Modular engineering of targeted dual-drug nano-assembles

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Biological and Medical Applications of Materials and Interfaces

Modular engineering of targeted dual-drug nanoassembles for cancer chemo-immunotherapy Tianyi Kang, Yang Li, Yong Wang, Jiao Zhu, Ling Yang, Yulan Huang, Meimei Xiong, Jinlu Liu, Shuai Wang, MeiJuan Huang, Xiawei Wei, and Maling Gou ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b11881 • Publication Date (Web): 06 Sep 2019 Downloaded from pubs.acs.org on September 6, 2019

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ACS Applied Materials & Interfaces

Modular Engineering of Targeted Dual-drug Nano-assembles for Cancer Chemoimmunotherapy Tianyi Kang 1, 2 †, Yang Li 1 †, Yong Wang 3 †, Jiao Zhu 4, Ling Yang 1, Yulan Huang 1, Meimei Xiong 1, Jinlu Liu 1, Shuai Wang 1, Meijuan Huang 4, Xiawei Wei 5, Maling Gou 1 * 1. State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu, 610041, P. R. China. 2. Department of Biomedical Engineering, Southern University of Science and Technology, Shenzhen, Guangdong, 518055, P. R. China 3. Department of Gastrointestinal Surgery, West China Hospital, Sichuan University, Chengdu, 610041, P. R. China. 4. Department of Thoracic Oncology, West China Hospital, Sichuan University, Chengdu, 610041, P. R. China. 5. State Key Laboratory of Biotherapy and Cancer Center, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu 610041, China. * Corresponding author: Maling Gou; E-mail: [email protected] † These authors contributed equally to this work.

Abstract Combination of chemotherapeutics and immunomodulators can generate synergistic anticancer efficacy, exerting efficient chemo-immunotherapy for cancer treatment. Nanoparticulate delivery system holds great promise to promote synergistic anticancer efficacy for the co-delivery of drugs. However, it remains challenges to precisely co-encapsulate and deliver combinational drugs at designed ratio due to the difference of compatibility between drugs and nanocarriers. In this study, co-assembled nanoparticles of lipophilic prodrugs (LPs) were designed to co-deliver chemotherapeutics and immunomodulators for cancer treatment. Such nano-assemblies (NAs) could act as a platform to ratiometrically co-encapsulate chemotherapeutics and immunomodulators. Based on this method, NAs formed by the self-assembly of iRGD peptide derivatives, paclitaxel

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(PTX) LPs and imiquimod (R837) LPs were demonstrated to target tumor at unified pharmacokinetics, further inducing the effective tumor inhibition and tumor recurrence prevention. This work provided an alternative to prepare chemo-immunotherapeutic NAs with advantages of ratiometric drug co-encapsulation and unified pharmacokinetics, which may advance the future cancer chemo-immunotherapy.

Keywords Nanoparticles, drug delivery, chemo-immunotherapy, lipophilic prodrug, modular engineering, breast cancer

Introduction Chemo-immunotherapy is a promising way for cancer treatment.

1, 2

At rational ratio, the

optimal synergy of chemotherapeutics and immunomodulators can be exerted. Therefore, precisely co-encapsulate and co-deliver combinational drugs at a designed ratio are crucial for improving synergistic effect. Nanoparticulate drug delivery systems (NDDS) show great promise in codelivery by overcoming pharmacokinetic difference between drugs. 3-5 The way of NDDS in drug loading often rely on physical encapsulation. Considering the difference of compatibility between drugs with carrier and the strong crystallinity of drugs, 6-8 it remains a challenge to co-encapsulate drugs ratiometrically for nanoparticle-mediated co-delivery. Recently, researchers have shown that hydrophobic drugs can be combined with hydrophobic elements, such as fatty acids and squalene, to obtain lipophilic prodrugs (LPs).

9

These highly

hydrophobic LPs can be dispersed in water and self-assembled into stable NAs without the aid of surface stabilizer.

10, 11

Lipophilic prodrugs nano-assembles (LP NAs) have many advantages,

including precisely defined chemical structure,

12

good thermodynamic stability and high drug

loading capability. 13 The LPs strategy has been used to deliver a variety of chemotherapeutics (e.g. paclitaxel, docetaxel, and camptothecin) to improve drug pharmacokinetics and significantly improve in vivo antitumor effects. 14-16 More importantly, these hydrophobic LPs can be assembled with each other at different proportions to form stable NAs due to the high compatibility provided by the coexistence of long alkyl chains. Functionalized components can be modularly embedded in the LP-NAs. Ligands that combine with the receptor highly expressed on tumor cells and peptides can be introduced to the LP-NAs for improving the tumor targeting efficacy.17 Here, iRGD peptide that binds with integrin receptors (e.g. αvβ3 and αvβ5) expressed on tumor cell membrane was used to form NAs with capability of tumor targeting. Meanwhile, stimuli-responsive linking groups can be inserted into the LPs to


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produce responsive release of drugs in the tumor microenvironment (such as pH and redox environment). Based on the difference in concentration of reductive glutathione (GSH) between intracellular and extracellular (intracellular concentration is nearly 1000 times higher than extracellular concentration),18, 19 disulfide bonds were used here to form stimulus-sensitive nanoassembles for responsive release of drugs. Paclitaxel is an important chemotherapeutics for the treatment of solid tumors in clinic.20, 21 It causes tumor cell death, producing tumor-associated antigens (TAAs) as well as damage associated molecular patterns (DAMPs). Imiquimod (R837) can enhance the presentation of TAAs by binding to the Toll-like receptors (TLRs) of DCs, initiating TAA-specific T cell immune response.22 PTX and R837 shows potential in synergistically treating cancer via chemo-immunotherapy.23 Here, we prepared NAs based on disulfide bond modified LPs to combine PTX and R837 for cancer treatment. iRGD derivative was applied to co-assemble with PTX LPs and R837 LPs for the formation of tumor targeted NAs. The obtained NA was characterized with high drug loading capacity, controllable drug ratios and reduction responsive drug release. Results indicated that the obtained NAs could efficiently target tumor and release drugs with unified pharmacokinetics, inducing suppression of tumor growth as well as prevention of tumor recurrence.

Experimental Section Materials Paclitaxel (PTX), Camptothecin (CPT), Curcumin (CUR) and Imiquimod (R837) were purchased from Meilun Biology Technology Co., LTD. (Dalian, China). Dithiodiglycolic acid and stearyl alcohol (SA) were purchased from TCI reagents (Tokyo, Japan). Dicyclohexylcarbodiimide (DCC) and N-Hydroxysuccinimide (NHS) were purchased from Alfa Aesar (MA, USA). 4dimethylaminopyridine (DMAP) was purchased from Adamas Reagent (Shanghai, China). The DSPE-mPEG2000 was purchased from Lipoid GmbH (Ludwigshafen, Germany). The iRGD peptide (H-Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys-NH2) was chemically synthesized by GL Biochem (Shanghai, China). Methylthiazolyldiphenyl-tetrazolium bromide (MTT) and coumarin-6 were purchased from Sigma-Aldrich (St Louis, USA). Cell Line and Animals. 4T1 murine breast cancer cell line was purchased from American Type Culture Collection (ATCC). Female BALB/c mice (6-8 weeks old) and male Sprague–Dawley (SD) rats (5~7 weeks old) were purchased from Laboratory Animal Center of Sichuan University (Chengdu, China) for in vivo research. Animals were maintained in laboratory animal room of specific pathogen-free (SPF) conditions and humanely treated. All animal procedures were approved and controlled by the



Institutional Animal Care and Treatment Committee of Sichuan University. Synthesis of LPs: PTX-acSS-SA, R837-acSS-SA, CPT-acSS-SA, CUR-acSS-SA. Synthesis routes of PTX-acSS-SA, R837-acSS-SA, CPT-acSS-SA, CUR-acSS-SA were shown in Figure 1A. Dithiodiglycolic acid (1 g) was dissolved in 40 mL dichloromethane (DCM). DCC (1.0 molar eq.), SA (1.0 molar eq.) and DMAP (~50 mg) were added and stirred at the room temperature overnight. After the DCM was removed under reduced pressure, 50 mL ethyl acetate (EA) was added and the solution was filtered to remove DCU. The filtrate was reduced under reduced pressure to obtain the crude products, which was further purified by silica gel column chromatography (200 mL petroleum ether (PE), 100 mL PE/EA 5:1, 100 mL PE/EA 4:1 and 200 mL PE/EA 2:1 (v/v)) to give SA-acSS-COOH. To synthesize PTX-acSS-SA, the obtained SA-acSS-COOH (200 mg) was dissolved in anhydrous DCM (20 mL). DCC (1.2 molar eq.), PTX (1.0 molar eq.) and DMAP (~1 mg) were added, stirred at room temperature overnight. After DCM was removed under reduced pressure, 50 mL EA was added and the solution was filtered to remove DCU. The filtrate was purified by silica gel column chromatography (400 mL PE/EA 20:1) to obtain the PTX-acSS-SA. The synthesis routes of CPT-acSS-SA, CUR-acSS-SA were the same as PTX-acSS-SA. To synthesize R837-acSS-SA, SA-acSS-COOH (200 mg), DCC (1.0 molar eq.) and NHS (1.0 molar eq.) were dissolved in anhydrous DCM (20 mL) and stirred at room temperature for 1 h. Then R837 (1.0 molar eq.) was added and stirred at room temperature overnight. R837-acSS-SA was obtained after purification by silica gel column chromatography (400 mL PE/EA 20:1). Co-assembly Ability of Immunomodulator with Different Chemotherapeutics. To explore the Co-assembly ability of different chemotherapeutics with immunomodulator, equal amount (2 mg) of PTX-acSS-SA, CPT-acSS-SA, CUR-acSS-SA were respectively codissolved with R837-acSS-SA (2 mg) in ethanol (400 µL) at 70 °C (10% DSPE-mPEG2000 were added). The obtained solution was added drop-wisely in 70 °C double-distilled water (2 mL) at 1200 rpm stirring, to form NA by self-assembly. Ethanol was removed through rotary evaporation at 45 °C. The final concentration of NA was adjusted to 2 mg/mL−1 by double-distilled water. The particle sizes of obtained PTX-R837 NA, CPT-R837 NA, CUR-R837 NA were detected by a dynamic light scattering (DLS) using a Zetasizer Nano ZS90 (Malvern Instruments, England). PTX and R837 were chosen as model drugs to study the co-assembly ability at different drug ratios. PTX-R837 NAs were prepared at ratios PTX-acSS-SA: R837-acSS-SA= 10: 0, 10: 2, 10: 5, 10: 10, 5: 10, 2: 10, 0: 10 under condition with or without DSPE-mPEG2000. Particle sizes of PTXR837 NAs were detected then. Preparation and Characterization of PTX-R837 NA and i-PTX-R837 NA.


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C18-PEG-iRGD was synthesized as described in our previous report.24 Briefly, stearic acid (0.03 mmol) and NH2-PEG-MAL (0.01 mmol) were mixed in dichloromethane. Then EDCI (0.03 mmol), NHS (0.03 mmol), triethylamine (2 μL) were added and stirred at room temperature overnight. C18-PEG-MAL was obtained after separated by silica gel column chromatography. iRGD (0.006 mmol) was dissolved in the mixture of PBS and DMSO. C18-PEG-MAL was dissolved in dimethyl sulfoxide and added dropwise into iRGD solution at 25 °C. After dialysis for 3 days, the supernatant was centrifuged at and lyophilized to obtain C18-PEG-iRGD. i-PTX-R837 NA were self-assembled from PTX-acSS-SA, R837-acSS-SA and C18-PEGiRGD. Briefly, Equal amount of PTX-acSS-SA (2 mg) and R837-acSS-SA (2 mg) were dissolved with 40 µL DSPE-mPEG2000 (10 mg/mL) in ethanol (360 µL) at 70 °C. C18-PEG-iRGD (0.2 mg) was dissolved by DCM and then mixed with above ethanol solute. The obtained solution was added in 70 °C double-distilled water (2 mL) dropwise with stirring to form the NA by self-assembly. Ethanol was removed through rotary evaporation at 45 °C. The final concentration of NA was adjusted to 2 mg/mL−1 by double-distilled water and stored at 4 °C. In addition, the fluorescence labeled i-PTX-R837 NA were prepared through mixing extra 0.1 mg of coumarin-6. The particle size and zeta potential of PTX-R837 NA and i-PTX-R837 NA were measured. The surface morphology of PTX-R837 NA and i-PTX-R837 NA were detected by Transmission Electron Microscope (Hitachi, Japan). Cellular Uptake Assay. To compare the cellular uptake of PTX-R837 NA and i-PTX-R837 NA by 4T1 cells, the coumarin-labeled PTX-R837 NA and i-PTX-R837 NA were respectively incubated with 4T1 cells for 2 h. The final concentration of NA in each well was 20 μg/mL. The fluorescent intensity of there two NAs by 4T1 cells was detected by flow cytometry (Beckman, USA). To detect the cellular uptake of i-PTX-R837 NA at different time points, 4T1 cells were precultured on the glass sheet and placed in 6-well plates before using, then coumarin-labeled i-PTXR837 NA were added into the 6-well plates. The cellular uptake pictures were captured at different incubation time (0.5 h, 2 h and 4 h). Briefly, glass sheets loaded with cancer cells were taken out respectively at different time and immobilized by 4% paraformaldehyde. Then DAPI (10 µg/mL) was applied to dye the cell nucleus. After washing with 1×PBS for several times, the glass sheets were dried and placed on the glass slide with 30% glycerinum to keep cellular morphology during observation under laser scanning confocal microscopy (Nikon, Japan). MTT Assay. 4T1 breast cancer cells were collected and re-planted in 96-well plates. Then cells were respectively treated with NS, PTX, PTX+R837, PTX-R837 NA and i-PTX-R837 NA of different



concentrations (the equivalent concentration of PTX was: 100 μg/mL, 20 μg/mL, 4 μg/mL, 0.8 μg/mL, 0.16 μg/mL, 0.032 μg/mL, 0.0064 μg/mL respectively). After 24 h incubation, MTT reagent was added and the OD values were detected by Microplate Reader (BioTek, USA). Responsive Drug Release Assay in vitro. PTX-acSS-SA and R837-acSS-SA solutions were diluted with 1mL PBS buffer (pH 7.4, 30 % ethanol (v/v), with or without 10 mM DTT). The equivalent concentration of PTX and R837 was 20 μg/mL respectively in the sample vials. Then sample vials were put into 37 °C water bath. 20 μL solution was collected at every time point (0h，2h，5h，10h，24h) for HPLC analysis (Agilent, USA). Biodistribution Assay. To study the biodistributions of PTX-R837 NA and i-PTX-R837 NA in vivo, 4T1 breast tumorbearing mice were used to perform the biodistribution assay. The coumarin-labeled PTX-R837 NA (2 mg/mL, 100 µL) and i-PTX-R837 NA (2 mg/mL, 100 µL) were intravenously injected through tail vein respectively. Mice treated with normal saline (NS) were taken as control group. At different time points (2h, 8h, 24h) after administration, mice were sacrificed to obtain tumor and normal tissues (heart, liver, spleen, lung and kidney). Live image analysis instrument (Caliper Life Sciences, USA) was used for fluorescent imaging, showing the drug biodistribution. Pharmacokinetics Assay. To study the pharmacokinetics of drugs in plasma, SD rats (4 rats/per group) were assigned to three groups. Each group received intravenous bolus of the i-PTX-R837 NA or combined injection of free PTX and R837 (PTX + R837) at an equivalent dose of 10 mg/kg (PTX) and 5 mg/kg (R837). 0.5 mL blood samples were taken and centrifuged to obtain the plasma sample at given time points. The concentrations of PTX, R837, PTX-acSS-SA, R837-acSS-SA in plasma were analyzed using HPLC with a wavelength of 227 nm and 248 nm for PTX and R837, respectively. To study the pharmacokinetics of drug in tumor site, tumor models were constructed through subcutaneously inoculated with 4T1 cancer cells (2 × 105) on the back of mice. After 15 days, animals were divided into two groups and each group received intravenous bolus of the i-PTX-R837 NA or combined injection of PTX-acSS-SA and R837-acSS-SA. After 48 h, tumors of each group were collected and grinded. HPLC was used to detect the content of PTX-acSS-SA and R837-acSSSA in each group. The ratios of PTX-acSS-SA/R837-acSS-SA were also calculated. Apoptosis Assay. To detect the cellular apoptosis, 4T1 cells were re-planted in 12-well plates before treated with NS, PTX, PTX+R837, PTX-R837 NA and i-PTX-R837 NA. The equivalent dose of PTX and R837 in each group was 32 μg/well and 16 μg/well respectively. After 24 h incubation, all the cells were


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collected and stained by Annexin V-FITC Apoptosis Detection Kit (BD biosciences, USA) according to the manufacturer’s instruction. Flow cytometry (Beckman-Coulter, USA) was applied to detect the apoptotic ratio. DCs Stimulation Assay. Dendritic cells derived from bone marrow (BMDCs) of BALB/c mice were used to investigate the DCs stimulation effect of i-PTX-R837 NA. 4T1 cells that treated by PTX, R837 and i-PTXR837 NA (The equivalent dose of PTX and R837 in each group was 32 μg/well and 16 μg/well) were co-incubated with BMDCs respectively. After 12 h incubation, BMDCs were collected and co-stained with anti-CD80 (APC), anti-CD86 (PE), anti-CD11c (FITC) antibodies. Flow cytometry was used to analyze the ratio of CD80+ CD86+ CD11C+ DCs. Hemolytic Test and Aggregation of Erythrocytes. To obtain red blood cells for the test, fresh blood was collected from SD rat and then washed by NS for three times. i-PTX-R837 NA (100 µL) solutions of different concentrations were incubated with 100 µL erythrocyte suspension (4%) respectively at 37 °C. Erythrocyte suspension added with NS or distilled water was taken as negative or positive control groups. The final equivalent concentration of PTX was 64 µg/mL, 32 µg/mL, 16 µg/mL, 8 µg/mL, 4 µg/mL in the blood. After 30 min incubation, all the groups were centrifuged at 3000 rpm for 3 min. To study the erythrocytes aggregation, 500 µL erythrocyte suspension (4%) were incubated with i-PTX-R837 NA of different concentrations (the final equivalent concentration of PTX was 128 μg/mL, 64 μg/mL, 32 μg/mL) in 12-well plates (Corning Costar, USA). After 2 h incubation at 37 °C, the status of erythrocytes was captured by an optical microscope (Olympus, Japan). Antitumor Assay in vivo. 4T1 cells (2 × 105 cells, 100 µL) suspension was injected subcutaneously into the back of mice (day 0). Tumor bearing mice were randomized divided into 5 groups on day 5. NS, PTX, PTX+R837, PTX-R837 NA and i-PTX-R837 NA were intravenously injected every other day for 5 times (The equivalent dose of PTX and R837 was 10 mg/kg and 5 mg/kg, respectively). The body weight and tumor volume of mice were recorded every other day from day 5. On day 19, all the mice were sacrificed to obtain tumors and heart, liver, spleen, lung, kidney. The tumor weight of mice was recorded on day 19. Histological Analysis and H&E Staining. The obtained tumors and tissues on day 19 were stored at -80 ℃ or fixed in neutral paraformaldehyde (4%) for at least 24 h, then embedded in OCT or paraffin. The obtained tumor frozen sections were stained with TUNEL kit (Vazyme, China) to detect the tumor apoptosis. The obtained tumor paraffin sections were stained with anti-CD31 rabbit polyclonal antibody (Abcam,



England) to detect vascular growth. To study the biological safety of i-PTX-R837 NA, paraffin sections of normal tissues (heart, liver, spleen, lung, kidney) were stained with Hematoxylin-eosin. Re-challenge Experiment. To study the tumor recurrence prevention by i-PTX-R837 NA, mice inoculated with 4T1 cells (2 × 105 cells, 100 µL) on the right back were pre-treated with NS, PTX+R837 and i-PTX-R837 NA every other day for 5 times (The equivalent dose of PTX and R837 was 10 mg/kg and 5 mg/kg respectively). Then mice were re-challenged with secondary tumor. On the other side of back, 4T1 cells (2 × 105 cells, 100 µL) were subcutaneously injected. The volume of secondary tumor was measured every other day. Mice were sacrificed on day 22 after re-inoculation and secondary tumors were obtained. To study the mechanism of i-PTX-R837 NA in tumor recurrence prevention, the secondary tumors were grinded, then the obtained cell suspension was co-stained by anti-CD3 with anti-CD8, or anti-CD3 with anti-CD4 antibodies. Flow cytometry was applied to detect the CD3+ CD8+ and CD3+ CD4+ T cells respectively. Statistical Analysis. Results are presented as mean ± standard deviation (SD). All the experiments were repeated at least three times. One-way ANOVA was applied to test the statistical significance of difference among three or more groups. Two-tailed student’s t-test was applied to test the statistical significance of difference between two groups. Statistical significance was set at *p < 0.05, ** p < 0.01 and *** p < 0.001, respectively.

Results Design of Chemo-immunotherapeutic NA Delivery Platform. To construct a chemo-immunotherapeutic NA delivery platform for co-delivering chemotherapeutics and immunomodulators, we combined the model immunomodulator: Imiquimod (R837) to combine with different chemotherapeutics: paclitaxel (PTX), camptothecin (CPT), curcumin (CUR) respectively. All the drugs were modified into corresponding LPs: PTX-acSS-SA, CPT-acSS-SA, CUR-acSS-SA, R837-acSS-SA by conjugating with stearyl alcohol (SA) via redoxresponsive linker of disulfanyl acetate (acSS) (Figure 1A). Their chemical structures were confirmed by 1H-NMR (Figure S1, Supporting Information). Aliphatic chain methyl (δ 0.88 ppm), aromatic hydrogen of PTX (δ 7-9 ppm), methyl of PTX (δ 0.9-2.44 ppm) and some other characteristic peaks were detected by 1H-NMR to confirm the synthesized PTX-acSS-SA. Aliphatic chain methyl (δ 0.88 ppm), aromatic hydrogen (δ 7-9 ppm), methyl of R837 (δ 1.04 ppm, δ 1.06 ppm) and some other characteristic peaks were detected by 1H-NMR to confirm the synthesized


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R837-acSS-SA. Characteristic peaks of CPT-acSS-SA and CUR-acSS-SA were also marked in their corresponding 1H-NMR spectrum to confirm the structure of products. The preparation schematic diagram was shown in Figure 1B, in which LPs co-assemble with each other to form chemo-immunotherapeutic NA. R837-acSS-SA could co-assemble with PTXacSS-SA, CPT-acSS-SA and CUR-acSS-SA respectively at the ratio of 1:1 to form NA with clarified profile (Figure 1C) and uniform particle size: PTX-R837 NA of 144 ± 5 nm, CPT-R837 NA of 195 ± 8 nm, CUR-R837 NA of 143 ± 2 nm (Figure 1D). The results indicated that the LPsbased method makes it possible to co-encapsulate immunomodulator with different chemotherapeutics, providing a platform for preparation of chemo-immunotherapeutic NAs. Then we studied the ratiometric co-encapsulation ability of chemotherapeutics and immunomodulators based on LPs. We mixed PTX-acSS-SA and R837-acSS-SA at different ratios (10: 0, 10: 2, 10: 5, 10: 10, 5: 10, 2: 10, 0: 10, w/w) under condition with (+) or without (-) DSPEmPEG2000, and dispersed mixtures in water respectively. The results showed that when lack of DSPE-mPEG2000, sizes of NAs became more and more large along with the increased proportion of R837-acSS-SA. When R837-acSS-SA was two times over PTX-acSS-SA, NAs were unable to be formed. However, when low dose of DSPE-mPEG2000 (10%) added, PTX-acSS-SA and R837-acSSSA could co-assemble into well dispersed NAs under various drug ratios (Figure 1E, F). Preparation and characterization of i-PTX-R837 NA. Based on the synergistic anticancer effect of PTX and R837, we prepared the chemoimmunotherapeutic NA of PTX and R837 for cancer treatment. PTX-acSS-SA, R837-acSS-SA and C18-PEG-iRGD peptide were self-assembled into i-PTX-R837 NA. As measured by DLS, the mean particle size of i-PTX-R837 NA was 140 ± 5 nm (PDI = 0.3) (Figure 2A), which was slightly larger than that of PTX-R837 NA (112 ± 8 nm) (PDI = 0.1) (Figure S2A). As shown in Figure 2B, the mean zeta potential of i-PTX-R837 NA was -23.0 ± 1.5 mV, showing no significant difference with that of PTX-R837 NA at -19.9 ± 2.2 mV (Figure S2B). Determined by transmission electron microscopy (TEM) analysis (Figure 2C), i-PTX-R837 NA had a vesicle like morphology with a mean particle size of ~80 nm, which was similar to that of PTX-R837 NA (Figure S2C). To study the cellular uptake efficiency of PTX-R837 NA and i-PTX-R837 NA by 4T1 cells, we incubated 4T1 cells with coumarin-6-labeled PTX-R837 NA and i-PTX-R837 NA, respectively. The cellular uptake of NAs was then detected the fluorescence intensity by flowcytometry. The results showed that the fluorescence intensity of cells incubated with i-PTX-R837 NA was much higher than that of PTX-R837 NA (Figure 2D). We next incubated 4T1 cells (marked with DAPI) with i-PTX-R837 NA (labeled with coumarin-6) and observed fluorescence under confocal microscopy at different time points. As shown in Figure 2E, the green fluorescence (coumarin-6)



surrounding the blue fluorescence (DAPI) became stronger along with incubation time and reached top at 2 h. The results indicated that iRGD could improve the cellular uptake of PTX-R837 NA by cancer cells. MTT assay was applied to detect the anticancer effect of i-PTX-R837 NA on breast cancer cells in vitro (Figure 2F). i-PTX-R837 NA showed anticancer activity with half-maximal inhibitory concentration (IC50) of 32.3 μg/mL, which was significantly lower than that of R837(512.4 μg/mL) and PTX-R837 NA (58.4 μg/mL), but slightly higher than that of free PTX (15.0 μg/mL) and PTX+R837(12.5 μg/mL). Both NAs showed inferior antitumor effect compared with free PTX or PTX+R837, probably attributing to the sustained release of drugs from NAs. The results also indicated that iRGD could improve anticancer effect through promoting cellular uptake by cancer cells. Assays of drug release, biodistribution and pharmacokinetics. To study the release profiles of R837-acSS-SA and PTX-acSS-SA, we incubated R837-acSSSA and PTX-acSS-SA in PBS with (+) or without (-) DTT, respectively. As shown in Figure 3A, 57.3% R837 was released in the presence of 10 mM DTT, which is significantly higher than that released in PBS (23.2%) from R837-acSS-SA within 24 h. As shown in Figure 3B, more than 90% PTX was released within 2 h upon incubation in 10 mM DTT from PTX-acSS-SA, whereas only 28.6% PTX was released in PBS within 24 h. These results indicated that R837-acSS-SA and PTXacSS-SA that both containing disulfide bond can responsively release drugs in reductive environment, showing promise in cancer treatment. Since PTX was linked to SA-acSS-COOH via an ester, whereas R837 was conjugated by an amide. As amide is much more difficult to be hydrolyzed than ester at the same condition, PTX could be released more easily than R837. To study the biodistributions of i-PTX-R837 NA and PTX-R837 NA, we intravenously injected coumarin-6-labeled i-PTX-R837 NA and PTX-R837 NA to tumor-bearing mice, then observe the fluorescent intensity of tumors and normal tissues at different time points. As shown in Figure 3C, the ﬂuorescence of both NAs in tumor sites were significantly higher than that in most normal tissues (heart, spleen, kidney, lung). The Average radiant efficiency of ROI from tumors were shown in Figure S3. Fluorescence in tumors administrated with i-PTX-R837 NA were significantly higher than that administrated with PTX-R837 NA. The results indicated that iRGD could effectively promote the tumor targeting effect of PTX-R837 NA. To study the pharmacokinetics profiles of i-PTX-R837 NA, we detected the content of drugs in plasma post intravenous injection of PTX+R837, i-PTX-R837 NA at different time points. As shown in Figure 3D, i-PTX-R837 NA exhibited a significantly prolonged drug circulation time with the AUC (PTX-acSS-SA, 326.4 μM/h; R837-acSS-SA, 559.6 μM/h) compared with that of


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PTX+R837 (PTX, 10.4 μM/h; R837, 6.0 μM/h). More importantly, the molar ratio of PTX-acSSSA/R837-acSS-SA in plasma was relatively stable along with time course. The ratio of PTX-acSSSA/R837-acSS-SA was nearly equal with the feed ratio (Figure 3E). Moreover, we detected the content of i-PTX-R837 NA in tumor site. After injected with iPTX-R837 NA, the contents of PTX-acSS-SA and R837-acSS-SA in tumors were detected and the ratio of PTX-acSS-SA/R837-acSS-SA was calculated. The molar ratio of PTX-acSS-SA/R837acSS-SA was found to be approximately equal to the feed ratio and that in plasma (Figure 3F). The results indicated that i-PTX-R837 NA could co-deliver drugs with unified pharmacokinetics. Considering the ratio of PTX-acSS-SA/R837-acSS-SA in tumor could determine ratio of released drugs for the release rate of drug was proportional to the concentration of prodrugs, the lipophilic prodrugs rather than free drugs were detected for this study. Anticancer effect and antigen presentation in vitro. To study the anticancer effect of i-PTX-R837 NA, we incubated 4T1 cells with different preparations. As shown in Figure 4A, the cellular apoptosis rates were 49.2％ ± 4.1％，55.9％ ± 4.4％，27.6％ ± 2.5％，43.8％ ± 3.9％ when treated with free PTX, PTX+R837, PTX-R837 NA and i-PTX-R837 NA, respectively. These results indicated that higher cellular apoptosis was induced by i-PTX-R837 NA than PTX-R837 NA because of the improved cellular uptake effect induced by iRGD. However, due to the sustained release of drugs from i-PTX-R837 NA, its effect on cellular apoptosis was lower than that of free PTX or PTX+R837. DCs could engulf and process antigens along with migration to nearby lymph nodes where they interact with T cells and B cells to initiate the adaptive immune response.25 Next, immature DCs would turn into mature DCs and the expression of co-stimulatory molecules CD40, CD80, CD86 and MHCII would be upregulated simultaneously. To explore DCs stimulation, we coincubated 4T1 cells that treated by free PTX, R837 and i-PTX-R837 NA with bone marrow-derived DCs (BMDCs) (Figure 4B). Then the expression of DCs surface antigens was detected. The results showed that, the ratio of CD11c+ CD80+ CD86+ DCs that co-incubated with 4T1 cells treated with i-PTX-R837 NA, PTX and R837 was 61.2% ± 4.1%, 45.5% ± 3.7%, 72.7%±7.2% respectively (Figure 4C). The results indicated that i-PTX-R837 NA enhanced DCs antigen presentation effect compared with free PTX, which was attributed to the enhancement of TAA immunity by R837 as an adjuvant. Antitumor efficacy in vivo. To study the antitumor effect of i-PTX-R837 NA in vivo, we constructed subcutaneous 4T1 tumor model of Balb/c mice and intravenously injected with i-PTX-R837 NA. Haemolysis test and erythrocyte agglutination assay were conducted to make sure the security of i-PTX-R837 NA for



intravenous administration. As shown in Figure S4A, B, i-PTX-R837 NA caused no obvious hemolysis and hemagglutination at experimental concentration, showing good bio-compatibility. After 5 treatments of NS, PTX, PTX+R837, PTX-R837 NA and i-PTX-R837 NA respectively every other day, mice were sacrificed to harvest the tumors and heart, liver, spleen, kidney, lung. As illustrated in Figure 5A, the growth of tumors in i-PTX-R837 NA treatment group was significantly slower than that of other groups. Tumor weight of each group was recorded on the last day. As shown in Figure 5B, control groups exhibited enlarged tumors (0.62 ± 0.11 g, NS; 0.42 ± 0.13 g, PTX; 0.39 ± 0.16 g, PTX+R837; 0.33 ± 0.23 g, PTX-R837 NA), i-PTX-R837 NA (0.13 ± 0.05 g) treated mice also exhibited a significant decrease in tumor weight, suggesting i-PTX-R837 NA had a better antitumor effect. (P < 0.05) To explore the underlying mechanisms of the antitumor effects of i-PTX-R837 NA, terminal deoxynucleotidyl transferase mediated dUTP nick end-labeling (TUNEL) and CD31 staining were carried out here. As shown in Figure 5D, tumor bearing mice treated with either PTX-R837 NA or i-PTX-R837 NA both showed higher proportion in the number of TUNEL-positive tumor cells compared with those treated with NS, PTX or PTX+R837. In addition, i-PTX-R837 NA treated tumor showed remarkable cell apoptotic induction and cell proliferation inhibition when compared with those treated with PTX-R837 NA. As shown in Figure 5E, the micro-vessel density assessed by CD31 staining markedly decreased in the PTX-R837 NA and i-PTX-R837 NA treatment groups when compared with other groups, indicating that PTX-R837 NA and i-PTX-R837 NA could inhibit angiogenesis in tumors. Furthermore, the anti-angiogenesis efficacy of i-PTX-R837 NA was better than PTX-R837 NA. As a result, i-PTX-R837 NA showed a superior apoptotic induction and angiogenesis inhibition compared with PTX, PTX+R837, PTX-R837 NA. In addition, i-PTX-R837 NA showed no obvious impact on the mice body weight during the treatment process (Figure 5C). From H&E staining result of normal tissues from i-PTX-R837 NA treated mice, no significant pathological changes were found in the heart, liver, spleen, lung or kidney. These results suggested that i-PTX-R837 NA had no obvious side effects on mice. (Figure 5F). The tumor recurrence prevention in vivo. We launched re-challenge experiment to evaluate the cancer recurrence prevention effect of iPTX-R837 NA. After treated by NS, PTX+R837 or i-PTX-R837 NA for 5 doses, the tumor-bearing mice were re-inoculated with 4T1 tumors on the other side of their back (Figure 6A). No treatments were administered to mice after the secondary tumor was inoculated. The tumor volume was measured at different time points in the following 22 days. As shown in Figure 6B, tumor-bearing mice that treated with i-PTX-R837 NA showed inhibited growth in the secondary tumor, at the


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meantime, those treated with NS or PTX+R837 exhibited fast growth rate in the secondary tumor. It suggested that i-PTX-R837 NA could exert immune protection effect to the re-challenged tumors. To study the immune protection mechanism of i-PTX-R837 NA, immune cells in secondary tumors were analyzed on day 22. Cytotoxic T lymphocytes (CTL) (CD3+ CD8+) could directly kill targeted cancer cells, helper T cells (CD3+ CD4+) play important roles in the regulation of adaptive immunities.26 As illustrated in Figure 6C, 4T1 tumor-bearing mice treated with NS were unable to initiate CD8+ CTL infiltration into the secondary tumors and mice with primary tumors treated with free PTX combined with R837 could cause slight CD8+ CTL in the secondary tumors (1.3％ ± 0.2%). On the contrary, the percentage of CD8+ CTL in the secondary tumors of mice after the iPTX-R837 NA treatment significantly increased to 6.8% ± 1.1%. At the same time, compared with those treated with NS or PTX+R837, tumor-bearing mice treated with i-PTX-R837 NA exhibited an increased percentage of CD4+ helper T cells in the secondary tumors (Figure 6D). Mice with first tumor treated by PTX+R837 exhibited a lower percentage of CD3+ CD4+ helper T cells (1.8% ± 0.4%) compared with those treated with i-PTX-R837 NA (3.8% ± 0.5%). The results indicated that i-PTX-R837 NA could successfully stimulate cellular immunity to protect mice from recurrence.

Discussion In this work, we demonstrated the chemo-immunotherapeutic nanomedicine based on coassembling of LPs for cancer treatment. Such NAs acted as a novel platform for the ratiometrically co-loading

of

chemotherapeutics

and

immunomodulators,

releasing

drugs

at

unified

pharmacokinetics. The i-PTX-R837 NA prepared by this method exhibits advantages, such as good blood compatibility, tumor targeting capability and reduction-triggered drug release. More importantly, this novel prodrug NAs significantly inhibited the growth of breast cancer and effectively prevented tumor recurrence, without causing obvious systemic toxicities. The described method shows great promise in optimizing future cancer chemo-immunotherapy. Nanotechnology has been widely used for cancer treatments because of advantages to improve solubility of hydrophobic drugs, facilitate target drug delivery and optimize pharmacokinetics.27 Chemo-immunotherapy plays an important role in the research of cancer treatment.28 Currently, nanoparticulate delivery systems have been widely used for the co-delivery of chemotherapeutics and immunomodulators. For example, Paul G. Tardi et al prepared a liposomal nanosystem for the co-deliver of irinotecan and cisplatin,29 which showed superior anticancer effect than that of freedrug cocktails. As synergistic effect of combinational therapeutics is significantly affected by the molar ratios of distinct drugs,30 rational integration and ratiometric co-delivery of combinational drugs are crucial to ensure the optimal combinational efficacy. Researchers have tried to use various



nanotechnologies for the co-delivery of chemotherapeutics. For instance, Lei Miao et al used dioleoyl phosphatidic acid (DOPA) to coat cisplatin and gemcitabine monophosphate (GMP), further ratiometrically prepared them into PLGA NA.31 The obtained PLGA NA showed synergistic anticancer effects in vivo at constant drug ratios. As these co-delivery strategies were constructed based on the carrier-assistant nanosystems, which suffered the severe drawback of low loading capacity. Therefore, these nanomedicines were usually subjected to a potential risk of side effects due to the introduction of large amounts of carrier materials. On the other hand, as a certain carrier always displayed varied compatibility towards different drugs with distinct physicochemical properties, it is still challenging to develop a nanosystem with the capacity for co-loading of different drugs at an arbitrary ratio. In this study, we developed a novel co-delivery nanosystem based on co-assembling LP NAs, showing the high drug loading capacity, precisely defined chemical structure, improved thermodynamic stability and promoted cancer therapy. Nanoparticles self-assembled form lipid prodrugs had been reported before. Wang Y et al had inserted disulfide bond as linkage between two hydrophobic drugs (such as PTX and vitamin E, doxorubicin and vitamin E) to find that synthesized molecules can self-assemble in water to form stable Nanocolloid solution.32 However, this reported research is different from ours. Here corresponding LPs of induvial drugs were synthesized firstly, then the obtained LPs of different drugs were mixed to form self-assembled nanoparticles. Furthermore, we found that co-assembling NAs could be prepared at various drug ratios with the help of DSPE-mPEG2000, which made them as potential candidate for the ratiometric co-delivery of combinational drugs. Therefore, we synthesized the LPs of poorly soluble PTX and R837 (i.e. PTX-acSS-SA and R837-acSS-SA) for the preparation of chemo-immunotherapeutic NAs. As expected, PTX-acSS-SA and R837-acSS-SA could be co-assembled at random ratios into uniform NA with the presence of DSPE-mPEG2000, providing the basis for ratiometric co-delivery of PTX and R837. Although disulfide bond was applied to form LPs, our previous study found that LPs NA containing disulfide bond exhibited no difference in drug release between the homogenate of tumor and normal tissues (heart, liver, kidney, lung and spleen).12 Therefore, co-delivery system with tumor targeting effect needs to be designed to improve the selective accumulation at tumor site. In this research, we used modified C18-PEG-iRGD to collaborate with disulfide bond for improved tumor targeting effect. C18-PEG-iRGD could co-assembled with PTX-acSS-SA and R837-acSS-SA into i-PTX-R837 NA with uniform size. Zeta potential of i-PTX-R837 NA was determined to be negative, indicating that stabilization of NA may be attributed to the electrostatic repulsion between NAs. The resulting i-PTX-R837 NA was proved to be effective in enhancing cellular uptake and having good tumor targeting effect. After intravenous injection of i-PTX-R837


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NA, the molar ratio of PTX-acSS-SA/R837-acSS-SA was detected and found to be constant with the feed ratio both in plasma and tumor site. It confirmed that LPs-based NA could ratiometrically co-encapsulate and co-deliver chemotherapeutics and immunomodulators successfully. Further experiments showed that i-PTX-R837 NA effectively induced apoptosis of tumor cells and inhibited the growth of breast tumor. The apoptotic tumor cells produced TAAs to collaborate with R837 for stimulating DCs and further activating tumor specific immune response, as well as preventing the tumor recurrence.

Conclusions This research provides a LPs-based method for preparing targeted dual-drug NA for cancer chemo-immunotherapy. This method is proved to have wide applicability in chemotherapeutics and immunomodulators. NA prepared by this method have characteristics of ratiometric drug coencapsulation and unified drug pharmacokinetics. Using this preparation method, we obtained a combinational preparation of PTX and R837 for cancer chemo-immunotherapy. The obtained iPTX-R837 NA has been demonstrated to ratiometrically release drugs at tumor site, efficiently inhibiting tumor growth. The therapeutic effect of tumor is achieved by inducing cellular apoptosis and inhibiting angiogenesis. Furthermore, tumor recurrence is prevented by enhancing specific antitumor immune responses. This research provides a method to solve problems of ratiometric coencapsulation and co-delivery of different hydrophobic drugs, showing promise in tumor chemoimmunotherapy.

Acknowledgment This project was supported by the following fundings: Sichuan Science and Technology Program (2019YJ0068); 1·3·5 project for disciplines of excellence, West China Hospital, Sichuan University (ZYYC08007，ZYJC18017) ; The Science and Technology Project of Chengdu (2018CY02-00041-GX). Thank the Core Facility of West China Hospital for providing the platform of HPLC and flow cytometry. Thanks very much for Prof. Cardon from Ghent University. He has provided a great help to improve the English writing.

Author Contributions M.G. designed and supervised the experiments; T.K., Y.L., Y.W. mainly performed the experiments; T.K. and Y.L. wrote the paper. All the authors supported performing experiments, analyzing data and revising paper.



Competing Interests The authors declare no competing financial interests.

Supporting Information The Nuclear magnetic resonance spectrum analysis, Characterization of PTX-R837 NA, Average radiant efficiency of ROI from tumor and Blood compatibility of i-PTX-R837 NA were supplied as Supporting Information.

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Figure 1. Preparation of LPs-based chemo-immunotherapeutic NAs. A) Synthesis route of CPT-acSSSA，CUR-acSS-SA, PTX-acSS-SA, R837-acSS-SA lipid prodrugs. B) Preparation diagram of chemoimmunotherapeutic NAs. C) Solution of PTX-R837 NA, CPT-R837 NA, CUR-R837 NA. D) Size distribution of PTX-R837 NA, CPT-R837 NA, CUR-R837 NA. E) Solution of PTX-R837 NA at different ratios (PTX-acSS-SA: R837-acSS-SA=10: 0, 10: 2, 10: 5, 10: 10, 5: 10, 2: 10, 0: 10 with or without DSPE-mPEG2000). F) Particle size of PTX-R837 NA versus different ratios (PTX-acSS-SA: R837-acSS-SA=10: 0, 10: 2, 10: 5, 10: 10, 5: 10, 2: 10, 0: 10 with or without DSPE-mPEG2000).



Figure 2. Characterization of i-PTX-R837 NA. A) Size distribution of i-PTX-R837 NA. B) Zeta potential of iPTX-R837 NA. C) Morphology of i-PTX-R837 NA under TEM. D) The fluorescence peak of cells after incubated with i-PTX- R837 NA, PTX-R837 NA (both dyed with coumarin-6) for 2 hours respectively on flow cytometry histogram. E) The cellular uptake results of 4T1 cells (marked with DAPI) after incubated with i-PTX-R837 NA (dyed with coumarin-6) at different time points (0.5h, 2h, 4h). F) Cell viability of 4T1 cells treated with iPTX-R837 NA at different concentrations. (Scale bar=50 μm)


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Figure 3. The drug release, biodistribution and pharmacokinetics study of i-PTX-R837 NA. A, B) Drug release of R837 A) or PTX B) from R837-acSS-SA or PTX-acSS-SA in buffer with or without DTT. C) The biodistributions of PTX-R837 NA and i-PTX-R837 NA in main organs and tumor site at different time points (2h, 8h, 24h). D) Pharmacokinetics of drugs in plasma. E) The mole ratio of PTX/R837 and PTX-acSSSA/R837-acSS-SA in plasma after injected with PTX+R837, i-PTX-R837 NA respectively. F) The mole ratio of PTX-acSS-SA/R837-acSS-SA at tumor site after injected with i-PTX-R837 NA.



Figure 4. The cellular apoptosis and DCs stimulation experiments. A) Representative dot plot for cellular apoptosis (upper right quadrant and lower right quadrant represent the apoptotic cells). B) The schematic diagram of co-incubation of apoptotic tumor cells and DCs. C) The antigen expression of DCs after incubated with 4T1 cells, which were treated by PTX, R837, i-PTX-R837 NA respectively (Proportion of CD80+CD86+ CD11C+ DCs were detected).


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Figure 5. The anti-tumor effect of i-PTX-R837 NA on breast cancer in vivo. A) Tumor volume growth trend of mice. B) Tumor weights of mice in each group. C) Mouse body weight-time change trend. D) Representative histological sections of tumors stained with TUNEL. E) Representative histological sections of tumors stained with anti-CD31. F) Representative histological sections of main organs stained with HE. (Scale bar=50 μm).



Figure 6. The immune protective effect. A) Schematic diagram of tumor cells re-inoculation and dosing schedule. B) The secondary tumor volumes-time curve. C, D) The typical dot plots of tumors from NS pretreated mouse, PTX+R837 pre-treated mouse and i-PTX-R837 NA pre-treated mouse. The numbers in the top right area indicate the percentage of CD3+ CD8+ C) or CD3+ CD4+ cells D).


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Modular engineering of targeted dual-drug nano-assembles

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