Polyphosphoester Based Nanocarrier for Combined Radio

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Controlled Release and Delivery Systems

Polyphosphoester Based Nanocarrier for Combined Radio-Photothermal Therapy of Breast Cancer Beibei Zhang, Congfei Xu, Chunyang Sun, and Chunshui Yu ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.9b00051 • Publication Date (Web): 18 Mar 2019 Downloaded from http://pubs.acs.org on March 18, 2019

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ACS Biomaterials Science & Engineering

Polyphosphoester Based Nanocarrier for Combined Radio-Photothermal Therapy of Breast Cancer Beibei Zhang†, Congfei Xu‡, Chunyang Sun†,¶,*, and Chunshui Yu†,* †

Department of Radiology and Tianjin Key Laboratory of Functional Imaging, Tianjin

Medical University General Hospital, Tianjin 300052, P.R. China ‡

Institutes for Life Sciences, School of Medicine and National Engineering Research

Center for Tissue Restoration and Reconstruction, South China University of Technology, Guangzhou, Guandong 510006, P. R. China ¶

State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin

300071, P.R. China *Corresponding Authors: Chunyang Sun, E-mail: [email protected]; Chunshui Yu, E-mail: [email protected]

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ABSTRACT: Recently, clinical research on tumor therapy has gradually shifted from traditional monotherapy toward combination therapy as tumors are complex, diverse and heterogeneous. Combination therapy may be essential for achieving the optimized treatment efficacy of tumors through distinct tumor-inhibiting mechanisms. At the same time, nanocarriers are emerging as an excellent strategy for delivering both drugs simultaneously. This work presents utilization of a polyphosphoester-based nanocarrier (NPIR/Cur) to achieve the codelivery of hydrophobic photothermal agent IR-780 and radiosensitizer curcumin (Cur). The IR-780 and curcumin coencapsulated NPIR/Cur exhibited adequate drug loading, a prolonged blood half-life, enhanced passive tumor homing, improved curcumin bioavailability, as well as combined therapeutic functions. Briefly, NPIR/Cur could not only achieve effective thermal ablation through the conversion of near-infrared light to heat but also give rise to a significant boosted local radiation dose to trigger promoted radiation damages, thus resulting in enhanced tumor cell growth inhibition. In conclusion, the as-prepared NPIR/Cur manifested excellent performance in facilitating combined photothermal and radiation therapy, thus expanding the application range of PPE-based carriers in nanomedicine, and also prompting exploration of their potential for other effective combination therapies. KEY WORDS: polyphosphoesters, nanomedicine, radiotherapy, photothermal therapy, combination therapy

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1. INTRODUCTION Currently, surgical resection, radiotherapy, chemotherapy and targeted therapy are the mainly clinical therapeutic approaches for cancer.[1-3] In addition to traditional treatment methods, photothermal therapy (PTT), as a controllable and noninvasive tumor therapy, has attracted significant interest in last decade. In a typical method based on photoabsorption agents, transdermal near-infrared (NIR) radiation is transformed into heat energy using a 700~1100-nm NIR laser to induce mild hyperthermia to kill cancer cells, thereby achieving selective local therapy and reducing the side effects on surrounding cells due to the high transmission of NIR light through biological tissues, blood and water.[4-6] For instance, indocyanine green is a U.S. Food and Drug Administration approved NIR dye for clinical applications.[7] However, it is arduous to completely eradicate tumors using PTT alone, especially deep tumors, as an inevitable depth-dependent decrease in NIR laser intensity. Considering this limitation, combination therapy with PTT is considered a promising strategy to achieve enhanced therapeutic effect.[8-11] In radiation therapy (RT), ionizing radiation(e.g., γ-rays, X-rays) is locally applied to a tumor to induce oxygen-centered radicals, causing DNA damages and subsequent tumor cell death without depth restriction.[12-14] However, the efficacy of clinically applied RT can be severely hindered by hypoxia-induced radiation resistance in some tumors, and high radiation dosages may damage healthy tissues and induce other toxicity and side effects.[15,16] Therefore, various radiosensitizers have been exploited to address the radioresistance of cancer cells, and drastically reduce the radiation dose 3



and the damages to adjacent healthy tissues. [17-19] More importantly, it has been found that a certain level of PTT-induced hyperthermia can promote intratumoral blood flow and subsequently ameliorate the oxygen level of tumor microenvironment, which may improve the sensitivity of cancer cells to RT, thus illustrating the potential for the clinical application of combination PTT and RT for cancer therapy.[20-24] Nanocarrier-based drug delivery promotes the bioactivity and biocompatibility of drugs and emerged as a promising strategy for cancer treatment.[25-27] The surface modification of nanoparticles with polyethylene glycol (PEG) can prevent the recognition by macrophages and prolong the pharmacokinetic behaviors of small molecular drugs, thus, these molecules can successfully accumulate into the tumor interstitium based on the enhanced permeability and retention (EPR) effect.[28,29] Moreover, nanocarriers can be loaded with photothermal agents and radiosensitizers simultaneously to achieve combined PTT and RT for cancer. For example, Liu et al. fabricated core-shell MnSe@Bi2Se3 nanoparticles and achieved a strong synergistic therapeutic effect partly due to increased oxygenation resulting from mild PTT-induced hyperthermia.[30] Zhao et al. synthesized bovine serum albumin (BSA) coated BiOI@Bi2Se3 semiconductor heterojunction nanoparticles with a good photothermal conversion efficiency and radiosensitization effects.[31] Polyphosphoester (PPE) is a biodegradable polymer with repeating phosphoester linkages that can be easily functionalized; PPE is potentially biocompatible and have attracted considerable attention in biomaterials research.[32-37] In recent years, Wang’s group and others have developed various delivery systems based on PPEs to 4


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encapsulate chemotherapeutic drugs, photothermal agents and photosensitizers.[38-41] To the best of our knowledge, PPE-based radiosensitizer-loaded carriers have not been reported. Meanwhile, curcumin is a polyphenolic compound extracted from roots of turmeric, and several studies have demonstrated that curcumin possesses potential radiosensitization property.[42-45] In this work, we successfully synthesized a PEGylated PPE to coencapsulate IR-780 and curcumin (Cur), which greatly boosted sensitivity of breast cancers to ionizing radiation. Specifically, the prepared NPIR/Cur was utilized as both a photothermal absorption agent and radiosensitizer for combined PTT and RT (Scheme 1). Compared to the use of PTT or RT alone, the as-prepared NPIR/Cur substantially limited the cancer cells proliferation and efficiently inhibited cancer cell growth via combined X-ray ionizing RT and NIR PTT.

Scheme 1. Illustration of utilizing NPIR/Cur for combined photothermal and radiation therapy. 2. MATERIALS AND METHODS 2.1 Materials. 2-chloro-2-oxo-1,3,2-dioxaphospholane (COP) was synthesized 5



according to previous studies in literature and was distilled twice under vacuum.[46] Monomethoxy poly(ethylene glycol) (PEG, Mn = 5000 g/mol; Sigma-Aldrich) was dried by azeotropic distillation twice with toluene. Curcumin, IR-780 iodide, anhydrous CH2Cl2 and 1,5,7-Triazabicylo[4.4.0]dec-5-ene (TBD) were purchased from SigmaAldrich. Triethylamine (TEA, Aladdin) was used after phthalic anhydride treatment and distillation. 2-Ethyl-1-butanol and tetrahydrofuran (THF) (Alfa Aesar, Shanghai) was used after distillation in vacuum. Since the IR-780 is insoluble in water, we first used DMSO to form a storage solution at a concentration of 10 mg/mL, and then diluted it to a specific concentration with H2O or PBS. All other undeclared reagents were used without treatment. 2.2 Characterization. The molecular weights were detected on a gel permeation chromatography (GPC) system. The proton nuclear magnetic resonance (1H NMR) spectra were recorded in CDCl3 on a 400-MHz spectrometer (Avance Ш, Bruker, Germany). The mean particle size, polydispersity and zeta potential were investigated by dynamic light scattering (DLS) using a Malvern Zetasizer (Nano-ZS, Malvern instruments, UK) performed at 25 °C. The morphology of nanoparticles was observed by transmission electron microscopy (TEM, Hitachi HT7700). The UV/Vis absorptions of nanoparticles in aqueous solution and drug loading contents were measured on an UV/Vis spectrophotometer (UV-3600 Shimadzu, Japan). 2.3 Cell Culture. NIH-3T3 cells, MCF-7 cells and MDA-MB-231 cells were obtained from American Type Culture Collection and cultured in Dulbecco’s modified Eagle’s medium (DMEM, HyClone, Logan, UT, USA) at 37 °C. The media were 6


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supplemented with 10% (v/v) fetal bovine serum (FBS, HyClone). 2.4 Synthesis of Di-Block Copolymer PEG-b-PBYP. 2-Ethylbutoxy-2-oxo-1, 3, 2dioxaphospholane (BYP) was synthesized via previous report.[38] PEGylated PBYP was synthesized by a PEG-initiated ring-opening polymerization. Typically, PEG (2.0000 g, 0.40 mmol) and BYP (5.2000 g, 25.00 mmol) were dissolved in anhydrous CH2Cl2 in a nitrogen-purified flask. Subsequently, TBD (55.6 mg, 0.40 mmol) was added to catalyze the polymerization followed by stirring for 20 min. The reaction was then carried out at 25 °C for 20 min in a glovebox (Vigor, Suzhou) with the water content below 0.1 ppm. The mixture was further purified by precipitating into cold diethyl ether/methanol mixture (10/1, v/v) twice. Finally, the product was dried under vacuum with a yield of 82.7%. 2.5 Preparation of IR-780/Cur Loaded Nanoparticles. The IR-780 and Cur loaded micelles were prepared through nanoprecipitation method. Briefly, PEG-b-PBYP (10.0 mg), IR-780 (0.5 mg) and Cur (1.0 mg) were dissolved in 1.0 mL of DMSO, and then the solution was added dropwisely into deionized water (10.0 mL) under stirring gently. Subsequently, DMSO and unloaded Cur and IR-780 were removed through dialysis (MWCO 3500) against distilled water and centrifugation at 3000 × g. Meanwhile, the micelles loaded with IR-780 or Cur were prepared similarly, respectively. 2.6 Drug Release from IR-780/Cur Loaded Nanoparticles. To determine curcumin release in vitro, NPIR/Cur, NPIR and NPCur (1.5 mL, [curcumin] = 200.0 μg/mL) suspensions were added to dialysis bags (MWCO 3500) and immersed in phosphate buffer (pH 7.4) or acetate buffer (pH 5.5) containing Tween-80 (0.5% w/w) in dark. 7



The dialysis bags were gently shaken at 37 °C at 80 rpm. At certain time intervals, the incubation solution (20.0 mL) was withdrawn and replaced with fresh buffer. The amount of released curcumin was determined by UV-Vis spectroscopy at 425 nm after lyophilization. Meanwhile, the amount of released IR-780 was determined by fluorescence emission intensity at 820 nm using fluorescence spectrophotometer (F7000, Hitachi). 2.7 Photothermal Efficacy In Vitro. The NPIR/Cur and NPIR suspensions at different concentrations ([IR-780] = 40, 20, 10 μg/mL) were placed in centrifuge tubes and exposed to 808-nm laser at 0.75 W/cm2 for 5 min, and PBS was used as a control. The temperature changes of the NPIR/Cur and NPIR solutions were monitored using an infrared camera (ICI7320, Infrared Camera, Inc.) and analyzed via thermal imaging analysis software (FLIR tools). 2.8 In Vitro Cellular Uptake. To quantitatively determine the cellular uptake of IR780, MDA-MB-231 cells were seeded in 24-well plates (50,000 cells per well) and incubated overnight. After further incubation the cells with NPIR/Cur and NPCur for 2 h, 6 h and 12 h, the cells were then washed twice with cold PBS and lysed with 1% Triton X-100 in PBS (250 μL) at 37 °C for 30 min, followed by three freeze-thaw cycles. The content of curcumin in the cell lysates was quantified by high performance liquid chromatography (HPLC) and then was normalized according to the total intracellular protein content examined by BCA protein assay kit. (Pierce, Rockford, IL). For the confocal laser scanning microscopy (CLSM) observations, MDA-MB-231 cells were seeded onto 10-mm coverslips in 12-well plates with 20,000 cells per well 8


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in 1.0 mL of DMEM. Then the cells were incubated with NPIR/Cur for 6 h. Subsequently, the cells were washed with cold PBS and fixed with 4% paraformaldehyde for 15 min at rt. The cells were then stained with Alexa Fluor 488 Phalloidin (Invitrogen, Carlsbad, CA) and 4-6-diamidino-2-phenylindole (DAPI) following manufacturer’s protocol before observation on a Zeiss LSM-800 confocal microscope. 2.9 DNA double strand breaks induction (γ-H2AX foci). MDA-MB-231 cells (5 × 104) were seeded on coverslips in 24-well plates and treated with NPIR/Cur and NPCur for 2 h. After exposure to X-rays at 4 Gy, the cells were washed twice with cold PBS and fixed with 4% paraformaldehyde for 10 min at rt. The cells were then blocked with 2% BSA in PBS for 1 h, after which mouse monoclonal anti-γ-H2AX antibody (Millipore) was added at a dilution of 1:200 in 1% BSA in PBS and incubated for 1 h at rt. The cells were then washed thrice in PBS before being incubated with goat anti-mouse FITC- and HRP-conjugated IgG (Santa Cruz Biotechnology, Dallas, TX) for 30 min to label γ-H2AX. The γ-H2AX level was then evaluated by CLSM. 2.10 Cell Viability. To investigate the cytotoxicity of the blank nanoparticles, MDAMB-231, MCF-7, and NIH-3T3 cells were seeded into a 96-well plate at 5,000 cells per well and then incubated at 37 °C overnight. Subsequently, the cells were incubated with DMEM containing different concentrations of nanoparticles for 48 h. Then, the cell culture was replaced with DMEM-containing 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT, 1 mg/mL) and incubation for another 2 h, followed by the adding of extraction buffer (100 µL, 20% SDS in 50% N,N-dimethylformamide, pH 4.7, prepared at 37 °C) and incubation overnight at 37 °C. The absorbance at 570 9



nm was detected using a Bio-Rad 680 microplate reader (Bio-Rad, USA). Cell viability was normalized using PBS as a control to represent 100% cell viability. To investigate the cell-killing efficacy of drug-loaded nanoparticles, MDA-MB-231 cells were seeded into a 96-well plate at the above-mentioned cell density and then incubated at 37 °C overnight. Following further incubation the cells with medium containing different concentrations of NPIR/Cur, NPIR or NPCur for 12 h, fresh complete DMEM was added to replace the medium and exposed to NIR (0.75 W/cm2, 10 min)/Xray (4 Gy, 0.8 Gy/min) irradiation. Next, the cells were further incubated at 37 °C for 24 h, and the cell viability was determined by MTT assay as described above. 2.12 Pharmacokinetic and Biodistribution Studies. Female ICR mice (18-20 g) were used to study the pharmacokinetics of NPIR/Cur, NPIR and free IR-780 in PBS (10 mM, pH 7.4) and these formulations were i.v. injected at an equivalent dose of 10 mg IR-780 per kg of mouse body weight (n = 4). At predetermined time intervals (0.167, 0.5, 1, 2, 6, 12, 24 and 48 h), blood samples were collected from the retro-orbital plexus, and 100 μL of plasma was obtained to analyse the concentration of IR-780. Furthermore, following i.v. injection of IR-780, NPIR or NPIR/Cur into nude mice bearing MDA-MB-231 xenografts for 6 h or 24 h, the mice were sacrificed, the solid tumor tissue and the major organs were collected to quantitative determine the biodistribution of IR-780. 2.12 Anticancer evaluation in vivo. MDA-MB-231 xenograft mice were assigned randomly to six groups and received an i.v. injection once a week when the tumor volume was approximately 50 mm3: (1) PBS; (2) free curcumin + X-Ray; (3) free IR10


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780 + laser; (4) NPIR + laser; (5) NPCur + X-Ray; (6) NPIR/Cur + X-Ray + laser ([curcumin] = 1.5 mg/kg; 808 nm, 0.75 W/cm2, 10 min; 4 Gy, 0.8 Gy/min). The tumor volume and body weight were simultaneous monitored every 3 days for the next 27 days, using a caliper to measure tumor diameters, and the tumor volume was estimated by the formula: tumor volume = 0.5 × length × width2. Then on the 28th day, tumor tissues were excised, fixed with 4% paraformaldehyde overnight at 4 °C and embedded by paraffin. The tissue sections (6 μm) were performed with hematoxylin and eosin (H&E) staining. Meanwhile, paraffin embedding tumor sections (6 μm) were also prepared for proliferating cell nuclear antigen (PCNA) immunohistochemical staining. Furthermore, in vivo toxicology of the nanomedicine was studied by routine blood analysis and H&E staining. Specifically, PBS, NPIR/Cur, NPIR or NPCur were i.v. injected at an equivalent dose of 2 mg IR-780 per kg of mouse body weight once a day for three days. At the fourth day, blood samples were collected from the retro-orbital plexus for routine blood analysis. Then the mice were sacrificed, and the major organs were collected for H&E staining. 2.13 Statistical analysis. The statistical significance of treatment outcomes was assessed using Student’s t-test (two-tailed); p < 0.05 was considered statistically significant in all analyses (95% confidence level). 3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization of IR-780 and Curcumin loaded nanoparticles. To prepare micelles for hydrophobic drug encapsulation, block

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copolymer PEG-b-PBYP was synthesized through ring-opening polymerization with PEG used as a macroinitiator. The product was characterized by GPC (Figure S1) and 1H

NMR spectrum (Figure S2). The forward GPC profile of PEG-b-PBYP compared

with that of PEG suggested the successful synthesis of the diblock copolymer. Meanwhile, most of the resonance peaks shown in Figure S2B could be attributed to protons of the PEG-b-PBYP, and the degree of polymerization of PBYP was 47, which was calculated based on the ratio of the area of the peak at 4.25 ppm (a) to that of the peak at 3.65 ppm (c). Due to the hydrophobicity of PBYP demonstrated by Wooley et al., amphiphilic PEG-b-PBYP is capable of encapsulating hydrophobic cargoes via hydrophobic interactions. We then loaded IR-780 (photothermal agent) and curcumin (radiosensitizer) into the hydrophobic core using a nanoprecipitation method, and the resulting nanoparticles are denoted as NPIR/Cur. Meanwhile, micelles loaded with IR780 (NPIR) or curcumin (NPCur) were prepared by a similar method. As shown in Figure 1A, the average size of these three nanoparticles determined by DLS was approximately 40 nm and polydispersity index (PDI) was 0.163, 0.172, 0.191, respectively. The morphology of NPIR/Cur, NPIR and NPCur observed by TEM (Figure 1B) exhibited a compact and spherical micellar structure with a diameter of ~40 nm. With similar PEG modifications and the polyphosphoester segments, these nanoparticles showed comparable and negative surface zeta potentials of -17.1 mV, -20.4 mV, and -21.5 mV, respectively.[47] The UV-Vis-NIR spectra of NPIR/Cur, NPIR and NPCur (Figure 1C) exhibited strong characteristic absorption bands of curcumin and IR-780 at about 425 nm and 790 nm, respectively. The IR-780 and curcumin loading content of NPIR/Cur was 12


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3.76% and 8.75%, respectively, similar to the corresponding loading content of NPIR and NPCur. Additionally, no obvious changes in nanoparticle size were observed after 7 days (Figure 1D) of incubation with DMEM containing 10% FBS at 37 °C, indicating the occurrence of PEGylation and superior nanoparticle stability in biological media. We next studied the degradation properties and drug release behaviors of PEG-bPBYP micelles at both pH 7.4 and pH 5.5, which mimicking the conditions of blood circulation and intracellular endo/lysosomes, respectively.[48] As shown in Figure S3, the degradation of PBYP segment was demonstrated by the characteristic peak shift in 31P

NMR spectra. It is worth noting the 31P resonance shifted more significantly (from

-1.008 ppm to -1.145 ppm) when samples were incubated at pH 5.5 for 96 h, suggesting the accelerated degradation in acidic condition.[34] On the other hand, the particle size of PEG-b-PBYP micelles showed a slight increase at pH 5.5 (Figure S4), which may be attributed to decreased hydrophobic-hydrophobic interaction after degradation. In contrast, the micellar nanoparticles of poor-degradable PEG-b-PCL was used for comparison and there was no obvious change could be observed from 13C NMR spectra and DLS measurement (Figure S3 and S5). Then we monitored the release profiles of curcumin from NPIR/Cur and NPCur at pH 7.4 or pH 5.5, respectively. As shown in Figure 1E, less than 20% of total curcumin was released from NPIR/Cur or NPCur after 72 h under neutral conditions. In contrast, we observed an apparent acidity-promoted curcumin release behavior at pH 5.5, and the total drug release over 72 h was 53.0% (NPIR/Cur) and 52.2% (NPCur), respectively. Meanwhile, similar accelerated IR-780 release pattern was also found at pH 5.5 (Figure S6), and we speculated that the rapid drug release may 13



be due to the degradation of PPEs under abundant H+ conditions.[34,49]

Figure 1. Preparation and characterization of NPIR/Cur, NPIR and NPCur. (A) Particle size distribution. (B) TEM images. The scale bar is 200 nm. (C) The UV-Vis-NIR absorption spectra. (D) Size changes of NPIR/Cur, NPIR and NPCur incubated in medium containing 10% FBS at different time. (E) The cumulative release of curcumin from NPIR/Cur and NPCur at pH 7.4 and 5.5. 3.2. Photothermal Effect in Vitro. It has been demonstrated that encapsulating IR780 in micelles enhances its photothermal properties because the nanoparticles can protect IR-780 from decomposition by oxygen.[50,51] To assess the photothermal ability of NPIR/Cur and NPIR, the temperature change of NPIR/Cur and NPIR at different IR-780 concentrations was monitored using an IR camera. NPIR/Cur or NPIR was irradiated with an 808-nm-wavelength laser (0.75 W/cm2), and PBS was used as a control. As shown in Figure 2A and 2B, only a negligible temperature increase (approximately 1.2 °C) was monitored in the PBS group after 5 min of irradiation. In contrast, concentrationdependent temperature changes were observed in both the NPIR/Cur and NPIR groups. The temperature of both the NPIR/Cur and NPIR solutions increased by ~33 °C (40 μg/mL), 14


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~21 °C (20 μg/mL) and ~11 °C (10 μg/mL), respectively. Because cancer cells are sensitive to high temperatures (41~48 °C),[52,53] the photothermal ability of NPIR/Cur and NPIR is sufficient for PTT in vivo.

Figure 2. (A) Temperature change curves of NPIR/Cur and NPIR under NIR irradiation (0.75 W/cm2, 808 nm, 5 min) at different concentrations of IR-780. (B) IR thermal images of NPIR/Cur and NPIR under laser irradiation. 3.3. In Vitro Cellular Uptake. Compared with PTT agents (e.g., IR-780), it is necessary for curcumin to be taken up by cells due to the mechanisms underlying its radioprotection and radiosensitization.[41,54] We next studied the intracellular drug content using a quantitative method. MDA-MB-231 cells were incubated with free curcumin, NPIR/Cur or NPCur for 2 h, 6 h, or 12 h, and then the curcumin uptake was determined by HPLC. As shown in Figure 3A, for cells treated with NPIR/Cur or NPCur, the intracellular curcumin content gradually increased with increasing incubation time. After incubation for 12 h, the NPIR/Cur totally facilitated the uptake of 1.91 μg of curcumin per mg of protein by MDA-MB-231 cells. Furthermore, the uptake of NPIR/Cur by MDA-MB-231 cells was confirmed by CLSM. After incubating NPIR/Cur 15



with MDA-MB-231 human breast cancer cells for 2 h, substantial fluorescence signals of NPIR/Cur were found in the cytoplasm, suggesting its effective cellular uptake (Figure 3B).

Figure 3 (A) Intracellular curcumin content after incubating MDA-MB-231 cells with NPCur and NPIR/Cur ([curcumin] = 0.4 µM) for different time. (B) Cellular uptake and subcellular distribution of IR-780 fluorescence after incubation with NPIR/Cur ([curcumin] = 0.4 µM) for 6 h observed by CLSM. The nucleus was counterstained with DAPI (blue) while the F-actin was counterstained with Alexa Fluor 488 phalloidin (green). The scale bar is 50 μm. 3.4. DNA Damage Assays. The enhancement of RT by curcumin is generally considered to be induced by DNA damage, which can be detected by immunofluorescence staining for γ-H2AX (a marker of double-strand DNA break).[55] To demonstrate this, we analyzed the γ-H2AX foci of MDA-MB-231 cells after incubation with NPCur, NP IR or NPIR/Cur and exposure to X-ray radiation. Compared with the PBS group, the other three groups showed significantly more γ-H2AX foci after X-ray radiation (4 Gy). While the drug-loaded nanoparticles alone induced 16


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negligible DNA damage, increased DNA damage was detected with the addition of radiation (NPCur + X-ray and NPIR/Cur + X-ray groups) (Figure 4).

Figure 4. Evaluation of DNA damages in different groups. The expression of γ-H2AX was stained with FITC (green), and cell nuclei were stained with DAPI (blue). The scale bar is 20 μm. 3.5. Combination PTT and RT in Vitro. As demonstrated by previous studies, PEGylated PPEs are considered promising candidates for drug delivery due to their biocompatibility and biosafety.[32,56] The cytotoxicity of the free NPs to MDA-MB-231, MCF-7, and NIH-3T3 cells was examined by MTT assay. No noticeable cytotoxicity of the nanoparticle formulations was observed in any of the cell lines, even at a concentration up to 800 μg/mL (Figure 5A). Following the encapsulation of IR-780, NPIR and NPIR/Cur were endowed with the capability of photothermal conversion. Therefore, we further assessed the cancer cell-killing efficacy of the formulations as photothermal agents against MDA-MB-231 cells. As illustrated in Figure 5B, treatment with NPIR and NPIR/Cur showed negligible anticancer effects in the absence of the 80817



nm laser. However, upon laser irradiation, both NPIR and NPIR/Cur exhibited an increased ability to inhibit tumor cell growth at each concentration, with a reduction in cell viability to ~47.3% (NPIR + laser) and ~44.5% (NPIR/Cur + laser, [IR-780] = 1.6 μM), respectively. On the other hand, similar results were found when NPCur and NPIR/Cur were examined as radiosensitizers. The combination of curcumin-loaded micelles and X-ray radiation decreased cell viability to ~68.1% (NPCur + X-ray) and ~69.5% (NPIR/Cur + X-ray) at the highest curcumin concentration (Figure 5C). Numerous researches have verified that mild hyperthermia can increase intratumoral blood perfusion to ameliorate oxygenation, thus overcoming the hypoxia-associated radioresistance and achieving excellent therapeutic effect.[30,57-60] Furthermore, to illustrate the combined effect of RT and PTT, we incubated MDA-MB-231 cells with NPIR/Cur and exposed the cells to both 808-nm laser and X-ray irradiation sequentially. As displayed in Figure 5D, the combination therapy substantially decreased cell viability to 24.17±2.72%, while treatment with NPCur + 808-nm laser or NPIR + X-ray irradiation only led to a moderate inhibition of cell proliferation, suggesting the more efficient combination outcome in vitro.

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Figure 5. (A) Quantitative analysis of viability of MDA-MB-231, MCF-7, and NIH3T3 cells treated with blank nanoparticles. (B) Quantitative analysis of the cell viability after the PTT treatment at various concentrations of IR-780. (C) Quantitative analysis of the cell viability after the RT treatment at various concentrations of curcumin. (D) Combination therapeutic effect of NPIR/Cur ([curcumin] = 0.4 µM) exposed to X-Ray and NIR irradiation (808 nm, 0.75 W/cm2, 10 min; 4 Gy). *p < 0.05. 3.6. Pharmacokinetics and Tumor Accumulation. The pharmacokinetics of NPIR/Cur, NPIR and free IR-780 were then evaluated in tumor-free ICR mice. As shown in Figure 6A, both NPIR/Cur and NPIR exhibited prolonged circulation in the bloodstream while free IR-780 was eliminated rapidly after the systemic injection. At 24 h postinjection, the IR-780 concentration in plasma was determined as 2.73% of injected dose in NPIR-treated mice and 2.52% of injected dose in NPIR/Cur-treated mice, respectively. 19



Additionally, the deposition of IR-780 in major organs and tumor tissues was measured by HPLC after systemic injection of the various formulations. As shown in Figure S7, a phenomenon of liver and spleen enrichment was observed in NPIR/Cur and NPIR groups at 24 hours post-injection, which was related to their blood clearance by reticuloendothelial system.[60] Meanwhile, treatment with nanosized NPIR/Cur or NPIR remarkably increased IR-780 deposition in tumor tissue compared with that of free IR780 (Figure 6B), which could be attributed to the prolonged blood circulation and the EPR effect.[61]

Figure 6 Blood circulation and tumor accumulation in vivo. (A) Pharmacokinetics study of different formulations. Data are presented as mean ± SD (n = 4). (B) Quantitative analysis of IR-780 distribution in tumor tissues. *p < 0.05. 3.7. Combined Photothermal and Radiation Sensitization Effect of NPIR/Cur in Vivo. To indicate the advantages of the combined effect of NPIR/Cur in vivo, the antitumor effects of the various formulations were examined. MDA-MB-231 tumorbearing mice were treated with the free drug or drug-loaded nanoparticles. At 24 h postadministration, the tumor tissues were irradiated with an 808-nm NIR laser at a power density of 0.75 W/cm2 for 10 min or with 4 Gy of X-rays, and the tumor growth was 20


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then monitored. As shown in Figure 7A, treatment with NPIR + L or NPCur + X-ray irradiation resulted in comparable tumor growth inhibition, and treatment with NPIR/Cur + L + X-ray irradiation resulted in the most significant tumor growth inhibitory effect among all groups. However, the anticancer efficacy was moderately improved after NIR or X-ray irradiation alone, which may be due to slight photothermal or radiative ablation of the tumor. Under the combined effect of X-Ray treatment and increased tumor volume, none of the groups had significant weight loss (Figure 7B). In addition, blood routine analysis and H&E staining of major organs were examined to study in vivo toxicology of NPIR/Cur. There was no significant difference in blood routine count or significant organ damage or inflammation among PBS, NPIR, NPCur and NPIR/Cur groups, suggesting their safety and biocompatibility in vivo (Table S1 and Figure S8). Meanwhile, proliferating cell nuclear antigen (PCNA) was used to analyze cell proliferation in tumor tissues (Figure 7C). As expected, NPIR/Cur + NIR + X-ray group manifested obvious tumor cell damage (H&E) and significantly reduced proportion of proliferating cells (PCNA-positive cells) in tumors.

21



Figure 7 (A) Tumor growth curves with different treatments. Free curcumin, free IR780, NPIR/Cur, NPIR and NPCur ([Curcumin] = 1.5 mg/kg) were intravenously injected, respectively. The injections were indicated by arrows. *p < 0.05. (B) Body weight changes of tumor-bearing mice at different time points. (C) H&E and PCNA analysis of tumor tissues at the final of treatments. All the data are shown as mean ± SD. In comparison with RT or PTT alone, our findings indicate that combination therapy mediated by NPIR/Cur lead to promoted antitumor activity. Meanwhile, pervious work has used engineered nanoagents, usually inorganic nanoparticles, to combine PTT and RT for highly effective cancer treatment (some classical literatures were summarized in Table S2).[24, 30, 62-68] These reports are definitely meaningful as they offered a new synthetic route of functional nanomaterials with multicomponent compositions and structures and broadened their possible applications for use in biomedicine field. In addition to inorganic nanoagents, several small molecules could also serve as 22


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radiosensitizers and photothermal agents, and the micellar nanocarriers could help them to overcome the limitations in clinical application like poor solubility and insufficient tumor accumulation. The results in this work give us a new point of view to fabricate an efficient nanocarrier to codeliver IR-780 and curcumin with good biosafety and biocompatibility on orthotopic human tumor xenograft. In comparison with the superior antitumor activity of those inorganic systems, more advanced designs, such as the introduction of targeting or responsive moieties, are necessary to improve the therapeutic effect in future studies. It also encourages the further integration of diagnosis (e.g. PET imaging) to achieve promising cancer theranostics. 4. CONCLUSION In conclusion, we synthesized PEGylated PPE-based nanocarrier to codeliver curcumin and IR-780 for the combination of PTT and RT. Our results demonstrated that NPIR/Cur can be applied simultaneously as effective photothermal agent and radiosensitizer for the treatment of cancer. Compared with PTT or X-ray radiation alone, the combination of RT and PTT achieved the most appreciable anticancer effect, which was verified by increased tumor inhibition and destruction via both in vitro and in vivo experiments. Moreover, no significant cell death was observed in group of NIR laser irradiation alone, indicating the safety of power density we applied in vitro. In addition, the synthesized NPIR/Cur has favorable biocompatibility and showed no obvious cytotoxicity at the concentration required for cancer cell therapy. Our research not only studied the utilization of PPE-based nanocarrier for efficient combined PTT and RT, but also expanded its application range, leading to exploration of PPEs for multiple 23



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combination therapies, which will potentially make contribution to their clinical translation. Supporting Information Gel permeation chromatography of PEG-b-PBYP, synthetic route for the PEG-bPBYP, 1H NMR spectrum of PEG-b-PBYP, degradable properties of the PEG-b-PBYP and PEG-PCL, release of IR-780 at different pH, IR-780 distribution and H&E stained images of major organs, routine blood analysis of mice with different treatments. Acknowledgments This work was supported by the National Natural Science Foundation of China (51603150),

Tianjin

Municipal

Science

and

Technology

Commission

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For Table of Contents Use Only Polyphosphoester Based Nanocarrier for Combined Radio-Photothermal Therapy of Breast Cancer Beibei Zhang, Congfei Xu, Chunyang Sun*, and Chunshui Yu*

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Polyphosphoester Based Nanocarrier for Combined Radio

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