer cell lines in vitro and the highly tumor-selective targeting “homing” to the homologous tumor in vivo even in the competition of another heterologous tumor. As the result, MNP@DOX@CCCM nanovehicle showed strong potency for tumor treatment in vivo and the MR imaging. This bio-inspired strategy shows great potential for precise therapy/diagnosis of various tumors merely by adjusting the cell membrane source accordingly on the nanoparticle surface. KEYWORDS: tumor self-targeting (TST), cancer cell self-recognition, magnetic iron oxide, cancer cell membrane, MRI

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The ultimate goal for cancer therapy and diagnosis is to produce a “magic bullet” that can evade the immune system (macrophage uptake and systemic clearance), enable specific targeting to tumors and efficient entry into cancer cells. So far, tumor-specific delivery vehicles have mostly been made within nanoscale dimension in order to benefit the passive tumor targeting owing to the enhanced permeability and retention (EPR) effect of tumor tissues.1,2 Furthermore, the conjugation with certain ligands permits the active targeting to tumor tissues/cells.3-7 However, the passive targeting depends on the varying degrees of tumor vascularization and the permeability associated with tumor types and development stages.3,8,9 The active targeting is largely limited by the recognition selectivity and the receptor density expressed in the target sites.3,10,11 These uncontrollable factors make the targeting performance far from satisfactory, and the targeting mechanisms vary tremendously for different tumor diseases. In addition, the ligand-associated targeting approach is highly restricted by the complex chemistry involved in the preparation. Biological entities possess special biofunctions that are hardly recreated by the tailormade materials. By directly utilizing natural materials, biomimetic strategy may endow the nanomaterials with new and improved functionalities beyond expectation.9,12-18 In view of the definite mechanism of the T antigen-mediated homotypic metastatic cell adhesive interaction,19 the cell membranes of highly metastatic human MDA-MB-435 cells have been recently chosen to coat the nanoparticles (NPs), which display homotypic recognition to the same cell lines with strong internalization in cellular levels.13 Though the mechanism regarding the homotypic affinity of other cancer cells has been almost unclear, it is known that tumor cells readily agglomerate with strong adhesion to constitute solid tumors owing to the presence of specific proteins (focal


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adhesion proteins, integrin, focal adhesion kinase and RHO family proteins) on surface.20-25 The downregulation of these adhesion components partly accounts for the escape/translocation of tumor cells and the resulting tumor metastasis.20,21 We surmise that this feature of strong adhesion among homotypic tumor cells may be utilized to realize the cell recognition to the same cancer cells. Furthermore, we would like to make clear the possibility if this self-recognition in cellular levels can lead to the effective targeting of NPs to the homologous tumors developed from the same cell lines. If established, this bio-inspired tumor self-targeting (TST) strategy would offer specific targeting to various tumors just by adjusting the cell membrane source accordingly. Moreover, cancer cells are capable of “immune escape” via the mechanisms of immune tolerance, immunosuppression and immunosenescence.26-30 Therefore, the surface decoration with cancer cell membranes may be favorable for the in vivo application of NPs. To verify this idea, we devised a magnetic iron oxide based nanoplatform that was coated with different types of cracked cancer cell membranes (CCCM) (Figure 1). For the first time, we showed that the biomimetic camouflage with CCCM towards the magnetic iron oxide NPs (MNPs) can achieve highly specific self-recognition to the source cell lines in vitro and the excellent self-targeting “homing” ability to the homologous tumor in vivo even in the competition of another heterologous tumor. In turn, the drug-loaded CCCM-coated nanovehicle showed strong potency for tumor treatment in vivo with high efficiency.


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Figure 1. Preparation of cancer cell membrane-cloaked magnetic nanoparticles and illustration of preferential cancer cell self-recognition and tumor self-targeting (TST). Clinically used doxorubicin hydrochloride (DOXHCI) was exploited as the model drug to electrostatically attach onto the negatively charged Fe3O4 MNP (~13 nm in dry state) via an amended nanoprecipitation method.31 Human squamous carcinoma (UM-SCC-7) cell lines developed at the University of Michigan were firstly selected as a model cancer cell, and the cell membrane fragments were collected by exploiting membrane protein extraction kit and differential centrifugation.32 The MNP@DOX NPs were mixed with UM-SCC-7 CCCM dispersion under vortex stirring and then extruded consecutively through a series of water-phase filters with reducing pore sizes (~2.0 µm, ~800.0 nm, ~450.0 nm). To provide the better stability, the membrane-to-core weight ratio was eventually optimized at 1:1 throughout the study (Figure S1). The MNP@DOX@CCCM NPs were collected by magnetic separation, finally giving the hydrodynamic diameter (Dh) of 102.6 ± 2.4 nm and § potential of -15.0 ± 3.7 mV measured in PBS (yield


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~83.6%) (Table S1). Transmission electron microscopy (TEM) revealed the superficial coverage of CCCM layer and the well-dispersion of MNP@DOX@CCCM nanospheres (Figure 2A). The Dh of MNP@DOX@CCCM NPs was fairly close to the mean particle size measured by TEM in dry state, indicating the compact coverage of CCCM in aqueous medium. MNP@DOX@CCCM dispersion exhibited high stability and remained always transparent with minimal changes of Dh under 10% serum-containing conditions over 7 days (Figure S2). Based on the analysis of protein ingredient using gel electrophoresis, it came out that the membrane proteins within the source cell membrane can be well retained during the treatment (Figure 2B). Western blotting analysis towards a series of membrane and intracellular protein markers indicated the well retention of cadherins and Na+/K+-ATPase, both as plasma membrane-specific markers, and glycoprotein 100 (gp100), a widely reported tumor-associated transmembrane protein.13 Conversely, intracellular protein markers of Histone H3, COX IV, and GAPDH for the nucleus, mitochondria, and cytosol were poorly present on the cracked cancer cell membrane, demonstrating the selective retention of membrane fragments after the treatment (Figure 2C). Immunofluorescence staining analysis was carried out to identify the orientation of membrane coating, using two monoclonal antiCXCR4 primary antibodies to bind the N-terminus and C-terminus of CXCR4

33,34

(a

kind of G-protein-coupled seven-span transmembrane receptor) located on the extracellular and intracellular regions, respectively (Figure S3). Based on the ratio of fluorescence intensity between the two antibodies, it was shown that the coating membrane was present in the right-side-out orientation. Furthermore, confocal laser scanning microscopy (CLSM) in Figure 2D showed the overlapping co-location of FITClabeled green CCCM and red DOX after 2 h co-culture of cells with the nanosystem,


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partly suggesting NPs are more likely taken up via endocytic rather than fusogenic pathway. To ensure better stability of MNP@DOX@CCCM NPs after drug loading (data not shown), the drug loading content (DLC) were eventually optimized as 16.8%. At pH=7.4 (mimicking blood plasma), the nanosystem displayed a fast release of less than 40% drug during the initial 10 hours followed by a stage with slow release (Figure 2E). The initial fast release was associated with the drug adsorption on CCCM periphery with weak binding force. The following slow release indicated the compact package of drugs inside NPs inner. Relatively, the release was apparently accelerated at pH 5.0 (mimicking endo/lysosomal environments). A desirable delivery system ought to possess favorable immunocompatibility. Antiphagocytosis is an important indicator reflecting the response of immune system to foreign substances. Murine hepatocellular carcinoma (H22) cell membrane coated MNP@DOX@H22 NPs exhibited negligible macrophage engulfment upon 3 h or 6 h coincubation with Raw264.7 murine macrophages based on CLSM observation (Figure S4). The resulting data showed that the ability to inhibit the macrophage uptake can be effectively enhanced once coating the NPs with either mouse embryonic fibroblasts (3T3) or cancerous H22 cell membranes.13 There seems no significant difference between the cancer cell and fibroblast membrane coated NPs in terms of their uptake by macrophage. The result manifested that the coating with cancerous cell membranes can afford the similar immune-evasion efficacy as that for normal cells.35 Interleukin (IL)1β, IL-6, IL-8 and tumor necrosis factor (TNF-α) are the common inflammatory cytokines, whose production would be notably elevated along with the inflammatory immune response.36 mRNA expression of these inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-8)


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in Raw264.7 murine macrophages cells after incubation with MNP@DOX and MNP@DOX@H22 for 6 h were measured by quantitative real-time PCR (RT-PCR). Lipopolysaccharide (LPS) was used as positive control. Compared with the group treated with MNP@DOX and LPS, the MNP@DOX@H22 administration induced obviously weaker expression of these inflammatory cytokines, indicating the low immunogenicity of MNP@DOX@H22 (Figure S5). In addition, the in vivo protein expression (Figure 2F) of these cytokines and their corresponding messenger ribonucleic acid (mRNA) expression (Figure 2G) in murine serum upon intravenous administration were measured. It was evident that the coating with cell membrane could dramatically reduce the expression of various inflammatory cytokines compared with the uncoated group.

Figure 2. (A) TEM images of MNP and MNP@DOX@CCCM NPs; (B) SDS-PAGE protein

analysis

of

source

cancer

cell

membrane


(I),

CCCM

(II)

and

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MNP@DOX@CCCM (III). Samples were stained with Coomassie Blue; (C) Western blotting analysis of cancer cell lysate (I) and CCCM (II) for membrane-specific and intracellular protein markers. Samples were run at equal protein concentration and immunostained against membrane markers including pan-cadherin, Na+/K+-ATPase, and gp100, and intracellular markers including histone H3 (a nuclear marker), cytochrome c oxidase (a mitochondrial marker), and glyceraldehyde 3-phosphate dehydrogenase (a cytosolic marker); (D) CLSM images of UM-SCC-7 cells upon 2 h coincubation with MNP@DOX@UM-SCC-7 NPs. Cracked UM-SCC-7 cell membrane was labelled green with FITC and nucleus was stained blue with Hoechst 33342. (E) Release profiles of DOX from MNP@DOX@CCCM at different pHs. Values were expressed as means ± SD; (F) Protein expression of inflammatory cytokines (IL-1β, TNF-α, IL-6, IL-8) in murine serum after intravenous injection with PBS (I), MNP@DOX@H22 (II) and MNP@DOX (III) assayed by western blotting; (G) The ratio of mRNA expression of inflammatory cytokines (IL-1β, TNF-α, IL-6, IL-8) of MNP@DOX@H22 group relative to that of MNP@DOX group (a) or LPS group (b) in murine serum. The data were obtained by quantitative real-time PCR (RT-PCR).

To prove our assumption that CCCM coating would provide cancer cell selfrecognition by homotypic cancer cells, cellular internalization of UM-SCC-7 and HeLa (human

cervix

carcinoma)

cell

membrane

coated

MNP@DOX

NPs

(termed

MNP@DOX@UM-SCC-7 and MNP@DOX@HeLa respectively) was studied upon 3 h co-incubation with four cell lines including UM-SCC-7, HeLa, HepG2 (Human hepatocellular carcinoma cell), and COS7 (African green monkey kidney cell) cells. An amazing outcome was found that the fluorescence intensity originating from two CCCM


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coated NPs was far superior in the corresponding source cells over those in heterotypic cells (Figure 3), which approximated to 12~18 folds in terms of the mean fluorescence intensity inside cells. The comparison with blank controls indicated that the uptake of MNP@DOX@CCCM by heterotypic cells was minimal in the observation period. Consistently, substantially more rapid nuclear internalization by the source cell membrane coated MNP@DOX NPs were found when the incubation period further prolonged (Figure S6). These results suggested the highly specific self-recognition affinity of MNP@DOX@CCCM to the source cells.

Figure 3. CLSM images and flow cytometric profiles of four cell lines including UMSCC-7, HepG2, HeLa and COS7 cells upon 2 h co-incubation with MNP@DOX@UMSCC-7 (A) and MNP@DOX@HeLa (B). DOX concentration was fixed at 1.5 µg/mL. Scale bars: 20 µm.

The encouraging in vitro results regarding the self-recognition to homotypic cancer cells stimulate us to evaluate the in vivo tumor self-targeting (TST) ability towards homologous tumors. To the end, two mouse models were established. Firstly, mice


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bearing a UM-SCC-7 tumor on the right hind limb were intravenously injected with MNP@DOX@CCCM NPs prepared with different cell membranes at an identical DOX dosage of 2.5 mg/kg (Figure 4A). At 24 h, the in vivo living fluorescence imaging (Figure 4B) and the ex vivo imaging (Figure S7A) towards major viscera (heart, liver, spleen, lung and kidneys) and tumors were performed. In the group intravenously injected

with

MNP@DOX@UM-SCC-7,

the

tumors

exhibited

much

stronger

fluorescence compared with other tissues except for liver, indicating the efficient tumor accumulation of MNP@DOX@UM-SCC-7. In contrast, intratumor DOX signals was barely

detectable

in

the

control

groups

treated

with

MNP@DOX@HeLa,

MNP@DOX@COS7 and free DOX. Quantitative analyses of fluorescence intensity in UM-SCC-7 tumors indicated that the intratumor content of MNP@DOX@UM-SCC-7 was at least 3 folds as the controls treated with heterotypic cell membrane coated NPs (Figure S7B). The quantification of Fe content in the tissues provided detailed data about

the

biodistribution,

reconfirming

the

stronger

tumor

accumulation

of

MNP@DOX@UM-SCC-7 (Figure 4C).


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Figure 4. (A) Schematic illustration of UM-SCC-7 tumor-bearing mouse model treated with DOX and various cell membrane cloaked MNP@DOX@CCCM; (B) In vivo fluorescence images at 24 h post intravenous injection with MNP@DOX@CCCM (a: @UM-SCC-7; b: @COS7; c: @HeLa) and DOX (d) with an equivalent DOX dosage (2.5 mg/kg); (C) Distribution of Fe element in tumors and tissues at 24 h after MNP@DOX@CCCM administration at a [Fe] dosage of 2.5 mg kg−1(in the presence/absence of MF). Values were expressed as means ± SD; (D) Schematic illustration of H22 and UM-SCC-7 dual-tumor bearing mouse model. In vivo fluorescence images and ex vivo images of tumors at 12 h post injection with MNP@DOX@H22 and MNP@DOX@UM-SCC-7; Average fluorescence signals in


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major organs and tumors at 12 h post tail-vein injection with MNP@DOX@H22 (E) and MNP@DOX@UM-SCC-7 (F).

To further validate this tumor self-targeting (TST) effect originating from the selfrecognition of MNP@DOX@CCCM to the homotypic cancer cells, we established another mouse model simultaneously bearing two different types of tumors (H22 tumor on the left hind limb and UM-SCC-7 tumor on the right). The dual-tumor bearing mice were intravenously administrated with MNP@DOX@H22. Both the in vivo living fluorescence imaging and ex vivo fluorescence imaging evidently indicated the overwhelmingly preferential DOX accumulation in the H22 tumor, as compared to the fluorescence intensity in UM-SCC-7 tumor (Figure 4D). Quantitative analysis on the biodistribution of DOX fluorescence reconfirmed the conclusion that MNP@DOX@H22 can actively recognize and “home” to the homologous H22 tumor while to a great extent “bypassing” the co-existing heterologous UM-SCC-7 tumor (Figure 4E). OF special note, the self-targeting to UM-SCC-7 tumor can be achievable in like manner just by substituting H22 cell membrane with human UM-SCC-7 cell membrane surrounding the MNP@DOX NPs, confirming the dependency of tumor self-targeting (TST) effect on the cell membrane sources (Figure 4F). Having proved the in vitro and in vivo self-recognizing tumor targeting of MNP@DOX@CCCM NPs, we subsequently studied their properties of MR imaging and magnetic targeting under external magnetic field (MF). As shown in Figure S8, T2weighted MRI of MNP@DOX@HeLa displayed a Fe concentration-dependent manner of the dark signal. The T2 relaxation rate (r2) was calculated to be 521.83 mM−1 s−1, significantly higher than that of the uncoated MNPs (420.55 mM−1 s−1). This was


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probably due to the enhanced magnetic liquid density and restricted water molecule mobility caused by membrane coating. In view of the ultrahigh in vitro r2 value, the in vivo T2-weighted MRI was conducted for the mice bearing two UM-SCC-7 tumors on opposite hind limbs. To avoid the interference with the targeting effect from cell membrane coating, MNP@DOX@HeLa NPs were used. The tumor attached with a magnet appeared as a dark area on the in vivo T2-MRI over time, as opposite to the very weak MRI signals in the non-magnet-attached tumor (Figure S9A). The strong MRI contrast and the magnetic targeting were represented more evidently upon pseudocolor treatment (Figure S9A). As compared with the organs and the control tumor, the abundance of prussian blue positive signals in the MF-treated tumor reconfirmed the MF-guided selective accumulation of Fe element (Figure S9B). Spleen is prussian blue positive due to the inherent enrichment of Fe content. Encouraged by the favorable outcomes concerning self-recognizing targeting, the antitumor efficacy was evaluated in vitro and in vivo. In vitro cytotoxicity of MNP@DOX@CCCM samples was firstly examined by MTT assay in UM-SCC-7 cells. Much higher cytotoxicity of MNP@DOX@CCCM was identified in the homotypic source cells, due to the self-selective cellular uptake (Figure 5A). For in vivo antitumor therapy, female nude mice bearing UM-SCC-7 tumor were intravenously injected with three MNP@DOX@CCCM samples, using PBS and free DOX as controls. As displayed in Figure 5B~5E and Figure S10, relative to the case using free DOX (2.5 mg/kg body weight), the tumor-inhibition efficacy of MNP@DOX@HeLa and MNP@DOX@COS7 were slightly enhanced despite the EPR effect. In comparison, tumor progression could be seriously depressed upon MNP@DOX@UM-SCC-7 injection. As expected, the antitumor efficacy can be further enhanced under MF guidance. No obvious abnormality


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was found in terms of body weight when the mice were treated with MNP@DOX@UMSCC-7 (Figure 5D). Furthermore, the immunofluorescence staining to Ki67 (a biomarker of cell proliferation) and caspase-3 (a marker of apoptotic cell death) was performed after various treatments (Figure 5F). The MNP@DOX@UM-SCC-7 administration led to apparently weaker Ki67 signal and stronger caspase 3 signal compared with the controls treated with other samples, demonstrating the relatively stronger ability of MNP@DOX@UM-SCC-7 for the inhibition of tumor cell proliferation (Figure 5G) and the induction of apoptotic cell death (Figure 5H). Consistently, through hisotological analysis using hematoxylin and eosin (H&E) staining to tissue slices, more marked reduction of tumor cells in MNP@DOX@UM-SCC-7 treated tumor was detected than other control groups (Figure 6). The H&E staining assay also indicated the minimal systematic toxicity of MNP@DOX@UM-SCC-7.


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Figure 5. (A) Cytotoxicity profiles in UM-SCC-7 cells obtained after 3 h and 6 h coincubation with DOX and various MNP@DOX@CCCM NPs coated with the membrane fragments from different cell lines (UM-SCC-7, COS7 and HeLa cells). #p < 0.05,

##

p

Preferential Cancer Cell Self-Recognition and Tumor Self-Targeting

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