Tumor-Homing and Penetrating Peptide-Functionalized

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Tumor-Homing and Penetrating Peptide-Functionalized Photosensitizer-Conjugated PEG-PLA Nanoparticles for Chemo-Photodynamic Combination Therapy of Drug-Resistant Cancer Xingye Feng,† Di Jiang,† Ting Kang,† Jianhui Yao,† Yixian Jing,† Tianze Jiang,† Jingxian Feng,† Qianqian Zhu,† Qingxiang Song,‡ Nan Dong,† Xiaoling Gao,*,‡ and Jun Chen*,† †

Key Laboratory of Smart Drug Delivery, Ministry of Education, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, P. R. China ‡ Department of Pharmacology, Institute of Medical Sciences, Shanghai Jiaotong University School of Medicine, 280 South Chongqing Road, Shanghai 200025, P. R. China S Supporting Information *

ABSTRACT: The combination of photodynamic therapy (PDT) and chemotherapy holds great potential in combating drug-resistant cancers. However, the major challenge that lies ahead is how to achieve high coloading capacity for both photosensitizer and chemo-drugs and how to gain efficient delivery of drugs to the drug-resistant tumors. In this study, we prepared a nanovehicle for codelivery of photosensitizer (pyropheophorbide-a, PPa) and chemo-drugs (paclitaxel, PTX) based on the synthesis of PPa-conjugated amphiphilic copolymer PPa-PLA-PEG-PLA-PPa. The obtained nanoparticles (PP NP) exhibited a satisfactory high drug-loading capacity for both drugs. To achieve effective tumor-targeting therapy, the surface of PP NP was decorated with a tumorhoming and penetrating peptide F3. In vitro cellular experiments showed that F3-functionalized PP NP (F3-PP NP) exhibited higher cellular association than PP NP and resulted in the strongest antiproliferation effect. In addition, compared with the unmodified nanoparticles, F3-PP NP exhibited a more preferential enrichment at the tumor site. Pharmacodynamics evaluation in vivo demonstrated that a longer survival time was achieved by the tumor-bearing mice treated with PP NP (+laser) than those treated with chemotherapy only or PDT only. Such antitumor efficacy of combination therapy was further improved following the F3 peptide functionalization. Collectively, these results suggested that targeted combination therapy may pave a promising way for the therapy of drug-resistant tumor. KEYWORDS: tumor-homing peptide, multidrug resistance, combination therapy, photodynamic therapy, nanoparticle

1. INTRODUCTION Multidrug resistance (MDR) represents the most common causes that lead to treatment failure in more than 90% patients with malignant tumor.1,2 P-glycoprotein (P-gp) which is encoded by the MDR-1 (ABCB1) gene was recognized as the dominant factor underlying MDR.3,4 Unfortunately, currently used antitumor chemotherapeutics including paclitaxel, doxorubicin, and vincristine are all the substrates of P-gp.5,6 As reported previously, the tumor penetration of PTX was seriously limited by the efflux transporter, leading to an unsatisfactory therapy effect.7 To combat drug resistance, one strategy is to codeliver P-gp inhibitor such as verapamil along with anticancer drugs.8 However, a non-negligible concern is that these inhibitors would also block the ABCB1-mediated drug efflux in normal cells, which then contribute to side effects of chemotherapy.9 Therefore, an advanced strategy with enhanced therapy efficiency and safety is necessary for treating drug-resistant cancer. © 2016 American Chemical Society

The combination of photodynamic therapy (PDT) and chemotherapy has exhibited superiority in the treatment of drug-resistant cancer through exploiting the synergistic effects.10−12 Such treatment strategy mainly based on that P-gp inhibition was not associated with the PDT, and ROS produced in the process of PDT could not only directly kill tumor cells but also induce photooxidation of endocytic membranes and finally lead to release of chemotherapeutics from endosomes to cytosol.13−15 To obtain such a synergistic effect, one of the widely used methods is to deliver the photosensitizer and chemotherapeutics using a single nanosystem. Nonetheless, such treatment strategy is impeded by the obstacles, such as Received: April 14, 2016 Accepted: June 22, 2016 Published: June 22, 2016 17817

DOI: 10.1021/acsami.6b04442 ACS Appl. Mater. Interfaces 2016, 8, 17817−17832

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ACS Applied Materials & Interfaces

a highly tumor-homing peptide that can offer an attractive strategy for mediating dual-targeting delivery to specific tumor cells and tumor angiogenesis and enabling deep penetration of drugs into the tumor parenchyma.25 In the present work, human colorectal cancer cells (HCT-15, a PTX-resistant cell) and human umbilical vein endothelial cells (HUVEC) were applied to evaluate the cellular association and cytotoxicity of F3-PP NP. Biodistribution of nanoparticles was further evaluated in vivo to study the efficiency of tumortargeting drug delivery. Finally, pharmacodynamics evaluation was performed both on cells and in tumor-bering mice in the presence/absence of laser to study the therapeutic effect of the combination treatment strategy presented in this study.

instability, drug leakage, and low entrapment efficiency that are mainly attributed to the steric hindrance between the photosensitizer and chemotherapeutics.16,17 As reported previously, coencapsulation of chemotherapeutics (Docetaxel) and photosensitizers (zinc-phthalocyanine) led to a sharp decrease in both the entrapment efficiency and loading capacity.17 Therefore, it is necessary to establish an advanced nanosystem with stable and efficient loading of both photosensitizer and chemotherapeutics. Chlorophyll-a-based photosensitizers are attracting increased attention in PDT due to the high optical activity. Pyropheophorbide-a (PPa), which belongs to the chlorophyll-a compounds, is one of the most widely used agents with the advantages of high singlet oxygen generation, low skin toxicity, and light absorbance above 650 nm.18,19 However, PPa has been characterized with poor solubility in water, which significantly limits its therapeutic efficacy in PDT.19 Furthermore, the physical encapsulation of PPa resulted in low loading capacity and significant leak of the drug that are not beneficial for in vivo treatment. Therefore, to build a desirable nanocarrier, we first conjugated PPa to HO-PLA-PEG-PLA-OH through an esterification reaction, and then loaded PTX into the nanoparticles prepared from the PPa-conjugated polymer (PPa-PLA-PEG-PLA-PPa) to form the PPa and PTX coloaded nanoparticles (PP NP). To accumulate both photosensitizer and chemotherapeutics at the tumor site simultaneously and precisely, and to achieve a more efficient tumor-growth inhibition with a minimum side effect, the F3 peptide was further decorated on the surface of PP NP. The F3 peptide could selectively bind to nucleolin that expressed on the surface of angioginic endothelial cells and tumor cells. In contrast, only the nucleus of normal cells was positive for the expression of nucleolin.20 In addition, previous studies reported that fluorescence-labeled F3 peptide could be efficiently internalized by the tumor cells.21,22 Furthermore, F3 peptide contains a CendR sequence, which possesses the ability of tumor penetration.23,24 Taken together, F3 peptide is

2. RESULTS AND DISCUSSION To establish a nanocarrier, which could effectively coload photosensitizers and chemotherapeutics, the commonly used ester bond was applied for synthesis of PPa-PLA-PEG-PLA-PPa. The PTX-loaded nanoparticles (PP NP) were developed as reported previously.25 The resulted PP NP exhibited satisfactory entrapment efficiency and loading capacity for both PTX and PPa. In addition, we modified the surface of PP NP with a nucleolintargeting peptide F3 for selectively transporting drugs from blood vessels to tumor sites after administration. In vitro cellular experiments showed that the peptide-modified nanoparticles exhibited highly selective cellular association and resulted in a stronger cytotoxic effect against both HUVEC cells and HCT-15 cells. Besides, the in vivo experiment demonstrated that F3-PP NP penetrated deep into tumor parenchyma, which could be mainly ascribed to the CendR sequences contained in F3 peptide. Furthermore, pharmacodynamics evaluation in tumor-bearing mice indicated that F3-PP NP-based combination of PDT and chemotherapy in this study achieved the best antitumor effect, as evidenced by the longest survival time of the tumor-bearing mice remedied by the F3-PP NP. 2.1. Synthesis of PPa-PLA-PEG-PLA-PPa. The synthesis scheme of PPa-PLA-PEG-PLA-PPa was shown in Scheme 1.

Scheme 1. Scheme for the Synthesis of PPa-PLA-PEG-PLA-PPa

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Figure 1. 1H NMR analysis of (A) PPa, (B) HO-PLA-PEG-PLA-OH, and (C) PPa-PLA-PEG-PLA-PPa. (D) HPLC graphs of PPa and PPa-PLAPEG-PLA-PPa. (E) Normalized UV−vis absorption spectra of PPa and PPa-PLA-PEG-PLA-PPa.

Table 1. Characterization of the Prepared Nanoparticlesa

The results of 1H NMR analysis were shown in Figure 1A−C, in which the signals at 12.15 ppm (peak a) represented the peak of −COOH in PPa (Figure 1A). In the case of HO-PLAPEG-PLA-OH (Figure 1B), the characteristic signals at 1.41.6 ppm (peak a) and 5.05.2 ppm (peak b) represented the peak of −CH3 protons and −CH protons in PLA segment, respectively, and that at 3.53.6 ppm (peak c) represented the

nanoparticles

particle size (mean ± SD, nm)

polydispersity index (PI)

zeta potential (mV)

PP NP F3-PP NP

109.81 ± 3.55 118.25 ± 4.43

0.102 ± 0.063 0.124 ± 0.078

−31.75 ± 2.76 −17.59 ± 4.73

a

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Data represent mean ± SD, n = 3. DOI: 10.1021/acsami.6b04442 ACS Appl. Mater. Interfaces 2016, 8, 17817−17832

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Figure 2. Particle size and TEM images of (A) PP NP and (B) F3-PP NP. The bar is 200 nm.

Figure 3. Profile of in vitro release of (A) PTX and (B) PPa from PP NP and F3-PP NP. Stability of PP NP and F3-PP NP in DMEM with 10% FBS and PBS, respectively. (C) The size of nanoparticles was measured, and (D) the appearance of F3-PP NP solutions was photographed each day during the inspection. The green and red tubes represent the F3-PP NP sample dissolved in PBS and DMEM containing 10% FBS, respectively.

peak of −CH2 protons in the PEG block of HO-PLA-PEGPLA-OH. In the case of PPa-PLA-PEG-PLA-PPa (Figure 1C), except the characteristic signals of CH, CH2, and CH3 of the

PLA and PEG segments, the signals of PPa which showed at 8.510.0 ppm (peak d) were also detectable in the conjugates while that of −COOH was undetectable. These results together 17820


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Figure 4. Qualitative analysis of the cellular uptake of PP NP/F3-PP NP in HUVEC cells and HCT-15 cells in the presence/absence of (A) F3 peptide analyzed by fluorescent microscopy. Quantitative analysis of cellular uptake of PP NP/F3-PP NP in (B) HUVEC cells and (C) HCT-15 cells analyzed via the Kinetic Scan HCS Reader. Red indicates nanoparticles visualized by the fluorescence of PPa. Original magnification: 20×. ***p < 0.001 as compared with that of F3-PP NP+F3 associated by cells.

PP NP and F3-PP NP was 2.40 ± 0.09% and 2.37 ± 0.14%, respectively. In addition, the results of XPS analysis showed that the surface nitrogen detected on F3-PP NP was 0.63% which was negligible on the PP NP. 2.3. In Vitro Release and Stability of Nanoparticles. As shown in Figure 3A, a similar release trend was observed for PP NP and F3-PP NP under the same condition, indicating that the release pattern of PTX did not change significantly post F3 peptide decoration. However, the cumulative release of PTX from the nanocarriers in the media containing rat plasma (81.60% and 84.2% for PP NP and F3-PP NP, respectively) is slightly faster than that in the Tween-80-containing media (72.94% and 73. 72% for PP NP and F3-PP NP, respectively), which could be mainly contributed to the various enzymes in the plasma.28 The release profiles of PPa from PP NP and F3-PP NP were also studied with the same methods to evaluate the stability of conjugation of PPa-PLA-PEG-PLA-PPa after the preparation of the nanoparticles. As shown in Figure 3B, cumulative release of PPa from nanoparticles in the media containing rat plasma (2.36% and 2.46% for PP NP and F3-PP NP, respectively) and in the Tween-80-containing media (1.92% and 2.05% for PP NP and F3-PP NP, respectively) were both below 2.5%, indicating a desirable stability for PPa-PLA-PEG-PLA-PPa. Good stability of the drug delivery system is always closely associated with the long circulation that is critical for its high accumulation at the tumor site.29 Figure 3C,D showed that both PP NP and F3-PP NP did not exhibit obvious aggregation phenomena during the determined days, indicating that the

demonstrated that the copolymer PPa-PLA-PEG-PLA-PPa was successfully synthesized. Such results were further confirmed by HPLC analysis (Figure 1D), in which the peak of free PPa was undetectable in the PPa-PLA-PEG-PLA-PPa sample. Additionally, the amount of PPa conjugated to 1 mg of HO-PLA-PEG-PLA-OH was 27.56 μg, which was close to that of the theoretical value of 28.5 μg. The analysis of the normalized UV−vis absorption spectra as shown in Figure 1E indicated that the optical activity of PPa did not change after the conjugation with HO-PLAPEG-PLA-OH. 2.2. Characterization of PP NP and F3-PP NP. As reported previously, for a nanosystem to take advantage of the enhanced permeability and retention (EPR) effect of tumor microenvironment, such a vehicle should be characterized with size of around 100 nm.26,27 Results in Table 1 exhibited that the sizes of nanoparticles were 109.81 ± 3.55 nm (PP NP) and 118.25 ± 4.43 nm (F3-PP NP), respectively, which both met the demands well. Zeta potentials of PP NP and F3-PP NP were −31.75 ± 2.76 and −17.59 ± 4.73 mV, respectively. The change was mainly ascribed to the electropositivity of F3 peptide due to several cationic amino acids contained in the peptide. In addition, the TEM images shown in Figure 2A,B displayed that both PP NP and F3-PP NP were generally spherical. Besides, for the loading of PTX, the EE of the developed nanoparticles were 71.07 ± 2.57% (PP NP) and 67 ± 3.05% (F3-PP NP), respectively. Besides, the LC of nanoparticles were 3.7 ± 0.27% (PP NP) and 3.41 ± 0.73% (F3-PP NP), respectively. For the loading of PPa, the LC of the optimized 17821


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HO-PLA-PEG-PLA-OH due to the hydrophobic properties of photosensitizer. 2.6. Colocalization Assay. It is widely accepted that endolysosomal compartments are always involved in the cellular endocytosis of nanoparticles and closely associated with the limited therapy effect by degrading the drug nanosystem and the encapsulated drugs.30,31 In this study, a lysotracker green was introduced to determine the colocalization of the nanoparticles and lysosomes. As shown in Figure 6, after incubating HUVEC cells and HCT-15 cells with PP NP and F3-PP NP for 3 h, the nanoparticles were mostly colocalized with the endolysosomal compartments, which demonstrated that the cellular uptake of those prepared nanoparticles was exactly endolysosome involved. 2.7. Photooxidation of Endocytic Membranes. As reported previously, photosensitizer-based endosomal escape could significantly potentiate the effect of chemotherapeutics through the mechanism of membrane rupture by the PDT.32 In this study, we evaluated the membrane rupture effect of F3-PP NP under different irradiation times. As shown in Figures 7 and 8, both cells incubated with F3-PP NP without irradiation exhibited the most distinct red fluorescence. In contrast, in the presence of the laser, the fluorescence signal of lysosomes was gradually decreased once the laser power was increased, and almost disappeared after irradiation at 1.0 J/cm2 for 90 s. 2.8. In Vitro Cytotoxicity of Nanoparticles. To clearly illustrate the antiproliferation effect of various therapy strategies, PTX-free nanoparticles PPa NP and F3-PPa NP and PPafree nanoparticles NP-PTX were prepared with the methods as

nanoparticles prepared in this study exhibited a satisfactory stability. 2.4. Cellular Association of Nanoparticles. We execute a cellular association assay in HUVEC cells and HCT-15 cells to demonstrate the F3 peptide-mediated cellular intake of nanoparticles. As shown in Figure 4, both HUVEC cells and HCT-15 cells incubated with F3-PP NP displayed the strongest fluorescence when compared with the cells exposed to PP NP. In addition, after pretreatment with excess F3 peptide, cellular association of F3-PP NP was seriously inhibited. Quantitative results in Figure 4B,C further demonstrated a peptide-mediated manner of cellular uptake of F3-PP NP, which was 1.21-fold higher in HUVEC cells and 1.18-fold higher in HCT-15 cells, respectively, when compared with that of PP NP. 2.5. Reactive Oxygen Species Production. After irradiating the cells at various laser power, the production of ROS in cells was evaluated through observing the green fluorescence of DCF via a fluorescence microscope. Results showed that, in contrast to the cells in the absence of irradiation, both HUVEC cells and HCT-15 cells that were exposed to a laser exhibited evident green fluorescence (Figure 5). In addition, the fluorescent signals in the irradiated cells were enhanced after increasing the laser power. For the comparison between PPa and PP NP, results shown in Figure S1 illustrated that, after incubation of cells with PPa (4.8 μg/mL) or PP NP (containing 4.8 μg/mL PPa) and irradiation of cells with an appropriate laser (0.5 J/cm2), the green fluorescence of PP NP was significantly higher than that of free PPa. Such results mainly ascribed to the observation that cellular internalization of PPa was significantly increased after being conjugated to

Figure 5. Evaluation of reactive oxygen species production in (A) HCT-15 cells and (B) HUVEC cells following exposure to 200 μg/mL of PP NP or F3-PP NP and irradiation at the laser power of 0 (control), 0.005, 0.05, and 0.5 J/cm2, respectively. Green represents the produced ROS tracked by DCF fluorescence. 17822


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Figure 6. Intracellular localization of nanoparticles after incubating HUVEC cells and HCT-15 cells with 200 μg/mL (A) PP NP and (B) F3-PP NP for 3 h. Blue shows nuclei stained with DAPI. Green shows endosomes labeled by Lysotracker Green. Red shows particles tracked by fluorescence of PPa.

obvious inhibition effect on cell proliferation in both HUVEC and HCT-15 cells compared with PP NP in the absence of irradiation, suggesting that the produced reactive oxygen during PDT was able to kill tumor cells and endothelial cells. Importantly, after incubating with F3-PP NP, the cell viabilities of HUVEC cells and HCT-15 cells were significantly decreased, which could be mainly ascribed to the elevated cellular association of nanoparticles that were mediated by F3 peptide. For quantitative analysis, we calculated the IC50 values by using the Graph Prism 5.0, and the IC50 in HUVECE cells were 122.1, 84.09, 41.21, and 17.0 ng/mL for Taxol, PP NP (−laser), PP NP (+laser), and F3-PP NP (+laser), respectively. The IC50 in HCT-15 cells were 426.7, 86.32, and 32.86 ng/mL for PP NP (−laser), PP NP (+laser), and F3-PP NP (+laser), respectively. In contrast, the IC50 of Taxol on HCT-15 cells was above 1000 ng/mL, confirming that HCT-15 cells were exactly PTXresistant. In combination therapy, the quantitative definitions of antagonism (CI > 1), additive effect (CI = 1), and synergism (CI < 1) were provided by the CI theorem.35 As calculated, the CI of PP NP in the presence of laser was less than 0.49 and

PP NP was developed. As shown in Figure 9A,B, PPa NP in the dark exhibited undetectable cytotoxicity against both HUVEC cells and HCT-15 cells, indicating that PPa exhibited noncytotoxicity without irradiation. In contrast, along with the increasing intensity of the irradiated laser, the cytotoxicity of each group except the NP-PTX group increased, suggesting that PDT was a laser dependent treatment. In the case of those cells treated with NP-PTX (+laser), cell viability displayed inconspicuous changes even at the irradiated laser intensity up to 3.0 J/cm2, indicating that the illumination intensity used in this study would not induce toxicity to cells. For further evaluate the cytotoxicity of PPa NP and F3-PPa NP, both cells were exposed to different concentrations of PPa NP and F3-PPa NP in the absence of irradiation. As shown in Figure 9C,D, inconspicuous cytotoxicity of both PPa NP and F3-PPa NP on HUVEC cells and HCT-15 cells was observed under all the nanoparticle concentrations without irradiation. Such results indicated that the prepared PPa NP was nontoxic in the dark and therefore could be used as a safe drug carrier. In the cases of PP NP and F3-PP NP, as shown in Figure 9E,F, in the presence of irradiation, PP NP showed an 17823


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Figure 7. Localization of F3-PP NP in HUVEC cells after irradiation at the laser intensity of 0 (control), 0.05, 0.5, and 1.0 J/cm2, respectively. Blue shows nuclei stained with DAPI. Green shows endosomes labeled by Lysotracker Green. Red shows particles tracked by fluorescence of PPa.

mediated by F3 peptide. Such hypothesis was clearly demonstrated in Figure 11 as much stronger fluorescence intensity of PPa was observed far from tumor blood vessels following the treatment with F3-PP NP compared with the treatment with PP NP. 2.11. Pharmacodynamic Evaluation in Vivo. The human colorectal adenocarcinoma-bearing mice models were established to evaluate the therapy effect of F3-PP NP in vivo. Results shown in Figure 12 indicated that the mice treated with F3-PP NP (+laser) displayed the best antitumor effect. As a typical PTX-resistant cancer, the mice treated with Taxol exhibited negligible tumor inhibition effect while the group given with PP NP in the absence of laser displayed a weak inhibitory effect. Such results might be mainly due to the encapsulation of PTX which could reduce the chance of interaction between PTX and P-gp.35 As reported previously, the reactive oxygen species produced during PDT did not exhibit tolerance to tumor cells, consistent with the results in this study that the tumor-bearing mice treated with PPa NP (+laser) showed a higher tumor inhibitory effect compared to those given with PP NP (−laser). Meanwhile, combining PDT with chemotherapeutics resulted in a stronger tumor-growth inhibition effect

0.202 for HUVEC cells and HCT-15 cells, respectively, indicating a strong synergistic effect of the combination therapy. 2.9. Biodistribution of Nanoparticles. The tumor cell and tumor vasculature dual-targeting was generally recognized as one of the most promising methods for cancer-targeting treatment.33,34 In this study, we decorated the surface of PP NP with an F3 peptide for dual-targeting drug delivery and evaluated the tumor-targeting effect in vivo. As results show in Figure 10A, consistent with the ex vivo images (Figure 10B), the tumorbearing mice treated with F3-PP NP displayed a higher tumor accumulation when compared with the unmodified ones. In addition, semiquantitative analysis (Figure 10C) of distribution of nanoparticles in the main organs further indicated that F3-PP NP exhibited a higher tumor-homing efficiency but similar level of distribution in other tissues as PP NP. 2.10. Intratumor Distribution of Nanoparticles. It was shown that a small quantity of unmodified PP NP was observed at the tumor site and only accumulated around the blood vessels (Figure 11). In contrast, the accumulation of F3-PP NP at the tumor site was significantly increased, which could be mainly ascribed to the tumor cells and tumor angiogenesis dualtargeting delivery and deep penetration into the tumor inner 17824


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Figure 8. Localization of F3-PP NP in HCT-15 cells after irradiated at the laser intensity of 0 (control), 0.05, 0.5, and 1.0 J/cm2, respectively. Blue shows nuclei stained with DAPI. Green shows endosomes labeled by Lysotracker Green. Red shows particles tracked by fluorescence of PPa.

2.12. Safety Evaluation of Nanoparticles in Vivo. Histopathology analysis was applied to evaluate the toxicity of PP NP and F3-PP NP on normal tissues. As shown in Figure 14, there was no obvious necrosis, hyperemia, or inflammation in normal brain or the main organs. Such results indicated that there was inconspicuous toxicity to normal tissues for PP NP and F3-PP NP under the therapeutic dosage of PTX or PPa, and the results further suggested that the combination system of PDT and chemotherapy established in this study was safe under the current dosage.

compared to any monotherapy. Such results were further demonstrated by the measurement of survival time after treatment with various formulations (Figure 11). It was shown that the mice treated with F3-PP NP (+laser) achieved the best survival situation (above the inspection days). Under the same experimental conditions, the mice given with saline, Taxol, PP NP (−laser), PPa NP (+laser), and PP NP (+laser) achieved the survival time of 22, 24, 30, 41, and 53 days, respectively. The evaluation of the inhibition rate of the tumor (Figure 12D) illustrated the highest inhibition rate for F3-PP NP (+laser) with a value of 79.92%, while the values of those treated with Taxol, PP NP (−laser), PPa NP (+laser), and PP NP (+laser) were 5.71%, 18.97%, 38.08%, and 54.68%, respectively. The results above demonstrated that the combination treatment strategy in this study could serve as an advanced program for the therapy of drug-resistant cancer. To study the apoptosis of cells in the tumor tissue, an H&E staining experiment was performed. It was shown that the F3-PP NP (+laser)-treated mice exhibited the highest level of cell apoptosis, while those treated with Taxol displayed inconspicuous differentiation compared to the control animals treated with saline (Figure 13), indicating that the colorectal cancer model established in this study was exactly PTX resistance.

3. CONCLUSION To overcome tumor drug resistance, ROS-based photodynamic therapy was introduced and combined with conventional chemotherapy in this study. A photosensitizer and chemotherapeutic double-loaded nanosystem was established and modified with F3 peptide to achieve targeted combination therapy. In vitro cellular experiments showed that F3-PP NP (+laser) exhibited the highest antiproliferation effect against both HUVEC cells and HCT-15 cells. Besides, the effect of PP NP in the presence of laser was superior to PP NP (−laser) and PPa NP (+laser), indicating that the strategy of combination 17825


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Figure 9. Evaluation of the cytotoxicity of nanoparticles in (A) HUVEC cells and (B) HCT-15 cells post-incubation with Taxol, PP NP, and F3-PP NP followed by irradiation at various laser power and incubation for another 24 h. Evaluation of the cytotoxicity of nanoparticles in (C) HUVEC cells and (D) HCT-15 cells post-incubation with PPa NP and F3-PPa NP without irradiation. Evaluation of the cytotoxicity of nanoparticles in (E) HUVEC cells and (F) HCT-15 cells post-incubation with Taxol, PP NP, and F3-PP NP in the presence/absence of irradiation at the laser intensity of 1.0 J/cm2 (20 mW/cm2 for 50 s) and incubation for another 24 h. Dojindo (Kumamoto, Japan) and Alexa Fluor 647 antimouse CD31 Antibody from Biolegend (San Diego, CA). Hoechst 33258 and Coumarin-6 were obtained from Sigma−Aldrich (St. Louis, MO). Lyso Tracker Green and 4,6-diamidino-2-phenylindole (DAPI) were obtained from Molecular Probes (Eugene, OR). F3 peptide was purchased from Shanghai Mocell Biotech Co., Ltd. (Shanghai, China). Reactive Oxygen Species Assay Kit was procured from Beyotime Biotechnology Co., Ltd. (Nantong, China). Pyropheophorbide-a (PPa) was purchased from Shanghai Xianhui Pharmaceutical Co., Ltd. (Shanghai, China). 4-Dimethylaminopyridine (DMAP) and 1,3-dicyclohexyl carbodiimide (DCC) were obtained from Aladdin Co., Ltd. (Shanghai, China). All other solvents were purchased from Sinopharm Chemical Reagent (Shanghai, China) and of analytical or chromatographic grade. The HCT-15 cell line and primary human umbilical vein endothelial cells (HUVEC) were obtained from the American Type Culture Collection (Manassas, VA) and Cell Institute of Chinese Academy of Sciences (Shanghai, China), respectively. Both cells were cultured under the standard condition as reported previously.28,37 4.2. Animal and Tumor Model. Specific pathogen-free male BALB/c nude mice (20 ± 2 g) were purchased from the BK Lab Animal Ltd. (Shanghai, China). The mice was raised under the standard conditions, and all animal experiments were preformed according to the principle as reported previously.25,28 The colorectal tumor model was established as previously reported.37 Briefly, trypsinized HCT-15 cells (2 × 106) were subcutaneously injected into the selected flanks of nude mice which were

therapy in this study was superior to PDT only or chemotherapy only, and such results were further demonstrated by the pharmacodynamic evaluation in vivo. In vivo targeting assay and tumor distribution experiment showed that F3-PP NP selectively accumulated at the tumor site and distributed not only around tumor angiogenesis but also into the interior of tumor parenchyma more efficiently when compared with the unmodified ones. In addition, the safety evaluation in vitro or in vivo showed that the paclitaxel unloaded-PPa NP and F3-PPa NP exhibited negligible cytotoxicity and toxicity to normal tissues in the absence of irradiation, indicating that the nanosystem prepared in this study was safe under the current dosage. In conclusion, the F3-PP NP-based combination therapy here holds great potential for the therapy of drugresistant tumor.

4. MATERIALS AND METHODS 4.1. Materials and Cells. Hydroxyl-poly(lactic acid)19000-poly(ethylene glycol)3000-poly(lactic racid)19000-hydroxyl (HO-PLA-PEGPLA-OH) and maleimide-poly(ethylene glycol) 3400 -poly(lactic acid)34000 (Male-PEG-PLA) were synthesized as previously described.36 Paclitaxel was obtained from Shanghai Jinhe BioTechnology Co., Ltd. (Shanghai, China), and clinical formulation Taxol was supplied by Bristol-Myers Squibb Pharmaceuticals (New York). Cell counting kit-8 (CCK-8) was purchased from 17826


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Figure 10. (A) Biodistribution of (a) PP NP and (b) F3-PP NP in vivo at 1, 6, 8, 12, and 24 h post-injection. (B) Ex vivo imaging of tissues and tumors from the above mice. (C) Semiquantitative analysis of major organs and tumors. ***p < 0.001 as compared with that of mice treated with PP NP.

Figure 11. Intratumor distribution of nanoparticles at 3 h after iv injection. Green represents the blood vessels stained with CD31. Blue signal represents the cell nuclei stained with DAPI. Red shows fluorescence of PPa as the indicator of the nanoparticles. raised in standard conditions. While tumors reached ∼100 mm3 in volume, mice were applied to pharmacological experiments because of the Gompertzian kinetics of solid tumor.38 4.3. Synthesis of PPa-PLA-PEG-PLA-PPa. PPa-PLA-PEGPLA-PPa was synthesized through the esterification reaction between PPa and HO-PLA-PEG-PLA-OH under the mediation of DCC and DMAP (Scheme 1). First, PPa (2.4 mg) was dissolved in anhydrous DMSO. Thereafter, 1.0 mol equiv of DMAP and DCC were added. After activation of the carboxyl of PPa at 25 °C for 1 h, HO-PLA-PEGPLA-OH (100 mg) was added into the mixtures and subjected to gentle stirring for 48 h under the atmosphere of nitrogen. The unconjugated PPa and DMSO were removed via equilibrium dialysis (MW cutoff: 3000 Da, dialyzed for 3 days). Finally, the solution was

subjected to flash-frozen dry conditions and lyophilized. In addition, the formed DCU (N,N′-dicyclohexylurea) was removed via the acetone precipitation method. Briefly, the synthesized products were dissolved by acetone and preserved in 4 °C for precipitation of DCU. Such a precipitation procedure was performed more than 4 times until no more precipitate formed. The purified copolymer was then obtained by evaporating the organic solvent via a ZXB98 rotary evaporator (Shanghai Institute of Organic Chemistry, China). 4.4. Preparation of PP NP and F3-PP NP. Nanoparticles were prepared as described elsewhere.39 Briefly, the blend of 45 mg of PPa-PLA-PEG-PLA-PPa, 5 mg of Mal-PEG-PLA, and 1.25 mg of PTX dissolved with the help of 1 mL dichloromethane. Thereafter, 2 mL of 1% sodium cholate solution was added, and then a probe sonicator 17827


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Figure 12. Antitumor effect of Taxol, PPa NP, PP NP, and F3-PP NP, respectively, with or without irradiation for 9.8 min with a 660 nm laser 170 mW/cm2. The mice given with saline were applied as the negative control. (A) Changes in tumor volume of mice during the 14-day experimental period. (B) Kaplan−Meier survival curve of mice. (C) Weight of dissected tumors. (D) Tumor-growth inhibition rate of the various treatment strategies.

Figure 13. Tumor morphology and H&E staining of tumor tissue sections (14 days post the last injection) in those tumor-bearing mice treated with Taxol, PPa NP, PP N,P and F3-PP NP, respectively, with or without irradiation for 9.8 min with a 660 nm laser 170 mW/cm2. The saline-treated mice were utilized as the negative control. 17828


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Figure 14. Histological analysis of main organs obtained from normal nude mice after given PP NP (−laser) and F3-PP NP (−laser), respectively. The saline-treated mice were utilized as the negative control. Drug release profiles were studied in vitro by the method of dialysis as reported previously.29 Briefly, 1 mL of nanoparticle formulations containing 0.1 mg PTX (diluted by different medium, respectively) was added into a dialysis bag. Then, the bags were immersed in 30 mL of release medium, and samples shook at the speed of 120 rpm under 37 °C away from light. The release sample was withdrawn, and the concentrations of PTX and free PPa were measured by HPLC.42 4.6. Cellular Uptake Experiment. HUVEC cells and HCT-15 cells were applied to study the cellular selectivity of F3-PP NP in blood endothelial cells and tumor cells, respectively. The cells were seeded in a confocal dish at a density of 1 × 104/cm2 and incubated for 20 4 h. Then, 1 mL of fresh culture medium containing different concentration of PP NP and F3-PP NP (ranging from 50 to 400 μg/mL) was added. One hour later, the cells were washed twice with cold PBS and fixed with 4% paraformaldehyde. Thereafter, cells were stained with Hoechst 33258 for 15 min to visualize the nucleus. Finally, the qualitative results were analyzed via confocal microscopy analysis (LSM710, Leica, Germany). For quantitative analysis, both cells were seeded in 96-well plates at a density of 5 × 103 per well. The cells were exposed 24 h later to PP NP and F3-PP NP at a nanoparticle concentration of 200 μg/mL. The cells were washed twice 1 h later with cold PBS. Before analysis via a KineticScan HCS Reader (Thermo Scientific), the cells were fixed with 4% paraformaldehyde for 15 min and stained with Hoechst 33258 for nuclei visualization. In addition, to verify the F3 peptide-mediated cellular association of nanoparticles, the cells were pretreated with 200 μg/mL F3 peptide before the exposure to F3-PP NP. Then the qualitative and quantitative experiments were performed as mentioned above. 4.7. Evaluation of Reactive Oxygen Species Production. The reactive oxygen species assay kit was introduced to evaluate the production of ROS.5 Briefly, HUVEC cells and HCT-15 cells were seeded in 6-well culture dishes. Both cells were exposed 24 h later to PP NP or F3-PP NP (concentration of nanoparticles was 200 μg/mL). After a 3 h incubation, 10 μmol/mL DCFH-DA, which could react with ROS to produce DCF with green fluorescence, was introduced and cultured with cells for 20 min. Thereafter, the cells were washed twice with PBS and irradiated with laser power of 0.005, 0.05, and 0.5 J/cm2, respectively, through a 660 nm fiber coupled laser system (ADR1860, Feimiao Scientific Co., Changchun, China). The intracellular distribution of DCF fluorescence was finally observed under a fluorescence microscope (Leica DMI4000 B, Germany) at the excitation wavelength 488 nm and emission wavelength 525 nm. Besides, the generation of singlet oxygen species was also detected for free PPa and compared with that of PP NP.

(Ningbo Scientz Biotechnology Co. Ltd., China) was applied to form nanoparticles. After ultrasonication (320 W, 2.8 min) under an ice bath, the resulting solution was diluted with 8 mL of 0.5% sodium cholate aqueous solution. After 5 min of rapid stirring, the solution of PTX-loaded PPa NP was evaporated to remove the dichloromethane, and the nanoparticles were collected through centrifugation (14 500 rpm, 1 h) under the help of a TJ-25 centrifuge (Beckman Counter). The F3 peptide was decorated on the surface of nanoparticles via the method reported previously.39 The unconjugated peptide was removed by centrifugation. 4.5. Characterization of PP NP and F3-PP NP. The synthesis of PPa-PLA-PEG-PLA-PPa was verified through a 1H NMR measurement with the help of a Varian NMR spectrometer (Varian) operating at 600 MHz. Furthermore, the normalized UV−vis absorption spectra analysis was applied to studying the changes of optical activity of PPa after the conjugation. In addition, the concentration of PPa and PPa-PLA-PEG-PLA-PPa were measured via a validated HPLC method. HPLC separation was performed on a Welchrom C18 column (200 mm × 4.6 mm, 5 μm, Welch Materials, Shanghai, China) maintained at room temperature. An isocratic mobile phase containing 0.2% formic acid in methanol was delivered at the flow rate of 1.2 mL min−1. Ultraviolet detection was set at 409 nm, and the injection volume was 20 μL. The calibration curve was linear from 0.2 to 100 μg mL−1. For the characterization of nanoparticles, we applied a dynamic light scattering detector (Zetasizer, Nano-ZS, Malvern, U.K.) to determine the particle size and zeta potential of nanoparticles. After negatively staining with sodium phosphotungstic solution, the morphologies of PP NP and F3-PP NP were determined via a transmission electron microscope (TEM) (H-600, Hitachi, Japan). X-ray photoelectron spectroscopy (XPS) was applied for the verification of peptide decoration of PP NP as described previously.40 In addition, the stabilities of PP NP and F3-PP NP were further studied in the media of DMEM containing 10% FBS and PBS (pH 7.4) after incubation for 7 days. The size of nanoparticles was examined once a day during the inspection. The amount of PTX in nanoparticles was detected via HPLC after being dissolved in acetonitrile.41 The EE (%) and LC (%) of nanoparticles were calculated as follows (n = 3). EE (%) =

amount of PTX in the nanoparticles × 100% total amount of PTX added

LC (%) =

amount of PTX in nanoparticles × 100% nanoparticle weight 17829


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4.12. Evaluation of Targeted Combination Therapy of DrugResistant Tumor in Vivo. To investigate the antitumor effect of the combination therapy in colorectal carcinoma-bearing mice, 36 tumorbearing mice were divided into six groups (n = 6) randomly and treated with Taxol, PPa NP (+laser), PP NP (−laser), PP NP (+laser), and F3-PP NP (+laser), respectively, with the dosages of PPa and PTX being 2.5 and 3.8 mg/kg, respectively. The mice treated with saline were applied as the negative control. The laser intensity used here was 100 J/cm2 (170 mW/cm2 for 9.8 min). At 0, 2, 4, 6 days postadministration, the tumor volume of each mouse was carefully measured and calculated (formula: volume = length × width2/2). After 14 days, all of the mice were sacrificed, and the obtained tumors of each mouse were weighed. In addition, the tumor inhibition rate (TIR %) of each group was calculated via the formula as shown below:

4.8. Colocalization Assay and Photooxidation of Endocytic Membranes. A colocalization assay was performed to investigate the intracellular location of PP NP and F3-PP NP. HUVEC cells and HCT-15 cells were seeded in a confocal dish at the density of 1 × 104/cm2. After being cultured for 24 h, the cells were exposed to 200 μg/mL PP NP and F3-PP NP for 3 h and then 50 nmol/L Lyso Tracker Green to indicate the endolysosomal compartments. After being stained for 30 min, the cells were fixed with 4% paraformaldehyde, and the cell nucleus was stained by Hoechst 33258. Images were observed under a confocal microscope (LSM710, Leica, Germany). For the evaluation of PDT-mediated endosome escape, HUVEC cells and HCT-15 cells were seeded in confocal dish at the density of 1 × 104/cm2. After 24 h of incubation, the cells were treated with 200 μg/mL F3-PP NP for 4 h followed by irradiation at the laser intensity of 0.05, 0.5, and 1.0 J/cm2, respectively. Thereafter, 50 nmol/L LysoTracker Green was introduced followed by processing as above, and the results were analyzed via a confocal microscope (LSM710, Leica, Germany). 4.9. In Vitro Cytotoxicity Evaluation. HUVEC cells and HCT-15 cells were seeded in 96-well plates at the density of 5 × 103 cells per well. Serum-free medium containing NP-PTX, PPa NP, PP NP, and F3-PP NP (concentrations of PTX and PPa were 100 and 200 ng/mL, respectively) were added 24 h later. The cells were irradiated 24 h later with laser intensity ranging from 0.005 to 3.0 J/cm2, and incubated for another 24 h with those cells in the dark used as the control. After that, 10 μL of CCK-8 was introduced, and the results were analyzed via a microplate reader (Thermo Multiskan MK3) at the wavelength of 450 nm. To study the safety of nanocarriers, the cytotoxicity of various concentrations of PTX-free PPa NP and F3-PPa NP was evaluated as well with the same method in the absence of irradiation. To evaluate the antiproliferation effect of nanoparticles on endothelial cells of angiogenesis and drug-resistant tumor cells, HUVEC cells and HCT-15 cells were seeded in 96-well plates (5 × 103 cells per well) and incubated under standard condition for 24 h. Then, the original medium in plates was replaced with free serum medium containing Taxol, PP NP, and F3-PP NP (PTX concentration ranged from 1 to 1000 ng/mL), respectively. After 4 h, the cells were irradiated at laser intensity of 1.0 J/cm2 (20 mW/cm2 for 50 s) and incubated for another 24 h. Then, the cells were exposed to 10 μL of CCK-8 solutions for 1 h with the cellular viability of HUVEC cells and HCT-15 cells analyzed via a microplate reader (Thermo Multiskan MK3), and the IC50 of various PTX formulations was calculated via the Graphd Prism 5.0 software. To further evaluate the effect of combination therapy with PDT and chemotherapy, the obtained IC50 values were applied for calculating the combination index (CI) via the third-generation software “CompuSyn” based on Chou−Talalay theory.43 4.10. Biodistribution of Nanoparticles. To evaluate the efficacy of F3 peptide-mediated tumor-targeting in vivo, six tumor-bearing mice were randomly divided into two groups (n = 3). Then the mice were intravenously given PP NP and F3-PP NP, respectively, via the caudal vein at the PPa dosage of 2.5 mg/kg. After that, the fluorescent signal of PPa was detected at 2, 6, 12, and 24 h, respectively, by using an In Vivo IVIS spectrum imaging system (PerkinElmer). Finally, the mice were sacrificed, and the major organs (heart, liver, spleen, lung and kidney) were harvested for ex vivo imaging followed by semiquantitative analysis. 4.11. Intratumor Distribution of Nanoparticles. Tumorbearing mice were intravenously injected with PP NP and F3-PP NP, respectively, at the PPa dosage of 2.5 mg/kg. Then the heart of each mouse was perfused with saline and 4% paraformaldehyde solution post 3 h, and the tumors were harvested. Fixed the obtained tumors with 4% paraformaldehyde and dehydrated them in 15%, 30% sucrose solution. Finally the tumors were frozen at −80 °C and then sectioned at 10 μm for further analysis. Immunostaining assay was performed to study the location of nanoparticles at the tumor sites as reported previously.40 The observation was performed under a confocal microscope ((LSM710, Leica, Germany).

TIR % =

Wcontrol − Wtreated × 100% Wcontrol

Wcontrol represents the average tumor weight of control group, and Wtreated represents the average tumor weight of treatment group. Another set of experiments was also implemented to study the antitumor effect of F3-PP NP, in which 36 mice were processed as above, and survival of the tumor-bearing mice was monitored and finally analyzed via a Kaplan−Meier nonparametric analysis. 4.13. Apoptosis of Cells in Drug-Resistant Tumor. The 18 tumor-bearing mice were divided into six groups (n = 3) randomly and treated with saline, Taxol, PPa NP (+laser), PP NP (−laser), PP NP (+laser), and F3-PP NP (+laser), respectively, every 3 days for 2 weeks. Importantly, the laser intensity here was 100 J/cm2 (170 mW/cm2 for 9.8 min). After that, all the mice were sacrificed with tumors harvested and fixed in 4% paraformaldehyde. Then the tumors were embedded in paraffin followed by sectioning 5 μm slides for hematoxylin and eosin (H&E) staining. 4.14. Safety Evaluation of Nanoparticles in Vivo. To study the toxicity of the PTX-loaded nanoparticles prepared in this study to normal tissues, 18 normal nude mice were divided into six group randomly and treated with PP NP and F3-PP NP (the dosage of PPa and PTX was 2.5 and 3.8 mg/kg), respectively, at 0, 2, 4, and 6 days. The mice treated with saline were utilized as the control. After that, the mice were sacrificed, and the brains and major organs were collected for for H&E staining. 4.15. Statistical Analysis. Multiple-group comparison was performed via one-way ANOVA analysis followed by Bonferroni tests. Statistical significance was defined as p < 0.05.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b04442. Cellular uptake of PPa and PP NP and generated efficacy of singlet oxygen species between the free photosensitizer and the PP NP (PDF)

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AUTHOR INFORMATION

Corresponding Authors

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

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the National Natural Science Foundation of China (81373353), Grants from Shanghai Science and Technology Committee (13NM1400500, 15540723700), Shanghai Talent Development Fund (201459), and the “Shu Guang” project supported by Shanghai Municipal Education Commission and Shanghai Education Development Foundation. 17830


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