Targeted Co-delivery of Docetaxel and Atg7 siRNA for Autophagy

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Targeted Co-delivery of Docetaxel and Atg7 siRNA for Autophagy Inhibition and Pancreatic Cancers Treatment Miaozun Zhang, Wei Zhang, Guping Tang, Hebin Wang, Min Wu, Weiming Yu, Zhenfeng Zhou, Yiping Mou, and Xingang Liu ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00764 • Publication Date (Web): 20 Feb 2019 Downloaded from http://pubs.acs.org on February 21, 2019

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Targeted Co-delivery of Docetaxel and Atg7 siRNA for Autophagy Inhibition and Pancreatic Cancers Treatment Miaozun Zhang,†, # Wei Zhang,‡, # Guping Tang, ⊥ Hebin Wang,§ Min Wu, ⊥ Weiming Yu,† Zhenfeng Zhou,∥ Yiping Mou,¶,* Xingang Liu⊥,* † Department

of General Surgery, Ningbo Medical center Lihuili Hospital, Ningbo, China ‡ Department of Gastroenterology, Ningbo No.2 Hospital, Ningbo, China ⊥ Department of Chemistry, Zhejiang University, Hangzhou, China ¶ Department

of General Surgery, Zhejiang Provincial People’s Hospital, Hangzhou, China College of Life Sciences, Tarim University, Alar 843300, China ∥ Department of Anesthesiology, Zhejiang Provincial People’s Hospital, Hangzhou, China §

ABSTRACT As the fourth leading cause of cancer-related deaths worldwide, pancreatic cancer has a higher basal level of autophagy as compared to other epithelial tumors. Recently, increasing evidence suggested that Docetaxel (DTX) triggered autophagy in pancreatic cancer cells which promoted cancer cell survival after chemotherapy. Therefore, we constructed an amphiphilic block copolymer that can form micelles to simultaneously co-delivery with siAtg7 and DTX for effective inhibition of tumor cells grown in vitro and in vivo. The iRGD peptide internalized on the surface of the vehicle could target to tumors and increase the penetration of vehicle into solid tumors. By downregulating the expression of Atg7 gene, the DTX-induced autophagy was inhibited, which improved the DTX therapeutic outcomes during the treatment of pancreatic cancer.

Keywords: Chemotherapy, Docetaxel, Autophagy, siRNA, Pancreatic cancers INTRODUCTION Autophagy is a process of intracellular lysosomal degradation and recycling of proteins and organelles, which has recently emerged as a significant mechanism in the development and treatment of malignancies.1-4 Pancreatic cancer is one of the most lethal cancers, with a dismal 5-year survival of ～ 6 % due to the advanced stage

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diagnosis and lack of effective treatment options.5 In pancreatic adenocarcinoma, autophagy was significantly induced compared to healthy pancreatic tissue in patients.6-7 The sustained activation of autophagy was associated with poor survival in pancreatic cancer.8-9 Early studies demonstrated that autophagy caused by certain cancer treatment would induce cancer cells against apoptosis.10 For example, the resistance to cytotoxic chemotherapies agents, like Docetaxel (DTX), is a main factor causing therapy failure of pancreatic cancer.11 During the chemotherapy process, DTX triggered autophagy in cancer cells and the autophagy promoted cancer cell survival, thus lead to the failure of chemotherapy.12-14 To address the challenges, the combination of autophagy inhibition with traditional antitumor approach could achieve improved therapy effectiveness, thus allowing for lower dose usage and reduced side effects1, 12, 14 Autophagy inhibitors like chloroquine (CQ) and 3-methyladenine (3-MA) were used to combine with the antitumor drugs for effective chemotherapy.15-17 Compared to autophagy inhibitors, RNA interference (RNAi) technology is a more temperate way to silence the target gene,18-20 which usually suppresses gene expression by small interfering RNAs (siRNA).21-23 As one of the Atg autophagy regulatory proteins family, Atg7 regulates the autophagosomes formation during the initiation of autophagy, which plays a predominant role in the maturation of autophagic vacuoles by engaging other proteins to the autophagosomal membrane.24-25 Thus we postulated that the inhibition of autophagy by siAtg7 could enhance the apoptosis of cancer cells when associated with DTX treatment. Based on our recent studies,19,

26-27

we constructed an amphiphilic block

copolymers PP6/iRGD which can delivery DTX and siRNA simultaneously inside the formed micelles to effectively inhibit tumor growth in vitro and in vivo. The co-delivery vehicle was essentially composed of a triblock copolymer Pluronic P123 with poly(ethylene oxide)-block poly(propylene oxide)-block-poly(ethylene oxide) structure as the backbone, which formed micelles to encapsulate the hydrophobic drugs inside the inner core.28-31 The low molecular weight polyethylenimine (PEI) rendered the vehicle cationic properties for binding the gene effectively. The cyclic


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peptide iRGD (CRGDK/RGPD/EC) internalized on the surface of the vehicle can target to tumors through binding to integrins (such as αv and β3/β5) which were overexpressed on the endothelium of tumor vessels.32-34 The peptide could also increase the penetration of vehicle into solid tumors significantly.35 siAtg7 and DTX which were loaded inside PP6/iRGD micelle reached the tumor tissue by active targeting and were released from the micelles after endo-lysosomal escape due to the “proton-sponge” effect.36-37 Thus, autophagy in tumor cells was inhibited by downregulating the expression of the Atg7 gene, which improved the DTX treatment outcomes in the therapeutic of pancreatic cancer.

EXPERIMENTAL SECTION Cells and animals PANC-1 cells were acquired from the American Type Culture Collection (ATCC, MD, USA) and were cultured in the DMEM containing 10 % fetal bovine serum in a CO2 incubators at 37 ℃ and 5 % CO2. Athymic male mice (BALB/c strain) (3~4 weeks old，12~14 g) were bought from the Zhejiang Chinese Medical University and All animal experiments were operated according to China Animal Protection Law. Synthesis of PP6 For the synthesis of PP6, 0.58 g Pluronic P123 (0.1 mmol), 0.17 g CDI (1.0 mmol) and 0.2 mL triethylamine were mixed in anhydrous DMSO (30 mL). After stirring at 37 ℃ for 6 h under nitrogen, 0.60 g PEI (1.0 mmol) was added dropwise into the mixture solution and stirred overnight. The reaction mixture was dialyzed using dialysis bags (10000 Da) in H2O and then freeze-dried. The yield is 65.8 %. 1H NMR (400 MHz, CDCl3) δ(TMS, ppm) 3.20-3.75 (370 H, m), 2.50-2.81 (109 H, m), 1.07 (210 H, s). Synthesis of PP6iRGD 3.92 mg SPDP (0.0125 mmol ） were dissolved in 5 mL DMSO:PBS = 1:1 mixture solution, and then added into 260 mg PP6 solution in 10 mL PBS. After



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stirring at 37 ℃ for 2 h under nitrogen, 15.84 mg iRGD (0.0166 mmol) in 5 mL PBS solution were added dropwise into the reaction solution and stirred for another 4 h. The reaction mixture was dialyzed using dialysis bags (10000 Da) in H2O and then freeze-dried. The yield is 57.2 %. Preparation of DTX-Loaded micelles We use thin-film hydration method to prepare the DTX-loaded micelles. First, 100 mg PP6iRGD and 15 mg DTX were mixed in acetonitrile. A solid thin-film of PP6iRGD/DTX was obtained by evaporating the solvent, which was hydrated by sonicating for 0.5 h in 15 mL H2O, then filter membrane (0.45 µm) was used to filter the unencapsulated DTX. UV-vis spectrum was used to determine the concentrations of DTX. The encapsulation ratio (ER %) and drug loading efficiency (DL %) was calculated according to the following two equations: Weight of the drug in micelles

𝐷𝐿 = Weight of the feeding polymer and drug × 100% 𝐸𝑅 =

Weight of the drug in micelles Weight of the feeding drug

× 100%

(1) (2)

Characterization of DTX-Loaded micelles The particle size and zeta potential of the free PP6, PP6/DTX, PP6iRGD/DTX, PP6/siRNA，PP6/DTX/siRNA and PP6iRGD/DTX/siRNAmicelles were analyzed at 25 ℃ by dynamic light scattering (DLS) on the Zetasizer 3000 (Malvern Instruments, Worcestershire, UK). The morphology was examined by transmission electron microscopy (TEM, HT-7700, Hitachi, Japan). Agarose gel electrophoresis Gel electrophoresis was performed according to methods reported before.18 In vitro DTX release The in vitro release experiment of the DTX-loaded micelles was performed according to methods reported before.18 Cell viability assay PANC-1 cells were seeded in 96-well plate at 1×104 cells/well and incubated for 20 h. Then cells were incubated with DTX, siAtg7, PP6/DTX, PP6iRGD/DTX,


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PP6/DTX/siAtg7, PP6iRGD/DTX/siAtg7 with various concentration for 48 h, culture medium was removed, and then the cytotoxicity was assessed using the MTT assay. Cellular uptake PANC-1 cells were seeded in 24-well plates at 5×104 cells per well and incubated for 24 h. The medium was replaced by serum-free culture medium containing PP6/FM-siRNA, PP6/DTX/FM-siRNA and PP6iRGD/DTX/FM-siRNA at N/P=30 and incubated for 4 h. After rinsed with PBS, the cells were immobilized using 4 % paraformaldehyde and stained with DAPI for 15 min. Scanning laser confocal microscope (Leica SPE) were used to acquire confocal images. Tumor therapy The nude mice were divided into six groups (six mice per group) and PANC-1 cells were implanted subcutaneously at the right-sided abdominal. When diameter of the tumors reached about 5 mm, each group was injected with (1) the PBS, (2) free DTX, (3) PP6/DTX, (4) PP6/DTX/si Atg7, (5) PP6iRGD/DTX and (6) PP6iRGD/DTX/siRGD via peritumoral, respectively. The DTX doses of each injection were 1.0 mg/kg and the N/P was fixed at 30. The tumor volume was detected by calipers each four days and the therapy was performed twice a week for 2 weeks. Tumor volume (V) was calculated according to the formula: V (mm3) = π/6 ×length (mm) ×width (mm)2. The mouse weights were also recorded. Histological assessment The experiments of histological analysis (H&E and TUNEL staining) were carried out by methods reported before.27 Immunohistochemistry The experiment of immunohistochemistry was carried out by methods reported before.27 Quantitative real-time PCR Quantitative reverse transcription (RT)-PCR was used in the analysis of the PANC-1 cell lines expression of genes encoding Atg7. The cells were treated with (1)



the PBS, (2) free DTX, (3) siAtg7, (4) PP6/DTX, (5) PP6/siAtg7, (6) PP6/DTX/si Atg7, (7) PP6iRGD/DTX/siAtg7, (8) PP6iRGD/siAtg7, (9) PP6iRGD/DTX, respectively. After incubated for 4 h, the medium was removed and the cells were incubated with fresh DMEM for another 48 h. Cells were collected to extract mRNA and reverse-transcribed into cDNA. The following pairs of primers were used (5'-3'): 5' -GCTTGGCTGCTACTTCTG -3' (sense), 5' - GGTTGGAGGCTCATTCATC -3'(antisense). Western blot analysis The experiment of western blot analysis was carried out by methods reported before.18

RESULTS AND DISCUSSION Synthesis and characterization of PP6iRGD To prepare the tumor-targeted drug delivery system, iRGD modified Pluronic P123-PEI (PP6iRGD) was synthesized according to the synthesis route showed in Scheme 1. P123 was first reacted with PEI 600 kD using 1,1’-carbonyl diimidazole (CDI) as the coupling agent, then the resulted PP6 containing PEI was further conjugated with iRGD by N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP) to give the product. Proton nuclear magnetic resonance (1HNMR) spectroscopy was used to analysis the chemical structure of PP6 and PP6iRGD (Figure 1A). The proton signals observed clearly between 3.3-3.8 ppm and 1.0 ppm were attributed to P123 in the PP6 spectrum, while the successful conjugation of PEI could be confirmed by the multiple peaks between 2.5 and 2.8 ppm. The molar ratio of P123 and PEI was about 1:2 calculated by the peak integration of P123 and PEI. In PP6iRGD spectrum, the characteristic peaks between 1.4 and 2.0 ppm were attributed to the methyl protons of iRGD. The 1HNMR results showed that these proposed compounds were synthesized successfully.


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Scheme 1. Synthetic routes of PP6iRGD and the schematic illustration which showed the co-delivery process of siRNA and DTX using PP6iRGD micelle.

Preparation and Characterization of DTX-loaded micells All hybrid micelles were prepared by thin-film hydration method. To investigate the hydrophobic drugs loading ability of PP6iRGD micelles, DTX was selected as the model drug. Different amount of DTX (10 mg, 15 mg, 20 mg) with 100 mg PP6iRGD were used to prepared DTX-loaded micelles. The maximum drug loading efficiency (DL) of PP6iRGD/DTX was calculated to be 9.15 %, which was much higher than those Pluronic polymers reported previously. As showed in Table 1, the size of blank PP6iRGD micelles had a hybrid diameter at 214.3 ± 13.0 nm, while the size increased to 238.0 ± 15.1 nm after DTX were encapsulated. When condensed with siRNA at N/P = 30, the particle size decreased while the zeta potential drop to +7.6 (±0.4) mV. Transmission electron microscopy (TEM) was used to observe the morphology of the micelles. As showed in Figure 1B, the PP6iRGD/DTX/siRNA micelles were spherical with a diameter of



about 150 nm, which was smaller than the hydrodynamic diameter from the DLS experiments. To investigate drug release kinetics, the release behavior of DTX was evaluated under different pH condition (pH=7.0 and 5.0) simulating the physiological condition and acidic endosome compartments over a total period of 50 h. As showed in Figure 1C, there were nearly 93 % DTX releasing from PP6iRGD/DTX/siRNA micelles in pH 5.0 buffer solutions, while only 49 % DTX released in pH 7.0 buffer solutions, which meant the DTX could DTX will be released at the endosomal acidic pH and less will be released in physiological condition to decrease drug-related side effects. According to the Chinese Pharmacopoeia, more than 40 % of the drug being released in 0.5 h is defined as a burst release. In our research, there were not more than 15 % of DTX released from the drug vectors within 0.5 h. Therefore, the drug release amount within 50 h wound not affect the drug safety.38 The iRGD peptide seemed to have no effect on the DTX release behavior.

Figure 1. (A) 1H NMR spectra of iRGD, PP6 and PP6iRGD (400 MHz, CDCl3). (B) TEM images of PP6 and PP6iRGD/DTX/siRNA micelles. (C) The cumulative DTX release profile of PP6/DTX/siRNA and PP6iRGD/DTX/siRNA at pH 5.0 and 7.0. Data are showed as mean ± SD (n=3).

The surface charge of PP6iRGD micelles was analyzed after drug loading. As


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showed in Table 1, the blank PP6iRGD micelles and DTX-loaded PP6iRGD micelles had a zeta potential at about +11 mV, which meant the encapsulation of DTX seldom affected the surface property of PP6iRGD micelles. The positive charge of the PP6iRGD micelles suggested that micelles had the potential to condense the negative charged siRNA. Next, the binding capacity of siRNA to PP6iRGD micelles was determined via agarose gel retardation assay at various N/P ratios (the mole ratios between the amine groups of cationic polymers and phosphate groups of DNA). As showed in Figure 2A, PP6iRGD micelles could fully retard migration of siRNA in the agarose gel at N/P ratio of 4:1. While the DTX loaded PP6iRGD/DTX micelles could bind to siRNA at higher N/P ratios of 6:1. This can be explained by after encapsulating the DTX inside the micelle, the particle size increased while the zeta potential decreased. Thus the encapsulating of DTX reduced the RNA retardation ability of the complex as a result of the decreased density of ethylenimine units, which had been reported by us and others.39-40

Table 1. Particle size and zeta potential of various micelles. PP6 PP6iRGD PP6iRGD/siRNA PP6iRGD/DTX PP6/DTX/siRNA PP6iRGD/DTX/siRNA

Particle size (nm)

zeta potential (mV)

199.0 ± 11.5 214.3 ± 13.0 178.7 ± 10.0 238.0 ± 15.1 199.0 ± 12.5 227.0 ± 11.5

14.4 ± 0.6 12.4 ± 0.8 8.3 ± 0.5 10.7 ± 0.3 7.4 ± 0.4 7.6 ± 0.4

Cellular uptake To more precisely elucidate the effect of N/P ratio on cellular internalization of the drug delivery vectors, flow cytometry analysis was used to quantify the cell uptake efficiency. First, the siRNA was labeled with fluorescein isothiocyanate (FITC), termed as FAM-siRNA. The percentage of the PP6iRGD/FAM-siRNA positive cells increased with the N/P ratio becoming higher, reaching the maximum at N/P=30. When the N/P increased to 40, no more obvious cellular uptake of



PP6iRGD/FAM-siRNA was found. Thus we chose N/P=30 as the optimal ratio in the

later cellular and in vivo experiment.

Figure 2. (A) Agarose gel electrophoresis retardation of siAtg7 complexed with PP6iRGD or PP6iRGD/DTX at various N/P ratios. (B) Quantitative determination of PANC-1cells incubated with PP6iRGD/FAM-siRNA at various N/P ratios by flow cytometry. (C) Confocal images of PANC-1 cellular uptake of PP6/siRNA, PP6/DTX/siRNA, PP6iRGD/DTX/siRNA. Plasma membrane is stained with WGA Alexa Fluor, siRNA is labeled with FITC (green), nucleus is stained with DAPI (blue).

Next, we evaluated the cellular uptake efficiency of the micelles in PANC-1 cell line by confocal image analysis to verify the active targeting ability. The iRGD peptide has been reported to target to tumors through binding to integrins (such as αv and β3/β5) and increase the penetration to solid tumors. Compared to PP6/siRNA and PP6/DTX/siRNA, the iRGD peptide conjugated PP6iRGD/DTX/siRNA showed an improved efficiency of cellular uptake (Figure 2C). The competitive inhibition results detected by flow cytometry also verified the effectiveness of targeted delivery (Figure


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S1). These results represented that iRGD peptide could enhance the intracellular uptake of the targeting micelles.41-45

Figure 3. In vitro cytotoxicity of (A) DTX, PP6/DTX, PP6iRGD/DTX, PP6/DTX/siAtg7, PP6iRGD/DTX/siAtg7 in PANC-1 cells with various DTX concentration for 48 h of incubation; (B) In vitro cytotoxicity of siAtg7, PP6/siAtg7, PP6/DTX/siAtg7, PP6iRGD/siAtg7 and PP6iRGD/DTX/siAtg7 with various siAtg7 concentration for 48 h of incubation.

In vitro cytotoxicity assays The blank PP6iRGD showed low in vitro cytotoxicity on PANC-1 cells after 48 h of incubation (Figure S2). Based on these results, we tested whether the co-delivery of DTX and siAtg7 by PP6iRGD would show more toxic on PANC-1 cells. As illustrated in Figure 3A, the DTX treated group cells were moderately inhibited and only 44 % cells were inhibited at 4 μg/mL of DTX, while 77 % cells growth were inhibited in the PP6iRGD/DTX/Atg7 treated group at the same concentration. Next, we investigated the siRNA impact on the cell viability. As showed in Figure 3B, naked siAtg7 showed almost no cytotoxicity against PANC-1 cell lines, even the PP6iRGD-mediate siAtg7 treated group showed low cytotoxicity. Only the combined DTX and siAtg7 treated group exhibited significant cytotoxicity. The significant inhibition of pancreatic cancer proliferation by PP6iRGD/DTX/siAtg7 was also found on Panc02 prostate cancer cell line (Figure S3, S4). The above results could be explained by that DTX could increase cell autophagic activity, which would impact the therapeutic effect. The suppressing of autophagy by siAtg7 would not cause cells apoptosis directly, but it could enhance the DTX-induced apoptosis in PANC-1 cells.



Figure 4. (A) Representative Atg7, LC3 I, LC3 II, Cleaved Caspase3 and β-actin protein expressions determined by western blot analysis at 48 h in PANC-1 cells. (B) Expression of Atg7 mRNA in PANC-1 determined by quantitative RT-PCR at 48 h. Analysis of light intensities of (C) Atg7, (D) LC3II/LC3I and (E) Cleaved Caspase3 protein expression in PANC-1 cells from Western blot results. #1 for PBS; #2 for DTX; #3 for PP6; #4 for PP6/DTX; #5 for PP6/siAtg7; #6 for PP6/DTX/siAtg7; #7 for PP6iRGD/DTX/siAtg7; #8 for PP6iRGD/siAtg7; #9 for PP6iRGD/DTX.

Inhibition of autophagy by siAtg7 in vitro To detect the autophagy and apoptotic level of cells after the treatment, we examined Atg7, LC3 and Cleaved Caspase3 protein expression by protein quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot (WB) analysis. First, we observed the increased expression of LC3-II protein in DTX, PP6/DTX and PP6iRGD/DTX treated group. LC3-II is an autophagy marker, which is distributed in the membranes of autophagosomal when autophagy is induced. Thus the boosted LC3-II/LC3-I ratio could represent the appearance of autophagy (Figure 4A, 4D).46-47 Next, the expression of Atg7 gene expression dramatically increased in DTX, PP6/DTX and PP6iRGD/DTX treated group both in qRT-PCR and WB (Figure 4B and 4C), which were consistent with the literature results. The above results indicated that the DTX treatment could induce autophagy of the PANC-1 cells.48 When siAtg7 was added into the system to form PP6iRGD/DTX/siAtg7 micelles and incubated with PANC-1 cells for 48 h, both Atg7 and LC3-II protein expression were inhibited. That meant the siAtg7 co-delivered by PP6iRGD/DTX micelles could effectively lower the autophagy level by decreasing the expression of Atg7 protein, which was coincident with what we expected. Increased apoptotic rates were observed in the PP6iRGD/DTX/siAtg7 treated group, which indicated that the inhibition of


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autophagy could enhance the sensitivity of PANC-1 cells to DTX and these results were in consistent with the MTT assay (Figure 4E).

Figure 5. Antitumoral therapeutic effects of PBS, PP6/DTX, PP6/DTX/siAtg7, PP6iRGD/siAtg7, DTX, PP6iRGD/DTX/siAtg7 complexes in BALB/c nude mice with PANC-1 xenografts: (A) Tumor volume and (B) Body weights during the treatment; (C) Digital images of tumor on nude mice with PANC-1 xenografts after the treatment; (D) H&E staining, Atg7, LC3, Cleaved caspase3 protein, and TUNEL assays of tumor tissues after treated with PBS, PP6/DTX, PP6/DTX/siAtg7, PP6iRGD/siAtg7, DTX, PP6iRGD/DTX/siAtg7 complexes. All scale bars represent 50 µm.

In vivo tumor therapy To evaluate the therapeutic efficacy of DTX and siAtg7 co-delivery for pancreatic cancer, the in vivo distribution of PP6iRGD/DTX/FAM-siRNA was first studied. As showed in Figure S5, the iRGD conjugated nanoparticles accumulated much more in the tumor than the non-iRGD conjugated nanoparticles, which demonstrated the active targeting effect of iRGD. Then, serial DTX and siRNA formulations were administered by peritumoral injection two times a week for two weeks into PANC-1 tumor-bearing mice. As showed in Figure 5A and 5C, the animals treated with the PBS showed rapid tumor growth, while mild inhibition was



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observed in the DTX and PP6/DTX treated animals. The most dramatical inhibition against tumor growth was the animals treated with PP6iRGD/DTX/siAtg7, which showed an enhanced inhibition than animals treated with PP6DTX/siAtg7 without the iRGD peptide. This meant that the maximal DTX therapeutic outcomes could be achieved only when DTX and siAtg7 were loaded by PP6 with the target peptide of iRGD. In addition, there was no significant body weight loss after various treatments of formulations (Figure 5B).

Figure 6. (A) Representative Atg7, LC3 I, LC3 II, Cleaved Caspase3 and β-actin protein expressions determined by western blot analysis in PANC-1 tumors 48 h after the last treatment. (B) Expression of Atg7 mRNA in PANC-1 tumor datected by quantitative RT-PCR at 48 h after the last treatment. Analysis of light intensities of (C) Atg7, (D) LC3II/LC3I and (E) Cleaved Caspase3 protein expression in PANC-1 tumor from Western blot results after last injection. #1 for PBS; #2 for PP6/DTX; #3 for PP6/DTX/siAtg7; #4 for PP6iRGD/siAtg7; #5 for DTX; #6 for PP6iRGD/DTX/siAtg7.

To detect the cell proliferation and apoptosis in the tumor tissues, hematoxylin and eosin (H&E) staining and TUNEL assay were carried out after treatment. As showed in Figure 5D, the mice treated with PP6iRGD/DTX/siAtg7 represented the least mounts of cancer cell but an increased apoptosis and necrosis ratio when compared with the other treated groups. Immunohistochemical assays were further analyzed to detect the expression of target proteins in the tumor tissues. Briefly, the treatment with DTX increased the expression of Atg7 and LC3 protein, and the cleaved caspase3 protein level seemed litter higher than the negative control group of PBS.

When

the

DTX

and

siAtg7

were

co-delivery

in

the

micelle,

PP6iRGD/DTX/siAtg7 dramatically decreased the Atg7 and LC3 protein expression


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and the cleaved caspase3 protein level was upregulated compared with the DTX (Figure 5D). To show the inhibition of cancer cell proliferation quantitatively, western blot was used to analyse the protein expression. As showed in Figure 6, the DTX treatment increased Atg7 and LC3 protein expression, representing the induction of autophagy during the process of therapy. The PP6iRGD/DTX/siAtg7 treated group dramatically decreased cell autophagy by inhibiting Atg7 and LC3 expression and had increased apoptosis which were proved by the upregulated expression of the cleaved caspase3 protein. Noteworthily, the PP6/DTX/siAtg7 treated mice showed a lower cleaved caspase3 expression, which meant the iRGD peptide conjugated on the micelles could help to increase cell apoptosis by targeting to tumors and increasing the penetration of vehicle to solid tumors.

CONCLUSIONS In conclusion, we developed a multifunctional drug delivery system to achieve the co-delivery of chemotherapeutic DTX and siAtg7 for pancreatic tumor therapy. The co-delivery vehicle was essentially composed of a triblock copolymer Pluronic P123 structure as the backbone, which formed micelles to encapsulate the hydrophobic drugs inside the inner core. The conjugated low molecular weight polyethylenimine (PEI) rendered the vehicle cationic properties for binding the gene effectively. The iRGD peptide internalized on the surface of the vehicle could target to tumors and increase the penetration of vehicle to solid tumors. This co-delivery system downregulate the DTX-induced autophagy by inhibiting the Atg7 and LC3 protein expression, thus improve the DTX therapeutic outcomes in the treatment of pancreatic cancer both in vitro and in vivo. The system we constructed offered an effective way for pancreatic tumor treatment and had the potential to deliver other pharmaceutics and gene for the combination therapy of carcinoma.



ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Quantitative determination of cellular uptake, cytotoxicity on Panc02 cells, in vivo distribution experimental results (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] *Email: [email protected] Author Contributions # These

authors contributed equally

ORCID Xingang Liu: 0000-0001-7071-0209 Guping Tang: 0000-0003-3256-740X Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS This work was supported by the Zhejiang Provincial Natural Science Foundation of China (LQ19E030011) and the National Science Foundation for Young Scientists of China (21807092). REFERENCES (1) Sharma, N.; Thomas, S.; Golden, E. B.; Hofman, F. M.; Chen, T. C.; Petasis, N. A.; Schönthal, A. H.; Louie, S. G., Inhibition of Autophagy and Induction of Breast Cancer Cell Death by Mefloquine, an Antimalarial Agent. Cancer Lett. 2012, 326, 143-154. (2) Zhang, X.; Li, W.; Wang, C.; Leng, X.; Lian, S.; Feng, J.; Li, J.; Wang, H., Inhibition of Autophagy Enhances Apoptosis Induced by Proteasome Inhibitor Bortezomib in Human Glioblastoma U87 and U251 Cells. Mol. Cell. Biochem. 2014, 385, 265-275. (3) Akar, U.; Chaves-Reyez, A.; Barria, M.; Tari, A.; Sanguino, A.; Kondo, Y.; Kondo, S.; Arun, B.; Lopez-Berestein, G.; Ozpolat, B., Silencing of Bcl-2 Expression by Small Interfering RNA Induces Autophagic Cell Death in MCF-7 Breast Cancer Cells. Autophagy 2008, 4, 669-679. (4) Jin, S.-M.; Jang, H. W.; Sohn, S. Y.; Kim, N. K.; Joung, J. Y.; Cho, Y. Y.; Kim, S. W.; Chung, J. H., Role of Autophagy in the Resistance to Tumour Necrosis Factor-related


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Targeted Co-delivery of Docetaxel and Atg7 siRNA for Autophagy

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