antiangiogenic combination therapy through improving targeting drug release

Accepted Manuscript pH-activatable Polymeric Nanodrugs Enhanced Tumor Chemo/Antiangiogenic Combination Therapy through Improving Targeting Drug Release Hui Xiong, Yuanyuan Wu, Zhijie Jiang, Jianping Zhou, Min Yang, Jing Yao PII: DOI: Reference:

S0021-9797(18)31233-5 https://doi.org/10.1016/j.jcis.2018.10.039 YJCIS 24194

To appear in:

Journal of Colloid and Interface Science

Received Date: Revised Date: Accepted Date:

27 July 2018 26 September 2018 15 October 2018

Please cite this article as: H. Xiong, Y. Wu, Z. Jiang, J. Zhou, M. Yang, J. Yao, pH-activatable Polymeric Nanodrugs Enhanced Tumor Chemo/Antiangiogenic Combination Therapy through Improving Targeting Drug Release, Journal of Colloid and Interface Science (2018), doi: https://doi.org/10.1016/j.jcis.2018.10.039

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pH-activatable Polymeric Nanodrugs Enhanced Tumor Chemo/Antiangiogenic

Combination

Therapy

through

Improving Targeting Drug Release Hui Xionga, Yuanyuan Wua, Zhijie Jianga, Jianping Zhoua, Min Yangb*, Jing Yaoa* a

State Key Laboratory of Natural Medicines and Jiangsu Key Laboratory of Druggability of Biopharmaceuticals, Department of Pharmaceutics, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China b

Jiangsu Institute of Nuclear Medicine, Molecular Imaging Center, Jiangsu Institute of Nuclear Medicine, 20 Qianrong Rd, Wuxi 214063, China *Corresponding Author: Prof. Jing Yao, E-mail: [email protected] (J. Yao); Min Yang, E-mail: [email protected] (M. Yang)

Keywords. pH-activatable, polymeric nanodrugs, low molecular weight heparin, ursolic acid, chemo/antiangiogenic combination therapy.

Abstract It was widely accepted that polymeric nanodrugs held superiority in enhancing antitumor efficacy, reducing side effect and achieving better long-term prognosis. However, there still existed disputes that whether their therapeutic efficiency was closely related to insure effective release of hydrophobic drug located in their hydrophobic core in tumor site. In order to investigate this controversy, we constructed two polymeric nanodrugs (pH-activatable sLMWH-UOA and non-sensitive LMWH-UOA) with low molecular weight heparin (LMWH) and ursolic acid (UOA) for chemo-and anti-angiogenic combination therapy in hepatocellular carcinoma. The degradation ratio of pH-activatable sLMWH-UOA increased by 33% compared with non-sensitive LMWH-UOA in in vitro degradation study. Besides, confocal microscopy captured that sLMWH-UOA could effectively release drug in acidic microenvironment of lysosome while LMWH-UOA nearly could not. More

importantly, in contrast with LMWH-UOA, sLMWH-UOA presented pH-dependent cytotoxicity, indicating that promoting drug release played a key role in enhancing the cytotoxicity of polymeric nanodrugs. Additionally, in vivo pharmacodynamic evaluation showed that although non-sensitive LMWH-UOA had benefited from enhanced tumor targeting drug delivery ability to achieve absolute advantage over free drug combination therapy in antitumor combination therapy, sLMWH-UOA could acquire further optimized combined therapeutic effect with better drug release in tumor. All above, application of tumor-triggered chemical bonds to construct polymeric nanodrugs held vast prospect for improving the therapeutic efficiency for tumor cells.

Introduction The use of polymer-drug conjugates-based nanosystems (or called polymeric nanodrugs) was an established approach for improvement of cancer combination therapy. There were a lot of polymers which not only showed adjuvant antitumor activities but also could form tumor specific nanosystems through being conjugated to hydrophobic antitumor drugs [1-3]. In this way, the pharmacokinetics of the drug attaching to the polymeric carrier could also be modulated [2, 4]. For example, the long-circulating effect could be realized by covalently bonding chemotherapeutic agents to polyethylene glycol (PEG) [5]. Besides, active tumor targeting accumulation could also be achieved by conjugating hyaluronic acid to antitumor drugs [6]. Early clinical trials showed several advantages of polymer-drug conjugates over the corresponding antitumor drugs, including enhanced antitumor efficacy, reduced side effects, ease of drug administration, improved patient compliance and better long-term prognosis [3]. However, there was a controversy whether the therapeutic efficiency of polymeric nanodrugs was closely related to insure effective release of hydrophobic drug located in the hydrophobic core of polymeric nanodrugs in tumor site. Some previous studies showed that polymeric nanodrugs without sensitive response linker for smart drug release also could achieve combination therapy effect as intact nanodrugs [7, 8]. Xiong et al. have conjugated doxorubicin (DOX) to polyethylene oxide-b-

polycaprolactone (PEO-b-PCL) core using the stable amide bonds to construct nanoparticles

decorated

with

αvβ3

intergrin-targeting

ligand(RGD)

called

RGD4C-PEO-b-P(CL-Ami-DOX) [9]. The results of cytotoxicity test showed that RGD4C-PEO-b-P(CL-Ami-DOX) could significantly enhance the cytotoxic response of DOX, suggesting that non-sensitive polymeric nanodrugs which might keep intact in tumor cells could still remain and even improve the antitumor activities of free drug [9]. Contrastively,

other

studies

showed

that

it

was

necessary to

introduce

microenviroment-sensitive linkers (e.g. pH-sensitive or enzyme-sensitive bonds) to connect polymer and hydrophobic drug for accelerating the drug release from polymeric nanodrugs in tumor site and thereby achieving prominent antitumor efficiency [10-15]. To explore the above controversy, we constructed a range of polymer-drug conjugate based nanoparticles with or without pH-sensitive linkers. Anti-angiogenesis therapy was considered to be a promising approach for antitumor therapy since that tumor vessels were necessary for transporting nutrient required for tumors growth [16, 17]. Inhibition of the pro-angiogenic growth factors such as vascular endothelial growth factor (VEGF) offered remarkable advantages to achieve effective antiangiogenic therapy for most tumor types [17]. Interestingly, drugs that specifically target VEGF pathway such as bevacizumab, sunitinib and aflibercept have shown remarkable therapeutic activity in cancer treatment [18]. However, it was also reported that monotherapy using anti-angiogenic drug alone could not achieve satisfactory antineoplastic efficacy to benefit the patients’ life in clinic [19]. Accordingly, it was necessary to develop therapeutic regimen by combining anti-angiogenic drug and chemotherapeutic agent for enhancing their anticancer efficiency through synergism [20]. Low molecular weight heparin (LMWH) is a water-soluble

natural

mucopolysaccharide

with

non-cytotoxic

and

good

biocompatibility [21, 22]. It was known that LMWH could inhibit tumor angiogenesis by interacting with growth factors such as the VEGF and basic fibroblast growth factor (bFGF) that could promote tumor angiogenesis [22]. In addition, modifying LMWH

with hydrophobic moiety was found to not only improve its anti-angiogenesis activities but also reduce its hemorrhagic risk [23]. Ursolic acid (UOA), a pentacyclic triterpenoid with pleiotropic biological effects, has demonstrated the capability to inhibit key steps of angiogenesis, including endothelial cell proliferation, migration and differentiation [24, 25]. Previous study illustrated that modifying LMWH with UOA could effectively improve the anti-angiogenesis activities and antitumor efficiency of nanodrugs in vivo and in vitro through combining UOA induced inhibition of the downregulation of matrix metalloproteinase (MMP) activity and LMWH caused VEGF signal pathway blockade [26]. Moreover, UOA also had strong cytotoxicity against tumor cells through blocking the G0/G1 cycle [27]. Especially, after modifying the 17-COOH of UOA, UOA derivatives further gained remarkably enhanced cytotoxicity against HepG2 cells through improving caspase-3 enzyme activity and strengthening tumor cell cycle blockage [28]. It was also known that the treatment efficiency of liver cancer was always hampered by microvascular invasion [29]. Accordingly, modifying LMWH with UOA and thereby forming polymeric nanodrugs will provide new opportunities for liver cancer treatment through simultaneously inhibiting tumor blood vessels and tumor cells proliferation. Furthermore, in order to investigate whether it could significantly strengthen the combination antitumor efficiency of LMWH and UOA through accelerating the drug release in tumor, a pH-activatable polymeric nanodrugs(sLMWH-UOA) and a non-sensitive polymeric nanodrug (LMWH-UOA) were prepared. Specifically, schiff base (-CH=N-) with pH-triggered hydrolysis properties and non-sensitive amido bond were employed to connect the LMWH and UOA to form sLMWH-UOA and LMWH-UOA respectively [30]. As displayed in Scheme 1, compared with non-sensitive LMWH-UOA, sLMWH-UOA remained stable before reaching tumor sites and was disassembled after entering into lysosomes with acid microenvironment to spontaneously release UOA and LMWH for exerting dual inhibition of angiogenesis and cytotoxicity. In this study, the structures, particle sizes and drug release behaviors

of both LMWH-UOA and sLMWH-UOA were characterized. Besides, we mainly compared the capacity of LMWH-UOA and sLMWH-UOA for inhibition of angiogenesis and tumor proliferation in vitro and in vivo to deduce the correlation between the drug release behaviors and the therapeutic efficiency of polymeric nanodrugs.

Scheme 1. Schematic design of LMWH-UOA and sLMWH-UOA. (A) The main components of LMWH-UOA and sLMWH-UOA: low molecular weight heparin (LMWH) and ursolic acid (UOA). (B) LMWH-UOA and sLMWH-UOA selectively accumulated in tumor site based on enhanced permeability and retention (EPR) effect. (C) LMWH-UOA and sLMWH-UOA with different degradation behavior in tumor cell could inhibit the vascular endothelial growth factor (VEGF) secretion of tumor cells at different degrees. (D) The anti-angiogenesis mechanism of LMWH-UOA and sLMWH-UOA. Experiments Materials and methods

UOA was purchased from Wuhan Yuancheng Co-created Technology Development Co. Ltd. (Wuhan, China) and LMWH (100 IU/mg, average molecular weight 5795 Da) was obtained from Nanjing University. p-Hydroxybenzaldehyde (PHBA) was acquired from

Aladdin

Industrial

Corporation

(Shanghai,

China).

1-ethyl-3-

(3-dimethylaminopropyl)-carbodiimide (EDC), N-Hydroxysuccinimide (NHS), N, N-dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) were provided by Sinopharm Chemical Reagent Co. Ltd. (Nanjing, China). Acetone was received from Nanjing Chemical Reagent Co. Ltd. (Nanjing, China). Dimethyl sulfoxide (DMSO), N,N-Dimethylformamide (DMF) and tetrahydrofuran (THF) were from Shanghai Lingfeng Chemical Reagent Co. Ltd. (Shanghai, China). All other chemicals were of analytical grade and were used without further purification. Cell culture Human hepatocellular carcinoma cell line HepG2 were cultured in Dulbecco's Modified Eagle Medium(DMEM) containing 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 mg/mL streptomycin at 37 °C using humidified 5% CO2 incubator (311, Thermo Scientific, USA). All cells grown in medium were sub-cultivated every 2 days by using 0.25% (w/v%) trypsin at a split ratio of 1:3. Animals Balb/c mice (5 weeks, 19-24 g) were received from Qinglong mountain animal breeding center (Nanjing, China). All animal experiments were conducted in accordance with a protocol approved by the guidelines of the Institutional Animal Care and Use Committee of China Pharmaceutical University (Nanjing, China). In order to get tumor-bearing mice, approximately 1×106 of Heps cells were inoculated subcutaneously in the right flanks region of the mice. The tumor growth was measured by the caliper and tumor volume was calculated by using the formula:

Synthesis and characterization of sLMWH-UOA

The sLMWH-UOA conjugates were synthesized by coupling UOA-OH with LMWH. Firstly, UOA dissolved in THF was reacted with DCC and NHS (molar ratio 1:1.2:1.2) for 24 h at room temperature under nitrogen atmosphere, then filtrated off the precipitated dicyclohexylurea(DCU) to obtain the activated UOA solution. The resulting solution was precipitated in n-hexane for 6 h, filtered and dried by vacuum dryer at room temperature. Subsequently, ethylenediamine was added dropwise into UOA solution which was dispersed in DMF and reacted for 6 h, hereafter, the reaction mixture was precipitated in saturated brine, filtered off, washed several times with distilled water to get UOA–NH2. Secondly, UOA–NH2 was dissolved in absolute ethyl alcohol and added dropwise into p-Hydroxybenzaldehyde which was also dissolved in absolute ethyl alcohol with appropriate glacial acetic acid as a catalyzer at 90°C in oil bath for 5h to obtain the mixture with vacuum rotary evaporation. The molar ratio of UOA–NH2 to p-Hydroxybenzaldehyde was 1:2. Afterwards, the mixture was washed with anhydrous ether, filtered off and dried to obtain UOA-OH. Finally, the mixed solution of LMWH, EDC, NHS was added dropwise into UOA-OH (8 mmol) which was dissolved in methanamide with DMAP and reacted for 24 h, then precipitated in excess of cold acetone, washed several times with acetone and filtered off to get sLMWH-UOA. The reactant was dialyzed against DI water for 36 h with a dialysis membrane (MWCO 3500), followed by freeze-drying to obtain sLMWH-UOA. Thereafter, sLMWH-UOA was obtained after solvent evaporation and stored at 4 ℃ for further use. The mole grafting ratio of UOA-OH to LMWH was determined by UV spectrophotometer. LMWH-UOA nanodrug was prepared according to previously reported method [26]. The critical association concentration (CAC) of sLMWH-UOA was determined by fluorescence spectroscopy. Briefly, 6.0×10−6 M pyrene was dissolved in 1 mL acetone to form a solution, and then transferred to a series of 10 mL brown volumetric flasks to evaporate all the acetone. After that , an appropriate amount of aqueous solution was added in sLMWH-UOA respectively to acquire the final nanodrug with concentration of 3.2, 6.4, 16, 32, 80, 160, 400, 1000, 2000 mg/mL, followed by

sonicating for 30 min at 100 W. Then, all the samples were incubated under 70 ℃ for 1h and placed to chill down overnight at room temperature. Pyrene fluorescence spectra were acquired by using a RF-5301 PC fluorescence spectrophotometer (Shimadzu, Japan). The CAC was estimated as the cross-point when extrapolating the intensity ratio I338/I333 at low and high concentration regions. LMWH-UOA and sLMWH-UOA nanodrug were characterized with respect to their particle sizes and polydispersity index (PDI) by dynamic light scattering (DLS) using a Malvern Zetasizer Nano-ZS90 (Malvern instruments, UK). All DLS measurements were performed at a concentration of 1.0 mg/mL at 25 ℃ and at a scattering angle of 90°. The morphology of sLMWH-UOA nanodrug was characterized by transmission electron microscopy (H-600, Hitachi, Japan). In vitro stability evaluation of sLMWH-UOA nanodrug The acid-sensitive property of sLMWH-UOA nanodrug was evaluated with qualitative

experiment

by

loading

1,1‘-dioctadecyl-3,3,3’,3‘-

tetramethylindotricarbocyanine iodide (DiR) (Figure 1A) and quantitative experiment by loading coumarin-6(C6) (Figure 1B). The particle size and PDI of sLMWH-UOA nanodrug dissolved in phosphate buffer solutions with different pH at different time were measured by DLS to investigate the stability of sLMWH-UOA nanodrug in vitro. 1 mL LMWH-UOA and sLMWH-UOA (containing 1mg UOA) was added into 3 mL plasma or 3 mL Heps tumor homogenate. The resulting solution was incubated at 37°C and 70-80 rpm shaking speed. At the pre-set time points (12, 24, 48, and 72 h), the samples were extracted with ethyl acetate for further measurement.

Figure 1. The chemical structure of DiR(A) and C6(B). In vitro cellular uptake study of nanodrug The cellular uptake of nanodrug was studied by using confocal laser scanning microscopy. Firstly, HepG2 cells were placed in the confocal dishes (NEST, China) at a density of 105cells/dish and incubated for 24 h. After that, the cells were treated with free C6, C6-loaded LMWH-UOA and C6-loaded sLMWH-UOA (the concentration of C6 is 300 ng/ml), respectively. At pre-determined intervals(2h, 6h, 24h), the cells were washed with PBS thrice, then stained the lysosome by 500μL Lyso-Tracker Red for 30 min, fixed with 4 % paraformaldehyde for 20 min followed by adding Hoechst 33258 to stain the cell nuclei. At last, the cells were observed using confocal laser scanning microscopy (Leica, Germany). In vivo imaging analysis When the tumor volume reached around 500 mm3, the Heps tumor-bearing mice were randomly divided into three groups and administered with free DiR, DiR-loaded LMWH-UOA nanodrug and DiR-loaded sLMWH-UOA nanodrug, respectively. At pre-determined time interval (1, 2, 4, 8, 12 and 24 h) after injection, fluorescence images of the mice were captured by in vivo imaging system (FX PRO, Kodak, USA). In vitro cytotoxicity studies The cytotoxicity of sLMWH-UOA nanodrug was performed using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. Briefly, the HepG2 cells were seeded at a density of 5×103 cells/well in a 96 well plate and incubated for 24 h. Subsequently, the cells were treated with LMWH, UOA, LMWH plus UOA, LMWH-UOA and sLMWH-UOA nanodrug (at equivalent LMWH concentration of 3.125, 6.25, 12.5, 25, 50, 100, 200, 400 μg/mL, respectively) for 48 h. Afterwards, MTT solution (2.5 mg/mL in PBS) was added to each well at dose of 40μL and the cells were incubated further for 4 h at 37 °C. Hereafter, the old media was taken off and DMSO was added to dissolve formazan crystals. Microplate reader (ELX800

Biotek®, USA) was employed to measure the absorbance at 490 nm and the rate of cell viability was calculated according to the following formula:

In vivo matrigel plug assay The matrigel plug angiogenesis assay is a in vivo technique to detect the newly formed blood vessels in the transplanted gel plugs in mice, which has been proved to be a relatively quick and easy method to evaluate both angiogenic and antiangiogenic compounds in vivo [31]. Accordingly, matrigel plug assay was performed to evaluate anti-angiogenic effect of LMWH-UOA and sLMWH-UOA in vivo. Firstly, Matrigel (growth factor-reduced and phenol redfree, BD Bioscience) was thawed at 4°C overnight. Secondly, 500 μL Matrigel was mixed with 50 μL bFGF (at a final concentration of 77 ng/mL) and 100 μL PBS containing UOA, LMWH, LMWH plus UOA, LMWH-UOA or sLMWH-UOA (at a final concentration of 308 ng LMWH/mL) to get the mixture which was injected subcutaneously into the flanks of mice (n=5). The whole operations above were performed under ice bath strictly. The groups of PBS and bFGF-PBS were used as negative and positive controls, respectively. After 10 days, mice were sacrificed, matrigel plugs were removed to take photo and then homogenized in hypotonic lysis buffer. After being centrifuged for 10 min at 10,000 rpm, a constant volume of supernatant was incubated in 0.5 mL of Drabkin's solution (Sigma, St. Louis, MO.) for 15 min at room temperature and measured absorbance at 540 nm with Drabkin's solution as a blank. The relative hemoglobin content was calculated according to the following formula:

In vivo anti-tumor activity Heps cells were injected s.c. into the right flank of male mice (5 to 6 weeks old) to obtain Heps tumor-bearing mice models. When tumor became palpable (about 50 mm3), these mice were randomly distributed into 6 groups and were administered one of the

following formulations via tail vein injection every two days: (i) saline, (ii) LMWH at 20 mg/kg, (iii)UOA, (iv)LMWH plus UOA at a 20 mg LMWH /kg, (v)LMWH-UOA at a 20 mg LMWH /kg and (vi)sLMWH-UOA at a 20 mg LMWH /kg. Tumor size and body weight were measured every day. After 10 days, blood samples were collected to determine the levels of biochemical markers of liver function (alanine aminotransferase (ALT) and aspartate aminotransferase (AST)). After that, the mice were sacrificed and tumor tissues were harvested and weighed. Some of tissue samples were fixed in 4% (v/v) formaldehyde in PBS (pH 7.4) for hematoxylin and eosin (H&E) staining and immunohistochemistry analysis. The tumor inhibition rate (IR), another indicator of antitumor efficacy, was calculated using the following formula:

Ws and Wf stand for the average tumor weight of control group and treatment group, respectively. Western-blot assay The tumor tissues excised from the mice were cut into pieces. Then 1 mL of radio immunoprecipitation

assay

buffer

(RIPA)

lysate

containing

1

mM

phenylmethanesulfonyl fluoride (PMSF) was added and the sample was lysed with a tissue disrupter (0 °C). After being ultrasounded for 10-15 s, this mixture solution was centrifuged at 12000 rpm for 10 min (4 °C) to get the supernatant. Bicinchoninic acid (BCA) protein assay kit (Bicinchoninic acid, Thermo, USA) was used to quantify

protein, subsequently samples were eluted with sodium dodecyl sulfate (SDS) buffer, separated on SDS- polyacrylamide gel electrophoresis (PAEG), and electroblotted onto polyvinylidene fluoride (PVDF) membranes. The membrane was placed into blocking buffer for 1 h, washed 3 times with tris buffered saline tween buffer (TBST), treated with primary antibody overnight. After being washed 3 times again, the membrane was dealt with horseradish peroxidase (HRP)-labeled secondary antibody for 1h at room temperature. Immunoreactive protein bands were checked by gel imaging system (Bio-Rad, USA).

Safety evaluation of sLMWH-UOA nanodrug Hemolysis assay was performed to access the safety of sLMWH-UOA nanodrug in vivo. Rabbit red blood cells (RBCs) were obtained by centrifuging rabbit whole blood at 3000g for 10 min, removing the supernatant, washing the RBC pellet with normal saline and repeating until the supernatant was clear. Then, the RBCs were resuspended with normal saline to a concentration of 2% (v/v). Subsequently, DMSO, UOA, LMWH, LMWH plus UOA, LMWH-UOA and sLMWH-UOA were diluted with normal saline to obtain different concentrations, incubated with 2% RBCs at 37 °C for 1 h, and then centrifuged at 3000g for 10 min. Absorbance of hemoglobin in the supernatant was measured at 540 nm by UV-Vis spectrophotometry. The observed hemolysis of RBCs in normal saline and water were respectively regarded as negative (0% hemolysis) and positive (100% hemolysis) controls. Statistical analysis The data are given as Mean

S.D. Statistical significance was tested by two-tailed

Student’s t-test or one ANOVA. Statistical significance was set at *P <0.05 while extreme significance was set at **P<0.01.

Results and discussion Synthesis and characterization of sLMWH-UOA LMWH-UOA was synthesized by chemically conjugating hydrophobic UOA to hydrophilic LMWH using non-pH sensitive ethylenediamine as the linker (Figure 2A) [26]. Besides, the amphiphilic pH-sensitive sLMWH-UOA conjugates were synthesized by chemically conjugating hydrophobic UOA to hydrophilic LMWH using ethylenediamine and p-hydroxybenzaldehyde as the linker (Figure 2B). The structures of LMWH-UOA and sLMWH-UOA were characterized using 1H-NMR as shown in Figure S1. Compared with LMWH, the spectrum of LMWH-UOA showed the newly formed amide linkage between LMWH and UOA (7.8 to 8.4 ppm) and the

characteristic peaks of UOA (1.0-1.5 ppm), verifying the successful synthesis of LMWH-UOA. Moreover, compared with LMWH-UOA, the 1HNMR spectrum of sLMWH-UOA further showed the newly formed pH-sensitive schiff base (-CH=N-) group appeared at 8.03 ppm and the proton peak of benzene ring of p-hydroxybenzaldehyde emerged at 6.92 ppm, indicating that UOA was successfully conjugated to LMWH through pH-sensitive linker. The degree of substitution (DS) of UOA in LMWH-UOA and sLMWH-UOA conjugates were controlled at 13.2% and 12.3 % (molar ratio) respectively to ensure that the structure of two graft copolymers was analogical. Furthermore, the particle size was tested by dynamic light scattering method and the results illustrated that the particle size of sLMWH-UOA was 223.1±0.9 nm which was slightly higher than that of LMWH-UOA (194.7 ± 3.7 nm). According to the previous study, nanoparticles with particle size of 100-300 nm always have similar in vivo tumor targeting accumulation behaviors , meaning that this difference in particle size between LMWH-UOA and sLMWH-UOA would not obviously affect their in vivo tumor distribution [32]. Besides, the CAC of two nanodrugs were tested. As showed in Figure S2, the CAC of sLMWH-UOA was 38.30 μg/mL which was remarkable lower than that of LMWH-UOA (269.15 μg/mL), indicating that sLMWH-UOA remained more stable than LMWH-UOA in dilute conditions, which might result in good stability after intravenous injection into the systemic circulation.

Figure 2. The synthesis route of sLMWH-UOA(A) and LMWH-UOA(B).

pH-activatable degradation behavior of sLMWH-UOA nanodrug in vitro Shiff base bond (-CH=N-) is unstable under acidic conditions such as tumor environment or endo-lysosome, which makes it is easily to be hydrolyzed into aldehyde or ketone and amine [30]. Accordingly, we assumed that hydrolysis of the shiff base bond connecting LMWH and UOA could induce the disintegration of the intact structure of LMWH-UOA-based nanodrug in tumor site and achieve tumor-triggered drug release. In order to investigate the pH-sensitive degradation behavior of sLMWH-UOA nanodrug, we firstly utilized TEM to observe the morphological changes of sLMWH-UOA nanodrug in different pH PBS. As shown in Figure 3A, sLMWH-UOA nanodrug displayed nearly spherical shape in PBS (pH 7.4). However, once sLMWH-UOA nanodrug was incubated in PBS (pH 4.5), the shape of sLMWH-UOA became irregular with remarkably increased particles size, indicating that the nanostructure of sLMWH-UOA undergo disintegration in acidic medium. This hydrolysis of shiff base linker in sLMWH-UOA triggered release of the UOA by inducing disassembly of hydrophobic UOA core in sLMWH-UOA nanodrug. We also used sLMWH-UOA to load DiR (a hydrophobic fluorescent dye) to verify its pH-activated profile. DiR-loaded sLMWH-UOA nanodrug was incubated in PBS at different pH. As depicted in Figure 3B, the solution of DiR-loaded sLMWH-UOA nanodrug in PBS (pH 7.4) still clarified after 24h. In contrast, after being incubated with PBS (pH 4.5), there were obvious blue DiR precipitates at the bottom of the PE tube. These results suggested that the loaded DiR was leaked in acidic medium, which might be caused by the pH-activated disintegration of intact structure of sLMWH-UOA under acidic medium. Figure 3C showed the fluorescence intensity changes of C6-loaded sLMWH-UOA that was incubated in PBS with different pH for 12 h. The fluorescence intensity of C6-loaded sLMWH-UOA that was incubated at pH 4.5 showed a 9.5-fold increase compared to that incubated at pH 7.4. This result further confirmed that sLMWH-UOA held pH-triggered disassembly behavior. It was revealed that once the balance between hydrophilic forces and hydrophobic forces were broken, the intact and stable structure of nanodrugs would be difficult to

maintain and thereby the particle size changed remarkably. Therefore, the changes in particle size of both LMWH-UOA and sLMWH-UOA in medium with different pH were investigated. As shown in Figure 3D and 3E, compared with sLMWH-UOA of which the particle sizes presented nearly sextuple increase after being incubated in PBS at acidic pH for 24h, the particle size of LMWH-UOA only showed slight fluctuations within 20 nm. Furthermore, both LMWH-UOA and sLMWH-UOA were incubated in plasma and tumor tissue homogenate and the particle sizes were tested to investigate the tumor-triggered nanostructure changes. As shown in Figure 3F, only about 8.88% of LMWH-UOA and 9.52% of sLMWH-UOA were degraded in plasma respectively, indicating that they could keep a stable and integral nanostructure in blood circulation system. In contrast, sLMWH-UOA showed significantly increased derogation ratio (P<0.05) than LMWH-UOA when they were incubated in tumor tissue homogenate. All these results suggested that once being distributed in acidic environment like tumor microenvironment, pH-activatable sLMWH-UOA would display a noticeable increased in particle size which is due to nanostructure changes as a result of shiff base bond linker hydrolysis, while non-sensitive LMWH-UOA remains intact. This different between sLMWH-UOA and LMWH-UOA would further lead to different UOA release pattern in tumor cells. In other words, pH-activatable sLMWH-UOA allowed for prompt release UOA in tumor and thereby promoting the UOA concentration to increase rapidly in tumor while LMWH-UOA showed a sustained release of UOA.

Figure 3. (A) TEM images of sLMWH-UOA nanodrug at pH=7.4(A-a and A-b) and pH=4.5(A-c) PBS solutions. The scale bar of A-a was 200 nm while the scale bars of A-b and A-c were 500 nm.(B) Photographs of DiR-loaded sLMWH-UOA at pH 7.4 and 4.5 for 0 h and 24 h. (C) Fluorescence intensity of C6-loaded sLMWH-UOA nanodrug at different pH phosphate buffers in a time dependent manner. Error bars indicate s.d. (n=3). (D)The particle size changes of LMWH-UOA at PBS with different pH. Error bars indicate s.d. (n=3). (E) The particle size changes of sLMWH-UOA at PBS with different pH. Error bars indicate s.d. (n=3). (F)The degradation ratio of LMWH-UOA and sLMWH-UOA in plasma and tumor tissues homogenate at different time. Error bars indicate s.d. (n=3).*P<0.05.

In vitro cellular uptake To investigate the cellular uptake and intracellular trafficking behavior of LMWH-UOA and sLMWH-UOA, C6, a dye with green fluorescence, was employed as fluorescent indicator to be loaded into the LMWH-UOA and sLMWH-UOA. The lysosomes were stained to show red fluorescence. As exhibited in Figure 4, the fluorescence intensity of both LMWH-UOA and sLMWH-UOA was intensified with time. Except that, compared with free C6, both LMWH-UOA and sLMWH-UOA group showed remarkable enhanced green fluorescence in cells at different time points, indicating that they promoted the cellular uptake of drugs. Furthermore, we also noted that compared with LMWH-UOA, sLMWH-UOA showed stronger fluorescence intensity within HepG2 at any given time point. Theoretically, since that both LMWH-UOA and sLMWH-UOA held same tumor cell targeting ability, there should be no difference in cell uptake efficiency. Presumably, the observed changes in intracellular intensity might be due to the pH-sensitive ability of sLMWH-UOA in triggered prompt release of C6. In details, compared with LMWH-UOA, sLMWH-UOA could easily disassemble in acidic lysosomes and thereby releasing C6 in cells. This inference was further attested by the observed stronger yellow fluorescence (the red fluorescent signal of lysosome overlapping with the green fluorescent signal of C6) intensity of sLMWH-UOA group than LMWH-UOA group. All above, LMWH-UOA could only promote the cellular uptake of C6 while sLMWH-UOA could further release C6 in accelerated manner owing to acidic environment that triggered intracellular disassembly and thereby resulting into high drug concentrated in tumor cells.

Figure 4. Confocal microscopy images of HepG2 cells treating with (I) free C6,(II) C6 loaded LMWH-UOA nanoparticles and (III) C6 loaded sLMWH-UOA nanoparticles after 1 h, 2 h and 6 h. All experiments were repeated three times (n=3). In vivo targeting evaluation

The tumor targetability of LMWH-UOA and sLMWH-UOA were investigated. After DiR-loaded LMWH-UOA (DiR/LMWH-UOA) and DiR-loaded sLMWH-UOA (DiR/sLMWH-UOA) were administrated intravenously into the Heps tumor xenograft mice, the in vivo biodistribution behavior was observed by non-invasive near infrared optical imaging technology. As shown in Figure 5A, obviously fluorescence signals of both DiR-loaded sLMWH-UOA and DiR-loaded LMWH-UOA could be trapped in the tumor site at 1 h and the strongest fluorescence signals appeared at 8 h post-injection, indicating that both of them held strong tumor targetability. Besides, compared with DiR group of which the fluorescence signal at tumor gradually disappeared with time, both LMWH-UOA and sLMWH-UOA group still showed remarkable fluorescence signal at 24 h, suggesting the continuous tumor retention effect of LMWH-UOA and sLMWH-UOA. Moreover, we also found that sLMWH-UOA group showed further enhanced fluorescence signal than LMWH-UOA group at various time-points. Especially, excised tumors of sLMWH-UOA group after 24 h post injection exhibited significantly stronger fluorescence intensity than that of LMWH-UOA group(P<0.05) (Figure. 5B and 5C). Theoretically, LMWH-UOA and sLMWH-UOA should have similar tumor targeting accumulation ability since they held similar physiochemical properties (i.e. surface chemical properties, nanostructure and particle size) that are considered as the main influencing factors that would impact the tumor targetability of nanoparticles. Accordingly, these differences in the intensity of fluorescent signals at the tumor site might be due to different drug release behaviors of LMWH-UOA and sLMWH-UOA. Compared with LMWH-UOA, sLMWH-UOA with pH-activatable drug release ability could release more loaded DiR at tumor site and thereby displaying significantly stronger fluorescent intensity.

Figure 5. In vivo targeting evaluation. (A) The biodistribution of free DiR (I), DiR/LMWH-UOA(II) and DiR/sLMWH-UOA (III) at 1h,2h,4h,8h,12h and 24h after injection. All experiments were repeated three times (n=3). (B) Ex vivo tumor distribution of free DiR (I), DiR/LMWH-UOA(II) and DiR/sLMWH-UOA (III) at 24h after drug injection. a,heart; b,liver; c,spleen; d,lung. All experiments were repeated three times (n=3). (C) Region-of-interest analysis of fluorescent signals from the tumors at 24h after i.v. injection. Error bars indicated s.d. (n = 3). *P<0.05. In vitro cytotoxicity study MTT assay was used to evaluate the cytotoxicity of LMWH-UOA and sLMWH-UOA against HepG2 cells. As shown in Figure 6A, LMWH had no obvious cytotoxicity against HepG2 cells while other groups with UOA showed different levels of cytotoxicity. This might because that UOA could arrest tumor cells in the G0/G1 phase and reduce the cells in S phase and thereby achieving strong cytotoxicity against tumor cells [27]. We also noted that UOA and LMWH+UOA group exhibited stronger cytotoxicity than sLMWH-UOA and LMWH-UOA, the reason of which might be that free UOA could exert its effects after entering into tumor cells by passive diffusion while nanodrugs (sLMWH-UOA and LMWH-UOA) required to further release UOA in tumor cells after being uptaken. This phenomenon is consistent with most

nanoparticles-based cellular uptake behavior [33]. Further to this, the IC50 of sLMWH-UOA (142.42 μg/mL) was 32.39% lower than LMWH-UOA (210.66 μg/mL), indicating that sLMWH-UOA with pH-activatable drug release ability showed remarkable advantages of cytotoxicity against LMWH-UOA with sustained drug release pattern. We also tested the cytotoxicity of LMWH-UOA and sLMWH-UOA in cell culture medium with different pH to further investigate whether the differences in drug release pattern would have an impact on cytotoxicity. The cell culture medium at pH 7.4 was used to simulate blood circulation and cell culture medium of pH 6.5 was employed to simulate the acidic environment of tumor [34, 35]. As shown in Figure 6B, compared with LMWH-UOA group which showed no remarkable differences of cytotoxicity when the pH of cell culture medium decreased to pH 6.5, the cytotoxicity of sLMWH-UOA was significantly enhanced(P<0.05) in cell culture medium of pH 6.5. Since that sLMWH-UOA held pH-activatable drug release property, this observed remarkable stronger cytotoxicity might be attributed to the fact that that sLMWH-UOA could release more UOA in acidic microenvironment. All these results suggested that although polysaccharide-nanodrug without tumor triggered drug release property (eg. LMWH-UOA) showed effective tumor cell proliferation inhibition effect, constructing polysaccharide-nanodrug using tumor triggered linker such as pH-sensitive shiff base bond (eg. sLMWH-UOA) could further significantly improve the treatment efficiency by accelerating the release of grafted small molecule chemotherapeutics in vivo.

Figure 6. (A) Cell viability of HepG2 cells after 48h treatment with LMWH,UOA,LMWH plus UOA,LMWH-UOA nanodrug and sLMWH-UOA

nanodrug with different concentration (3.125,6.25,12.5,25,50,100,200 and 400 μg/mL equivalent of LMWH). Error bars indicated s.d. (n=6). (B) Cell viability of HepG2 cells after 48h treatment with LMWH-UOA nanodrug and sLMWH-UOA nanodrug dissolved at pH 7.4 and pH 6.5 culture medium. Data were represented as mean±SD (n=6). *P<0.05. In vivo matriel plug assay Antiangiogenesis treatment could inhibit the growth of tumor by blocking its nutrient transportation channels. In our study, antiangiogenesis treatment was one of the two important components of combination therapy. Therefore, matrigel plug assay, a simple and effective method to evaluate the anti-angiogensis ability of drugs, was conducted to test the anti-angiogenesis effect of LMWH-UOA and sLMWH-UOA [36, 37]. bFGF was used as angiogenesis-promoting agent (positive control group) to motivate vascular proliferation [37-39]. Figure 7A showed the images of dissected matrigel plugs. The negative control group which was treated with physiological saline showed white facade while the positive control group exhibited obvious red color, suggesting the bFGF had induced hyperactive vascular proliferation in vivo. Besides, compared with the positive control groups, other group s showed different anti-angiogenic effect, of which the strongest anti-angiogenic effect was induced by sLMWH-UOA. This might be due to the fact that both LMWH and UOA could bind with the bFGF and thereby blocking the vascular proliferation. Besides, LMWH-UOA and

sLMWH-UOA

showed

further

enhanced

anti-angiogenic

effect

than

LMWH+UOA due to additive synergistic effect of LMWH and UOA when conjugated. The hydrophobic structure modification of LMWH could further enhance the anti-angiogenic activity of LMWH due to the increased steric hindrance of LMWH could enhance the binding site of LMWH and bFGF [40]. Notably, sLMWH-UOA showed significantly enhanced anti-angiogenic effect than LMWH-UOA (P<0.05) (Figure

7B),

suggesting

that

sLMWH-UOA

could

further

improve

the

antiangiogenesis treatment efficiency. The possible reason was that pH-activatable linker between LMWH and UOA was longer than non-pH-sensitive ethylenediamine

linker, which could reduce the space hindrance between LMWH and UOA and thereby facilitating the binding of LMWH derivatives and bFGF to further promote anti-angiogenic treatment efficacy.

Figure 7. (A) Representative isolated matrigel plugs images of different groups. All experiments were repeated three times (n=3). (B) Relative hemoglobin content in isolated matrigel plugs： Matrigel containing only bFGF (+control), Matrigel containing bFGF with LMWH, UOA, LMWH+UOA, LMWH-UOA nanodrug and sLMWH-UOA nanodrug. Data were represented as Mean±SD (n=3). *P<0.001 vs (+)control. #P<0.001 vs LMWH+UOA group. $P<0.001 vs LMWH-UOA group.

In vivo anti-tumor activity To investigate the feasibility of LMWH-UOA and sLMWH-UOA nanodrug for hepatic carcinoma therapy in vivo, Heps tumor xenograft mice were used to evaluate the antitumor efficiency. As exhibited in Figure 8A-8C, compared with the negative

control group, monotherapy that used LMWH with antiangiogenesis effect or UOA with tumor cell proliferation inhibition effect could only slightly inhibit the proliferation of tumors while combination therapy based on free LMWH and free UOA (LMWH+UOA group) remarkably curtailed the increase of tumor volume (P<0.05), indicating that LMWH and UOA could synergistically inhibit tumor growth. Moreover, the tumor inhibition rates of LMWH-UOA and sLMWH-UOA nanodrug were 2.04-fold and 1.52-fold than that of LMWH+UOA respectively, suggesting that LMWH-UOA and sLMWH-UOA nanodrug could further enhance the antitumor efficiency of LMWH+UOA. This might because that compared with LMWH+UOA group, both LMWH-UOA and sLMWH-UOA nanodrug could locate to tumor resulting into increased drug accumulation in tumor through EPR-effect induced passive tumor targeting ability and LMWH-mediated endocytosis [41]. More importantly, it was noted that sLMWH-UOA showed further significantly enhanced antitumor efficiency than LMWH-UOA (P<0.01). On Day 10, tumor volumes of mice in sLMWH-UOA group were 63.83% of that in the LMWH-UOA group. Besides, the tumor inhibition rate of sLMWH-UOA was significantly higher than LMWH-UOA group (P<0.05). These findings were further confirmed by the results of H&E-staining of tumor tissue sections. As exhibited in Figure 8D, compared with other groups especially LMWH-UOA group, the tumor cells of sLMWH-UOA group showed remarkably enhanced cancer cell remission such as tumor coagulative necrosis, nuclei fragmentation and intercellular blank. In summary, sLMWH-UOA showed significantly improved antitumor efficiency than non-sensitive LMWH-UOA. That is, although non-sensitive LMWH-UOA had benefited from enhanced tumor targeting drug delivery ability to achieve absolute advantage over LMWH+UOA in antitumor combination therapy in vivo, further optimized combined therapeutic effect could be acquired by sLMWH-UOA through accelerating the drug release in tumor cells. These results illustrated that the therapy efficiency of polymeric nanodrugs was closely related to effective drug release in

tumor site. Applying tumor-triggered chemical bonds to construct polymeric nanodrugs could significantly improve their therapeutic efficiency against cancer cells.

Figure 8. In vivo anti-tumor activity. (A) Tumor volume observed in Heps bearing mice treated with different groups after a schedule of multiple doses. Arrows indicated the days of injection of each formulation. The data were expressed as Mean±SD (n=5). *

P<0.05 vs. control； **P<0.01 vs. control； ##P<0.01 vs. LMWH-UOA. (B)Tumor

weight of Heps bearing mice treated with different groups. The data were expressed as Mean±SD (n=5). *P<0.05 vs. control； **P<0.01 vs. control； ##P<0.01 vs. LMWH-UOA. (C) Tumor inhibition rate of Heps bearing mice treated with different groups. The data were

expressed

as

Mean±SD

(n=5).

*

P<0.05

vs.

control； **P<0.01

vs.

control； ##P<0.01 vs. LMWH-UOA. (D) HE staining of tumor sections from Heps tumor-bearing mice treated with 0.9% NaCl solution, LMWH, UOA, LMWH+UOA, LMWH-UOA and sLMWH-UOA.

The in vivo study of anti-angiogenesis mechanisms In order to investigate the anti-angiogenic effect of sLMWH-UOA and LMWH-UOA, CD31 which was involved in the adhesion cascade between endothelial cells during angiogenesis was used to mark the cytoplasmic endothelial cells [42]. After the antitumor efficiency study, the tumor sections from different groups were stained with anti-CD31 antibody for microvessel density (MVD) analysis. As shown in Figure 9A, compared with the control group, all formulations had showed different anti-angiogenesis effect. Besides, the density of anti-CD31 positive microvessels (brown color) of LMWH+UOA group was further lower than that of either free LMWH or UOA, indicating that LMWH and UOA had additive synergistic effects on inhibition of angiogenesis. Moreover, as depicted in Figure 9B, further statistical analysis of MVD showed that sLMWH-UOA and LMWH-UOA had significantly improved anti-angiogenesis effect than LMWH+UOA groups, which might be benefited from the enhanced tumor targeting accumulation of LMWH and UOA that was induced by EPR effect. Notably, sLMWH-UOA with pH-activatable drug release property showed remarkably enhanced anti-angiogenesis effect than LMWH-UOA, suggesting that effective drug release at tumor site could further facilitate the combination therapy based on LMWH and UOA. Vascular endothelial growth factor (VEGF) could interact with the receptor on the surface of endothelial cells and thereby inducing angiogenesis [43, 44]. Accordingly, we also tested the VEGF expression after in vivo antitumor efficiency by western blotting assay to further analyze the anti-angiogenesis mechanisms. Consistent with the above MVD assay , the results of VEGF expression ( Figure 9C and 9D) showed that sLMWH-UOA group held lower VEGF expression compared with other group (P<0.05 or P<0.01), indicating that the intense anti-angiogenesis effect of sLMWH-UOA was attained by its VEGF down regulation effect. Taken together, compared with monotherapy of LMWH or UOA, the combination of LMWH and UOA could synergistically inhibit the angiogenesis of tumor through down regulating the VEGF expression synergistically thus blocking the supply of

nutrients to tumor cells. Moreover, this combination therapy efficiency could be further enhanced by LMWH-UOA and sLMWH-UOA with tumor targeting drug accumulation effect. More importantly, we also found that this synergetic anti-angiogenesis based on LMWH and UOA combination therapy could be further strengthened by incorporating a pH sensitive moiety that enhanced the targetability release of drug at tumor site.

Figure 9. Antiangiogenic effect of LMWH, UOA, LMWH plus UOA, LMWH-UOA and sLMWH-UOA nanoparticles in tumor bearing mice. (A) Microphotographs of anti-CD31 antibody immunostaining against microvessels on the isolated tumor tissue sections. All experiments were repeated three times (n=5). (B) The microvessels density assay of tumor tissues. Data were expressed as Mean±SD (n=5). *

P<0.05;**P<0.01. (C) Western-blot assay was performed to examine the expression of

VEGF. All experiments were repeated three times (n=3). (D) The integrated density ratio of VEGF and GAPDH. Data were expressed as Mean±SD (n=3). *

P<0.05;**P<0.01.

In vivo preliminary toxicity evaluation and hemolysis test assessment Hepatotoxicity was one of the most common adverse reactions during chemotherapy and anti-angiogenesis treatment, which would reduce the compliance of patients and thus resulting in treatment failure. Accordingly, we also preliminarily evaluated the potential toxicity of different formulations, especially the hepatotoxicity after in vivo antitumor efficiency study. As shown in Figure 10A, no abnormal body weight reductions were observed in any group during the treatment, indicating low toxicity of all the formulations. Besides, hepatotoxicity was evaluated by testing the serum levels of AST and ALT. The increasing levels of ALT and AST always indicated liver impairment as described previously [45]. As shown in Figure 10B and 10C, compared with the control group, all tested groups showed no significant differences of AST and ALT levels. Moreover, the AST and ALT levels of all tested groups were lower than that of control group, indicating that LMWH and UOA as well as nanosytems constructed with them possessed unnoticeable side effects on liver. Further to this, the hemolysis test was conducted to assess the safety of intravenous administration of LMWH derivatives. As shown in Figure 10D, the hemolytic rates of all groups were less than 3%, indicating that all the tested materials, especially the LMWH derivatives (LMWH-UOA and sLMWH-UOA), posed no detectable disturbance to erythrocyte membranes as well as no hemolysis risk [45]. Besides, we also noted that the hemolytic rate of LMWH was negative. These might because that LMWH could inhibit the complement activation and thereby inhibiting hemolysis [46]. Overall, LMWH-UOA and sLMWH-UOA exhibited excellent antitumor efficacy as well as low toxicity.

Figure 10. In vivo toxicity evaluation and hemolysis test assessment. (A) Body weight observed in Heps bearing mice treated with different groups. The data were expressed as Mean+SD (n=5). (B)ALT values of the serum from tumor bearing mice treated with LMWH, UOA, LMWH+UOA, LMWH-UOA, sLMWH-UOA. Data were expressed as Mean±SD (n=5). (C) AST values of the serum from tumor bearing mice treated with LMWH, UOA, LMWH+UOA, LMWH-UOA, sLMWH-UOA. Data were expressed as Mean±SD (n=5). (D) The hemolysis ratio of LMWH, UOA, LMWH+UOA, LMWH-UOA and sLMWH-UOA. Data were represented as Mean±SD (n=3).

Conclusions A lot of polymeric nanodrugs prepared through connecting

hydrophobic

chemotherapeutics to multifunctional polymers incorporating either stimuli-responsive or non-sensitive covalent linkers had shown encouraging combination therapy effect in vivo [47, 48]. However, previous studies did not enunciate whether the therapeutic efficiency of polymeric nanodrugs was closely related to their drug release behavior

in tumor site [49-51]. In this study, in order to explore this issue, we synthesized two different amphiphilic polymer-drug conjugates-based nanodrugs (sLMWH-UOA and LMWH-UOA) with LMWH and UOA. Among them, sLMWH-UOA was synthesized through connecting LMWH and UOA with acid-sensitive schiff base linker while LMWH-UOA was constructed by connecting LMWH and UOA with non-sensitive linkers. In vitro pH-activatable degradation study showed that sLMWH-UOA could be effectively hydrolyzed and release drug in lysosome with acidic microenvironment while LMWH-UOA could not. In in vitro and in vivo pharmacodynamics evaluations, sLMWH-UOA showed further significantly enhanced tumor inhibition effect as well as anti-angiogenesis therapy efficiency than LMWH-UOA, indicating that promoting targeting drug release of polymeric nanodrugs played a crucial role in enhancing the combination therapy efficiency of polymer and hydrophobic chemotherapeutics. Besides, it is also important to note that LMWH-UOA showed its considerable advantages than simple combination therapy of free LMWH and UOA, reminding us non-sensitive polymeric nanodrugs might take effect as a structurally complete nanodrug and could keep the activities of polymer and chemotherapeutics. In summary, our study gives a new angle on thinking about the design of polymeric nanodrugs. That is, further benefits for combination therapy efficiency of polymeric nanodrugs could be obtained through intelligent drug release design and thereby achieving additive synergistic treatment efficiency. Besides, balance between complex stimuli-responsive chemical conjugate designs cost and pharmacodynamic benefits should be also weighed cautiously when non-sensitive polymeric nanodrugs could already achieve expected therapy efficiency.

Acknowledgments This work was supported by the National Natural Science Foundation of China (No.81773655), the 12th of Six Talent Peak Foundation of Jiangsu Province (YY-001), the “333” Project Talent Training Fund of Jiangsu Province (BRA2017432), the Open

Project of Jiangsu Key Laboratory of Druggability of Biopharmaceuticals (JKLDBKF201702), and the Project Program of State Key Laboratory of Natural Medicines, China Pharmaceutical University (JKGQ201107, SKLNMZZJQ201605). We appreciate Dickson Pius Wande for editing the manuscript.

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Graphical abstract