Doxorubicin and anti-PD-L1 antibody conjugated gold nanoparticles

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Doxorubicin and anti-PD-L1 antibody conjugated gold nanoparticles for colorectal cancer photo-chemotherapy Fakhrossadat Emami, asmita banstola, Alireza Vatanara, Sooyeun Lee, Jong Oh Kim, Jee-Heon Jeong, and Simmyung Yook Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b01157 • Publication Date (Web): 30 Jan 2019 Downloaded from http://pubs.acs.org on January 31, 2019

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Molecular Pharmaceutics

Doxorubicin and anti-PD-L1 antibody conjugated gold nanoparticles for colorectal cancer photo-chemotherapy Fakhrossadat Emami a, Asmita Banstola b, Alireza Vatanara a, Sooyeon Lee b, Jong Oh Kim c, JeeHeon Jeong c,**, Simmyung Yook b,* a

College of Pharmacy, Tehran University of Medical Science, Tehran, Iran

b College

c

of Pharmacy, Keimyung University, Daegu 42601, Republic of Korea

College of Pharmacy, Yeungnam University, Gyeongsan, Gyeongbuk 38541, Republic of Korea

KEYWORDS. Gold nanoparticles, anti-PD-L1 antibody, colorectal cancer, doxorubicin.

ACS Paragon Plus Environment

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ABSTRACT. Colorectal cancer (CRC) is the third leading cause of cancer-related death worldwide, and prognosis and overall survival are known to be significantly correlated with the overexpression of PD-L1. Because combination therapies can significantly improve therapeutic efficacy, we constructed doxorubicin (DOX) conjugated and anti-PD-L1 targeting gold nanoparticles (PD-L1-AuNP-DOX) for the targeted chemo-photothermal therapy of CRC. DOX and anti-PD-L1 antibody were conjugated to α-terminal end group of lipoic-acid polyethylene glycol N-hydroxysuccinimide (LA-PEG-NHS) using an amide linkage, and PD-L1-AuNP-DOX was constructed by linking LA-PEG-DOX, LA-PEG-PD-L1 and short PEG chain on the surface of AuNP using thiol-Au covalent bonds. Physicochemical characterizations and biological studies of PD-L1-AuNP-DOX were performed in the presence of near infrared (NIR) irradiation (biologic studies were conducted using cellular uptake, apoptosis and cell cycle assays in CT-26 cells). PDL1-AuNP-DOX (40.0 ± 3.1 nm) was successfully constructed and facilitated the efficient intracellular uptake of DOX as evidenced by pronounced apoptotic effects (66.0%) in CT-26 cells. PD-L1-AuNP-DOX treatment plus NIR irradiation significantly and synergistically suppressed the in vitro proliferation of CT-26 cells by increasing apoptosis and cell cycle arrest. The study demonstrates that PD-L1-AuNP-DOX in combination with synergistic targeted chemophotothermal therapy has a considerable potential for the treatment of localized CRC.


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INTRODUCTION Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the third leading cause of cancer-related mortality worldwide.1-3 An annual report on the status of CRC (issued by American cancer society) documented five-year survival rates ranging from 88.1% (stage I) to only 12.6% (stage IV).4 The most effective therapeutic strategy for all stages of localized CRC is surgical resection.5, 6 However, surgery may not eliminate all cancerous cells and around fifty percent of advanced-stage CRC patients experience tumor regrowth and recurrence following surgery.5 Therefore, the prognosis of patients with advanced CRC after surgery still remains poor.5, 7

Adjuvant treatments administered after surgery, such as, chemotherapy have reduced recurrence

and increased survival.8 Chemotherapeutics like doxorubicin (DOX), fluorouracil, cisplatin, and mitomycin are usually used to kill residual CRC cells after surgery,7 DOX is an anthracycline that inhibits nucleic acid synthesis9 and inhibits the proliferation CRC cells6,

10, 11

but current

chemotherapeutic monotherapies are unsatisfactory due to the ability of CRC cells to develop multidrug resistance. On the other hand, combinatorial strategies, such as, chemotherapy with immunotherapy,12 radiation therapy,13, 14 targeted therapy15, 16 or photothermal therapy (PTT)17 have been reported to cooperatively suppress cancer progression and to minimize side effects.18 Gold nanoparticles (AuNP) exhibit characteristic surface plasma resonance (SPR) absorption in the near infrared (NIR) region and are viewed as excellent agents for cancer PTT.19-21 AuNP-based PTT is based on the conversion of NIR light energy into heat and the generation of reactive oxygen species (ROS), which can be utilized to ablate tumors.22 Cytotoxic drugs can also be bound to AuNP platforms,23 which enable drugs and heat to be delivered specifically and simultaneously to tumor microenvironments.18, 24 DOX loading onto NP offers a promising approach to control drug release for cancer treatment because of synergism and tunable NIR absorption characteristics of


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AuNP.1, 23-26 However, high intensity NIR irradiation can damage surrounding normal tissues, and thus, targeted delivery is required to enhance photosensitizer delivery and minimize adverse effects.22,

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In particular, the nonspecific distribution of DOX in vivo is associated with

cardiotoxicity.25, 28, 29 Therefore, targeted drugs that are taken up by receptors uniquely expressed in CRC are required to overcome drug delivery issues.21,

26, 30, 31

Preclinical studies have demonstrated PD-L1 is

overexpressed by CRC cells.32-34 PD-L1 (B7-H1 or CD274) is a type I transmembrane protein that was recently implicated in the etiology of CRC and shown be a biomarker of CRC.35 Losa et al.36 and Schirripa et al.37 showed some CRC subtypes, especially microsatellite instability-high (MSIH) CRC (a highly immunogenic cancer), exhibit PD-L1 upregulation on cell surfaces. Furthermore, PD-L1 overexpression is known to be significantly associated with prognosis and overall survival in curatively resected CRC patients,38, 39 and currently the safety and efficacy of MEDI4736 or MPDL3280A (both anti-PD-L1 antibodies) plus cetuximab combination therapy are undergoing phase II trials in advanced CRC.34 This study was conducted to develop AuNP-modified with anti-PD-L1 antibody and drugcovalent conjugation to lipoic-acid polyethylene glycol N-hydroxy succinimide (LA-PEG-NHS) as a novel drug delivery system for combined delivery of a drug and heat to CRC cells. DOX and anti-PD-L1 antibody were attached to the α-terminal end group of LA-PEG-NHS through an amide linkage. PEGylated DOX (LA-PEG-DOX) and PEGylated anti-PD-L1 antibody (LA-PEG-PDL1) were covalently attached to the surfaces of AuNP by dithiol-Au covalent bonds. AuNP were further coated with short PEG-SH chains to improve nanoparticles (NP) stability.23 The antitumor activity of PD-L1-AuNP-DOX plus NIR was evaluated in CT-26 cells.


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EXPERIMENTAL SECTION Murine colorectal cancer cells CT-26 murine CRC cells were provided by the Korean Cell Line Bank (Seoul, Republic of Korea). CT-26 cancer cells were cultured in Dulbecco Modified Eagle Medium (DMEM, GibcoInvitrogen, USA) supplemented with 10% fetal bovine serum (FBS, Gibco-Invitrogen, USA) and 100 U/mL of penicillin, 100 µg streptomycin (Gibco-Invitrogen, USA) at 37°C in a 5% CO2 atmosphere.

Figure 1. Schematic illustration of the construction of PD-L1-AuNP-DOX. (A) LA-PEG-NHS was derivatized with DOX. (B) Anti-PD-L1 antibody was modified with LA-PEG-NHS. (C) Reaction scheme for the conjugation of LA-PEG-DOX, LA-PEG-PD-L1, and PEG-SH onto the surfaces of AuNP.


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Synthesis and characterization of DOX and anti-PD-L1 antibody conjugated gold nanoparticles (PD-L1-AuNP-DOX) The synthesis and characterization of PD-L1 targeted and DOX conjugated AuNP (PD-L1AuNP-DOX) are described in the Supporting Information (SI). LA-PEG-DOX (Figure 1A) was synthesized by reacting the amine group of DOX with LA-PEG-NHS40-43 and characterized as described in SI (Figure S1). LA-PEG-DOX was characterized by Fourier transform infrared (FTIR) spectroscopy (SI, Figure S1A) and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) (SI, Figure S1B). Fluorescence and UV spectra of LAPEG-DOX confirmed the presence of conjugated DOX in LA-PEG-DOX (SI, Figure S1C and D). LA-PEG-PD-L1 was prepared as described in Figure 1B. Briefly, LA-PEG-PD-L1 was prepared by reacting the ε-amino groups on the lysine residue of anti-PD-L1 antibody with LA-PEG-NHS (5 kDa with a NHS terminal group).44, 45 PEGylation of anti-PD-L1 antibody was confirmed by SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) and a TNBSA (2, 4, 6trinitrobenzene sulfonic acid) assay (SI, Figure S2A and B). Size-exclusion chromatography (SEC) confirmed the PEGylation of anti-PD-L1 antibody (SI, Figure S2C) and the molecular mass of LA-PEG-PD-L1 was determined by MALDI-TOF-MS (SI, Figure S2D). The synthesis of PD-L1AuNP-DOX is described in the SI (Figure 1). Briefly, PD-L1-AuNP-DOX was prepared by first adding 10 μg of LA-PEG-PD-L1 to AuNP (30 μg/mL). Then, LA-PEG-DOX (1 μg) was added to AuNP solution (30 μg/mL).45, 46 Finally, 5 μL of 200 μM of PEG-SH was added to stabilize AuNP, as previously described 44. PD-L1-AuNP was prepared by adding LA-PEG-PD-L1 and PEG-SH to AuNP solution, whereas PD-L1 non-targeted (NT-AuNP) was prepared in a similar manner without adding LA-PEG-PD-L1 to AuNP.


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To evaluate retention of PD-L1 immunoreactivity following PEGylation, different ratios of LAPEG-NHS to anti-PD-L1 antibody (5:1, 25:1 and 50:1) were prepared and then their bindings to PD-L1 overexpressing CT-26 cells were evaluated using a FACSCaliburTM flow cytometer (BD Biosciences, CA, USA). Briefly, CT-26 cells (2 × 105 cells/well) were cultured in 12-well plates with 20 ng/mL of recombinant mouse interferon gamma (IFN-ɤ, Gibco, ThermoFisher Scientific, CA, USA) overnight. Cells were then trypsinized, blocked with 2% FBS and 0.5% bovine serum albumin (BSA, Sigma-Aldrich, St Louis, MO, USA) in phosphate buffer saline (PBS, Gibco, USA). They were then incubated with unmodified anti-PD-L1 antibody or LA-PEG-PD-L1 conjugates for 2 h at room temperature. After washing with PBS, cells were incubated at 4 °C for 1 h with 8 μg/mL of AlexaFluor 488 anti-rat IgG (Thermo Fischer Scientific, Rockford, USA), rinsed three times, and dispersed in FACS buffer (PBS containing 0.1% BSA and 0.05% sodium azide) and analyzed by flow cytometry. Untreated CT-26 cells and cells treated with AlexaFluor 488 anti-rat IgG but not pretreated with anti-PD-L1 antibody were used as controls. Confocal laser scanning microscopy (CLSM) was used to compare the immunoreactivities of LA-PEG-PD-L1 conjugates prepared using different LA-PEG-NHS to anti-PD-L1 antibody ratios (5:1, 25:1, or 50:1) and different amounts of anti-PD-L1 antibody conjugated on AuNP (5, 10, 20, or 40 μg). CT-26 cells (5 × 105 cells/well) were plated on cover glass slips in 6-well plates, pretreated with IFN-ɤ and cultured overnight at 37°C. After rinsing with PBS, cells were further incubated with 10 μg of anti-PD-L1 antibody and LA-PEG-PD-L1 conjugates overnight. Finally, cells were fixed with 4% para-formaldehyde, rinsed, incubated with 8 μg/mL of AlexaFluor 488 anti-rat IgG, and then with 10 μg/mL of DAPI (4, 6 –diamidino–2–phenylindole; Sigma-Aldrich, St Louis, MO, USA) to stain nuclei. Slides were then mounted in Vectashield mounting medium


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(Vector laboratories, Inc; USA) and sealed. Images were obtained by CLSM (Carl Zeiss LSM5, Carl Zeiss Inc, Germany). Immunogold–TEM analysis was used to estimate the number of anti-PD-L1 antibodies on the surfaces of PD-L1-AuNP. PD-L1-AuNP or NT-AuNP conjugates were incubated with anti–rat IgG–5 nm gold–conjugate (Cytodiagnostics, Burlington, CA, USA) for 1 h. Following conjugation, AuNP were centrifuged at 7,000 × g to separate 20 nm AuNP from secondary antibody conjugated–5 nm AuNP. For TEM imaging, 5 μL of AuNP conjugates were deposited on a copper-coated grid. Particle sizes, zeta potentials and UV-vis spectra, as determined by dynamic light scattering (DLS, Brookhaven instruments Corp, NY, USA) and UV-vis spectroscopy, respectively, were obtained at each stage of the AuNP bioconjugation procedure.

Cellular Uptake and intracellular distribution The cellular uptakes and intracellular distributions of PD-L1-AuNP-DOX, NT-AuNP-DOX, and free DOX were observed by CLSM. CT-26 cells (5 × 105 cells/well) were seeded on coverslips placed in 6-well plates, pretreated with IFN-ɤ and incubated for 24 h. Cells were then treated for 1, 2, 12, or 24 h with NT-AuNP, PD-L1-AuNP, DOX, NT-AuNP-DOX, or PD-L1-AuNP-DOX (0.5 μg/mL DOX), rinsed with PBS, fixed with 4% para-formaldehyde, and incubated with 8 μg/mL of AlexaFluor 488 anti-rat IgG and 10 μg/mL of DAPI. Cells were mounted on slides in Vectashield mounting medium and images were obtained by CLSM. To compare the cellular uptakes of PD-L1-AuNP-DOX, free DOX, or NT-AuNP-DOX, CT-26 cells (2 × 105 cells/well) were seeded in a 12-well plate overnight with pretreatment of IFN-ɤ, incubated for 2 h with native DOX, NT-AuNP-DOX, or PD-L1-AuNP-DOX at a DOX concentration of 0.5 μg/mL. Additionally, to evaluate the time dependent intracellular


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distributions, cells were treated with PD-L1-AuNP-DOX for 0.5, 1, 2, 12, or 24 h at 37 °C, washed with PBS, trypsinized, resuspended in 1 mL FACS buffer, and subjected to flow cytometric analysis.

In vitro cytotoxicity assay Viabilities of cells were determined using the colorimetric cell counting kit-8 (Dojindo Molecular Technology Inc., Rockville, MD). CT-26 cells (1 × 104 cells/well) were seeded in 96well plate with a pretreatment of IFN-ɤ and incubated overnight at 37 °C. The growth medium was then replaced with formulations containing DOX, NT-AuNP-DOX, or PD-L1-AuNP-DOX (0, 0.01, 0.1, 0.5, 1, 2.5, 10 or 25 μg/mL of DOX) for 24 h. In addition, PD-L1-AuNP-DOX-induced cancer cell death was performed by 3 min of PTT (808-nm laser, 2.5 W/cm2) after 2 h cellular uptake of NT-AuNP, PD-L1-AuNP, DOX, NT-AuNP-DOX and PD-L1-AuNP-DOX (0.5 μg/mL DOX) followed by 24 h of chemotherapy. Cells were then washed and incubated with 10 μL of CCK-8 solution for 4 h at 37 °C. Formazan absorption was measured at 450 nm using an Infinite™ M200 PRO microplate reader. We also assessed the long-term cytotoxic effect of PD-L1-AuNP-DOX on the clonogenic survival (CS) of PD-L1 overexpressing CRC cells. CT-26 cells (5 × 105 cells/well) were cultured with pre-treatment of IFN-ɤ overnight in a 6-well plate, incubated with NT-AuNP, PD-L1-AuNP, DOX, NT-AuNP-DOX, or PD-L1-AuNP-DOX (0.5 μg/mL DOX) for 2 h and then irradiated or not with NIR, incubated at 37 °C for 24 h. After treatment, CT-26 cells were rinsed with PBS, trypsinized, seeded at 500−1000 cells/well in a 6-well plate and cultured for 7-10 days. After washing with PBS, colonies (> 50 cells) were stained with methylene blue (Sigma-Aldrich, St Louis, MO, USA) and counted. Plating efficiencies (PE) were calculated by dividing numbers of


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colonies generated by the number of cells cultured. CS was estimated by dividing the PE values of treated CT-26 cells by that of control cells. Membrane–integrity was assessed using a kit containing calcein acetoxymethyl (calcein AM) and ethidium homodimer–1(EthD–1) (Live/Dead Viability/Cytotoxicity assay; Invitrogen, Carlsbad, CA). CT-26 cells (2 × 105 cells/well) were seeded in a 12-well plate with pre-treatment of IFN-ɤ, incubated overnight, treated with NT-AuNP, PD-L1-AuNP, NT-AuNP-DOX, PD-L1AuNP-DOX, or native DOX (0.5 μg/mL) for 2 h, NIR-irradiated or not and incubated at 37 °C for 24 h. The cells were stained with 2 μM of calcein-AM and 4 μM of EthD-1. Images were obtained using a fluorescence microscope (IX71, Olympus, TKY, Japan).

Apoptosis assay The FITC-annexin V apoptosis kit (BD Biosciences, San Diego, CA, USA) was used to evaluate the apoptotic effects of NT-AuNP, PD-L1-AuNP, NT-AuNP-DOX, PD-L1-AuNP-DOX, or free DOX plus NIR irradiation on CT-26 cells. Briefly, CT-26 cells (2× 105 cells/well) were seeded in 12-well plates, pre-treated with IFN-ɤ and incubated overnight at 37 °C. They were then treated with NT-AuNP, PD-L1-AuNP, DOX, NT-AuNP-DOX, or PD-L1-AuNP-DOX (0.5 μg/mL DOX) with or without NIR, incubated for 24 h, rinsed, detached from well surfaces by trypsinization, dispersed in 500 μL annexin V binding buffer, treated with 5 μL of FITC-annexin and propidium iodide (PI), and incubated in the dark for 15 min. Finally, cells were rinsed twice with PBS, 1 mL of annexin V binding buffer was added, and analyzed by flow cytometry.

Intracellular ROS generation


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CT-26 cells (2× 105 cells/well) were seeded in 12-well plates, pretreated with IFN-ɤ and incubated overnight at 37 °C, treated with NT-AuNP, PD-L1-AuNP, DOX, NT-AuNP-DOX, or PD-L1-AuNP-DOX (0.5 μg/mL DOX), irradiated or not with NIR, and incubated for 24 h. Cells were then trypsinized, treated with 10 μM of freshly prepared DCFH-DA solution (2′,7′dichlorofluorescin diacetate; Sigma-Aldrich, St Louis, MO, USA) for 1 h and subjected to flow cytometry. In addition, the effect of ROS generation and subsequent apoptosis were evaluated by western blotting. CT-26 cells (2 ×105 cells/well) were incubated with NT-AuNP, PD-L1-AuNP, NTAuNP-DOX, PD-L1-AuNP-DOX, or free DOX (0.5 μg/mL DOX) and NIR irradiated or not, incubated for 24 h. The extract of cells were harvested in RIPA buffer (Thermo Scientific, Rockford, USA), supplemented with protease and phosphatase inhibitors (Thermo Scientific, Rockford, USA). Soluble protein concentrations were determined using bicinchoninic acid reagent (BCA, Pierce, Rockford, IL, USA). Cell lysates with equivalent amounts of protein were loaded onto 10% SDS-PAGE gels, and separated proteins were electrophoretically transferred to PVDF (polyvinylidene difluoride) membranes (transfer membranes; Merck Millipore Ltd, Germany). Membranes were incubated with rabbit primary antibody (Cell Signaling technology, MA, USA) including catalase, superoxide dismutase (SOD2) and GAPDH (1:1,000 dilution) overnight then with anti-rabbit IgG-horseradish peroxidase-linked secondary antibody (Cell Signaling technology, MA, USA) for 1 h at 25 °C. Proteins of interest were visualized by incubation with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, USA) for 5 min and then membranes were exposed to film.

Cell cycle analysis


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Cell cycle changes induced by PD-L1-AuNP-DOX treatment were studied by flow cytometry. CT-26 cells (5×105 cells/well) were seeded in 6-well plates, pre-treated with IFN-ɤ, incubated for overnight and treated for 24 h with NT-AuNP, PD-L1-AuNP, DOX, NT-AuNP-DOX, or PD-L1AuNP-DOX (0.5 μg/mL of DOX) and irradiated or not with NIR. Cells were harvested by trypsinization and fixed in 70% ethanol and treated with PI solution (50 μg/mL of PI and 100 μg/mL of RNase A) (Sigma–Aldrich, St. Louis, MO, USA) for 2 h. The cell cycle status of treated CT-26 cells were analyzed with flow cytometer.

Statistical analysis Intergroup comparisons were performed using the unpaired t-test. Results are expressed as means ± SDs, and P values of < 0.05 were considered statistically significant.

RESULTS Synthesis and characterization of PD-L1-AuNP-DOX The results of all characterization studies for PD-L1-AuNP-DOX are provided in SI. The successful conjugation of LA-PEG-NHS with the amine group of DOX was confirmed by the disappearance of the FT-IR peak at 1713 cm-1 and the appearance of characteristic peaks at 1650 cm-1, which was attributed to amide bond formation (-CO-NHR) (SI, Figure S1A). Additionally, the conjugation of LA-PEG-NHS and DOX was confirmed by MALDI-TOF-MS, which showed a molecular weight increase of ~ 500 Da (SI, Figure S1B). The components of LA-PEG-DOX were investigated by UV-vis spectroscopy (SI, Figure S1C). Furthermore, the presence of conjugated DOX in LA-PEG-DOX was demonstrated by fluorescence spectroscopy (SI, Figure S1D).


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SDS-PAGE showed that increasing the LA-PEG-NHS:anti-PD-L1 antibody ratio resulted slight enhancement in the molecular weight of anti-PD-L1 antibody, representing more PEG substitution (SI, Figure S2A). A 5:1 ratio of LA-PEG-NHS to anti-PD-L1 antibody was chosen and this resulted in 12.0 ± 3.1% modification of the ε-amino groups on anti-PD-L1 antibody by LA-PEGNHS (SI, Figure S2B). SEC analysis and the MALDI-TOF-MS spectra of LA-PEG-PD-L1 conjugates showed double peaks attributable to LA-PEG-PD-L1 and unmodified anti-PD-L1 antibody, respectively (SI, Figure S2C and D). Flow cytometry analysis (Figure 2A) of LA-PEG-PD-L1 exhibited increased binding when LAPEG-NHS:anti-PD-L1 molar ratio was decreased. When LA-PEG-PD-L1 conjugates were prepared by reacting anti-PD-L1 antibody with a 50-fold molar excess of LA-PEG-NHS, the immunoreactivity of anti-PD-L1 antibody was only 50%. Flow cytometry of LA-PEG-PD-L1 immunoconjugates indicated PD-L1 binding affinity was > 90% for anti-PD-L1 antibody reacted with a 5-fold molar excess of LA-PEG-NHS. CLSM confirmed a reduction in cell binding when a 50-fold molar excess of LA-PEG-NHS was reacted with anti-PD-L1 antibody (Figure 2B). On the other hand, when anti-PD-L1 antibody was incorporated at LA-PEG-NHS:anti-PD-L1 molar ratios of 5:1 or 25:1 cell binding was satisfactory, and thus, a molar ratio of 5:1 was used for further experiments. Mono-, di- or multi-thiol-containing LA-PEG-NHS can interact with AuNP to form covalent bonds 45, 47. The amount of anti-PD-L1 antibody used (10 mg) was selected because it resulted in good PD-L1-AuNP immunoreactivity in CT-26 cells (SI, Figure S3). TEM images of PD-L1AuNP and NT-AuNP (Figure 3A) indicated binding of gold-conjugated secondary anti-rat IgG antibody to anti-PD-L1 antibody localized on AuNP. TEM results showed NT-AuNP were well separated (Figure 3A). PEG-SH coatings were observed as faint coronas on AuNP (Figure 3A).


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The mean distance between two crossed antibodies conjugated on AuNP was 40.0 ± 3.1 nm, which meant the mean distance from AuNP surfaces to antibody conjugated with LA-PEG was ~11.0 ± 1.5 nm (Figure 3A). The approximate number of LA-PEG-PD-L1 conjugated via the LA-PEG per AuNP was around 8.0 ± 2.0 (Figure 3A).

Figure 2. Immunoreactivity of LA-PEG-PD-L1. (A) Flow cytometry results for the fluorescences of LA-PEG-PD-L1 conjugates associated with PD-L1-overexpressing CT-26 cells. Cells were treated with anti-PD-L1 antibody and LA-PEG-PD-L1 at different molar ratios (5:1, 25:1, and 50:1). (B) CLSM images of PD-L1-overexpressing CT-26 cells incubated with anti-PD-L1 antibody and LA-PEG-PD-L1 produced using different molar ratios of LA-PEG-NHS to anti-PDL1 antibody (5:1, 25:1, and 50:1). Cells were incubated with AlexaFluor 488-anti- rat IgG (green), which visualizes anti-PD-L1 antibody. Cell nuclei were stained with DAPI (blue). Scale bar: 10 μm.


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Figure 3. Verification of anti-PD-L1 antibody conjugation on the surfaces of AuNP. (A) TEM images of AuNP, NT-AuNP and PD-L1-AuNP reacted with 5 nm-gold labeled anti-rat IgG. (B) Hydrodynamic diameters and (C) zeta potentials of AuNP, NT-AuNP, PD-L1-AuNP, and PD-L1AuNP-DOX as determined by DLS.

The successful anchoring of LA-PEG-DOX and LA-PEG-PD-L1 on the surfaces of citratestabilized AuNP was confirmed by hydrodynamic size and zeta potential changes that occurred during conjugation. The mean hydrodynamic size of AuNP was 23.1 ± 1.2 nm, whereas those of NT-AuNP, PD-L1-AuNP, and PD-L1-AuNP-DOX were 29.4 ± 1.4 nm, 54.3 ± 1.3 nm and 62.0 ± 1.2 nm, respectively (Figure 3B). The surface charge of native AuNP was −29.6 ± 2.3 mV, while the surface charges of NT-AuNP, PD-L1-AuNP, and PD-L1-AuNP-DOX were −20.5 ± 2.5 mV, −11.5 ± 2.8 mV and −11.1 ± 2.7 mV, respectively (Figure 3C).


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Cellular Uptake and intracellular distribution study Green fluorescence originating from AlexaFluor 488 revealed the receptor binding of PD-L1AuNP and its receptor-mediated endocytosis. CLSM images of AlexaFluor 488 and DAPI stained CT-26 cells showed that PD-L1-AuNP bound strongly to cells, whereas NT-AuNP did not (Figure 4A). Intense red DOX fluorescence colocalized with DAPI in nuclei. Confocal images indicated that PD-L1-AuNP-DOX was efficiently taken up by CT-26 cells and was localized in nuclei after 2 h of incubation. After 1 or 2 h of incubation, the cellular uptake of PD-L1-AuNP-DOX by CT26 cells was obviously greater than that of NT-AuNP-DOX or free DOX. In the present study, lower intracellular DOX concentrations were detected in free DOX treated cells than in NT-AuNPDOX treated cells. CLSM images at 12 and 24 h displayed higher DOX fluorescence intensities in NT-AuNP-DOX and free DOX treated CT-26 cells than at 1 or 2 h. Interestingly, at 12 and 24 h post-treatment, PD-L1-AuNP-DOX internalization remained greater than those of NT-AuNP-DOX or free DOX. The cellular uptake of PD-L1-AuNP-DOX was also quantitatively evaluated by flow cytometry. As illustrated in Figure 4B, NT-AuNP-DOX-treated CT-26 cells displayed DOX fluorescence intensities two times higher than free DOX treated cells (P < 0.001) after incubation for 2 h, and after exposure for 2 h, DOX fluorescence intensity in PD-L1-AuNP-DOX treated cells was significantly higher than in DOX (7.6- fold; P < 0.001) or NT-AuNP-DOX (3.8- fold; P < 0.001) treated cells. Furthermore, DOX fluorescence intensity was increased 60-fold when CT-26 cells were treated with PD-L1-AuNP-DOX for 12 h as compared with 0.5 h, but increased by only 1.3fold when cells were treated for 24 h as compared with 12 h. This phenomenon was confirmed by PD-L1-AuNP-DOX cellular uptakes determined by flow cytometry after different incubation times.


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Figure 4. Cellular uptake of PD-L1-AuNP-DOX by CT-26 cells. (A) CLSM images of untreated cells and of cells treated with NT-AuNP, PD-L1-AuNP, DOX, NT-AuNP-DOX, or PD-L1-AuNPDOX (0.5 µg/mL). Cells were incubated with AlexaFluor 488-anti-rat IgG (green), which visualizes anti-PD-L1 antibody. Nuclei were counterstained with DAPI (blue). Scale bar: 20 μm. (B) Flow cytometry analysis of the cellular uptakes of DOX, NT-AuNP-DOX, and PD-L1-AuNPDOX (0.5 µg/mL of DOX). To investigate the time-dependent cellular uptake of PD-L1-AuNPDOX, cells were incubated at different times (0.5, 1, 2, 12, and 24 h). Results are expressed as means ± SD (n=3).


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In vitro cytotoxicity assay As shown in Figure 5A, the cell proliferation inhibition efficacies of PD-L1-AuNP-DOX, NTAuNP-DOX, and free DOX were strongly dose-dependent after culture for 24 h. As expected, the IC50 value of PD-L1-AuNP-DOX was 0.25 μg/mL, which was significantly lower than IC50 values of free DOX or NT-AuNP-DOX (0.5 and 0.4 μg/mL, respectively; P < 0.001) (Figure 5A). The anti-tumor activities of different formulations are compared in Figure 5B. NT-AuNP and PD-L1AuNP had negligible impacts on cell proliferation (cell viability ˃ 95%), whereas NT-AuNP or PD-L1-AuNP plus NIR irradiation showed less cell viability (91.2 ± 1.1%; P < 0.05) and (82.2 ± 2.2%, P < 0.001), respectively. CT-26 cells exposed to PD-L1-AuNP-DOX plus NIR laser irradiation were markedly less viable (21.4 ± 0.75%; P < 0.001). To more quantitatively evaluate therapeutic efficacies, the CS of CT-26 cells exposed to PD-L1AuNP-DOX for 24 h and then cultured for 10 days were evaluated as measures of long-term toxicity (Figure 5C). Treatment of CT-26 cells with NT-AuNP or PD-L1-AuNP had no significant effect on survival (98.9 ± 0.8%, 98.5 ± 1.3%, respectively; P > 0.05). Furthermore, CT-26 cells exposed to NT-AuNP plus NIR or NT-AuNP has similar survivals (94.6 ± 1.1%, 98.9 ± 0.8%, respectively; P > 0.05). However, cells treated with PD-L1-AuNP plus NIR exhibited significantly lower survivals (82.2 ± 2.2%; P < 0.001) as were the CS of CT-26 cells exposed to PD-L1-AuNPDOX (22.2 ± 1.7%), and this CS was significantly lower than those of cells treated with NT-AuNPDOX (47.5 ± 2.8%; P < 0.001) or DOX (55.0 ± 2.6%; P < 0.001). Exposure of CT-26 cells to PDL1-AuNP-DOX plus NIR irradiation significantly reduced CS to 10.5 ± 1.9% as compared with 22.2 ± 1.7% for cells exposed to PD-L1-AuNP-DOX (P < 0.001) (Figure 5C). The anti-tumor effects of PD-L1-AuNP-DOX on CT-26 cells was further verified using live/dead calcein–AM and EthD–1 assay (Figure 5D); in this assay live and dead cells fluoresce


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green and red, respectively. Untreated controls, NT-AuNP or PD-L1-AuNP treated cells showed high green fluorescence, whereas PD-L1-AuNP plus NIR treated cells showed high intensity of red fluorescence indicating obvious decrease in cell viability (Figure 5D). On the other hand, most CT-26 cells treated with NT-AuNP-DOX, DOX, or PD-L1-AuNP-DOX died, which was revealed by strong red fluorescence; conversely, lower intensity of green–stained (live) cells were visualized. Furthermore, enhanced cell death was observed clearly in PD-L1AuNP-DOX especially, with NIR irradiation compared to NT-AuNP-DOX or free DOX (Figure 5D).

Apoptosis assay In Figure 6A, the left and right lower quarters contain live cells and cells in early apoptosis, respectively, whereas, the right and left upper quarters contain cells in late apoptosis and necrosis, respectively. AuNP without DOX caused little or no CT-26 cells apoptosis (1.4%) (Figure 6A), whereas free DOX induced early and late apoptosis (44.0%) (Figure 6A). In NT-AuNP-DOX or PD-L1-AuNP-DOX treated cells, necrotic cell proportions were significantly higher than in free DOX treated cells, whereas early apoptotic proportions were decreased. In PD-L1-AuNP-DOX treated cells increased the proportions of apoptotic and necrotic cells. Furthermore, NT-AuNP and PD-L1-AuNP plus NIR irradiation increased the proportion of apoptotic CT-26 cells as compared with NT-AuNP or PD-L1-AuNP treated cells. NIR irradiation did not increase the effect of free DOX, but did increase apoptotic and necrotic proportions when administered with NT-AuNPDOX or PD-L1-AuNP-DOX.


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Figure 5. The cytotoxic effect of PD-L1-AuNP-DOX on CT-26 cells. (A) In vitro viability of CT26 cells exposed to PD-L1-AuNP-DOX, NT-AuNP-DOX, or free DOX for 24 h. (B) In vitro viability of CT-26 cells exposed to NT-AuNP, PD-L1-AuNP, NT-AuNP-DOX, PD-L1-AuNPDOX, or DOX (0.5 µg/mL) for 24 h treated with or without NIR irradiation. Results are expressed as means ± SDs (n = 4). (C) Clonogenic survival of CT-26 cells exposed to NT-AuNP, PD-L1AuNP, NT-AuNP-DOX, PD-L1-AuNP-DOX or DOX (0.5 µg/mL) for 24 h with or without NIR irradiation then cultured for 10 days. Results are expressed as means ± SDs (n=3). (D) Live/dead staining assay results of the effects of NT-AuNP, PD-L1-AuNP, NT-AuNP-DOX, PD-L1-AuNPDOX, or DOX (0.5 µg/mL) treated for 24 h with or without NIR. Live cells were stained with calcein (green) and dead cells were stained with ethidium homodimer (red). Scale bar: 400 μm.


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Figure 6. In vitro evaluations of apoptosis and intracellular ROS generation. (A) FACS analysis showed apoptosis and necrosis of CT-26 cells treated or not with NT-AuNP, PD-L1-AuNP, NTAuNP-DOX, PD-L1-AuNP-DOX or DOX (0.5 µg/mL) for 24 h with or without NIR radiation. (B) Intracellular ROS generation in CT-26 cells treated with different formulations with or without NIR radiation as determined by FACS analysis. (C) Determination of the levels of ROS detoxification enzymes (SOD2 and catalase) and of GAPDH in CT-26 cells treated with the different formulations.

Intracellular ROS generation CT-26 cells treated with NT-AuNP or PD-L1-AuNP shown similar patterns of ROS generation as untreated cells, whereas NT-AuNP or PD-L1-AuNP plus NIR increased ROS generation (1.2– 1.6- fold; P < 0.001) (Figure 6B). Exposure of CT-26 cells to DOX also caused considerable ROS


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generation (Figure 6B). As was expected, NT-AuNP-DOX and PD-L1-AuNP-DOX caused 1.3and 2.6- fold increases in ROS formation, respectively, versus DOX in CT-26 cells (P < 0.001). Furthermore, DOX plus AuNP-NIR irradiation interacted synergistically in terms of increasing ROS generation (Figure 6B). In PD-L1-AuNP-DOX plus NIR treated cells, intracellular ROS generation was 12.5-fold that observed in untreated controls (P < 0.001). Additionally, western blotting was performed to assess SOD2 and catalase expression level as a ROS marker protein in CT-26 cells. As seen in Figure 6C, NT-AuNP and PD-L1-AuNP showed similar patterns as untreated cells. Furthermore, ROS markers were down-regulated in CT-26 cells treated with free DOX, NT-AuNP-DOX or especially PD-L1-AuNP-DOX. In addition, a visible decrease in SOD2 and catalase expression under NIR irradiation was evident. The GAPDH protein band, which was used as a control, was relatively consistent across all groups.

Cell cycle analysis Cell cycle analysis showed NT-AuNP-DOX and PD-L1-AuNP-DOX caused arrest in the S and G2/M phase and significantly decreased in the proportion of cells in the G0/G1 phase (13.4% and 19.9%, respectively; P < 0.001) (Figure 7). In addition, cell cycle analysis showed DOX interaction with DNA caused G2/M phase cell cycle arrest (15.6%) and significantly reduced the proportion of cells in the G0/G1 phase (Figure 7). NT-AuNP-DOX and PD-L1-AuNP-DOX both significantly decreased the proportion of cells in the G0/G1 phase. Exposure of CT-26 cells to NT-AuNP or PD-L1-AuNP plus NIR radiation increased the proportions of cells in the S and G2/M phase. Additionally, exposure of cells to NT-AuNP-DOX or especially PD-L1-AuNP-DOX plus NIR induced obvious shifts toward the S and G2/M phases.


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Figure 7. Effect of PD-L1-AuNP-DOX on cell cycle stage in CT-26 cells. Flow cytometry results of cell cycle distributions after cells were exposed to NT-AuNP, PD-L1-AuNP, DOX, NT-AuNPDOX or PD-L1-AuNP-DOX (0.5 µg/mL) for 24 h with or without NIR irradiation. Results are expressed as means ± SD (n=3).

DISCUSSION In this study, a novel chemo-photothermal approach was devised by linking 20.4 nm diameter AuNP to LA-PEG-NHS (5 kDa) derivatized with DOX and anti-PD-L1 antibody to caused specific PD-L1-AuNP-DOX binding with and taking up by PD-L1-positive CT-26 CRC cells. The surfaces


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of AuNP were also coated with short PEG chains (2 kDa) to prevent aggregation in solution. The results of all characterization studies for PD-L1-AuNP-DOX are provided in SI. The PEGylated DOX was synthesized by covalently conjugating LA-PEG-NHS with 1.5- fold molar excess of DOX in the presence of TEA as a catalyst.41-43 Modification of εamino groups on the lysine residues of anti-PD-L1 antibody using LA-PEG-NHS provided LA-PEG-PD-L1 conjugate with different degree of PEG substitution. Previous studies21, 48 have demonstrated that PEGylation of antibodies resulted in the loss of 70-90% of antigen binding efficiency. In our study, CLSM and flow cytometry analysis indicated a significant reduction (50%) in cell binding when a 50-fold molar excess of LA-PEG-NHS was reacted with anti-PD-L1 antibody. On the other hand, when anti-PD-L1 antibody was incorporated at LA-PEGNHS:anti-PD-L1 molar ratios of 5:1 or 25:1 cell binding was satisfactory (> 90%), and thus, a molar ratio of 5:1 was used for further experiments. This result indicates that PEGylation with 5 kDa LA-PEG-NHS did not generate any non-specific effect on anti-PD-L1 antibody immunoreactivity.21 The reduced receptor-binding affinity of PEGylated anti-PD-L1 antibody (50:1) can be explained by the steric effect of PEG on anti-PD-L1 antibody. Mono-, di- or multi-thiol-containing LA-PEG-NHS can interact with AuNP to form covalent bonds.45, 47 TEM results showed NT-AuNP were well separated, indicating they were effectively stabilized by PEG-SH. TEM images of PD-L1-AuNP indicated binding of gold-conjugated secondary anti-rat IgG antibody to anti-PD-L1 antibody localized on AuNP. The attachment of several LA-PEG-PD-L1 molecules to an AuNP has been reported to enhance interactions with PDL1 on CRC cells through multivalent interaction.49-51 The successful anchoring of LA-PEG-DOX and LA-PEG-PD-L1 on the surfaces of AuNP was confirmed by hydrodynamic size changes that occurred during conjugation. As reported


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previously,52 the increase in hydrodynamic size of AuNP formed from PD-L1-AuNP-DOX due to the existence of a swollen PEG corona around AuNP was larger than that determined by TEM. The observed enhancement in zeta potential indicated the successful replacement of LA-PEGDOX, LA-PEG-PD-L1, and PEG-SH with negatively charged citrate on AuNP. The hydrophilic PEG-SH shielded the outermost layer of the conjugate and increased PD-L1-AuNP-DOX stability. In addition, the incorporation of short and flexible long chain PEG provided physically stable AuNP with PD-L1 binding ability. Additionally, the presence of chemically conjugated anti-PDL1 antibody on AuNP rather is more predictable and probably enhances the control of biological responses.53 CLSM images of AlexaFluor 488 and DAPI stained CT-26 cells showed that PD-L1-AuNP bound strongly to cells, whereas NT-AuNP did not, demonstrating that PD-L1 binding was mediated by the anti-PD-L1 antibody modification. CLSM images showed that PD-L1 targeting played an important role in enhancing the cellular uptake of DOX-loaded AuNP. It has been previously shown different modes of cellular uptake may influence the intracellular distribution and retention of a drug.54 Confocal images indicated that PD-L1-AuNP-DOX was efficiently taken up by CT-26 cells and was localized in nuclei after 2 h of incubation. PD-L1-AuNP-DOX uptake occurred through a PD-L1-mediated process, whereas NT-AuNP-DOX and DOX were taken up by a non-specific endocytic pathway and by passive diffusion, respectively. Furthermore, the slow intracellular release of DOX from PD-L1-AuNP-DOX would be expected to result in sustained drug release and higher intracellular DOX concentrations.54 Following the uptake of PD-L1AuNP-DOX, a broad distribution of DOX was observed in the cells, suggesting that the conjugated AuNP are deconstructed during intracellular trafficking, thereby leading to DOX release. The endocytic compartments as well as lysosome could provide a proteolytic activity together with


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acidic environments sufficient for amide bonds cleavage and subsequent DOX release.11, 55 Study reported by Farooq et al.55 have demonstrated that acidic pH triggered drug release for DOX conjugated AuNP.55 Similarly, Vandana and Sahoo41 synthesized PEGylated gemcitabine with amide bond formation. They have demonstrated that at acidic pH, similar to endo-lysosomal environment, amide bond easily was cleaved within few minutes by various lysosomal enzymes. However, in more complex realistic situation in vivo a tumor environment is characterized not only by acidic pH, but also by presence of trypsin, enzymes capable of amid bond scission. Moreover, drug release can be further triggered using NIR laser light as an external stimulus.55 After 1 or 2 h of incubation, the cellular uptake of PD-L1-AuNP-DOX by CT-26 cells was obviously greater than that of NT-AuNP-DOX or free DOX, indicating that anti-PD-L1 antibody binding to PD-L1 receptors triggered the fast internalization of PD-L1-AuNP-DOX in to CT-26 cells. Due to lower levels of DOX-loaded AuNP uptake, cells treated with NT-AuNP-DOX had lower DOX fluorescence intensities than PD-L1-AuNP-DOX. Ye et al.56 reported that the intracellular accumulation of DOX in folic acid targeting-DOX-hydrazone-PEG NP in epidermal KB cells in the oral cavity was much greater than those of free DOX or non-targeting DOXhydrazone-PEG NP. In the present study, lower intracellular DOX concentrations were detected in free DOX treated cells than in NT-AuNP-DOX treated cells. This result indicates DOX penetrated cellular and nuclear membranes by passive diffusion, which depends on the concentration gradient, whereas NT-AuNP-DOX has a unidirectional transfer into cells,57 which agrees with the finding of Sun et al.58 who reported greater cellular uptake of AuNP-coated pluronic-b-poly (L-lysine) NP than of free DOX. In addition, they reported the positive charges of DOX-loaded NP aided adsorption by cell membranes and the unidirectional transfer of NP by MDA-MB-231 human breast cancer cells. This suggests greater intracellular accumulation of PD-


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L1-AuNP-DOX are promoted by accelerating the uptake kinetics of PD-L1 mediated endocytosis.59 Previous studies have demonstrated targeting NP facilitate rapid internalization due to antibody-receptor interactions.59-62 Interestingly, at 12 and 24 h post-treatment, PD-L1-AuNP-DOX internalization remained greater than those of NT-AuNP-DOX or free DOX, which may have been due to the exocytosis of DOX from cytoplasm.63, 64 However, this process may be inhibited by the conjugation of DOX in DOX-loaded AuNP. Furthermore, the slow release DOX from AuNP localized inside cell is expected to result in sustained DOX release.54 Besides the above mechanisms, in PD-L1-AuNPDOX due to targeting moiety higher amount of DOX is delivered inside the cell.63, 65 Similarly, Acharya et al.63 have demonstrated that higher fluorescence intensities were seen in cells exposed for 2 h to EGFR-rapamycin-NP than in cells exposed to non-targeting NP or free rapamycin, which reinforces the notion that targeted drug-containing NP achieve higher intracellular drug concentrations. In addition, they demonstrated cellular uptake was enhanced further by extending incubation times to 24 h or 48 h when targeting NP also achieved higher fluorescence intensities. Flow cytometry (Figure 4B) also supported our previous results that PD-L1-AuNP-DOX uptake by CT-26 cells was greater as than those by NT-AuNP-DOX or free DOX after treatment for 2 h. This phenomenon was confirmed by PD-L1-AuNP-DOX cellular uptakes determined by flow cytometry after different incubation times. Further cellular toxicity study demonstrated that enhanced nuclear uptake of DOX corresponded to greater cellular toxicity. The lower IC50 value of PD-L1-AuNP-DOX as compared to NTAuNP-DOX or free DOX, indicates the greater cytotoxicity of targeted group. Conjugating of anti-PD-L1 antibody to AuNP-DOX can enhance the targeting efficiency by reducing the probable partial loss of drug concentration to kill CT-26 cells,21 thereby improve its cytotoxicity


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in CRC cells 47. Drug cytotoxicity is strongly correlated with the concentration and duration of its intracellular retention.54 For a free drug, uptake occurs via diffusion and intracellular concentration increases to a saturation level,54 whereas for cell treated with NT-AuNP-DOX or PD-L1-AuNPDOX, higher levels of DOX were uptaken remained inside cells and DOX was released in a sustained manner,54 which suggests PD-L1-AuNP-DOX is likely to have greater therapeutic efficiency. Sahoo et al.54 investigated the anti-proliferative activity of paclitaxel-loaded NP conjugated to transferrin in MCF-7 breast cancer cells. It was found transferrin-targeted paclitaxelloaded NP had greater therapeutic efficiency than free paclitaxel or 1 ng/mL of non-targeting paclitaxel-loaded NP and that greater therapeutic efficiencies were correlated with higher cellular uptake and intracellular retention. NT-AuNP and PD-L1-AuNP had negligible impacts on cell proliferation, whereas NT-AuNP or PD-L1-AuNP plus NIR irradiation showed less cell viability. NIR PTT acts by causing protein denaturation, cell membrane disruption, cellular signaling disturbances, stimulating the activities of heat-shock-related proteins, and impairing DNA synthesis by doing so can induce cancer cell death.66 CT-26 cells exposed to PD-L1-AuNP-DOX plus NIR laser irradiation were markedly less viable. This result indicates that PD-L1-AuNP-DOX and NIR irradiation act synergistically to enhance cell death. Liao et al.67 synthesized WS2 quantum dots (WQDs)-coated DOX-loaded periodic mesoporous organosilicas (PMOs-DOX@WQDs) NP for treatment of HCT-116 colon cancer cells. They also demonstrated that this nanomedicine with significant synergistic effect of chemo-PTT, exhibited a greater cytotoxic effect in HCT-116 colon cancer cells. Similarly, Lee et al.68 developed the chemo-photothermal platform (DR4-DOXPLGA-AuH-S NP) consisted of DOX-loaded-polylactic-co-glycolic acid-Au half-shell NP with targeting agent of anti-death receptor-4 antibody. They have demonstrated that DR4-DOX-PLGAAuH-S NP formulation plus NIR irradiation significantly enhanced the in vitro anti-tumor activity


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over single therapy in DLD-1 colon cancer cells. As reported previously,69, 70 this synergistic effect may be due the greater sensitivity of NIR exposed cells to DOX. Park et al.70 demonstrated that selective photothermal heating of AuNP can increase the accumulation of targeted AuNP that enhanced the overall hyperthermia and chemotherapeutic anti-tumor activity in local tumor microenvironment. To more quantitatively evaluate therapeutic efficacies, long-term anti-tumor activity of the PDL1-AuNP-DOX were investigated (Figure 5C). No cytotoxicity was observed in CT-26 cells treated with NT-AuNP or PD-L1-AuNP, whereas cells were incubated with DOX, the survival decreased, and decreased further with NT-AuNP-DOX treatment. Interestingly, CT-26 cells treated with PD-L1-AuNP-DOX had a lower fraction of CS than NT-AuNPDOX or DOX, suggesting that targeted NP system provides a promising approach for cancer treatment.71 These results mean that targeted NP had a greater long-term cytotoxic effect.63 For PD-L1-AuNP-DOX, since more DOX were accumulated, can prevent CRC recurrence. Exposure of CT-26 cells to PD-L1-AuNP-DOX plus NIR irradiation significantly reduced CS as compared with cells exposed to PD-L1-AuNP-DOX (Figure 5C). Cooperative, synergistic therapies using dual therapeutic strategies, such as, simultaneous chemotherapy and AuNP-induced hyperthermia, could significantly reduce anti-cancer drug dosages and more effectively eradicate cancers.24, 70, 71 The synergistic photo-chemotherapy effects of PD-L1-AuNP-DOX plus NIR on CT-26 cells was further verified using live/dead calcein–AM and EthD–1 assay. Untreated controls, NT-AuNP or PD-L1-AuNP treated cells showed high green fluorescence, whereas PD-L1-AuNP plus NIR treated cells showed some evidence of cell death. On the other hand, most CT-26 cells treated with NT-AuNP-DOX, DOX, or PD-L1-AuNP-DOX died. Furthermore, treated with PD-L1-AuNP-


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DOX showed higher levels of cell death than NT-AuNP-DOX or DOX treated cells. Furthermore, the effective DOX cytotoxicity and photothermal ablation of CT-26 cells induced by NIR irradiation was observed clearly by enhanced cell death (Figure 5D). DOX induced early and late apoptosis. Similarly, Gupta et al.72 reported DOX induced MCF-7 breast cancer cell and A549 lung cancer cell death by early and late apoptosis. Furthermore, it is believed ROS generated by DOX activates caspases which serve as the primary mediators of apoptosis.73, 74 In NT-AuNP-DOX or PD-L1-AuNP-DOX treated cells, necrotic cell proportions were significantly higher than in free DOX treated cells, whereas early apoptotic proportions were decreased. Similarly, Kenerson et al.75 reported increasing doses of rapamycin increased the proportions of necrotic tumor cells and reduced those of apoptotic cells. In PD-L1-AuNP-DOX treated cells efficient cellular uptake resulted in increases in the proportions of apoptotic and necrotic cells. Furthermore, NT-AuNP and PD-L1-AuNP plus NIR irradiation increased the proportion of apoptotic CT-26 cells as compared with NT-AuNP or PD-L1-AuNP treated cells. NIR irradiation did not increase the effect of free DOX, but did increase apoptotic and necrotic proportions when administered with NT-AuNP-DOX or PD-L1-AuNP-DOX. As reported previously, high energy NIR-responsive AuNP (T > 45°C) can induce necrosis, while low energy radiation (T < 45°C) induce cells death by triggering apoptosis.76, 77 Li, and Gu78 demonstrated that laser-induced PTT employing transferrin-targeted gold nanorods induced the apoptosis of HeLa cells, and Agarwal et al.79 designed a thermosensitive liposome-gold nanorod carrier containing DOX as a combination therapy. NIR irradiation of U87 human glioblastoma treated with a thermosensitive NP via a synergistic chemo-PTT at 43°C showed a significant increase in apoptosis and necrosis.


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CT-26 cells treated with NT-AuNP and PD-L1-AuNP plus NIR shown enhanced ROS generation versus non-treated cell. PTT-induced cell death is predominantly due to oxidative stress caused by singlet oxygen generated by the radiolysis of water.80 When AuNP are exposed to NIR irradiation, they immediately generate ROS, which react with nucleic acid to initiate radiation-induced apoptosis. ROS causes protein oxidation, lipid peroxidation, and DNA damages, which result in irreversible cellular damage and even cell death.81, 82 Exposure of CT-26 cells to DOX also caused considerable ROS generation, which has been reported to be a major cause of DOX induced apoptosis and necrosis.83 As was expected, NT-AuNP-DOX and PD-L1-AuNP-DOX caused 1.3and 2.6- fold increases in ROS formation, respectively, versus DOX in CT-26 cells. Furthermore, CT-26 cells treated with PD-L1-AuNP-DOX plus NIR irradiation greatly enhance intracellular ROS generation, which suggested a synergistic effect. Similarly, Yang et al.84 demonstrated that when SN-38 drug was encapsulate into nanoporphyrin micelles (SN-NPM), it served as a highly potent chemotherapeutic agent together with a photosensitizer for PTT in HT-29 colon cancer treatment. SN-NPM formulation plus NIR irradiation significantly enhanced the in vitro ROS formation over single therapy in HT-29 colon cancer cells. In addition, Zheng et al.85 prepared DOX-loaded gold nanosponge-liposome bilayer for the aptamer-based targeted therapy of MCF7 breast cancer cells, and found targeted delivery of DOX and photosensitizer plus NIR enhanced ROS production in MCF-7 cells as compared with non-targeted, chemo- or photothermal treatments. PD-L1-AuNP-DOX induced cellular damage either via considerable ROS generation effect or extensive temperature increase and the subsequent release of the energy in the form of small shock waves, as reported previously.82, 86 Western blotting was performed to assess SOD2 and catalase levels in CT-26 cells. ROS markers were down-regulated in CT-26 cells treated with free DOX, NT-AuNP-DOX or especially PD-L1-


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AuNP-DOX. DOX is known to suppress the expressions of anti-oxidant proteins,87 and our findings suggest that the downregulations of ROS scavenger enzymes (SOD2 and catalase) induced apoptosis. In addition, PD-L1-AuNP-DOX plus NIR suppressed the expressions of SOD2 and catalase and increased ROS levels. Cell cycle analysis showed NT-AuNP-DOX and PD-L1-AuNP-DOX caused arrest in the S and G2/M phase and significantly decreased in the proportion of cells in the G0/G1 phase, indicating sustained DOX release inhibited cell cycle progression. In addition, cell cycle analysis showed DOX interaction with DNA caused G2/M phase cell cycle arrest and significantly reduced the proportion of cells in the G0/G1 phase.88,

89

NT-AuNP-DOX and PD-L1-AuNP-DOX both

significantly decreased the proportion of cells in the G0/G1 phase, demonstrating both were more effective at controlling CT-26 growth than DOX. In PD-L1-AuNP-DOX, higher amount of DOXloaded NP interacted with the DNA in the nucleus and resulted in a reduced cell proliferation 63, 90, 91.

Similar observations were reported for rapamycin-loaded NP, which increased the proportion

of human vascular smooth muscle cells (VSMCs) in the G0/G1 phase more than rapamycin.91 In the present study, exposure of CT-26 cells to NT-AuNP or PD-L1-AuNP plus NIR irradiation increased the proportions of cells in the S and G2/M phase. By generating heat PTT denatures intracellular proteins and DNA.58 Additionally, exposure of cells to NT-AuNP-DOX or especially PD-L1-AuNP-DOX plus NIR induced obvious shifts toward the S and G2/M phases. Our results suggest that the mechanism responsible for AuNP-responsive PTT (S and G2/M phase cell cycle arrest) inhibition differed from that of DOX (G2/M phase cell cycle arrest), which indicates PDL1-AuNP-DOX and NIR irradiation combination therapy might cooperatively suppress cancer progress, and thus, be used to minimize the side effects of DOX.


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NP-based drug delivery systems have the potential to enhance therapeutic efficacies by minimizing undesired side effects through the targeted localization of drugs in tumors and by enabling active intracellular uptake.92 Continuous ROS generation inside a cell induced by PDL1-AuNP-DOX in the presence of irradiation can induce cancer cell cycle arrest, necrotic, and apoptosis.93 Since, chemotherapeutics have been shown to be more cytotoxic at elevated temperatures,94-96 combinations of chemotherapeutics and hyperthermia can decrease IC50 values and reduce dose-dependent side effect.94 Labeling of AuNP with other clinically useful chemotherapeutics could also permit the treatment of CRCs with different clinical cancer phenotypes. Anti-PD-L1 antibody was used in the present study to take advantage of the overexpression of PD-L1 receptors on CRC cells. However, this antibody could be replaced with other antibodies according to molecular phenotype. For example, human epidermal growth factor receptor 2 (HER2) targeting trastuzumab could be used in HER2 positive CRC patients.97, 98 Thus, the combination of a suitable antibody, AuNP, and NIR irradiation could be used to develop personalized treatments that address the heterogeneous nature of CRC.

CONCLUSION This study demonstrates that the conjugation of anti-PD-L1 antibody and DOX to citratestabilized AuNP via LA-PEG-NHS provides stable AuNP with high PD-L1 affinity for PD-L1 overexpressing CT-26 cells. The efficient intracellular uptake of DOX was evidenced by pronounced apoptotic effects on CT-26 cells caused by ROS generation. Intracellular uptake via PD-L1 receptors was found to improve intracellular retention, and hence the therapeutic efficacy of DOX. PD-L1-AuNP-DOX plus NIR through co-administration of DOX and AuNP induced cell cycle arrest and the apoptosis and necrosis of CT-26 cells, which can synergistically inhibit cell


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growth. We believe this novel nanomedicine offers a potential means for treating PD-L1 overexpressing CRC.

SUPPORTING INFORMATION (SI) Additional experimental details and data of synthesis and characterization of PD-L1-AuNPDOX; stability of PD-L1-AuNP-DOX; photothermal effect of PD-L1-AuNP DOX (PDF) AUTHOR INFORMATION Corresponding Authors * Tel: +82–53–580–6656. Fax:

E–mail: [email protected].

** Tel: +82-53-810-2822. Fax: E-mail: [email protected]. ORCID Fakhrossadat Emami: 0000-0003-2954-3829 Author Contributions All authors contributed to the writing of the manuscript, and all authors have read and approved to the final version of the manuscript.

Conflicts of interest The authors declare no competing financial interest. ACKNOWLEDGMENT


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This research grant was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (Grant Nos: NRF2018R1D1A1B07040858 and NRF-2016R1A6A1A03011325).

ABBREVIATIONS CRC, colorectal cancer; DOX, doxorubicin; LA-PEG-NHS, lipoic acid polyethylene glycol Nhydroxysuccinimide; PTT, photothermal therapy; AuNP, gold nanoparticles; NIR, near infrared; ROS, reactive oxygen specious; MSI-H, microsatellite instability-high; NP, nanoparticles; MALDI-TOF-MS, matrix assisted laser desorption ionization-time of flight-mass spectrometry; SEC, size exclusion chromatography; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TNBSA, 2, 4, 6- trinitrobenzene sulfonic acid; DAPI, 4, 6 –diamidino–2– phenylindole; IFN-ɤ, interferon gamma; PI, propidium iodide; DCFH-DA, 2′,7′-dichlorofluorescin diacetate; calcein AM, calcein acetoxymethyl; EthD–1, ethidium homodimer–1; CS, clonogenic survival; PE, plating efficiency.


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Doxorubicin and anti-PD-L1 antibody conjugated gold nanoparticles

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