Phototheranostics: Active Targeting of Orthotopic Glioma Using

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Phototheranostics: Active Targeting of Orthotopic Glioma Using Biomimetic Proteolipid Nanoparticles Yali Jia, Xiaobing Wang, Dehong Hu, Pan Wang, Quanhong Liu, Xuanjun Zhang, Jingying Jiang, Xin Liu, Zonghai Sheng, Bin Liu, and Hairong Zheng ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b06556 • Publication Date (Web): 21 Dec 2018 Downloaded from http://pubs.acs.org on December 21, 2018

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Phototheranostics: Active Targeting of Orthotopic Glioma Using Biomimetic Proteolipid Nanoparticles Yali Jia,†,‡,⊥ Xiaobing Wang,‡,⊥ Dehong Hu,† Pan Wang,‡ Quanhong Liu,‡ Xuanjun Zhang,# Jingying Jiang,† Xin Liu,† Zonghai Sheng,†,* Bin Liu,§,* and Hairong Zheng†,* †Paul

C. Lauterbur Research Center for Biomedical Imaging, Institute of Biomedical and Health

Engineering, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China.

‡Key

Laboratory of Medicinal Resources and Natural Pharmaceutical Chemistry, Ministry of

Education, College of Life Sciences, Shaanxi Normal University, Xi’an 710119, China.

§Department

of Chemical and Biomolecular Engineering, National University of Singapore, 4

Engineering Drive 4, Singapore 117585, Singapore.

#Faculty

of Health Sciences, University of Macau, Taipa, Macau SAR, China.

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KEYWORDS: biomimetic engineering, phototheranostics, glioma, blood-brain barrier, photothermal therapy

ABSTRACT: Advances in phototheranostics revolutionized glioma intraoperative fluorescence imaging and phototherapy. However, the lack of desired active targeting agents for crossing the blood brain barrier (BBB) significantly compromises the theranostic efficacy. In this study, biomimetic proteolipid nanoparticles (NPs) with U.S. Food and Drug Administration (FDA)-approved indocyanine green (ICG) were constructed to allow fluorescence imaging, tumor margin detection and phototherapy of orthotopic glioma in mice. By embedding glioma cell membrane proteins into NPs, the obtained BLIPO-ICG NPs could cross BBB and actively reach glioma at the early stage thanks to their specific binding to glioma cells due to their excellent homotypic targeting and immune escaping characteristics. High accumulation in the brain tumor with a signal to background ratio of 8.4 was obtained at 12 h post injection. At this time point, the glioma and its margin were clearly visualized by near-infrared fluorescence imaging. Under the imaging guidance, the glioma tissue could be completely removed as a proof of concept. In addition, after NIR laser irradiation (1 W/cm2, 5 min), the photothermal effect exerted by BLIPO-ICG NPs efficiently suppressed glioma cell proliferation with a 94.2% tumor growth inhibition. No photothermal damages of normal brain tissue and treatment-induced side effects were observed. These results suggest that the biomimetic proteolipid NP is a promising phototheranostic nanoplatform for brain-tumor-specific imaging and therapy. 2


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Glioblastoma multiforme (GMB) is one of the most aggressive intracranial tumors with an extremely poor prognosis, high risk of recurrence and mortality.1-3 Despite great advances in the past years, GMB treatment remains a great challenge in oncology.4 So far, surgery is one of the first line treatment strategies that significantly alleviate symptoms and prolong the survival of glioma patients.5,6 However, the heterogeneous and infiltrating characteristics of glioma cells prevent accurate resection of tumors.7 Therefore, fluorescence imaging, a highly sensitive, real-time and low-cost characterization tool is widely used by surgeons to identify tumor margins and guide tumor resection.8,9 Clinical studies demonstrated that intraoperative fluorescence imaging using fluorescein,10 5-aminolevulinic acid (5-ALA)11 and indocyanine green (ICG)12,13 enabled tumor resection optimization and improved progression-free survival of glioma patients. Furthermore, 5-ALA and ICG can be used as drugs for inducing reactive oxygen species production and hyperthermia for cancer photodynamic therapy (PDT) and photothermal therapy (PTT).14-17 The synergetic phototheranostic agents integrated with fluorescence imaging and phototherapy show a significant superiority in neurosurgery.18-20 However, the lack of active targeting and crossing the blood brain barrier

(BBB)

abilities

extremely

limits

their

applications

to

glioma

phototheranostics.21 To address these limitations, different bioconjugates between poly (ethylene glycol) (PEG) and target ligands including human H-ferritin,22 chlorotoxin,23 cyclic 3


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arginine-glycine-aspartic acid (c-RGD),24 and D-peptide,25 have been developed to cross BBB and serve as active targeting theranostics.26-31 For example, Lu et al. reported RGD-targeted hollow gold nanospheres for photoacoustic imaging and PTT of GMB.32 Zhang and co-workers reported chlorotoxin-labeled magnetic NPs as drug and gene delivery carriers for magnetic resonance image-guided glioma therapy.26,27 These inorganic NPs generally have poor biodegradability with long-term safety concerns, which limits further clinic translation and applications. Moreover, it is well known that ligand-mediated active targeting and receptor-mediated transcytosis are highly dependent on the corresponding receptor expression density on glioma cells, which generally exhibits individual differences among patients.33-35 In addition, PEG may activate the immune system, leading to fast removal of the PEGlated NPs by the reticuloendothelial system/mononuclear phagocyte system.36 Therefore, it is of practical importance to develop a kind of NPs with the prospect of clinical translation, active targeting, crossing BBB and cloaking functions feasible for phototheranostics of gliomas. Recently, cancer cell membrane-based bioengineering approach gained much attention and was consequently applied in the development of biomimetic NPs possessing homotypic binding and immune escaping functions.37-40 Unlike ligandbased active targeting, the self-recognition function of biomimetic NPs is primarily dependent on the interaction between natural membrane proteins and target cells,38,39,41 thus avoiding the limitations of receptor expression density variations. Moreover, the 4


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immune system has difficulties in recognizing membrane-camouflaged NPs, because they are perceived as source cells with long circulation.41 Since the first report by Zhang and co-workers,37 the cancer cell membrane-based biomimetic technology has been applied for molecular imaging,43,44 drug delivery,45,46 immunotherapy47,48 and theranostics49-51 in various cancer models (Table S1). However, to the best of our knowledge, biomimetic NPs for active targeting of glioma phototheranostics are rarely reported. In this work, we developed membrane proteins camouflaged liposome-ICG (LIPO-ICG) NPs for crossing BBB and active targeting phototheranostics of orthotopic gliomas in mice (Figure 1). The biomimetic ICG-loaded liposome (BLIPO-ICG) NPs have several advantages. (i) Biosafety: The BLIPO-ICG NPs were constructed by U.S. Food and Drug Administration (FDA)-approved drug nanodelivery system (liposome) and imaging agent (ICG), exhibiting excellent biocompatible and biodegradable features. (ii) Deformability: Unlike the cell membrane-coated poly (lactic-coglycolicacid) (PLGA) “hard” NPs, the cell membrane proteins are easy to be embedded into phospholipid bilayer of “soft” LIPO-ICG NPs, which could load simultaneously with hydrophilic and hydrophobic ingredients. (iii) Phototheranostics: The BLIPO-ICG NPs can be used for active targeting, fluorescence imaging and tumor margin detection, which offer guidance to precise surgery and localized PTT. RESULTS AND DISCUSSION Preparation and Characterization of BLIPO-ICG 5


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LIPO-ICG were synthesized by the thin layer evaporation method.34 Proteins derived from the membrane of C6 glioma cells were efficiently incorporated into LIPO-ICG to form a uniform BLIPO-ICG after successive extrusion and dialysis treatment (Figure 1a). Transmission electron microscopy (TEM) images showed that BLIPO-ICG possessed spherical shape (Figure 2b). Dynamic light scattering (DLS) results revealed that BLIPO-ICG had a size of 104 ± 3 nm (PDI = 0.164), which was 1.4–fold smaller than that for lipid NPs (151 ± 4 nm, PDI = 0.094, Figure 2c), as a result of the cell membrane protein (CMP) incorporation into the phospholipid bilayer by successive extrusion. The decreased diameter of BLIPO-ICG is due to higher deformability of the phospholipid bilayer after protein fusion.33 The successful incorporation of CMP onto the NP liquid bilayer was also reflected by the clear zeta potential change from -18.5 mV to -15.6 mV (Figure S1). To further validate this result, proteins in the CMPs and BLIPO-ICG were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting (WB). The results showed similar protein bands in both CMP and BLIPO-ICG (Figure 2e and 2f), demonstrating the successful incorporation of glioma CMP into BLIPO-ICG. The amount of protein in each BLIPO-ICG NP was calculated to be 5.36 ag (Figure S2). Conformation changes of the proteins after incorporated into the liposomes were investigated using circular dichroism (CD) spectroscopy. As shown in Figure 2i, CD spectrum of BLIPO-ICG was similar with that of CMPs, indicating that the secondary structures of membrane proteins, including random coil, β sheet and α helix, were not obviously changed by

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embedding in the surface of BLIPO-ICG. The result indicates that the preparation of BLIPI-ICG do not affect the bioactivity of CMP. The absorption and emission spectra of BLIPO-ICG and LIPO-ICG were similar to these of free ICG (Figures 2d and 2g). However, the fluorescence of BLIPO-ICG and LIPO-ICG was decreased to 62% and 64% of the same amount of free ICG, respectively, as a result of aggregation-caused fluorescence quenching.52 Fortunately, the decreased fluorescence could favor the enhanced photothermal performance of BLIPO-ICG and LIPO-ICG. To verify this speculation, NPs-mediated photothermal effect (upon NIR laser irradiation, 808 nm, 1 W/cm2) was measured at different times. As shown in Figure 2h, the temperature in BLIPO-ICG and LIPO-ICG rapidly reached 54.4 °C and 54.0 °C after 5 min laser irradiation, which is higher than that of free ICG (52.8 °C). Moreover, the stability of BLIPO-ICG in H2O, phosphate buffer solution (PBS) and fetal bovine serum (FBS) was evaluated by measuring the particle size changes and drug release behaviors, respectively. As shown in Figure 2j and S3, the particle sizes of BLIPO-ICG were changed from 104 nm to 121 nm in H2O, from 107 nm to 128 nm in PBS, and from 100 nm to 141 nm in FBS. Although the slight alteration in particle size in different solution, the drug accumulative leakage was lower than 15% in PBS and H2O, and below 30% after 6 h incubation in FBS (Figure 2k), suggesting a certain stability of BLIPO-ICG in bloods circulation. In addition, BLIPO-ICG NPs significantly improved the stability of ICG dye. As shown in Figure 2l and S4, the fluorescence intensity of free ICG was rapidly decreased, however, it was decreased 7


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slowly for BLIPO-ICG under the same conditions and kept approximately above 70% after 1 week storage. These results confirmed the good stability of BLIPO-ICG. One of the possible reasons could be that modification of CMP reduced the nonspecific protein conglutination of liposomes in serums. Additionally, the uniform structure of BLIPOICG obtained by the successive extrusion during its preparation might also contribute to its good stability. Active targeting and immune escape characteristics of BLIPO-ICG The inherent homologous adhesion property of the surface antigens in cancer cells allows excellent active-targeting abilities.41, 53, 54 To validate if these abilities are also present in BLIPO-ICG, the NPs were incubated with several cell lines, including HepG2 (human hepatocellular carcinoma cell), B16 (murine melanoma cell), MCF-7 (human breast cancer cell), bEnd.3 (murine normal brain microvascular endothelial cell), U87 cells (human glioma cell) and C6 (rat glioma cell). The flow cytometry results revealed that ICG fluorescence intensity in C6 cells was 3.9- to 7.9-fold stronger than that of other heterotypic cells (Figures 3a and 3b), suggesting its good selfrecognition property to homologous cancer cells. BLIPO-ICG cell uptake was further investigated by confocal laser scanning microscope (CLSM) imaging. After incubation of the cells with BLIPO-ICG for 2 h, BLIPO-ICG were mainly localized in the cytoplasm of C6 cells, and exhibited brighter red fluorescence than that of LIPO-ICGtreated cells (Figure 3c). Quantitative analysis by flow cytometry indicated that the fluorescence intensity in BLIPO-ICG-treated cells was 1.7-fold stronger than that of 8


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LIPO-ICG-treated cells (Figure 3d and S5), revealing its excellent active targeting ability. In addition, to test its antiphagocytic properties, the internalization of BLIPOICG by RAW264.7 macrophages was evaluated by CLSM imaging.41,55 As shown in Figure 3e, only a very small number of BLIPO-ICG was internalized by RAW264.7 macrophages, which exhibited 2.6-fold weaker fluorescence as compared to that for LIPO-ICG treated cells (Figures 3f and S6), suggesting the good immune escape ability of BLIPO-ICG. In vitro active targeting and PTT Prior to the photothermal treatment study, the toxicity of BLIPO-ICG was evaluated using a standard cell counting kit-8 (CCK-8) assay. As shown in Figures S7 and S8, cell viability of more than 94% was observed in C6 glioma cells and bEnd.3 cells after incubation with BLIPO-ICG for 24 h, even at a high ICG concentration of 30 μg/mL, revealing its good biocompatibility. After incubation for 4 h, BLIPO-ICG induced local hyperthermia upon 808 nm laser irradiation (1 W/cm2, 5 min), which could specifically kill glioma cells. This result was visually examined by the co-staining assay, in which dead cells were stained by PI (red fluorescence) and live cells were stained by calceinAM (green fluorescence) (Figure 4a). The control, laser-, LIPO-ICG-, and BLIPO-ICGtreated cells exhibited green fluorescence, indicating that laser or treatment with BLIPO-ICG alone caused no cell death. C6 glioma cells showed stronger red fluorescence after treated with BLIPO-ICG+laser than that for LIPO-ICG+laser treatment, indicating the effectiveness of the former in the treatment of C6 glioma cells. 9


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The results were further quantified by a standard CCK-8 assay. As shown in Figure 4b, cell viability decreased with increasing ICG concentration under laser treatment, revealing a concentration-dependent photothermal behavior. After laser irradiation, cell viability was 38 ± 3% and 16 ± 2% in LIPO-ICG and BLIPO-ICG ([ICG] = 30 μg/mL) groups (Figure 4b), respectively, indicating a much higher PTT effect exerted by BLIPO-ICG. In addition, the in vitro PTT effect was also dependent on the laser irradiation time. As shown in Figure 4c, cell viability after treatment with BLIPO-ICG ([ICG] = 20 μg/mL) under 2, 5 and 10 min laser irradiation was 53 ± 4%, 29 ± 3%, and 11 ± 1%, respectively, suggesting an irradiation time-dependent toxicity. These results suggest the necessity to optimize ICG dosage and laser parameters to achieve the best cancer ablation efficiency. In vivo fluorescence imaging in subcutaneous glioma model Encouraged by the good biocompatibility and homotypic binding abilities of BLIPOICG in vitro, the feasibility of BLIPO-ICG for in vivo imaging was investigated in subcutaneous glioma-bearing mice. After injection of BLIPO-ICG and LIPO-ICG (0.5 mg/kg each mice) via tail vein, respectively, fluorescence images at different time points were obtained using IVIS fluorescence imaging system (excitation at 710 nm and emission at 800 nm). As shown in Figures 5a and 5b, fluorescent signals in the tumor region increased gradually over time, reaching the maximum at 12 h postinjection, indicating that the high accumulation of both NPs in the tumor site. The reason for the accumulation of NPs might be its passive targeting based on the enhanced 10


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permeability and retention (EPR) effect. It has been reported that the fenestrations in tumor capillaries ranged from 100 to 1, 000 nm under different tumor microenvironment, depending on cell types, localization and growth stage.56,57 Therefore, long-circulating NPs with size in 100 nm to 200 nm are able to extravasate from vessels.57 Herein, the diameters of BLIPO-ICG and LIPO-ICG were both below 200 nm, leading to the successful extravasation from vessels for passive targeting to tumor. However, it was important to note that the BLIPO-ICG-treated mice showed 6.5-fold higher fluorescence intensity in the tumor region than that in the LIPO-ICGtreated mice (Figure 5b). The signal to background (S/B) ratio in BLIPO-ICG-treated mice was 17.7, which was almost twice higher than that for the LIPO-ICG-treated mice (Figure S9). The smaller size of BLIPO-ICG might improve its further diffusion through the interstitial space to reach the tumor site. Additionally, the excellent homotypic targeting ability also contributes to the active delivery of BLIPO-ICG into tumor tissue, finally leading to the higher S/B ratio by fluorescence imaging in BLIPOICG group. The ex vivo images showed that both BLIPO-ICG and LIPO-ICG were mainly accumulated in the liver and tumor, and a small amount was accumulated in the kidney (Figure 5c), suggesting a liver and kidney metabolism pathway and a high tumor accumulation of BLIPO-ICG and LIPO-ICG, which was agreed with the semiquantitative analysis results (Figure 5d). In vivo active-targeting fluorescence imaging in orthotopic glioma model

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To further evaluate the imaging performance of BLIPO-ICG, an orthotopic glioma mice model was established. Luciferase reporter-gene labeled C6 (C6-Luc) glioma cells were used, allowing us to evaluate the glioma cell activity via bioluminescence imaging. In addition, DTPA-Gd enhanced T1-weight magnetic resonance imaging (MRI) indicated that the glioma average volume was 8.7 mm2 and the depth was 4.5 mm after 14 days of tumor cell inoculation (Figure 6a). These C6 glioma cells possessed high proliferative activity as demonstrated by the high bioluminescence intensity in the brain (Figure 6b). At this time point, the BBB was locally disrupted by the rapid growth of C6 glioma cells into the brain,58,59 as evidenced by the Evans blue (EB) staining glioma tissue (Figure 6c). Once the BLIPO-ICG and LIPO-ICG were intravenously injected into orthotopic glioma-bearing mice after 14 days of tumor cell inoculation, fluorescence images were recorded at different time points. The fluorescent signal in the glioma area increased over time and reached the maximum at 12 h post injection (Figures 6d and 6e), suggesting that both BLIPO-ICG and LIPO-ICG could diffuse through the disrupted BBB and accumulate in the glioma tissue. At this time point, the average fluorescence intensity and S/B ratio in the tumor of BLIPO-ICG-treated mice was 1.8- and 1.9-fold higher than that of LIPO-ICG-treated mice (Figures 6e and S10), respectively, indicating the superior homotypic targeting ability of BLIPO-ICG to glioma. The ex vivo fluorescence images indicated the main distribution of both NPs in liver, kidney and tumor (Figures 6f and 6g), which was consistent with the in vivo

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results. These results demonstrate that BLIPO-ICG is superior to conventional LIPOICG in crossing the disrupted BBB and reaching the glioma site. On the other hand, the detection of tiny glioma is of great importance for early diagnosis and intraoperative-guided neurosurgery. Previous studies demonstrated that the BBB intact structure and function were partly preserved in the early stage of glioma.56,57 In order to evaluate BLIPO-ICG performance in the diagnosis of glioma at the early stage, a tiny orthotopic glioma tumor in mice was considered by performing our evaluation in the brain after 7 days of C6 tumor cell inoculation. DTPA-Gd enhanced T1-weight MRI imaging showed a tumor volume of 2.9 mm2 (Figure 7a), and bioluminescence images with weak signals revealed a small number of C6 glioma cells in the brain (Figure 7b). EB staining images of brain showed a pale blue in the glioma region (Figure 7c), indicating that BBB was only slightly disrupted in the early stage of glioma. Both BLIPO-ICG and LIPO-ICG were intravenously injected into the tiny glioma-bearing mice. No fluorescent signal in the brain was detected in the LIPO-ICGtreated mice (Figure 7d), however, a distinct fluorescent signal in the brain with an S/B ratio of 8.4 was observed in BLIPO-ICG-treated mice (Figures 7d, 7e and S11), suggesting that the EPR effect was not dominant in the early stage of tumor. In contrast, BLIPO-ICG with high homotypic targeting and immune escape characteristics was able to penetrate the slightly disrupted BBB and bind to the homotypic C6 glioma cells. The ex vivo fluorescence images indicated a clear fluorescent signal in the brain glioma (Figure 7f), which was consistent with the in vivo results. A brain tissue section showed 13


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a strong ICG fluorescent signal distributed in the tumor but not in the normal brain tissue, highlighting a clear tumor margin (Figure 7g), which was consistent with the vivid boundary between normal brain tissue and glioma detected by 4′, 6-Diamidino-2phenylindole (DAPI) staining due to an intense tumor cell proliferation. These results indicate that BLIPO-ICG is not only able to visualize tiny glioma, but also able to detect tumor margin, allowing for precise neurosurgery. Under the precision guidance of NIR fluorescence imaging, a complete removal of glioma tissue was performed as a proof of concept. As shown in Figure 8a and 8b, the intact mouse brain was removed from C6 glioma-bearing mice for simulating open operating environment at 12 h post-injection of BLIPO-ICG and LIPO-ICG. After resection of the glioma step by step, the fluorescent signal in the glioma area gradually decreased and finally disappeared (Figures 8c and S12). To further confirm the removal results, hematoxylin and eosin (H&E) staining was used to examine glioma and normal cells for evidence of precision excision. For LIPO-ICG group, although no normal brain cells existed in the resected glioma tissue section (Figure 8e), a small amount of glioma cells still existed in the brain tissue section (Figure 8d). The results suggested that achieving the complete removal of glioma tissue under the guidance of LIPO-ICG combined with NIR fluorescence imaging was still far from satisfactory. However, after sequential resections in BLIPO-ICG group, no residual glioma cells existed in the brain tissue section (Figure 8d), and no normal brain cells existed in the resected glioma section (Figure 8e), indicating a precision excision guided by BLIPO-ICG-fluorescence 14


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imaging. The better outcome for BLIPO-ICG might be due to the superior homotypic targeting ability of BLIPO-ICG with the average fluorescence intensity and S/B ratio in the tumor of BLIPO-ICG-treated mice being 1.8- and 1.9-fold higher than that of LIPO-ICG-treated mice in in vivo fluorescence imaging (Figure 7). The results indicate that BLIPO-ICG can be used as a potential fluorescent probe for image-guided neurosurgery. In vivo PTT in subcutaneous C6 glioma model Encouraged by the good PTT performance in vitro, and active targeting in vivo, BLIPOICG was injected in subcutaneous glioma-bearing mice to investigate the in vivo PTT efficacy. Thirty subcutaneous glioma-bearing mice were randomly divided into five groups (n = 6 in each group): (1) control (injected with PBS), (2) BLIPO-ICG, (3) laser, (4) LIPO-ICG+laser, and (5) BLIPO-ICG+laser. Under the guidance of NIR fluorescence imaging, the optimal treatment time (12 h post-injection) and laser irradiation region were established and used in the following experiments. The temperature at the tumor site in BLIPO-ICG-treated group rapidly increased to 57.2 °C, as compared to 51.5 °C and 38.9 °C in the LIPO-ICG- and PBS-treated groups after laser irradiation (808 nm, 1 W/cm2, 5 min) (Figures 9a and S13), suggesting the high photothermal effect of BLIPO-ICG. H&E staining was subsequently performed for the evaluation of the PTT effect. The results showed more cell necrosis and karyolysis in BLIPO-ICG+laser group compared to LIPO-ICG+laser group (Figure 9b). No photothermal damage was observed in adjacent normal tissue (Figure S14), indicating 15


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good selectivity and biosafety of BLIPO-ICG-mediated PTT. TUNEL staining images further confirmed the apoptosis effect (Figure 9c). The PTT efficiency was quantitatively evaluated by recording the tumor growth every 3 days after treatment (Figures 9d, 9e and S15). Mice in control, BLIPO-ICG- and laser-treated groups exhibited a rapid tumor growth (Figure 9d) resulting in 0% survival on day 19, 20 and 19, respectively (Figure 9e), which indicated that these treatments could not exert any therapeutic effect. On the other hand, LIPO-ICG+laser could partly suppress tumor growth, extending the mice life for several more days as compared to the previously mentioned mice groups, resulting in 0% survival rate on day 32. Surprisingly, the tumors completely disappeared in the BLIPO-ICG+laser group, resulting in 100% survival after 50 days, with no tumor recurrence observed in this group during this time interval, demonstrating its superior anticancer effect. In addition, no variations in the body weight were recorded in all groups during the treatment process (Figure 9f), and H&E images of organs such as heart, liver, spleen, lung, and kidney in all the treated groups revealed no abnormality or lesion compared to control group (Figure S16), indicating low side effects. Therefore, BLIPO-ICG is a very promising PTT agent to combat glioma. In vivo PTT in orthotopic glioma model On the basis of the promising results and treatment efficacy obtained in subcutaneous C6 glioma model, the feasibility of BLIPO-ICG for PTT in orthotopic glioma-bearing mice was further investigated. The NIR laser spot was pointed on the glioma site (bright 16


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lateral: 2.0 mm, bregma: 1.8 mm) and the photothermal temperature of the brain consequent to the laser activation was recorded by an infrared thermal imaging camera. As shown in Figures 10a and S17, after 5 min laser irradiation (1 W/cm2), glioma reached a maximum temperature of 47.9 °C in BLIPO-ICG-treated group, which exceeded the threshold value (42 °C) of PTT, leading to efficient thermal ablation of glioma. In contrast, the LIPO-ICG-treated group revealed a mild temperature increase up to 43.7 °C. Compared to the PTT in subcutaneous C6 glioma model, the BLIPOICG-mediated PTT effect in orthotopic C6 glioma model was weaker due to laser energy loss through skull. This shortcoming could be overcome by implanting fibers for direct irradiation on the glioma under the precise MRI-guided temperature detection system.60 To visualize the treatment effect, bioluminescence imaging was performed. As shown in Figure 10b, luminescence intensity in tumors of mice treated with BLIPOICG+laser was lower than control, BLIPO-ICG alone and LIPO-ICG+laser treated group after 15-day treatment. Quantitative analysis showed an inhibition of tumor growth by BLIPO-ICG+laser, with an inhibition rate of 94.2%, more effective than LIPO-ICG+laser treatment (69.1%) and BLIPO-ICG alone (5.8%) (Figure 10c). The result was further confirmed by H&E (Figure 10d) and TUNEL staining (Figure 10e). Indeed, BLIPO-ICG+laser group showed the smallest tumor size as compared to the other treated groups after 15-day treatment. These results were consistent with the bioluminescence images, demonstrating that BLIPO-ICG-mediated PTT could significantly inhibit glioma growth. TUNEL staining images at 4 h post treatment

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indicated an extensive cell apoptosis in glioma but not in normal brain tissue for the BLIPO-ICG+laser group (Figure 10e), confirming good selectivity of BLIPO-ICG. CONCLUSIONS In this paper, proteolipid NPs with biomimetic functions, high homotypic binding and BBB crossing abilities were developed for phototheranostics of glioma with activetargeting effects. The BLIPO-ICG was prepared through CMPs embedding into liposome NPs, providing an alternative strategy for biomimetic functions of clinically used NPs. Through active targeting based on homologous binding mechanism, the proteolipid NPs enhanced delivery of ICG crossing the BBB and into glioma cells. The brain tumor margin could be clearly observed for imaging-guided surgery and phototherapy. The BLIPO-ICG-mediated PTT significantly improved the therapeutic effect compared to LIPO-ICG-mediated PTT, leading to superior glioma inhibition. These findings provide insights into the development of biomimetic nanoplatforms for precise diagnosis and therapy of glioma. MATERIALS AND METHODS Materials 1,2-Distearoyl-sn-glycero-3-phosphocholine

(DSPC),

cholesterol

(Chol),

1,2-

dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3phosphocholine (DOPC) were purchased from Avanti (Alabaster, AL, USA). DAPI and ICG were purchased from Sigma-Aldrich (St Louis, MO, USA). Penicillin18


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streptomycin and high-glucose DMEM were obtained from Hyclone (USA). Trypsin EDTA and FBS were purchased from Gibco Life Technologies (AG, USA). PI and calcein-AM were purchased from Yeasen (Shanghai, China). CCK-8 was purchased from Dojindo Laboratories (Shanghai, China). Antibody against Na+/K+-ATPase was purchased from Cell signaling technology (MA, USA). Enhanced chemiluminescence WB substrate, coomassie blue, RIPA lysis buffer and bicinchoninic acid protein kit (BCA) were obtained from Beyotime biotechnology (China). Cell culture C6-Luc glioma cells, bEnd.3 brain microvascular endothelial cells, B16 melanoma cells, U87 glioma cells, MCF-7 human breast cells, HepG2 hepatocellular carcinoma cells and C6 glioma cells were cultured in high-glucose DMEM containing 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin and 2 mM l-glutamine and were incubated at 37 °C in an incubator with humidified atmosphere and 5% CO2. Preparation and Characterization of BLIPO-ICG C6 CMPs were obtained using hypotonic lysing, mechanical disruption, and centrifugal separation methods as previously reported.40,61 The protein concentration in the purified cell membrane fragments was determined by the BCA assay. Lipid NPs were prepared by a thin layer evaporation method. Briefly, appropriate amounts of DPPC, DSPC, DOPC and Chol were dissolved in chloroform at the molar ratio of 5:1:3:1, and then the organic solvent was removed under nitrogen flow until a thin lipid film was formed. 19


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Subsequently, the obtained lipid film was dried for 3 h under vacuum. The thin dried phospholipid blends were hydrated at 65 °C in PBS and freezing-thawing for 5 cycles using liquid N2 and a water bath at 65 °C. The formed liposome dispersion was extruded 20 times through a polycarbonate membrane of 200 nm pores to produce small vesicles. For the preparation of ICG-loaded lipid NPs (LIPO-ICG), a certain amount of ICG was added to the hydrated solution at a 1:40 ICG to phospholipid weight ratio. After repeated freezing-thawing cycles, LIPO-ICG solution was dialyzed overnight in a dialysis bag (MWCO 1000) to remove unencapsulated ICG. The lipid NPs covered with cancer cell membrane protein (BLIPO-ICG) were fabricated by a one-step extrusion method. The CMP was added to LIPO-ICG solution at a 1:300 protein to phospholipid weight ratio, mixed uniformly and extruded through a polycarbonate membrane of 400 and 200 nm pore sizes. The obtained BLIPO-ICG was dialyzed overnight at 4 °C to remove the excess of CMP. The hydrodynamic size and zeta potential of the obtained NPs (LIPO-ICG and BLIPO-ICG) were detected by DLS measurement (Zetasizer Nano ZS, Malvern Instrument, UK). The morphology of BLIPO-ICG was examined after negative stain with 2% phosphotungstic acid solution using TEM (FEI Tecnai G2 F20 S-Twin, USA). CMP and BLIPO-ICG protein profiles were analyzed by SDS-PAGE and the membrane protein marker (Na+/K+-ATPase) was detected by WB. CD spectra of BLIPO-ICG and CMPs were measured by a Model Jasco J-715 CD spectrometer. The

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amount of protein incorporated in BLIPO-ICG was calculated using the following equation:

The Cpro was measured according to the standard curve of bovine serum albumin (BSA) detected by BCA method. The Npar was obtained using qNano analyzer (IZON Sciences, Christchurch, New Zealand). The fluorescence spectra of free ICG, LIPOICG and BLIPO-ICG were collected using a FL spectrophotometer (F900, Edinburgh Instruments, Ltd., U.K) and the absorption spectra were measured by a UV-vis spectrometer (PerkinElmer LAMBDA 25, USA). The ICG concentration was quantified at the absorbance of 792 nm. Free ICG, LIPO-ICG and BLIPO-ICG solutions (CICG = 0.2 mg/mL) were irradiated using an 808 nm laser (1 W/cm2) for 5 min. The temperature profiles were recorded by an infrared thermal camera (Fluke, USA). The stability of BLIPO-ICG in H2O, PBS and 10% FBS was recorded by monitoring the diameter changes by DLS measurement. The drug leakage of BLIPOICG in H2O, PBS and FBS was also evaluated. Cell uptake BLIPO-ICG was incubated with different cell lines, including HepG2 cells, bEnd.3 cells, MCF-7 cells, U87 cells, B16 cells and C6 cells. In brief, 100000 cells in 500 L of medium were plated in a 24-well plate. After 12 h incubation, cell culture medium 21


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was replaced by fresh medium containing BLIPO-ICG (CICG = 20 μg/mL). After another 4 h, cells were washed with PBS and collected for flow cytometer analysis. C6 cells in 24-well plate at the same concentration mentioned above were also incubated with LIPO-ICG and BLIPO-ICG for 2 h, washed with PBS, fixed with 4% paraformaldehyde, stained with DAPI, and finally imaged under CLSM. Macrophage RAW264.7 cells were incubated with LIPO-ICG and BLIPO-ICG (CICG = 20 μg/mL) for 2 h and the cellular uptake was measured using CLSM and flow cytometry. In vitro PTT To evaluate BLIPO-ICG toxicity, bEnd.3 and C6 cells were seeded in a 96-well plate (1×104 cells in 100 L of medium for each well). After 12 h incubation, cells were treated with BLIPO-ICG at various ICG concentrations from 5 μg/mL to 30 μg/mL. After further 24 h incubation, the cell viability was measured by CCK-8 assay. For calcein AM/PI co-staining assay, C6 cells in 96-well plate (1×104 cells in 100 L of medium for each well) were treated with LIPO-ICG and BLIPO-ICG (CICG = 20 μg/mL) for 4 h, irradiated with 808 nm laser (1 W/cm2, 5 min), incubated for another 4 h, stained by calcein AM/PI, and then imaged using Leica DMI6000 inverted microscope. The photothermal toxicity was also measured by CCK8 assay. C6 cells (1×104 cells in 100 L of medium for each well) in a 96-well plate were incubated with LIPO-ICG and BLIPO-ICG (containing 10, 20, 30 μg/ml ICG) for 4 h at 37 °C, washed with PBS, and irradiated using an 808 nm laser (1 W/cm2, 5 min) before measurements. In addition,

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cells incubated with 20 μg/mL of ICG were irradiated by 808 nm laser (1 W/cm2) for different time (2, 5 and 10 min). Animals and tumor model Female BALB/c nude mice (18-22 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (China). Animals had free access to food and water and housed under sterile conditions at room temperature with a 12 h light/dark cycle. Animal experiments were carried out under the protocol approved by Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences Animal Care and Use Committee. To establish a glioma subcutaneous xenotransplanted tumor model, C6 cells (1 × 106 cells in 100 μL of PBS) were subcutaneously injected in the mice hind paw. Tumorbearing mice were used for the experiments when the tumor volume reached 50-100 mm3. To establish a glioma orthotopic implantation tumor model, C6-Luc cells were implanted into the mouse brain striatum. Briefly, animals were anesthetized with 1% pentobarbital sodium and immobilized on a stereotactic frame. Next, approximately 5.0 × 105 C6-Luc cells in 5 μL of serum-free medium were inoculated into the right striatum (bright lateral: 2.0 mm, bregma: 1.8 mm, depth: 3.5 mm) of nude mice using a mouse adaptor (RWD Life Science, Shenzhen, China). The growth of intracranial glioma cells was monitored by MRI and bioluminescence imaging. 23


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In vivo fluorescence imaging LIPO-ICG or BLIPO-ICG (CICG = 0.5 mg/kg) was intravenously injected into gliomabearing mice, and fluorescent images were acquired at different time points (2 h, 12 h and 24 h) using an in vivo fluorescence imaging system (IVIS Spectrum, PerkinElmer). At 24 h post injection, mice were euthanized and the main organs (liver, kidney, spleen, heart and lung), tumors and brains were collected for ex vivo fluorescence imaging. In vivo PTT To investigate the PTT effect in vivo on glioma, subcutaneous glioma-bearing mice were randomly divided into five groups (n = 6 in each group) as follows: (1) control (treated with PBS); (2) laser irradiation; (3) BLIPO-ICG (1 mg ICG/kg) without laser; (4) LIPO-ICG (1mg ICG/kg)+laser; (5) BLIPO-ICG (1 mg ICG/kg)+laser. After 12 h post injection, laser irradiation was performed by an 808 nm laser (1 W/cm2, 5 min), and thermal imaging was used to monitor the tumor temperature. The body weight and tumor volume were recorded every 3 days. In addition, tumors were collected at 4 h post treatment for H&E staining using a standard protocol and TUNEL immunofluorescent kit (Beyotime biotechnology, China). To evaluate the treatment effect on orthotopic glioma tumor, nude mice bearing orthotopic glioma were randomly divided into four groups as follows (n = 6 in each group): (1) control (treated with PBS); (2) BLIPO-ICG (1 mg ICG/kg) without laser; (3) LIPO-ICG (1 mg ICG/kg)+laser; (4) BLIPO-ICG (1 mg ICG/kg)+laser. For laser 24


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irradiation, the scalp of mice was removed and the laser irradiation was applied by 808 nm laser (1 W/cm2, 5 min). The spot of laser beam was adjusted according to small burr hole in the skull. After different treatments, mice were subjected to bioluminescence imaging at day 5, 10 and 15 to measure tumor growth. At 4 h post treatment, mice were sacrificed and their brains were collected for TUNEL immunofluorescence staining. Statistical Analysis SPSS 13 software (IBM, Endicott, NY) was used for statistical analysis. Results were expressed as mean±SD and Student t-test was used to evaluate the significance between two groups. P < 0.05 was considered statistically significant. ASSOCIATED CONTENT The authors declare no competing financial interest. AUTHOR INFORMATION Corresponding Author *E-mail:

[email protected]

*E-mail:

[email protected]

*E-mail:

[email protected]

Author Contributions ⊥Yali

Jia and Xiaobing Wang contributed to this work equally. 25


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ACKNOWLEDGMENT The authors gratefully acknowledge support for this research from Natural Science Foundation of China (81472846, 81527901, 81327801, 81827807), Major State Basic Research Development Program of China (973 Program) (2015CB755500), Singapore National Research Foundation (R279-000-483-281), Fundamental Research Funds for the Central Universities (Grant No. 2016TS059, GK201802002), Academic Leaders and Academic Backbones, Shaanxi Normal University (16QNGG012), and Shenzhen key laboratory of ultrasound imaging and therapy. Supporting Information Supporting Information Available: table of currently explored cancer cells for biomimetic nanoparticles; additional figures: Zeta potentials, diameter changes of BLIPO-ICG in different solutions detected by DLS, fluorescence spectra of BLIPOICG and free ICG in different solutions, flow cytometry data for cells incubated with BLIPO-ICG, cytotoxicity data of BLIPO-ICG, the signal-to-background ratio of tumor regions for fluorescence imaging, temperature-increase profiles after laser irradiation, representative photographs of subcutaneous C6 tumor-bearing mice during the treatment, H&E staining of major organs; supporting references. This material is available free of charge via the Internet at http://pubs.acs.org. REFERENCES

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46. Zhang, N.; Li, M.; Sun, X.; Jia, H.; Liu, W. NIR-Responsive Cancer Cytomembrane-Cloaked Carrier-Free Nanosystems for Highly Efficient and SelfTargeted Tumor Drug Delivery. Biomaterials 2018, 159, 25-36. 47. Kroll, A. V.; Fang, R. H.; Jiang, Y.; Zhou, J.; Wei, X.; Yu, C. L.; Gao, J.; Luk, B. T.; Dehaini, D.; Gao, W.; Zhang, L. Nanoparticulate Delivery of Cancer Cell Membrane Elicits Multiantigenic Antitumor Immunity. Adv Mater. 2017, 29, 1703969. 48. Noh, Y.; Kim, S.; Kim, J., Kim, S.; Ryu, J.; Kim, I.; Lee, E.; Um, S.; Lim, Y. Multifaceted Immunomodulatory Nanoliposomes: Reshaping Tumors into Vaccines for Enhanced Cancer Immunotherapy. Adv. Funct. Mater. 2017, 1605398. 49. Li, S. Y.; Xie, B. R.; Cheng, H.; Li, C. X.; Zhang, M. K.; Qiu, W. X.; Liu, W. L.; Wang, X. S.; Zhang, X. Z. A Biomimetic Theranostic O2-Meter for Cancer Targeted Photodynamic Therapy and Phosphorescence Imaging. Biomaterials 2018, 151, 1-12. 50. Yu, B.; Goel, S.; Ni, D.; Ellison, P. A.; Siamof, C. M.; Jiang, D.; Cheng, L.; Kang, L.; Yu, F.; Liu, Z.; Barnhart, T. E.; He, Q.; Zhang, H.; Cai, W. Reassembly of (89) ZrLabeled Cancer Cell Membranes into Multicompartment Membrane-Derived Liposomes for PET-Trackable Tumor-Targeted Theranostics. Adv Mater. 2018, 201704934. 51. Cai, L.; Sheng, Z.; Hu, D.; Xue, M.; He, M.; Gong, P. Indocyanine Green Nanoparticles for Theranostic Applications. Nano-Micro Lett. 2013, 5, 145-150. 52. Huang, P.; Gao, Y.; Lin, J.; Hu, H.; Liao, H. S.; Yan, X.; Tang, Y.; Jin, A.; Song, J.; Niu, G.; Zhang, G.; Horkay, F.; Chen, X. Tumor-Specific Formation of Enzyme-

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Figure 1 Schematic illustration of biomimetic proteolipid BLIPO-ICG for crossing the BBB and active targeting delivery of orthotopic glioma. (a) Preparation process of BLIPO-ICG. (b) Schematic of BLIPO-ICG for crossing BBB and active targeting imaging.

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Figure 2 Characterization of biomimetic proteolipid NPs. TEM images of LIPOICG (a) and BLIPO-ICG (b) after negatively staining with 2% phosphotungstic acid solution. (c) Hydrodynamic diameters of LIPO-ICG and BLIPO-ICG detected by DLS measurement. PDI = polydispersity index. (d) UV-vis absorption spectra of free ICG, LIPO-ICG and BLIPO-ICG. (e) SDS-PAGE analysis of cell membrane proteins extracted from C6 glioma cells and BLIPO-ICG. (f) WB results of C6 cell lysate, CMP and BLIPO-ICG. (g) Fluorescence spectra of free ICG, LIPO-ICG and BLIPO-ICG. (h) Time-dependent temperature profiles of free ICG, LIPO-ICG and BLIPO-ICG after NIR laser irradiation (808 nm, 1 W/cm2, 5 min) CICG = 0.2 mg/ml. (i) CD spectra of BLIPO-ICG and CMPs. (j) Hydrodynamic diameters and (k) drug release profiles of BLIPO-ICG in H2O, PBS and FBS after 24 h incubation. (l) Fluorescence decrease of BLIPO-ICG and free ICG in FBS after 1 week storage.

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Figure 3 In vitro evaluation of homotypic targeting and immune escape abilities of BLIPO-ICG. (a) Flow cytometry analysis of MCF-7, HepG2, B16, bEnd.3, U87 and C6 cells treated with BLIPO-ICG for 4 h. CICG = 20 μg/mL. (b) ICG mean fluorescence intensity of different cells. (c) CLSM images and (d) ICG mean fluorescence intensity detected by flow cytometry in C6 glioma cells after incubation with LIPO-ICG and BLIPO-ICG for 2 h. Cfree ICG= CBLIPO-ICG=ICG 20 μg/ml. Scale bar = 10 μm. (e) CLSM images of RAW264.7 cells treated with LIPO-ICG and BLIPO-ICG for 2 h and (f) ICG mean fluorescence intensity. CLIPO-ICG = CBLIPO-ICG=20 μg/ml of ICG. Scale bar = 10 μm.

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Figure 4 In vitro photothermal cytotoxicity of BLIPO-ICG. (a) Live/dead staining of C6 cells after different treatments. CICG = 5 μg/mL. Viable cells (green) were stained with calcein-AM, and dead cells (red) were stained with PI. (b) Viability of C6 cells treated with different concentrations of LIPO-ICG and BLIPO-ICG under 808 nm laser irradiation (1 W/cm2, 5 min). *p < 0.05, **p < 0.01 versus control. (c) Viability of C6 cells treated with LIPO-ICG and BLIPO-ICG at ICG concentration of 20 μg/ml with laser irradiation time from 2 to 10 min (808 nm, 1 W/cm2). *p < 0.05, **p < 0.01 versus control.

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Figure 5 In vivo fluorescence imaging of BLIPO-ICG in subcutaneous C6 tumorbearing mice. (a) Real-time fluorescent images of subcutaneous tumor bearing mice before and after intravenous injection of LIPO-ICG and BLIPO-ICG. CLIPO-ICG= CBLIPOICG=ICG 0.5 mg/kg. Red circles show tumor regions. (b) Semiquantitative fluorescence intensity analysis at the tumor site at different time intervals. *p < 0.05 versus LIPOICG. (c) Ex vivo fluorescent images of major organs and tumors at 24 h post injection. (d) Semiquantitative biodistribution of LIPO-ICG and BLIPO-ICG in major organs and tumors. *p < 0.05 versus LIPO-ICG.

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Figure 6 In vivo fluorescence imaging of BLIPO-ICG in orthotopic C6 tumorbearing mice. (a) Coronal (left) and transverse (right) section view by MRI of C6-Luc glioma-bearing mice after 14 days tumor cell inoculation. (b) In vivo bioluminescence images of glioma-bearing mice after 14 days tumor cell inoculation. (c) EB dye staining of brain tissue after 14 days tumor cell inoculation. Left: an aerial view of a whole brain; Right: brain tissue section. (d) Real-time fluorescence imaging of tumor-bearing mice. CLIPO-ICG = CBLIPO-ICG = ICG 0.5 mg/kg. Red circles show tumor regions. (e) Semiquantitative fluorescence analysis in the tumor site at different time points after treatment with LIPO-ICG and BLIPO-ICG. *p < 0.05 versus LIPO-ICG. (f) Ex vivo fluorescent images and (g) corresponding semiquantitative fluorescent analysis of major organs and tumors at 24 h post injection. *p < 0.05 versus LIPO-ICG.

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Figure 7 In vivo fluorescence imaging of BLIPO-ICG in tiny orthotopic gliomabearing mice. (a) Coronal (left) and transverse (right) section view by MRI of C6-Luc glioma-bearing mice after 7 days tumor cell inoculation. (b) In vivo bioluminescence images of glioma-bearing mice after 7 days tumor cell inoculation. (c) EB dye staining of brain tissue after 7 days tumor cell inoculation. Left: an aerial view of a whole brain; Right: brain tissue section. (d) Real-time fluorescence imaging of the whole body. CLIPO-ICG= CBLIPO-ICG=ICG 0.5 mg/kg. Red circles show tumor regions. (e) Semiquantitative fluorescence analysis in the tumor site at different time points. *p < 0.05, **p < 0.01 versus LIPO-ICG. (f) Ex vivo fluorescent images in major organs and brains at 24 h post injections. (g) Brain tissue section at 24 h post injection. Red: BLIPO-ICG; Blue: cell nuclei stained with DAPI. White lines show the boundary between glioma and normal brain tissue. Scale bar = 100 μm.

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Figure 8 Ex vivo fluorescence imaging-guided surgery. (a) Tumor resection step by step after 12 h post injection of LIPO-ICG. Top: digital photographs; Bottom: fluorescence images. The black dashed circle indicates the tumor site. (a) Tumor resection step by step after 12 h post injection of BLIPO-ICG. Top: digital photographs; Bottom: fluorescence images. The black dashed circle indicates the tumor site. (c) Semiquantitative fluorescence intensity at the tumor site during sequential tumor resection. H&E staining of section from (d) brain tissue after sequential resections and (e) resected tumor tissue. The black dashed circle indicates the extracted glioma tumor tissue. The red arrow points to the residual glioma tissue in brain.

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Figure 9 In vivo PTT of BLIPO-ICG in subcutaneous glioma-bearing mice. (a) Representative in vivo infrared thermal images of mice before and after 808 nm laser irradiation (1 W/cm2, 5 min). CLIPO-ICG= CBLIPO-ICG= ICG 1 mg/kg. (b) H&E staining and (c) TUNEL staining of tumor sections at 4 h post laser treatment. Scale bar = 50 μm. (d) Relative tumor volume curves in mice after different treatments. **p < 0.01 versus control. ##p< 0.01 versus LIPO-ICG+laser. (e) Mice survival rate curves. (f) Mice body weight curves under different treatments.

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Figure 10 In vivo PTT of BLIPO-ICG in orthotopic glioma-bearing mice (a) Representative in vivo infrared thermal images of the brain region before and after 808 nm laser irradiation (1 W/cm2, 5 min). CLIPO-ICG= CBLIPO-ICG=ICG 1 mg/kg. (b) Representative bioluminescent images of C6-Luc glioma-bearing mice in different groups. (c) Semiquantitative bioluminescent signal intensity in the brain. **p < 0.01 versus control. #p< 0.05 versus LIPO-ICG+laser. (d) H&E staining of brain sections of orthotopic glioma-bearing mice in all groups. (e) TUNEL staining of brain tumors. Scale bar = 200 μm.

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Phototheranostics: Active Targeting of Orthotopic Glioma Using

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