Matrix Metalloproteinase Cleavable Nanoparticles for Tumor

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Matrix Metalloproteinases Cleavable Nanoparticles for Tumor Microenvironment and Tumor Cells Dual-Targeting Drug Delivery Zhenliang Sun, Ruihong Li, Ji Sun, You Peng, Linlin Xiao, Xingxing Zhang, Yixin Xu, and Man Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 02 Nov 2017 Downloaded from http://pubs.acs.org on November 3, 2017

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ACS Applied Materials & Interfaces

Matrix Metalloproteinases Cleavable Nanoparticles for Tumor Microenvironment and Tumor Cells Dual-Targeting Drug Delivery Zhenliang Sun1,2#，Ruihong Li3#，Ji Sun4# , You Peng1,

Linlin Xiao1，

Xingxing Zhang1, Yixin Xu5*，Man Wang1* 1

Shanghai University of Medicine & Health Sciences Affiliated Sixth People's

Hospital South Campus, Shanghai 201499，China 2

Department of General Surgery, Shanghai Tenth People’s Hospital Affiliated to

Tongji University, No. 301, Yan-Chang Road, Shanghai 200072, China; 3

Hangzhou Normal University Qianjiang College，HangZhou 310036, China

4

Shanghai University of Medicine & Health Sciences, Shanghai 201318, China

5

School of Pharmacy, Shanghai University of Medicine & Health Sciences, Shanghai

201318, China *Correspondence to: Man Wang, email: [email protected] Yi-Xin Xu, email: [email protected] #

All of the authors contributed equally to this work.

ACS Paragon Plus Environment


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ABSTRACT Matrix metalloproteinases (MMPs), mostly abundant in 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 could achieve site-specific drug delivery and satisfactory tumor treatment effect. Herein, we rationally developed a novel tumor microenvironment and tumor cells dual-targeting nanoparticle by integrating a chemotherapeutics-loaded drug-loaded carrier and a versatile

polypeptide-LinTT1-PVGLIG-TAT

(LPT)

which

composed

by

a

multi-targetingpeptide-LinTT1 and a cell penetrating 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. Besides, cellular experiments demonstrated that cell penetrating ability of TAT was significantly shielded by the addition of LinTT1 to the fourth lysine of the TAT via a MMPs 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 unselectively 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 longest survival time than other groups. Collectively, this study provides a novel robust nanoplatform which could simultaneously target tumor microenvironment and tumor cells 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 was 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 fatal disease globally.1-3 To address the drawbacks that drugs poorly differentiating between cancer and normal cells, polymeric nanoparticles-based site-specific drug delivery systems have initiated a new trend in oncology.4,5 Among these strategies, the covalent


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conjugation of a tumor targeting moiety to the surficial PEG chain terminal of 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 tumor.9 In such indirect way, the nutritional channels indispensable for tumor progression and metastasis are cut off, thus leading to a tumor inhibition. To make a further improvement, tumor cells and vascular endothelial cells dual-targeting therapy was developed as an alternative option.10-12 Unfortunately, such strategies are significantly impaired by the lack of efficiently cellular internalization within tumors and finally lead to an unsatisfactory therapy effect. Therefore, it is still urgent to seek a super approach, which could target deliver drugs to tumor tissues and further to the target sites of action, to get away from this dilemma. Cell penetrating peptides (CPPs) have many advantages such as smaller in size, straightforward and more biocompatible in nature over antibodies or other small molecular targeted agents.13,14 Importantly, efficient cell penetration composed 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 HIV-TAT protein, TAT includes an N-terminal pentateptide (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

chemotherapeutics-loaded nanovehicles.18 However, TAT-based modification would leads to cell unspecificity which finally results in a serious systemic toxicity. Recently studies showed that conjugation of tumor targeting peptides (TTPs) and CPPs seems to be a potential strategy for tumor targeting therapy since it perfectly makes a combination of tumor specificity of TTPs and penetrating capacity of CPPs.19-21 Although the combinational target therapy could improve the treatment efficiency to a certain extent, increasing evidences suggested that chemotherapy was always severely impaired by the tumor microenvironment.22,23 As the prominent components of tumor microenvironment, tumor-associated macrophages (TAMs) always act as a



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critical modulator in tumor development.24 Furthermore, sufficient evidences showed that TAMs were able to initiate a chemo-resistance 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 newly 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 super tumor-penetrating function.31 More importantly, it was reported that LinTT1 also had a specific affinity to tumor-associated macrophages affinity and resulted in a substantial overlap with TAM in tumor tissues after conjugated to a carrier.28 In this study, we made a description of a novel PLA nanoparticle-based therapy agent delivery system for tumor cells and tumor microenvironment dual-targeting drug delivery via a polypeptide-LPT modification. Such polypeptide was constructed through incorporation of TAT and LinTT1 by a matrix metalloproteinases-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 MMPs sensitive linker will decrease the transmembrane transport capacity of TAT to a large extent.33 Consequently, the cell penetrating ability of TAT remain sealed until they are recognized by the MMPs which are abundant in tumor cells, vascular cells and tumor extracellular matrix;

32,34

3) distinct with the traditional tumor cells and tumor

vasculature dual-targeting strategies, such developed drug delivery nanoplatform was also supposed to could target the TAMs which is closely related to tumor growth and progress.35-37 In a word, such prepared drug delivery system was supposed to be a promising strategy for malignant cancer therapy in clinic. 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


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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 anti-mouse CD31 antibody

was

obtained

from

Thermo

(USA),

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, USA). 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/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 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 cells 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 the standard condition with free access to food and water. 2.2. Preparation of LPT-Modified Nanoparticles. For the preparation of LPT



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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 dichloromethane (DCM). Then 4 mL of 1% (w/v) sodium cholate solution was added into the mixture under gently stirring by a magnetic stirrer at room temperature. Ten minutes later, 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, Germany) under the condition of 10,000 × g at 4°C for 30 min. To form the peptide-modified nanoparticles, the resulting NP-PTX were resuspended in 2 mL distilled water and added into a penicillin bottle. After that, LPT peptide was introduced into the NP-PTX suspension at 1.2:1 molar ratio of peptide to maleimide and the conjugation reaction were 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 14000 rpm. The LinTT1-NP-PTX, TAT-NP-PTX and coumarin-6-loaded or Cy5-labeled nanoparticles were developed use the similar method as above. 2.3. Characterization of Prepared Nanoparticles. To examine the particle size of nanoparticles, NP-PTX or LPT-NP-PTX (10 mg) were resuspended with 1 mL of distilled water and finally determined by the dynamic light scattering (DLS) (Delsa Nano C Zetasizer, Beckman Coulter, USA). For the evaluation of zeta potential of NPs, nanoparticle suspension were prepared as above and the zeta potential was examined by the electrophoretic light scattering (Delsa Nano C Zetasizer, Beckman Coulter, USA). The morphologies of NP-PTX and LPT-NP-PTX were further examined, respectively. Briefly, 100 µL of nanoparticle suspension (50 µg/mL) was negatively stained using the sodium phosphotungstate solution before being measured by the H-600 transmission electron microscopy (TEM) (Hitachi, Japan). In addition, the encapsulation efficiency (EE) and drug loading capacity (LC) were also


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investigated by the high performance liquid chromatography (HPLC) as previously described.11 To determine whether the plasma affect the release behavior, PBS with the pH value of 7.4 containing 10% rat plasma (v/v) was applied to investigate the PTX release pattern of PTX from NP-PTX and LPT-NP-PTX. Typically, 1 mL of nanoparticles 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 the predetermined time intervals, 100 µL of the nanoparticle suspension samples were suck up and supplemented with an equal volume of fresh medium in meanwhile. 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, 200mm, Diamonsil, Dikma) with the 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 NP-PTX and LPT-NP-PTX, respectively. In brief, 10 mg nanoparticles were suspended by 2 mL of above culture solution. Then it was preserved in dark place at room temperature for 30 days. The particles size of nanoparticles were then determined every five days in the total experimental period. 2.4. Sensitivity of Polypeptide to MMPs. Tumor cells were seeded in the glass dishes with the 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 MMPs inhibitors (MMP-2/9 inhibitors, 1 µM). Finally, the cells were washed with PBS and the image results were observed via a confocal laser scanning microscopy (CLSM, Zeiss LSM710, Germany) after cells being stained with DAPI for visualization of nuclei. In addition, to clearly examine whether sealing the fourth lysine of TAT would lead



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to an obviously 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 coumarin-6-loaded nanoparticles. Thereafter, the PTAT modified nanoparticles were incubated with HUVEC cells and tumor cells, for 1 h, respectively. Importantly, both the cells had been seed in 12-well plates at the density of 1× 104 cells per well with or without MMPs inhibitors prior the treatment. The quantitative results were obtained through a 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 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, respectively. 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. One hour later, cells were washed by cold PBS and stained with DAPI to visualize the nucleus. Finally, the results were observed under a confocal laser scanning microscopy. Furthermore, to examine whether the prepared nanoparticles is specificity to normal cells, the Beas2B cells (human bronchial epithelial cells) was introduced and the cellular uptake assay was performed as above. In addition, to investigate whether the MMPs inhibitors affect the cellular uptake of nanoparticles, cellular association of NP-C6 was also performed in HUVEC cells, J774A.1 cells, and A549 cells, respectively, with or without the presence of MMPs inhibitors. To quantitatively investigate the cellular targeting ability of nanoparticles, HUVEC cells and A549 cells were cultured in the six-well plates with the density of 1 × 105 cells per well. Twenty-four hours later, each well of plates was added with 1 mL of nanoparticle suspension (200 µg/mL). Then the cells were coincubated with nanoparticles and washed using cold PBS post 1 h incubation to remove the untouched nanoparticles. Finally, the cells were trypsinized and collected by centrifugation before being determined by a flow cytometer (FCM). To make a thorough inquiry of the mechanism of the cellular internalization of


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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 the 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 One hour later, 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, collected the cells by centrifugation post trypsinized and performed the quantitative analysis via the FCM analysis. 2.6. Lysosomes Co-Localization. To clearly trace 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) were added into the dishes and allowed to interact with cells for 0.5 h or 1 h. Then the Lyso Tracker Red was introduced to stain the lysosomes of cells as reported previously.39 Finally, the co-localization evaluation was determined via a confocal laser scanning microscopy post 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 to HUVEC cells and A549 cells. For determination, 4 × 103 cells were seeded in each well of 96-well plates. After 24 h incubation, the cells were exposed to various formulations (Taxol®, NP-PTX, LinTT1-NP-PTX, TAT-NPPTX and LPT-NP-PTX) at the concentrations of PTX was ranging from 1 to 1 × 103 ng/mL and then 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 control group) was evaluated using the microplate reader (Thermo Multiskan MK3). The IC50 values of each PTX formulations were calculated by the GraphPad Prism5.0 software. More importantly, blank nanoparticles at the same concentrations range as NP-PTX or



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peptide modified ones were also tested to evaluate the cytotoxicity of nanocarrier. 2.8. Tumor Targeting Experiments. The established A549 lung cancer-bearing mice were applied to investigate the biodistribution of nanoparticles. When the tumor volume increasing to approximate 250 mm3, twelve mice were randomly grouped (n=3). Then they were intravenously injected with DiR-loaded NP, LinTT1-NP, TAT-NP, and LPT-NP, respectively, 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, MA, USA). Briefly, the filter for emission was set at 780 nm post 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 the WinLight 32 software. To clearly observe the nanoparticle distribution in organs, the tumor-bearing mice were subjected to euthanasia at 24 h post injection 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 with tumor penetrating, LinTT1 peptide was hence supposed to mediate a tumor inner drug delivery. To make a clarification, the randomly

grouped

(n=6)

tumor-bearing

mice

were

administrated

with

coumarin-6-labled NP, LinTT1-NP, TAT-NP or LPT-NP. Six hours later, 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 post the samples being frozen for 1 h at 80°C. To visualize the vessels within tumors, the Alexa Fluor 594-conjugated anti-mouse CD31 antibody was incubated with slides for 24 h. The results were obtained under the confocal microscope after staining the nuclei with DAPI. The remaining section was subjected to homogenizing on ice with 10 mL of deionized water. After addition of 10 µL of daunorubicin with the concentration of 10 µg/mL, the samples were processed as previously reported.40 Finally the levels of


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coumarin-6-labeled nanoparticles in tumor site were determined via the 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 TAMs model.41 For qualitative determination, cells were seeded in a glass dish with the density of 1 × 103 cells/cm2. After 24 h 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 co-incubation for one hour, 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 applied an antibody against F4/80 to incubate with the cells for overnight at 4°C. To visualize the TAMs, an Alexa 594-labelled secondary antibody (Invitrogen) was used to incubate for 1 h and subsequently stained with DAPI for observation of the nuclei. Finally, the results were determined under a confocal laser scanning microscopy. The quantitative evaluation was performed with the similar method described for HUVEC cells or A549 cells, and finally determined via a FCM analysis. For further investigation of TAM targeting capacity of LPT-NP-C6 in tumor site, six tumor bearing-mice were randomly grouped (n=3). Subsequently, the mice were systematic treated by NP-C6 and LPT-NP-C6, respectively. Six hours later, the treated mice were all executed with tumor samples were obtained and subsequently subjected to fast 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, performed the experiments as the same method as the immunofluorescence staining of TAM and analysis the distribution of nanoparticles under a confocal laser scanning microscopy. 2.10. Plasma Pharmacokinetics Study. In the determination of drug blood retention effect of nanoparticles prepared in this study, the plasma pharmacokinetics (PK) of PTX was examined. Briefly, fifteen male SD rats (200 ± 20 g) were randomly grouped (n = 3) and i.v injected with Taxol®, NP-PTX, LinTT1-NP-PTX, TAT-NP-PTX and LPT-NP-PTX, respectively, via the tail vein with the PTX dosage



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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) post-injection and centrifuged immediately at 3000 rpm for 10 min. To analysis the collected samples, 150 µL of methanol was added followed by centrifuging at 12000 rpm for 10 min. Then an equal volume of deionized water was added into the supernatant followed by vortexing for 5 min. Finally, the pharmacokinetic data analysis was conducted via the 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 extract 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 the voltage of 80 V. Proteins were then transferred onto a nitrocellulose membrane using a wet transfer method. And the membrane was blocked with 5% non-fat milk for 1 h, followed by being incubated overnight at 4°C with anti-MMPs 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 horse-radish 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 performed as above to use as the control. 2.12. Therapeutic Efficacy of Nanoparticles. In the anti-tumor efficacy experiments, tumor-bearing mice were established as above and randomly grouped (n=6). When the tumor volume increasing to approximate 100 mm3, the mice were intravenously injected with Taxol®, NP-PTX, LinTT1-NP-PTX, TAT-NP-PTX, and LPT-NP-PTX, respectively, with the PBS treated group as the control. Importantly, all of the mice were injected with PTX formulations every two days for a total of four injections with PTX dosage of 5 mg/kg. Then the body weight of each mouse was carefully monitored every 2 days over a whole period of 14 days and the tumor volume was


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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 tumors 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 the tumor cell necrosis and apoptosis. For the 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 multi-group comparison. Statistical significance was noted as follows: (*) p < 0.05; (**) p < 0.01; (***) p < 0.001. 3. RESULTS AND DISCUSSION 3.1. Characterization of Prepared Nanoparticles. The TEM photographs of nanoparticles exhibited that both NP-PTX and LPT-NP-PTX showed a generally spherical morphology and uniformly dispersed (Figure 1B). The average size 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. Besides, the polydispersity index (PDI) of NP-PTX and LPT-NP-PTX were both below 0.2 with the 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 zeta potential of -26.41 mV for NP-PTX and -21.52 mV for LPT-NP-PTX, respectively. 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-NP-PTX being 56.44 ± 2.13% and 54.12 ± 3.31%, respectively. What’s more, the



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physicochemical properties of LinTT1-NP-PTX and TAT-NP-PTX were also examined. Results showed in Table 1 suggesting that modification of peptide affect negligible 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 NP-PTX and LPT-NP-PTX exhibited a controlled release pattern when compared with Taxol®, which released about 90% of PTX within 6 h. Besides, 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 indistinctive difference was mainly contributed to the existing enzymes in plasma.39 Drug leaky during circulating 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 preconditions of drug delivery systems to achieve an efficient tumor-targeting therapy.42,43 In this study, the stability of drug-loaded nanoparticles was evaluated in the medium of DMEM containing 10% FBS. As shown in Figure 1E, both nanoparticles did not show a significant size change during the experiments, indicating that the prepared drug carrier was stable enough to deliver agents to specific sites in vivo. Table 1. Determination of the properties of PTX-loaded nanoparticles by the DLS.


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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. 3.2. Sensitivity of Polypeptide to MMPs. To examine 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 un-pretreated with MMPs inhibitors exhibited an almost negligible signal of red fluorescence while obvious co-localization of green and red signals was observed in the cells pretreated with excess MMPs inhibitors (Figure 2A). Furthermore, cells incubated with PTAT-NP-C6 in the presence of MMPs inhibitors displayed a negligible difference in cellular



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uptake of nanoparticles from the NP-C6 treated ones. However, the signal of fluorescence was significantly improved after cells co-incubating with only PTAT-NP-C6, suggesting that the cell penetrating capacity of TAT was mostly recovered (Figure 2B, 2C and 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.

Figure 2. (A) Confocal images of tumor cells after being treated with coumarin-6 and Cy5 dual-labeled LPT-NP with or without MMPs inhibitors. (B) Images of cellular uptake of PTAT-NP-C6 in the presence or absence of MMPs 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 a flow cytometry. 3.3. Cellular Uptake Experiments. To examine whether there is difference between the cellular association of NPs and LPT-NPs, both A549 lung cancer cells and tumor


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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 significant higher fluorescence signal compared 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 negligible difference for NP-C6, indicating that LPT-NP-C6 was not specificity to normal cells (Figure S1). Besides, cellular uptake of nanoparticles was obviously increased after nanoparticles being modified with TAT peptide, indicating that TAT could mediate a super cell penetration. In addition, pretreatment of cells with MMPs inhibitors significantly restricted the cellular association of LPT-NP-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 the investigation of effect of MMPs inhibitors on cellular internalization of nanoparticles, results showed in Figure S2 exhibited that there was no obvious difference for cellular association of NP-C6 with or without the presence of MMPs inhibitors, indicating that the inhibitors did not affect the endocytosis process. Such results were further proved by the quantitative analysis (Figure 3B and 3C). Moreover, cellular internalization of both NP-C6 and LPT-NP-C6 exhibited a concentration-depended manner and showed the biggest difference at the nanoparticle concentration of 200 µg/mL as demonstrated in Figure 3D and 3E. In conclusion, these results together suggested that the excellent affinity to tumors cells and angiogenesis of LPT-NP-C6 was mainly contributed by the properties of Lin-TT1 molecule. Besides, after clearing away the sensitive linker by MMPs, the TAT peptide further led to an efficient cell membrane crossing.



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Figure 3. (A) Images of cells post 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. To examine the mechanisms of cellular internalization of nanoparticles, cells were incubated with various endocytosis inhibitors for 1 h before being exposed to LPT-NP-C6. Results in Figure 4A displayed that the uptake of LPT-NP-C6 in


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HUVEC and A549 cells was obviously restricted by NaN3, filipin, genistein, M-β-CD, and moneisin, indicating that the endocytosis was an energy-dependent, lipid raft- and endosomes-mediated process. Besides, 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 microtubule was involved in the cellular association of nanoparticles in A549 cells (Figure 4B). For the subcellular co-localization investigation, a Lyso Tracker Red was applied to visualize the endosomes in cytoplasm of tumor cells. Results in Figure 4C and D illustrated that nanoparticles was co-localized well with endosomes after incubation for 0.5 h. However, when the time 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.



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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 h or 1 h. (D) Intracellular localization of C6-labeled LPT-NP after being incubated with A549 cells for 0.5 h or 1 h. Blue represents nuclei stained with DAPI. Green represents nanoparticles tracked by coumarin-6; red represents endosomes labeled by


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Lysotracker Red. 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 CCK-8 method. As illustrated in Figure 5A and B, cells treated with LPT-NP-PTX exhibited the lowest cell viability when compared with 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 super targeting performance and lead to an efficient cell penetrating 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 the cell viability of about 95% for HUVEC and A549 cells, suggesting a good biocompatibility for the prepared drug delivery vehicle. In addition, to qualitatively study the in vitro apoptosis-inducing capacity of these drug-loaded nanoparticles, the images of nucleus after the treatment of various PTX formulations with cells were obtained by a fluorescence microscope. Results in Figure 5C showed that both cells incubated with LPT-NP-PTX exhibited the most fragments while the control group displayed an intact spherical morphology.



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Figure 5. Cell viability of HUVEC cells (A) and A549 cells (B) post treatment by various nanoparticle formulations with the cells incubated by drug-free 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. 3.5. Tumor Targeting Ability of Nanoparticles. After the lung cancer-bearing mouse model being 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 tumor site when compared with TAT-NP. Importantly, the mice administered with TAT-NP showed an accumulation in tumor site as well as the normal tissues due to the lack of specificity of TAT peptide. For further evaluation of 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 semi-quantitative fluorescence analysis as displayed in Figure 6C and 6D.


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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 post injection of DiR-loaded NP (a), LinTT1-NP (b), TAT-NP (c) and LPT-NP (d). (C) & (D) Semi-quantitative investigation of the fluorescent intensity of DiR-labeled nanoparticles in different organs and tumors. 3.6. Distribution of Nanoparticle in Tumor Site. The distribution of nanoparticles in tumor site was 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 distributed into the interior of tumor. Besides, the TAT decorated nanoparticles also displayed a super tumor affinity and penetrating than that of 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 coumarin-6-loaded nanoparticles were homogenized and the nanoparticle concentrations accumulated in tumors were determined by the



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HPLC analysis. As depicted in Figure 7B, the distribution of nanoparticles was perfectly in accordance with the above results, suggesting a super targeting capacity of LPT-NP.

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 the HPLC analysis. 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 super TAM targeting capacity of LinTT1 peptide. Moreover, after conjugation with TAT moiety, the cellular uptake of nanoparticles was significantly increased due to the super cell penetrating ability of TAT peptide. These results were further confirmed by the quantitative investigation as shown in Figure 8B. To further evaluate 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


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sacrificed 3 h after injection with tumor tissues being then obtained. Thereafter, an immunofluorescence experiment was performed to verify the co-localization of TAM with nanoparticles in tumors. As shown in Figure 8C, a large amount of LPT-NP-C6 was co-localized well with TAM within tumor tissues while only a little amount of NP-C6 was detectable in tumor tissues with poor co-localization with TAM within tumors. The results above suggested that LPT peptide-modified nanoparticles were exactly a super tumor associate macrophages targeting drug delivery system.

Figure 8. Evaluate the TAM targeting capacity of LPT-NP-C6 through the 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, respectively. (B) Quantitative study of TAM uptake of different nanoparticles determined by a flow cytometer. (C) Co-localization 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



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signal represents F4/80 marked TAMs. 3.8. Therapeutic Efficacy of Nanoparticles. As encouraged by the above investigation, the anti-tumor efficacy of those nanoparticle formulations was further investigated. Firstly, the expression level of MMPs in tumor tissues was determined by a western blot assay. As shown in Figure 9A, a high level of MMPs expression was detectable in tumor while a negligible signal was determined in the normal tissues. Next, we investigated the pharmacokinetic of different formulations in vivo. As shown in the plasma concentration-time curves (Figure 9B), Taxol® exhibited the rapidest 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 times 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 post a programmatic therapy by various PTX formulations. As depicted in Figure 9B, 9C, 9D, 9E and 9F, passive targeting therapy of NP-PTX only showed a moderate inhibition of tumor growth with 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 anti-tumor effect after modification of active targeting peptide. Besides, 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 decrease in body weight ascribed to the toxicity of chemotherapeutics. In addition, the survival curve in Figure 9G showed that tumor-bearing mice 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 days to 20 days (NP-PTX), 32 days (TAT-NP-PTX) and 41 days (LinTT1-NP-PTX), respectively. To further evaluate 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-PTX


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treated mice showed the largest area of apoptosis, indicating the best tumor toxicity of LPT-NP-PTX compared with other formulations.

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 the tumors post dissected. (D) Relative body weight changes of tumor-bearing mice during the treatment. (E) Images of tumor tissues 14d 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 post treated by various PTX-loaded nanoparticles while the PBS group used as the control. 4. CONCLUSION For the treatment of lung cancer, we rationally developed a robust nanoplatform by intergrading 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



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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 tumor penetrating capacity as confirmed by the in vivo targeting assay. Tumor inhibiting, H&E and tunel assay confirmed that the tumor growth inhibition property can be significantly expanded by the combination therapy. All these results together implied a considerable potential of LPT-NP-PTX for clinical treatment of lung cancer. 5. 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). Supporting information Cellular uptake of NP-C6 or LPT-NP-C6 in different cells; Cellular uptake of NP-C6 in different cells with or without the presence of MMPs inhibitors.

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Table of Contents Graphic


Matrix Metalloproteinase Cleavable Nanoparticles for Tumor

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