Reversal of Ovarian Cancer Multidrug Resistance by a Combination of

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Reversal of ovarian cancer multidrug resistance by a combination of LAH4-L1-siMDR1 nanocomplexes with chemotherapeutics Nana Guo, Chen Gao, Jing Liu, Jun Li, Nan Liu, Yanli Hao, Lei Chen, and Xiaoning Zhang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00031 • Publication Date (Web): 05 Apr 2018 Downloaded from http://pubs.acs.org on April 7, 2018

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Molecular Pharmaceutics

Reversal of ovarian cancer multidrug resistance by a combination of LAH4-L1-siMDR1 nanocomplexes with chemotherapeutics Nana Guo‡abf, Chen Gao‡c, Jing Liua, Jun Lid, Nan Liue, Yanli Haoe, Lei Chenbf*, Xiaoning Zhangae*

a

Collaborative Innovation Center for Biotherapy, Tsinghua University, Beijing 100084, China

b

Anhui Medical University, Hefei, Anhui 230032, China

c

College of Life Science, Hebei Normal University, Shijiazhuang, Hebei 050024, China

d

School of Life Science, Jiangsu Normal University, Xuzhou, Jiangsu 221116, China

e

School of Medicine, Tsinghua University, Beijing 100084, China

f

Department of gynaecology and obstetrics, PLA Navy General Hospital, Beijing 100037, China

KEYWORDS: ovarian cancer, MDR, siRNA, pH responsive peptides, nanocomplex

ACS Paragon Plus Environment

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GRAPHICAL ABSTRACT

ABSTRACT The mortality of ovarian cancer stably ranks first in gynecological malignancies due to the lack of specific symptoms and diagnostic methods at an early stage. For most patients, the cancer cells had metastasized before they were diagnosed. As a result, 90% of them died of multidrug resistance to chemotherapeutics. In our study, RNAi technology was introduced and applied to overcome this big problem. LAH4-L1, an amphipathic cationic polypeptide, was reported to


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have high transfection efficiency and was firstly selected by us to deliver siMDR1 for overcoming ovarian cancer cells MDR. In this research, LAH4-L1-siRNA nanocomplexes(LSCs) delivery system was designed via electrostatic interactions. The nanocomplexes could realize 87.3% MDR1 gene silence and 85% P-gp down-regulation on SKOV-3 cells. What’s more, with the combination of chemotherapeutics, SKOV-3 cells growth inhibition can reach to 82.9%. We have also found that there was about 50% reduction on cells migration when MDR1 gene was down-regulated. Besides what have been mentioned above, physicochemical characteristics, cytotoxicity, pH responsivity, cells cycle, cellular uptake and endosomal escape abilities were also studied in this research. In conclusion, lower cytotoxicity, higher down-regulation of targeted gene and great cells inhibition when combined with chemotherapeutics, all these advantages show great potential of LSCs for the reversal of multidrug resistance on SKOV-3 cells in the future.

INTRODUCTION As one of the three commonest gynecological malignancies, ovarian cancer has the highest mortality and worst prognosis. Its 5-year RS was only 45. 6% in 2007-2013 in US (1). This kind of disease mainly occurs in postmenopausal women, diagnosed at a median age of 63 years old, accompanied by abdominal pain and distension (2). Because of no specific symptoms and detection at early stages (3, 4), for at least 75% of patients, cancer cells had metastasized (FIGO stages 3/4) when they were diagnosed, in which stage tumor tissues cannot be completely removed by surgery (5). Although the combination of surgery with platinum/taxane-based


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chemotherapy regimens can achieve a short disease-free interval, over 75% of patients with advanced stages would relapse and be resistant to chemotherapy. After that, patients would have only 16-21 months survival and eventually died of a series of complications followed by metastasis (6, 7). Approximately 90% of deaths of advanced patients (stages 3/4) were caused by MDR (8). Not only ovarian cancer but many other tumors eventually become incurable due to multidrug resistance. Thus, it is emergent to solve the problem of cancers multidrug resistance. MDR refers to the phenomenon that a cross-resistance against to some other antitumor drugs with different structures and mechanisms when they are resistant to the initial one. Among various mechanisms of MDR, P-gp is the most widely investigated. Although there were some P-gp inhibitions which have been developed trying to overcome MDR, yet toxicity, side effects, interactions with other chemotherapeutics and no improvement in survival rate have limited its access to clinic (9, 10). RNAi technology was first realized by Andrew Fire and Craig Mello (11) and has developed dramatically in medical field especially in the area of cancers with gene dysregulation (12-14) since the promising results of silence Fas gene on mice with fulminant hepatitis in 2003 (15). Therefore, we planned to reduce the expression of P-gp by silencing its predominantly encoding gene MDR1, which was located on chromosome 7q21 (16). siRNA is a wonderful choice for RNAi technology on account of higher selectivity, easier to be packaged and delivered and safer than other nucleotide-based methods (17). However, naked siRNA cannot arrive at the destination triumphantly by reason of serum endonucleases and renal clearance (18).


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Having been developed until now, there are at least five delivery systems including viral vectors, lipid-based nanoparticles, peptide-based nanoparticles, polymer-based nanoparticles and inorganic small molecules (19). Most of them have been eliminated due to difficulties of cells uptake or endosomal escape or toxicity. LAH4-L1 is one of peptide-based vectors which shows high transfection efficiency both in DNA and siRNA gene deliveries (21). It is an isomer of LAH4 with angle of 80° subtended by the positively charged histidine residues (20). The protonation of its imidazole groups of the histidine residues account for transfection process and endosomal escape (21). We prepared self-assembled LAH4-L1-siRNA nanocomplexes (LSCs) via electrostatic interaction. The effect of gene silencing, following expression of corresponding proteins, reversal of SKOV-3 cells MDR by drugs combination were the focuses of the research. Besides, characterization, pH responsivity, idiographic mechanisms of endocytosis and endosomal escape of this nanocomplexes were also evaluated. SKOV-3 cells are from ascites of ovarian cancers patients, with resistance to tumor necrosis factor and to several cytotoxic drugs [ATCC]. The schematic representation was shown in Figure 1. [Figure 1 near here]

MATERIALS AND METHODS Materials LAH4-L1

with

peptide

sequence

KKALLAHALHLLALLALHLAHALKKA-NH2

was

purchased from SciLight Biotechnology(Beijing, China) at 99. 55% purity and was used as provided. Cy5-labeled siRNA was purchased from Suzhou Ribo Life Science Co. , Ltd (Kunshan,


5


China),

and

the

sequences

were

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as

5’-UUCUCCGAACGugUCACGUdTdT-3’;

follows:

sense

antisense

strand, strand,

5’-ACGugACACGUUCGGAGAAdTdT-3’. Cy5 fluorophore was labeled at 5’ of the sense strand. And, MDR1 targeted siRNA (siMDR1) from the same company has the sequences as follows:

sense

strand,

5’-CAGAAAGCUUAGUACCAAAdTdT-3’;

antisense

strand,

5’UUugGUACUAAGCUUUCugdTdC-3’. The fluorescent dye Hoechst 33342 was purchased from Fanbo Biochemical Co (Beijing, China). Cell Counting Kit-8(CCK8) was from Dojindo Laboratories(Kumamoto, Japan). Annexin V-FITC/PI Apoptosis Detection Kit was got from Yeasen Biotech (Shanghai, China). Paclitaxel injections were purchased from Haikou Pharmaceutical Factory Co. , Ltd (Haikou, China). All other reagents were obtained from the Biodee Reagent Company (Beijing, China). Cell culture and transfection Human multidrug-resistant ovarian cancer cell line (SKOV-3) was obtained from the cell bank of Chinese Academy of Sciences (Beijing, China) and cultured in RPMI 1640 medium (Gibco, USA),

supplemented

with

10%

fetal

bovine

serum

(FBS)

and

100

unit/mL

penicillin/streptomycin. The cells were incubated at 37℃ in a humidified atmosphere containing 5% CO2. LAH4-L1-siRNA nanocomplexes (LSCs) were prepared at a required weight ratio by adding aqueous solution of siRNA to an equal volume of LAH4-L1 that were both prediluted in 0. 9% NaCl (154 mM), followed by incubation for 20 min at room temperature. Characterization of LSCs: size, charge, morphology, and siRNA encapsulation


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The zeta potential of LSCs was measured by a Zetasizer Nano ZS90 (Malvern Instruments Ltd, Malvern, UK). All experiments were performed in triplicate. The size was detected by Dynamic light scattering (DLS) and the morphologic characteristics of LSCs were determined by transmission electron microscope (TEM, H-7650B, Hitachi, Japan). For siRNA binding assay, LSCs with different weight ratios were evaluated by agarose gel retardation assay. Twenty microliters of nanocomplexes containing solution with 0.5 ug siRNA was electrophoresed on the 1% (w/v) agarose gel containing ethidium bromide with Tris-acetate-EDTA (TAE) running buffer at 100 V for 50 min. siRNA was visualized on a Vilber Lourmat UV transilluminator. Silencing efficiency of the target MDR1 gene 2×10^5 SKOV-3 cells were seeded in 2 ml RPMI-1640 medium in 6-well plates and incubated for 24 h at 37℃ in 5% CO2. Then the cells were transfected with LSCs dissolved in 1 ml of opti-MEM medium (2 ug siMDR1/well), After co-incubation for 6 h, the supernatant was replaced by 2 ml fresh RMPI-1640 medium containing 10% FBS for another 42 h incubation. After 48 h transfection, total RNA was extracted with TransZolTM Up RNA kit (TransGen Biotech, Beijing, China) and reversely transcribed into complementary DNA (cDNA) with TransScript First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). UV-Vis spectrophotometer (Quawell Technology, USA) was used to detect the concentration and integrity of total RNA. A 20 µl of reaction mix consisting of synthesized cDNA, forward and reverse primers, EasyTaq PCR Supermix and RNA-free water, was subjected for the RT-PCR with the thermal cycle programme as below: 95°C for 5 min,

95°C for 30 s, 56°C /60°C for 30

s, and 30 cycles at 72°C for 30 s, 72℃ for 15 min, and final step at 10°C for 10 min. Sequences


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of MDR1 primers were as follows: forward (5’-ATATCAGCAGCCCACATCAT-3’) and reverse

(5’-GAAGCACTGGGATGTCCGGT-3’).

GAPDH

primers

were:

forward

(5’-TTCACCACCATGGAGAAGGC-3’) and reverse (5’-GGCATGGACTGTGGTCATGA-3’). The PCR products were loaded onto 1% agarose gel with 0. 1‰ DuRed (Bridgen Biotech, Beijing, China) staining at 110 V for 45 min, and the results were analyzed by using gel imaging and analysis system (Eastwin Life Science, Beijing). Cell Counting Kit-8(CCK8) assay CCK8 assay can be used to examine the cytotoxicity of LSCs and therapy efficacy via analysis cell growth. For cytotoxicity test, SKOV-3 cells were seeded in 96-well plates at a density of 10, 000 cells/well in 100 ul of RPMI 1640 medium. After incubation for 24 h, 100 ul opti-MEM solutions of LSCs (0.2 ug siMDR1) were added into each well. After 6 h, 100 ul fresh complete RPMI 1640 medium took the place, and transfection proceeded for an additional 42 h in the presence of 10% FBS. Then, 5 ul CCK8 in 95 ul completed medium was added to each well with further incubation for 1 h~4 h. As for therapy efficacy test, gene transfection was used to combine with chemotherapeutics to determine the reversal of drug resistance. 4000 SKOV-3 cells were seeded per well in 96-well plates and transferred according to the above method for 72 h before media with different concentrations of chemotherapy drugs took the place. The control groups were also set at the same time. After another 24 h of drugs action, 5 ul CCK8 in 95 ul completed media was added to each well, and further incubated for 1 h~4 h. The absorbance was measured at 450 nm by a microplate reader (Tecan Infinite200 PRO, Switzerland). The cells viability rate can be calculated as follows: cell viability rate = (As-Ab)/(Ac-Ab), where (As) is


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the experimental group containing cells, media, gene transfection, chemotherapeutics and CCK8; (Ac) is the control group containing cells, media, and CCK8; (Ab) is the blank group only containing media and CCK8. Cellular uptake and endocytosis mechanism To investigate the cellular uptake of LSCs, we used Cy5 labelled siRNA instead of siMDR1. SKOV-3 cells were incubated with the LSCs for 1h - 6h and uptake efficacy was analysed by using ﬂow cytometry. And the mechanisms of SKOV-3 cells uptake LSCs was explored by applying endocytosis inhibitors (chlorpromazine, M-β-CD, filipin, amiloride) 1h before added LSCs. Nonspecific effects as a result of toxicity were eliminated by control experiments at concentrations in the circumstances which inhibitions were specific. After another 1 h, the medium was washed off, cells were detached by trypsin. Cells suspension was then washed twice with PBS. Finally, 250 ul PBS was added to resuspend the cells as specimens. The mean fluorescence intensity and percentage of Cy5-si positive cells were quantitated by fluorescence-activated cell sorting (FACS) machine. The endocytosis of SKOV-3 cellular also can be verified by confocal laser scanning microscopy. Endosomal escape and its mechanism were detected by Confocal laser scanning microscopy (CLSM) SKOV-3 cells were plated at 3*10^4 cells/well in a 20 mm cell culture dish for 24 h before transfection with LSCs. Then the culture media were replaced by 1 ml opti-MEM and every sample contained 2 ug Cy5-si. The two incubation durations 3 h and 6 h were conducted at 37°C in 5% CO2 humidified atmosphere. After that the transfection solution was aspirated and


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substituted, the cells were washed with PBS for three times before being fixed by 4% paraformaldehyde for 10 min at room temperature, then washed three times with PBS before endosomes were stained with LysoTracker green (Life Technologies, USA) for 30 min at 37°C. Subsequently, cells were incubated with Hoechest 33342 dye for another 10 min and cells are thoroughly cleaned before being observed with a confocal laser scanning microscope LSM 710 (Zeiss, Germany). As for studies in mechanism, Bafilomycin A1 was added 1 h before LSCs. After 6 h transfection, cells were prepared according to the above method before observation. Apoptosis and cell cycle analysis SKOV-3 cells were plated in a 24-well plate (3. 6*10^4 cells per well) and transfected as described above. After incubation for 72 h, the media were replaced by 7.5 µg/ml PTX. The control groups were also set at the same time. Another 24 h of drugs action was taken, after that the cells were collected and re-suspended in 100 ml of Binding buffer, 5 µl Annexin V-FITC and 10 µl PI were added. Furthermore, the stained cells were incubated at room temperature for 20 min in the dark, and analyzed by a flow cytometer (BD FACS CaliburTM, San Jose, USA) and the data was processed by flowjo 7.6 software. For cell cycle analysis, the cells were digested and washed before being fixed with 75% ethanol over night at 4°C. Subsequently, cells were washed again and incubated with RNaseA for 30 min at 37°C. Finally, cells were stained with 400 µl PI solution for approximately 30 min followed by analysis on a cell-flow cytometer (BD FACSCaliburTM, San Jose, USA). The data was analyzed by using Modfit software. Wound healing and transwell migration assays


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The wound healing assay was performed by scratching the cell monolayer with a 10 µl sterile pipette tip resulting in a straight scratch on the center of the 6-well plates. Following that, the samples were washed with PBS to remove the impaired cells before adding LSCs. At the meantime, cells of each group were photographed at indicated time points (0 h, 6 h, 12 h, 24 h) using a phase contrast Microscope (Olympus IX-73, Tokyo, Japan). The images were analyzed by corresponding software. The migration rate was expressed as percentage of scratch closure and was calculated as follows: % of scratch closure = (a-b)/a, where (a) is a distance between edges of the wound, and (b) is the distance which remained cell-free during cell migration to close the scratch. Cell migration was measured using transwell chamber (Corning, New York, NY, USA). SKOV-3 cells were plated in a 6-well plate (2*10^5 cells per well) and transfected as described above. After incubation with treated samples for 48 h, the treated cells were washed, trypsinized and transferred to the upper transwell chambers at a density of 20, 000 cells/well in 200 µl serum-free medium and 500 µl medium containing 10% FBS was added to the lower chamber. Cells were incubated for another 24 h. At last, cells remaining on the upper chamber were wiped off with cotton swab and the cells that migrated into the lower chambers were fixed with 4% paraform-aldehyde, followed by staining with 0. 1% crystal violet for 10 min. Cells remained were cleaned with PBS and air dried before being imaged with a microscope and the number of migrated cells was determined by Image pro plus software. Western blot assay


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We used scraper to collect those cells with cold PBS which have already been transferred by LSCs for 72 h. The collected supernatant with cells were centrifuged at 300 g for 4 min, keep the centrifuged cells deposit, adding cell lysis buffer containing 5 µl Protease Inhibitor Cocktail to the pellet, then incubating on ice for 10 min. After centrifugation, the supernatant contained the final protein of the cells, the concentration of which was determined by the BCA assay. The supernatant was added with 5× protein loading buffer prior to be boiled at 100°C for 5 min and run on 12% SDS-PAGE for 1 h. Subsequently, the protein was transferred from the gel to the PVDF membrane. The membrane was blocked with 5% skim milk powder in 1× TBST for 2 h at room temperature prior to incubation with 1:1000 dilutions of rabbit monoclonal primary antibody (GAPDH, Novus) in blocking buffer overnight at 4°C. The membrane was washed with 1× TBST three times, each time for 10 min, and then incubated with secondary antibody (HRP AffiniPure goat anti-rabbit IgG, 1:10000, EarthOx) for 8 h at 4°C. P-gp protein was extracted by using ProteoExtract Transmembrane Protein Kit. Procedures of extracting transmembrane protein referenced to the specific protocol. Total protein was separated by 6% SDS-PAGE and the primary antibody was mouse-originated with dilution ratio 1:1000, secondary antibody was goat anti-mouse IgG with dilution ratio 1:10000. Other steps remained unchanged. Transparent plastic wrap was used to keep the membrane moist after interaction with chemiluminescent solution. Images were acquired by using molecular imaging system for chemiluminescence (ImageQuant LAS 4000, GE). Statistical analysis


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Statistical significance between two samples was performed using one sample Student’s t-tests. A value of P < 0. 05 was considered as statistically significant, and P-value < 0. 01 was considered as highly significant.

RESULTS Characterization of LSCs LSCs was prepared by a gentle method with different weight ratio. Combined results of the agarose gel retardation assay (Figure 2A) with zeta-potential detection (Figure 2C) we can see that the peptide can protect siRNA from migrating into the gel when the weight ratio of LAH4-L1/siRNA was 3. Since a positive surface charge helps nanocomplexes bind to the cell surface with negative charge. However, excessive positive charge can result in non-specific binding and significant toxicity (22). And efficiencies of MDR1 gene silencing at different weight ratios were detected (Figure 3A, B), the efficiency was the highest when the weight ratio of LAH4-L1/siRNA was at 3 and finally chosen. According to reports, LAH4-L1 is a pH responsive polypeptide (23). Three representative pH points have been set, pH 7. 4 stands for blood homeostasis, pH 6. 5 for tumor microenvironment and pH 5 for endosomal/lysosomal acidity. Shown in Figure 2B, LSCs successfully protected siRNA in blood and tumor microenvironment and released in endosome/lysosome when pH became lower, which suggested a passive targeting characteristic of LSCs. To obtain more information about the nanocomplexes, the particle size and morphology were tested by TEM (Figure 2D). DdH2O was applied here since salt crystals which were formed by physiological saline during samples preparation would


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interfere the observation of LSCs. From the images we can see that LAH4-L1 polypeptide can compress siRNA into a circular/spherical shape nanoparticle with a size smaller than 200 nm. Since the collapse of particles during the sample preparation of TEM, size of LSCs detected by DLS (Figure 2E) was bigger than that of TEM (24). A good delivery system is also required to have lower toxicity, while achieving high down-regulating of target gene. Lower cytotoxicity of LSCs was determined at 48 h after transfection (Figure 2F). [Figure 2 near here] MDR1 gene silence and corresponding protein expression Reverse transcription PCR was applied to measure the MDR1 gene silence (Figure 3A, B). From the results we can see that 87.3% MDR1 gene down-regulation can be achieved when the LAH4-L1/siMDR1 weight ratio at 3, which represented 5.6 times higher than naked siMDR1 with 15.6% knockdown (Figure 3A, B). While human P-gp production is encoded by MDR1 gene and responsible for multidrug resistance (25, 26), the alteration on protein aspect was studied. Follow the results we can see that the expression of P-gp was down-regulated about 85% (Figure 3C, D). Reversal of multidrug resistance in ovarian cancer after MDR1 gene silence Theoretically, the accumulation of chemotherapeutics in SKOV-3 cells could increase after MDR1 gene silence. Method of combined with gene transfection and chemotherapy drugs was applied to detect the reversal of multidrug resistance. Paclitaxel is the basis of numerous chemotherapeutics, so as in ovarian cancer (8). Three different concentrations of PTX were used simultaneously in CCK8 assay (Figure 3E). From the results we can see that the apoptotic cells of dosing group without transfection at 3 µg/ml, 7. 5 µg/ml, 15 µg/ml of PTX were 7. 1%, 17.


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4% and 27. 9% respectively. However, the apoptotic cells of group with LSCs transfection can reach to 52. 5%, 60. 8% and 73. 7%. The lethal effect of paclitaxel chemotherapy drugs on tumor cells have increased by 43. 4%~45. 8% after being transferred. Results of apoptotic experiments were also in line with the CCK8 assay (Figure 6C). Besides PTX, another chemotherapeutic Doxorubicin (DOX) was used too. 82.9% cells inhibition can be reached at 10 µg/ml DOX concentration after MDR1 gene down-regulating, while only 37.4% without transfection (Figure 3F). All of those demonstrated a significant reversal of SKOV-3 cells multidrug resistance. [Figure 3 near here] Mechanisms of cellular uptake and endosomal escape Entering into cells and escaping from lysosomes are the prerequisites for any effectively delivery system (27-29). Either MFI aspect or Cy5-si positive cells we can conclude that LSCs not only significantly increased the uptake of siRNA in SKOV-3 compared to naked siRNA, but also reached the highest level at the incubation for 1h (Figure 4A, B). In order to explore the specific mode of ovarian cancer cells SKOV-3 uptake LSCs, a series of endocytosis inhibitors for various pathways were adopted here, chlorpromazine(CPZ) for clathrin mediated endocytosis (30), amiloride for Sodium-Proton (31), MβCD as a nonspecific inhibition of several internalization pathways by means of acute cholesterol depletion (32), and filipin as a selective inhibitors of the lipid raft/caveolae pathway (33, 34). The consequences illustrated that MβCD reduced SKOV-3 cells uptake dramatically and filipin showed slight inhibition (Figure 4C). Hence, besides lipid raft/caveolae pathway there are some other pathways that were responsible for SKOV-3 endocytosis since MβCD was also reported to block internalization of transferrin-mediated


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clathrin pathway and fluid-phase endocytosis in epithelial and endothelial cells besides the lipid raft/caveolae pathway (35-38). What’s more, 4℃ was set to detect whether the endocytosis process of LSCs is energy-dependent. The method of increasing the intracellular energy consumption by reducing the ambient temperature cut down ATP inside cellular which can be supplied for endocytosis indirectly. The result showed that the uptake of LSCs by SKOV-3 cells was an energy-dependent process. Based on the outcomes provided by FCM, CLSM, the more visual approach, was adopted to reconfirm the results of the two obvious elements (Figure 4D). We can see that the red fluorescence significantly decreased after cells stayed with MβCD or 4℃. CLSM was used to observe lysosomal escape ability of LSCs in SKOV-3 cells, 2 occations (3 h/6 h) were set after cells had been transfected. Exhibited in Figure 5A, the yellow region which stands for the colocalization of Cy5-siRNA (red) and endosomes/lysosomes (green) appeared when co-incubation for 3 h, and disappeared when time prolonged to 6 h, which suggested that LSCs can protect siRNA in lysosome and release into cytoplasm. Bafilomycin A1, an H+-ATPase inhibitor, which can inhibit endosomal acidification was applied to verify the role of protonation in lysosomal escape (Figure 5B). Yellow areas remarkably increased after the application of bafilomycin A1, which means that protonation takes some responsibility for lysosomal escape. Moreover, with the endosomal acidified, imidazolyl group in the histidine residue of LAH4-L1 become protonated. The structure of peptides transformed into disordered, causing the dissociation of complexes and LAH4-L1 release. While those released LAH4-L1 adopt an in-plane alignment in the lysosomal membrane resulting in membrane destabilization


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thus further promoting the rest nanocomplexes or siRNA to escape from endosomes (23, 39-41). Result of RT-PCR (Figure 5C) also assured the importance of lysosomal escape ability in gene knockdown. [Figure 4 near here] [Figure 5 near here] Effect of transfection on cells cycle Some treatments that induced cells apoptosis can destroy the cell cycle in some way (42). Compared with control group, cells transfected with LSCs showed an increase in the proportion of S phase (Figure 6D). So we can consider SKOV-3 cells were arrested in this stage. When combining LSCs with PTX, cells were blocked both at S and G2 phase. Affections on the ability of cells migration Migration is a special property of tumors, which raises arithematical facts of treatments and makes the tumor become incurable. Follow the results we can see that, those cells which were transfected with LSCs have lower capability of migration comparing with control group. The numbers of cells that have migrated in transwell migration assays reduced from 722 to 312, approximately 57% decreased (Figure 6A). This result is also confirmed in scratch experiment (Figure 6A, B). [Figure 6 near here]

CONCLUSION In summary, LSCs are a kind of round-like nanoparticle with low cytotoxicity and weakly positive charge on their surface. Its pH responsiveness can protect siRNA in blood and tumor microenvironment and release it inside tumor cytoplasm. The higher efficiency of MDR1 gene silence and P-gp down-regulating achieved by LSCs had reversed the multidrug resistance of


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ovarian cancer cell cline SKOV-3 which is from ascites in patients with metastasis. Cells inhibition increased from 7.1% to 52.5%, 17.4% to 60.8%, 27.9% to 73.7% when combined gene transfection with three different concentrations of PTX (3 µg/ml, 7.5 µg/ml, 15 µg/ml respectively) and from 37.4% to 82.9% combined with DOX, another chemotherapy drug at 10 µg/ml concentrations. What’s more, MDR1 gene down-regulation can reduce 50% migration ability of cells. And as a further study, experiments in vivo with animal models are also needed to confirm the gene silence efficiency of LSMCs delivery system in multidrug resistance ovarian cancer SKOV-3 cells in vitro. All in all, overcoming multidrug resistance of ovarian cancer SKOV-3 cells by using LSCs to silence MDR1 gene combined with chemotherapy is one of the most promising strategies.

FIGURES


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Figure 1. Schematic representation of LAH4-L1-siMDR1 nanocomplexes and their intracellular delivery processes for targeted MDR1 gene silence to overcome multidrug resistance (MDR) on SKOV-3 cells.


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Figure 2. Characterization of LSCs. A: Agarose gel retardation assay of LSCs at different LAH4-L1/siRNA weight ratios. B: pH-dependent siRNA released from LSCs (LAH4-L1/siRNA weight ratio is 3/1). C: Zeta potentials of complexes at different weight ratios. Results were expressed as mean ± standard deviation (n = 3). D: TEM of LSCs (LAH4-L1/siRNA weight ratio is 3/1). Scale bar is 200nm. E: Particle size of LSCs (LAH4-L1/siRNA weight ratio is 3/1) by DLS. F: cell viability measured by CCK8 assay after transfection. Results were expressed as mean ± standard deviation (n = 5).


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Figure 3. Down-regulation of MDR1. A: mRNA level of MDR1 expression (1: control, 2: siMDR1, 3-5: LSCs (wLAH4-L1/wsiRNA were 3/1, 5/1 and 7/1 respectively)) in SKOV-3 cells by RT-PCR; B: Semi-quantitative analysis of A based on normalization with internal control gene GAPDH by gray intensity analysis. C: P-gp down-regulation in LSCs treated cells detected by Western blotting assay (1: control, 2: siMDR1, 3: LSCs (wLAH4-L1/wsiRNA=3/1) and its quantification (D). E/F: CCK-8 assay for MDR reversal test. Cell growth inhibition induced by combination of LSCs with different concentrations PTX (E) or DOX (F). Error bars indicate s. d. (n = 5). **P < 0. 01, *P

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