Tumor-Homing and Penetrating Peptide-Functionalized

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Tumor-Homing and Penetrating Peptide-Functionalized Photosensitizer-Conjugated PEG-PLA Nanoparticles for Chemophotodynamic 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 ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b04442 • Publication Date (Web): 22 Jun 2016 Downloaded from http://pubs.acs.org on June 23, 2016

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ACS Applied Materials & Interfaces

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, PR China ‡

Department of Pharmacology, Institute of Medical Sciences, Shanghai Jiaotong

University School of Medicine, 280 South Chongqing Road, Shanghai, 200025, PR China Corresponding Authors' E-mail addresses: [email protected] (J. Chen), [email protected] (XL. Gao)

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 co-loading 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 co-delivery 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 tumor-homing 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 anti-proliferation effect. In addition, compared with the unmodified nanoparticles, F3-PP NP exhibited a more preferential enrichment at the tumor site. Pharmacodynamics evaluation in vivo 1

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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 anti-tumor 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 dominate factor underlying MDR.3,

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Unfortunately, currently used anti-tumor

chemotherapeutics including paclitaxel, doxorubicin, and vincristine are all the substrates of P-gp.5,

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As reported previously, the tumor penetration of PTX was

seriously limited by the efflux transporter, leading to unsatisfactory therapy effect.7 To combat drug resistance, one strategy is to co-deliver 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. 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 synergistic effect, one of the widely-used methods is to deliver the photosensitizer and chemotherapeutics using a single nanosystem. Nonetheless, such 2


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treatment strategy is impeded by the obstacles, such as instability, drug leakage and low entrapment efficiency that is mainly attributed to the steric hindrance between the photosensitizer and chemotherapeutics.16, 17 As reported previously, co-encapsulation of chemotherapeutics (Docetaxel) and photosensitizers (zinc-phthalocyanine) leaded to a sharp decrease in both the entrapment efficiency and loading capacity.17 Therefore, it is necessary to establish an advanced nanosystem with a stable and efficient loading of both photosensitizer and chemotherapeutics. Chlorophyll-a-based photosensitizers are attracting increased attentions 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 limit 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 firstly 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 co-loaded 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 sequences, which possesses the ability of tumor penetration.23, 24 Taken together, F3 peptide is 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 3



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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. Bio-distribution of nanoparticles was further evaluated in vivo to study the efficiency of tumor-targeting drug delivery. Finally, pharmacodynamics evaluation was performed both on cells and in tumor-bering mice in presence/absence of laser to study the therapeutic effect of the combination treatment strategy presented in this study. 2. Results and Discussion To establish a nanocarrier, which could effectively co-load 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 on the surface of PP NP with a nucleolin targeting 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, 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 anti-tumor 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 showed in scheme 1. The results of 1H NMR analysis were showed in Fig 1A, B and C, in which the signals at 12.15 ppm (peak a) represented the peak of -COOH in PPa (Fig 1A). In the case of HO-PLA-PEG-PLA-OH (Fig 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 4


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PLA segment, respectively, and that at 3.5—3.6 ppm (peak c) represented the peak of -CH2 protons in the PEG block of HO-PLA-PEG-PLA-OH. In the case of PPa-PLA-PEG-PLA-PPa (Fig 1C), except the characteristic signals of CH, CH2 and CH3 of PLA and PEG segment, the signals of PPa which showed at 8.5—10.0 ppm (peak d) was also detectable in the conjugates while that of -COOH was undetectable. These results together demonstrated that the copolymer PPa-PLA-PEG-PLA-PPa was successfully synthesized. Such results were further confirmed by HPLC analysis (Fig 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 theoretical value of 28.5 µg. The analysis of normalized UV/Vis absorption spectra as shown in Fig 1E indicated that the optical activity of PPa did not change after the conjugation with HO-PLA-PEG-PLA-OH.

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

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Figure 1.

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H-NMR analysis of PPa (A) ， HO-PLA-PEG-PLA-OH (B) and

PPa-PLA-PEG-PLA-PPa (C). HPLC graphs of PPa and PPa-PLA-PEG-PLA-PPa (D). Normalized UV/Vis absorption spectra of PPa and PPa-PLA-PEG-PLA-PPa (E). 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 vehicle 6


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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 electro positivity of F3 peptide due to several cationic amino acids contained in the peptide. In addition, the TEM images showed in Fig 2A and 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 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. Table 1. Characterization of the prepared nanoparticles (Data represent mean ± SD, n = 3). Nanoparticles PP NP F3-PP NP

Particle size (mean ± SD, nm) 109.81 ± 3.55 118.25 ± 4.43

Polydispersity index (P.I.) 0.102 ± 0.063 0.124 ± 0.078

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Zeta potential (mV) –31.75 ± 2.76 –17.59 ± 4.73


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Figure 2. Particle size and TEM images of PP NP (A) and F3-PP NP (B). The bar is 200 nm. 2.3 In vitro release and stability of nanoparticles As shown in Fig 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-contained 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 profile 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 Fig 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-contained 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. A good stability of drug delivery system is always closely associated with its long 8


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circulation that is critical for its high accumulation at the tumorsite.29 Fig 3C and D showed that both PP NP and F3-PP NP did not exhibit obvious aggregation phenomenon during the determined days, indicating that the nanoparticles prepared in this study exhibited a satisfactory stability.

Figure 3. Profile of in vitro release of PTX (A) and PPa (B) from PP NP and F3-PP NP. Stability of PP NP and F3-PP NP in DMEM with 10% FBS and PBS, respectively. The size of nanoparticles (C) was measured and the appearance of F3-PP NP solutions (D) was photographed each day during the inspection. The green and red tube represents the F3-PP NP sample dissolved in PBS and DMEM containing 10% FBS, respectively. 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 Fig 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 pretreated with excess F3 peptide, cellular association of F3-PP NP was 9



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seriously inhibited. Quantitative results in Fig 4B and C further demonstrated a peptide-mediated manner of cellular uptake of F3-PP NP, which was 1.21 folds higher in HUVEC cells and 1.18 folds higher in HCT-15 cells, respectively, when compared with that of PP NP.

Figure 4. Qualitative analysis of cellular uptake of PP NP/F3-PP NP in HUVEC cells and HCT-15 cells in the presence/absence of F3 peptide (A) analyzed by fluorescent microscopy. Quantitative analysis of cellular uptake of PP NP/F3-PP NP in HUVEC cells (B) and HCT-15 cells (C) analyzed via the Kinetic Scan HCS Reader. Red: 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. 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 an evident green fluorescence (Fig 5). In addition, the fluorescent signals in the irradiated cells 10


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were enhanced after increasing the laser power. For the compare between PPa and PP NP, results showed in Fig S1 illustrated that after incubated cells with PPa (4.8 µg/mL) or PP NP (containing 4.8 µg/mL PPa) and irradiated 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 that cellular internalization of PPa was significantly increased after conjugated to HO-PLA-PEG-PLA-OH due to a hydrophobic properties of photosensitizer.

Fig 5. Evaluation of reactive oxygen species production in HCT-15 cells (A) and HUVEC cells (B) 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. 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 11



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nanoparticles and lysosomes. As shown in Fig 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.

Fig 6. Intracellular localization of nanoparticles after incubating HUVEC cells and HCT-15 cells with 200 µg/mL PP NP (A) and F3-PP NP (B) for 3 h. Blue: nuclei stained with DAPI. Green: endosomes labeled by Lysotracker Green. Red: particles tracked by fluorescence of PPa. 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 12


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F3-PP NP under different irradiation time. As shown in Fig 7 and 8, both cells incubated with F3-PP NP without irradiation exhibited the most distinct red fluorescence. In contrast, in the presence of laser, the fluorescence signal of lysosomes was gradually decreased once increasing the laser power and almost disappeared after irradiated at 1.0 J/cm2 for 90 s.

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

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Fig 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: nuclei stained with DAPI. Green: endosomes labeled by Lysotracker Green. Red: particles tracked by fluorescence of PPa. 2.8 In vitro cytotoxicity of nanoparticles To clearly illustrate the anti-proliferation effect of various therapy strategies, PTX-free nanoparticles PPa NP and F3-PPa NP and PPa-free nanoparticles NP-PTX were prepared with the methods as PP NP was developed. As shown in Fig 9A and B, PPa NP in the dark exhibited undetectable cytotoxicity against both of HUVEC cells and HCT-15 cells, indicating that PPa exhibited non-cytotoxicity without irradiation. In contrast, along with the increasing intensity of the irradiated laser, the cytotoxicity of each group except NP-PTX group increased, suggesting that PDT was a laser 14


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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 evaluating 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 Fig 9C and 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 non-toxic in the dark and therefore could be used as a safe drug carrier. In the case of PP NP and F3-PP NP, as shown in Fig 9E and F, in the presence of irradiation, PP NP showed an 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 viability of HUVEC cells and HCT-15 cells were significantly decreased, which could be mainly ascribed to the elevated cellular association of nanoparticles that 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 ng/mL, 84.09 ng/mL, 41.21 ng/mL 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 ng/mL, 86.32 ng/mL 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 was exactly PTX resistance. 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 0.202 for HUVEC cells and HCT-15 cells, respectively, indicating a strong synergistic effect of the combination therapy. 15



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Fig 9. Evaluation of the cytotoxicity of nanoparticles in HUVEC cells (A) and HCT-15 cells (B) post incubated with Taxol, PP NP and F3-PP NP followed by irradiation at various laser power and incubation for another twenty-four hours. Evaluation of the cytotoxicity of nanoparticles in HUVEC cells (C) and HCT-15 cells (D) post incubated with PPa NP and F3-PPa NP without irradiated. Evaluation of the cytotoxicity of nanoparticles in HUVEC cells (E) and HCT-15 cells (F) post incubated 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 twenty-four hours. 2.9 Bio-distribution of nanoparticles The tumor cell and tumor vasculature dualtargeting was generally recognized as one of the most promising ways for cancer targeting treatment.33, 34 In this study, we decorated on the surface of PP NP with an F3 peptide for dual targeting drug delivery and evaluated the tumor targeting effect in vivo. As results shown in Fig 10A, consistent with the ex vivo images (Fig 10B), the tumor-bearing mice treated with F3-PP NP displayed a higher tumor accumulation when compared with the 16


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unmodified ones. In addition, semi-quantitative analysis (Fig 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.

Fig 10. (A) Bio-distribution of PP NP (a) and F3-PP NP (b) 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) Semi-quantitative analysis of major organs and tumors. ***p < 0.001 as compared with that of mice treated with PP NP. 2.10 Intra-tumor distribution of nanoparticles It was showed that a small quantity of unmodified PP NP was observed at tumor site and only accumulated around the blood vessels (Fig 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 dual-targeting delivery and deep penetration into the tumor inner mediated by F3 peptide. Such hypothesis was clearly demonstrated in Fig 11 as much stronger fluorescence intensity of PPa was observed far from tumor blood vessels following the treatment with F3-PP NP compared with that treated with PP NP. 17



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Fig 11. Intra-tumor distribution of nanoparticles at three hours after i.v injection. Green represents the blood vessels stained with CD31. Blue signal represents the cell nuclei stained with DAPI. Red: fluorescence of PPa as the indicator of the nanoparticles. 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 Fig 12 indicated that the mice treated with F3-PP NP (+laser) displayed the best anti-tumor 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 compared to any monotherapy. Such results were further demonstrated by the measurement of survival time after treated with various formulations (Fig 11). It was shown that the mice treated with F3-PP NP (+laser) achieved the best survival situation (above the inspection days). 18


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Under the same experimental conditions, the mice given with saline, Taxol®, PP NP (-laser), PPa NP (+laser), PP NP (+laser) achieved the survival time of 22, 24, 30, 41 and 53 days, respectively. The evaluation of inhibition rate of tumor (Fig 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), PP NP (+laser) were 5.71%, 18.97%, 38.08% and 54.68%, respectively. 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, 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 unconspicuous differentiation compared to the control animals treated with saline (Fig 13), indicating that the colorectal cancer model established in this study was exactly PTX resistance.

Fig 12. Anti-tumor 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. 19



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(C) Weight of dissected tumors. (D) Tumor-growth inhibition rate of the various treatment strategies.

Fig 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 NP 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. 2.13 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 Fig 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 it further suggested that the combination 20


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system of PDT and chemotherapy established in this study was safe under the current dosage.

Figure 14. Histological analysis of main organs obtained from normal nude mice after given with PP NP (-laser) and F3-PP NP (-laser), respectively. The saline-treated mice were utilized as the negative control. 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 anti-proliferation 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 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 21



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under the current dosage. In conclusion, the F3-PP NP-based combination therapy here holds great potential for the therapy of drug-resistant tumor. 4. Materials and methods 4.1 Materials and cells Hydroxyl-poly (lactic acid) 19000 -

19000

- poly (ethylene glycol)

3000

- poly (lactic racid)

Hydroxyl (HO-PLA-PEG-PLA-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 Bio-Technology Co., Ltd. (Shanghai, China) and Clinical formulation Taxol® was supplied by Bristol-Myers Squibb Pharmaceuticals (NY, USA). Cell counting kit-8 (CCK-8) was purchased from Dojindo (Kumamoto, Japan) and Alexa Fluor® 647 anti-mouse CD31 Antibody from Biolegend (San Diego, CA, USA). Hoechst 33258 and Coumarin-6 were obtained from

Sigma–Aldrich

(St.

Louis,

MO,

USA).

Lyso

Tracker

Green

and

4,6-diamidino-2-phenylindole (DAPI) were obtained from Molecular Probes (Eugene, OR, USA). 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 the 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) was 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 condition and all animal experiments were preformed according to the principle as 22


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reported previously.25, 28 Colorectal tumor model was established as previously reported.37 Briefly, subcutaneously injected the trypsinized HCT-15 cells (2 × 106) into the selected flanks of nude mice and raised them in standard condition. While tumors reached ~100 mm3 in volume, mice were applied to pharmacological experiment because of the Gompertzian kinetics of solid tumor.38 4.3 Synthesis of PPa-PLA-PEG-PLA-PPa PPa-PLA-PEG-PLA-PPa was synthesized through the esterification reaction between PPa and HO-PLA-PEG-PLA-OH under the mediation of DCC and DMAP (Scheme 1). Firstly, dissolved PPa (2.4 mg) in anhydrous DMSO. Thereafter, added 1.0 molar equivalent of DMAP and DCC. After activated the carboxyl of PPa at 25 ºC for 1 h, HO-PLA-PEG-PLA-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 cut-off: 3000 Da, dialyzed for three days). Finally, the solution was subjected to flash-frozen dry 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 4ºC for precipitation of DCU. Such precipitation procedure was performed for more than 4 times until no more precipitate formed. The purified copolymer was then obtained by evaporating the organic solvent via a ZXB98 rotavapor (Shanghai Institute of Organic Chemistry, China). 4.4 Preparation of PP NP and F3-PP NP Nanoparticles were prepared as described elsewhere.39 Briefly, dissolved the blend of 45 mg PPa-PLA-PEG-PLA-PPa, 5 mg Mal-PEG-PLA and 1.25 mg PTX with the help of 1 mL dichloromethane. Thereafter, added 2 mL of 1% sodium cholate solution, and then a probe sonicator (Ningbo Scientz Biotechnology Co. Ltd., China) was applied to form nanoparticles. After ultrasonication (320 W, 2.8 min) under ice bath, the resulting solution was diluted by 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 collected the nanoparticles through centrifugation 23



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(14,500 rpm, 1 h) under the help of a TJ-25 centrifuge (Beckman Counter, USA). The F3 peptide was decorated on the surface of nanoparticles via the method as 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 under the help of a Varian NMR spectrometer (Varian, USA) 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 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, UK) to determine the particle size and zeta potential of nanoparticles. After negatively staining with sodium phosphotungstic solution, the morphology 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 descripted previously. 40 In addition, the stability 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 seven days. The size of nanoparticles was examined once a day during the inspection. The amount of PTX in nanoparticles was detected via HPLC after dissolved in acetonitrile.41 The EE (%) and LC (%) of nanoparticles were calculated as follows (n=3). EE(%) =

Amountof PTX in thenanoparticles ×100% Totalamountof PTX added 24


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LC(%) =

Amount of PTX in nanoparticles ×100% nanoparticles weight

Drug release profiles were studied in vitro by the method of dialysis as reported previously.29 Briefly, one milliliter of nanoparticle formulations containing 0.1 mg PTX (diluted by different medium, respectively) was added into a dialysis bag. Then immersed the bags in 30 mL of release medium and samples were shook at the speed of 120 rpm under 37°C away from light. The release sample was withdrawn and the concentration 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 cell and tumor cells, respectively. The cells were seeded in confocal dish at the density of 1×104/cm2 and incubated for twenty four hours. Then 1 mL of fresh culture medium containing different concentration of PP NP and F3-PP NP (ranged from 50 µg/mL to 400 µg/mL) was added. One hour later, washed the cells twice with cold PBS and fixed them 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 the density of 5 × 103 per well. Twenty-four hours later, exposed the cells to PP NP and F3-PP NP at the nanoparticle concentration of 200 µg/mL. One hour later, the cells were washed twice with cold PBS. Before analyzed via a KineticScan HCS Reader (Thermo scientific, USA), 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 ROS5. Briefly, HUVEC cells and HCT-15 cells were seeded in 6-well culture dishes. 25



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Twenty-four hours later, both cells were exposed to PP NP or F3-PP NP (concentration of nanoparticles was 200 µg/mL). After a three-hour 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 a laser of 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.

4.8 Colocalization assay and photooxidation of endocytic membranes 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 confocal dish at the density of 1×104/cm2. After cultured for 24 h, the cells were exposed to 200 µg/mL PP NP and F3-PP NP for three hours and then 50 nmol/L Lyso Tracker Green to indicate the endolysosomal compartments. After staining 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 endosomes escape, HUVEC cells and HCT-15 cells were seeded in confocal dish at the density of 1 × 104/cm2. After 24-h 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 J/cm2, 0.5 J/cm2, and 1.0 J/cm2, respectively. Thereafter, 50 nmol/L LysoTracker Green was introduced followed by processed as above and 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. Twenty-four hours later, serum free medium containing NP-PTX, PPa NP, PP NP and F3-PP NP (concentration of PTX and PPa were 100 ng/mL and 200 ng/mL, respectively) were added. Twenty-four hours later, the cells were 26


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irradiated at the laser intensity ranged from 0.005 J/cm2 to 3.0 J/cm2, and incubated for another twenty-four hours with those cells in dark were used as the control. After that, 10 µL CCK-8 was introduced and the results were analyzed via a microplate reader (Thermo Multiskan MK3, USA) 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 anti-proliferation 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 ng/mL to 1000 ng/mL), respectively. Four hours later, the cells were irradiated at laser intensity of 1.0 J/cm2 (20 mW/cm2 for 50 s) and incubated for another twenty-four hours. Then the cells were exposed to 10 µL 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, USA) and the IC50 of various PTX formulations 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 Bio-distribution 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 given intravenously with 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 (Perkin Elmer, USA). Finally, the mice were sacrificed and the major organs (heart, liver, spleen, lung and kidney) were harvested for ex vivo imaging followed by semi-quantitative analysis. 27



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4.11 Intra-tumor distribution of nanoparticles Tumor-bearing mice were intravenously injected with PP NP and F3-PP NP, respectively, at the PPa dosage of 2.5 mg/kg. Then perfused the heart of each mouse with saline and 4% paraformaldehyde solution post 3 h and harvested the tumors. Fixed the obtained tumors with 4% paraformaldehyde and dehydrated them in 15%, 30% sucrose solution. Finally frozen the tumors at −80ºC and sectioned then 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).

4.12 Evaluation of targeted combination therapy of drug resistant tumor in vivo To investigate the anti-tumor effect of the combination therapy in colorectal carcinoma-bearing mice, thirty-six tumor bearing 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, at the dosage of PPa and PTX 2.5 mg/kg 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 post-administration, the tumor volume of each mouse was carefully measured and calculated (formula: Volume = length × width2/2). Fourteen days later, sacrificed all of the mice and weighted the obtained tumors of each mouse. Besides, calculated the tumor inhibition rate (TIR%) of each group via the formula as showed below:

Wcontrol − Wtreated × 100% Wcontrol Wcontrol represents the average tumor weight of control group, Wtreated TIR% =

represents the average tumor weight of treatment group. Another set of experiment was also implemented to study the anti-tumor effect of F3-PP NP, in which thirty-six 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 Eighteen tumor-bearing mice were divided into six groups (n=3) randomly and 28


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treated with saline, Taxol®, PPa NP (+laser), PP NP (-laser), PP NP (+laser) and F3-PP NP (+laser), respectively, every three days in two 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, eighteen 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 mg/kg 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.

Acknowledgment 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 “Shu Guang” project supported by Shanghai Municipal Education Commission and Shanghai Education Development Foundation.

Supporting information Cellular uptake of PPa and PP NP; Generate efficacy of singlet oxygen species between the free photosensitizer and the PP NP.

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Tumor-Homing and Penetrating Peptide-Functionalized

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