Abstract

Subscriber access provided by UNIVERSITY OF TOLEDO LIBRARIES

Applications of Polymer, Composite, and Coating Materials

Overcoming multidrug resistance by co-delivering of MDR1 targeting siRNA and doxorubicin using EphA10-mediated pH-sensitive lipoplexes#In vitro and in vivo evaluation Jiulong Zhang, Zhouqi Du, Shuang Pan, Menghao Shi, Jie Li, Chunrong Yang, Haiyang Hu, Mingxi Qiao, Dawei Chen, and Xiuli Zhao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b01806 • Publication Date (Web): 25 May 2018 Downloaded from http://pubs.acs.org on May 25, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Overcoming multidrug resistance by co-delivering of MDR1 targeting siRNA and doxorubicin using EphA10-mediated pH-sensitive lipoplexes：：In vitro and in vivo evaluation Jiulong Zhanga, Zhouqi Dua, Shuang Pana, Menghao Shia, Jie Lib, Chunrong Yangc, Haiyang Hua, Mingxi Qiaoa, Dawei Chena, Xiuli Zhaoa* a

School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang, Liaoning,

PR China, 110016 b

Mudanjiang Medical University, Tongxiang Street No.3, Mudanjiang, Heilongjiang, PR China,

157011 c

College Pharmacy of Jiamusi University, 148 Xuefu Street, Jiamusi, Heilongjiang, PR China, 154007

Corresponding author: [email protected] (Zhao.X)

Keywords: gene therapy; multidrug resistance; lipoplexes; combination therapy, systematic evaluation; pH-sensitive

Abstract The therapeutic efficacy of chemotherapy is dramatically hindered by multidrug resistance (MDR), which is induced by overexpression of P-glycoprotein (P-gp). Co-delivery of antitumor drug and siRNA is an effective strategy recently applied in overcoming P-gp related MDR. In this study, a multifunctional drug delivery system with both pH-sensitive feature and active targetability was designed, in which MDR1-siRNA and DOX were successfully loaded. The resulting carrier Eph A10 antibody conjugated pH-sensitive doxrobicin (DOX), MDR1 siRNA co-loading lipoplexes (shorten as DOX+siRNA/ePL) with high serum stability had a favorable psychemical properity. DOX+siRNA/ePL exhibited incremental cellular uptake, enhanced P-gp downregulation efficacy as well as a better cell cytotoxicity in human breast cancer cell line/adriamycin drug resistance (MCF-7/ADR) cells. The results of intracellular co-localization study indicated DOX+siRNA/ePL possessed the ability of pH-responsive rapid endosome escape in a time-dependent characteristic. Meanwhile, in vivo antitumor activities suggested that DOX+siRNA/ePL could prolong circulation time as well as specifically accumulate in tumor cells via receptor-mediated endocytosis after intravenous administration into blood system. Histological study further demonstrated that DOX+siRNA/ePL could inhibit the proliferation, induce apoptosis effect and downregulate the P-gp expression in vivo. Altogether, DOX+siRNA/ePL was expected to be a suitable co-delivery system for overcoming MDR effect.

1

Introduction

Breast cancer, the most common malignant disease in females, is accountable for mortality and morbidity worldwide1-3. Although several advances have been achieved in the field of radiotherapy and surgery, chemotherapy still makes a critical difference in traditional treatment of cancer4-5. However, dose-limiting toxicity, occurrence of multidrug resistance (MDR) and non-specific distribution throughout the body dramatically limit its clinical application6. The presence of MDR, intensely impeded the process of anticancer drugs-based treatment, is the main reason for recurrence and failure of chemotherapeutics7-8 . Overexpression of the drug efflux transporters P-glycoprotein (P-gp) has been known to be associated with the occurrence of MDR9. Several strategies have been applied in overcoming MDR, including

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

functional chemical inhibitors10-12, immunotherapy13-14, and gene therapy15-16. Of all the listed approaches, RNA interference (RNAi) is a suitable strategy for its superior ability for knocking down the target gene17 . RNAi could induce the degradation of target messenger RNA (mRNA) and possess knockdown capability18-20. As a negatively charged macromolecule, however, siRNA suffers poor cellular uptake, rapid renal clearance as well as degradation under ribonuclease (RNase) in bloodstream after intravenous administration21-22. Therefore, it is necessary for researchers to find a promising carrier for the efficient delivery gene agent to tumor regions. Nowadays, many non-viral drug delivery systems have been applied to genes and/or drugs to tumor sites including liposomes, dendrimers, micelles, peptides, and inorganic nanoparticles. Due to high affinity to cell membrane and excellent biocompatibility, cationic liposomes have been extensively studied23-24. Furthermore, easy to be modified is another advantage for its clinical application. However, systematic toxicity arose from high positive charge and easily taken up by reticuloendothelial system (RES) become the major barrier for its in vitro and in vivo application25. Therefore, shielding the surface charge and increasing the in vivo circulation time is becoming one of the most important problem to be solved. Typically, the attachment of polyethylene glycol (PEG) is employed to shield positive charge of the formulation. Meanwhile nonspecific interactions between liposomes and serum proteins could be reduced. Last but not least, PEGylated liposomes could significant avoid the recognition of RES and increase its in vivo circulation time in blood system26-28. However, after reaching target tissue, PEG chain becomes the major obstacle for the treatment of cancer, there would be a weak repulsion between the cell membrane and the liposomes. This phenomenon would result in poor intracellular accumulation and cytotoxicity against tumor cells. On the one hand, the existence of PEG shell would significant decrease the release rate of antitumor drug from the liposomes to the tumor environment. Therefore, considerable efforts should be devoted to surmount this handicap. On the other hand, unsatisfactory target delivery efficiency is another puzzle cationic liposomes faced. As is mentioned above, PEGylated liposomes would form a weak hydrophilic shell and decrease its affinity between liposomes and cell membrane. Although these liposomes could target tumor regions via enhanced permeability and rention (EPR) effect, intracellular drug concentration still remains in a low value29. To overcome this drawback, a variety of ligands have been explored for targeting delivery via receptor-mediated endocytosis pathway, such as monoclonal antibodies30-31, , peptides32-34, and some small molecular35-36 . It has been reported that Eph receptor A10 is a unique breast cancer marker37-38. Anti-EphA10 plays an important part in tumor progression and metastasis. Furthermore, compared with normal cells, EphA10 is highly expressed in breast cancer cell39. Based on these characteristics, we hypothesized that EphA10 antibody modified liposomes could bind breast cancer cells and reach its active targetability. In our previous study, we have constructed a multifunctional drug delivery system (named as EPSLR) for intracellular delivery of siRNA with Schiff based bond mediated pH-responsive characteristic, EphA 10 antibody mediated active targetability and endo/lysosomal escape capability3. EPSLR owned small particle size with narrow size distribution. Meanwhile, EPSLR showed an excellent serum stability, minor cell cytotoxicity and high transfection efficiency. In vivo biodistribution assay also indicated that EPSLR could specifically accumulate into tumor site. However, whether it owes excellent in vitro/vivo antitumor activity has not been investigated. Therefore in this paper, we used this drug delivery system for co-delivering MDR1 siRNA and chemotherapeutical drug doxorubicin (DOX), which named DOX+siRNA/ePL. As is illustrated in Scheme 1, antitumor agent DOX was incorporated into the phospholipid bilayer of the liposomes and positive charge of the carrier could bind siRNA via electrostatic


Page 2 of 23

Page 3 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


interaction. PEG was used to shield the positive charge and increase its stability. When DOX+siRNA/ePL was administrated into the body of mice, they could accumulate in tumor regions via EPR effect and sufficient be internalized into tumor cells via active targetability. Afterwards, in endo/lysosome with even lower pH, the removal of PEG could facilitate endosome escape and released siRNA to cytoplasm in the degradation of target mRNA and following P-gp down regulation. Meanwhile, DOX efflux would be significant decreased and intracellular DOX concentration would be increased. This synergistic effect would facilitate its therapeutical effect and overcome MDR effect. Physicochemical properties involving size, zeta potential, morphology, and siRNA-loading capacity were characterized. Cell cytotoxicity, cellular uptake, P-gp silencing capability, in vivo antitumor activity was also investigated in this paper to evaluate its antitumor efficiency.

2

Method and materials

2.1

Materials

Doxorubicin hydrochloride (DOX·HCl) was supplied by Beijing Huafeng Co. Ltd. (China).Soybean phospholipid (SPC) was obtained from Shanghai Taiwei Phamacy Co.Ltd (China). 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioleoyl Phosphoethanolamine (DOPE) and cholesterol (CHOL) were obtained from A.V.T Pharmaceutical Co,. Ltd. (Shanghai, China). DiR, (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and trypsin, were purchased from Sigma-Aldrich. Hoechst 33258 and Lyso-Tracker Red were supplied by Shanghai Beyotime Biotechnology Co,. Ltd. (Suzhou, China). The siRNA used in this study was custom synthesized and purified with HPLC by Shanghai GenePharma Co. Ltd (China), including NC-siRNA, MDR1-siRNA and fluorescence labeled FAM-siRNA. The sequence of MDR1-siRNAsense was as follows: (antisence strand of 5’-CAC CCA GGC AAU GAU GUA UTT-3’ and 5’-AUA CAU CAU UGC CUG GGU GTT-3’). Ki-67 primary antibody were supplied by Abcam (England). P-gp primary antibody (ab103477) was purchased from Abcam (England). FITC-labelled goat-anti rabbit IgG, immunofluorescence staining kit with Cy3-labeled goat anti-rabbit IgG and hematoxylin–eosin (H&E) staining kit were all bought from Shanghai Beyotime Biotechnology (China). DAB Kit, biotin-streptavidin HRP detection Kit were supplied from Beijing Zhongshan Goldenbridge (China). Ultrasensitive streptavidin-peroxidase immunohistochemistry Kit was obtained from Jiangsu Maixin biotechnology (China).TUNEL kit was bought from Roche. Other chemical reagents used in this study were analytical grade and supplied from Concord Technology Co.Ltd. (Tianjin, China). 2.2

Cell line and culture

DOX-resistant human breast cancer line MCF-7/ADR were supplied by Nanjing keyGen Biotech Co. Ltd (China) and cultured using RPMI-1640 medium (Gibco, NY, USA) with 20% fetal bovine serum (FBS, Gibco, NY, USA), 100 units mL-1 penicillin, 100 µg mL-1 streptomycin at 37℃ in humidified atmosphere containing 5% CO2. To maintain their MDR property, culture medium contain 1 µg mL-1 DOX. The cells in exponential growth phase were used in the following experiments. For transfection of siRNA, formulations at the final concentration of siRNA of 100nM were dispersed in RPMI-1640 medium without FBS, penicillin, and streptomycin. 2.3

Synthesis of COOH-PEG-Schiff base-cholesterol derivant (PSC)

The synthesis pathway of PSC has been described in the previous report and the detail operation could be seen in the reference

29

.For the synthesis of PSC, HOOC–PEG2000–COOH and Chol–Schiff base–NH2 was reacted in

DCM with the molar ratio was 1.5:1. HATU, DIPEA and EDCI were added in the mixture and stirred at room temperature for 48h to allow react. The organic solvent was removed, by-product was removed through washing with distilled water. The residue was the purified and PSC was obtained.



2.4

Page 4 of 23

Preparation of different drug-loaded liposomes

2.4.1 Preparation of DOX base DOX·HCl (50 mg 0.085 mmol) was dissolved in 4 mL methanol/acetone (1:1, v/v, 4 mL) mixed solution and 25 µL triethylamine was added. The mixture was allowed to react for 12 h and solvent was removed using rotary evaporation.DOX base was obtained after lypophilization. 2.4.2 Preparation of DOX-loaded liposomes Different liposomes were prepared using simple thin-film hydration method according to our previous report with minor modification3. Briefly, DOX, SPC, DOPE, DOTAP, cholesterol and PSC (weight ratio of 2:2:2:4:2:1.5) were dissolved in 5 mL of DCM. The organic solvent was removed and thin film was obtained. The thin film was hydrated and resultant suspension was sonicated for 4 minutes with a probe-type sonicator (Scientz-1200E, Ningbo, China) and filtered. DOX-loaded liposomes were obtained (named DOX/PL). Non-PEGylated liposomes were prepared using the same method (named as DOX/L). 2.5

Preparation of DOX+siRNA/ePL lipoplexes

The attachment of anti-EphA10 antibody on DOX/PL was prepared as described previously3. In brief, EDCI and sulfo-NHS was dissolved in PBS (pH 7.4). DOX/PL was added to react for 2 h. 200 µL antibody solution was mixed and stirred over 4oC overnight. Excess antibody was removed using Sepharose 4B column and DOX/ePL was obtained. For the DOX+siRNA/ePL lipoplexes, DOX/ePL and siRNA were gently mixed and incubated at room temperature. DOX+siRNA/PL and DOX+siRNA/L were prepared using the same procedure except DOX/ePL was replaced by DOX/PL and DOX/L, respectively. 2.6

Characterization of liposomes and lipoplexes

2.6.1 Determination of encapsulation efficiency (EE %) of DOX and siRNA DOX was determined using a UV-Vis spectrophotometer at 481 nm and encapsulation efficiency of DOX was determinated using cation exchange resin-mini column centrifugation method. The EE % of different drug-loaded liposomes were determinated using this formula below:

(1) The amount of siRNA adsorbed onto the surface of liposomes was determined by ultra-filtrating method using Amicon® Ultra-4 centrifugal filter devices. FAM-siRNA loaded liposomes were added into filter devices, after thorough ultra-filtration with a centrifugal speed of 4000 rpm for 10 min, free FAM-siRNA solution was collected and quantified using a standard curve (data were not shown). The fluorescence intensity of FAM-siRNA was measured and EE % of siRNA was calculated as the formula below:

EE%=

(2)

100

Ct and Cu were defined as the concentration of total and unloaded siRNA, respectively. 2.7

Particle size and zeta potential measurement


Page 5 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Dynamic light scattering instrument (Zetasizer Nano ZS) was used to measure the particle size and zeta-potential of different formulations. Prior to measurement, the samples were diluted as least 10 fold with deionized water. The analysis was performed at 25℃ and done in triplicate. 2.7.1 Transmission electron microscope (TEM) study The morphology of different formulations were observed by TEM (JEM-1230, Japan) and the detail procedure of operation procedure could be seen in our previous report40. 2.8

Agarose gel electrophoresis analysis

Gel electrophoresis was performed in 2% (w/w) agarose gel (in the presence of ethidium bromide in gel) with a current of 100 V for 20 min in Tris-Borate-EDTA buffer. Lipoplexes with different N/P ratios were simultaneously electrophoresed in the agarose gel, naked siRNA was set as negative control. The retardation of siRNA was visualized and pictures were taken under UV light using Tanon 2500R automatic digital gel image analysis system. For heparin decomplexation assay, heparin were added into lipoplexes (fixed at N/P ratio of 10) at different weight ratios (heparin/siRNA, IU/µg) to allow for siRNA desorption, followed by vortex and then incubation for 15 min. Then all the samples were analyzed using electrophoresis. For serum stability assay, after adding serum at a 50% concentration, lipoplexes (fixed at N/P ratio of 10) were incubated at 37℃ and 10 µL sample was taken at different time point. siRNA was incubated with excess heparin for 15 min and analyzed using 2% agarose gel. 2.9

In vitro release of siRNA and DOX from lipoplexes

Dialysis method was used to investigate in vitro DOX release profile. Briefly, different lipoplexes were added into a dialysis bag and put into a conical flask with 100 mL PBS solution containing 0.5% Tween 80 with different pH (7.4 and 5.0). The flasks was then put into shaking incubator (stirring speed: 100 rpm in 37℃).At scheduled time point, sample was taken out and equal medium was added. The amount of DOX in the medium was measured and analyzed. siRNA release from different carriers at different pH was measured according to the reference previously41. Released siRNA from the carrier was detected with ES-2 spectrophotometer. 2.10 Internalization of lipoplexes Internalization of siRNA was studied by quantitative and qualitative analysis with fluorescence-labeled FAM-siRNA. For quantitative analysis. Cells were seeded in 6-well plate at a density of 5×105 cells per well overnight and incubated with different FAM-siRNA loaded preparations for 4 h. The cells rinsed twice with pre-cooled PBS and collected after trypsinization, subsequently suspended in 0.5 mL pre-cooled PBS. Then supernatant was abandoned after centrifugation and re-suspended in 0.5 mL PBS. The samples were finally analyzed using BD flow cytometer. For qualitative assay, cells were added on coverslips placed in 6-well plate at a density of 2×105 cells per well overnight and then treated as described above. After incubation period, medium was removed and rinsed the cells with pre-cooled PBS. The sample was then fixed for 15 min using 4% paraformaldehyde at RT, followed by staining cell nucleus with Hoechst 33258 (10 µg mL-1). Afterwards, the coverslips were mounted on microscope slides, then the distribution of FAM-siRNA in cells was observed and imaged using laser scanning confocal microscope. 2.11 P-gp silencing efficiency The P-gp silencing effect of different MDR1-siRNA-loaded formulations was investigated by Western blotting



Page 6 of 23

method. Briefly, cells were seeded in 6-well plate and different formulations were added to allow transfection. Then the cells were washed using PBS and solubilized with lysis buffer. The protein was obtained and determinate the concentration. The protein were separated using SDS-PAGE and transferred to PVDF membrane. The membrane was blocked, incubated with primary antibody, secondary antibody and finally visualized using ECL. 2.12 In vitro cytotoxicity assays Cell cytotoxicity of different formulations were determinated using MTT assay. Briefly, cells were seeded in 96-plate well with 5×103 cells per well and incubated overnight to allow attachment. Then different formulations with were added and incubated. After incubation, 20 µL MTT solution (5 mg mL-1) were added and further incubated for 4 h. The supernatants were then removed and 100µL DMSO were added and measured. 2.13 Intracellular trafficking of lipoplexes The intracellular localization of DOX+siRNA/ePL was investigated by LSCM with the green fluorescence property of FAM-siRNA. Cells were seeded on coverslips placed in 6-well plate and then incubated with DOX+siRNA/ePL at predetermined time intervals. Chloroquine (8 µg mL-1) was added and incubated for 1 h before the addition of DOX+siRNA/ePL to stimulate the environment of endo/lysosome. Afterwards, the cells were fixed with 4% paraformaldehyde, stained with Lyso Tracker Red and Hochest 33258, respectively. Finally, the cells were observed using LSCM. 2.14 Animal study 2.14.1

Animal tumor xenograft models

Female nude mice were bought from Shenyang Changsheng biotechnology Co.Ltd. All the animal experiments were carried out according to the Experimental Animal Administrative Committee of Shenyang Pharmaceutical University. Female nude mice (body weight of 18~20 g) were injected MCF-7/ADR cell suspension (approximately 1×107 cells per 200 µL PBS) into the left backs of mice under anesthesia. When the tumor grew to approximately 100 mm3, the mice were separated into several groups for further study. 2.14.2

In vivo antitumor efficacy

Tumor bearing mice were divided into several groups with different treatment by injecting 0.2 mL different formulations (5 mg kg-1 of DOX). Tumor size and body weight were measured every 2 days and calculate the tumor volume as follows: V=a×b2/2

(3)

a: biggest diameter; b: shortest diameter of the tumor At the end of the trail, mice were killed and tumor was obtained to calculate the inhibition rate (IR %) using this formula below: IR%=

(4)

100

Wt: tumor weight of drug-treated group; Wn: tumor weight of control group 2.15 Histological study Tumor tissue were obtained from 2.14 and fixed, embedded and cut into 5 µm slides. The slides were treated using H&E staining kit, Ki-67 immunohistochemistry, TUNEL and P-gp immunofluorescence according to the kits. 2.16 In vivo toxicity


Page 7 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Healthy BALB/c mice (18±2 g) were divided several groups and used to evaluate the in vivo toxicity of formulations. Then different preparations were administrated (5mg kg-1 of DOX) for every 2 days. The administration would remain for 4 times and animal behavior were assessed. Body weight of the mice would be calculated during this period. Finally, organs of mice was obtained and stained using H&E.

3

Result and discussion

3.1

Physicochemical characteristics of liposomes and lipoplexes

In this paper, we constructed a multifunctional liposomes-based drug delivery system for co-delivery of chemotherapeutics agent DOX and MDR1-siRNA. As was illustrated in Table.1, EE % of different DOX-loaded liposomes were all above 60 %, demonstrating DOX was successfully inserted into the phospholipid bilayer of the liposomes. All the liposomes showed the relative low polydispersity index (PDI), suggesting the liposomes were uniform. The zeta-potential of different liposomes were all ~30 mV, indicating there were a large amount of positive charge presented on the liposomes. This characteristic could be used to further bind siRNA via electrostatic interaction on the surface of the carrier to prepare lipoplexes. DOX-loaded liposomes and siRNA were co-incubating to prepare different lipoplex formulations with varied N/P ratio. The EE % of different lipoplexes have been optimized and the data has been shown in Fig.1A. All lipoplexes were able to bind no less than 70% siRNA according to the result of EE % determination, indicating all the cationic liposomes have a good performance in siRNA encapsulation. The EE % increased as N/P ratio increased, presumably due to stronger binding capability with siRNA as the addition of more positively charged amine groups. It was also found that EE % of DOX+siRNA/PL and DOX+siRNA/ePL at the same weight ratio was slightly less than that of DOX+siRNA/L. It is supposed that PEG shell showed a significant influence on surface charge of liposomes, subsequently the electrostatic interaction of liposomes and siRNA may be interfered to some extent, resulting in a reduced siRNA binding capacity. For in vivo delivery of siRNA, the small size of lipoplexes ensured penetration into tumor cells from the blood stream by EPR effect instead of renal clearance from the body. Particle size of the lipoplexes is a major factor for the efficient delivery drug/gene to tumor site. Therefore, the optimization of particle size was investigated with different N/P ratio in this study. As shown in Fig.1B, particle size of DOX+siRNA/L decreased significantly from 227.1 nm to 129.3 nm as N/P ratio varied from 3 to 15 and the optimized particle size of 118.6 nm was achieved at ratio of 10, both indicating siRNA adsorption exerted profound effect on particle size of liposomes. Meanwhile, zeta-potential of DOX+siRNA/L dramatically declined from 30.6 mV to 10.5 mV when N/P ratio was up to 3, demonstrating successful loading of siRNA through electrostatic interaction. As was predicted, an increase N/P ratio induced the augment of zeta-potential, due to the presence of relatively more positive charge provided by DOTAP. Taking all these results into consideration, lipoplexes with 10 N/P ratio was prepared for further investigation. As shown in Table.2, the average diameter of all lipoplexes was about 120 nm with a narrow polydispersity index (PDI). Meanwhile, the EE % of siRNA was suitable for further investigation. Generally speaking, strong zeta potential would increase non-specific interaction of nanocarrier to cells through electrostatic interactions. However, excessive density of cation would directly contact with cell membrane, which would probably cause systematic cytotoxicity. Zeta-potential of all lipoplexes were below ~20 mV, indicating these preparations were safe for intravenous administration.



TEM was employed for determinating the morphology of formulations. As shown in Fig.1 C and D, lipoplexes both exhibited a spherical shaped particle with phospholipid bilayer. Additionally, particle sizes of TEM were in consistent with the DLS data, indicating that particle size of lipoplexes are larger than that of liposome due to siRNA adsorption. 3.2

Agarose gel electrophoresis

As cationic carriers can deprive the motility of siRNA in the electric field, gel retardation assay was used to further investigate the siRNA binding ability of lipoplexes. As was illustrated in Fig.2 A, there were no siRNA stripe visualized as the N/P ratio exceeded 1 for DOX+siRNA/L group, indicating full neutralization of liposomes with siRNA. For DOX+siRNA/PL and DOX+siRNA/ePL groups, complete retardation of siRNA could be observed when N/P ratio was up to 3. This might be ascribed to positive charge shielding of PEG outer shell, which weaken the binding capacity of liposomes with siRNA and more positive charge was required for sufficient electrostatic interaction. Obviously, all liposomes with higher N/P ratio exhibited stronger binding ability, which was similar with results of encapsulation efficiency of siRNA. The presence of polyanionic in human body may compete with siRNA for adsorption with liposomes, resulting in desorption of siRNA Two more electrophoresis analysis were conducted to investigate the capacity of liposomes to prevent siRNA from decomplexation and degradation. In heparin decomplexation assay, all siRNA was released from lipoplexes when heparin was added with heparin/siRNA weight ratio of 4 (Fig. 2 B), indicating all lipoplexes have a good resistance to the polyanionic and excellent stability in the blood system Due to rapid degradation of siRNA in bloodstream, a suitable gene delivery system should owe the capability to protect the carrier from decomposing in serum environment. Therefore, serum stability assay was carried out in this study. As shown in Fig. 2 C, when incubated at 37℃with 50% FBS, naked siRNA suffered pronounced degradation within 2 h and almost degraded thoroughly at 4 h. In contrast, formulation groups could survive for 12 h when siRNA was loaded with liposomes and even for 24 h in DOX+siRNA/PL and DOX+siRNA/ePL. These results demonstrated that all lipoplexes could effectively protect siRNA from degradation of serum environment. Overall, all above results indicated that all lipoplexes with proper size and zeta-potential demonstrated tremendous siRNA binding capacity, which laid the foundation for further application. 3.3

In vitro drug/gene release study

It could observed that siRNA could bind with liposomes and the lipoplexes owes high stability in blood system. However, in order to exert the antitumor activity, drug/gene must release from the carrier. It is well known the pH of normal tissue regions (pH 7.4) was different from tumor area (pH 5.0~6.8). Therefore, pH-sensitive carrier could increase release into tumor site and decrease its non-specific toxicity. In this study, pH-responsive drug/gene release were both investigated at pH 7.4 and 5.0 at 37oC, respectively. Fig.3A and 3B illustrated that all the lipoplexes showed a significant pH-responsive release behavior. As shown in Fig.3A, the accumulative siRNA release profiles were below 30 % for all the lipoplexes groups in pH 7.4, indicating all the lipoplexes were relative stable in normal environment. It was noted that the accumulative siRNA release profile of DOX+siRNA/L group was a little higher than both PEGylated lipoplexes groups. This phenomenon demonstrated that PEG modified liposomes could facilitate the stability of the lipoplexes and shield the drug release from the carrier. In comparison, fast siRNA release (about ~80 % of siRNA release) could be obviously observed in acidic pH for all lipoplexes groups. Interestingly, the accumulative siRNA release profile of DOX+siRNA/L was a slight higher than both PEGylated lipoplexes in the first 4 h, which was attributed to the leakage of PEG shell would hinder the siRNA release from the carrier. In order to investigate whether DOX and


Page 8 of 23

Page 9 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


siRNA could release into the medium at the same time. DOX release from lipoplexes was also investigated and the result has been shown in Fig.3B. Hopefully, similar release behavior could also be observed at different pH value. It seems that both PEGylated lipoplexes showed a slight lower release profiles at pH 5.0 in the first 24 h due to the existence of pH-sensitive PEG shell compared with DOX+siRNA/L group. Taking all these results into consideration, the lipoplexes were stable in circulation, while it would possess rapid release behavior in acidic tumor microenvironment. These characteristics could decrease the systematic toxicity arose from DOX but showed a high drug accumulation into cytoplasm of tumor cells due to its pH-sensitive property and facilitate its antitumor activity. 3.4

Internalization of lipoplexes

From the result above we could find DOX+siRNA/ePL was a suitable carrier for the efficient co-delivering antitumor drug DOX and siRNA. However, only when the lipoplexes were internalized into tumor cells can the therapeutical agent exert its antitumor activity. Therefore, cellular uptake of different lipoplexes were investigated using laser confocal microscopy (LSCM). It is noted that MDR1-siRNA was replaced by FAM-siRNA and used it to prepare different DOX-free lipoplexes. As shown in Fig.4A, green and blue fluorescence represents siRNA and nucleus, respectively. There was nearly no green fluorescence could be observed for naked siRNA group. This was attributed to its high hydrophilic characteristic. This feature reduce the interaction between siRNA and hydrophobic cell membrane, which resulted in the insufficient internalization. In comparison, siRNA/L group showed a significant higher green fluorescence intensity. Two reasons could perfectly explain this phenomenon: (1) high affinity between lipoplexes and cell membrane; (2) relative higher surface charge of lipoplexes would increase the interaction between the carrier and cell membrane. However, high positive surface charge would arise no-specific toxicity. In order to solve this problem, PEGylated lipoplexes were prepared. There was a slightly weaker fluorescence intensity could be observed for siRNA/PL group, which might due to the existence of repulsion between PEG shell and cell membrane, and this phenomenon would result in a lower internalization efficiency. In order to overcome this drawback, EphA 10 antibody was modified on lipoplexes to achieve active targetability. siRNA/ePL group possessed the strongest green fluorescence, which was because EphA 10-mediated endocytosis. Flow cytometry was used to quantitatively evaluate the internalization capability of different lipoplexes, as well. As shown in Fig.4.B and C, no significant difference between control group and naked siRNA group could be observed, indicating only limited siRNA was internalized into tumor cells. In contrast, the mean fluorescence intensity (MFI) of siRNA/L group (MFI was 206.2) was significant higher than siRNA group, suggesting lipoplexes based drug delivery system could efficiently delivery therapeutical agent to tumor cells. The MFI of siRNA/PL was 0.70-fold lower than siRNA/L group, indicating PEG shell could hinder the cellular uptake. However, MFI of siRNA/ePL was the highest among all reference groups via receptor-mediated endocytosis. All these data was in the same trend with LSCM assay. 3.5

P-gp silencing efficiency

In cellular uptake assay we could find all lipoplexes could deliver siRNA to cytoplasm in different degree. In order to investigate whether siRNA could downregulate the expression of P-gp. Western blotting assay was used in this assay. We used Lipfectamine 2000 as a positive control to transfer siRNA. As shown in Fig. 4.D, control and blank group possessed significant dark strip, suggesting there was no significant influence for carrier itself in expression of P-gp. No obvious downregulation of P-gp could be found for MDR1 group, indicating only a minor siRNA was internalized into tumor cells. In comparison, significant downregulation of P-gp could be observed for siRNA/L group, indicating siRNA was efficient delivered into tumor cells and transfected, which resulted in a



downregulation of P-gp. For siRNA/PL group, the knockdown efficiency was slightly lower than siRNA/L, suggesting PEG domain have an influence on cellular uptake. siRNA/ePL possessed the best transfection efficiency among all the groups due to the existence of Eph A10. 3.6

MTT assay

Based on the results above, these lipoplexes was confirmed to have an excellent capability for delivering siRNA and DOX to tumor cells. Therefore, we measured the cell cytotoxicity of different lipoplexes in this section with different cell lines. As illustrated in Table.3, IC50 of 24 h was significant higher than IC50 in 48 h for all groups against both sensitive and multidrug resistance cell lines, respectively, indicating the cytotoxicity was time-dependent. In detail, IC50 in 24 h of free DOX was 22.5 µg mL-1 for MCF-7 cell lines. However, for MCF-7/ADR cell lines, IC50 increased to about 100 µg mL-1, indicating the cells showed a good resistance against DOX. There was almost no difference between DOX and DOX+siRNA group, which was probably because only a minor amount of siRNA was internalized into the tumor cells, which resulted in insufficient overcoming MDR effect. However, significant decrease in IC50 of DOX+siRNA /L group could be observed in both cell lines, which was 0.71-fold and 0.50-fold lower than DOX+siRNA group for sensitive and multidrug resistance cell lines, respectively. For sensitive cell lines, the decrease of IC50 was mainly attributed that lipoplexes based drug delivery system could increase cellular uptake of antitumor agent into cytoplasm. For MCF-7/ADR cell lines, MDR1 siRNA was efficient internalized into cytoplasm and expressed in tumor cells, which resulted in a downregulation of P-gp. This phenomenon could decrease the efflux of DOX and increase DOX accumulation into cytoplasm. There was a slight increase of IC50 for DOX+siRNA/PL group in both cell lines, indicating PEG shell would hinder the cellular uptake, which resulted in a lower cytotoxicity. Hopefully, DOX+siRNA/ePL showed the highest cytotoxicity against both cell lines. It is noted that IC50 of 48 h was in the same trend with IC50 in 24 h for different groups. All the cell cytotoxicity study indicated DOX+siRNA/ePL could efficiently deliver antitumor drug DOX and MDR1-siRNA into cytoplasm to exert its synergistic antitumor effect. 3.7

Intracellular co-localization of lipoplexes

Intracellular co-localization was carried out using laser confocal microscopy (LSCM) to evaluate whether the acidic environment of endo/lysosomal could trigger rapid siRNA release from lipoplexes. Chloroquine was used to regulate the pH value of acidic organelles. FAM-siRNA was used to bind on the surface of DOX-free liposomes to prepare siRNA/ePL. As shown in Fig.5, whether in the presence or in the absence of chloroquine, green fluorescence always could be observed, which implied the presence of chloroquine have no influence to cells. At 0.5h, most siRNA was accumulate into endo/lysosomal when Cells without incubation of chloroquine, as characterized by the appearance of yellow fluorescence (colocalization of green and red fluorescence, indicated siRNA and endo/lysosomes, respectively). However, the yellow fluorescence gradually disappeared, suggesting most siRNA entered into cytoplasm. These results was mainly attributed to two reasons: (1) pH-sensitive PEG derivate would remove from the lipoplexes in acidic environment of endo/lysosomes and expose liposomes in the endo/lysosome; (2): the introduction of DOPE in the formulation of the lipoplexes would increase the endosome escape and deliver siRNA into cytoplasm. Interestingly, green signal increased gradually with time increasing, demonstrating the lipoplexes could be internalized via receptor-mediated active targetability. In contrast, in the presence of chloroquine, yellow fluorescence appeared in the 0.5h and didn’t disappear with an increase of time, indicating this carrier possess owes favorable pH-sensitive characteristics. This was in the similar trend with in vitro release assay. All these results indicated that the lipoplexes could maintain the structure in normal environment with the rapid release profiles and endosome escape characteristic in the acidic microenvironment. 3.8

In vivo antitumor activity


Page 10 of 23

Page 11 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


In vitro results indicated DOX+siRNA/ePL possessed a satisfied cell cytotoxicity and overcome MDR effect against MCF-7 cells. However, an unsatisfied result could be observed: PEGylated lipoplexes showed less cytotoxicity against tumor cells. It seemed that PEG would influence the efficient antitumor agent delivery. In fact, PEGylated liposomes is necessary for the in vivo antitumor treatment. Thus, we observed the in vivo therapeutical efficacy of different formulations. Fig.6A and B showed that DOX group possessed a moderate antitumor efficacy with the IR % was 8.32 %, compared with control group. This was probably because the noon-specific distribution of DOX and poor internalization of DOX against MDR tumor cells. DOX+siRNA group showed the similar antitumor activity compared with free DOX. This phenomenon was probably because naked siRNA suffers rapid renal clearance as well as degradation under ribonuclease (RNase) presented in bloodstream after intravenous administration, which resulted in poor knockdown capability. However, DOX+siRNA/L group showed a relative higher antitumor activity (IR % was 35.8%), which was probably because the liposome based drug delivery system could efficient deliver therapeutical agent to tumor tissue. When the DOX and siRNA was internalized into cytoplasm, MDR1-siRNA showed knockdown capability for P-gp and decreased the efflux of DOX arose from P-gp, which resulted in a higher accumulation of DOX and exert excellent antitumor activity.

However, rapid

clearance in bloodsteam is the major barrier need to overcome for this lipoplexes. To overcome this drawback, PEGylated lipoplexes (DOX+siRNA/PL) was prepared. Significant antitumor activity could be observed for this group with the IR % was 50.13 %. This result was mainly resulted in the existence of PEG. PEGylated liposomes could facilitate the in vivo circulation time of the lipoplexes in blood environment, which resulted in higher antitumor activity. As we mentioned above, PEG shell could shield cellular uptake in some degree and EphA 10 antibody modified lipoplexes were prepared to reach active targetability. Hopefully, DOX+siRNA/ePL group possessed the highest therapeutical effect and this was probably because the synergistic effect arose from EphA10-mediated specific recognition, enhanced circulation time from PEG, P-gp knockdown capability of MDR1-siRNA and high accumulation of DOX into cytoplasm. In order to evaluate whether these preparations showed a significant toxicity against mice model. Body weight of the mice were also weighted. As shown in Fig. 6.C, both DOX group and DOX+siRNA group showed a significant decrease in body weight, indicating free DOX do exist non-specific distribution and arise systematic toxicity. However, no obvious weight change could be observed for other lipoplexes groups, suggesting this DDS possessed favorable safety. 3.9

Histological study

From the above results we could find DOX+siRNA/ePL lipoplexes could efficient inhibit the growth of tumor and overcome MDR effect. Histological study was also carried out to evaluate the therapeutical effect in histological level. Tumor tissues were obtained from the mice after different treatment. As shown in Fig.7, H&E staining assay was carried out firstly. Small voids in tumor section means the absence of dead tumor cells. It could be observed that DOX+siRNA/ePL owes more voids compared with other groups, demonstrating DOX+siRNA/ePL could efficient deliver DOX and siRNA to tumor site to overcome MDR effect and reach higher antitumor efficiency. The anti-proliferation effect was investigated by Ki-67 immunohistochemistry (IHC). As shown in Fig.7, There are many brown pixel dots for control group, indicating the tumor was in a good proliferation level. In comparison, drug-treated groups possessed lower Ki-67 expression, demonstrating DOX could inhibit the proliferation level against tumor regions. DOX+siRNA/ePL showed the lowest expression of Ki-67, indicating these lipoplexes could efficiently delivery drug and nucleic acid to tumor sites. The synergistic effect could inhibit tumor proliferation to prevent the growth of tumor. Although DOX+siRNA/ePL could efficiently inhibit the tumor proliferation, we are also interested to whether the



lipoplexes owes apoptosis inducing effect. Therefore, TUNEL assay was carried out in histological level. As shown in Fig. 8, almost no apoptosis cells (green fluorescence) appeared in control group, indicating the tumor tissues were kept in a high proliferation status. Among all the groups, DOX+siRNA/ePL possessed highest apoptosis inducing effect, which was probably because DOX+siRNA/ePL could efficiently intracellular deliver therapeutical agents via receptor-mediated endocytosis. MDR1 siRNA could downregulate P-gp expression to enhance the accumulation of DOX into cytoplasm and reach higher therapeutical effect. From the above mentioned study we have demonstrated that our lipoplexes could downregulate P-gp expression in cellular level. Whether lipoplexes could also efficiently knockdown the P-gp expression in vivo has not been investigated. Therefore, in vivo P-gp expression was carried out with immunofluorescence (IF) assay. As shown in Fig. 9, red fluorescence and blue fluorescence indicated the P-gp and nucleus, respectively. Control group possessed high P-gp expression level, indicating tumor tissues owe a good resistance. No significant difference between DOX group and DOX+siRNA group could be found, indicating naked siRNA was difficult to be internalized into tumor cells when it was administrated in blood system. Different lipoplexes groups showed a significant decrease of P-gp expression in different degree, demonstrating lipoplexes could efficiently deliver siRNA into cytoplasm. Hopefully, DOX+siRNA/ePL showed the highest knockdown capability against P-gp among all the reference groups. This result was in alignment with previous histological assay and in vivo assay, further demonstrating DOX+siRNA/ePL could efficiently co-delivering DOX and MDR1 siRNA to cancerous cells and overcome MDR effect. 3.10 In vivo toxicity It has been reported that cancer chemotherapeutical would result in toxicity to normal tissue. Therefore, it is necessary to evaluate the toxicity of formulations using healthy mice. Change of body weight and clinical signs associated with toxicity were both recorded during the experiment period. Meanwhile, major organs were obtained from the mice after treatment of lipoplexes and examined with H&E. Fig. 10 A depicted the body weight, no significant body weight change occurred for different lipoplexes. As illustrated in Fig.10 B, no obvious histological change could be found, as well. Meanwhile, no signs of systematic toxicity like locomotor impairment, anorexia, dehydration could be observed during this period. All these results indicated this carrier was a suitable drug delivery system for the treatment of cancer.

4

Conclusion

In this paper, a multifunctional drug delivery system was constructed with pH-sensitive and EphA 10 antibody mediated active targetability for the efficiently co-delivering antitumor drug DOX and MDR1-siRNA to overcome MDR effect. The lipoplexes could efficiently load both DOX and MDR1-siRNA. The resulting lipoplexes could form a stable nanosized particles and protect siRNA from degradation. DOX+siRNA/ePL showed a strong ability for co-deliver two therapeutical agents into cytoplasm in order to escape the degradation from endosomes. Gene silencing study further demonstrated that DOX+siRNA/ePL owes the capability for downregulating P-gp expression. In vitro and in vivo studies all demonstrated DOX+siRNA/ePL was a suitable carrier for overcoming MDR effect.

5

Acknowledgements

The authors are grateful for the financial support of the National Natural Science Foundation of China (81202483, 81773668 and 81302721), Liaoning Natural Science Foundation for Excellent Talents in university (LR 2015020543), Science and Technology project of Shenyang (F15-139-9-06, F15-199-1-24).


Page 12 of 23

Page 13 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


6

Conflicts of interest

There are no conflicts to declare.

Notes and references (1)

Harbeck,

N.;

Gnant,

M.

Breast

Cancer.

The

Lancet

2017,

389

(10074),

1134-1150,

DOI:

https://doi.org/10.1016/S0140-6736(16)31891-8. (2)

Loibl,

S.;

Gianni,

L.

Her2-Positive

Breast

Cancer.

The

Lancet

2017,

389

(10087),

2415-2429,

DOI:

https://doi.org/10.1016/S0140-6736(16)32417-5. (3) Zang, X.; Ding, H.; Zhao, X.; Li, X.; Du, Z.; Hu, H.; Qiao, M.; Chen, D.; Deng, Y.; Zhao, X. Anti-Epha10 Antibody-Conjugated Ph-Sensitive Liposomes for Specific Intracellular Delivery of Sirna. International journal of nanomedicine 2016, 11, 3951-67, DOI: 10.2147/IJN.S107952. (4) Liu, Y.; Qiao, L.; Zhang, S.; Wan, G.; Chen, B.; Zhou, P.; Zhang, N.; Wang, Y. Dual Ph-Responsive Multifunctional Nanoparticles for Targeted Treatment of Breast Cancer by Combining Immunotherapy and Chemotherapy. Acta Biomaterialia 2018, 66, 310-324, DOI: https://doi.org/10.1016/j.actbio.2017.11.010. (5) Wilhelmsson, A.; Roos, M.; Hagberg, L.; Wengström, Y.; Blomberg, K. Motivation to Uphold Physical Activity in Women with Breast Cancer

During

Adjuvant

Chemotherapy

Treatment.

European

Journal

of

Oncology

Nursing

2017,

29,

17-22,

DOI:

https://doi.org/10.1016/j.ejon.2017.03.008. (6) Zhang, Y.-K.; Zhang, X.-Y.; Zhang, G.-N.; Wang, Y.-J.; Xu, H.; Zhang, D.; Shukla, S.; Liu, L.; Yang, D.-H.; Ambudkar, S. V.; Chen, Z.-S. Selective Reversal of Bcrp-Mediated Mdr by Vegfr-2 Inhibitor Zm323881. Biochemical Pharmacology 2017, 132, 29-37, DOI: https://doi.org/10.1016/j.bcp.2017.02.019. (7) Kim, J.; Yung, B. C.; Kim, W. J.; Chen, X. Combination of Nitric Oxide and Drug Delivery Systems: Tools for Overcoming Drug Resistance in Chemotherapy. Journal of Controlled Release 2017, 263, 223-230, DOI: https://doi.org/10.1016/j.jconrel.2016.12.026. (8) Chakravarty, G.; Mathur, A.; Mallade, P.; Gerlach, S.; Willis, J.; Datta, A.; Srivastav, S.; Abdel-Mageed, A. B.; Mondal, D. Nelfinavir Targets Multiple Drug Resistance Mechanisms to Increase the Efficacy of Doxorubicin in Mcf-7/Dox Breast Cancer Cells. Biochimie 2016, 124, 53-64, DOI: https://doi.org/10.1016/j.biochi.2016.01.014. (9) Shen, J.; Wang, Q.; Hu, Q.; Li, Y.; Tang, G.; Chu, P. K. Restoration of Chemosensitivity by Multifunctional Micelles Mediated by P-Gp Sirna to Reverse Mdr. Biomaterials 2014, 35 (30), 8621-8634, DOI: https://doi.org/10.1016/j.biomaterials.2014.06.035. (10) Joshi, P.; Vishwakarma, R. A.; Bharate, S. B. Natural Alkaloids as P-Gp Inhibitors for Multidrug Resistance Reversal in Cancer. European Journal of Medicinal Chemistry 2017, 138, 273-292, DOI: https://doi.org/10.1016/j.ejmech.2017.06.047. (11) Cheon, J. H.; Kim, K. S.; Yadav, D. K.; Kim, M.; Kim, H. S.; Yoon, S. The Jak2 Inhibitors Cep-33779 and Nvp-Bsk805 Have High P-Gp Inhibitory Activity and Sensitize Drug-Resistant Cancer Cells to Vincristine. Biochemical and Biophysical Research Communications 2017, 490 (4), 1176-1182, DOI: https://doi.org/10.1016/j.bbrc.2017.06.178. (12) Huo, X.; Liu, Q.; Wang, C.; Meng, Q.; Sun, H.; Peng, J.; Ma, X.; Liu, K. Enhancement Effect of P-Gp Inhibitors on the Intestinal Absorption and Antiproliferative Activity of Bestatin. European Journal of Pharmaceutical Sciences 2013, 50 (3), 420-428, DOI: https://doi.org/10.1016/j.ejps.2013.08.010. (13) Curiel, T. J. Immunotherapy: A Useful Strategy to Help Combat Multidrug Resistance. Drug Resistance Updates 2012, 15 (1), 106-113, DOI: https://doi.org/10.1016/j.drup.2012.03.003. (14) Owyong, M.; Hosseini-Nassab, N.; Efe, G.; Honkala, A.; van den Bijgaart, R. J. E.; Plaks, V.; Smith, B. R. Cancer Immunotherapy Getting Brainy: Visualizing the Distinctive Cns Metastatic Niche to Illuminate Therapeutic Resistance. Drug Resistance Updates 2017, 33-35, 23-35, DOI: https://doi.org/10.1016/j.drup.2017.10.001. (15) Chen, Q.; Yu, Q.; Liu, Y.; Bhavsar, D.; Yang, L.; Ren, X.; Sun, D.; Zheng, W.; Liu, J.; Chen, L.-m. Multifunctional Selenium Nanoparticles: Chiral Selectivity of Delivering Mdr-Sirna for Reversal of Multidrug Resistance and Real-Time Biofluorescence Imaging. Nanomedicine: Nanotechnology, Biology and Medicine 2015, 11 (7), 1773-1784, DOI: https://doi.org/10.1016/j.nano.2015.04.011.



Page 14 of 23

(16) Yhee, J. Y.; Song, S.; Lee, S. J.; Park, S.-G.; Kim, K.-S.; Kim, M. G.; Son, S.; Koo, H.; Kwon, I. C.; Jeong, J. H.; Jeong, S. Y.; Kim, S. H.; Kim, K. Cancer-Targeted Mdr-1 Sirna Delivery Using Self-Cross-Linked Glycol Chitosan Nanoparticles to Overcome Drug Resistance. Journal of Controlled Release 2015, 198, 1-9, DOI: https://doi.org/10.1016/j.jconrel.2014.11.019. (17) Shen, J.; Zhang, W.; Qi, R.; Mao, Z.-W.; Shen, H. Engineering Functional Inorganic-Organic Hybrid Systems: Advances in Sirna Therapeutics. Chemical Society Reviews 2018, 47 (6), 1969-1995, DOI: 10.1039/C7CS00479F. (18) Hua, J.; Mutch, D. G.; Herzog, T. J. Stable Suppression of Mdr-1 Gene Using Sirna Expression Vector to Reverse Drug Resistance in a Human Uterine Sarcoma Cell Line. Gynecologic Oncology 2005, 98 (1), 31-38, DOI: https://doi.org/10.1016/j.ygyno.2005.03.042. (19) Creixell, M.; Peppas, N. A. Co-Delivery of Sirna and Therapeutic Agents Using Nanocarriers to Overcome Cancer Resistance. Nano Today 2012, 7 (4), 367-379, DOI: https://doi.org/10.1016/j.nantod.2012.06.013. (20) Misra, R.; Das, M.; Sahoo, B. S.; Sahoo, S. K. Reversal of Multidrug Resistance in Vitro by Co-Delivery of Mdr1 Targeting Sirna and Doxorubicin Using a Novel Cationic Poly(Lactide-Co-Glycolide) Nanoformulation. International Journal of Pharmaceutics 2014, 475 (1), 372-384, DOI: https://doi.org/10.1016/j.ijpharm.2014.08.056. (21) Goldshtein, M.; Forti, E.; Ruvinov, E.; Cohen, S. Mechanisms of Cellular Uptake and Endosomal Escape of Calcium-Sirna Nanocomplexes. International Journal of Pharmaceutics 2016, 515 (1), 46-56, DOI: https://doi.org/10.1016/j.ijpharm.2016.10.009. (22) Wang, J.; Ayano, E.; Maitani, Y.; Kanazawa, H. Enhanced Cellular Uptake and Gene Silencing Activity of Sirna Using Temperature-Responsive Polymer-Modified Liposome. International Journal of Pharmaceutics 2017, 523 (1), 217-228, DOI: https://doi.org/10.1016/j.ijpharm.2017.03.035. (23) Inoh, Y.; Nagai, M.; Matsushita, K.; Nakanishi, M.; Furuno, T. Gene Transfection Efficiency into Dendritic Cells Is Influenced by the Size of Cationic Liposomes/DNA Complexes. European Journal of Pharmaceutical Sciences 2017, 102, 230-236, DOI: https://doi.org/10.1016/j.ejps.2017.03.023. (24) Inoh, Y.; Haneda, A.; Tadokoro, S.; Yokawa, S.; Furuno, T. Cationic Liposomes Suppress Intracellular Calcium Ion Concentration Increase Via Inhibition of Pi3 Kinase Pathway in Mast Cells. Biochimica et Biophysica Acta (BBA) - Biomembranes 2017, 1859 (12), 2461-2466, DOI: https://doi.org/10.1016/j.bbamem.2017.09.025. (25) Tian, H.; Lin, L.; Jiao, Z.; Guo, Z.; Chen, J.; Gao, S.; Zhu, X.; Chen, X. Polylysine-Modified Polyethylenimine Inducing Tumor Apoptosis as an Efficient Gene Carrier. Journal of Controlled Release 2013, 172 (2), 410-418, DOI: https://doi.org/10.1016/j.jconrel.2013.06.026. (26) Wolfram, J.; Suri, K.; Yang, Y.; Shen, J.; Celia, C.; Fresta, M.; Zhao, Y.; Shen, H.; Ferrari, M. Shrinkage of Pegylated and Non-Pegylated

Liposomes

in

Serum.

Colloids

and

Surfaces

B:

Biointerfaces

2014,

114,

294-300,

DOI:

https://doi.org/10.1016/j.colsurfb.2013.10.009. (27) Jøraholmen, M. W.; Basnet, P.; Acharya, G.; Škalko-Basnet, N. Pegylated Liposomes for Topical Vaginal Therapy Improve Delivery of

Interferon

Alpha.

European

Journal

of

Pharmaceutics

and

Biopharmaceutics

2017,

113,

132-139,

DOI:

https://doi.org/10.1016/j.ejpb.2016.12.029. (28) Ando, H.; Abu Lila, A. S.; Kawanishi, M.; Shimizu, T.; Okuhira, K.; Ishima, Y.; Ishida, T. Reactivity of Igm Antibodies Elicited by Pegylated Liposomes or Pegylated Lipoplexes against Auto and Foreign Antigens. Journal of Controlled Release 2018, 270, 114-119, DOI: https://doi.org/10.1016/j.jconrel.2017.12.002. (29) Chen, Q.; Ding, H.; Zhou, J.; Zhao, X.; Zhang, J.; Yang, C.; Li, K.; Qiao, M.; Hu, H.; Ding, P.; Zhao, X. Novel Glycyrrhetinic Acid Conjugated Ph-Sensitive Liposomes for the Delivery of Doxorubicin and Its Antitumor Activities. RSC Advances 2016, 6 (22), 17782-17791, DOI: 10.1039/C6RA01580H. (30) Al-Ahmady, Z. S.; Chaloin, O.; Kostarelos, K. Monoclonal Antibody-Targeted, Temperature-Sensitive Liposomes: In Vivo Tumor Chemotherapeutics in Combination with Mild Hyperthermia. Journal of Controlled Release 2014, 196, 332-343, DOI: https://doi.org/10.1016/j.jconrel.2014.10.013. (31) Ohradanova-Repic, A.; Nogueira, E.; Hartl, I.; Gomes, A. C.; Preto, A.; Steinhuber, E.; Mühlgrabner, V.; Repic, M.; Kuttke, M.; Zwirzitz, A.; Prouza, M.; Suchanek, M.; Wozniak-Knopp, G.; Horejsi, V.; Schabbauer, G.; Cavaco-Paulo, A.; Stockinger, H. Fab Antibody Fragment-Functionalized Liposomes for Specific Targeting of Antigen-Positive Cells. Nanomedicine: Nanotechnology, Biology and Medicine 2018, 14 (1), 123-130, DOI: https://doi.org/10.1016/j.nano.2017.09.003.


Page 15 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(32) Mallick, S.; Thuy, L. T.; Lee, S.; Park, J., II; Choi, J. S. Liposomes Containing Cholesterol and Mitochondria-Penetrating Peptide (Mpp) for Targeted Delivery of Antimycin a to A549 Cells. Colloids and Surfaces B: Biointerfaces 2018, 161, 356-364, DOI: https://doi.org/10.1016/j.colsurfb.2017.10.052. (33) Suga, T.; Fuchigami, Y.; Hagimori, M.; Kawakami, S. Ligand Peptide-Grafted Pegylated Liposomes Using Her2 Targeted Peptide-Lipid Derivatives for Targeted Delivery in Breast Cancer Cells: The Effect of Serine-Glycine Repeated Peptides as a Spacer. International Journal of Pharmaceutics 2017, 521 (1), 361-364, DOI: https://doi.org/10.1016/j.ijpharm.2017.02.041. (34) Chun, J.-Y.; Min, S.-G.; Jo, Y.-J. Production of Low Molecular Collagen Peptides-Loaded Liposomes Using Different Charged Lipids. Chemistry and Physics of Lipids 2017, 209, 1-8, DOI: https://doi.org/10.1016/j.chemphyslip.2017.10.003. (35) Li, L.; An, X.; Yan, X. Folate-Polydiacetylene-Liposome for Tumor Targeted Drug Delivery and Fluorescent Tracing. Colloids and Surfaces B: Biointerfaces 2015, 134, 235-239, DOI: https://doi.org/10.1016/j.colsurfb.2015.07.008. (36) Drummond, D. C.; Hong, K.; Park, J. W.; Benz, C. C.; Kirpgtin, D. B. Liposome Targeting to Tumors Using Vitamin and Growth Factor Receptors. In Vitamins & Hormones; Academic Press: 2000; pp 285-332. (37) Kamada, H.; Taki, S.; Nagano, K.; Inoue, M.; Ando, D.; Mukai, Y.; Higashisaka, K.; Yoshioka, Y.; Tsutsumi, Y.; Tsunoda, S.-i. Generation and Characterization of a Bispecific Diabody Targeting Both Eph Receptor A10 and Cd3. Biochemical and Biophysical Research Communications 2015, 456 (4), 908-912, DOI: https://doi.org/10.1016/j.bbrc.2014.12.030. (38) Surawska, H.; Ma, P. C.; Salgia, R. The Role of Ephrins and Eph Receptors in Cancer. Cytokine & Growth Factor Reviews 2004, 15 (6), 419-433, DOI: https://doi.org/10.1016/j.cytogfr.2004.09.002. (39) Nagano, K.; Maeda, Y.; Kanasaki, S.-i.; Watanabe, T.; Yamashita, T.; Inoue, M.; Higashisaka, K.; Yoshioka, Y.; Abe, Y.; Mukai, Y.; Kamada, H.; Tsutsumi, Y.; Tsunoda, S.-i. Ephrin Receptor A10 Is a Promising Drug Target Potentially Useful for Breast Cancers Including Triple Negative Breast Cancers. Journal of Controlled Release 2014, 189, 72-79, DOI: https://doi.org/10.1016/j.jconrel.2014.06.010. (40) Zhang, J.; Yang, C.; Pan, S.; Shi, M.; Li, J.; Hu, H.; Qiao, M.; Chen, D.; Zhao, X. Eph A10-Modified Ph-Sensitive Liposomes Loaded with Novel Triphenylphosphine-Docetaxel Conjugate Possess Hierarchical Targetability and Sufficient Antitumor Effect Both in Vitro and in Vivo. Drug delivery 2018, 25 (1), 723-737, DOI: 10.1080/10717544.2018.1446475. (41) Zheng, W.; Yin, T.; Chen, Q.; Qin, X.; Huang, X.; Zhao, S.; Xu, T.; Chen, L.; Liu, J. Co-Delivery of Se Nanoparticles and Pooled Sirnas for Overcoming Drug Resistance Mediated by P-Glycoprotein and Class Iii Beta-Tubulin in Drug-Resistant Breast Cancers. Acta Biomater 2016, 31, 197-210, DOI: 10.1016/j.actbio.2015.11.041.



Page 16 of 23

Table 1: characterization of different DOX-loaded liposomes Particle size (nm)

PDI

EE(%) of DOX

Zeta potential (mV)

Blank L

81.6±2.3

0.26±0.07

-

33.5±0.8

DOX/L

93.56±2.4

0.27±0.08

66.3±2.2

30.63±2.1

DOX/PL

112.35±2.2

0.23±0.09

65.4±1.5

29.15±0.6

DOX/ePL

121.36±1.8

0.22±0.07

64.5±1.8

28.86±1.3

Table 2: Characteristics of liposomes and lipoplexes at N/P ratio was 10 Particle size (nm)

PDI

EE(%) of siRNA

Zeta potential (mV)

DOX+siRNA/L

118.6±2.4

0.27±0.08

88.3±2.2

18.3±2.3

DOX+siRNA/PL

136.5±2.2

0.23±0.09

83.2±1.5

14.5±1.5

DOX+siRNA/ePL

141.0±1.8

0.22±0.07

82.8±1.8

14.3±2.2

Table 3: cell cytotoxicity of different formulations against MCF-7 and MCF-7/ADR cell lines. IC50 (μg mL-1)of 24 h

IC50 (μg mL-1)of 48 h

Preparations MCF-7

MCF-7/Adr

MCF-7

MCF-7/Adr

DOX

22.5

103.2

18.6

86.7

DOX+siRNA

21.8

100.5

17.8

85.3

DOX+siRNA/L

15.4

50.2

12.4

40.1

DOX+siRNA/PL

17.3

55.3

13.5

42.5

DOX+siRNA/ePL

12.9

34.2

9.7

21.3


Page 17 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig.1: encapsulation efficiency of different lipoplexes with different N/P ratio (A); particle size and zeta potential of DOX+siRNA/ePL with different N/P ratio (B); TEM images of DOX+siRNA/PL (C) and DOX+siRNA/ePL (D) with N/P ratio was 10. Scale bars represent 100 nm.



Fig.2: agarose gel electrophoresis assay of different formulations with different N/P ratio. (A) Binding capability assay of different formulations with different N/P ratio; (B) heparin stability assay of different formulations with different N/P ratio; (C) serum stability of different formulations with different N/P ratio.

Fig.3: In vitro siRNA (A) and DOX (B) release from different formulations in different pH at 37℃. (n=3)


Page 18 of 23

Page 19 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig.4: Fluorescence microscopy images of MCF-7/Adr cells incubated with different FAM-siRNA loaded lipoplexes (A). Green and blue fluorescence indicated siRNA and nucleic, respectively. Scale bars represent 50 μm. Flow cytometry measurement of cellular uptake of different formulations (B and C). Western blotting assay of MDR1 expression against different formulations (D).

Fig.5: Confocal laser scanning microscope (CLSM) images of MCF-7/Adr cells incubated with FAM-siRNA/ePL in the presence and absence of chloroquine. Green, red and blue fluorescence indicated FAM-siRNA, endo/lysosome and nucleic, respectively. Scale bars represent 20 μm.



Fig.6: in vivo antitumor activity. The tumor volume changes (A), tumor inhibition rate (B) and body weight changes (C) of different formulations in nude mice bearing MCF-7/Adr solid tumor cells.(n=6)


Page 20 of 23

Page 21 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig.7: Morphological evaluation in H&E sections of tumor sites (blue and pink pixel dots represent nucleic and cytoplasm, respectively). Ex vivo evaluation of tumor proliferation level by Ki-67 immunohistochemistry (proliferation cells shown brown pixel dots). Scale bars represent 50 μm.

Fig.8: In situ cell death detection of tumor tissue (TUNEL) treated with saline (a), DOX (b), DOX+siRNA (c), DOX+siRNA/L (d), DOX+siRNA/PL (e) and DOX+siRNA/ePL (f), respectively. Blue and green fluorescence represent nucleic and apoptosis cells, respectively. Scale bars represent 50 μm.

Fig.9: ex vivo evaluation P-gp expression using P-gp immunofluorescence assay treated with saline (a), DOX (b), DOX+siRNA (c), DOX+siRNA/L (d), DOX+siRNA/PL (e) and DOX+siRNA/ePL (f), respectively. Blue and red fluorescence represent nucleic and P-gp expression, respectively. Scale bars represent 50 μm.



Fig.10: In vivo toxicity assay in normal mice treated with different formulations. Body weight curves of different treatments (A). H&E staining of main organs of normal mice treated with different formulations (B). Scale bars represents 50 μm.

Scheme 1: schematic illustration of the approach to overcome MDR by multifunctional DOX+siRNA/ePL lipoplexes.


Page 22 of 23

Page 23 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table of Contents Graphic


Abstract

Recommend Documents