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Biological and Medical Applications of Materials and Interfaces

Injectable, Biodegradable, Thermosensitive Nanoparticlesaggregated Hydrogel with Tumor-specific Targeting, Penetrating and Releasing for Efficient Postsurgical Prevention of Tumor Recurrence Huiming Liu, Xiaoguang Shi, Di Wu, Frewein Kahsay Khshen, Liandong Deng, Anjie Dong, Weiwei Wang, and Jianhua Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b01987 • Publication Date (Web): 09 May 2019 Downloaded from http://pubs.acs.org on May 9, 2019

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ACS Applied Materials & Interfaces

Injectable,

Biodegradable,

Thermosensitive

Nanoparticles-aggregated

Hydrogel with Tumor-specific Targeting, Penetrating and Releasing for Efficient Postsurgical Prevention of Tumor Recurrence Huiming Liu†, Xiaoguang Shi†, Di Wu†, Frewein Kahsay Khshen †,‡, Liandong Deng†, Anjie Dong†,§, Weiwei Wang*,∥, Jianhua Zhang*,†, ‡

†Department

of Polymer Science and Engineering, Key Laboratory of Systems Bioengineering

of the Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China ‡Tianjin

Key Laboratory of Membrane Science and Desalination Technology, Tianjin

University, Tianjin 300072, China §Collaborative

Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin

300072, China ∥

Tianjin Key Laboratory of Biomaterial Research, Institute of Biomedical Engineering,

Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300192, China

ACS Paragon Plus Environment

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ABSTRACT: High locoregional recurrence of breast cancer after surgery remains a clinically appealing

challenge.

Local

chemotherapy,

especially

sustainable

delivery

of

chemotherapeutics at tumor site by implantable hydrogels, has shown great potential to prevent cancer recurrence. However, the applications of conventional hydrogels are often limited by their intrinsic poor drug penetration into solid tumors and non-specific drug accumulation in adjacent normal tissues. Herein, we developed a novel modular coassembly strategy to prepare a kind of pH-sensitive, tumor-specific targeting and penetrating peptide (CRGDK)-modified, DOX-based prodrug nanoparticles (PDNPs), whose aqueous dispersion can undergo sol-gel transition after in vivo injection by thermo-induced self-aggregation to in situ form biodegradable hydrogel depot (PDNPs-gel), anchoring high amounts of PDNPs at tumor site. Due to CRGDK-mediated targeting to overexpressed Nrp-1 receptors on tumor vessels and tumor cells, PDNPs released out from PDNPs-gel can effectively penetrate into tumor tissues, specifically enter tumor cells and finally realize intracellular acid-triggered drug release. In an in vivo incomplete resection of breast cancer model, a single peritumoral administration of PDNPs-gel can achieve high inhibition efficacy against tumor recurrence. In addition, the administration of PDNPs-gel only involves simple redispersion of PDNPs in water without any pretreatment for gelation, providing great convenience for storage, dosage and prescription in practical use. Collectively, the reported multifunctional nanoparticles self-aggregated hydrogel system possesses great potential for efficient postsurgical prevention of tumor recurrence. KEYWORDS: Injectable hydrogel, Modular coassembly nanoparticles, Tumor-specific targeting and penetrating peptide, Cancer recurrence, Local chemotherapy


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1. INTRODUCTION Surgical resection is a main treatment modality for most solid tumors at early stage, especially for breast cancer.1 Nevertheless, postsurgical recurrence, mainly due to the residual microtumors, leads to treatment failure and even high mortality. Postsurgical chemotherapy is often used to prevent cancer recurrence. Systemic chemotherapy for locoregional recurrence treatment was often limited by poor bioavailability and severe toxicity due to rapid body clearance and nonspecific tissue biodistribution.2-3 Despite great efforts to develop various nanoparticles (NPs) for improved delivery efficiency, most of the intravenously injected dose of the nanomedicines (> 99%) were demonstrated to be captured or eliminated by mononuclear phagocytic and renal systems.4-7 Tumor local chemotherapy, especially using an injectable polymer hydrogel as drug depot, has been proven to be an effective approach to achieve tumor site-specific drug delivery, minimize excessive drug circulation, decrease the required drug dosage, reduce the frequency of drug administration, provide long-lasting therapeutic benefit, thus presenting great potential for efficient postsurgical prevention of tumor recurrence.8-20 However, despite the great potential of conventional polymer hydrogels for local chemotherapy, several challenges still exist in many aspects. First, owing to high water content and inherent large pore sizes of hydrogels, the drug molecules dispersed in the hydrogel network often exist initial burst release and uncontrolled drug diffusion.21-22 In addition, the inevitable drug crystallization or precipitation in the hydrogel, particularly for hydrophobic drugs, often cause poor formulation stability and low drug-loading capacity.21-23 Moreover, the entrapped drugs are often naked, and thus drugs released from hydrogel still confront the delivery barriers of tumor tissues and cells.8, 18, 23 Furthermore, the in vivo applications of polymer hydrogels are often impeded by their poor biocompatibility and low biodegradability as well as the tedious pretreatment for gelation and undesirable effect of crosslinkers.8, 11, 22


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To overcome above problems of conventional polymer hydrogel for local drug delivery, during the last decade, in situ thermosensitive hydrogels consisted of self-aggregated nanoparticles (NPs) networks generated from concentrated aqueous solutions of some amphiphilic block polyester and polyether have been developed as injectable local drug delivery systems.23-31 The amphiphilic block copolymers solutions undergo spontaneous solgel transition with temperature increase after injection into body, free of any chemical reaction. Compared with the nonbiodegradable amphiphilic polyether, PEGylated amphiphilic polyester has achieved significant success. For example, ReGel, a triblock copolymer consisted of polyethylene glycol (PEG) and poly(D,L-lactide-co-glycolide) (PLGA) with the chain structure of PLGA-PEG-PLGA, has been used for paclitaxel local delivery formulation in a number of clinical studies.32 Recently, a novel thermosensitive hydrogel based on poly(εcaprolactone-co-1,4,8-trioxa[4.6]spiro-9-undecanone)-PEG-poly

(ε-caprolactone-co-1,4,8-

trioxa[4.6]spiro-9-undecanone) copolymer (PECT) NPs was developed for locoregional drug delivery.24,

28-29

Compared with ReGel, PECT hydrogel is more convenient for clinical

operations, as it can avoid the need for long-term incubation or pre-quenching. Apparently, The NPs self-aggregated hydrogel combined the merits of NPs in drug loading, release and cellular internalization with the advantages of hydrogels for local treatments, which are expected to achieve desired therapeutic effects in local tumor therapy. However, most hydrogels used for local cancer therapy, even combining the NPs functions into hydrogels, still face some challenges for clinical translation, such as the high drug concentration gradient between the drug depot and surrounding tissue, higher interstitial fluid pressure and denser extracellular matrix in the tumors than normal tissues.33-34 These obstacles resulted in random drug diffusion into adjacent normal tissue and poor penetration into the extravascular tumor tissue, limiting the efficacy of hydrogels in cancer treatments.35-36 However, so far, very few studies have been conducted to address these key issues.


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To improve the penetration and targeting of drugs into the tumor, the active ligands are urgently required to increase the tumor vascular and tissue permeability in a tumor-specific manner. Neuropilin-1 (Nrp-1), a transmembrane receptor glycoprotein, is significantly and selectively overexpressed in tumor vessels and tumor cells in the tumor region.37-41 In particular, tumor-specific targeting and penetrating peptide (Cys-Arg-Gly-Asp-Lys, CRGDK) can actively recognize the Nrp-1. This kind of CRGDK-Nrp-1 specific recognition has been widely used to enhance the affinity for tumor antigen and facilitate the CRGDK-mediated nanomedicines to be effectively internalized into tumor cells via ligand-receptor mediated endocytosis. Moreover, CRGDK-mediated active targeting has been demonstrated to enhance the nanoparticles penetration into tumor tissues.35-36, 41-43 Inspired by these previous findings, herein we designed and developed a kind of novel tumor-specific prodrug nanoparticles self-aggregated hydrogel (PDNPs-gel) to increase tumorspecific targeting, penetration and release for tumor local precision chemotherapy to prevent tumor recurrence. The PDNPs-gel was derived from the concentrated aqueous solutions of freeze-dried powder of pH-responsive, high-targeting doxorubicin (DOX)-loaded prodrug nanoparticles (PDNPs). As shown in Scheme 1, the PDNPs were readily prepared by a modular coassembly of a demonstrated thermosensitive amphiphilic triblock copolymer PECT, acidcleavable PEGylated polymeric prodrug of DOX (DOX-conjugated PECT via acid-cleavable hydrazone linkages, DOX-PECT-DOX) and CRGDK-modified poly(ethylene glycol)-poly(εcaprolactone) (CRGDK-PEG-PCL). The PDNPs are expected to have several desirable features: (I) High stability and low leakage owing to the combined effect of hydrophobic interactions and chemical conjugations; (II) Tunable loading capacity; (III) Enhanced tumor penetration and cell endocytosis by the active targeting of CRGDK; (IV) Intracellular controlled DOX release by acid-triggered degradation; (V) In situ thermosensitive self-gelation without need of any pretreatments; (VI) Biocompatibility and biodegradation for safe use and


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avoiding surgical removal. The PDNPs were fully evaluated to demonstrate their functions. The freeze-dried powder of PDNPs can be well re-dispersed in water to obtain NPs aqueous dispersion at room temperature. After peritumor injection, the PDNPs aqueous dispersion can undergo spontaneous sol-gel transition to form PDNPs-gel at 37 °C and thus anchor high amounts of PDNPs at tumor site. A single peritumoral injection of PDNPs aqueous dispersion was proven to be able to achieve a high inhibition effect on tumor recurrence in an incomplete tumor resection model. Collectively, the design presented here provides a promising nanomedicine platform for local cancer therapy.

Scheme 1. Schematic illustrations of the modular coassembly to construct a multifunctional prodrug nanoparticles (PDNPs) with the capability of in situ self-gelation, CRGDK-mediated tumor targeted delivery and intracellular controlled release for tumor local drug delivery.


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2. EXPERIMENTAL SECTION 2.1 Materials Poly (ethylene glycol) (PEG, Mn = 1500 Da), tert-butyl carbazate (Boc-NHNH2, 99%) and εcaprolactone (CL, 99%) were provided by Aladdin Reagent Company (Shanghai, China). CL was dried using calcium hydride for 2 days at room temperature before use. 4-Nitrophenyl chloroformate (NPC, 99%), trifluoroacetic acid (CF3COOH, 99%) and trimethylamine (TEA, 99%) were provided by J&K Chemical (Beijing, China). Doxorubicin (DOX, 99%) was purchased from Dalian Meilun biotechnology Co., Ltd. Meta-chloroperoxybenzoic acid (mCPBA) was purchased from Tianjin Hainachuan Chemical Co., Ltd. (China). Stannous octoate

(Sn(Oct)2,

99%)

was

provided

by

Sigma-Aldrich.

1,4-Cyclohexanedione

monoethylene acetal was purchased from Hebei Huage Chemical Co., Ltd. (China). 1,4,8Trioxa[4.6]spiro-9-undecanone (TOSUO) was prepared through a Baeyer–Villiger oxidation of 1,4-cyclohexanedione monoethylene acetal using mCPBA as previously described.30 The synthesis pathway and 1H-NMR characterization of TOSUO were shown in Figure S1 in Supporting Information. TOSUO was purified through repeated recrystallization in diethyl ether before use. Cys-Arg-Gly-Asp-Lys (CRGDK, 99%) peptide was purchased from Shanghai GL Biochem. Ltd. (China). Maleimide-poly(ethylene glycol)-hydroxy (Mal-PEG-OH, Mn = 2000 g/mol) was provided by J&K Chemical (Beijing, China). Dimethyl sulfoxide (DMSO, 99%), dichloromethane (DCM, 99%) and tetrahydrofuran (THF, 99%) were purchased from Damao Chemical Co., Ltd. (Tianjin, China). The Neuropilin-1 overexpressed breast cancer cells (4T1) were purchased from Procell Life Science and Technology Co., Ltd. (Wuhan, China). HPLC grade THF was purchased from Merck (Darmstadt, Germany). CRGDKconjugated poly (ethylene glycol)-b-poly (ε-caprolactone) (CRGDK-PEG-PCL) as active targeting module was synthesized according to our previous study.44


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2.2 Synthesis and characterization of DOX-PECT-DOX Poly(ε-caprolactone-co-1,4,8-trioxa[4.6]spiro-9-undecanone)-poly(ethylene caprolactone-co-1,4,8-trioxa[4.6]spiro-9-undecanone)

triblock

glycol)-poly(ε-

copolymers

(P(CL-co-

TOSUO)-PEG-P(CL-co-TOSUO), defined as PECT) with well-defined structure and composition was synthesized through the ring-opening copolymerization of CL and TOSUO in the presence of poly(ethylene glycol) as an initiator and Sn(Oct)2 as catalyst.28, 30 According to a modified procedure, the hydrazone-linked DOX polymeric prodrug DOX-hydrazoneP(CL-co-TOSUO)-PEG-P(CL-co-TOSUO)-hydrazone-DOX

(DOX-PECT-DOX)

was

synthesized in two steps,23 including (I) hydrazide-functionalizing both ends of PECT; (II) conjugating DOX to both ends of PECT. The pre-prepared PECT (P(CL16.5-co-TOSUO1.65)PEG34-P(CL16.5-co-TOSUO1.65), Mn ≈ 5830 g/mol, demonstrated by 1H-NMR and GPC in Figure S2) was hydrazide-functionalized as follows. Firstly, TEA (83.4 µL, 0.6 mmol) and PECT (1.16g, 0.2 mmol) were dissolved in 10 mL THF in a flask. 96.7 mg NPC was dissolved in 5 mL THF and then the mixture was slowly dropwise added into the flask at approximately 0 °C. A filtration was used to remove the resultant salts after 8 h reaction. Subsequently, the NPC-activated PECT was obtained by precipitation in cold ether and then drying under vacuum overnight. The obtained NPC-activated PECT (1.24 g, 0.2 mmol) and Boc-NHNH2 (66.1 mg, 0.5 mmol) were dissolved in DMF and reacted at room temperature for 12 h. To obtain hydrazide-functionalized PECT (NH2NH-PECT-NHNH2), the mixture was stirred for 4 h at room temperature in the presence of TEA (2.5 mL) to remove -COOC(CH3)3. A dialysis membrane with MWCO of 3500 g/mol was used to removed DMF by dialysis in water, the hydrazide-functionalized PECT as a white solid was obtained by lyophilization. Finally, 0.272 g DOX (0.5 mmol) and hydrazide-functionalized PECT (1.19 g, 0.2 mmol) were dissolved in DMSO (20 mL). The mixture was reacted at 30 °C for 24 h after adding 0.5 mL of acetic acid as catalyst. The remaining unreacted DOX was removed by dialysis against DMSO for 24 h.


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Then DMSO and other impurities were removed by dialysis in PBS buffer (pH 7.4) for 24 h. DOX-PECT-DOX as red solid was obtained after lyophilization at a yield of about 90%. All reactions were carried out under dark condition. The obtained DOX-PECT-DOX was confirmed by nuclear magnetic resonance spectroscopy (1H-NMR). 1H-NMR spectra were recorded in DMSO-d6 using a Varian Inova 500MHz NMR spectrometer (Varian, USA). An Agilent 1100 series gel permeation chromatography (GPC) analyses (Agilent Technologies, Palo Alto, CA, USA) was used to further confirm the successful preparation of DOX-PECT-DOX. HPLC grade THF was used as eluent at a flow rate of 1.0 mL/min at 30 °C. The content of conjugated DOX in DOXPECT-DOX was determined by WFZ-26A ultraviolet-visible (UV-Vis) spectrophotometer (Science Instrument Plant, Tianjin, China) at 485 nm. 2.3 Preparation and characterization of multifunctional PDNPs The pH-sensitive, high-targeting PDNPs were prepared by modular coassembly using solvent displacement method. Briefly, PECT (2.0 g) as gel module, DOX-PECT-DOX (0.9 g) as drug module, and CRGDK-PEG-PCL (0.1 g) as targeting module were completely dissolved in 50 mL DMSO. Under magnetic stirring, the mixture was dropwise added into 500 mL deionized water. DMSO was removed by dialysis in water at room temperature with a dialysis bag (MWCO = 3500 g/mol). Finally, the PDNPs were obtained after lyophilization. With similar procedure, PDNPs with different composition and drug loading were prepared by adjusting the content of DOX-PECT-DOX and PECT. In addition, PECT NPs were also prepared by using only PECT. With similar procedure, nontargeting PDNPs (NT-PDNPs) were also prepared by using DOX-PECT-DOX NPs and PECT NPs without CRGDK-PEG-PCL. The detailed formulations were given in Table 1. The size and shape of NPs were measured by dynamic light scattering (DLS) (Malvern Zetasizer Nano ZS, Malvern, UK) and transmission electron microscopy (TEM) (JEM-100CX


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II). The samples for TEM observation were prepared by adding few drops of the NPs dispersion on the TEM copper grid. According to a previous work, the hydrazone bonds between DOX and PECT in PDNPs were hydrolyzed under acidic conditions,23 and then the amount of DOX in the PDNPs was quantified using UV-Vis spectrophotometer using a standard curve method. 2.4 Thermosensitive properties and hydrogel behaviors of PDNPs aqueous solution The freeze-dried powders of PDNPs were re-dispersed in water at room temperature with a concentration of 25% (w/w). The viscosity change of the PDNPs aqueous dispersion as a function of temperature was detected by Fluids Rheometer (Stress Tech, Rheological Instruments AB). The PDNPs aqueous dispersion was placed between parallel plates of 25 mm diameter and a gap of 0.5 mm. The temperature range was 20 °C~60 °C and the heating rate was 0.5 °C/min. The data were collected under a controlled stress (0.01 Pa) at a frequency of 1.0 rad/s. The sol-gel transition of PDNPs aqueous dispersion was also evaluated by the test tube inverting method. The transition temperatures were measured by a flow (sol)–no flow (gel) criterion when the tube was inverted with a temperature increment of 1 °C per step. For each measurement, the tube was equilibrated for 3 min. SEM (S-4800, Hitachi) was employed to investigate the interior morphology of the PECT hydrogel (25 wt%). The PECT hydrogel sample was quickly frozen in liquid nitrogen and lyophilized for 72 h. The freeze-dried hydrogel was then carefully fractured and the interior morphology of hydrogel was visualized. 2.5 In vitro drug release from PDNPs and PDNPs-gel The release of drugs from PDNPs was studied using the dialysis method. The dialysis membrane tube (MWCO = 3500 g/mol) containing 5 mg of PDNPs was immersed in 20 mL of PBS release medium with different pH values (pH = 5.0, 6.5 and 7.4) at 37 °C under continuous stirring (70 rpm). 10 mL of release medium was withdrawn at pre-


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determined time intervals and replaced with equivalent fresh medium. The amount of DOX in the release medium was determined by UV-Vis spectrophotometer at 485 nm. The aqueous dispersion (25 wt%, 1 mL) of PDNPs was placed in a test tube and immersed in a water bath at 37 °C for 3 min to form PDNPs-gel. After gelation, 10 mL of buffer saline (pH = 6.5 and 7.4) was added into the test tube. After incubation at 37 ºC for predefined time intervals, the release media was withdrawn from the test tube for determining the released amount of drug and replaced with an equal volume of pre-warmed fresh media. The amount of DOX in the release medium was determined by UV-Vis spectrophotometer. 2.6 In vitro degradation of PDNPs-gel The in vitro degradation of PDNPs-gel in a PBS solution with pH 7.4 corresponding to physiological pH level in normal tissue and pH 6.5 corresponding to pH in tumor tissues was investigated. In brief, the aqueous dispersion (25 wt%, 1 mL) of PDNPs was placed in a series of test tubes and immersed in a water bath at 37 °C for 3 min to form PDNPs-gel. And then 2 mL of pH 7.4 PBS solution or pH 6.5 PBS solution was added on top of the gel in each test tube. After incubation at 37 °C for predetermined time intervals, the remaining solid hydrogels were collected, dried and weighed. All experiments were carried out in triplicate. 2.7. In vitro cytotoxicity and endocytosis MTT assay was carried out to evaluate the cytotoxicity of free DOX, NT-PDNPs and CRGDKmediated PDNPs against Nrp-1 overexpressed 4T1 breast cancer cells. The 4T1 cells were seeded into the 96-well plates at 8000 cells per well, cultured in 100 μL complete dulbecco's modified eagle medium (DMEM) and incubated at 37 °C with 5% CO2. After incubation for 24 h, the supernatants were removed and the cells were washed twice with PBS (pH 7.4). Free DOX, NT-PDNPs and CRGDK-mediated PDNPs were placed into the wells at a particular DOX concentration range of 0.125 μg/mL to 40 μg/mL. The cells treated with pure culture medium severed as control group. After incubation for 24 h, 20 μL MTT dye (5 mg/mL) was


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added. After incubation for 4 h at 37 °C, the medium was replaced by 150 μL DMSO to dissolve the resulting formazan crystals. A microplate reader (Thermo Scientific Varioskan Flash, Waltham, MA, USA) was used to record the absorption at 570 nm. The experiments were performed in triplicate and the results were presented as the average ± standard deviation. To observe cellular uptake of free DOX, NT-PDNPs and CRGDK-mediated PDNPs, The 4T1 cells were seeded into a 12-well plate at a density of 1.0 × 105 cells per well and incubated for 24 h at 37 °C. And then the cells were incubated with 4',6-diamidino-2-phenylindole (DAPI) for 60 min. The cells were washed three times with PBS and then incubated with fresh medium containing free DOX, NT-PDNPs and PDNPs. The concentration of different formulations was at an equivalent DOX dosage of 10 μg/mL. The cells were incubated at 37 °C for 4 h. After washing with PBS, a Leica Microsystems confocal laser scanning microscope (Leica, Heidelberg, Germany) was applied to investigate the cellular uptake under excitation of 480 nm. The gray value was used for qualitative analyses using software Image-Pro Plus 6.0 (Media Cybernetics, Inc., USA). In addition, to verify the role of CRGDK in cellular uptake, CRGDK competitive cellular uptake study was conducted by pretreating cells with free CRGDK for 30 min before incubation with CRGDK-mediated PDNPs. To quantify the cellular uptake, we performed the flow cytometry. 4T1 cells were seeded in 6-well plates at a density of 2 × 105 cells per well and incubated at 37 °C for 24 h. The original medium was discarded and washed twice with PBS. Then fresh medium containing free DOX, NT-PDNPs and CRGDK-mediated PDNPs were added. Then, the cells were incubated for 4 h at 37 °C, and then washed three times with cold PBS, and harvested by trypsin treatment. The harvested cells were suspended in PBS and centrifuged at 1000 g for 5 min at 4 °C. The supernatants were discarded and the cell pellets were washed with PBS to remove the background fluorescence in the medium. After two cycles of washing and centrifugation, cells were resuspended with 200 μL of PBS for analyses using a FACS Calibur flow cytometer (BD


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Biosciences, U.S.A.) with an argon laser excitation at 488 nm (Becton Dickinson) and fluorescence (FL2) was detected. Cells treated with PBS were used as control. 2.8 Inhibition efficiency of local cancer recurrence All animal experiments were conducted in accordance with the People’s Republic of China national standard (GB/T 16886.6-1997) and approved by the Animal Research Committee of Institute of Biomedical Engineering, Chinese Academy of Medical Sciences and Peking Union Medical College. The inhibition effect of local cancer recurrence of PDNPs-gel after peritumoral administration in an incomplete tumor resection cavity was evaluated according to previous studies.14,

45

A mouse model of local cancer recurrence was firstly established. Briefly,

luciferase-tagged 4T1 cells were subcutaneously injected in male Balb/c mice (6-7 weeks) offered by Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). The tumors were surgically removed when tumors reached about 100 mm3 after 10 days. About 5% of the original tumor mass was left in situ to mimic the presence of residual microtumors on the surgical bed.14, 45 After acclimatization for 6 days, the mice were randomly divided into the following five groups: (1) control group (no treatment); (2) free DOX; (3) PECT NPs gel; (4) NT-PDNPs gel; (5) PDNPs-gel; DOX formulation was given by intravenous injection (i.v.) via the tail vein for three doses (days 16, 23 and 30, equivalent to 20 mg/kg of DOX). The gel groups (equivalent to 20 mg/kg of DOX) were administrated by peritumoral injection of various NPs aqueous dispersion (25 wt%), only once on day 16. Changes in cancer tissue were detected by fluorescence imaging. Body weight of each animal was recorded every 3 days. Each experimental group consisted of eight mice. 2.9 Statistical analysis The results are presented as mean ± standard deviation (SD) of minimum three replicates unless indicated otherwise. Differences with a P-value of less than 0.05 were considered to be statistically significant by Student’s t-test.


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3. RESULTS AND DISCUSSION 3.1 Preparation and characterization of coassembly modules The amphiphilic triblock polymer PECT with excellent thermal gelation behavior was selected as hydrogel module. The improved dispersibility in water and controlled gelation behavior of PECT and its freeze-dried nanoparticle powder were due to the introduction of cyclic ether pendant groups on the PCL backbone, which not only can increase the hydrophilicity of the hydrophobic phase but also decrease the crystallinity of PCL.24, 28, 30 PECT was synthesized by a one-pot ring-opening polymerization as shown in Figure 1(A) according to our previous study.30 The obtained PECT was characterized and shown in Figure S2. And then pH responsive PECT-based DOX prodrug as drug module and CRGDK-PEG-PCL as active targeting module were prepared, as shown in Figure 1(B) and 1(C). For obtaining DOX-PECTDOX, the hydroxyl groups in both terminal positions of PECT chain were firstly converted to hydrazide moieties by sequential reactions with NPC, Boc-NHNH2 and CF3COOH. Finally, DOX-PECT-DOX were prepared via the reaction between the ketonic groups of DOX and hydrazide groups of PECT. CRGDK-PEG-PCL was synthesized via a simple and effective thiol-maleimide coupling reaction between maleimide group of Mal-PEG-PCL and thiol group of CRGDK at room temperature.44

Figure 1. Synthesis routes of (A) PECT, (B) DOX-PECT-DOX and (C) CRGDK-PEG-PCL.


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The obtained DOX-PECT-DOX was characterized by 1H-NMR, as shown in Figure 2(A). Compared with the 1H-NMR spectrum of original PECT in Figure S2, no significant variations or differences in the characteristic peaks corresponding to PCL segments at approximately 1.02.2 ppm, PEG at about 3.6 ppm and cyclic ether at 3.91 ppm were observed. Moreover, the characteristic signals of DOX at about 4.5 and 7.5-8.0 ppm in the spectrum of DOX-PECTDOX can be found. GPC curves of PECT and DOX-PECT-DOX were shown in Figure 2(B). Both PECT and DOX-PECT-DOX curves were unimodal with relatively narrow molecular weight distributions. Compared with PECT, the GPC peak of the DOX-PECT-DOX was observed to move to lower retention time, suggesting a significant molecular weight increase and thus successful conjugation of DOX to PECT. The peak intensities of phenyl protons of DOX moieties (at approximately 8.0 ppm) and ethylene protons of the PEG (at about 3.6 ppm) were applied to evaluate the DOX conjugation efficacy in DOX-PECT-DOX. The content of conjugated DOX in DOX-PECT-DOX was estimated to be around 12.7 wt% (the molar conjugation efficacy is about 92%). Further evidence for the formation of DOX-PECT-DOX has been obtained from FTIR spectra in Figure S3 in Supporting Information. It can be seen that DOX-PECT-DOX showed the characteristic peaks of PECT, such as methylene (-CH2-) at about 2870 cm-1, carbonyl (-C=O) at 1730 cm-1 and the ether stretch (C-O-C) of PEG at 1150 cm-1. On inspection of the FTIR spectra, DOX-PECT-DOX presented a characteristic peak of imine (-C=N-) at about 1590 cm-1, a peak of the skeleton vibration of the ring at about 1410 cm-1 and aromatic C-H out-of-plane deformation in DOX molecule at about 840 cm-1. These results indicated the successful conjugation of DOX to PECT chain via hydrazone bonds. Moreover, the UV spectrum of DOX-PECT-DOX in Figure 2(C) exhibited a characteristic absorption peak of DOX at about 490 nm, further indicating the successful preparation of DOX-PECT-DOX. The peak intensity can be also used to estimate the DOX conjugation efficacy of about 90%, which was quite close to the result of 1H-NMR data.


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Figure 2. Characterizations of DOX-PECT-DOX and CRGDK-PEG-PCL. (A) 1H-NMR spectra of DOX (up) and DOX-PECT-DOX (down) in DMSO-d6; (B) GPC curves of PECT and DOX-PECT-DOX; (C) UV spectra of PECT (0.1 mg/mL), DOX (10 μg/mL), and DOX-PECT-DOX (0.7 mg/mL) in DMSO.

3.2 Preparation and characterization of coassembled multifunctional PDNPs The coassembly of two or more different components has been proven to be an easy and effective way to prepare multifarious nanostructures with well-defined structures and functions for biomedical and pharmaceutical applications.46-51 PECT, DOX-PECT-DOX and CRGDKPEG-PCL are all amphiphilic and thus they are able to self-assemble or coassemble into NPs. Apparently, the integration of PECT as hydrogel module, DOX-PECT-DOX as drug module and CRGDK-PEG-PCL as active targeting module via coassembly approach will generate pHresponsive, high-targeting doxorubicin (DOX)-loaded PDNPs. PECT, DOX-PECT-DOX and CRGDK-PEG-PCL can coassemble into one nanoparticle, as all of them have PCL-based


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hydrophobic segments that act as the main driving force behind the coassembly of amphiphilic copolymers in water. By ratiometric coassembly of different module, a series of PDNPs with different drug loading and thermal gelation behavior were successfully prepared. Their physicochemical properties, such as size and drug loading, were characterized and shown in Table 1. The drug loading in PDNPs can be well ratiometrically controlled by adjusting the content of PECT and DOX-PECT-DOX. Obviously, the increase in the ratio of DOX-PECTDOX content can be used to prepare the PDNPs with higher drug loading, which is desirable to reduce administration dosage or dosing frequency. However, the too high content of DOXPECT-DOX in final PDNPs will inevitably cause an undesirable thermal gelation behavior, which will be demonstrated below. In addition, the results in the Table 1 indicated that all of the PECT NPs, NT-PDNPs and PDNPs by modular coassembly exhibited particle sizes of about 110~130 nm and low PDI (below 0.2), depending on their composition. Table 1. Characterizations of PDNPs Weight ratio in feed solution Samples PECT NPs

Size a PDI a

(PECT : DOX-PECT-DOX: CRGDK-PEG- (nm) PCL) 100 : 0 : 0 130 ± 12 0.15

DOX content b

DOX loading c

(wt%)

(wt%)

0

0

NT-PDNPs d

66.7 : 33.3 : 0

121 ± 15 0.17

4.9

4.7 ± 0.13

PDNPs

66.7 : 30 : 3.3

118 ± 9 0.14

4.4

4.2 ± 0.09

PDNPs-I

50 : 45 : 5

117 ± 11 0.12

6.6

6.1 ± 0.17

PDNPs-II

20 : 72 : 8

114 ± 5 0.11

10.6

9.8 ± 0.15

a Measured

by DLS Calculated by the ratio of the weight of DOX in DOX-PECT-DOX to the total weight of PECT, DOX-PECT-DOX and CRGDK-PEG-PCL in feed c Determined by UV–vis spectrophotometer using a standard curve method d NT-PDNPs stands for nontargeting PDNPs consisted of PECT and DOX-PECT-DOX b

Typically, the PDNPs prepared by ratiometric coassembly of PECT, DOX-PECT-DOX and CRGDK-PEG-PCL at a predetermined dose ratio (66.7:30:3.3, w/w/w) with drug loading of 4.4% were characterized for their morphology and size. As shown in Figure 3(A) and 3(B), PDNPs presented good spherical shape and fairly uniform size distribution. Figure 3(C)


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showed the DOX release profiles from PDNPs in a release medium with pH 7.4 corresponding to pH value of normal tissue, pH 6.5 corresponding to the acidic environment of extracellular tumor tissue and pH 5.0 corresponding to lyso/endosomal condition. A significantly pHdependent release behavior can be seen. The release of conjugated DOX from PDNPs was very low at pH 7.4. Only about 9% of DOX was released in 24 h, due to the stable hydrazone bond under physiological condition. However, DOX release rate was notably enhanced under acidic condition. The cumulative release of DOX in 24 h at pH 6.5 was about 51.6%. Comparatively, the release rate of DOX at pH 5.0 was further increased to 77.5%. The significantly accelerated drug release could be ascribed to the combinated effect of acid-triggered hydrolysis of hydrazone bonds and concomitantly promoted disintegration of PDNPs. In addition, the accelerated DOX release under acidic condition might also be beneficial from enhanced solubility of DOX. Apparently, the tumor intracellular acid-triggered drug release of PDNPs was highly beneficial for increasing cancer therapy efficacy and reducing the side effects of DOX.

Figure 3. Characterizations of PDNPs. (A) TEM image; (B) Size histogram; (C) In vitro drug release profiles at different pH condition

3.3 Thermosensitive self-gelation behaviors of PDNPs


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Concentrated aqueous solutions of the freeze-dried powders of PECT NPs were demonstrated to be able to undergo a reversible sol-gel transition upon heating,27-30 as shown in Figure S4, due to the NPs aggregation by forming a “bridge” of two hydrophobic end blocks of PECT inserted into two NPs.31,

52

Concentrated aqueous solutions of the freeze-dried powders of

PDNPs derived from the coassembly of PECT, DOX-PECT-DOX and CRGDK-PEG-PCL also exhibited sol-to-gel transitions with the increase of temperature, as demonstrated in Figure 4(A). Test tube inversion method was used to comparatively study the phase transition behaviors of PDNPs-gel, PDNPs-gel (I) and PDNPs-gel (II), which were derived from PDNPs, PDNPs-I and PDNPs-II in Table 1, respectively. It can be found that all PDNPs aqueous dispersions (20-30 wt%) showed a sol-to-gel transition (lower transition) and gel-toprecipitation transition (upper transition). Moreover, all gel window contained 37 °C, corresponding to the body temperature. These results indicated that the coassembly of DOXPECT-DOX and CRGDK-PEG-PCL with PECT can preserve the thermosensitive gelation behavior of PECT. However, the introduction of DOX-PECT-DOX and CRGDK-PEG-PCL affected the thermo gelation behavior of PECT NPs. Compared with the phase transition behavior of PECT NPs in Figure S4, the gel window of PDNPs was found to be increased. Moreover, the gel-to-sol transition (upper transition) of PECT NPs was changed into gel-toprecipitation transition, which is probably because the dispersion stability of PECT NPs in water was decreased due to introducing DOX-PECT-DOX and CRGDK-PEG-PCL. Compared with phase transition behavior of PDNPs-gel, most sol-to-gel transitions of PDNPs-gel (I) and PDNPs-gel (II) were found to occur below room temperature (25 °C), due to lower PECT content in PDNPs-I and PDNPs-II. Apparently, PDNPs-gel exhibited a more desirable gelation behavior for biomedical applications, as its sol-to-gel transition temperature lies between room temperature and body temperature.


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Figure 4. Characterizations of PDNPs-gel. (A) Phase transition behaviors of PDNPs aqueous dispersion; (B) Temperature-dependent viscosity of PDNPs aqueous dispersion (25 wt%); (C) Images of gelation transition of PDNPs aqueous dispersion (25 wt%) at 25 °C and body temperature 37 °C; (D) Representative SEM images of the PDNPs-gel; PDNPs-gel, PDNPs-gel (I) and PDNPs-gel (II) were derived from PDNPs, PDNPs-I and PDNPs-II, respectively.

The desirable gelation behavior of PDNPs-gel was further confirmed by its temperaturedependent viscosity, as presented in Figure 4(B). The PDNPs aqueous dispersion (25 wt%) presented a good flowability with a viscosity of about 100 Pa·s at room temperature (< 30 °C), and then its viscosity was sharply increased with temperature increase to about 35 °C and reached a maximum at about 37 °C. As shown in Figure 4(C), PDNPs aqueous dispersion (25 wt%) was an injectable fluid at 25 °C but can spontaneously form gel after incubation at 37 °C for several minutes. The resultant PDNPs-gel was then lyophilized to investigate its microscopical morphology by SEM. It can be observed from Figure 4(D) the heterogeneous


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porous microstructures with pore sizes of about 2.0-5.0 μm, a typical polymer-based hydrogel structure. Further inspection reveals that PDNPs-gel was consisted of aggregated PDNPs. 3.4 In vitro degradation and drug release of PDNPs-gel Biodegradation properties of implantable hydrogels are of very importance for their clinical applications, as it can not only avoid the need for surgical removal after treatment, but also affect the drug release profiles. The in vitro biodegradation of PDNPs-gel was first estimated by direct observation of the appearance change of the PDNPs-gel samples incubated under tumor mildly acidic environment (pH 6.5) and normal physiological condition (pH 7.4) for different periods. As presented in Figure 5(A), no obvious changes were observed for PDNPsgel incubated in pH 7.4 PBS for 3 days. And then a very slight red turbidity was observed. However, when incubated with pH 6.5 PBS for only 1 day, a very slight red turbidity was observed, which can be ascribed to the appearance of PDNPs or DOX in the supernatant. No matter incubated with pH 7.4 PBS or pH 6.5 PBS, it can be seen that PDNPs-gel can maintain its shape after incubation for 7 days. These results suggest a slight acid-accelerated degradation of PDNPs-gel, due to the acid-triggered hydrolysis of hydrazone linkage and disintegration of PDNPs. The inset in Figure 5A confirmed that PDNPs can be dissociated and released out from PDNPs-gel along with the gel degradation. The in vitro biodegradation behavior of PDNPs-gel was further evaluated by monitoring the weight changes of remaining hydrogels incubated under different pH conditions (Figure 5(B)). The PDNPs-gel incubated with pH 6.5 PBS showed much quicker erosion than that incubated with pH 7.4 PBS, further indicating the pH-sensitivity of PDNPs-gel, due to introduction of pH responsive DOX-PECT-DOX into PECT NPs. The released DOX in incubation medium can reflect the detachment rate of PDNPs from PDNPs-gel and the disintegration rate of hydrogel. As shown in Figure 5(C), PDNPs-gel showed faster DOX release at pH 6.5 than that at pH 7.4. In the mildly acidic environment (pH 6.5), PDNPs-gel released out about 20% of the initially loaded DOX for 7 days. However,


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under pH 7.4 (normal physiological environment), only approximately 10% DOX was released within the same period of time. The release of DOX from PDNPs-gel seems to be quite relative to hydrogel degradation, which may be due to that the mildly acidic environment can not only enhance the hydrolysis of hydrazone linkage to release DOX, but also facilitate the degradation of PECT. These results indicated the accelerated erosion of PDNPs-gel in the mildly acidic environment and sustained release of PDNPs and subsequent DOX, due to acidtriggered cleavage of hydrazone linkage and enhanced PECT degradation. Considering the mildly acidic environment in tumor tissue, the mildly acid-accelerated release of PDNPs was desirable for effective treatment of cancer.

Figure 5. In vitro degradation and release of PDNPs-gel. (A) Photographs of PDNPs-gel incubated with pH 7.4 PBS and pH 6.5 PBS at 37 ºC; The inset is TEM image of NPs collected from release medium; Scale bar is 100 nm; (B) In vitro decomposition of PDNPs-gel when incubated in pH 7.4 PBS and pH 6.5 PBS solution


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at 37 ºC. Weight loss was calculated from the weight of PDNPs-gel samples collected at different time points; (C) DOX release from PDNPs-gel under different pH conditions.

3.5 In vitro cell uptake and cell toxicity Efficient and specific drug uptake by tumor cells has an important role in both enhancing chemotherapy efficacy and reducing side effects. CRGDK-mediated targeting can actively recognize the neuropilin-1 receptor (Nrp-1) overexpressed on the surface of tumor vessels and tumor cells.37-41 The cellular uptake of free DOX, nontargeting NT-PDNPs and PDNPs in Nrp1 overexpressed 4T1 cells was monitored by confocal laser scanning microscope. DAPI was employed to label nucleus (blue). As presented in Figure 6(A), the red DOX fluorescence can be evidently seen in the cytoplasm and nuclei of 4T1 cells treated with free DOX and DOXloaded NPs. Further inspection of Figure 6(A), it can be observed the relative weak fluorescence in the cytoplasm of cells treated with free DOX, indicating rapid passive diffusion of free DOX. When the cells were incubated with NT-PDNPs and PDNPs, the fluorescence signals mainly appeared in the proximity of nuclei of cells. The detected fluorescence within the cells incubated with PDNPs was much higher in the nuclei than that in the cells treated with nontargeting NT-PDNPs. To further investigate the role of CRGDK in the cellular uptake of CRGDK-mediated PDNPs, 4T1 cells were pretreated with free CRGDK to saturate the overexpressing Nrp-1 receptor and then incubated with PDNPs. The results showed that, after incubation with PDNPs, the DOX fluorescence intensity in CRGDK-pretreated cells was much lower than that in normal cells, which demonstrated the specific binding of CRGDK-mediated PDNPs to 4T1 cells. Flow cytometry was performed to quantify the cellular uptake, as shown in Figure 6(B) and 6(C). Compared with free DOX treatment, the cellular uptake for 4T1 cells showed a better uptake efficiency after incubation with the various nanomedicines. Significantly, PDNPs showed a greater targeting efficiency than that of non-targeting NTPDNPs. Moreover, the cellular uptake of PDNPs by 4T1 cells was evidently inhibited when the 4T1 cells were pretreated by excess free CRGDK. By competitive binding to Nrp-1 on the


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surface of 4T1 cells, the excess free CRGDK was found to be able inhibit the cell binding and uptake of PDNPs, further confirming the role of Nrp-1-mediated process in the cellular uptake of PDNPs. These results not only indicated significantly increased cellular uptake and considerably enhanced nucleus localization cells treated with PDNPs, but also suggested the receptor-mediated endocytosis of NPs and efficient intracellular drug release. Apparently, CRGDK surface decorated PDNPs possess very high binding activity with Nrp-1 overexpressed tumor cells, and thus have great potential to achieve cell-specific drug delivery. We incubated free DOX, nontargeting NT-PDNPs and PDNPs at various DOX concentrations for 24 h to evaluate the cell viability by MTT assay, as shown in Figure 6(D). All formulations gradually increased their cytotoxicity with increasing concentrations. NTPDNPs and PDNPs exhibited comparable cytotoxicity to 4T1 cells with free DOX. However, 4T1 cells showed highest sensitivity to the treatment with PDNPs. The IC50 of PDNPs was measured to be about 8.1 μg/mL (Figure 6(E)), which was lower than that of free DOX (10.5 μg/mL) and NT-PDNPs (11.2 μg/mL).The IC50 of CRGDK-modified PDNPs was much lower than that of nontargeting NT-PDNPs, which may be ascribed to the improved targeting efficacy of the CRGDK-modified PDNPs, further indicating that the CRGDK ligand played an important role in the enhanced cytotoxicity. In addition, we also evaluated the cytotoxicity of empty PECT NPs to avoid the cytotoxicity caused by PECT NPs themselves. The result was shown in Figure S5, which indicated that empty PECT NPs had no toxicity to 4T1 cells at the concentration from 10 up to 2000 μg/mL. Hence, lower IC50 of PDNPs was not due to the toxic effect of PECT component, mainly by enhanced cellular uptake of DOX when delivered by PDNPs.


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Figure 6. In vitro cell uptake and cell toxicity. (A) Representative fluorescence microscopy images of Nrp1 overexpressed 4T1 cells incubated with free DOX, NT-PDNPs, PDNPs and PDNPs + CRGDK at equivalent DOX concentration of 10 μg/mL for 4 h; Scale bar is 25 μm; (B) Flow cytometric analysis of 4T1 cells treated with free DOX, NT-PDNPs, PDNPs and PDNPs + CRGDK; (C) Quantitative measurement of DOX uptake in 4T1 cells; (D) Cell viability of Nrp-1 overexpressed 4T1 cells incubated with free DOX, NT-PDNPs and PDNPs at various concentrations for 24 h; (E) Half-maximal inhibitory concentration (IC50) value of free DOX, NT-PDNPs and PDNPs.

3.6 In vivo inhibition of tumor locoregional recurrence Surgery resection was the major treatment strategy for various cancer, especially for breast cancer. However, very high local recurrence rate of breast cancer after surgical resection remains a clinically fatal problem. Based on the desirable function of PDNPs-gel, it can be expected that PDNPs-gel could effectively inhibit tumor locoregional recurrence after resection. The therapeutic effects of PDNPs-gel were demonstrated by an incomplete tumor


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resection model. As shown in Figure 7(A), the antitumor efficacies of PDNPs-gel injected into the tumor resection cavity and repeated intravenous administration of DOX were examined. Drug free PECT NPs gel, nontargeting NT-PDNPs gel and no treatment were used control groups. Tumor regrowth with time was monitored by bioluminescence signals from luciferasetagged cancer cells at predesigned time after treatment (Figure 7(B)). Mice treated with single injection of PDNPs-gel and NT-PDNPs gel showed significant inhibition of tumor regrowth. The total bioluminescence signals of the mice treated with PDNPs-gel and NT-PDNPs gel were significantly lower than that of the other groups (Figure 7(C)). Especially, the single injection of PDNPs-gel showed highest antitumor efficacy at the tumor sites after resection. Free DOXtreated mice showed an inhibition activity at early stage but no inhibition activity against tumor regrowth at late stage. In addition, the systemic toxicities of the treatments were assessed by monitoring the body weights of the mice treated with different therapies. As shown in Figure 7(D), the systemic toxicity of free DOX resulted in a significant decrease in body weight. However, during the experimental period for mice treated by other formulations, no significant body weight loss was observed, implying the decreased systemic toxicities by using hydrogelbased local chemotherapy.14, 16, 53-54 Survival study for mice treated with different therapies was carried out, as shown in Figure 7(E). The results suggested that the survival rate of mice treated with DOX was much lower than that of all the groups, which can be ascribed to the high systemic toxicities of the repeated administration of DOX. Apparently, the treatment with PDNPs-gel exhibited the highest mouse survival rate. The survival rates exhibited similar trend with the inhibition of tumor regrowth in Figure 7(C). These results indicated that the PDNPsgel was more efficient in preventing tumor locoregional recurrence.


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Figure 7. In vivo inhibition of locoregional tumor recurrence using an incomplete tumor resection model via luciferase-tagged tumor cells implantation. (A) Experimental design; (B) Representative live bioluminescence images of mice treated with different formulations at different time; Bioluminescence signals from luciferase-tagged cancer cells were used to monitor tumor growth; (C) Quantified bioluminescence for tumors in mice treated with different formulations; (D) Body weight changes and (E) survival rates of mice treated with different formulations. The gel groups were administrated by peritumoral injection of corresponding NPs aqueous dispersion (25 wt%) once on day 16. DOX solutions were intravenously injected (3 dose on day 16, the day 23 and day 30). The concentration of drug was 20 mg/kg (DOX-equivalent). Data are presented as mean ± SD. (n = 8 per group, * p < 0.05).

Briefly, the peritumoral injection of PDNPs-gel was demonstrated to efficiently inhibit locoregional tumor recurrence, which can be attributed to the combined advantages of hydrogel-based local chemotherapy and CRGDK-targeting nanomedicine: (I) Forming a long-


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acting depot to achieve sustained release nanomedicine in the vicinity of tumor, avoiding a series of physiological barriers of systemic administration; (II) CRGDK-mediated targeting overexpressed Nrp-1 on the tumor vessels and tumor cells to achieve enhanced NPs penetration within tumors and effective endocytosis of tumor cells; (III) Intracellular acidic endo/lysosomal environment-triggered drug release via acid-cleavable hydrazone linkages. These features make PDNPs-gel highly desirable for local chemotherapy to effectively inhibit locoregional tumor recurrence. 4. CONCLUSIONS In this study, a simple and efficient modular coassembly approach was developed to prepare a kind of pH-sensitive, CRGDK-targeting, DOX-based prodrug nanoparticles (PDNPs). The freeze-dried powder of PDNPs with good dispersibility in water can easily be reconstructed into injectable NPs aqueous dispersion at ambient temperature and undergo thermo-induced self-aggregation after in vivo injection to in situ form biodegradable hydrogel depot (PDNPsgel), anchoring high amounts of PDNPs at tumor site. Especially, due to specific binding activity with overexpressed Nrp-1 receptors on vessels and tumor cells, PDNPs sustainably released out from PDNPs-gel were demonstrated to effectively penetrate into tumor tissues, specifically enter tumor cells and finally realize intracellular acid-triggered drug release. The in vivo studies demonstrate that a single peritumoral injection of PDNPs aqueous dispersion can in situ form a long-acting depot to sustainably release nanomedicine PDNPs in tumor for over 3 weeks and achieve high inhibition efficacy against tumor recurrence. In addition, the administration of the PDNPs-gel only involves simple redispersion of PDNPs in water without any pretreatment for gelation, providing great convenience for storage, dosage and prescription in practical use. Collectively, the reported multifunctional nanoparticles self-aggregated hydrogel system possesses great potential for efficient locoregional chemotherapy.


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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Preparation process and 1H-NMR characterization of TOSUO; 1H-NMR

spectrum and GPC curve of PECT;

FTIR spectra of PECT and DOX-PECT-DOX; Phase transition behavior of PECT NPs aqueous dispersions (25 wt%) In vitro cell viability of PECT NPs against 4T1 cells. AUTHOR INFORMATION Corresponding Authors * Email: [email protected] * Email: [email protected] ORCID Jianhua Zhang: 0000-0001-7833-9715 Weiwei Wang: 0000-0003-0333-0868 Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS


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This work was supported by the National Natural Science Foundation of China (No. 31470925, No. 31670977 and No. 51703246) and Tianjin Research Program of Application Foundation and Advanced Technology (No. 15JCQNJC03000).


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