Photothermal, Targeting, Theranostic Near-Infrared Nanoagent with

Article pubs.acs.org/molecularpharmaceutics

Photothermal, Targeting, Theranostic Near-Infrared Nanoagent with SN38 against Colorectal Cancer for Chemothermal Therapy Ming-Hsien Tsai,† Cheng-Liang Peng,‡ Shu-Jyuan Yang,*,§ and Ming-Jium Shieh*,†,⊥ †

Institute of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, Taipei City 10051, Taiwan ‡ Isotope Application Division, Institute of Nuclear Energy Research, Taoyuan City 32546, Taiwan § Gene’e Tech Co. Ltd. 2F., No.661, Bannan Rd., Zhonghe Dist., New Taipei City 235, Taiwan ⊥ Department of Oncology, National Taiwan University Hospital and College of Medicine, #7, hung-Shan South Road, Taipei 100, Taiwan S Supporting Information *

ABSTRACT: Cancer research regarding near-infrared (NIR) agents for chemothermal therapy (CTT) has shown that agents with specific functions are able to inhibit tumor growth. The aim of current study was to optimize CTT efficacy for treatment of colorectal cancer (CRC) by exploring strategies which can localize high temperature within tumors and maximize chemotherapeutic drug uptake. We designed a new and simple multifunctional NIR nanoagent composed of the NIR cyanine dye, polyethylene glycol, and a cyclic arginine-glycine-aspartic acid peptide and loaded with the anti-CRC chemotherapeutic agent, 7-ethyl-10-hydroxy-camptothecin (SN38). Each component of this nanoagent exhibited its specific functions that help boost CTT efficacy. The results showed that this nanoagent greatly strengthens the theranostic effect of SN38 and CTT against CRC due to its NIR imaging ability, photothermal, enhanced permeability and retention (EPR) effect, reticuloendothelial system avoidance, and angiogenic blood vessel-targeting properties. This NIR nanoagent will help facilitate development of new strategies for treating CRC. KEYWORDS: chemothermal therapy, SN38, IR780, cRGD, colorectal cancer

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INTRODUCTION

drug-loaded nanoagents to easily pass through loose vessels within tumors, thereby increasing retention time and accumulation of the drug at the target site.10−12 Some antiCRC nanomedicines have good anticancer efficacy,13 and have been authorized for use as new investigational drugs.14 For example, 7-ethyl-10-hydroxy-camptothecin (SN38), an active metabolite of clinical CPT-11, 15 loaded nanomedicine (NK012) has already passed Phase I and is entering Phase II clinical trials for CRC patients.16 In addition, numerous NIR nanoagents designed to exploit the EPR effect have been shown

Combination therapy has acquired significant prominence in colorectal cancer research and clinical practice in the past decade because it can prolong patient survival more efficiently and significantly.1−3 Specifically, chemothermal therapy (CTT) has shown promise with induction of cancer cell death and tumor inhibition.4,5 Photothermal therapy (PTT) involving near-infrared (NIR) light with high penetration depth6 is a controllable, noninvasive therapy that only produces heat at the target site, reducing damage to normal tissue,7 thereby making it an ideal choice for CTT. With the advent of nanotechnology, many studies have shown that loading chemo drugs into nanoagents boosts drug efficacy by enhancing permeation and retention (EPR) effects (passive targeting).8,9 This affect is attributed to the ability of © XXXX American Chemical Society

Received: April 17, 2017 Revised: June 26, 2017 Accepted: June 30, 2017

A

DOI: 10.1021/acs.molpharmaceut.7b00315 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Article

Molecular Pharmaceutics

Scheme 1. Efficient Accumulation of the Designed Drug-Loaded NIR Nanoagent in the Tumor Site after Intravenous Injection and Administration of PTT To Enhance Efficacy of CTT

to result in good PTT efficacy against cancer.17,18 In particular, cyanine dye-based NIR nanoagents can also be detected by NIR imaging, enabling confirmation of its in vivo localization, accumulation, and cellular uptake.19,20 Initiating PTT upon maximal nanoagent accumulation within the tumor highly enhances its efficacy. Thus, cyanine dye-based NIR nanoagents are a potential candidate for enhancing CTT. Nanoagent modifications, such as adding polyethylene glycol (PEG) and cyclic Arg-Gly-Asp (cRGD) peptides, impart nanoagents with specific functions and can be an effective approach for improving antitumor efficacy.21 PEG has been demonstrated to prevent rapid clearance by the reticuloendothelial system (RES), thereby prolonging their circulation time and increasing the probability of tumor uptake and drug delivery.22,23 The cRGD peptide is an active targeting ligand demonstrated to have high affinity for integrins αvβ3 on angiogenic blood vessels in various tumors24,25 Thus, the cRGD peptide enables nanoagents to specifically bind tumor vessels expressing αvβ3 (active targeting), further facilitating targeted uptake.26−28 Herein, we desgined a new and simple multifunctional NIR nanoagent (cRGDIRNANOSN38) composed of NIR cyanine dye, IR780, PEG, and cRGD peptide and loaded with anticolorectal cancer chemotherapeutic (chemo) drug, SN38. Each component of this nanoagent exhibited its specific functions that optimize the efficacy of CTT treatment of CRC. With its nanoscale size and added NIR imaging, EPR (passive targeting), RES avoidance, and active angiogenic blood vesseltargeting properties, we posit cRGDIRNANOSN38 will ensure delivery of a substantial amount of SN38 and significantly

strengthen the PTT effect on CRC tumors. Moreover, this agent will allow us to confirm whether initiation of PTT in CTT upon maximum drug accumulation within the tumor is reached (Scheme 1). To validate each function of our nanoagent, cRGDIRNANO without SN38, IRNANOSN38 without the cRGD peptide, and IRNANO without SN38 and the cRGD peptide were also evaluated in vitro with different CRC cell lines and in vivo with tumor-bearing mice. First, the basic nanoagent characteristics were assessed, including absorption and fluorescence (FL) properties, critical micelle concentration (CMC), size, zeta potential (ZP), loading efficiency (LE), and drug content (DC). Then, in vitro PTT effects were evaluated, along with thermal stability and drug release profiles to verify SN38 release efficiency. SN38, PTT, and CTT cytotoxicity in human CRC, HCT116, Caco2, and DLD-1 cells was evaluated to determine whether this nanoagent effectively kills CRC cells. Finally, in vivo pharmacokinetics (PK) was used to evaluate RES avoidance, and NIR imaging measured targeted SN38 and IR780 uptake. Thermal imaging camera was used to evaluate in vivo PTT effect, and the localization of temperature in tumor. The antitumor effects of chemo, PTT, and CTT were also evaluated in vivo to estimate the effectiveness of this nanoagents with and without SN38 for tumor growth inhibition.

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EXPERIMENTAL SECTION Materials. Methoxy PEG thiol (mPEG-SH; MW, 5000) and carboxymethyl-PEG-thiol (CM-PEG-SH; MW, 5000) were obtained from Laysan Biotechnology, Inc. (Arab, AL, USA). 2-[2-[2-Chloro-3-[(1,3-dihydro-3,3-dimethyl-1-(2-hydroxyeth-

B


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Figure 1. (A) Synthesis of IR780-mPEG and IR780-PEG-cRGD and their self-assembly into the SN38-loaded multifunctional NIR nanoagent, IR IR IR IR IR cRGD NANOSN38. (B) Size distribution analysis of NANO, cRGD NANO, NANOSN38, and cRGD NANOSN38. (C) Absorption spectra of IR780, IR IR IR IR NANO and NANOSN38 in PBS. (D) FL spectra of NANO and NANOSN38 in PBS. (E) In vitro photothermal effect. The increase in PBS temperature with IRNANOSN38 at a specific concentration (0, 0.125, 0.25, 0.2, 1, and 10 mg/mL) after 60 s of NIR irradiation (808 nm, 1 W/cm2). (F) DLS size and PdI of IRNANOSN38 at 25 °C, 37 °C, 45 °C, 50 °C, and 60 °C. (G) SN38 release profile. SN38 release from IRNANOSN38 with and without a 60-J/cm2 PTT after 24 h.

yl)-2H-indol-2-ylidene)ethylidene]-1-cycloxen-1-yl]-ethenyl]3,3-dimethy-1-(2-hydroxyethyl)-1H-indoliumperchlorate (ADS780HO, IR780) was purchased from American Dye

Source, Inc. (Quebec, Canada). Dichloromethane, methanol (MeOH), dimethyl sulfoxide (DMSO), and pyridine were obtained from Sigma-Aldrich (Milwaukee, WI, USA). TetrahyC


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amount of SN38-loaded nanoagents. CMC was evaluated using pyrene.32 Absorption and FL Properties of IRNANO and IRNANOSN38. The absorption and FL properties of IRNANO and IR NANOSN38 were evaluated based on absorption and FL spectra obtained using ultraviolet−visible and FL spectroscopy (SpectraMax M2Multi-Mode Microplate Reader, BioTek Instruments, Italy). For this analysis, IRNANO, IRNANOSN38, and IR780 were dissolved in PBS and DMSO (about 0.05 mg/ mL of IR780) and their FL and absorption spectra were measured. In addition, SN38 was also dissolved in DMSO and PBS for comparison. In Vitro Photothermal Effect. In vitro photothermal effects achieved by IRNANO and IRNANOSN38 were evaluated using a temperature sensor with a micro thermocouple and a 1-W/cm2 NIR laser (808 nm) (TangYu, Ltd. Kaohsiung, Taiwan). Each well in a 96-well plate was filled with 100 μL PBS containing IR NANO SN38 or IR NANO at a specific concentration IR ( NANOSN38: 10, 1, 0.5, 0.25, 0.125, and 0 mg NIR nanoagent/mL; IRNANO: 1, 0.5, 0.25, and 0 mg NIR nanoagent/mL). The temperature of the media was recorded per second under NIR irradiation. Temperature increases were calculated by subtracting the temperature before PTT from that recorded during PTT. Thermal Stability. Thermal stability of IRNANOSN38 at the concentration of 0.25 mg/mL was evaluated by analyzing its size and PdI via DLS at 25, 37, 45, 50, and 60 °C, respectively, using Zetasizer NANO-ZS90. Besides, 1 mg/mL IRNANOSN38PBS dispersions treated with a 60 J/cm2 dose of NIR light was monitored using infrared thermal imaging camera and then observed under TEM. Drug Release Profile. Drug release profiles of IRNANOSN38 treated with and without PTT were evaluated using a dialysis tube technique.33 A dialysis tube with a membrane (MWCO, 8000 Da) containing about 1 mg/mL IRNANOSN38 (0.008 mg SN38) was suspended in 50 mL of PBS containing 0.1% (w/w) Tween-80 in a closed glass bottle containing a magnetic mixer and incubated at 37 °C. For PTT, the dialysis tube was taken out and treated with a 60 J/cm2 NIR light at 24 h. After light treatment, the dialysis tube was suspended in PBS and incubated at 37 °C again, immediately. Aliquots (0.5 mL) were taken at select time intervals. Finally, the SN38 concentration of each aliquot was determined using a high performance liquid chromatography (HPLC) system (Waters e2696) with an FL detector (Waters 2475, Ex/Em: 427/557 nm) and a C18 column (XBridge). Cell Culture. All cells used in this study were cultured in an humidified incubator at 5% CO2 and 37 °C. HCT116 human CRC cells were cultured in McCoy’s 5A medium (Gibco BRL, Gaithersburg, MD, USA) supplemented with 10% heatinactivated fetal bovine serum (FBS), 1.5 mM L-Gln, 1% (v/ v) penicillin−streptomycin-amphotericin B solution (Gibco BRL), and 2.2 g/L NaHCO3. Caco2 human CRC cells were cultured in Minimal Essential Medium (Gibco BRL) supplemented with 20% FBS, 2 mM L-Gln, nonessential amino acids, 1 mM sodium pyruvate, 1% (v/v) penicillin− streptomycin-amphotericin B solution (Gibco BRL), and 1.5 g/ L NaHCO3. DLD-1 human CRC cells were cultured in RPMI 1640 medium (Gibco BRL) containing 10% heat-inactivated FBS, 10 mM HEPES, 2 mM L-Gln, 4.5 g/L L-glucose, 1.5 g/L NaHCO3, and 1% (v/v) penicillin−streptomycin-amphotericin B solution (Gibco BRL).

drofuran for gel permeation chromatography (GPC) was purchased from Avantor Performance Materials (Center Valley, PA, USA). N-Hydroxysulfosuccinimide sodium salt (sulfoNHS), 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC.HCl), triethylamine (TEA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT), pyrene,phosphate-buffered saline (PBS), and other chemicals used in this study were purchased from Sigma-Aldrich. SN38 was purchased from ScinoPharm Taiwan, Ltd. (Tainan, Taiwan). Irinotecan (CPT-11) was purchased from Pfizer Inc. (NY, USA). The cRGD-3-aminomethylbenzoyl peptide was purchased from OmicsBio, Ltd. (Taipei, Taiwan). Ethidium homodimer-1 (EthD-1), Hoechst 33342 were purchased from Thermo Fisher Scientific (Carlsbad, CA, USA). DeadEnd fluorometric terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) system was purchased from Promega Corp. (Madison, WI, USA). Synthesis of mPEG-IR780 and cRGD-PEG-IR780. Methoxypolyethylene glycol−IR780 (mPEG-IR780) was synthesized via thiosubstitution reaction.29,30 mPEG-SH (0.02 mmol) was reacted with excess IR780 (0.2 mmol) in dichloromethane (5 mL) with TEA (50 μL) for 48 h to obtain mPEG-IR780 (Figure 1A). Next, a dialysis with a membrane (MWCO, 3500 Da) was used to purify mPEG-IR780 with MeOH. In addition, cRGD-modified PEG−IR780 (cRGDPEG-IR780) was synthesized via two chemical reactions: thiosubstitution and EDC-HCl/sulfo-NHS reaction (Figure 1A). CM-PEG-SH (0.02 mmol) was reacted with excess IR780 (0.2 mmol) for 48 h to obtain CM-PEG-IR780. CM-PEGIR780 was purified by dialysis with MeOH. Subsequently, the purified CM-PEG-IR780 (0.01 mmol) was reacted with excess cRGD peptide (0.05 mmol) for 6 h in the presence of sulfoNHS (0.1 mmol) and EDC-HCl (0.1 mmol) to obtain cRGDPEG-IR780. Finally, the purified cRGD-PEG-IR780 was obtained by dialysis with a membrane (MWCO, 3500 Da) and deionized water. mPEG-IR780 and cRGD-PEG-IR780 were characterized by 1HNMR and GPC.31 Preparation and Characteristics of I R NANO, IR IR NANOSN38, and cRGDIRNANOSN38. The uncRGD NANO, IR loaded ( NANO and cRGD IRNANO) and SN38-loaded (cRGDIRNANOSN38 and IRNANOSN38) NIR nanoagents were prepared using a lyophilization−hydration method.31 IRNANO was prepared using mPEG-IR780, while cRGDIRNANO was prepared using a mixture of 80% (w/w) mPEG-IR780 and 20% (w/w) cRGD-PEG-IR780. For IRNANO and cRGDIRNANO, 10 mg of mPEG-IR780/cRGD-PEG-IR780 was dispersed in 1 mL of PBS after lyophilization−hydration. For cRGDIRNANOSN38 and IRNANOSN38, 10 mg of mPEG-IR780/cRGD-PEG-IR780 and 1 mg of SN38 were dispersed in 1 mL of PBS after lyophilization−hydration. Subsequently, the nanoagents were ultrasonicated and then sterilized by filtration using a 0.22-μm filter. Finally, all of the nanoagents were characterized by dynamic light scattering (DLS) using Zetasizer NANO-ZS90 (Malvern instruments Ltd., UK) and transmission electron microscopy (TEM) in terms of size and electrophoretic light scattering for ZP. The nanoagents were stained with 1% phosphotungstate acid before being observed by TEM. In addition, LE and DC were determined according to an SN38 calibration curve, which was obtained using the maximum absorption of SN38 in DMSO. LE (%) was calculated by dividing the amount of SN38 loaded in the nanoagents by the total amount of SN38 used. DC (%) was determined by dividing the amount of SN38 loaded in the nanoagents by the D


Article

Molecular Pharmaceutics In Vitro Cytotoxicity of Chemo, PTT, and CTT. In vitro cytotoxicity was evaluated using MTT assay and EthD-1 assay.34,35 For chemo, cells were seeded onto 96-well plates (DLD-1:1 × 105 cells/well; Caco2:1 × 105 cells/well; HCT116:7.5 × 104 cells/well) and incubated for 24 h before being treated with different concentrations (0.01, 0.1, 1, 10, and 100 μg SN38/mL) of free SN38, and IRNANOSN38 and IR cRGD NANOSN38 for 1.5 and 24 h. Cells were washed with PBS and cultured in fresh medium for another 24 h before MTT assay involving the use of a scanning multiwell ELISA reader (Microplate Autoreader EL311, BioTek Instruments Inc., Winooski, VT, USA). In addition, the cytotoxicity of IRNANO and cRGDIRNANO at 0.1, 1, 10, 100, and 1000 μg NIR nanoagent/mL were also evaluated. MTT assay results were used to determine the IC25 and IC50 of each. For PTT, cells were seeded onto 96-well plates (HCT116:7.5 × 104 cells/well) and incubated for 24 h before being treated for 1.5 h with different concentrations of IR NANO (0, 0.25, and 1 mg NIR nanoagent/mL). During the 1.5 h treatment, cells were treated with different NIR light doses (0, 20, 40, and 60 J/cm2) involving a 1-W/cm2 NIR laser at 808 nm. After the 1.5 h treatment, cells were washed and cultured in fresh medium for another 24 h before MTT assay. In addition, the other group of cells treated with IRNANO combined with NIR light were immediately stained with EthD1 (Ex/Em: 495/635 nm) and visualized with a FL microscope (X51 Olympus Optical Co., Tokyo, Japan). For CTT, cells were seeded onto 96-well plates (HCT116:7.5 × 104 cells/well) and incubated for 24 h before being treated for 1.5 h with IRNANOSN38 at the IC25 and IC50. During the 1.5 h treatment, cells were further treated with different NIR light doses (0, 20, 40, and 60 J/cm2) involving a 1-W/cm2 NIR laser at 808 nm. Then, cells were washed and cultured in fresh medium for another 24 h before MTT assay. In Vivo Animal Model. Approval for all in vivo animal protocols was obtained from the National Taiwan University College of Medicine and the College of Public Health Institutional Animal Care and Use Committee. BALB/ cAnN.Cg-Foxnlnu/CrlNarl female mice, aged about 4 weeks, were obtained from the National Laboratory Animal Center of Taiwan. HCT116 cells (3 × 106 cells) dispersed in PBS were injected subcutaneously into mice to model in vivo tumor development. When tumor sizes were 100−150 mm3 (tumor size: 1/2 × length × width2), treatments were started. PK Analysis. PK analysis was conducted by determining the concentration of SN38 in plasma using HPLC. Normal mice were divided into three groups (n = 3 each) and intravenously injected with 10 mg/kg of SN38/CPT-11 with CPT-11, IR NANOSN38, or cRGDIRNANOSN38, respectively. Retro-orbital blood samples were taken at different times (0.5, 1, 3, 6, 12, and 24 h), collected in blood collection tubes containing heparin, and centrifuged at 3500 rpm for 8 min. SN38 in plasma was extracted with MeOH. The amount of SN38 in plasma was determined using HPLC. In Vivo NIR Imaging and Tumor Accumulation. In vivo accumulation and biodistribution were evaluated using a NIR FL (NIRF) imaging and HPLC. When tumor sizes reached 100−150 mm3, the two groups of tumor-bearing mice were given IRNANOSN38 or cRGDIRNANOSN38 loaded with 10 mg/kg SN38. Then, mice were observed using a IVIS imaging system (Xenogen IVIS Spectrum In Vivo Imaging System, Caliper Life Sciences Inc., Hopkinton, USA; Ex/Em: 745/800 nm) at select

time points (6, 12, 24, 48, and 72 h). In addition, two groups of tumor-bearing mice (n = 4 each) were given IRNANOSN38or IR cRGD NANOSN38 loaded with 10 mg/kg SN38. Mice were sacrificed when maximum accumulation of I R NANOSN38/cRGDIRNANOSN38 in tumors was observed using an IVIS image system after intravenous injection. Tumors were then removed, weighed, and milled. SN38 accumulated in each tumor was extracted using MeOH, and the amount accumulated was determined by HPLC. In Vivo Photothermal Effect. In vivo photothermal effects were evaluated using an infrared thermal imaging camera. When tumor sizes reached 100−150 mm3, four groups of tumor-bearing mice (n = 4 each) were treated with saline, IR IR IR cRGD NANO, NANOSN38, or cRGD NANOSN38 (100 mg NIR nanoagent/kg each). When maximal tumor accumulation of the nanoagents occurred (observed by IVIS image system), tumors were given a 60 J/cm2 NIR light dose using a 1-W/cm2 NIR laser at 808 nm. The temperature in the tumor site was measured using an infrared thermal imaging camera (Thermo Shot F30, Nippon Avionics Co., Ltd., Japan). Thermal images were captured every 6 s, and the temperature in each tumor was determined. In Vivo Antitumor Efficacy of Chemo, PTT, and CTT. In vivo antitumor efficacy was evaluated by observing changes in tumor size and TUNEL assay.36 Treatments started when tumor sizes were 100−150 mm3 (designated as day 0). For chemo, four groups of tumor-bearing mice (n = 6 each) were intravenously injected on days 0, 3, and 6 with saline (200 μL), CPT-11 (3 doses of 10 mg/kg each; 30 mg/kg total), IR NANOSN38 (3 doses of 10 mg SN38/kg each; 30 mg SN38/kg total), or cRGDIRNANOSN38 (3 doses of 10 mg SN38/ kg each; 30 mg SN38/kg total). For PTT, six tumor-bearing mice were intravenously injected with a single dose of IR cRGD NANO (100 mg NIR nanoagent/kg) on day 0, and then tumors were treated with a 60-J/cm2 NIR light dose using 1-W/cm2 NIR light (808 nm) on day 1. For CTT, six tumorbearing mice were intravenously injected with a single dose of IR cRGD NANOSN38 (100 mg NIR nanoagent/kg; 10 mg SN38/ kg) on day 0, and then tumors were treated with a 60-J/cm2 NIR light dose using a 1-W/cm2 NIR light at 808 nm on day 1. Body weight and tumor size were measured and recorded every 3 d from the day 0. In addition, apoptotic cells in tumors excised on day 2 were stained with Hoechst 33342, subjected to TUNEL assay, and observed with FL microscopy. Furthermore, in vivo toxicity was evaluated using hematoxylin and eosin (H&E) staining. At the end of chemo experiments, normal tissues from organs, such as heart, liver, spleen, liver, and kidney, were excised and stained with H&E. Analysis of the Interaction of PTT and Chemo in Vitro. In vitro synergistic effects in CTT were evaluated using the coefficient of drug interaction (CDI) of chemo and PTT, which is frequently used to verify the synergistic effect of two drugs.37 CDI was calculated as follows: CDI = AB/(A × B). AB = relative cell viability of CTT; A = relative cell viability of chemo; B = relative cell viability of PTT. CDI < 1 indicates synergism, CDI = 1 indicates additivity, and CDI > 1 indicates antagonism. Statistical Analysis. All data in the current study were expressed as the mean ± standard deviation. To determine statistically significant differences between groups, a Student’s t test was utilized. * indicates P < 0.05 and ** indicates a P < 0.01. E


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RESULTS AND DISCUSSION Characteristics of the Designed Nanoagents. mPEGIR780 and cRGD-PEG-IR780, which self-assembled into the new multifunctional NIR nanoagent, were successfully synthesized via thio-substitution reaction (Figure 1A). The self-assembly mechanism might be the same as that resulting in micelles.29,30 Nanoparticles formed as IR780s, the hydrophobic part, aggregated in the center of nanoparticles and PEGs, the hydrophilic part, suspended outside of nanoparticles. The hydrophobic SN38 would be entrapped in the hydrophobic core of our designed nanoparticles. 1HNMR and GPC were used to verify the structures of mPEG-IR780 and cRGD-PEGIR780. The 1HNMR spectrum of mPEG-IR780 showed IR780 peaks [δ 1.35(d), δ 2.72(c), δ 4.12(j), δ 4.25(k), δ 4.61(i), δ 7.00−7.72 (benzyl group, e−h)] and mPEG [δ 3.37, δ 3,62(b)] peaks (Figure S1), indicating IR780 was successfully added to mPEG via thiol substitution. 1HNMR spectrum of cRGD-PEGIR780 showed IR780 [δ 1.35(d), δ 2.72(c), δ 4.12(j), δ 4.25(k), δ 4.61(i), benzyl group: δ 7.00−7.72(e−h), PEG [δ 3.37, δ 3,62(b)], and cRGD [δ 4.31(αH backbone, k), δ 8.81− 9.2(NH, l)] peaks (Figure S2), confirming the cRGD peptide and IR780 were successfully added to PEG. In addition, GPC data shown in Table 1 and Figure S3 indicate that mPEG-

distribution of all nanoagents was narrow (PdI < 0.3). These results confirmed that IRNANO, cRGDIRNANO, IRNANOSN38, and cRGDIRNANOSN38 could be self-assembly formed from mPEG-IR780 and cRGD-PEG-IR780 similar to micelle formation,24 and their sizes were nanoscale and uniform. The LEs of IRNANOSN38 and cRGDIRNANOSN38 indicated that SN38 could be efficiently loaded into IRNANO and cRGDIRNANO. As shown in Table 2, the LEs of IRNANOSN38 (LE, 85.1%; DC, 7.8%) and cRGDIRNANOSN38 (LE, 80.6%; DC, 7.4%) were high, denoting a good load ability of SN38 in the nanoagents. Furthermore, the ZP and CMC of the designed nanoagents were measured. As shown in Table 2, ZPs of IRNANO, IR IR IR cRGD NANO, NANOSN38, and cRGD NANOSN38 were −7.15, −4.73, −4.03, and −1.46 mV, respectively. The variances in zeta potential between IRNANOSN38 and IRNANO might be caused by the fact that the SN38 facilitates nanoparticles under the presence of low amount of mPEG-IR780 exhibiting negative charge (alkoxy group) to become more stable. The variances between IRNANOSN38 and cRGDIRNANOSN38 might be attributed to cRGD peptides with positive charge. The CMCs of IRNANO and cRGDIRNANO were 0.071 and 0.092 mg/mL, respectively (Figure S5). In view of the data mentioned above which indicate the nanoagents created could be efficiently loaded with SN38, our nanoscale design is ideal and thus suggests effective EPR effect. Ultraviolet−visible absorption spectra of IRNANO and IR NANOSN38 in PBS (Figure 1C) showed a broad absorption peak between about 600 and 900 nm for both IRNANO and IR NANOSN38, which was attributed to IR780. However, the absorption spectra of free IR780 showed very little absorption near 780 nm due to its low solubility in PBS. When IRNANO and IRNANOSN38 were suspended in DMSO and detected by the ultraviolet−visible spectroscopy, there was no significant change in absorption spectra among IRNANO, IRNANOSN38, and IR780 in DMSO (Figure S6), indicating that dissembled IR NANO and IRNANOSN38 have the same ability to absorb NIR light as free IR780. It is noted that there was no significant SN38 absorption peak in DMSO, indicating that SN38 does not induce PTT effect. These findings might be conjectured that these NIR nanoagents with IR780 can absorb NIR light and then generate heat under NIR irradiation, bringing about good photothermal effect in PTT and CTT. Moreover, fluorescence emission spectra of IRNANO and IR NANOSN38 showed these agents will emit FL light between 730 and 860 nm after excitation (Figure 1D), indicating the light emitted from IRNANO and IRNANOSN38 could be detected by NIRF imaging. This confirms IRNANO and IR NANOSN38 have NIR imaging function due to inclusion of IR780 in their design. Hence, these results suggest the best time to initiate PTT and improve CTT efficacy is when the

Table 1. Molecular Characteristics of mPEG-5000, mPEGIR780, and cRGD-PEG-IR780 polymer

Mna

MWb

Mpc

PDd

mPEG-5000 mPEG-IR780 cRGD-PEG-IR780

7508 8836 9132

7967 9348 9862

8421 9881 10230

1.06 1.06 1.08

a c

Number-average molecular weight. bMass-average molecular weight. Peak molecular weight. dPolydispersity.

IR780 had a molecular weight (MW) of 9881 g/mol (Mp) and a PD of 1.06, while cRGD-PEG-IR780 had a MW of 10 230 g/ mol (Mp) and a PD of 1.08. The MW of cRGD-PEG-IR780 was larger than that of mPEG-IR780, further confirming successful conjugation of cRGD onto mPEG-IR780. The characteristics of NIR nanoagents with and without SN38 loaded were evaluated by dynamic light scattering (DLS) and transmissin electron microscopy (TEM). As shown in Table 2, the DLS sizes of IRNANO and cRGDIRNANO were about 180 nm, while those of the SN38 loaded nanoagents, IR NANOSN38 and cRGDIRNANOSN38, were both about 150 nm. The decrease in size could be attributed to the condensed hydrophobic core of SN38-loaded nanoagents.33 TEM images (Figure S4) showed nanoagent sizes consistent with those determined by DLS. Polydispersity index (PdI; Table 2) and size distribution data (Figure 1B) indicate that the size Table 2. Characteristics of IRNANO, IRNANOSN38, nanoagent

D/Pa (mg/mg)

NANO IR NANO NANOSN38 IR cRGD NANOSN38

0/10 0/10 1/10 1/10

IR

cRGD IR

size (nm)b 180.1 187.2 148.1 149.7

± ± ± ±

3.2 4.1 2.1 2.3

cRGD

IR

NANO, and

PdIb 0.22 0.23 0.21 0.19

± ± ± ±

cRGD

IR

LE (%)c

0.04 0.06 0.02 0.08

85.1 ± 6.2 80.6 ± 5.8

NANOSN38 DC (%)d

7.83 ± 0.6 7.46 ± 0.6

ZP (mV)e −7.15 −4.73 −4.03 −1.46

± ± ± ±

1.07 0.22 0.04 0.02

CMC (mg/mL)f 0.071 0.092

a Drug/polymer feeding ratio. bSize (nm) and polydispersity index (PdI) were determined by dynamic light scattering. cLoading efficiency (%) = amount of SN38 loaded in nanoagents/total amount of SN38 used. dDrug content (%) = amount of SN38 loaded in nanoagents/amount of SN38loaded nanoagents. eZeta potential (mV), determined by electrophoretic light scattering. fCritical micelle concentration (mg/mL), determined using pyrene.

F


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Figure 2. In vitro cytotoxicity of chemo, PTT, and CTT. (A) Viability of HCT116, Caco2, and DLD-1 cells after incubation with free SN38, IR NANOSN38, or cRGDIRNANOSN38, respectively, for 1.5 and 24 h. (B) Viability of HCT116 cells treated for 1.5 h with IRNANO at a different concentrations (0, 0.25, and 1 mg/mL) in combination with different NIR light doses (0, 20, 40, and 60 J/cm2). (C) Viability of HCT116 cells treated with IRNANOSN38 for 1.5 h at IC25 and IC50 in combination with different NIR light doses (0, 20, 40, and 60 J/cm2), respectively. *P < 0.05; **P < 0.01.

temperature by 5.4 °C after 60 s. Likewise, IRNANO without SN38 (Figure S7) could also efficiently heat up PBS under NIR irradiation. However, NIR irradiation (0 mg NIR nanoagents/ mL) only increased the temperature of PBS by 2 °C after 60 s. Previous reports have shown that effective PTT of cancer requires an in vivo tumor temperature above 42 °C to kill cancer cells.38 Current in vitro results indicate that tumor cell death can be effectively induced by increasing tumor temperature with these nanoagents via NIR irradiation after significant accumulation.

nanoagent has maximal accumulation in the tumor site, which can be determined by measuring the FL intensity of emitted light using NIRF imaging. After measuring the increase in temperature of PBS under 808 nm NIR irradiation, IRNANO and IRNANOSN38 were able to rapidly and efficiently generate heat. As shown in Figure 1E, a high concentration of IRNANO (10 mg/mL) in IRNANOSN38 rapidly increased the temperature by 5 °C in 5 s and 30 °C (total) in 60 s. In contrast, a low concentration of IRNANO (0.125 mg/mL) in IRNANOSN38 was only able to increase the G


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Molecular Pharmaceutics As expected, high temperatures render IR NANO SN38 relatively unstable. Thermal stability results showed the DLS size and PdI of IRNANOSN38 will significantly change at temperatures over 45 °C. As shown in Figure 1F, the DLS size and PdI of IRNANOSN38 indicated large particles at temperature over 45 °C. TEM image of IRNANOSN38 treated with a specific NIR light dose which allowed the PBS temperature to increase by 52 °C also showed larger particles (Figure S8B). These findings suggest that photothermal effect will cause IRNANOSN38 disintegration and result in aggregation. Our previous studies have demonstrated that the instability of loaded nanoagents facilitates drug release33,39 and drug release efficiency of IRNANOSN38 treated with and without PTT was evaluated next. SN38 release profiles showed the release efficiency of IR NANOSN38 treated with a 60 J/cm2 PTT was 10-times higher than that without PTT. As shown in Figure 1G, less than 3% of SN38 was released from IRNANOSN38 without PTT after 144 h, demonstrating the long-term stability of IRNANOSN38 at temperatures below 45 °C. On the other hand, about 30% of SN38 was released after 120 h with 60-J/cm2 PTT, increasing the death cancer cells. Notably, about 10% of SN38 was released from IRNANOSN38 in 24 h after 60-J/cm2 PTT, indicating a large amount of SN38 can be quickly released after PTT and begin eliminating cancer cells within 24 h. The above results support the notion that our drug-loaded nanoagent could be a candidate in CTT since its drug release efficiency could be enhanced after PTT. In Vitro Cytotoxicity of Chemo, PTT, and CTT. The biocompatibility of cRGDIRNANO and IRNANO without SN38 were determined to be moderate. As shown in Figure S9, IR NANO and cRGDIRNANO alone did not significantly induced HCT116, Caco2, or DLD-1 cytotoxicity after a 1.5 h incubation. However, a decrease in viability was observed after 24 h incubation with 1000 μg/mL concentrations of IR NANO and cRGDIRNANO. These results indicate the low degree of cytotoxicity of these nanoagents without SN38. Assessment of SN38 cytotoxicity showed the both IR IR NANOSN38 brought about great cRGD NANO SN38 and effectiveness in inducing human CRC cell death. As shown in Figure 2A, regardless of incubation time, the viability of each cell type decreased with increasing SN38 concentration in IR NANOSN38, cRGDIRNANOSN38, and free SN38, indicating SN38 induced cell death in a dose-dependent manner. Interestingly, compared to IRNANOSN38, significantly lower DLD-1 viability was observed for cRGDIRNANOSN38 at the SN38 concentration of 10 μg/mL regardless of incubation time. DLD-1 cytotoxicity after 24 h incubation revealed the 50% inhibitory concentrations (IC50) of cRGDIRNANOSN38, IRNANOSN38, and free SN38 to be 3.1 ± 0.6, 5.8 ± 1.15, and 11.1 ± 1.2 μg/mL, respectively (Table 3). This great ability to kill

DLD-l cells might be because cRGD on cRGDIRNANOSN38 is shown to efficiently bind to αvβ1 integrins that DLD-1 cells overexpress and enhance amount of SN38 uptake by DLD-l cells.40 In addition, the IC50 of IRNANOSN38, cRGDIRNANOSN38, and free SN38 for Caco2 cells were 7.2 ± 0.63, 6.2 ± 0.65, and 4.6 ± 0.76 μg/mL while for HCT116 cells were 0.057 ± 0.007, 0.066 ± 0.007, and 0.045 ± 0.006 μg/mL, indicating SN38 loaded in the designed nanoagents can successfully kill CRC cell. These results suggest that NIR nanoagents with or without cRGD peptide could effectively boost the efficacy of SN38 against CRC. In vitro cytotoxicity assays also showed that without PTT agents, CRC cells cannot be efficiently killed with NIR irradiation. As shown in Figure 2B, no significant change in cell viability was observed after HCT116 cells were treated with 0 mg/mL IRNANO and 20, 40, or 60 J/cm2 NIR light. On the other hand, IRNANO (NIR nanoagent >0 mg/mL) could efficiently kill CRC cells under NIR irradiation. As shown in Figure 2B, when NIR light doses increased, a slight decrease in cell viability with 0.25 mg/mL IRNANO and a significant decrease with 1 mg/mL IRNANO. Higher concentrations of IR NANO could efficiently kill HCT116 cells when used in combination with NIR light doses over 40 J/cm2. Moreover, EthD-1 assay results confirmed the ability of IR NANO to kill CRC cells with NIR irradiation. As shown in Figure S10, the FL intensity of cells treated with IRNANO significantly increased with the dose of NIR light. As expected, the strongest FL intensity was observed in cells treated with IR NANO in combination with 60-J/cm2 NIR light, in agreement with MTT assay results. Based on the cytotoxicity of PTT, IRNANO significantly increases the efficacy of PTT in CRC treatment. Therefore, delivery of a large amount of our NIR nanoagent to tumors should localize the high temperatures therein via PTT, efficiently killing tumor cells. In vitro CTT cytotoxicity assays showed IRNANOSN38 was adept at eliminating CRC cells and demonstrated a synergistic effect of PTT and chemo. As shown in Figure 2C, significantly lower viability was only found in cells treated with the 25% inhibitory concentration (IC25) of IRNANOSN38 and 60-J/cm2 NIR light. It is assumed that the increased temperature during PTT would trigger IRNANOSN38 to release more SN38 (enhanced SN38 release efficiency) and thus induce more cancer cell death, resulting in enhanced chemotherapeutic efficacy. The CTT cytotoxicity of IRNANOSN38 at IC50 showed cell viability significantly decreased with increasing NIR light dose. This was attributed to the excellent photothermal effect of IR NANOSN38 at IC50 under NIR irradiation. In addition, CDI analysis showed the in vitro synergistic effect of PTT and chemo (SN38) was achieved using IR NANOSN38. Table S1 shows the CDI calculated for IRNANOSN38 at IC25 combined with 60-J/cm2 NIR light was 0.92, clearly indicating synergism (CD1 < 1). Synergism was also observed when cells were treated with IRNANOSN38 at IC50 combined with NIR light doses of 20 J/cm2 (CDI, 0.9) and 40 J/cm2 (CDI, 0.5). However, the CDI for IRNANOSN38 at IC50 combined with 60-J/cm2 NIR light was 2.0 due to the tremendous photothermal effect resulting in relatively low cell viability. Based on CTT cytotoxicity results, the presence of both IR780 (for PTT) and SN38 (for chemo) in our nanoagents improves the in vitro efficacy of CTT. It was expected that the in vivo efficacy of CTT for CRC treatment

Table 3. IC50 Values of Free SN38, IRNANOSN38, and IR cRGD NANOSN38 (in Equivalent μg/mL SN38) on Colorectal Cancer Cell Lines cytotoxicity (IC50) in μg/mL for 24 h incubation drug-loaded NIR nanoagent IR

NANOSN38 IR cRGD NANOSN38 free SN38

DLD-1

CaCO2

HCT116

5.8 ± 1.1 3.1 ± 0.6 11.1 ± 1.2

7.2 ± 0.63 6.2 ± 0.65 4.6 ± 0.76

0.057 ± 0.007 0.066 ± 0.007 0.045 ± 0.006 H


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Molecular Pharmaceutics can be heightened with the use of the designed SN38-loaded multifunctional NIR nanoagent. PK Analysis. cRGDIRNANOSN38 and IRNANOSN38 designed with PEG (for RES-avoidance) lengthens the circulation time of SN38 and reduces the SN38 distribution in nontargeting tissues. PK results showed the mean retention time and the distribution of SN38 from cRGDIRNANOSN38 and IRNANOSN38 were significantly better than that of SN38 from CPT-11 (Irinotecan), a conventional anti-CRC drug. As shown in Figure 3, the SN38 concentration decayed faster when using

were 511.9, 134.4, and 376.2 mL, respectively, indicating IR NANOSN38 and cRGDIRNANOSN38 can prevent rapid dissemination of SN38 to other bodily tissues, lowering the risk of adverse effects. Thus, PK results suggest the RES-avoidance function (via PEG) of IRNANOSN38 and cRGDIRNANOSN38 efficiently increases accumulation of SN38 in tumors and decreases the risk of adverse effects. In Vivo NIR Imaging and Tumor Accumulation. IR IR NANOSN38 can both be successfully cRGD NANOSN38 and detected for at least 72 h using NIRF imaging, with clear in vivo distribution and accumulation after intravenous injection in mice via FL intensity (Figure 4A). Moreover, the FL intensity of both cRGDIRNANOSN38 and IRNANOSN38 in tumors sites gradually increased after intravenous injection, reaching maximums at 24 and 48 h, respectively. These results demonstrate that the time it takes to reach maximal tumor uptake of cRGD IR NANO SN38 and IR NANO SN38 can be determined using noninvasive NIRF imaging. Moreover, it is expected that application of PTT at that time optimizes the efficacy CTT. In addition, quantification of FL intensities at tumor sites (Figure 4B) revealed that the amount of cRGDIRNANOSN38 in tumors at 24 h was significantly higher than that of IRNANOSN38 at 48 h, which could be attributed to the integrin αvβ3targeting function of cRGD peptides.41 Based on the finding that IR780 can be more rapidly and efficiently accumulated in tumors via cRGDIRNANOSN38, it is expected that high and localized tumor temperatures can be achieved if PTT is initiated 24 h after injection of cRGDIRNANOSN38. Moreover, the amount of SN38 in tumors from cRGDIRNANOSN38 after 24 h was 3-times higher than that from IRNANOSN38 at 48 h (Figure 4C), demonstrating SN38 delivery to tumors is more efficient with cRGD IR NANO SN38 . However, it was found that IR cRGD NANOSN38 also accumulated in other organs, which could be attributed to the shielding effect of protein corona on the surface of cRGDIRNANOSN38 and thus compromised its targeting ability. When the designed nanoparticles enter into a biological environment, a new interface onto the surface of nanoparticles will be developed, so-called “bionano interface”. The bionano interface also is referred to the protein corona since protein is rich in the biological environment.42,43 The protein corona will alter the size and interfacial composition of the designed nanoparticles, giving it a new biological identity, and consequently altering the ability of active targeting.44 Based on in vivo NIR imaging and tumor accumulation results, we concluded that cRGDIRNANOSN38 with passive (nanoscale, EPR) and active (cRGD peptide) targeting functions in combination with RES avoidance (PEG) results in greater IR780 and SN38 uptake. Furthermore, the best time to apply PTT can be verified using NIRF imaging (IR780). Application of PTT at this time increases the chances of achieving greater CTT efficacy during CRC treatment. In Vivo Photothermal Effect. Considering high tumor uptake of NIR nanoagents is a key to efficiently generating heat within, PTT was applied using cRGDIRNANO and IR IR cRGD NANOSN38 24 h and NANOSN38 48 h after intravenous injection based on in vivo NIR imaging and tumor accumulation status. In vivo PTT results showed that cRGDIRNANOSN38 was most effective in heating the tumor under NIR irradiation. As shown in Figure 5A, the tumor temperature rapidly increased with use of cRGDIRNANOSN38 and NIR irradiation, reaching a maximum temperature of about 52 °C in 60 s. This

Figure 3. PK analysis of SN38 after mice were injected with free CPT11, IRNANOSN38, or cRGDIRNANOSN38 (SN38/CPT-11, 10 mg/kg).

CPT-11, indicating quicker RES elimination. However, relatively slow SN38 concentration decay was seen with the use of IRNANOSN38 and cRGDIRNANOSN38. Because the SN38 concentration at 12 h was as low as that at 24 h, the PK parameters were determined from the data at 0.5, 1, 3, 6, 12 h. The PK parameters listed in Table 4 showed the mean Table 4. Pharmacokinetic Parameters parametersa

units

T1/2 AUC0‑∞ CL Vd MRT

h μg·h/mL/kg mL/h mL h

IR

NANOSN38 2.3 5.0 40.4 123.4 3.0

IR cRGD NANOSN38

CPT-11

2.1 2.3 87.4 376.2 2.8

1.5 1.1 177.9 511.5 1.7

a

T1/2, terminal half-life; AUC, area under the concentration−time curve from zero up to t based on the sum of exponential terms; CL, total blood clearance of SN38 after intravenous administration; Vd, volume of distribution.

retention time for CPT-11, IRNANOSN38, cRGDIRNANOSN38 were 1.7, 3.0, and 2.8 h, respectively, while the total blood clearance for each was 177.9, 40.4, and 87.4 mL/h/kg, respectively. The above-mentioned results demonstrate IRNANOSN38 and cRGDIRNANOSN38 can prolong the circulation of SN38 in blood, avoiding rapid uptake by the RES. The cRGD peptide conjugation would decrease the mean retention time of prepared nanoagents by the RES. In addition, the volumes of distribution for CPT-11, IRNANOSN38, and cRGDIRNANOSN38 I


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Figure 4. In vivo NIR imaging and tumor accumulation. (A) Representative NIRF images of tumor-bearing mice after injection with IRNANOSN38 or IR cRGD NANOSN38 at 0, 6, 12, 24, 48, and 72 h. (B) Quantitative analysis of FL intensity in tumors using FL images in panel A. (C) Amount of SN38 from cRGDIRNANOSN38 accumulated in tumors at 24 h and from IRNANOSN38 at 48 h. *P < 0.05; **P < 0.01.

photothermal effect induced by cRGDIRNANOSN38 was superior, suggesting that cRGDIRNANOSN38 boosts the efficacy of PTT and CTT in CRC treatment, while cRGDIRNANO could not. In Vivo Antitumor Efficacy of Chemo, PTT, and CTT. Chemo treatment of mice involved SN38, CPT-11, IRNANOSN38, and cRGDIRNANOSN38 (Figure 6A) and the saline control. Tumors treated with saline grew quickly (Figure 6B), growing nearly 10-times their initial volume (day 0) by day 21 (Figure 6C), showing no therapeutic effect. However, tumors treated with CPT-11 or IRNANOSN38 grew slowly, reaching a volume of about 400 mm3 by day 21 (Figure 6B,C), indicating their good ability to inhibit tumor growth. Notably, growth of tumors treated with cRGD IRNANO SN38 was significantly inhibited and only reached a volume of about 250 mm3 by day 21 (Figure 6B,C), revealing remarkable tumor inhibitory ability. This is attributed to the presence of PEG, the cRGD peptide, and the nanoscale size of cRGDIRNANOSN38 enabling efficient delivery of SN38 to tumors, thereby eliminating tumor cells. These results showed cRGDIRNANOSN38 has outstanding tumor inhibition ability, making it a valuable candidate for effective CRC-chemo treatment. Despite the possible shielding effect caused by protein corona on cRGDIRNANOSN38, adequate therapeutic effect was achieved using cRGDIRNANOSN38.

temperature was significantly higher than the maximum temperature (44 °C) of tumors in mice treated with IRNANOSN38 and NIR irradiation for 60 s. This was attributed to the relatively high tumor uptake of IR780 achieved by IR cRGD NANOSN38, which possessed RES-avoidance, as well as active- and passive-targeting, functions. However, the in vivo temperature in tumors without any agents slowly increased under NIR light irradiation, reaching to 38 °C in 60 s. This increased temperature was likely due to the slow blood flow in tumors, which did not allow the heat generated from NIR light to be carried away.45 This increased temperature indicates the used NIR light irradiation does not induce cancer cell death.46 The maximum tumor temperature in mice treated with IR cRGD NANO was about 42 °C with 60 s of NIR irradiation, which was not as high as that achieved by cRGDIRNANOSN38. It is posited that without hydrophobic SN38, cRGDIRNANO might be unstable and thus disassemble when circulated in blood, resulting in a lower tumor uptake in the absence of an EPR effect. In addition, thermal images (Figure 5B) showed this significant increase in temperature was only observed in the tumor, indicating that PTT generation of heat specifically within tumors reduces the risk of damaging normal cells and tissues, and hence, is ideal for CTT. Overall, the in vivo J


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Figure 5. In vivo photothermal effect. (A) Time−temperature profile of tumors in tumor-bearing mice 24 h post-treated with cRGDIRNANOSN38, IR IR cRGD NANO, and saline (control group) and tumors in tumor-bearing mice 48 h post-treated with NANOSN38 under NIR irradiation for 60 s (808 nm, 1 W/cm2). (B) Thermal images of tumor-bearing mice under NIR irradiation (808 nm, 1 W/cm2). Color bar indicates relative temperature level. *P < 0.05.

cells in CTT. The strongest FL intensity was observed in tumors treated with CTT on day 2 (Figure 6D), indicating that CTT with cRGDIRNANOSN38 induces CRC cell death more efficiently than chemo administration of cRGDIRNANOSN38, IR NANOSN38, or CPT-11. Hence, cRGDIRNANOSN38 is preferred for CTT over chemo when treating CRC. However, antitumor efficacy results of PTT showed no therapeutic effect on tumors after mice were treated with 2 IR cRGD NANO combined with a 60 J/cm NIR light. Mice were IR injected on day 0 with cRGD NANO and treated with a 60 J/ cm2 of PTT on day 1 (Figure 6A). As shown in Figure 6B, the tumor grew rapidly in size after PTT and measured to be about 850 mm3 on day 21 (Figure 6C). There was no significant difference in tumor volume between cRGDIRNANO and 60 J/ cm2 PTT combination treatment and saline after statistical analysis because tumors with low cRGDIRNANO accumulation cannot be efficiently heated via NIR irradiation. These results suggest that PTT involving cRGD IRNANO only is not recommended. Furthermore, in vivo toxicity of cRGDIRNANOSN38 was higher than that of IRNANOSN38 group, contributing from the higher IR cRGD NANOSN38 accumulation in tumor tissue, possibly. No noticeable change in body weight or H&E staining of normal tissues was found after chemo treatment. Weight loss percentage in all mice was less than 20% over the 21 d period (Figure 7A), indicating that neither IR NANO SN38 or IR cRGD NANOSN38 caused serious harm even if mice were treated with a total dose of 30 mg SN38/kg and a total dose of 300 mg NIR nanoagent/kg (3 doses of 100 mg/kg). Moreover, there was no significant toxic effect of IRNANOSN38 and IR cRGD NANOSN38 on normal organs by day 21 according to H&E stained sections of heart, liver, spleen, lung, and kidney among mice treated with saline, IR NANO SN38 , and IR cRGD NANOSN38 (Figure 7B). Based on these results, it was concluded that the dose of cRGDIRNANOSN38 and IRNANOSN38 was well-tolerated.

Results of CTT antitumor efficacy assays showed significant tumor inhibition with cRGDIRNANOSN38 combined with a 60 J/ cm2 NIR light. In CTT, mice were treated with only one dose of cRGDIRNANOSN38 on day 0 and given a 60 J/cm2 NIR dose of light dose on day 1 (Figure 6A), based on the results of in vivo tumor accumulation which showed maximal tumor accumulation of cRGDIRNANOSN38 24 h after intravenous injection. The volume of the tumor treated with cRGDIRNANOSN38 together with 60-J/cm2 PTT was measured to be zero on day 3 (Figure 6B). By day 9, tumor regrowth was observed, and the tumor volume continued to slowly increase, reaching nearly 220 mm3 by day 21 (Figure 6B,C). Notably, a high degree of burn damage on the tumor was found on day 2 (Figure S11), which was not observed with cRGDIRNANO and 60 J/cm2 PTT administration (data not shown). This was attributed to the severe hyperthermia that resulted from the photothermal effect induced by cRGDIRNANOSN38,39 which brought about tumor elimination by day 3. Moreover, the slow increase in tumor volume after day 9 was thought to result from SN38 delivery to the tumor that inhibited tumor growth. Compared with saline, IR cRGD NANOSN38 displayed significantly better CTT antitumor efficacy. The enhanced CTT antitumor efficacy can be attributed to the targeting function of cRGDIRNANOSN38, which help increase the accumulation of cRGDIRNANOSN38 containing PTT and chemo drugs in tumor site. In addition, localized and elevated temperature can be achieved during PTT, which is thought not only to efficiently kill localized tumor cells but also successfully trigger the release of chemo drugs from our designed nanoparticles. Our study demonstrated that cRGDIRNANOSN38 would be a good candidate for effective CTT for CRC. On day 21, we noticed there was no significant difference in tumor volume between administration of cRGDIRNANOSN38 via chemo and CTT. Despite this equivalent antitumor efficacy, use of cRGDIRNANOSN38 is preferred in CTT to treat CRC because the dose of cRGDIRNANOSN38 used in CTT is lower (one-third the total chemo dose), lowering the risk of adverse effects from SN38. Furthermore, TUNEL assay results showed the effectiveness of cRGDIRNANOSN38 for elimination of tumor K


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Figure 6. Antitumor efficacy. (A) Protocols for chemo, PTT, and CTT. (B) Relative tumor volume of tumor-bearing mice treated with chemo involving CPT-11 (3 doses of 10 mg/kg each, 30 mg/kg total), IRNANOSN38 (3 doses of 10 mg SN38/kg each, 30 mg SN38/kg total), or IR IR cRGD NANOSN38 (3 doses of 10 mg SN38/kg each, 30 mg SN38/kg total); mice treated with PTT involving cRGD NANO (100 mg NIR nanoagent/ IR 2 2 kg) in combination with a 60 J/cm NIR light dose (808 nm, 1 W/cm ); mice treated with CTT involving cRGD NANOSN38 (10 mg SN38/kg; 100 mg NIR nanoagent/kg) in combination with a 60 J/cm2 NIR light dose. *P < 0.05; **P < 0.01. (C) Representative photos of tumor-bearing nude mice on day 21 of treatment. (D) TUNEL assay of tumor tissues on day 2 from tumor-bearing mice treated with saline, CPT-11, IRNANOSN38, IR IR 2 cRGD NANOSN38, and cRGD NANOSN38 in combination with a 60 J/cm NIR light. Nuclei of cells were stained with Hoechst 33342 (blue). Scale bar: 50 μm.

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CONCLUSION

IR780 and loaded with the anti-CRC drug SN38 was successfully created. The functionality of cRGDIRNANOSN38 was proven to help achieve outstanding antitumor efficacy of both chemo and CTT in CRC treatment. RES-avoiding

In the current study, a new multifunctional NIR nanoagent, IR cRGD NANOSN38,

composed of a cRGD peptide, PEG, and L


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Figure 7. (A) Relative body weight of tumor-bearing mice treated with the designed chemo, PTT, or CTT. (B) H&E sections from normal organs of mice on day 21 after treatment with saline, IRNANOSN38 (3 doses of 10 mg SN38/kg each, 30 mg SN38/kg total), or cRGDIRNANOSN38 (3 doses of 10 mg SN38/kg each, 30 mg SN38/kg total). Scale bars: 100 μm. 1

function (PEG), angiogenic blood vessels-targeting function (cRGD peptide) and passive targeting function (EPR effect, nanosize) of cRGDIRNANOSN38 could successfully help delivery IR cRGD NANOSN38 to tumor, significantly increasing tumor uptake of cRGDIRNANOSN38. Furthermore, photothermal function (IR780) of c R G D I R NANO S N 3 8 could allow IR cRGD NANOSN38 to exhibit ability to generate heat in tumor under NIR irradiation in PTT. NIR imaging function (IR780) of cRGDIRNANOSN38 could help verify the optimal time to apply NIR light. Based on these findings, it is believed that this NIR nanoagent will be helpful in the development of new strategy for curing CRC in the future.

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H NMR spectra and GPC analysis, TEM images, CMC, absorption spectra in DMSO, in vitro photothermal effect, in vitro cytotoxicity, EthD-1assay, representative photos of tumor-bearing mice, and CDI analysis. (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Shu-Jyuan Yang: 0000-0002-4881-0040 Notes

The authors declare no competing financial interest.

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ASSOCIATED CONTENT

ACKNOWLEDGMENTS We appreciate the help of staff from the National Taiwan University College of Medicine. This research was primarily funded by the Ministry of Science and Technology of the Republic of China (NSC102-2320-B-002-038-MY3, 105-2622-

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.7b00315. M


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B-002-016-CC2), the Ministry of Health and Welfare (MOHW105-TDU-B-211-134005) and National Taiwan University Hospital (106-S3583).

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ABBREVIATIONS CTT, chemothermal therapy; PTT, photothermal therapy/ treatment; cRGD, cyclic arginine-glycine-aspartic acid; NIR, near-infrared; DLS, dynamic light scattering; CMC, critical micelle concentration; EPR effect, enhanced and permeation retention effect; TEM, transmission electron microscopy; GPC, gel permeation chromatography; ZP, zeta potential; LE, loading efficiency; DC, drug content; SN38, 7-ethyl-10-hydroxycamptothecin; HPLC, high-performance liquid chromatography; EthD-1, ethidium homodimer-1

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