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Folate Engineered Microvesicles for Enhanced Target and Synergistic Therapy towards Breast Cancer Lian Zhu, Di Dong, Zi-Li Yu, Yi-Fang Zhao, Dai-Wen Pang, and Zhi-Ling Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b14633 • Publication Date (Web): 20 Jan 2017 Downloaded from http://pubs.acs.org on January 21, 2017

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ACS Applied Materials & Interfaces

Folate Engineered Microvesicles for Enhanced Target and Synergistic Therapy towards Breast Cancer Lian Zhu, † Di Dong, † Zi-Li Yu, ‡ Yi-Fang Zhao, ‡ Dai-Wen Pang, † Zhi-Ling Zhang*,† †

College of Chemistry and Molecular Sciences, Key Laboratory of Analytical Chemistry for

Biology and Medicine (Ministry of Education), Wuhan University, Wuhan 430072, China. ‡

School and Hospital of Stomatology, Key Laboratory of Oral Biomedicine (Ministry of

Education) and Department of Oral and Maxillofacial Surgery, Wuhan University, Wuhan 430079, China.

KEYWORDS: Microvesicles, phospholipid substitution, tumor targeting, synergistic therapy, in vivo imaging

ABSTRACT: As an ideal nanovector candidate, microvesicles (MVs) have been gradually utilized for packaging kinds of functional molecules for effective tumor diagnosis and therapy; however, the deficiency of their tumor targeting influenced their therapy efficacy. Through a facile phospholipid substitution strategy, MVs based drug delivery system (DDS) was apparently endowed with high tumor targeting towards breast cancer thanks to the modified folate onto the membrane of MVs, simultaneously possessed of synergistic anti-tumor effect, and in vivo tumor imaging attributed to the SA-QDs labeling. Tumor killing effect could be improved up to 15 percentages with the help of the improved tumor targeting ability.

ACS Paragon Plus Environment

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1. INTRODUCTION

Confronted with the increasing threaten of tumor, establishing a versatile DDS for effective tumor diagnosis and therapy, especially, the DDS endowed with high tumor targeting capability towards specific tumor, has always been a challenging project. Fortunately, with the rapid development of nanoscience and advanced production strategies, several kinds of DDS platforms including inorganic materials composites,1-10 polymer assembly,11-16 liposome as well as hydrogel et al have been successfully established and applied for tumor diagnosis and therapy during the past decades.16-18 As pioneers of DDS, outstanding achievements have been achieved based on these classical DDS. They really have paved the ways for the further development of novel DDS except for their inherent flaws including complicated functionalization procedures, stability needed to be improved within the blood circulation as well as potential metal toxicity.1920

There is an urgent demand to exploit more versatile DDS possessed of excellent biocompatibility, composed of multifunctional elements engineered by facile and bio-friend preparation procedures. It should be pointed out that achieving multifunction on single biocompatible DDS based on a facile approach is of great importance regardless of a great challenge. MVs, attributed to their distinctive cell origin, have been gradually utilized as novel kinds of DDS for tumor diagnosis and therapy. Similarly, exosomes (with a size distribution ranging from 50 to 150 nanometers), as another kind of cell-derived vesicles, have also been gradually developed as a type of DDS for tumor therapy.

MVs directly shed from plasma membrane with a broad size distribution ranging from 100 nm to 1 µm. Due to the distinctive origin and biochemical features of MVs, they are exceedingly stable


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within the blood circulation and impervious to the immune surveillance. In combination with these features, they have been gradually used as potential DDS candidate to effectively deliver functional molecules and therapeutic agents.21-22 In 2012, Huang and coauthors incubated tumor cells with methotrexate (MTX) to produce MVs packaged with MTX for following tumor therapy and obtained apparent anti-tumor expectation. Based on the our previous research,23 biotinylated MVs labeled with SA-QDs, simultaneous packaged with epidermal growth factor receptor (EGFR) siRNA was successfully developed for further in vivo tumor diagnosis and imaging.24 Recently, high resolution real time tracking of the MVs in vivo was realized with the package of ultra-small magnetically engineered Ag2Se quantum dots.25 Obviously, these researches further confirmed the practicability of MVs as a perfect nanovector for functional molecules loading for in vitro/vivo tumor therapy and imaging. Most importantly, these researches initiated a new DDS field over the traditional models mentioned above and further flourished the development of different DDS towards tumor therapy. However, without additional surface modification, cell derived MVs commonly don’t possess tumor targeting capability to relevant tumor which, to some extent, may influence their therapy efficacy during the complicated therapy procedures. The deficiency of effective modifications strategies greatly influenced the exploitation of multifunctional MVs based DDS for effective tumor therapy.26-28

In consideration of the existent problem, based on our previous reports, a smart MVs based DDS possessed of high tumor targeting capability was successfully established (detailed procedures seen in Scheme 1). Based on a facile phospholipid substitution strategy, MVs with the outer membrane were simultaneously modified with biotin and folate (BFMVs). Under the aid of electroporation, Bcl-2 siRNA and paclitaxel were subsequently packaged into the cavity of BFMVs. Herein, the multifunctional MVs was especially endowed with high tumor targeting to


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breast cancer thanks to the modified folate, simultaneously possessed of synergistic anti-tumor effect, in vivo imaging was realized under the labeling of SA-QDs. Excellent anti-tumor efficacy could be achieved based on the constructed multifunctional DDS. Tumor killing effect could be improved up to 15 percentages with the aid of the folate modified MVs. We expect that more versatile DDS will be exploited with the flexible selection of phospholipid derivatives, which will certainly boost and flourish the development of new kinds of DDS towards tumor diagnosis and therapy.

Scheme 1. Procedures of the production of the BFMVs based DDS and its further applications both in vitro/vivo enhanced target and synergistic tumor therapy as well as in vivo tumor imaging. 2. MATERIALS AND METHODS


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2.1. Materials. PMI 1640 (Gibco), Dulbecco’s modified Eagle’s Medium (DMEM, Gibco), 10% FBS (Gibco), Calcein&AM (Sigma, 1 mM) and propidium iodide (PI, Sigma, 0.15 mM), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene

glycol)-2000]

ammonium salt (DSPE-PEG-Biotin) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N[folate(polyethylene glycol)-2000] ammonium salt (DSPE-PEG-Folate, Alabaster, AL, USA), streptavidin-Cy3 (SA-Cy3, Sigma), Dylight 649 (Abbkine), DiO (BeyotimeR Biotechnology, 2.3 mM), CellMaskTM (Thermo Fisher Scientific), Bcl-2 siRNA and paclitaxel (Shanghai), goatanti mouse β-tubulin antibody (Invitrogen), Mammalian Protein Extraction Reagent (M-PER, Thermo Scientific), Halt Protease Inhibitor cocktail (Pierce), goat anti-mouse secondary antibodies (Pierce), chemiluminescence kit (Thermo Scientific), SA-QDs (CdSe/ZnS, λex=605 nm, 1 mM, Wuhan Jiayuan Quantum Dots Co. Ltd). 2.2. Cell culture. MDA-MB-231 and Cal 27 cells were respectively cultured with RPMI 1640 and DMEM medium supplemented with 1% penicillin-streptomycin and 10% FBS under 5% CO2 atmosphere at 37 ℃. In general, the two cell lines were passaged about three times a week. 2.3. Immunofluorescence. During the collection procedures of multifunctional MVs, Cal 27 cells were firstly cultured with DMEM culture medium supplemented with 1% DSPE-PEGBiotin and 1% DSPE-PEG-Folate. After passaging for three times, Cal 27 cells were divided into two groups, in the experimental group, Cal 27 cells were firstly incubated with streptavidin-Cy3 for 15 minutes, and the cells was then incubated with the secondary antibody goat-anti mouse Dylight 649 for 45 minutes after they have been incubated with the folate primary antibody at 37 ℃ for 90 minutes. While in the control group, Cal 27 cells were directly incubated with the


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secondary antibody goat-anti mouse Dylight 649 after incubated with the SA-Cy3 for 15 minutes. Fluorescence images were collected with an inverted fluorescence microscope (Ti, Nikon, Japan). 2.4. Cell viability. Cal 27 cells were respectively cultured with the common DMEM, DMEM medium supplemented with 1% DSPE-PEG-Biotin; DMEM culture medium supplemented with 1% DSPE-PEG-Biotin and 1% DSPE-PEG-Folate. Then, mixtures of Calcein&AM and PI were used to incubate with Cal 27 cells for 15 minutes, and then washed with phosphate buffer solution (PBS) for three times, and then pictures were collected under an inverted fluorescence microscope, the investigation of the cell viability continued to two weeks. 2.5. Collection and characterization of BFMVs. After three times passaging of Cal 27 cells with DMEM supplemented with DSPE-PEG-Biotin and DSPE-PEG-Folate, the culture medium was replaced with fresh DMEM mentioned above but without FBS, Cal 27 cells were starved cultured for another two days. Afterwards, the supernatant was collected and further centrifuged with 2500 g for 15 minutes, the collected supernatant was further under supercentrifugation with 31000 g for 70 minutes, the pellet was then dispersed with PBS and stored under -70 ℃ ultralow temperature freezer (HARRIS ELT) until use. TEM, (Hitachi HT7700) and DLS (Malvern Nano-ZS ZEN 3600) were then used to characterize the morphology and size and zeta potential of the collected BFMVs. Then, the collected BFMVs were firstly incubated with streptavidin-Cy3 for 15 minutes, and then incubated with secondary antibody goat-anti mouse Dylight 649 after they have been incubated with the folate primary antibody at 37 ℃ for 90 minutes. Fluorescence images were collected with an inverted fluorescence microscope.


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2.6. DiO labeled BFMVs entering into MDA-MB-231 cells. During the collection procedures, the supernatant was incubated with DiO for 15 minutes with a dilution ratio 1:300 (V/V). After that, the DiO labeled BFMVs were successfully collected as the procedures depicted above. The DiO labeled BFMVs were incubated with MDA-MB-231 cells for different periods (0.5 h, 1 h, 2 h, 3 h and 4 h) respectively under 37 ℃. FCM (FACSAria III) was used to investigate the relative number of MDA-MB-231 cells contained the DiO labeled BFMVs and fluorescence intensity of MDA-MB-231 cells. In addition, the corresponding fluorescence images of DiO labeled BFMVs entering into MDA-MB-231 cells were collected with inverted fluorescence microscope under different time periods mentioned above. 2.7. Dynamic tracking the trajectory of the DiO labeled BFMVs. The cells were incubated with CellMaskTM for 3 minutes. Then, the DiO labeled BFMVs were incubated with MDA-MB-231 cells under 4 ℃ for 15 minutes. Then, the cells were cultured on a CO2 on line cell culture system (INUBG2-PI) for further dynamic imaging, each image was collected every 800 milliseconds. Series of fluorescence images were automatically collected under the confocal microscope (Andor Revolution XD) equipped with an EMCCD (Andor iXon DV885K). Images were analyzed with Andor IQ software. Then, confocal microscope Z scanning was used to investigate the distribution of DiO labeled BFMVs within the cytoplasm, the scanning scope was from the bottom to the top of the MDA-MB-231 cells with a △Z of 0.2 µm.

2.8. Bcl-2 siRNA and paclitaxel package efficiency. The FAM labeled Bcl-2 siRNA and paclitaxel were packaged into BFMVs with an electroporation apparatus (Gene Pulse MXcell) under the circumstance of 250 voltages, 350 µF capacitance and infinitely-great resistance. The volume of the whole electroporation system was 500 microliters; the sample was under


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electroporation for about 25 milliseconds. Then, based on the FCM, the packaging efficiency and fluorescence intensity under different initial Bcl-2 siRNA concentration were respectively calculated. The supernatant was collected with a 6 minutes centrifugation of the samples after electroporation. Then, the supernatant was under a spin dryer for abandoning the solvent. Precipitate was mixed with the selected mobile phase acetonitrile and water (50:50, V/V) and detected under High performance liquid chromatography (HPLC, Essentia LC-15C) equipped with an ultraviolet detector at a flow rate of 1.0 mL/min with column temperature steed at 25 ℃.

2.9. In vitro therapy towards MDA-MB-231 cells. MDA-MB-231 cells were firstly cultured in 25 cm2 culture plate respectively. Paclitaxel and Bcl-2 siRNA were synchronously packaged into BFMVs with series of dose and concentration. Then, the different formats of the preprocessed MVs were firstly incubated with MDA-MB-231 cells for 4 hours, then washed with PBS to remove the redundant DDS, further continuously incubated with MDA-MB-231 cells for another 24 hours. Calcein&AM and PI were then used to label the cells for 15 minutes, and then washed for 3 times with PBS. After that, fluorescence images were taken with a CCD camera (Nikon digital sight DS-U3) mounted on an inverted fluorescence microscope (Ti, Nikon, Japan). 2.10. Characterization of the synergistic effect among paclitaxel and Bcl-2 siRNA. After treated with different formats of therapy models, firstly, the cells in each group were washed twice with PBS and then incubated with 4% paraformaldehyde for 15 minutes and following incubated with 0.1% triton-X 100 for 15 minutes respectively. The pretreated cells were incubated with 1% BSA for half an hour. After that, the cell was incubated with goat-anti mouse β-tubulin antibody at 37 ℃ for 90 minutes, subsequently; goat-anti-mouse cy3 IgG was used to labeled the primary antibody under 37 ℃ for 45 minutes. Finally, the prepared sample


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was pictured under a spinning-disk confocal microscope. In the meantime, series of groups namely

blank,

BFMVs,

BFMVs@Nc@Paclitaxel,

BFMVs@GAPDH@Paclitaxel,

BFMVs@Bcl-2 siRNA@Paclitaxel and BFMVs@Bcl-2 siRNA were utilized to incubate with MDA-MB 231 cells for 24 hours. Each group has three duplicates. Firstly, total RNA was collected with the aid of RNA trizol and related protocol, the total RNA concentration was detected with a Nano spectrometry at 260 nm. Then, the relative gene expression level was characterized based on real time quantitative PCR under designed parameters. After that, the cells of other groups were lysed with Mammalian Protein Extraction Reagent supplemented with Halt Protease Inhibitor cocktail. Then, the total protein was collected, corresponding concentration was determined with bicinchonininc acid (BCA) protein assay kit. Subsequently, 10 microliter pretreated protein sample was utilized during the 10% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred onto the polyvinylidene fluoride (PVDF) membranes (Millipore Corporation, Billerica, MA). After that, the membrane was blocked with 5% dried skimmed milk for about 45 minutes. Then, primary anti-Bcl-2 and anti-GAPDH antibodies (Danvers, MA, USA) were incubated with the blocked membrane for whole night at 4 ℃. The immunoblots were probed with horseradish peroxidase conjugated goat anti-mouse secondary antibodies for 1 h at room temperature. Finally, the protein was investigated with a chemiluminescence kit and photographed. 2.11. Animal experiments. All animal procedures were approved by the Animal Care and Use Committee of Wuhan University. 45 nude mice (BALB/C, Hunan SJA Laboratory Animal Co.，Ltd) were inoculated subcutaneously with exponentially growing MDA-MB-231 cells (107 in 200 µL refrigerant PBS). After two weeks, 45 nude mice were uniformly divided into 9 groups. Different formats of MVs based DDS were intravenously injected in female mice (18–


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22 g) via the tail vein, during the therapy course, the weight of nude mice in each group was recorded every four day; tumor size was measured with electronic digital caliper. After thirty days of the therapy course, three of the nude mice in each group were respectively sacrificed, and then, the excised tumors were taken pictures according to the different groups. Firstly, BFMVs, BMVs were under a 20000 g centrifugation after respectively incubated with SA-QDs for 20 minutes. Fluorescence images were collected with an in-vivo imaging system (Woburn, MA, USA) within different time span. After 24 hours in vivo imaging, all the mice were sacrificed, then the main organs (heart, liver, spleen, lung and kidney) and tumors were collected. The main organs and tumor were further for fluorescence imaging in-vivo imaging system. After several course of therapy, about 30 microliters blood was respectively extracted from the tail of each nude mouse in each group, then, the blood was collected in the anti-coagulation tube, each group for five duplicates. Immediately, the blood was investigated under the corresponding apparatus. At the end of the therapy course, two mice in each group were sacrificed, main organs including heart, liver, spleen, lung and kidney were carefully collected and then under a series of procedures (organs fixation, dehydration, ceresin wax embedding, hematoxylin and eosin stain and collection of tissue slice). Then the images were collected under the panoramic viewer (3DHISTECH Ltd). 3. RESULTS AND DISCUSSION 3.1 Modification and Characterization of Multifunctional MVs. Achieving the relevant modification towards MVs would endure them with more versatile functions towards tumor therapy. Realizing the biotinylation of cell membrane has been successfully achieved in our work through a facile phospholipid substitution technique. Our strategy was skillfully taking advantage


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of the relatively mild phospholipid substitution between Cal 27 cells and the DMEM culture medium supplemented with both certain concentration of 1,2-distearoyl-sn-glycero-3phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] ammonium salt (DSPE-PEGBiotin) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethylene glycol)2000] ammonium salt (DSPE-PEG-Folate). The cell membrane will be dynamically substituted with the complementary phospholipid derivatives during the cell division and proliferation process. In consideration of the distinctive origin, the membrane of the following collected MVs will be naturally simultaneously engineered with both biotin and folate. Intuitively fluorescence images of Cal 27 cells were collected and displayed in Figure S1. The results revealed that the membrane surface of Cal 27 cell was indeed simultaneously successfully engineered with both biotin and folate after several times passaging, which guaranteed the following successful collection of BFMVs. Transmission electron microscope (TEM), dynamic light scattering (DLS), Flow Cytometry (FCM) as well as confocal microscopy were respectively used to characterize the morphology, size and modification efficiency of different formats of MVs. As shown in Figure 1a, BFMVs displayed an irregular morphology with an apparent membrane structure. According to Figure 1b, c, the mean hydrodynamic size of MVs, DSPE-PEG-Biotin modificatory MVs (BMVs) and BFMVs calculated in DLS statistics were 625, 773 and 792 nm, respectively. TEM images of MVs and BMVs were shown in Figure S2a, b. The polydispersity index (PDI) of BFMVs was about 0.568, in accordance with their wide size distribution. As shown in Figure 1b, from MVs to BFMVs, the red column clearly depicted the tendency of the zeta potential, namely zeta potential firstly increased and then decreased. Attributed to the modification of biotin on the MVs surface, the zeta potential of BMVs was relatively more positive in comparison with MVs. And then, the modification of folate onto the surface made the


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zeta potential of BFMVs more negative than BMVs due to the introduced carboxyl groups. These results, together with the confocal fluorescence images of the collected BFMVs (Figure S3a-c) proved the successful modification of biotin and folate on the surface of MVs. In the meantime, the membrane modification efficiency was simultaneously characterized through FCM technique (Figure S3d). The labeling efficiency of biotin and folate were respectively 60.2% and 54.9%. Besides, in order to confirm whether the modification strategy would affect the viability of Cal 27 cells or not, we here continuously tracked six generations of Cal 27 cells with the aid of cell viability kit. Compared with the control group, the results disclosed that Cal 27 cells could still keep relatively good viability even continuously cultured for as long as two weeks (Figure S4), which proved the phospholipid substitution based modification strategy was mild, and further guaranteed the massive collection of multifunctional MVs.


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Figure 1. Characterization of different formats of MVs. a) Transmission electron microscopy (TEM) image of BFMVs. b, c) Mean size and zeta potential of MVs, BMVs as well as BFMVs.

3.2 BFMVs Entering into the MDA-MB-231 Cells. The therapeutic agents contained in the BFMVs based DDS included paclitaxel and Bcl-2 siRNA. Therefore, the successful entering of BFMVs into MDA-MB-231 cells will greatly contribute to the delivery of therapeutic agents into recipient cells, fulfilling its therapy goal. Figure 2a-d intuitively depicted that the DiO labeled BFMVs could enter into MDA-MB-231 cells within 4 hours incubation under 37 ℃, which initially proved BFMVs could be served as a nanovector potentially used for therapeutic agents delivery. In order to further confirm whether the DiO labeled BFMVs really entered into MDA-MB-231 cells or just merely adsorbed on their surface, confocal microscopy Z scanning technique was utilized to characterize the distribution of the DiO labeled BFMVs in cells (Figure 2e). The intuitive images in combination with analysis of both YZ and XZ sections in Figure 2e proved that the DiO labeled BFMVs indeed entered into the cytoplasm of MDA-MB-231 cells, which guaranteed the successful delivery of the therapeutic agents into recipient cells. Furthermore, the DiO labeled BFMVs were incubated with MDA-MB-231 cells within different time (0.5, 1, 2, 3 and 4 hours respectively). FCM results (Figure S5a, b) reflected that the DiO labeled BFMVs could enter into almost 90% MDA-MB-231 cells after incubation for 2 hours. Simultaneously, the fluorescence intensity of MDA-MB-231 cells reached to the peak after 4 hours incubation, which, together with the confocal fluorescence results (Figure S6), showed that the numbers of BFMVs entering into the MDA-MB-231 cells apparently increased with the incubation time. Endocytosis and membrane fusion dominated the two main entry approaches of MVs into recipient cells.22,

29-30

Furthermore, ultrathin electron microscope technique was utilized to


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investigate the possible entry approaches of BFMVs into MDA-MB-231 cells. The picture in Figure 2f and its partial enlarged view (Figure 2g) clearly disclosed that BFMVs entered into MDA-MB-231 cells with an apparent embolus occurred on the membrane surface of MDA-MB231 cells, which was usually believed as the typical endocytosis approach.31-32 Further, confocal microscopy was used to dynamically track the trajectory of the DiO labeled BFMVs entering into the cell (Figure S7, Movie S1). 10

Figure 2. Characterization of BFMVs entering into MDA-MB-231 cells. a-d) Fluorescence images of the DiO labeled BFMVs entering into MDA-MB-231 cells, the blue, green and red channel referred to nucleus, BFMVs and cell membrane of MDA-MB-231 cells, respectively. Scale bars referred to 10 µm; e) Confocal microscopy Z scanning image of the distribution of the DiO labeled BFMVs inside the cytoplasm. △Z is 0.2 µm, the whole scanning scope is 6.2 µm. Scale bar referred to 5 micrometers; f-g) TEM images of BFMVs entering into MDA-MB-231 cells, and the local magnification of the dashed box in Figure 2f, the black arrows indicated single BFMVs.


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As depicted in the series captured images, the DiO labeled BFMVs (as the white arrow indicated) initially presented an apparent co-localization with the CellmaskTM labeled cell membrane (displayed as a yellow BFMVs), then, the yellow BFMVs strode over the cell membrane and entered into cytoplasm instead of fusing with the membrane of recipient cells, finally, the yellow BFMVs turned into green again as the time elapsed, which together with the consequence of Figure 2f, g confirmed the endocytosis dominated entry of BFMVs into MDAMB-231 cells. 3.3. Characterization of the Packaging Efficiency of Paclitaxel and Bcl-2 siRNA. Electroporation was usually used to package biomolecules into cells, bacterial et al.33-34 Bcl-2 siRNA and paclitaxel are two of the therapeutic agents packaged into the BFMVs based DDS for the synergistic therapy. Series of concentrations of FAM labeled Bcl-2 siRNA and paclitaxel were simultaneously electroporated into BFMVs. Packaging efficiency and fluorescence intensity were used to optimize the Bcl-2 siRNA concentration. The relative ration of the BFMVs containing FAM labeled Bcl-2 siRNA increased with the increased concentration of FAM labeled Bcl-2 siRNA, and then almost reached to the maximum value at 600 nM (Figure S8a). Similarly, the fluorescence intensity increased evidently with the increasing concentration of FAM labeled Bcl-2 siRNA, and then kept placid as the Bcl-2 siRNA concentration reached to 2400 nM (Figure S8b). Paclitaxel displayed an ultraviolet absorption at 227 nm. According to the HPLC results (Figure S9a, b), the package capability of paclitaxel in each BFMV increased with the increased initial concentration of paclitaxel under the same electroporation condition (Table S1). 0.77 microgram of the initial 1.02 microgram could be successfully packaged into the whole BFMVs under the maximum initial concentration of 2400 nM. In combination with the total


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numbers of BFMVs, it could be calculated that each BFMVs contained about 0.136 femtogram of paclitaxel. 3.4. In Vitro Enhanced Target and Synergistic Therapy Towards MDA-MB-231 Cells. The engineered folate and the packaged therapeutic agents made BFMVs a potential DDS towards enhanced target and synergistic tumor therapy. Different formats of MVs based DDS were used for MDA-MB-231 cells therapy. Fluorescence images were collected on an invert fluorescence microscope based on cell viability kit. The anti-tumor efficiency (cell viability) was calculated with the formula (Numbers of the green cells/ Numbers of the total cells). Almost all kinds of the DDS demonstrated apparent therapeutic agent dose dependent anti-tumor efficiency (Figure 3a). Most importantly, the anti-tumor efficacy of BFMVs@Bcl-2 siRNA@Paclitaxel occupied an overwhelming advantage over other formats of DDS. Especially, compared with either

BFMVs@Bcl-2

siRNA

or

BFMVs@Paclitaxel

DDS

alone,

BFMVs@Bcl-2

siRNA@Paclitaxel possessed more excellent anti-tumor efficacy under the same concentration of therapeutic units. The respective cell viability evidently decreased from 78.18% and 64.02% to 41.8%, declining up to 36.4 and 20.3 percentages mainly attributed to the synergistic effect between Bcl-2 siRNA and paclitaxel, the consequences were in accordance with the previous reports.35-36 Besides, the anti-tumor efficacy of BFMVs@Bcl-2 siRNA@Paclitaxel was more obvious over the BMVs@Bcl-2 siRNA@Paclitaxel system. Cell viability directly dropped from 56.9% to 41.8%, dropping up to 15.1 percentages, which further demonstrated that the engineered folate to the outer membrane of MVs greatly contributed to the enhanced target therapy of MDA-MB-231 cells.


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Figure 3. a) Anti-tumor efficacy towards MDA-MB-231 cells of the different formats of DDS. Each group for three duplicates; each group from the left to the right referred to the blank, Bcl-2 siRNA, BFMVs@Bcl-2 siRNA,

paclitaxel,

BFMVs@Paclitaxel,

BMVs@Bcl-2

siRNA@Paclitaxel

and

BFMVs@Bcl-2

siRNA@Paclitaxel, respectively. b) Anti-tumor efficacy of BFMVs@Bcl-2 siRNA@Paclitaxel applied for the therapy of MDA-MB-231, A549 as well as HEK-293T cells respectively, red columns referred to the control group, blue groups referred to experimental group. Each group for three duplicates. (* in Figure 3b means the p

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