Matrix Metalloproteinase Cleavable Nanoparticles for Tumor

Research Article Cite This: ACS Appl. Mater. Interfaces 2017, 9, 40614-40627

www.acsami.org

Matrix Metalloproteinase Cleavable Nanoparticles for Tumor Microenvironment and Tumor Cell Dual-Targeting Drug Delivery Zhenliang Sun,†,‡,# Ruihong Li,§,# Ji Sun,∥,# You Peng,† Linlin Xiao,† Xingxing Zhang,† Yixin Xu,*,⊥ and Man Wang*,† †

Shanghai University of Medicine & Health Sciences Affiliated Sixth People’s Hospital South Campus, Shanghai 201499, China Department of General Surgery, Shanghai Tenth People’s Hospital Affiliated to Tongji University, No. 301, Yan-Chang Road, Shanghai 200072, China § Hangzhou Normal University Qianjiang College, HangZhou 310036, China ∥ Shanghai University of Medicine & Health Sciences, Shanghai 201318, China ⊥ School of Pharmacy, Shanghai University of Medicine & Health Sciences, Shanghai 201318, China ‡

S Supporting Information *

ABSTRACT: Matrix metalloproteinases (MMPs), mostly abundant in the tumor extracellular matrix (ECM), tumor cells, and tumor vasculatures, are closely correlated with tumor progression and metastasis. In this case, making use of MMPs was supposed to achieve site-specific drug delivery and a satisfactory tumor treatment effect. Herein, we rationally developed a novel tumor microenvironment and tumor cell dual-targeting nanoparticle by integrating a chemotherapeutic-loaded drug-loaded carrier and a versatile polypeptide-LinTT1-PVGLIG-TAT (LPT) which is composed of a multitargeting peptide-LinTT1 and a cellpenetrating peptide-TAT. The functionalized nanoparticles exhibited a superior affinity to A549 lung-cancer cells and microenvironment including angiogenesis and tumor-associated macrophages (TAMs) in our study. In addition, cellular experiments demonstrated that the cell-penetrating ability of TAT was significantly shielded by the addition of LinTT1 to the fourth lysine of the TAT via an MMP cleavable linker PVGLIG and could be recovered under the catalysis of MMPs. This design was supposed to efficiently decrease the toxicological risk to normal tissues induced by the unselectivity of TAT. The finally treatment effect investigation showed that tumor-bearing mice treated with LPT-modified nanoparticles achieved an enhanced efficacy for inhibiting tumor growth and the longest survival time as compared to other groups. Collectively, this study provides a novel robust nanoplatform which could simultaneously target the tumor microenvironment and tumor cell drug delivery for increasing the efficacy of cancer therapy. KEYWORDS: matrix metalloproteinases, site-specific drug delivery, tumor microenvironment, dual-targeting, nanoparticle

1. INTRODUCTION Chemotherapy of cancer has been significantly impaired by many factors such as inefficient tumor specificity, limited penetration, and unsatisfactory association of agents in tumor cells, and therefore, the malignant tumor is still one of the globally fatal diseases.1−3 To address the drawbacks of drugs poorly differentiating between cancer and normal cells, polymeric nanoparticle-based site-specific drug delivery systems have initiated a new trend in oncology.4,5 Among these strategies, the covalent conjugation of a tumor-targeting moiety © 2017 American Chemical Society

to the surficial PEG chain terminal of a drug-loaded vehicle has gained the most popularity.6−8 For example, Hatakeyama et al. developed an angiogenic-targeting drug delivery system to kill cancer cells through disrupting the vascular systems within the tumor.9 In such an indirect way, the nutritional channels indispensable for tumor progression and metastasis are cut off, Received: August 4, 2017 Accepted: November 2, 2017 Published: November 2, 2017 40614

DOI: 10.1021/acsami.7b11614 ACS Appl. Mater. Interfaces 2017, 9, 40614−40627

Research Article

ACS Applied Materials & Interfaces

Figure 1. (A) Scheme of targeted delivery of drugs to tumor sites. (B) TEM images of NP-PTX (a) and LPT-NP-PTX (b). The scale bar represents 100 nm. (C) Size distribution of NP-PTX (the black curve) and LPT-NP-PTX (the red curve) determined by the DLS. (D) Drug release pattern from nanoparticles evaluated in the medium of PBS containing 10% rat plasma. (E) Stability of the prepared nanoparticles in the medium of DMEM containing 10% FBS.

HIV-TAT protein, TAT includes an N-terminal pentapeptide (GRKKR) which might be the dominant factor mediating the cell membrane crossing.17 TAT peptide conjugation-based tumor-targeting therapy has been shown to significantly enhance the tumor accumulation of chemotherapeutic-loaded nanovehicles.18 However, TAT-based modification would lead to cell unspecificity which finally results in a serious systemic toxicity. Recently, studies showed that conjugation of tumortargeting peptides (TTPs) and CPPs seems to be a potential strategy for tumor-targeting therapy since it perfectly makes a combination of the tumor specificity of TTPs and the penetrating capacity of CPPs.19−21 Although the combinational target therapy could improve the treatment efficiency to a certain extent, increasing evidence suggested that chemotherapy was always severely impaired by the tumor microenvironment.22,23 As the prominent components of the tumor microenvironment, tumor-associated

thus leading to tumor inhibition. For a further improvement, tumor cell and vascular endothelial cell dual-targeting therapy was developed as an alternative option.10−12 Unfortunately, such strategies are significantly impaired by the lack of efficient cellular internalization within tumors and finally lead to an unsatisfactory therapy effect. Therefore, it is still urgent to seek a superior approach, which could target-deliver drugs to tumor tissues and, further, to the target sites of action, to avoid this dilemma. Cell-penetrating peptides (CPPs) have many advantages such as being of smaller size, straightforward, and more biocompatible in nature over antibodies or other small molecular targeted agents.13,14 Importantly, efficient cell penetration comprised the main reasons for the wide application of CPP in drug delivery.15 Among those natural or artificial CPPs, one of the first and widely used CPPs was the TAT peptide.16 As derived from the C-terminal end of the 40615


Research Article


mL penicillin, and 100 μg/mL streptomycin at 37 °C and 5% CO2. Male Sprague−Dawley (SD) rats (200 ± 20 g) and male BALB/c nude mice (20 ± 2 g) were obtained from the Shanghai Laboratory Animal Center (Chinese Academy of Sciences). Care of the animals and handling of the corresponding experiments were approved by the Ethical Committee of Shanghai Jiao Tong University School of Medicine. The subcutaneous lung-cancer models were established by injecting 100 μL of A549 cell suspension with the density of 1.0 × 104/μL into the right flank of BALB/c mice. Then, the tumor-bearing mice were kept under standard conditions with free access to food and water. 2.2. Preparation of LPT-Modified Nanoparticles. For the preparation of LPT peptide-conjugated nanoparticles (LPT-NP-PTX), the NP-PTX was developed first by the emulsion and solvent evaporation method as previously reported.38 In brief, the blend of PTX (3 mg), Mal-PEG-PLA (3 mg), and MPEG-PLA (27 mg) was dissolved in 2 mL of dichloromethane (DCM). Then, 4 mL of 1% (w/ v) sodium cholate solution was added into the mixture under gentle stirring by a magnetic stirrer at room temperature. After 10 min, the mixture was subjected to sonication at 230 W for 40 s (Xin zhi Biotechnology Co., Ltd., China) to form the oil/water emulsion. The resulted solution was then diluted by 50 mL of 0.5% sodium cholate solution and was gently stirred at room temperature for 10 min. Thereafter, a rotary vacuum was introduced to evaporate the organic solvent, and the NP-PTX was finally concentrated by centrifugation (Sigma 3K18 centrifuge) under the condition of 10 000g at 4 °C for 30 min. For formation of the peptide-modified nanoparticles, the resulting NP-PTX were resuspended in 2 mL of distilled water and added into a penicillin bottle. After that, LPT peptide was introduced into the NPPTX suspension at a 1.2:1 molar ratio of peptide to maleimide, and the conjugation reaction was kept for 4 h under room temperature. Finally, the LPT-conjugated nanoparticles were collected, and the unreacted peptide in the supernatant was removed by centrifuging for 30 min at the speed of 14 000 rpm. The LinTT1-NP-PTX, TAT-NP-PTX, and coumarin-6-loaded or Cy5-labeled nanoparticles were developed using a similar method as above. 2.3. Characterization of Prepared Nanoparticles. For examination of the particle size of nanoparticles, NP-PTX or LPTNP-PTX (10 mg) were resuspended with 1 mL of distilled water and finally determined by dynamic light scattering (DLS; Delsa Nano C Zetasizer, Beckman Coulter). For the evaluation of ζ potential of NPs, nanoparticle suspensions were prepared as above, and the ζ potential was examined by electrophoretic light scattering (Delsa Nano C Zetasizer, Beckman Coulter). The morphologies of NP-PTX and LPTNP-PTX were further examined, separately. Briefly, 100 μL of nanoparticle suspension (50 μg/mL) was negatively stained using sodium phosphotungstate solution before being measured by H-600 transmission electron microscopy (TEM; Hitachi, Japan). In addition, the encapsulation efficiency (EE) and drug loading capacity (LC) were also investigated by high-performance liquid chromatography (HPLC) as previously described.11 For determination of whether the plasma affects the release behavior, PBS with a pH value of 7.4 containing 10% rat plasma (v/v) was applied to investigate the PTX release pattern from NP-PTX and LPT-NP-PTX. Typically, 1 mL of nanoparticle suspension (PTX concentration was diluted to 100 μg/mL by the release medium) was added into a centrifuge tube and incubated in PBS (pH 7.4) containing 10% plasma. Then, the tubes were translocated in a gas bath at 37 °C and shaken at 120 rpm. At predetermined time intervals, 100 μL of the nanoparticle suspension samples was sucked up and supplemented with an equal volume of fresh medium at the same time. Finally, the supernatant containing the drugs was analyzed by HPLC. Each measurement was made in triplicate. The HPLC experiment was carried out with a C18 analytical column (5 μm, 200 mm × 4.6 mm, 200 mm, Diamonsil, Dikma) with methanol containing distilled water (75:25, v/v) as the mobile phase. The flow rate and the ultraviolet detection were set at 1.2 mL/min and 227 nm, respectively. For the investigation of nanoparticulate stability, DMEM supplemented with 10% FBS was introduced to incubate with NPPTX and LPT-NP-PTX, separately. In brief, 10 mg of nanoparticles

macrophages (TAMs) always act as a critical modulator in tumor development.24 Furthermore, sufficient evidence has shown that TAMs were able to initiate a chemoresistance due to its immunosuppression of cytotoxic T cells through complex mechanisms.25−27 Therefore, it is essential to overcome the fatal drawbacks induced by TAMs for cancer treatment. LinTT1 peptide is a new tumor-homing peptide which has a specific affinity to the p32/gC1qR receptor that was abundant in many cancer cells and vascular cells.28−30 In addition, the conveyed tumor-penetrating activity (CendR motif) endowed this molecule with a superior tumor-penetrating function.31 More importantly, it was reported that LinTT1 also had a specific affinity to tumor-associated macrophages and resulted in a substantial overlap with TAM in tumor tissues after being conjugated to a carrier.28 In this study, we described a novel PLA nanoparticle-based therapy agent delivery system for tumor cell and tumor microenvironment dual-targeting drug delivery via a polypeptide-LPT modification. Such a polypeptide was constructed through incorporation of TAT and LinTT1 by a matrix metalloproteinase-sensitive linker-PVGLIG.32 The prepared drug delivery system was characterized with the following properties: (1) The constructed polypeptide is able to cross the plasma membrane, penetrate into tumor inner, as well as selectively guide the drug to the target sites of action (Figure 1A). (2) Addition of the peptide to the TAT via the MMPsensitive linker will decrease the transmembrane transport capacity of TAT to a large extent.33 Consequently, the cellpenetrating ability of TAT remains sealed until it is recognized by the MMPs which are abundant in tumor cells, vascular cells, and the tumor extracellular matrix.32,34 (3) Distinct with the traditional tumor cells and tumor vasculature dual-targeting strategies, such a developed drug delivery nanoplatform was also supposed to be able to target the TAMs, which is closely related to tumor growth and progress.35−37 In summary, such a prepared drug delivery system was supposed to be a promising strategy for clinical malignant cancer therapy.

2. EXPERIMENTAL SECTION 2.1. Materials, Cell Lines, and Animals. Methoxy-poly(ethylene glycol)-poly(lactic acid) (MPEG-PLA) with a molecular weight of 33 000 Da and maleimide-poly(ethylene glycol)-poly(lactic acid) (Mal-PEG-PLA) with a molecular weight of 33 400 Da were obtained from Adamas Corporation (Shanghai local agent, China). Paclitaxel (PTX) was bought from Knowshine Pharmachemicals (Shanghai, China). Taxol was purchased from Bristol-Myers Squibb Company. Coumarin-6, cell counting kit-8 (CCK-8) and the cellular uptake inhibitors were bought from Sigma-Aldrich (St. Louis, MO). The Alexa Fluor 594-conjugated antimouse CD31 antibody was obtained from Thermo, and 1,10-dioctadecyl-3,3,30,30-tetramethyl indotricarbocyanine iodide (DiR) was from Abcam (Hong Kong). The LysoTracker Red was purchased from Beyotime. Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), penicillin/ streptomycin, and trypsin−EDTA solution (0.25%, trypsin with 0.53 mM EDTA) were obtained from Life Technologies Co. (Grand Island, NY). The LinTT1 (AKRGARSTAC) peptide or Cy5-labled correspondence, TAT (GRKKRRQRRRC) peptide, and the polypeptide LinTT1-PVGLIG-TAT (LPT) were synthesized by China Peptides Co., Ltd. (Shanghai, China). All other chemicals and reagents were provided by Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China), unless mentioned otherwise. HUVEC and A549 lung-cancer cells were bought from Life Technologies. J774A.1 macrophage cells were from American type Culture Collection (ATCC, Manassas, VA). All of the cells were cultured in DMEM containing 10% FBS supplemented with 100 U/ 40616


Research Article


previously.39 Finally, the colocalization evaluation was determined via confocal laser scanning microscopy after the cell nuclei were stained with DAPI. 2.7. Cytotoxicity Assay in Vitro. The CCK-8 method was applied to examine the cytotoxicity of different PTX formulations against HUVEC cells and A549 cells. For determination, 4 × 103 cells were seeded in each well of 96-well plates. After 24 h of incubation, the cells were exposed to various formulations (Taxol, NP-PTX, LinTT1-NPPTX, TAT-NPPTX, and LPT-NP-PTX) at the concentrations of PTX ranging from 1 to 1 × 103 ng/mL, and then were cultured for 48 h. Thereafter, 20 μL of CCK-8 was added and allowed to interact with cells for 4 h. Finally, the cell viability (cell viability = ODex/ODcon; ODex represents the average optical density of cells treated with PTX formulations; ODcon represents the average optical density of the control group) was evaluated using the microplate reader (Thermo Multiskan MK3). The IC50 values of each PTX formulation were calculated by GraphPad Prism5.0 software. More importantly, blank nanoparticles at the same concentration range as NP-PTX or peptidemodified ones were also tested to evaluate the cytotoxicity of the nanocarrier. 2.8. Tumor-Targeting Experiments. The established A549 lungcancer-bearing mice were applied to investigate the biodistribution of nanoparticles. When the tumor volume increased to approximate 250 mm3, 12 mice were randomly grouped (n = 3). Then, they were intravenously injected with DiR-loaded NP, LinTT1-NP, TAT-NP, and LPT-NP, separately, at an identical DiR dosage of 1.0 mg/kg. The fluorescence signal of mice was then measured in the next 24 h through using the CRi in vivo imaging system (CRi). Briefly, the filter for emission was set at 780 nm after the tumor-bearing mice were placed in the light-tight chamber. Thereafter, the photon emission was integrated for 5 s, and the images were merged by WinLight 32 software. For clear observation of the nanoparticle distribution in organs, the tumor-bearing mice were subjected to euthanasia at 24 h postinjection with DiR-loaded formulations. Thereafter, all major organs (heart, liver, spleen, lung, and kidney) and tumor tissues were collected, and the adhesive bloodstain was cleared away by saline. Finally, the images of organs and tumors were photographed by the in vivo imaging system. As it was characterized as tumor-penetrating, the LinTT1 peptide was hence supposed to mediate a tumor inner drug delivery. For clarification, the randomly grouped (n = 6) tumor-bearing mice were administrated with coumarin-6-labled NP, LinTT1-NP, TAT-NP, or LPT-NP. After 6 h, the mice were sacrificed, and half of them were subjected to an immunofluorescence test as reported previously.39 In brief, the collected tumor tissues were fixed in 4% formaldehyde for 24 h followed by being dehydrated in graded sucrose solution. Then, the slides were prepared by sectioning tumor samples at 14 μm after the samples had been frozen for 1 h at 80 °C. For visualization of the vessels within tumors, the Alexa Fluor 594-conjugated antimouse CD31 antibody was incubated with slides for 24 h. The results were obtained under a confocal microscope after staining the nuclei with DAPI. The remaining section was subjected to homogenizing on ice with 10 mL of deionized water. After the addition of 10 μL of daunorubicin with a concentration of 10 μg/mL, the samples were processed as previously reported.40 Finally the levels of coumarin-6labeled nanoparticles in the tumor site were determined via HPLC analysis. 2.9. TAM-Targeting Experiments. To examine the specificity of the prepared nanoparticles to TAMs, the J774A.1 cells were used as the TAM model.41 For qualitative determination, cells were seeded in a glass dish with a density of 1 × 103 cells/cm2. After 24 h of incubation under the condition of 37 °C and 5% CO2, cell culture medium of the dish was replaced with 2 mL of various nanoparticles (NP, LinTT1-NP, and LPT-NP) traced by coumarin-6 (200 ng/mL). After coincubation for 1 h, the treated cells were washed using PBS followed by being fixed with 4% paraformaldehyde. Thereafter, the cells were blocked in 2% bovine serum albumin for 1 h, and subsequently an antibody was applied against F4/80 to incubate with the cells overnight at 4 °C. For visualization of the TAMs, an Alexa 594-labeled secondary antibody (Invitrogen) was used to incubate for

was suspended by 2 mL of the above culture solution. Then, it was preserved in a dark place at room temperature for 30 days. The sizes of nanoparticles were then determined every 5 days in the total experimental period. 2.4. Sensitivity of Polypeptide to MMPs. Tumor cells were seeded in glass dishes with a cell concentration of 2 × 103/cm2 and cultured for 24 h. Then, fresh culture medium containing 100 μg/mL of LPT-NP-Cy5/C6 was added into each well of the corresponding plates. Thereafter, the cells were incubated with nanoparticles for 1 h with or without MMP inhibitors (MMP-2/9 inhibitors, 1 μM). Finally, the cells were washed with PBS, and the image results were observed via confocal laser scanning microscopy (CLSM, Zeiss LSM710) after the cells were stained with DAPI for visualization of nuclei. In addition, for clear examination of whether sealing the fourth lysine of TAT would lead to an obvious restriction of cell-penetrating ability of TAT, the PVGLIG was covalently linked with TAT to form a PTAT peptide, which was then decorated on the surface of coumarin6-loaded nanoparticles. Thereafter, the PTAT-modified nanoparticles were incubated with HUVEC cells and tumor cells, for 1 h, separately. Importantly, both of the cells had been seeded in 12-well plates at a density of 1× 104 cells per well with or without MMP inhibitors prior the treatment. The quantitative results were obtained through flow cytometry analysis (BD Biosciences, San Jose, CA), and the results were photographed with a fluorescence microscope after the cell nuclei were visualized by DAPI. 2.5. Cellular Uptake. For a study of cellular uptake of nanoparticles, coumarin-6 was introduced as a fluorescence probe. Briefly, the fluorescent dye-loaded NP, LinTT1-NP, TAT-NP, and LPT-NP were diluted to 200 μg/mL by the FBS-free DMEM, separately. Then, the nanoparticle suspensions were incubated with HUVEC cells or A549 cells cultured in a 24-well plate (8 × 103 cells/ well) at 37 °C overnight. After 1 h, cells were washed by cold PBS and stained with DAPI to visualize the nucleus. Finally, the results were observed through confocal laser scanning microscopy. Furthermore, to examine whether the prepared nanoparticles have specificity to normal cells, the Beas2B cells (human bronchial epithelial cells) were introduced, and the cellular uptake assay was performed as above. In addition, for investigation of whether the MMP inhibitors affect the cellular uptake of nanoparticles, cellular association of NP-C6 was also performed in HUVEC cells, J774A.1 cells, and A549 cells, separately, with or without the presence of MMP inhibitors. For quantitative investigation of the cellular-targeting ability of nanoparticles, HUVEC cells and A549 cells were cultured in six-well plates with a density of 1 × 105 cells per well. After 24 h, 1 mL of nanoparticle suspension (200 μg/mL) was added to each well of the plates. Then, the cells were coincubated with nanoparticles and washed using cold PBS after 1 h of incubation to remove the untouched nanoparticles. Finally, the cells were trypsinized and collected by centrifugation before being determined by flow cytometry (FCM). For a thorough inquiry of the mechanism of the cellular internalization of nanoparticles, various endocytosis inhibitors (including 10 μg/mL chlorpromazine, 5 μg/mL BFA, 5 μg/mL filipin, 10 mM NaN3, 4 μg/mL colchicines, 2.5 μg/mL genistein, 2.5 mM methyl-β-cyclodextrin (M-β-CD), 200 nM monensin, 10 μg/mL cyto-D, and 20 μM nocodazole) were introduced.11 HUVEC cells and A549 cells were seeded in 12-well plates at a density of 1 × 104 cells/ well and cultured for 24 h. Then, the above endocytosis inhibitors were added at an appropriate concentration as reported previously.38 After 1 h, the cells were washed using cold PBS and coincubated with 200 μg/mL of LPT-NP-C6 for another 1 h. After that, cells were washed using PBS and fixed by 4% formaldehyde for 10 min. Finally, the cells were collected by centrifugation after being trypsinized, and the quantitative analysis was performed via FCM. 2.6. Lysosome Colocalization. For a clear trace of the nanoparticles after they were internalized by cells, A549 cells and HUVEC cells were seeded in confocal dishes. When the cells reached 80% confluency, 1 mL of LPT-NP-C6 (50 μg/mL) was added into the dishes and allowed to interact with cells for 0.5 or 1 h. Then, Lyso Tracker Red was introduced to stain the lysosomes of cells as reported 40617


Research Article

ACS Applied Materials & Interfaces Table 1. Determination of the Properties of PTX-Loaded Nanoparticles by the DLS nanoparticle NP-PTX LinTT1-NP-PTX TAT-NP-PTX LPT-NP-PTX

particle size (nm) 97.31 106.51 104.44 112.47

± ± ± ±

2.92 1.97 2.62 3.21

polydispersity index (PI) 0.119 0.125 0.131 0.137

± ± ± ±

0.036 0.053 0.037 0.042

ζ potential (mv) −26.41 −23.18 −19.25 −21.52

± ± ± ±

2.33 2.87 3.01 3.16

LC (%) 2.81 2.51 2.63 2.47

± ± ± ±

0.42 0.71 0.54 0.83

EE (%) 56.44 55.01 53.66 54.12

± ± ± ±

2.13 3.14 2.87 3.31

Figure 2. (A) Confocal images of tumor cells after being treated with coumarin-6 and Cy5 dual-labeled LPT-NP with or without MMP inhibitors. (B) Images of cellular uptake of PTAT-NP-C6 in the presence or absence of MMP inhibitor obtained by the fluorescence microscope. (C, D) Quantitative results of cellular internalization of PTAT-NP-C6 in HUVEC cells and A549 cells, respectively, analyzed via flow cytometry. and analysis of the distribution of nanoparticles was performed with confocal laser scanning microscopy. 2.10. Plasma Pharmacokinetics Study. In the determination of the drug blood retention effect of nanoparticles prepared in this study, the plasma pharmacokinetics (PK) of PTX was examined. Briefly, 15 male SD rats (200 ± 20 g) were randomly grouped (n = 3) and intravenously injected with Taxol, NP-PTX, LinTT1-NP-PTX, TATNP-PTX, and LPT-NP-PTX, separately, via the tail vein with a PTX dosage of 5 mg/kg. After that, the blood samples were obtained from the retinal vein plexus at predetermined time points (0.083, 0.25, 0.5, 1, 2, 3, 4, 6, 8, 12, and 24 h) postinjection and centrifuged immediately at 3000 rpm for 10 min. For analysis of the collected samples, 150 μL of methanol was added followed by centrifuging at 12 000 rpm for 10 min. Then, an equal volume of deionized water was added into the supernatant followed by vortexing for 5 min. Finally, the

1 h and was subsequently stained with DAPI for observation of the nuclei. Finally, the results were determined under confocal laser scanning microscopy. The quantitative evaluation was performed with a similar method described for HUVEC cells or A549 cells, and was finally determined via FCM. For further investigation of the TAM-targeting capacity of LPT-NPC6 in a tumor site, six tumor-bearing mice were randomly grouped (n = 3). Subsequently, the mice were systematically treated by NP-C6 and LPT-NP-C6, separately. After 6 h, the treated mice were all executed, and tumor samples were obtained and subsequently subjected to being quickly frozen at −20 °C. Then, slides of 10 mm in thickness were prepared and fixed by 4% paraformaldehyde for 10 min under room temperature. Finally, the experiments were performed with the same method as the immunofluorescence staining of TAM, 40618


Research Article


Figure 3. (A) Images of cells after being incubated with different coumarin-6-loaded nanoparticles obtained by a fluorescence microscope. (B) Quantitative analysis of cellular internalization of different nanoparticles in HUVEV cells. (C) Quantitative determination of cellular uptake of different nanoparticles in A549 cells. (D) Flow cytometry (FCM) analysis of cellular uptake of various concentrations of NP-C6 and LPT-NP-C6 in HUVEC cells at 1 h. (E) FCM examination of cellular association of various concentrations of NP-C6 and LPT-NP-C6 in A549 cells at 1 h. followed by being incubated overnight at 4 °C with anti-MMP primary antibody (1:1000; sc-67163, Santa Cruz). After being washed three times with Tris-buffer containing Tween 20, the membrane was subsequently incubated with a horseradish peroxidase-labeled secondary antibody for 1 h. After a second wash process as above, the membranes were visualized with an ECL kit. Blots probed with anti-β-actin antibody (1:5000; Santa Cruz) were used as the control. Importantly, the normal lung tissue was also analyzed as above to use as the control.

pharmacokinetic data analysis was conducted via liquid chromatography−tandem mass spectrometry (LCMS/MS) analysis. 2.11. Western Blot Assay. The expression of MMPs was determined via a Western blot experiment. In brief, the tumor-bearing mice were executed, and the tumors were collected and immediately subjected to extraction of proteins using the RIPA buffer. Subsequently, the protein samples were loaded onto an SDS-PAGE gel and allowed to run for 2 h under a voltage of 80 V. Proteins were then transferred onto a nitrocellulose membrane using a wet transfer method, and the membrane was blocked with 5% nonfat milk for 1 h, 40619


Research Article


Figure 4. Cellular uptake of C6-labeled LPT-NP in the presence of various endocytosis inhibitors in HUVEC cells (A) and A549 cells (B). (C) Intracellular localization of C6-labeled LPT-NP after being incubated with HUVEC cells for 0.5 or 1 h. (D) Intracellular localization of C6-labeled LPT-NP after being incubated with A549 cells for 0.5 or 1 h. Blue represents nuclei stained with DAPI; Green represents nanoparticles tracked by coumarin-6; red represents endosomes labeled by Lyso Tracker Red. 2.12. Therapeutic Efficacy of Nanoparticles. In the antitumor efficacy experiments, tumor-bearing mice were established as above and randomly grouped (n = 6). When the tumor volume increased to approximate 100 mm3, the mice were intravenously injected with

Taxol, NP-PTX, LinTT1-NP-PTX, TAT-NP-PTX, and LPT-NP-PTX, separately, with the PBS-treated group as the control. Importantly, all of the mice were injected with PTX formulations every 2 days for a total of four injections with a PTX dosage of 5 mg/kg. Then, the body 40620


Research Article


Figure 5. Cell viability of HUVEC cells (A) and A549 cells (B) after treatment by various nanoparticle formulations with the cells incubated by drugfree medium as the control. (C) Images of HUVEC cell nuclei and A549 cell nuclei stained by DAPI after the incubation of cells with various PTX formulations for 24 h. weight of each mouse was carefully monitored every 2 days over a whole period of 14 days, and the tumor volume was photographed and calculated using the formula: V = (L × W2)/2 (L, the longest dimension; W, the shortest dimension). All mice were sacrificed on day 14 after the first treatment, and all of the tumor xenografts were excised and weighed. For further examination, tumors were fixed with 4% paraformaldehyde for 48 h and embedded in paraffin followed by being sectioned at 3 μm thickness. Finally the slices were subjected to hematoxylin and eosin (H&E) analysis and Tunel experiments for determination of tumor cell necrosis and apoptosis. For survival monitoring, the randomly grouped tumor-bearing mice (n = 6) were treated with various PTX formulations as above, and the overall survival was carefully recorded. 2.13. Statistical Analysis. All data were provided as the mean ± standard error of mean (SEM). Significant differences were computed with one-way analysis of variance (ANOVA), followed by post hoc Dunnett tests for a multigroup comparison. Statistical significance was noted as follows: * p < 0.05; ** p < 0.01; *** p < 0.001.

mean value of 0.119 and 0.137, respectively, indicating that both nanoparticles had a narrow size distribution (Figure 1C). Furthermore, the PTX-loaded nanoparticles were both negatively charged with an average ζ potential of −26.41 mV for NP-PTX and −21.52 mV for LPT-NP-PTX. The drug loading capacity and encapsulation efficiency were determined by an Agilent 1260 infinity HPLC system with the LC value of NP-PTX and LPT-NP-PTX being 2.81 ± 0.42% and 2.47 ± 0.83%, respectively, and the EE value of NP-PTX and LPT-NPPTX being 56.44 ± 2.13% and 54.12 ± 3.31%, respectively. Moreover, the physicochemical properties of LinTT1-NP-PTX and TAT-NP-PTX were also examined. Results shown in Table 1 suggesting that modification of the peptide has a negligible effect on the properties of NP-PTX. The in vitro drug release pattern of nanoparticles was studied using the dialysis method. As displayed in Figure 1D, both NPPTX and LPT-NP-PTX exhibited a controlled release pattern when compared with Taxol, which released about 90% of PTX within 6 h. In addition, the PTX release from LPT-NP-PTX was slightly higher than that of NP-PTX with the values being 79.86% and 75.23% at 72 h, respectively. Such an indistinctive difference was mainly contributed to the existing enzymes in plasma.39 Drug leakage during circulation in vasculature systems is always correlated with toxicity to normal tissues and would decrease the curative effect to a large extent. Therefore, a good stability is the most important precondition of drug delivery systems to achieve an efficient tumor-targeting therapy.42,43 In this study, the stability of drug-loaded nanoparticles was

3. RESULTS AND DISCUSSION 3.1. Characterization of Prepared Nanoparticles. The TEM photographs of nanoparticles showed that both NP-PTX and LPT-NP-PTX showed a generally spherical morphology and were uniformly dispersed (Figure 1B). The average sizes of LPT peptide-decorated nanoparticles and the unmodified ones were all determined by the DLS. Results in Table 1 illustrated that NP-PTX showed a mean diameter of about 97 nm and slightly increased to around 112 nm after being functioned with LPT peptide. In addition, the polydispersity index (PDI) values of NP-PTX and LPT-NP-PTX were both below 0.2 with a 40621


Research Article


Figure 6. (A) Biodistribution of DiR-loaded NP, LinTT1-NP, TAT-NP, and LPT-NP in tumor-bearing mice photographed at 24 h. (B) Ex vivo images of dissected organs 24 h after injection of DiR-loaded NP (a), LinTT1-NP (b), TAT-NP (c), and LPT-NP (d). (C, D) Semiquantitative investigation of the fluorescence intensity of DiR-labeled nanoparticles in different organs and tumors.

vasculature mimic cells HUVEC were applied to perform the experiments with C6 as the fluorescence probe. As shown in Figure 3A, confocal laser microscopy images of cells showed that both A549 and HUVEC cells treated with LPT-NP-C6 exhibited a significantly higher fluorescence signal compared to those incubated with NP-C6. More importantly, cellular association of LPT-NP-C6 in HUVEC cells, J774A.1 cells, and A549 cells was obviously higher than that in Beas2B cells while there was a negligible difference for NP-C6, indicating that LPT-NP-C6 did not have specificity to normal cells (Figure S1). In addition, cellular uptake of nanoparticles was obviously increased after nanoparticles were modified with TAT peptide, indicating that TAT could mediate a superior cell penetration. In addition, pretreatment of cells with MMP inhibitors significantly restricted the cellular association of LPTNP-C6, which showed similar fluorescence intensity to the LinTT1-NP-C6, suggesting that the cell-penetrating capacity of TAT was perfectly sealed by the linker. For an investigation of the effect of MMP inhibitors on cellular internalization of nanoparticles, results shown in Figure S2 show that there was no obvious difference for cellular association of NP-C6 with or without the presence of MMP inhibitors, indicating that the inhibitors did not affect the endocytosis process. Such results were further proven by quantitative analysis (Figure 3B,C). Moreover, cellular internalization of both NP-C6 and LPT-NPC6 exhibited a concentration-dependant manner and showed the biggest difference at the nanoparticle concentration of 200 μg/mL as demonstrated in Figure 3D,E. In conclusion, these

evaluated in the medium of DMEM containing 10% FBS. As shown in Figure 1E, neither nanoparticles showed a significant size change during the experiments, indicating that the prepared drug carrier was stable enough to deliver agents to specific sites in vivo. 3.2. Sensitivity of Polypeptide to MMPs. For examination of the catalytic activity of MMPs, the LinTT1 peptide was labeled with Cy5, and coumarin-6 (C6) was loaded into the inner core of nanoparticles to trace the integration of LPT-NP. Results obtained by the laser scanning confocal microscopy showed that cells not pretreated with MMP inhibitors exhibited an almost negligible signal of red fluorescence while obvious colocalization of green and red signals was observed in the cells pretreated with excess MMP inhibitors (Figure 2A). Furthermore, cells incubated with PTAT-NP-C6 in the presence of MMP inhibitors displayed a negligible difference in cellular uptake of nanoparticles as compared to the NP-C6treated ones. However, the signal of fluorescence was significantly improved after cells coincubated with only PTAT-NP-C6, suggesting that the cell-penetrating capacity of TAT was mostly recovered (Figure 2B−D) in the presence of MMPs. These results together indicated that the synthesized peptide was sufficiently sensitive to the MMPs, and the additional linker to the fourth lysine of TAT could exactly result in a satisfactory seal of cell-penetrating function. 3.3. Cellular Uptake Experiments. For examination of whether there is a difference between the cellular association of NPs and LPT-NPs, both A549 lung-cancer cells and tumor 40622


Research Article


Figure 7. (A) Distribution of nanoparticles in tumor sites 3 h after injection of various formulations. Blue represents DAPI-visualized cell nuclei; green is coumarin-6-traced nanoparticles; and the red signal represents CD31-labeled blood vessels. (B) Quantitative analysis of distribution of nanoparticles in tumor sites using HPLC analysis.

other formulations. The IC50 values of Taxol, NP-PTX, LinTT1-NP-PTX, TAT-NP-PTX, and LPT-NP-PTX were 318.1, 257.9, 128.8, 93.69, and 57.59 ng/mL, respectively, for HUVEC cells and 349.5, 289.7, 146.5, 119.5, and 66.99 ng/mL, respectively, for A549 cells. These results indicated that LPT could mediate a superior targeting performance and lead to efficient cell penetration in the presence of MMPs, resulting in higher toxicity to cells. In comparison, the cells incubated with nanoparticles without PTX showed a negligible cytotoxicity with a cell viability of about 95% for HUVEC and A549 cells, suggesting a good biocompatibility for the prepared drug delivery vehicle. In addition, for a qualitative study of the in vitro apoptosis-inducing capacity of these drug-loaded nanoparticles, the images of the nucleus after the treatment of various PTX formulations with cells were obtained by a fluorescence microscope. Results in Figure 5C show that both cells incubated with LPT-NP-PTX exhibited the most fragments while the control group displayed an intact spherical morphology. 3.5. Tumor-Targeting Ability of Nanoparticles. After the lung-cancer-bearing mouse model was established with A549 cells, the tumor-targeting efficiency of LPT-modified nanoparticles was evaluated in vivo with DiR as a probe. As shown in Figure 6A, after being injected with an equivalent volume of DiR-labeled LinTT1-NP, TAT-NP, LPT-NP, or undecorated nanoparticles via the tail vein, the mice injected with LPT-NP displayed the highest tumor-targeting efficiency. In addition, the LinTT1-NP group showed stronger signals at the tumor site when compared with TAT-NP. Importantly, the mice administered with TAT-NP showed an accumulation in

results together suggested that the excellent affinity to tumors cells and angiogenesis of LPT-NP-C6 was mainly contributed by the properties of the Lin-TT1 molecule. In addition, after the sensitive linker was cleared away by MMPs, the TAT peptide further led to an efficient cell membrane crossing. For an examination of the mechanisms of cellular internalization of nanoparticles, cells were incubated with various endocytosis inhibitors for 1 h before being exposed to LPT-NPC6. Results in Figure 4A show that the uptake of LPT-NP-C6 in HUVEC and A549 cells was obviously restricted by NaN3, filipin, genistein, M-β-CD, and monensin, indicating that the endocytosis was an energy-dependent, lipid-raft- and endosomes-mediated process. In addition, quantitative results in A549 cells showed that cellular association of LPT-NP-C6 was also inhibited by cyto-D to a decrease of 30% compared with the control, suggesting that the microtubule was involved in the cellular association of nanoparticles in A549 cells (Figure 4B). For the subcellular colocalization investigation, Lyso Tracker Red was applied to visualize the endosomes in the cytoplasm of tumor cells. Results in Figure 4C,D illustrate that nanoparticles were colocalized well with endosomes after incubation for 0.5 h. However, when the time was extended to 1 h, most of the signal of nanoparticles departed from the red fluorescence, indicating the occurrence of endosome escape during the incubation of cells and nanoparticles. 3.4. In Vitro Cytotoxicity Evaluation. Both HUVEC cells and A549 cells were incubated with different nanoparticles for 48 h, and the cytotoxicity was then evaluated through the CCK8 method. As illustrated in Figure 5A,B, cells treated with LPTNP-PTX exhibited the lowest cell viability when compared with 40623


Research Article


Figure 8. Evaluation of the TAM-targeting capacity of LPT-NP-C6 through in vitro and in vivo experiments. (A) Images of J774A.1 cells after being treated by NP-C6, LinTT1-NP-C6, and LPT-NP-C6, separately. (B) Quantitative study of TAM uptake of different nanoparticles determined by a flow cytometer. (C) Colocalization of LPT-NP-C6 with TAM in tumor sites compared with NP-C6. Blue represents DAPI-visualized cell nuclei; green is coumarin-6 traced nanoparticles; and the red signal represents F4/80 marked TAMs.

the tumor site as well as the normal tissues due to the lack of specificity of TAT peptide. For further evaluation of the biodistribution of nanoparticles, tumor-bearing mice treated with DiR-loaded LinTT1-NP, TAT-NP, LPT-NP, or undecorated nanoparticles were sacrificed, and the main organs were isolated 24 h after injection. Results determined by the in vivo imaging system showed that more DiR was efficiently delivered into tumor tissue by the LPT-NP when compared with other types of nanoparticle formulations (Figure 6B). This result was further confirmed by the semiquantitative fluorescence analysis as displayed in Figure 6C,D. 3.6. Distribution of Nanoparticles in the Tumor Site. The distribution of nanoparticles in the tumor site is illustrated in Figure 7A; the fluorescence intensity of NP exhibited the weakest signal, and only a few nanoparticles accumulated around the tumor blood vessels. In comparison, the LinTT1 peptide-functionalized nanoparticles not only exhibited a stronger vascular accumulation, but also were distributed into the interior of the tumor. In addition, the TAT-decorated nanoparticles also displayed a superior tumor affinity and penetration than NP-C6 but weaker than LinTT1-functionalized nanoparticles. More importantly, after a combination of LinTT1 and TAT, the green fluorescence intensity was significantly increased in tumor sites, suggesting that the

polypeptide possessed the highest tumor-targeting effect. For further analysis, the tumor tissues containing various coumarin6-loaded nanoparticles were homogenized, and the nanoparticle concentrations accumulated in tumors were determined by HPLC analysis. As depicted in Figure 7B, the distribution of nanoparticles was perfectly in accordance with the above results, suggesting a superior targeting capacity of LPT-NP. 3.7. TAM-Targeting Experiments. The J774A.1 cells were applied as the tumor-associated macrophage model to investigate the TAM-targeting ability of LPT-NP. As shown in Figure 8A, a stronger intensity of red fluorescence was detectable in the LinTT1-NP-C6-treated cells while only a weak signal was detected in the NP-C6-treated cells, indicating a superior TAM-targeting capacity of LinTT1 peptide. Moreover, after conjugation with the TAT moiety, the cellular uptake of nanoparticles was significantly increased because of the superior cell-penetrating ability of the TAT peptide. These results were further confirmed by the quantitative investigation as shown in Figure 8B. For further evaluation of the in vivo colocalization of TAM and LPT-NP-C6, randomly grouped tumor-bearing mice were treated with NP-C6 or LPT-NP-C6, and were sacrificed 3 h after injection with tumor tissues being then obtained. Thereafter, an immunofluorescence experiment was performed to verify the colocalization of TAM with 40624


Research Article


Figure 9. (A) Examination of the expression levels of MMPs in tumor tissues via a Western blot assay. (B) Pharmacokinetic study of various PTX formulations in vivo. (C) Weighted tumors after being dissected. (D) Relative body weight changes of tumor-bearing mice during the treatment. (E) Images of tumor tissues 14 days after a programmatic therapy. (F) Changes of tumor volume in mice during the experimental period. (G) Kaplan− Meier survival curve of mice. (H) H&E staining and Tunel study of tumor tissue sections in the mice after being treated by various PTX-loaded nanoparticles while the PBS group was used as the control.

nanoparticles in tumors. As shown in Figure 8C, a large amount of LPT-NP-C6 was well colocalized with TAM within tumor tissues while only a little amount of NP-C6 was detectable in tumor tissues with poor colocalization with TAM within tumors. The above results suggested that LPT peptide-modified nanoparticles were exactly a superior tumor-associated macrophage-targeting drug delivery system. 3.8. Therapeutic Efficacy of Nanoparticles. As encouraged by the above investigation, the antitumor efficacy of those nanoparticle formulations was further investigated. First, the expression level of MMPs in tumor tissues was determined by a Western blot assay. As shown in Figure 9A, a high level of MMP expression was detectable in the tumor while a negligible signal was determined in the normal tissues. Next, we investigated the pharmacokinetics of different formulations in vivo. As shown in the plasma concentration−time curves (Figure 9B), Taxol exhibited the most rapid clearance when compared with other formulations with the PTX concentration undetectable at 12 h. In comparison, the PTX-loaded nanoparticles displayed a higher concentration at all time

points, indicating that the prepared nanoparticles could significantly prolong the elimination half-life and decreased clearance rate. Thereafter, we evaluated the cancer treatment efficacy by observing the tumor volumes, weights, and relative body weight change after programmatic therapy by various PTX formulations. As depicted in Figure 9B−F, passive targeting therapy of NP-PTX only showed a moderate inhibition of tumor growth with an inhibition rate of approximately 32.4% compared with the PBS group at the end of treatment. In contrast, the mice treated with LPT-NP-PTX showed 90.27% tumor suppression versus the control group, indicating an excellent antitumor effect after modification of active targeting peptide. In addition, the visually observed tumor tissues confirmed the remarkable efficacy of LPT-NP-PTX in suppressing tumor growth. More importantly, the treatment of LPT-NP-PTX did not significantly influence the body weight of the mice while the Taxol group exhibited varying degrees of decreases in body weight ascribed to the toxicity of chemotherapeutics. In addition, the survival curve in Figure 9G shows that tumor-bearing mice 40625


Research Article


(2) Jonsson, B.; Bergh, J. Hurdles in anticancer drug development from a regulatory perspective. Nat. Rev. Clin. Oncol. 2012, 9, 236−243. (3) Hernández-Pedro, N. Y.; Rangel-López, E.; Magaña-Maldonado, R.; de la Cruz, V. P.; del Angel, A. S.; Pineda, B.; Sotelo, J. Application of Nanoparticles on Diagnosis and Therapy in Lung carcimas. BioMed Res. Int. 2013, 2013, 351031. (4) Yu, D. H.; Lu, Q.; Xie, J.; Fang, C.; Chen, H. Z. Peptideconjugated biodegradable nanoparticles as a carrier to target paclitaxel to tumor neovasculature. Biomaterials 2010, 31, 2278−2292. (5) Ruan, H.; Chen, X.; Xie, C.; Li, B.; Ying, M.; Liu, Y.; Zhang, M.; Zhang, X.; Zhan, C.; Lu, W.; Lu, W. Stapled RGD Peptide Enables Glioma-Targeted Drug Delivery by Overcoming Multiple Barriers. ACS Appl. Mater. Interfaces 2017, 9, 17745−17756. (6) Gao, H.; Yang, Z.; Zhang, S.; Cao, S.; Shen, S.; Pang, Z.; Jiang, X. Ligand modified nanoparticles increases cell uptake, alters endocytosis and elevates lung cancer distribution and internalization. Sci. Rep. 2013, 3, 2534. (7) Gao, H. Perspectives on Dual Targeting Delivery Systems for Brain Tumors. J. Neuroimmune Pharmacol. 2017, 12, 6−16. (8) Soler, M.; Feliu, L.; Planas, M.; Ribas, X.; Costas, M. Peptidemediated vectorization of metal complexes: conjugation strategies and biomedical applications. Dalton Trans. 2016, 45, 12970−12982. (9) Hatakeyama, S.; Sugihara, K.; Shibata, T. K.; Nakayama, J.; Akama, T. O.; Tamura, N.; Wong, S. M.; Bobkov, A. A.; Takano, Y.; Ohyama, C.; Fukuda, M.; Fukuda, M. N. Targeted drug delivery to tumor vasculature by a carbohydrate mimetic peptide. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 19587−19592. (10) Zhang, Q.; Lu, L.; Zhang, L.; Shi, K.; Cun, X.; Yang, Y.; Liu, Y.; Gao, H.; He, Q. Dual-functionalized liposomal delivery system for solid tumors based on RGD and a pH-responsive antimicrobial peptide. Sci. Rep. 2016, 6, 19800. (11) Feng, X.; Gao, X.; Kang, T.; Jiang, D.; Yao, J.; Jing, Y.; Song, Q.; Jiang, X.; Liang, J.; Chen, J. Mammary-Derived Growth Inhibitor Targeting Peptide-Modified PEG-PLA Nanoparticles for Enhanced Targeted Glioblastoma Therapy. Bioconjugate Chem. 2015, 26, 1850− 1861. (12) Gao, H.; Yang, Z.; Cao, S.; Xiong, Y.; Zhang, S.; Pang, Z.; Jiang, X. Tumor cells and neovasculature dual targeting delivery for glioblastoma treatment. Biomaterials 2014, 35, 2374−2382. (13) Vivès, E.; Schmidt, J.; Pèlegrin, A. Cell-penetrating and celltargeting peptides in drug delivery. Biochim. Biophys. Acta, Rev. Cancer 2008, 1786, 126−138. (14) Koren, E.; Torchilin, V. P. Cell-penetrating peptides: breaking through to the other side. Trends Mol. Med. 2012, 18, 385−393. (15) Heitz, F.; Morris, M. C.; Divita, G. Twenty years of cellpenetrating peptides: from molecular mechanisms to therapeutics. Br. J. Pharmacol. 2009, 157, 195−206. (16) Vivès, E.; Brodin, P.; Lebleu, B. A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J. Biol. Chem. 1997, 272, 16010− 16017. (17) Ruben, S.; Perkins, A.; Purcell, R.; Joung, K.; Sia, R.; Burghoff, R.; Haseltine, W. A.; Rosen, C. A. Structural and functional characterization of human immunodeficiency virus tat protein. J. Virol. 1989, 63, 1−8. (18) Qin, Y.; Chen, H.; Zhang, Q.; Wang, X.; Yuan, W.; Kuai, R.; Tang, J.; Zhang, L.; Zhang, Z.; Zhang, Q.; Liu, J.; He, Q. Liposome formulated with TAT-modified cholesterol for improving brain delivery and therapeutic efficacy on brain lung cancer in animals. Int. J. Pharm. 2011, 420, 304−312. (19) Myrberg, H.; Zhang, L.; Mäe, M.; Langel, U. Design of a tumorhoming cell-penetrating peptide. Bioconjugate Chem. 2008, 19, 70−75. (20) Regberg, J.; Srimanee, A.; Langel, U. Applications of cellpenetrating peptides for tumor targeting and future cancer therapies. Pharmaceuticals 2012, 5, 991−1007. (21) Essler, M.; Ruoslahti, E. Molecular specialization of breast vasculature: a breast-homing phage-displayed peptide binds to aminopeptidase P in breast vasculature. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 2252−2257.

administered with LPT-NP-PTX achieved the longest median survival time of 55 days, while other formulations prolonged the survival time of mice from 14 to 20 (NP-PTX), 32 (TATNP-PTX), and 41 (LinTT1-NP-PTX) days, respectively. For further evaluation of the toxicity of nanoparticles to cells within tumor tissue, the H&E staining assay and Tunel experiment were carried out. As exhibited in Figure 9H, the LPT-NP-PTXtreated mice showed the largest area of apoptosis, indicating the best tumor toxicity of LPT-NP-PTX compared with other formulations.

4. CONCLUSION For the treatment of lung cancer, we rationally developed a robust nanoplatform by integrating a tumor-homing peptide and cell-penetrating peptide in this study. This formulation has been designed to inhibit tumor growth by not only directly killing tumor cells, but also destructing the tumor microenvironments including tumor-associated macrophages and angiogenesis. Experimental results demonstrated that the prepared drug delivery systems had a high affinity to tumor cells, vascular cells, and TAMs which in turn led to a significant cytotoxicity. In combination with cell-penetrating peptide, the LPT-modified nanoparticles significantly increased its tumorpenetrating capacity as confirmed by the in vivo targeting assay. Tumor inhibition, H&E, and Tunel assay confirmed that the tumor growth inhibition property can be significantly expanded by combination therapy. All of these results together implied a considerable potential of LPT-NP-PTX for the clinical treatment of lung cancer.

■

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b11614. Additional figures showing cellular uptake of NP-C6 and LPT-NP-C6 (PDF)

■

AUTHOR INFORMATION

Corresponding Authors

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

Man Wang: 0000-0003-0859-3501 Author Contributions #

Z.S., R.L., and J.S. contributed equally to this work.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The research is supported by Shanghai Fengxian District Science and Technology Project (20151205 and 20141001), Shanghai Municipal Health and Family Planning Commission Project (201540027), The seed fund program of Shanghai University of Medicine & Health Sciences (HSMF-17-22-031), Excellent Young Medical Expert of Shanghai (2017YQ048), and China Postdoctoral Science Foundation (2017M610278).

■

REFERENCES

(1) Hait, W. N. Anticancer drug development: the grand challenges. Nat. Rev. Drug Discovery 2010, 9, 253−254. 40626


Research Article

ACS Applied Materials & Interfaces (22) McMillin, D. W.; Negri, J. M.; Mitsiades, C. S. The role of tumour-stromal interactions in modifying drug response: challenges and opportunities. Nat. Rev. Drug Discovery 2013, 12, 217−228. (23) Klemm, F.; Joyce, J. A. Microenvironmental regulation of therapeutic response in cancer. Trends Cell Biol. 2015, 25, 198−213. (24) Franklin, R. A.; Liao, W.; Sarkar, A.; Kim, M. V.; Bivona, M. R.; Liu, K.; Pamer, E. G.; Li, M. O. The cellular and molecular origin of tumor-associated macrophages. Science 2014, 344, 921−925. (25) Jinushi, M.; Chiba, S.; Yoshiyama, H.; Masutomi, K.; Kinoshita, I.; Dosaka-Akita, H.; Yagita, H.; Takaoka, A.; Tahara, H. Tumorassociated macrophages regulate tumorigenicity and anticancer drug responses of cancer stem/initiating cells. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 12425−12430. (26) Mantovani, A.; Allavena, P. The interaction of anticancer therapies with tumor-associated macrophages. J. Exp. Med. 2015, 212, 435−445. (27) Ni, Y. H.; Ding, L.; Huang, X. F.; Dong, Y. C.; Hu, Q. G.; Hou, Y. Y. Microlocalization of CD68+ tumor-associated macrophages in tumor stroma correlated with poor clinical outcomes in oral squamous cell carcinoma patients. Tumor Biol. 2015, 36, 5291−5298. (28) Sharma, S.; Kotamraju, V. R.; Mölder, T.; Tobi, A.; Teesalu, T.; Ruoslahti, E. Tumor-Penetrating Nanosystem Strongly Suppresses Breast Tumor Growth. Nano Lett. 2017, 17, 1356−1364. (29) Agemy, L.; Kotamraju, V. R.; Friedmann-Morvinski, D.; Sharma, S.; Sugahara, K. N.; Ruoslahti, E. Proapoptotic peptide-mediated cancer therapy targeted to cell surface p32. Mol. Ther. 2013, 21, 2195− 2204. (30) Sánchez-Martín, D.; Cuesta, A. M.; Fogal, V.; Ruoslahti, E.; Alvarez-Vallina, L. The multicompartmental p32/gClqR as a new target for antibody-based tumor targeting strategies. J. Biol. Chem. 2011, 286, 5197−5203. (31) Pang, H. B.; Braun, G. B.; Friman, T.; Aza-Blanc, P.; Ruidiaz, M. E.; Sugahara, K. N.; Teesalu, T.; Ruoslahti, E. An endocytosis pathway initiated through neuropilin-1 and regulated by nutrient availability. Nat. Commun. 2014, 5, 4904. (32) Chau, Y.; Tan, F. E.; Langer, R. Synthesis and characterization of dextran-peptide-methotrexate conjugates for tumor targeting via mediation by matrix metalloproteinase II and matrix metalloproteinase IX. Bioconjugate Chem. 2004, 15, 931−941. (33) Liu, Z.; Xiong, M.; Gong, J.; Zhang, Y.; Bai, N.; Luo, Y.; Li, L.; Wei, Y.; Liu, Y.; Tan, X.; Xiang, R. Legumain protease-activated TATliposome cargo for targeting tumours and their microenvironment. Nat. Commun. 2014, 5, 4280. (34) Ke, W.; Zha, Z.; Mukerabigwi, J. F.; Chen, W.; Wang, Y.; He, C.; Ge, Z. Matrix Metalloproteinase-Responsive Multifunctional PeptideLinked Amphiphilic Block Copolymers for Intelligent Systemic Anticancer Drug Delivery. Bioconjugate Chem. 2017, 28, 2190. (35) Quail, D. F.; Joyce, J. A. Microenvironmental regulation of tumor progression and metastasis. Nat. Med. 2013, 19, 1423−1437. (36) Qian, B. Z.; Pollard, J. W. Macrophage diversity enhances tumor progression and metastasis. Cell 2010, 141, 39−51. (37) Pollard, J. W. Tumour-educated macrophages promote tumour progression and metastasis. Nat. Rev. Cancer 2004, 4, 71−78. (38) Yu, D. H.; Liu, Y. R.; Luan, X.; Liu, H. J.; Gao, Y. G.; Wu, H.; Fang, C.; Chen, H. Z. IF7-Conjugated Nanoparticles Target Annexin 1 of Tumor Vasculature against P-gp Mediated Multidrug Resistance. Bioconjugate Chem. 2015, 26, 1702−1712. (39) Feng, X.; Yao, J.; Gao, X.; Jing, Y.; Kang, T.; Jiang, D.; Jiang, T.; Feng, J.; Zhu, Q.; Jiang, X.; Chen, J. Multi-targeting PeptideFunctionalized Nanoparticles Recognized Vasculogenic Mimicry, Tumor Neovasculature, and Glioma Cells for Enhanced Anti-glioma Therapy. ACS Appl. Mater. Interfaces 2015, 7, 27885−27899. (40) Pang, Z.; Gao, H.; Yu, Y.; Guo, L.; Chen, J.; Pan, S.; Ren, J.; Wen, Z.; Jiang, X. Enhanced intracellular delivery and chemotherapy for glioma rats by transferrin-conjugated biodegradable polymersomes loaded with doxorubicin. Bioconjugate Chem. 2011, 22, 1171−1180. (41) Niu, M.; Naguib, Y. W.; Aldayel, A. M.; Shi, Y. C.; Hursting, S. D.; Hersh, M. A.; Cui, Z. Biodistribution and in vivo activities of

tumor-associated macrophage-targeting nanoparticles incorporated with doxorubicin. Mol. Pharmaceutics 2014, 11, 4425−4436. (42) Moghimi, S. M.; Hunter, A. C.; Andresen, T. L. Factors controlling nanoparticle pharmacokinetics: an integrated analysis and perspective. Annu. Rev. Pharmacol. Toxicol. 2012, 52, 481−503. (43) Ernsting, M. J.; Murakami, M.; Roy, A.; Li, S. D. Factors controlling the pharmacokinetics, biodistribution and intratumoral penetration of nanoparticles. J. Controlled Release 2013, 172, 782−794.

40627


Matrix Metalloproteinase Cleavable Nanoparticles for Tumor

Recommend Documents