Targeting cancer-associated fibroblasts by dual-responsive lipid-albumin nanoparticles to enhance drug perfusion for pancreatic tumor therapy

Journal Pre-proof Targeting cancer-associated fibroblasts by dual-responsive lipidalbumin nanoparticles to enhance drug perfusion for pancreatic tumor therapy

Qianwen Yu, Yue Qiu, Jianping Li, Xian Tang, Xuhui Wang, Xingli Cun, Shanshan Xu, Yayuan Liu, Man Li, Zhirong Zhang, Qin He PII:

S0168-3659(20)30130-9

DOI:

https://doi.org/10.1016/j.jconrel.2020.02.040

Reference:

COREL 10193

To appear in:

Journal of Controlled Release

Received date:

21 November 2019

Revised date:

1 February 2020

Accepted date:

25 February 2020

Please cite this article as: Q. Yu, Y. Qiu, J. Li, et al., Targeting cancer-associated fibroblasts by dual-responsive lipid-albumin nanoparticles to enhance drug perfusion for pancreatic tumor therapy, Journal of Controlled Release (2020), https://doi.org/10.1016/ j.jconrel.2020.02.040

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© 2020 Published by Elsevier.

Journal Pre-proof Targeting cancer-associated fibroblasts by dual-responsive lipid-albumin nanoparticles to enhance drug perfusion for pancreatic tumor therapy 1

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Qianwen Yu , Yue Qiu , Jianping Li , Xian Tang , Xuhui Wang , Xingli Cun , Shanshan Xu , Yayuan Liu1 , Man Li1 , Zhirong Zhang1 , Qin He1,* 1

Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry, Sichuan

Engineering Laboratory for Plant-Sourced Drug and Sichuan Research Center for Drug Precision Industrial Technology, West China School of Pharmacy Sichuan University, Chengdu, 610064 (P.R. China) * Corresponding author: Qin He, E-mail address: [email protected], Tel/Fax: +86-028-85502532.

Abstract:

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Pancreatic ductal adenocarcinoma (PDAC) is rich in cancer-associated fibroblasts (CAFs), which participate in the formation of tumor stroma. However, the dense tumor stroma of PDAC presents

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major barriers to drug delivery, resulting in an obstacle for PDAC therapy. Considering the special tumor microenvironment of PDAC, we constructed a novel nanoparticle which is responsive to the

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membrane biomarker FAP-α on CAFs and near-infrared (NIR) laser irradiation. Small sized albumin nanoparticle of paclitaxel (HSA-PTX) with strong tumor-penetration ability was encapsulated into the CAP-(a FAP-α responsive cleavable amphiphilic peptide) modified thermosensitive liposomes (CAP-TSL). Moreover, IR-780, a photothermal agent, was

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incorporated into CAP-TSL to afford CAP-ITSL. The designed HSA-PTX@CAP-ITSL increased

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the drug retention of HSA-PTX in solid tumor and HSA-PTX was released via FAP-α (specifically expresses on CAFs) triggered. Under sequential stimulation of NIR laser irradiation, IR-780 produced hyperthermia to kill tumor cells and expand the tumor interstitial space at the same time, which further promoted the release of small sized HSA-PTX in deep tumor regions.

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Consequently, the excellent antitumor efficacy of HSA-PTX@CAP-ITSL was demonstrated in Pan 02 subcutaneous and orthotopic tumor mouse models. Therefore, HSA-PTX@CAP-ITSL well

Keywords:

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combined chemotherapy with photothermal therapy, providing a promising drug delivery strategy for PDAC treatment.

Pancreatic tumor; thermosensitive liposomes; cancer-associated fibroblasts; deep penetration; chemo-photothermal therapy.

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Journal Pre-proof 1. Introduction Pancreatic cancer is one of malignant tumors with the worst prognosis [1]. Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer (that accounts for more than 80%), and the 5-year survival rate for PDAC is less than 5% [2]. Pancreatic tumors always contain multiple extracellular matrix (ECM), cancer-associated fibroblasts (CAFs), inflammatory cells, blood and lymphatic vessels, which form a dense tumor stroma impeding the delivery of drugs. The tumor stroma results in reduced tumor-targeting performance and weakened antitumor efficacy of traditional nanopartic les [3]. Therefore, strategies to overcome this physical barrier, increase the penetration of drugs and improve therapeutic efficiency in pancreatic tumors are urgently needed. For the tumor targeted drug delivery, characteristics of nanoparticles, including stability [4], surface properties [5], size [6] and shape [7], play critical roles. Among these factors, the size

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effects have been extensively studied. Nanoparticles with large size (100-150 nm) were often used for tumor targeted therapy because of their long blood circulation time and increased tumor

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accumulation [8]. However, nanoparticles with large size (100 nm) had poorer tumor penetration than the small ones (30 nm), resulting in poor therapeutic efficacy [9]. Moreover, it is more

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difficult to penetrate into pancreatic tumors than other solid tumors due to dense tumor stroma and high tumor interstitial pressure [10]. Conversely, nanoparticles with small sizes could diffuse further away from the blood vessels and penetrate deeper into tumors, but were cleared more rapidly from the blood circulation leading to poor tumor accumulation [11]. To overcome these

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obstacles, size-adjustable nanoparticles were more suitable to achieve both efficient penetration

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and retention in pancreatic tumors. Albumin, a component of serum proteins, is one of the most attractive natural nanocarriers for drug delivery because of its good biocompatibility and low immunogenicity [12]. Abraxane (nab-paclitaxel) has already been approved by the U.S. Food and Drug Administration (FDA) in

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2013 for the treatment of PDAC. Interestingly, this 130 nm nanoparticle form of paclitaxel can rapidly disassemble into small albumin-paclitaxel complexes (less than 10 nm) after intravenous

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injection [13], which may be beneficial to tumor penetration in pancreatic tumor. However, like other nanoparticles with small size, nab-paclitaxel exhibited poor tumor accumulation and drug blood retention [14], resulting in application of adjuvant chemotherapy. Recently, a strategy to encapsulate small human serum albumin ( HSA) nanoparticles into the thermosensitive liposomes (TSL) significantly enhanced drug blood retention and rapidly released drugs with the treatment of tumor local heating (42-45 °C) [15]. However, the distribution of TSL was not selective for tumor targeting. Besides, this heat-responsive nanocarrier required heating tumors in 43 °C water bath for over 1 h, which was a tedious process and limited its clinical application. In pancreatic tumor, dense fibrotic tissues expand and surround the tumor caused by a prominent fibrotic reaction [16]. CAFs is an essential part of pancreatic fibrotic tissues, and participate in tumor-stroma biological interaction by secreting diverse cytokines and ECM proteins such as α-smooth muscle actin (α-SMA) [17] and fibroblast-activated protein-α (FAP-α) for promoting tumor proliferation and protecting cancer cells [18]. FAP-α, a membrane-bound serine protease, always specifically expresses on the surface of CAFs [19]. Ji et al. designed a FAP-α responsive cleavable amphiphilic peptide (CAP) self-assembled nanoparticle to load hydrophobic chemotherapeutic drugs, resulting in disruption of the stromal barrier and enhanced local drug accumulation at tumor sites [20]. Moreover, it was reported that a matrix 2

Journal Pre-proof metalloproteinase-2 (MMP-2) responsive cleavable amphiphilic peptide-hybrid liposome could specifically release drug at MMP-2-rich ECM via MMP-2 triggered [21]. This MMP-2 responsive cleavable amphiphilic peptide had a similar structure to CAP. Consequently, it might be a promising strategy to overcome the tumor stromal barrier and specifically release drug by targeting to CAFs using FAP-α responsive CAP. Photothermal therapy (PTT) refers to the usage of near-infrared (NIR) laser that efficiently induced the heat to kill cancer cells for the treatment of cancer [22]. This promising anti-cancer treatment can be controlled spatio-temporally, which prevents damage to un-irradiated tissues [23]. PTT induced strong hyperthermia can expand the tumor interstitial space when killing cancer cells, which is beneficial to deep penetration of released small sized nanoparticles. Moreover, the combined of photothermal therapy and chemotherapy not only reduces chemotherapeutic agent concentration and minimizes side effects, but also significantly promotes therapeutic efficiency

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[24, 25]. Therefore, it may be a superior strategy, using photothermal heating to stimulate the thermosensitive liposomes release small s ized nanoparticles of chemotherapeutic drugs, instead of

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the water bath heating. In this study, IR-780 iodide, a lipophilic dye was used as a NIR light-absorbing agent with a

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high NIR fluorescence intensity and stable fluorescence [26]. IR-780 was incorporated into the lipid bilayer of thermosensitive liposomes for photothermal therapy. Considering the key role of CAFs in pancreatic tumor stroma, a novel CAF-responsive thermosensitive liposomes loading IR-780 (CAP-ITSL) was designed to be specifically responsive to FAP-α. CAP-TSL was

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constructed by co-assembly of a FAP-α responsive CAP with a phospholipid (DPPC). Moreover,

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small albumin-paclitaxel complexes ( HSA-PTX) were prepared using a self-assembly method and then encapsulated in CAP-ITSL to form HSA-PTX@CAP-ITSL (Scheme 1). HSA-PTX@CAP-ITSL firstly specifically accumulated at the tumor stroma site, then released HSA-PTX response to the special expression of FAP-α on abundant CAFs. Subsequently, NIR

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laser irradiation that induced strong hyperthermia could not only kill tumor cells but also further enhance HSA-PTX release for tumor deep penetration. In addition, the short circulation time and

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poor tumor retention of small nanoparticles were solved by loading HSA-PTX into CAP-ITSL. Excellent antitumor efficacy of HSA-PTX@CAP-ITSL was demonstrated both in vitro and in

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Scheme 1. Diagram of dual-responsive lipid-albumin nanoparticles (HSA-PTX@CAP-ITSL) for

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release for tumor deep penetration.

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deep tumor penetration and combined therapy against pancreatic tumors. (A) HSA-PTX@CAP-ITSL was administrated and specifically released its payloads (HSA-PTX) via the cleavage of FAP-α responsive CAP in tumor stroma. (B) Subsequently, NIR laser irradiation induced strong hyperthermia could not only kill tumor cells but also further enhance HSA-PTX

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Journal Pre-proof 2. Experimental Section 2.1 Materials Dipalmitoyl phosphatidylcholine (DPPC) and DSPE-PEG2K -OMe were obtained from Shanghai Advanced Vehic le Technology (AVT) Ltd. (Shanghai, China). CAP peptide (AC-Ala-Thr-Lys(C18)-Asp-Ala-Thr-Gly-Pro-Ala-Lys(C18)-Thr-Ala-NH2 ) was synthesized by ZheJiang Ontores Biotechnologies Co., Ltd. (Hangzhou, China). Paclitaxel (PTX) and IR-780 iodide were purchased from Melonepharma (Dalian, China) and J&K Scientific Ltd. (Beijing, China), respectively. Human serum albumin (HSA) was purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Recombinant human fibroblast activation protein (FAP-α) was purchased from Sino Biological Inc. (Beijing, China). Anti- FAP-α antibody was purchased from Abcam Ltd. (Cambridge, UK). Transforming growth factor -β (TGF-β) was

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purchased from Peprotech Inc. (New Jersey, USA).

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2.2 Cell lines and animals

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Mouse pancreatic cancer (Pan 02) cell lines and mouse embryonic fibroblast (NIH 3T3) cell lines were cultured in DMEM medium at 37 °C in a 5% CO 2 humidified environment incubator

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(Thermo Fisher Scientific, USA). DMEM media contained 10% FBS (10099-141, Gibco, Thermo Fisher Scientific, USA), 100 U/mL penicillin, and 100 mg/ml streptomycin. Pan 02 cells stably transfected with the luciferase gene (Pan 02-luc cells) were obtained from Key Laboratory of Drug-Targeting and Drug Delivery System, Sichuan University.

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Female C57BL/6 mice (6- to 8-week-old) were purchased from Chengdu Dashuo Biological

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Institute (Chengdu, China). All the animal experiments were approved by the specialty committee on the ethics of animal experimentation of Sichuan University. 2.3 Preparation of HSA-PTX@CAP-ITSL

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HSA-PTX was prepared by a self-assembly method with the help of PTX. PTX was dissolved in ethanol at 2 mg/mL, then mixed to HSA (at 4 mg/mL dissolved in PBS) at an HSA/PTX molar

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ratio of 1:4 and stirred slightly for 3 h. After that, the complex was filtered using ultra-filtration tubes (10 kDa), and the free PTX and ethanol were removed by centrifugation at 6000 rpm for 30 min. HSA-PTX complex was freeze-dried next, and dissolved in PBS before use. 10 mg HSA was dissolved in NaHCO3 (pH 8.5, 50 mM), then 1 mg Rhodamine B-NHS was added and stirred overnight at 0 °C. Then, the complex was filtered using ultra-filtration tubes (10 kDa), and Rhodamine B-labeled HSA (Rho-HSA) was obtained after freeze drying. HSA-PTX and IR-780 loaded CAP-modified thermosensitive

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(HSA-PTX@CAP-ITSL) was prepared using the thin film hydration method. 2.5 mg of DPPC, 0.1 mg of CAP, 0.51 mg of DSPE-PEG2K -OMe, and 0.2 mg of IR-780 were dissolved in 1 mL of the mixture solvent (chloroform/methanol, v/v = 2:1). After that, the organic solvent was evaporated by rotary evaporation under a vacuum, and the film was formed and hydrated in 1 mL HSA-PTX with PTX concentration of 0.5 mg/mL at 55 °C for 30 min. Then, the suspension was extruded 5 times through 200 nm membranes using an Avanti mini extruder, and free HSA-PTX was removed using sepharose CL-4B. Likewise, FITC-labeled CAP-TSL was prepared by adding 400 μL of FITC (100 μg/mL) instead of IR-780 to the organic solution before the lipid film was formed.

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Journal Pre-proof 2.4 Characterization of HSA-PTX@CAP-ITSL The hydrodynamic diameters and zeta potentials of HSA-PTX, CAP-ITSL and HSA-PTX@CAP-ITSL were detected using Malvern Zetas izer Nano ZS90 (Malvern Instruments Ltd., Malvern, UK). The morphology of HSA-PTX, CAP-ITSL and HSA-PTX@CAP-ITSL was observed under a transmission electronic microscope (TEM) (JEM 100CX, JEOL Ltd., Tokyo, Japan). The entrapment efficiency of PTX and IR-780 was determined by high performance liquid chromatography (HPLC) (Agilent 1200, USA) at 227 nm and UV–Vis spectrophotometer (Varian, USA) at 780 nm, respectively. Fluorescence resonance energy transfer (FRET) was conducted with two probes. FITC-probe was used as donor while Rhodamine B-probe as acceptor. FITC-CAP-TSL was assembled with Rho-HSA at different concentrations to form Rho-HSA@FITC-CAP-TSL with Rho/FITC ratios of 1:1 and 2:1. The emission spectra were recorded by a fluorescence spectrometer (Shimadzu, Japan)

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with 470 nm as the excitation wavelength. To visualize the combination of HSA and CAP-TSL, Rho-HSA@FITC-CAP-TSL was immobilized in 2% agarose gel and observed via a confocal microscope (CLSM) (LSM800, Carl Zeiss, Germany). IR-780 was dissolved in methanol at the concentration of 5, 10 and 20 μg/mL, and the solution

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was exposed to an 808 nm laser of 0.5 W/cm2 for 5 min. Temperature of the solution was measured every 0.5 min, and ΔT was calculated as follows: ΔT = T s -T 0 (Ts and T 0 represented the temperature of samples and beginning, respectively). CAP-ITSL (10 μg/mL IR-780) with CAP/Lipid ratios of 1:20, 1:30 and 1:50 was similarly exposed to an 808 nm laser of 0.5 W/cm 2

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for 5 min, and temperature of the solution was measured every 0.5 min.

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In vitro PTX release study was performed with a dialys is method [27], and salicylate sodium (pH 7.4, 1M) was used as the release media. HSA-PTX@ITSL and HSA-PTX@CAP-ITSL were 2 exposed to an 808 nm laser of 0.5 W/cm for 5 min before dialys is, representing HSA-PTX@ITSL (L+) and HSA-PTX@CAP-ITSL (L+). Different formulations were placed into dialysis tubes

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(Mw = 100 kDa) and incubated at 37 °C with gently oscillating for 24 h. At predetermined time points, 200 μL release media was sampled and analyzed by HPLC. To test the response to FAP-α,

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different formulations were incubated with FAP-α (200 ng/mL) during dialysis. 2.5 The expression of FAP-α in TGF-β activated fibroblasts and pancreatic tissues The expression levels of FAP-α and α-SMA in Pan 02 and NIH 3T3 cells were measured using western blotting. Cells were incubated with TGF-β (10 ng/mL) for 24 h, then harvested and lysed by cell lysis buffer (Beyotime, China). Polyvinylidene fluoride (PVDF) films were incubated with anti-rabbit FAP-α, α-SMA or β–actin primary antibodies (1:1000). After 24 h, the membranes were incubated with HRP-labeled goat anti-rabbit secondary antibodies (1:5000) and detected by Immobilon Western HRP Substrate (Millipore, Billerica, USA) on ChemiScope 6000 Touch System (Shanghai, China). The expression levels of FAP-α in pancreatic tissues were measured by western blotting and immunohistochemical assay. One week after the establishment of tumor model, the orthotopic tumor-bearing mice were euthanized and pancreases were collected. Then total protein was harvested and western blotting was performed. Pancreas histology was performed after FAP-α staining. 2.6 Cellular uptake and exocytosis 6

Journal Pre-proof TGF-β activated NIH 3T3 and Pan 02 cells were separately seeded on 6-well plates and incubated overnight. Free IR-780, ITSL and CAP-ITSL were added at a final IR-780 concentration of 4 μg/mL, and Rho-HSA, Rho-HSA@TSL and Rho-HSA@CAP-TSL were added at a final Rhodamine B concentration of 3 μg/mL. After incubation for 1 h, 4 h and 6 h, cells were washed twice with phosphate buffered saline (PBS), and were trypsinized and resuspended in 0.3 mL PBS. The fluorescence intensity of cells was measured by flow cytometry (FACSCelesta, BD, USA). To visualize the cellular uptake, cells treated as before were observed via CLSM (LSM800, Carl Zeiss, Germany). For the evaluation of exocytosis, Rho-HSA, Rho-HSA@TSL and Rho-HSA@CAP-TSL were incubated with TGF-β activated NIH 3T3 or Pan 02 cells. After 4 h incubation, the culture medium was replaced with fresh DMEM medium, and cells were cultured for further time to measure the exocytosis of different formulations. After further incubation for 1 h, 2 h, 4 h and 6 h,

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the fluorescence intensity of cultural supernatant was measured by a fluorescence spectrometer (Shimadzu, Japan).

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2.7 Cytotoxicity study

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CCK-8 assay was used to evaluate the cytotoxicity of different formulations. TGF-β activated NIH 3T3 and Pan 02 cells were separately seeded on 96-well plates and incubated overnight. HSA-PTX, HSA-PTX@TSL and HSA-PTX@CAP-TSL at predetermined PTX concentrations were added into plate for 24 h. By the end of the incubation, 10 μL CCK-8 solution was added

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into each well and cells were further incubated for 2 h. Then the samples were measured by a

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microplate reader (Varioskan LUX, Thermo Fisher Scientific, Waltham, USA) at 450 nm. To investigate the expression of FAP-α interfering by drug loaded nanoparticles, the expression levels of FAP-α in TGF-β activated NIH 3T3 cells and Pan 02 cells were incubation with HSA-PTX, HSA-PTX@TSL and HSA-PTX@CAP-TSL (10 μg/mL PTX) for 24 h, cells were harvested and

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the FAP-α protein were detected by western blotting. To evaluate the cytotoxicity of IR-780 and NIR laser irradiation, free IR-780 (dissolved in

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ethanol-Cremophor ELP35 mixture, v/v = 1:1) and CAP-ITSL at predetermined IR-780 concentrations were added into plate. After 4 h, cells were exposed to an 808 nm laser of 0.5 W/cm2 for 3 min, and further incubated until 24 h. The samples were processed as described above. To further evaluate the cytotoxicity of different formulations after NIR laser irradiation, HSA-PTX, CAP-ITSL, HSA-PTX@ITSL and HSA-PTX@CAP-ITSL (5 μg/mL PTX and 1 μg/mL IR-780) were added into the plate. The samples were processed as described above. 2.8 In Vitro penetration and growth inhibition of tumor spheroids To prepare the tumor three-dimensional spheroids, Pan 02 cells were seeded onto 96-well plate coated with 2% (w/v) low melting point agarose. When the diameter of tumor spheroids was approximately 300 μm, the uniform spheroids were selected. Rho-HSA (3 μg/mL Rhodamine B), Rho-HSA@CAP-TSL and Rho-HSA@CAP-TSL pre-incubation with FAP-α for 1 h were added into the plate for 4 h. The spheroids were washed and fixed with 4% paraformaldehyde, then subjected to CLSM analysis (LSM800, Carl Zeiss, Germany). Transwell chambers were used to establish pancreatic cancer model in vitro. TGF-β activated NIH 3T3 cells were seeded onto the upper chamber, and Pan 02 cells were seeded onto the under 6-well plate. After incubation for 24 h, Rho-HSA, Rho-HSA@ITSL and Rho-HSA@CAP-ITSL (3 7

Journal Pre-proof μg/mL Rhodamine B and 4 μg/mL IR-780) were added into the plate. After 4 h, NIH 3T3 cells on 2

upper chamber were exposed to an 808 nm laser of 0.5 W/cm for 3 min, and further incubated for 6 h. By the end of the incubation, cells were washed and fixed with 4% paraformaldehyde, then subjected to CLSM analysis (LSM800, Carl Zeiss, Germany). To evaluate the growth inhibition of tumor spheroids, a suspension of Pan 02 cells and TGF-β activated NIH 3T3 cells (2:1) were seeded onto ultra low attachment spheroid microplate (Corning, USA). When the diameter of tumor spheroids was approximately 300 nm, HSA-PTX, CAP-ITSL, HSA-PTX@ITSL, HSA-PTX plus CAP-ITSL and HSA-PTX@CAP-ITSL (5 μg/mL PTX and 1 μg/mL IR-780) were added into plate. After 4 h, tumor spheroids were exposed to an 808 nm laser of 0.5 W/cm2 for 3 min, and further incubated for 5 days. The morphology of tumor spheroids was observed and captured using optical microscopy (DM2000 LED, Leica, Germany).

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2.9 Tumor penetration in vivo The subcutaneous tumor model was established by subcutaneous injection of a suspension of 5

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1×10 Pan 02 cells and 5×10 NIH 3T3 cells (100 μL) into the right back of C57BL/6 mice. When subcutaneous tumors grew to about 80 mm 3, the tumor-bearing mice were intratumorally injected

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with Rho-HSA, Rho-HSA@ITSL and Rho-HSA@CAP-ITSL at a fixed needle insertion depth. After 4 h, the tumors were exposed to an 808 nm laser of 0.8 W/cm2 for 3 min. After 24 h, mice were euthanized via cardiac perfusion with PBS and 4% paraformaldehyde. Then the tumors were sliced at different depths from the top of the tumor and cryosectioned at a thickness of 10 μm

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using a freezing microtome. The sections were stained with DAPI (0.5 μg/mL) and imaged by CLSM (LSM800, Carl Zeiss, Germany).

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2.10 Bio-distribution and infrared thermal imaging in vivo Rho-HSA, Rho-HSA@TSL and Rho-HSA@CAP-TSL were injected into the subcutaneous

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tumor-bearing mice via the tail vein. At predetermined time points, mice were euthanized and organs were imaged using the IVIS Spectrum system (Caliper Life Sciences, Hopkinton, MA,

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USA). Then, tumors were cryosectioned at a thickness of 10 μm. The sections were firstly stained with primary anti-CD34 antibody and then with FITC-labeled secondary antibody. All the sections were stained with DAPI and imaged by CLSM (LSM800, Carl Zeiss, Germany). For evaluating the bio-distribution of IR-780 in vivo, free IR-780 and CAP-ITSL were injected into the subcutaneous tumor-bearing mice via the tail vein. At predetermined time points, mice were euthanized and tumors were imaged using the IVIS Spectrum system (Caliper Life Sciences, Hopkinton, MA, USA). To test the photothermal effect in vivo, PBS, HSA-PTX, CAP-ITSL, HSA-PTX@ITSL, HSA-PTX plus CAP-ITSL and HSA-PTX@CAP-ITSL (1 mg/kg PTX and 0.5 mg/kg IR-780) were injected into subcutaneous tumor-bearing mice. After 4 h, the tumors were exposed to an 808 2

nm laser of 0.8 W/cm for 3 min. An infrared thermal camera (Fotric 220, Texas, USA) was used to monitor the surface temperature change of mice during laser irradiation. The orthotopic tumor-bearing mice were treated as described above. 2.11 Evaluation of antitumor activity in vivo The subcutaneous tumor-bearing mice were randomized into 6 groups (9 mice/group): PBS, HSA-PTX, CAP-ITSL, HSA-PTX@ITSL, HSA-PTX plus CAP-ITSL and HSA-PTX@CAP-ITSL 8

Journal Pre-proof (1 mg/kg PTX and 0.5 mg/kg IR-780). When tumors grew to about 80 mm 3, mice were injected with different formulations every 3 days for 3 cycles. 4 h after injection, the tumors were exposed to an 808 nm laser of 0.8 W/cm2 for 3 min. Animal body weight and tumor volumes were th monitored every 3 days. On the 18 day, 3 mice were euthanized and their tumors were collected. The survival time of each group was continuously recorded. The expressions of α-SMA and Caspase-3 in tumors were measured by western blotting. Tumor histology was performed after hematoxylin and eosin (H&E), α-SMA and Caspase-3 staining. The luciferase labeled orthotopic tumor model was established by injection of 5×10 5 Pan 02-luc cells into the pancreas head of anaesthetic C57BL/6 mice. After 1 week, mice were injected with different formulations and treated as described above. Bioluminescence images of mice were captured using the IVIS Spectrum system (Caliper Life Sciences, Hopkinton, MA, USA) on day 0, day 8 and day 18. Animal body weight was monitored every 3 days. On the 18th day, 3 mice were

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euthanized and their pancreases were collected. The survival time of each group was continuously recorded. Then the expressions of α-SMA and Caspase-3 were measured as described above.

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To stress the antitumor effect of simply FAP-α responsive drug release, the Pan 02 subcutaneous and orthotopic tumor-bearing mice were respectively divided into 3 groups (5 mice/group): PBS,

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HSA-PTX@ITSL (L-) and HSA-PTX@CAP-ITSL (L-). Mice were treated as described above, except laser irradiation. Animal body weight and subcutaneous tumor volumes was monitored th every 3 days. On the 18 day, all mice were euthanized and their tumors were collected.

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2.12 Preliminary toxicity of formulations in vivo

2.13 Statistical analysis

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To evaluate the preliminary toxicity of different formulations, mice were randomized into 6 groups as treatment experiment in vivo. Mice were euthanized after treatment, and main organs and blood samples were collected. The organs were performed after H&E staining. The blood samples were tested for a blood cell assay.

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All the data were expressed as mean ± SD. The two-tailed Student’s t-test was used to compare the two groups (GraphPad Prism, V5.01, GraphPad, La Jolla, CA, USA). Significant differences between the groups were indicated by ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001, respectively.

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Journal Pre-proof 3. Results and Discussion 3.1 Characterization of HSA-PTX@CAP-ITSL To obtain small albumin-paclitaxel complexes, PTX was mixed with HSA at molar ratio of 0, 1:1, 2:1, 4:1, 5:1 and 10:1. When the ratio of PTX/HSA reached 5:1, the size of HSA-PTX complex rapidly increased to around 30 nm (Table S1), which might be difficult to release from TSL. Therefore, a relatively small diameter (7.9 ± 0.95 nm) of HSA-PTX complex was selected as the optimal formulation (PTX/HSA = 4:1). Different mass ratios of CAP/Lipid were mixed to form CAP-ITSL, and the size gradually increased with increasing mass ratio of CAP (Table S2). After loaded with HSA-PTX, the particle diameter of liposomes was slightly larger than unloaded liposomes (Table S3 and Figure S1). Considering the most appropriate diameter (smaller than 150 nm) of nanoparticles for the enhanced permeability and retention (EPR) effect [8], HSA-PTX@CAP-ITSL (123.9 ± 1.9 nm) at

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CAP/Lipid (mass/mass) ratio of 1:30 was selected as the optimal formulation (Figure 1A-B). The drug-loading efficiency of PTX and IR-780 for HSA-PTX@CAP-ITSL was 43.70% ± 3.40 and

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98.08% ± 2.07. Fluorescence resonance energy transfer (FRET) was used to demonstrate that HSA can be

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loaded to the liposomes. As we know, FRET is an energy transfer process between a fluorescent donor and an acceptor within 1−10 nm, which widely applies to nanotechnology [28]. Here, FITC as a donor was conjugated with CAP-TSL, while Rhodamine B as an acceptor was conjugated with HSA. The fluorescence intensity from the acceptor (Rho, 580 nm) increased with an

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increased concentration of Rho-HSA, while the donor (FITC) fluorescence at 525 nm decreased

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(Figure 1C), indicating the existence of FERT effect. Furthermore, the combination of HSA and CAP-TSL was also validated by the colocalization of the fluorescence signals of Rhodamine B in Rho-HSA and FITC in FITC-CAP-TSL (Figure S2). Figure 1D shows strong NIR absorbance of IR-780, resulting in rapid heating under NIR laser

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irradiation (808 nm). Even 5μg/mL IR-780 can rise about 10 °C in 3 min, and the effect of CAP modification on the photothermal efficiency of ITSL is negligible (Figure 1E). Interestingly, the

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photothermal efficiency ITSL (10 μg/mL IR-780) was higher than free IR-780 dissolved in methanol, showing that loading IR-780 into liposomes was beneficial to its photothermal

3.2 FAP-α/NIR dual-responsive drug release of HSA-PTX@CAP-ITSL To identify if the liposomes could release HSA-PTX under irradiation stimuli, PTX release behaviors of liposomes under NIR laser irradiation were measured. The results showed a fast drug release of HSA-PTX@ITSL and HSA-PTX@CAP-ITSL under NIR laser irradiation, while less than 40% of PTX was released from unirradiated groups (Figure 1F). The presence of IR-780 in the lipid bilayer might make the lipid shell very susceptible to disruption by NIR laser induced heat, allowing the encapsulated drugs to escape from the liposomes [29]. In addition, more than 60% of PTX was released from HSA-PTX@CAP-ITSL in the presence of FAP-α, and the PTX release efficacy was similarly increased under NIR laser irradiation (Figure 1G). However, no obvious change of PTX release efficacy was observed from HSA-PTX@ITSL in the presence of FAP-α, indicating that HSA-PTX@CAP-ITSL exhibited limited FAP-α responsive behavior. This might be caused by a lipid membrane disturbance when the CAP was cleaved by FAP-α.

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Figure 1. (A) The hydrodynamic sizes of HSA-PTX, CAP-ITSL and HSA-PTX@CAP-ITSL. (B) TEM images of HSA-PTX, CAP-ITSL and HSA-PTX@CAP-ITSL. (C) Fluorescence emission spectra of Rho-HSA@FITC-CAP-TSL with Rho/FITC mass ratios of 1:0, 0:1, 1:1 and 2:1. Excitation wavelength: 470 nm. (D) Temperature curves of IR-780 with different concentrations

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under NIR laser irradiation. (E) Temperature curves of CAP-ITSL with CAP/Lipid mass ratios of 0:1, 1:50, 1:30 and 1:20 under NIR laser irradiation. (F) The PTX release curves of different

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formulations. L+ represents NIR laser exposure before dialysis. (G) The PTX release profiles of different formulations incubated with FAP-α during dialysis. 3.3 Endocytosis and exocytosis of Rho-HSA@CAP-ITSL under FAP-α stimuli Cancer-associated fibroblasts (CAFs), one type of the stromal cells, form a main barrier that makes nanomedicines more difficult to deliver into solid tumors [30]. Normal fibroblasts can differentiate into CAFs under the stimulation of growth factors like TGF-β [31]. Figure 2A shows the FAP-α expression of NIH 3T3 cells (activated by TGF-β) was significantly increased, however Pan 02 cells expressed no FAP-α. The α-SMA expression of NIH 3T3 cells was higher than that of Pan 02 cells, and the α-SMA expression of both cells increased under TGF-β stimuli. After incubation of different formulations for 1 h, 4 h and 6 h, the time-dependent uptake was measured by flow cytometry (Figure 2B and 2C). Rho-HSA group showed strong fluorescence intensity in TGF-β activated NIH 3T3 cells and Pan 02 cells at each time point, due to faster endocytosis of smaller nanoparticles. More important, the cellular uptake of Rho-HSA form Rho-HSA@CAP-TSL group was more than Rho-HSA@TSL group in TGF-β activated NIH 3T3 cells, but no significant differences were observed from Pan 02 cells. This result revealed the presence of CAP peptide could effectively promote cellular uptake of Rho-HSA in TGF-β 11

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NIH 3T3 cells,

it was because Rho-HSA could

efficiently

release from

Rho-HSA@CAP-TSL in TGF-β activated NIH 3T3 cells (the positive expression of FAP-α). For visual observation, TGF-β activated NIH 3T3 cells incubated with different formulations for 4 h were imaged by CLSM. The fluorescence intensity of Rhodamine B from Rho-HSA@CAP-ITSL group was similar as Rho-HSA group and stronger than Rho-HSA@ITSL group (Figure 2D). However, there was no differences between Rho-HSA@CAP-ITSL group and Rho-HSA@ITSL group in Pan 02 cells (Figure S3). Size-dependent exocytosis of nanoparticles has been extensively investigated in various cell lines [32]. Serda et al. investigated the intracellular trafficking of porous silicon microcarriers with different sizes [33]. The results indicated that smaller nanoparticles were more favorable for exocytosis. Thus, cells were pre-incubated with different formulations for 4 h, then the culture media was replaced and re-incubated for 6 h (Figure 2E and 2F). The fluorescence intensity of

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Rho-HSA group was the highest at each time point in both TGF-β activated NIH 3T3 cells and Pan 02 cells. This might be attributed to the faster exocytosis of Rho-HSA with smaller size (~8

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nm) than Rho-HSA@TSL with a larger size (~90 nm). However, the fluorescence intensity of Rho-HSA@CAP-TSL group was significantly stronger than Rho-HSA@TSL group in TGF-β

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activated NIH 3T3 cells, while no significant differences were observed from Pan 02 cells. It indicated that small particles (Rho-HSA) was indeed released from Rho-HSA@CAP-TSL. Furthermore, the fluorescence intensity of Rho-HSA@CAP-TSL group was lower than Rho-HSA group after 2 h re-incubation (Figure 2E), indicating that Rho-HSA@CAP-TSL partly decreased

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the exocytosis of Rho-HSA and prolonged the residence time in TGF-β activated NIH 3T3 cell compared to Rho-HSA.

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3.4 In vitro chemothermal, photothermal and chemo-photothermal treatments CCK-8 assay was used for evaluating the cytotoxicity of different formulations against TGF-β

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activated NIT 3T3 cells and Pan 02 cells. All of the HSA-PTX loaded liposomes and HSA-PTX exhibited concentration-dependent inhibition effects (Figure S4). Among all the groups,

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HSA-PTX and HSA-PTX@CAP-TSL induced the strong anti-proliferation effect in NIH 3T3 cells, indicating that more drugs were delivered into the cells. However, the liposome carriers without PTX did not show significant cytotoxicity in cells. To investigate the expression of FAP-α interfering by drug loaded nanoparticles, the expression levels of FAP-α in TGF-β activated NIH 3T3 cells and Pan 02 cells were measured using western blotting. The results showed that no significant change of the FAP-α expression levels were observed in both TGF-β activated NIH 3T3 cells and Pan 02 cells (Figure S5). To evaluate the cytotoxicity of IR-780 and NIR laser irradiation, different formulations with increasing concentrations of IR-780 were incubated with TGF-β activated NIT 3T3 cells and Pan 02 cells (Figure S6). The strongest cytotoxicity was observed in free IR-780 group, but the cytotoxicity of IR-780 decreased after being loaded into CAP-TSL. It might attribute to the higher cellular uptake of free IR-780 than liposomes (Figure S7). Furthermore, NIR laser irradiation induced significant anti-proliferation effect in both NIT 3T3 cells and Pan 02 cells, and this effect was also shown from Figure 2G and 2H. Furthermore, Pan02/NIH 3T3 co-culture tumor spheroids were prepared to evaluate the growth inhibition of tumor spheroids (Figure S8). The results showed Rho-HSA@CAP-ITSL after irradiation induced the strongest spheroid growth delay, but the growth inhibition of tumor 12

Journal Pre-proof spheroids by NIR laser irradiation alone was not obvious. It indicated that the combination of

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photothermal and chemotherapy had a better effect on inhibiting tumor spheroid growth.

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Figure 2. (A) The expression of FAP-α and α–SMA in Pan 02 cells and NIH 3T3 cells. (+) and (-) represent pre-incubation with or without TGF-β for 24 h, respectively. Cellular uptake of different formulations in (B) TGF-β activated NIH 3T3 cells and (C) Pan 02 cells at different time points. (D) CSLM images of cellular uptake after TGF-β activated NIH 3T3 cells incubated with different

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formulations for 4 h. Scale bar, 20 μm. Exocytosis of different formulations in (E) TGF-β

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activated NIH 3T3 cells and (F) Pan 02 cells at different time points. Cells were incubated with different formulations for 4 h, and cultured for further time after replaced with the fresh culture media. Cytotoxicity of different formulations in (G) TGF-β activated NIH 3T3 cells and (H) Pan 02 cells. L+ and L- represent NIR laser exposure or not after incubation for 4 h, respectively. 3.5 In Vitro penetration effect of Rho-HSA@CAP-ITSL under FAP-α/NIR stimuli

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The tumor three-dimensional spheroids were prepared to evaluate the penetration ability in vitro. Compared to Rho-HSA@CAP-TSL group, Rho-HSA group showed the a stronger penetration in deep owing to the increased diffusional ability of smaller particles [34]. However, the fluorescence intensity of Rho-HSA@CAP-TSL group significantly increased at each depth after pre-incubation with FAP-α for 1 h, which was attributed to FAP-α-triggered release of Rho-HSA from Rho-HSA@CAP-TSL (Figure 3A and Figure S9). Transwell chambers were used to evaluate the penetration ability under NIR laser irradiation and cells were treated as shown in Figure 3B. In the upper chamber, TGF-β activated NIH 3T3 cells irradiated by NIR laser (L+ groups) showed larger intercellular space than Rho-HSA group. Moreover, cellular uptake obvious ly enhanced after NIR laser irradiation (Figure 3C). In the lower chamber, cellular uptake of Rho-HSA in Pan 02 cells after irradiation (Rho-HSA@ITSL and Rho-HSA@CAP-ITSL groups) was increased compared to Rho-HSA group (Figure 3D). The results indicated that the intercellular space of fibroblasts was expanded and more carriers could penetrate the fibroblast barrier to increase tumor cell uptake after NIR laser irradiation. Furthermore, cellular uptake of Rho-HSA@CAP-ITSL group was higher than Rho-HSA@ITSL group due to more released Rho-HSA from Rho-HSA@CAP-ITSL group after dual FAP-α/NIR stimuli. 13

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Figure 3. (A) Tumor spheroids uptake after incubation with Rho-HSA, Rho-HSA@CAP-TSL and Rho-HSA@CAP-TSL (pre-incubation with FAP-α for 1 h). Yellow represents Rhodamine B. Scale bar, 200 μm. (B) Scheme of transwell model. CSLM images of cellular uptake in (C) TGF-β activated NIH 3T3 cells on upper chamber and (D) Pan 02 cells on under chamber. Blue and

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yellow represent nuclei and Rhodamine B, respectively. Scale bar, 20 μm. L+ represents NIR laser exposure after incubation for 4 h.

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3.6 In vivo tumor penetration after photothermal treatments Firstly, the FAP-α expression of pancreas with or without tumors was detected using

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immuno-histochemical staining and western blotting. High expression levels of FAP-α were detected from sections of pancreatic tumor stroma and surrounding tissues (Figure 4C), while

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relatively low expression levels of FAP-α were observed from normal pancreas. Figure 4D shows the same results as immunohistochemical staining. To further demonstrate the tumor penetration ability of different formulations in vivo, the subcutaneous tumor-bearing mice were intratumorally injected with Rho-HSA, Rho-HSA@ITSL and Rho-HSA@CAP-ITSL at a fixed needle insertion depth. After 4 h, tumors were exposed to an 2

808 nm laser of 0.5 W/cm for 3 min. The results showed that Rho-HSA@ITSL was distributed in the edge of tumor (Figure 4A and Figure S10). However, Rho-HSA could penetrate into the deep regions of tumor, even at 3000 μm inside the tumor caused by stronger penetration ability of small nanoparticles [35]. Moreover, the fluorescence intensity of Rho-HSA@CAP-ITSL was greater than

Rho-HSA@ITSL in

each

tumor section.

After

irradiation,

the distribution

of

Rho-HSA@CAP-ITSL and the tumor interstitial space increased (Figure 4A-B), indicating that Rho-HSA might be further released from Rho-HSA@CAP-ITSL and penetrate into the deeper regions of tumor triggered by photothermal heating.

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Figure 4. (A) In vivo tumor penetration of Rho-HSA, Rho-HSA@ITSL, Rho-HSA@CAP-ITSL

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and Rho-HSA@CAP-ITSL (exposed to NIR laser after injection for 4 h) into tumor tissues of subcutaneous tumor-bearing mice. Red represents Rhodamine B. Scale bar, 50 μm. (B) Rhodamine B distribution of different formulations in the edge and core of pancreatic tumors. Red represents Rhodamine B. Dotted lines show the boundary between edge and core of tumor tissue. Scale bar, 100 μm. (C) FAP-α staining of pancreatic tissues (positive cells shown in brown). a and b represent pancreatic tumor and normal pancreas, respectively. Scale bar, 100 μm. (D) The expression levels of FAP-α in pancreatic tissues by western blotting. 3.7 Bio-distribution and infrared thermal imaging study in vivo It has been documented that nanoparticle sizes and molecular masses could influence the EPR effect [36]. Wang et al. found that the nanoparticle circulation time and tumor accumulation increased with the increasing diameters [9]. To evaluate the bio-distribution of different formulations in vivo, tumor-bearing mice were injected with Rho-labeled or IR-780-loaded formulations. After different time points, mice were euthanized and organs were imaged using the IVIS Spectrum system. The results showed that most Rho-HSA was fast eliminated after 4 h (Figure 5A). They rapidly accumulated to tumor tissues after 1 h, yet most diffused away from tumor tissues after 8 h (Figure 5B). However, Rho-HSA loaded liposomes (Rho-HSA@TSL and 15

Journal Pre-proof Rho-HSA@CAP-TSL) exhibited longer circulation time and more accumulation in tumor in vivo. Compared to free IR-780, a stronger fluorescence signal of tumor was observed in the CAP-ITSL group after 4 h (Figure S11), which was attributed to longer circulation time of PEGylated liposomes. After different time points, cryosections of the edge and deep regions of tumors were prepared, and neovessels were stained with FTIC-labeled anti-CD34 antibody. After 1 h, the fluorescence intensity of Rho-HSA group was stronger than other groups in both edge and deep regions of tumors, because small sized Rho-HSA could rapidly accumulate to tumor tissues (Figure S12). However, the fluorescence intensity of Rho-HSA@CAP-TSL group was higher than Rho-HSA group in deep regions of tumors after 4 h (Figure S13), 8 h (Figure S14) and 24 h (Figure 5E-F and Figure S15), which was consistent with the result of ex vivo imaging (Figure 5B). The result indicated that Rho-HSA could release from CAP-TSL and penetrated into the deep regions of

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tumors lacking in neovascularization. In addition, CLSM imaging also showed that the accumulation of all formulations in the tumor edge regions was higher than those in the deep

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regions, indicating that the delivery of nanoparticles into the deep tumor regions was generally impeded in pancreatic tumors.

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When the tumor accumulation of Rho-HSA@CAP-TSL reached its peaked level (4 h), tumors were exposed to an 808 nm laser of 0.5 W/cm2 for 3 min, and the surface temperature change of mice during laser irradiation was monitored by an infrared thermal camera (Figure 5C and G). Tumor surface temperatures of mice injected with formulations including IR-780 rapidly increased

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to ~47 °C under laser irradiation. In comparison, tumor surface temperatures of mice injected with

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PBS or HSA-PTX only increased to ~37 °C, which did not induce cell death [37]. For orthotopic tumor-bearing mice, similar results were also observed (Figure S16 and 6D), indicating the strong photothermal effect induced by CAP-ITSL.

Figure 5. (A) Ex vivo images of organs and tumors collected from subcutaneous tumor-bearing mice after injection with Rho-HSA, Rho-HSA@TSL and Rho-HSA@CAP-TSL at different time 16

Journal Pre-proof points. (B) Ex vivo images and semiqualitative study of fluorescence intensity of tumors. (C) The subcutaneous tumor temperature changes based on IR thermal imaging. a = PBS, b = HSA-PTX, c = CAP-ITSL, d = HSA-PTX@ITSL, e = HSA-PTX+CAP-ITSL and f = HSA-PTX@CAP-ITSL. (D) The orthotopic tumor temperature changes based on IR thermal imaging. Rhodamine B distribution of different formulations in the (E) edge and (F) deep region of tumors after 24 h. Blue, green and red represent nuclei, CD34 staining and Rhodamine B, respectively. Scale bar, 100 μm. (G) IR thermal images of subcutaneous tumor-bearing mice injected with different formulations under NIR laser irradiation. 3.8 Antitumor activity in subcutaneous and orthotopic model To evaluate the antitumor activity in vivo, different formulations were exploited for treatment on the subcutaneous and orthotopic pancreatic tumor-bearing mice.

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No obvious changes in body weight were observed during the treatment on the subcutaneous tumor model (Figure 6A). At the end of administration (day 8), groups under irradiation exhibited

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the significant tumor suppression effects by photothermal ablation (Figure 6B), indicating the excellent antitumor activity of PPT. However, except HSA-PTX@CAP-ITSL, mice treated with

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other photothermal groups developed tumor recurrence on day 18 (Figure 6B-C). This was because hyperthermia could ablate shallow tumors, but deep residual tumor cells rapidly proliferated after treatment, leading to tumor recurrence. Furthermore, HSA-PTX@CAP-ITSL plus irradiation significantly prolonged the survival time even no mice died until the day 52

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(Figure 6D), longer than other groups treated with PBS (41 days) and HSA-PTX (47 days). H&E

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staining images showed that most tumor cells were destroyed after treatment with HSA-PTX@CAP-ITSL plus irradiation (Figure 6E). Immunohistochemical staining of tumor sections and western blotting of tumor tissues were shown in Figure 6E-F, separately. Tumors treated with PBS group exhibited the high expression level of α-SMA (a component of tumor

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stroma) and low expression level of caspase-3 (an apoptosis marker). However, the expression level of α-SMA decreased after treatment with HSA-PTX@CAP-ITSL plus irradiation, and

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significant tumor apoptosis was induced, leading to the optimal therapeutic effect (Figure S17).

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Journal Pre-proof Figure 6. (A) The body weight of subcutaneous tumor-bearing mice during the treatment. (B) Representative photographs of one mouse from each group at different time points. (C) The tumor growth curves and (D) Kaplan-Meier survival curves of mice. (E) H&E staining, α-SMA staining and Caspase-3 staining of tumor tissues (positive cells shown in brown). Scale bar, 100 μm. (F) The expression levels of α-SMA and Caspase-3 in tumor tissues by western blotting. The numbers shown with the western blots represent the gray value normalized to β-actin. a = PBS, b = HSA-PTX, c = CAP-ITSL (L+), d = HSA-PTX@ITSL (L+), e = HSA-PTX+CAP-ITSL (L+) and f = HSA-PTX@CAP-ITSL (L+). L+ represents NIR laser exposure to tumors after administration for 4 h. To further mimic pancreatic tumor microenvironment in vivo, Pan 02-luc cells were injected into the pancreas head of anaesthetic C57BL/6 mice. Because orthotopic pancreatic tumors were

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not easy to be observed as subcutaneous tumors, Pan 02 cells were labeled with luciferase, which could be detected by bioluminescence imaging. No significant changes in body weight were

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observed during the treatment (Figure 7A). The luminescence of Pan 02 cells from the PBS group rapidly increased during the experimental period, while HSA-PTX@CAP-ITSL plus irradiation

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group significantly inhibited tumor growth (Figure 7B-C). Unlike the remarkable antitumor activity on subcutaneous tumors, groups under irradiation except HSA-PTX@CAP-ITSL showed different degrees of residual tumor cells at the end of administration (day 8), most likely due to high invasive ability of orthotopic tumor cells. In addition, HSA-PTX@CAP-ITSL plus irradiation

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also prolonged the survival time (Figure 7D). H&E staining of pancreases showed invasive growth

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of tumor cells, especially PBS group. However, our ideal formulation could reduce the expression level of α-SMA and increase the tumor apoptosis, resulting in significant tumor growth suppression (Figure 7E-F).

Figure 7. (A) The body weight of orthotopic tumor-bearing mice during the treatment. (B) Bioluminescence images of one mouse from each group at different time points. (C) Semiqualitative study of fluorescence intensity of tumors. (D) Kaplan-Meier survival curves of mice. (E) H&E staining, α-SMA staining and Caspase-3 staining of tumor tissues (positive cells 18

Journal Pre-proof shown in brown). Scale bar, 200 μm. (F) The expression levels of α-SMA and Caspase-3 in tumor tissues by western blotting. The numbers shown with the western blots represent the gray value normalized to β-actin. a = PBS, b = HSA-PTX, c = CAP-ITSL (L+), d = HSA-PTX@ITSL (L+), e = HSA-PTX+CAP-ITSL (L+) and f = HSA-PTX@CAP-ITSL (L+). L+ represents NIR laser exposure to tumors after administration for 4 h. In addition, the antitumor effect of the FAP-α responsive drug release were also evaluated in Pan 02 subcutaneous and orthotopic tumor mouse models. The results suggested that HSA-PTX@CAP-ITSL had a better effect on inhibiting tumor growth than HSA-PTX@ITSL (Figure S18 and Figure S19), which was attributed to FAP-α-triggered drug release in presence of CAP peptide. H&E staining images of main organs showed no obvious histological damage compared to PBS

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group (Figure S20). To further evaluate the preliminary toxicity of different formulations, the blood samples of mice were tested for a blood cell assay (Table S4). The results demonstrated no

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obvious decrease in white blood cells (WBC), red blood cells (RBC) and neutrophile granulocyte (Gran) compared to the PBS group, while HSA-PTX slightly increased the levels of WBC and

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Gran. Nevertheless, all values were within the normal reference range. All these results certificated that the ideal antitumor activity and absence of obvious side effects of HSA-PTX@CAP-ITSL plus irradiation group in vivo.

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Journal Pre-proof 4. Conclusion In summary, we successfully developed a novel CAF-responsive nanoparticle with dual-responsive drug release to modulate the tumor microenvironment and enhance the therapeutic efficacy for pancreatic tumor. HSA-PTX@CAP-ITSL was composed of CAF-responsive thermosensitive liposomes, photothermal agent and chemotherapeutic drug. When administrated systemically, HSA-PTX@CAP-ITSL with large size could specifically accumulate in the tumor stroma site and release HSA-PTX via cleavable CAP responsive to FAP-α in the tumor microenvironment. After NIR laser irradiation, photothermal therapy induced hyperthermia expanded the tumor interstitial space and promoted the small sized HSA-PTX releas ing in deep tumor regions. HSA-PTX@CAP-ITSL exhibited an excellent antitumor efficacy on different types of pancreatic tumor models. Furthermore, it is feasible to encapsulate other hydrophobic drugs into the albumin via noncovalent binding, such as anticancer drugs,

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anti-inflammatory drugs and immunomodulating drugs, providing a practical platform for PDAC treatment.

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Competing Interests

The authors have declared that no competing interest exists.

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Acknowledgement

The work was funded by the Major projects of the National Natural Science Foundation of China

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(grant number 81690261) and the National Natural Science Foundation of China (grant number

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81773658).

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Highlights Lipid-albumin nanoparticles released drug via specifically responsive to the FAP-α on CAFs. Lipid-albumin nanoparticles released drug under NIR laser irradiation.

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Hyperthermia induced by photothermal therapy expanded the tumor interstitial space. Small sized albumin nanoparticles exhibited tumor deep penetration.

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