Manganese Dioxide

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Erythrocyte-Membrane-Coated Prussian Blue/Manganese Dioxide Nanoparticles as H2O2 Responsive Oxygen Generator to Enhance Cancer Chemotherapy/Photothermal Therapy JinRong Peng, Qian Yang, Wenting Li, Liwei Tan, Yao Xiao, Chen Lijuan, Ying Hao, and Zhiyong Qian ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b17022 • Publication Date (Web): 06 Dec 2017 Downloaded from http://pubs.acs.org on December 6, 2017

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ACS Applied Materials & Interfaces

Erythrocyte-Membrane-Coated Prussian Blue/Manganese Dioxide Nanoparticles as H2O2 Responsive Oxygen Generator to Enhance Cancer Chemotherapy/Photothermal Therapy Jinrong Peng, † Qian Yang, ‡ Wenting Li, § Liwei Tan, § Yao Xiao, † Lijuan Chen, † Ying Hao, † Zhiyong Qian*†

† State Key Laboratory and Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University, No. 17, Section 3, Southern Renmin Road, Chengdu, Sichuan, P. R. China ‡ School of Pharmacy, Chengdu Medical College, No. 783, Xindu Avenue, Xindu District, Chengdu, Sichuan China. § Department of pharmacy, West China Second University Hospital, Chengdu, Sichuan, P. R. China KEYWORDS hypoxia, combinational therapy, long circulation, chemotherapy, photothermal therapy

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ABSTRACT

Due to the non-targeting release of anticancer drug, conventional chemotherapy results in serious side effects and poor therapeutic outcome. And hypoxia situation in tumor microenvironment also promote the growth and metastasis of tumor. Multifunctional nanocarriers with stimuliactivation and hypoxia relieving properties can help to overcome some of these limits. In this study, we have constructed a nanocarrier which is named as PBMn-DOX @ RBC. Prussian blue/manganese dioxide nanoparticle (PBMn) is used as an oxygen precursor or catalyzer for H2O2 activation, and red blood cell (RBC) membrane is used to increase the loading capacity of doxorubicin (DOX) and prolong the circulation time in vivo. H2O2 is overproduced in tumor tissue and tumor cells. It can be used as stimulus to activate drug release. In the presence of H2O2, the hypoxia inside tumor is relieved by administration of PBMn-DOX@RBC. The generated oxygen disrupts the RBC coated on the surface of PBMn, which accelerate the release of DOX. RBC also prolongs the circulation time of the nanometer system in vivo. Combining with the photothermal therapy, the tumor growth inhibition mediated by PBMn-DOX @ RBC is further enhanced. PBMn-DOX@RBC enables the demands to relieve tumor hypoxia and enhance cancer chemotherapy / photothermal therapy.


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Introduction Cancer is one of the serious threats to human health. Due to the non-targeting release of anticancer drugs in vivo and their low enrichment in tumor site, conventional chemotherapy results in serious side effects and poor therapeutic outcome. 1-3 The emergence and development of multifunctional nanostructures provide more suitable strategies to improve the problem. 4 In particular, the nanocarriers with stimuli-activation properties can not only achieve the activated release of drugs, but also be combined easily with other therapeutic treatments, such as hyperthermia, photothermal therapy (PTT), vaccination, radiotherapy, immunotherapy, etc., to overcome some of the limits of chemotherapy, to achieve co-therapy. 5-11 Therefore, the design and construction of multifunctional nanosystem with stimuli activation have attracted enormous attention, which is highlighted in nanomedicine. 12-16 Kinds of stimuli, including the ex vivo stimuli (magnetic field, light, electronic, etc.) and in vivo stimuli (temperature, pH, or cytokines, etc.) have been used to acheive activated therapy. 17-19 Most of the in vivo stimuli are connected with the tumor microenvironment, including high concentration of GSH, H2O2, etc. and tumor acidity. 20, 21 In the tumor microenvironment, due to rapid tumor growth and accumulation of a large number of metabolites, resulting in tumor tissue with acidic environment, relatively high concentrations of H2O2 and hypoxic environment, etc. 22-24

These specific characters can be used to trigger the release of therapeutic agents, and achieve

activated chemotherapy. 25 Besides, these characters, hypoxia in particular, which may promote tumor growth. The regulation of hypoxia situation in tumor microenvironment can regulate the tumor growth and metastasis. 26-30 Several reports have claimed the hypoxic environment inside tumor can be relieved by delivering oxygen to the tumor site via nanocarriers or by the catalysis


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of multifunctional nanoparticle itself. The relieve of hypoxic environment favors to enhance the anticancer efficacy of some therapeutic manners, including radiotherapy, immunotherapy, etc. 31 Perfluorocarbon (PFC) is widely used to deliver oxygen. The delivery efficacy, however, may be limited by its large particle size. It makes the multifunctional nanoparticle which can produce oxygen under the triggering of the stimuli inside tumor microenvironment become an alternative choice. 30, 32, 33 In our previous study, we constructed a Prussian blue/manganese dioxide (PBMn) nanocomposite. This nanocomposite can efficiently catalyze H2O2 to produce O2 and effectively improve the oxygen environment of the tumor site. In addition, PBMn has strong absorption in infrared region, with high photo-thermal conversion efficiency, can be used as an ideal photothermal therapy carrier. 34, 35 Moreover, we have confirmed that the introduction of PTT can enhance the tumor growth inhibition of chemotherapy in vivo. 36-38 But PBMn is of a nanocrystal, the drug can be only adsorbed on its surface, it is difficult to achieve the effective loading and delivery of drugs to tumor site. In order to improve the accumulation of drugs in the tumor site, further surface modification is needed. The red blood cell (RBC) membrane is an ideal surface modification material. Similar to many other cells, RBC surface expresses CD47 protein, which is a self-recognition protein to reticuloendothelial system (RES) in vivo. 39 Therefore, RBC membrane coating can provide the nanoparticle with long circulation properties. 40-49

And long circulation can enhance the enrichment of nanoparticle in tumor site by enhanced

permeability and retention (EPR) effect, which will further enhance the therapeutic effect in vivo. Besides, the expansion of the oxygen, similar with some other gases which are reported elsewhere, 50-52 generated by the catalysis of nanoparticle can also disrupt the coating structure, which may accelerate the release of therapeutic agents. With this property, activated drug release may be achieved. 53-57


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So, in this study, we plan to construct a drug delivery nanosystem which is composed by PBMn, DOX and RBC membrane to relieve the hypoxia of solid tumor and enhance the tumor growth inhibition by the co-therapy of chemotherapy and PTT, as shown in Scheme 1. PBMn is used as H2O2 responsive oxygen generator, DOX is used as model chemodrug, and RBC is used to enhance the drug loading efficacy of PBMn and prolong the circulation time in vivo. And MCF-7 breast cancer model was established to evaluate the performance of PBMn-DOX@RBC in relieving hypoxia and combinational therapy of chemotherapy and PTT in vitro and in vivo. RESULTS AND DISCUSSIONS Preparation and structural characterization. In our previous research, we successfully prepared PBMn (particle size is smaller than 50 nm) and used it as tri-model imaging contrast agents for imaging-guided photothermal therapy of breast cancer. [33] Herein, we use a reported protocol and extract the RBC membrane from the whole blood of the mice (Figure S1). By coextruding with PBMn and DOX, we can obtain PBMn-DOX@RBC. The morphology of the PBMn and PBMn-DOX@RBC was observed by TEM (Figure 1A and B). A clear bilayer structure was coated on the surface of PBMn, indicating the RBC membrane was successfully fused onto PBMn. Then we identified the proteins extracted from the PBMn-DOX@RBC and compared with the freshly extracted RBC membrane vesicles by Coomassie staining of the SDSPAGE, for visualization (Figure S2). The protein contents and compositions which extracted from PBMn-DOX@RBC is similar with the RBC membrane vesicles. And we further identified the CD47 remained on the surfaces of the extracted RBC membrane and the RBC membrane coated on PBMn-DOX by Western-blotting assay. Clear bands with similar grayscale were found on all groups, which indicates the existence of CD47 and equal proportion (Figure 1C). The size of the PBMn-DOX@RBC was 67±4 nm, which is about 18 nm larger than the PBMn


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(Figure 1 D). And the PDI of the PBMn-DOX@RBC is 0.077±0.012, which is similar with the PBMn (0.062±0.006. It demonstrated that the PBMn-DOX@RBC has a uniform particle size distribution. The zeta potential of the particles further proved the successful coating of RBC/DOX onto PBMn (Figure 1E). The zeta potential of PBMn is -31.7±2.9 mV, and the zeta potential of RBC membrane vesicles were -23.7±3.6mV. After being co-extruded of PBMn and RBC membrane vesicles, the obtained PBMn@RBC has a zeta potential of -27.6±2.7mV, which is higher than the PBMn and lower than the RBC membrane vesicles. The results are similar with some previous reports. 25 Further, after the co-extruding of PBMn and RBC membrane vesicle and DOX, the obtained PBMn-DOX@RBC has a higher zeta potential, which is -20.6±3.2 mV. The further increase of the zeta potential of PBMn-DOX@RBC is mainly ascribed to the low positive charge of DOX. Moreover, the PBMn-DOX@RBC exhibits well stability in PBS and DMEM culture medium for 4 weeks, respectively (Figure S3). Optical properties of PBMn-DOX@RBC. The optical absorption of the PBMn-DOX@RBC was measured by UV-Visible spectrometer (Figure 1F). The concentration of PBMn in different samples are settled at 50µg/mL. In Figure 1F, the UV-visible spectrum of DOX (curve d) is representing the absorption of DOX aqueous solution before being loaded by PBMn@RBC. And PBMn has low absorption at the range from 450 to 550nm, which favor to identify the concentration of DOX after being loaded by PBMn@RBC. After the coating of RBC membrane vesicle onto the surface of PBMn, the absorption peak at ~400 nm which is ascribing to the characterized peak of hemoglobin still can be found, although a little blue shift was also observed. The results indicate the existence of hemoglobin. It may be the hemoglobin inserted in the RBC membrane which was not washed out during the separation of membrane from RBC. The blue shift also indicates the interaction between RBC membrane and the surface of PBMn.


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After the loading of DOX, a characteristic peak of DOX at 485 nm can be found at the UVvisible spectrum of PBMn-DOX@RBC. It indicated the DOX is successfully loaded into the PBMn@RBC. By calculation, the loading capacity of PBMn@RBC to DOX is 4.7±0.3%. Furthermore, either the PBMn-DOX@RBC or PBMn@RBC was having a strong absorption peak at ~750 nm. The intensity and characteristic peak are equal to PBMn. It demonstrates the PBMn-DOX@RBC may maintain the NIR absorption of PBMn, which can also be used as PTT nanocarriers. 58 Therefore, we evaluated the photo-thermal conversion of PBMn-DOX@RBC, PBMn@RBC in vitro, and compared it with the PBMn (Figure 1G). After the irradiation by 808 nm laser for 5min at the laser power of 1.5W/cm2, the ∆T of the PBMn-DOX@RBC dispersion was ~30 oC, the same as PBMn@RBC and PBMn. It demonstrated the PBMn-DOX@RBC can be used a promising candidate for PTT. Oxygen production and Drug release triggered by H2O2. In our previous report, we have confirmed that PBMn is a promising catalyzer or generator to produce oxygen from H2O2. 33 In here, we further evaluated the oxygen production of PBMn-DOX@RBC and PBMn@RBC (Figure 2A). The oxygen produced by PBMn-DOX@RBC and PBMn@RBC in water can reach to 15.3mg/(L water) and 16mg/(L water), respectively, in 1min, which had the similar results with PBMn, and very clear gas bubble can be seen during the catalyzing (Figure 2B). It indicates that the PBMn-DOX@RBC or PBMn@RBC is also an oxygen generator, which is H2O2 responsive. Moreover, it is obvious that the generated oxygen can be expanded. The expansion of oxygen can disrupt the RBC coating. After being immersed in the buffer with the presence of H2O2 (100µM) for 1h, the surface morphology of PBMn-DOX@RBC changed from smooth coating to irregular surface (Figure 2C and D). It indicates the RBC coating was totally disrupted, which also demonstrates the generated oxygen can disrupt the structure of PBMn-DOX@RBC.


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Gas are used to realize the activated drug release in some previous research. Most of these systems are loaded with gas precursor, e.g. ammonium bicarbonate or sodium bicarbonate etc., or are directly loaded with gas (such as oxygen), which can be expanded by the stimulation of acidic environment or the sonication of ultrasound. These systems are promising, however, the large size or ex vivo stimuli dependent are still the challenges which need to overcome. The PBMn-DOX@RBC nanocarriers may provide a new choice. The PBMn can catalyze the H2O2 to generate oxygen, which can be used as driving force to activate drug release, and the RBC membrane and PBMn themselves all can be used as the carriers of DOX. Therefore, we further investigated the release behavior of PBMn-DOX@RBC, in particular, in the presence of H2O2. By optimizing the DOX loading procedure, the loading capacity of PBMn@RBC to DOX can reach to 4.7% which maintaining the stability of PBMn. Four kinds of release mediums, including pH=7.4 PBS, pH=6.0 PBS, pH=7.4 PBS plus with 100µM of H2O2 and pH=6.0 PBS plus with 100µM of H2O2 were used to investigate the release behavior of PBMn-DOX@RBC (Figure 2E). In 5 hours, the DOX release from the PBMn/RBC/DOX in pH=7.4 PBS is 6.3%, which are 21.6%, 49.3% and 71.3% in pH=6.0 PBS, pH=7.4 PBS with 100µM of H2O2, and pH=6.0 PBS plus with 100µM of H2O2, respectively. While the pH of the release medium reached to 5.0 with the absence of H2O2, the release rate of DOX is similar with that in the condition of pH=7.4 with the presence of H2O2. The results indicate the DOX release from the PBMn-DOX@RBC is H2O2 activated and tumor acidity triggering. The accelerated release of DOX from PBMn-DOX@RBC is mainly ascribed to the oxygen gas which generated from the PBMn catalyzing of H2O2. Based on these results, we further evaluated the drug release behavior of PBMn-DOX@RBC in alternate release mediums (Figure 2F). While the PBMn-DOX@RBC was placed in pH=7.4 PBS, the DOX released very slowly, 11.3% in 24 hours. While the release


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medium is adjusted to pH=6.0 PBS plus with 100µM of H2O2, over 75% of DOX was released in 12 hours. Burst release took place in the pH=6.0 PBS plus with 100µM of H2O2. The UV-visible spectrums of PBMn-DOX@RBC before and after DOX release in the presence of H2O2 were further proved the H2O2 activated drug release behavior of PBMn-DOX@RBC (Figure S4). It demonstrated the PBMn-DOX@RBC nanoparticles exhibited H2O2 and tumor acidity triggered release of DOX. The PBMn-DOX@RBC can be used as activating drug release carriers. Cellular Uptake and Cytotoxicity. To estimate the controlled release behavior of PBMnDOX@RBC in vitro, we evaluated the cellular uptake of PBMn-DOX@RBC by MCF-7. Most of the DOX are localized in the nucleus of the MCF-7 cells which was co-incubated with free DOX (Figure 3A, upper row). And in pH=7.4 DMEM culturing medium, the DOX delivered by PBMn-DOX@RBC was mainly localized in the cytoplasm (Figure 3A, middle row). While the culturing medium was adjusted to pH=6.0 DMEM plus with 100µM of H2O2, the DOX delivered by PBMn-DOX@RBC was distributed all over the cell (Figure 3A, bottom row). In order to quantitatively prove the H2O2/tumor acidity triggered release behavior of PBMn-DOX@RBC, flow cytometry assay was performed (Figure 3B). The fluorescence intensity of the MCF-7 cells which incubated with free DOX is 1.5 folds of which incubated with PBMn-DOX@RBC in pH=7.4 medium, and only 56% of which incubated with PBMn-DOX@RBC in pH=6.0 DMEM plus with 100µM of H2O2 (Figure 3C). The results demonstrated that the PBMn-DOX@RBC has H2O2/tumor acidity triggered drug release properties in cellular level. Then we evaluated the cell survival after the treatment with PBMn-DOX@RBC and chemotherapy/PTT co-therapy in vitro (Figure 4A). The IC50 of the free DOX, PBMn-DOX@RBC, PBMn-DOX@RBC+Laser were 14.5 µg/mL, 11.2 µg/mL, and 2.1 µg/mL, respectively. It proved the efficient tumor cell growth inhibition of PBMn-DOX@RBC and the chemotherapy/PTT co-therapy of breast cancer in vitro.


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The PTT performance of PBMn@RBC in vitro was also visualized by co-staining the MCF-7 cells with Calcein and PI (Figure 4B). The MCF-7 cells were co-incubated with PBMn@RBC for 4 hours before the culturing medium was replaced with PBS. After the cells were raised by PBS for two times, the laser irradiation was introduced (808 nm, 5min, 1.5W/cm2). About 99.9% of the cells which were co-cultured with PBMn@RBC emit strong red fluorescence, which indicated most of the cells were dead after the irradiation. The results demonstrated the PBMn@RBC is a promising candidate for PTT. We further evaluated the cytotoxicity of PBMnDOX@RBC in normal cells (HUVEC and 3T3 cells). The results show PBMn@RBC is biocompatible to normal cells. After being loaded with DOX, the survivals of HUVEC and 3T3 cells decrease as the concentration of DOX increase (Figure S5). It may be ascribed to the nonspecific cytotoxic of DOX. Long Circulation and Tumor Targeting of PBMn-DOX@RBC. Long circulation is a significant properties of RBC. For the existence of CD47 on the RBC membrane, the extracted RBC membrane can be coated on the surface of the nanoparticles to avoid/reduce the recognition of macrophage in vivo. From previous research, in order to maintain the outer layer of the RBC membrane after coating, the surface charge of the nanoparticles must be negative. The surface charge of PBMn is negative. Furthermore, we identified the CD47 existence in the PBMnDOX@RBC nanosystems (Figure 1C). It indicated the potential of PBMn-DOX@RBC for long circulation delivery. We measured the Mn content in the blood to investigate the long circulation properties of PBMn-DOX@RBC. After the intravenously injection of PBMn-DOX@RBC or PBMn into the SD rats, the Mn contents in the blood of PBMn treated groups dropped dramatically in 2 hours, and further dropped to normal level in 4 hours, which indicated the PBMn has been fast distributed into the organs (Figure 5A). On the opposite, in the PBMn-


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DOX@RBC treated group, the Mn content also had a significant drop in the first two hours. But the Mn concentration in the blood is still 3 folds higher than the PBMn treated group. In 24 hours, the Mn concentration in PBMn-DOX@RBC treated group is more than 100 folds higher than the PBMn treated group. The results indicated the PBMn-DOX@RBC remained the long circulation properties of RBC. Furthermore, we established MCF-7 cancer model in Balb/c-nu mice to investigate the biodistribution of PBMn-DOX@RBC in vivo. Mn content was also used as the indicator. In 24 hours after the injection of PBMn-DOX@RBC or PBMn, the Mn contents in the live tissue of the PBMn-DOX@RBC treated mice are much lower than in the PBMn treated group (p

Manganese Dioxide

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