Matrix Metalloproteinase Responsive ... - ACS Publications

Subscriber access provided by University of Otago Library

Article

Matrix Metalloproteinase Responsive Nanoparticles for Synergetic Treatment of Colorectal Cancer via Simultaneous Anti-Angiogenesis and Chemotherapy Leilei Shi, Yi Hu, Ang Lin, chuan ma, Chuan Zhang, Yue Su, Linzhu Zhou, Yumei Niu, and Xinyuan Zhu Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/ acs.bioconjchem.6b00643 • Publication Date (Web): 21 Nov 2016 Downloaded from http://pubs.acs.org on November 25, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Bioconjugate Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Bioconjugate Chemistry

Matrix Metalloproteinase Responsive Nanoparticles for Synergistic Treatment of Colorectal Cancer via Simultaneous Anti-Angiogenesis and Chemotherapy Leilei Shi,† Yi Hu,† Ang Lin,§ Chuan Ma,‡ Chuan Zhang,†* Yue Su,† Linzhu Zhou,† Yumei Niu,‖* and Xinyuan Zhu†*

†

School of Chemistry and Chemical Engineering, State Key Laboratory of Metal

Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, People’s Republic of China ‡

School of Dental Medicine, Shanghai Jiao Tong University, 227 South Chongqing Road, Shanghai, 200025, P. R. China §

Department of Medicine, Immunology and Allergy Unit, Karolinska Institute, Stockholm, SE 17176, Sweden

‖

Department of Endodontics, The First Affiliated Hospital of Harbin Medical University, 143 Yiman Street, Harbin 150001, P. R. China

*Corresponding author. E-mail: [email protected], [email protected], [email protected]; Telephone: +86-21-54746215; Fax: +86-21-54741297.

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Colorectal cancer (CRC) is one of the most commonly diagnosed cancers worldwide, especially in developed countries. Although patients’ overall survival has been improved by either conventional chemotherapy or newly developed anti-angiogenesis treatment based on its highly vascularized feature, the relatively low therapeutic efficacy and severe side effects remain as big problems in clinical practice. In this study, we describe an easy method to construct a novel matrix metalloproteinase-2 (MMP-2) responsive nanocarrier, which can load hydrophobic agents (camptothecin and sorafenib) with high efficiency to exert synergistic efficacy for CRC treatment. The drug-containing nanoparticles can particularly respond to the MMP-2 and realize the controlled release of payloads at the tumor site. Moreover, both in vitro and in vivo studies have demonstrated that this responsive nanoparticle exhibits much higher therapeutic efficacy than those of single antitumor agents or combined drugs co-administrated in traditional ways.

INTRODUCTION Colorectal cancer (CRC) ranks fourth in the numbers of the most commonly diagnosed cancer worldwide, especially in developed countries.1 It is estimated that around 1.2 million people are diagnosed with CRC each year with 50% death cases inside.2 Although patients’ overall survival has been improved by either conventional chemotherapy or newly developed anti-angiogenesis treatment based on its highly vascularized feature, the relatively low therapeutic efficacy and severe side effects remain as big problems in clinical practice.3 To give a better therapeutic effect, people have explored combination therapy that co-administrates anti-angiogenesis and chemotherapeutic agents for CRC treatment, but the enhancement of therapeautic efficacy and survival benefits, thus far, is rather modest.4,5 In a combination CRC treatment, synergistic effect using above two types of antitumor agents is often limited by their different solubility, administration route, pharmacodynamic properties, and


Page 2 of 31

Page 3 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


targeting capability. It is worthy noting that chronic and high-dose angiogenesis inhibition administrated by conventional oral intake or vein injection can significantly reduce the exposure of co-administered chemotherapeutic agents, reducing the overall efficacy.6 To address these issues, nanoparticle-based drug delivery system has been proposed as a promising way for CRC combinatorial therapy since it has shown many advantages in a wide range of cancer treatment, such as enhanced solubility, extended circulating time, altered biodistribution and bioavailability, reduced cellular toxicity, and passive or active targeting capability.7 In general, an ideal nanoparticle delivery system should keep the nanocarrier and its payload intact before reaching the tumor tissue and release the effective agents after shooting their target. Taking this into account, developing smart nanoparticles equipped with synergistic antitumor agents and responsive capability to the specific tumor micro-environment is highly motivated for CRC therapy.8-14 Similar to many other solid tumors, CRC has a distinctive architecture and peripheral micro-environment which isolate them from the normal tissues. CRC micro-environment is highly specific due to the extensive secretion of various proteinases, for example, matrix metalloproteinase (MMPs).15 Growing evidences have shown that MMPs, especially MMP-2 and MMP-9, are highly related to CRC tumor metastasis, invasiveness and angiogenesis.16,17 To obtain an improved CRC treatment efficacy, in this study, smart nanocarrier that can respond to such micro-environment is particularly designed to enhance the synergistic effect of simultaneous anti-angiogenesis and chemotherapy. As shown in Figure 1, we synthesize a novel MMP-2 responsive nanocarrier assembled by amphiphilic polyethylene glycol (PEG)-peptide diblock copolymer (PPDC) for simultaneously loading

both

hydrophobic

antitumor

agents.

Upon

exposing

to

CRC

micro-environment, the peptide segments could be digested by overexpressed MMP-2 and then the pre-loaded therapeutics could be released for CRC suppression via cooperative anti-angiogenesis and chemotherapeutic mechanism. To the best of our knowledge, there are few examples of co-delivery of anti-angiogenesis agent and



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

chemotherapeutics via MMP-2 responsive nanoparticles for CRC synergistic therapy. RESULTS AND DISCUSSION Synthesis and Characterization of PPDC. Firstly, methoxyl PEG (mPEG, Mw: 2 kDa, red segment of PPDC) was reacted with succinic anhydride to obtain carboxyl terminated PEG (mPEG-COOH). Then the product was further conjugated with a hydrophobic peptide (CPLGLAGG, blue segment of PPDC) which contained a substrate for MMP-2 digestion (Figure 1) to form PPDC via amide bond formation. The synthesized polymer was characterized using 1H nuclear magnetic resonance (1H NMR), infrared (IR) spectroscopy, and high performance liquid chromatograph (HPLC) (Figure 2, Figure S1, Supporting Information), by which the successful synthesis of PPDC was confirmed.

Figure 1. The synthesis of MMP-2 responsive amphiphilic polyethylene glycol


Page 4 of 31

Page 5 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(PEG)-peptide diblock copolymer (PPDC), nanoparticle assembly, and subsequent disassembly in response to MMP-2 proteinase, then blocking the formation of neovascularization and promoting the apoptosis.

Figure 2. 1H NMR analysis of the synthesized polymers. (upper panel) 1H NMR spectrum of mPEG-COOH. (400 MHz, DMSO-d6); (bottom panel) 1H NMR spectrum of PPDC. (400 MHz, DMSO-d6).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fabrication of Camptothecin (CPT), Sorafenib-Loaded Nanoparticles and PPDC Nanoparticles. Due to its amphiphilic feature, PPDC could assemble into nanosized spherical micelles with a hydrophobic peptide core inside and hydrophilic PEG corona surrounding the core. Therefore, antitumor drugs could be loaded into the core area through hydrophobic interaction. Upon exposing to MMP-2, the peptide segment would be digested by the proteinase and the micellar structure gradually became unstable, triggering the release of therapeutics at the tumor site (Figure 1). In this study, ultrasonication method was applied to fabricate the drug-containing PPDC micelle nanoparticles. Briefly, 5 mg sorafenib (a typical anti-angiogenesis agent), 5 mg CPT (a representative chemotherapeutics), and 10 mg PPDC were suspended in the ultrapure water and sonicated for 2 min via ultrasonic cell disruptor. Then, the non-encapsulated drugs were removed by centrifugation since they remained as large aggregates in the aqueous solution. After preparation, the size and morphology of as-synthesized drug-containing nanoparticles were characterized using dynamic light scattering (DLS) and transmission electron microscopy (TEM). As shown in Figure 3, the average hydrodynamic size of the responsive nanoparticles loaded CPT and sorafenib is about 105 nm based on DLS measurement. Comparatively, nanoparticle size in the TEM image is ~ 90 nm in diameter, which is slightly smaller than that of DLS result due to the drying and shrinking effect under high vacuum condition. However, the average hydrodynamic size of the responsive nanoparticles without loading drugs is about 55 nm which is smaller than that of drug-loaded nanoparticles based on DLS measurement.

Figure 3. TEM image of drug loaded MMP-2 responsive nanoparticles and amiphilic polymer nanoparticles, the scale bar is 100 nm (A) and representative DLS measurement (B, C).


Page 6 of 31

Page 7 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


In Vitro Drug Release. With drug-loaded nanoparticles, we first tested their capability of responsive and controllable drug release. In physiological condition without external stimuli, the CPT-containing nanoparticles are stable and the accumulative release of loaded antitumor therapeutics is about 30% in 72 h (Figure 4). In contrast, when incubated with MMP-2 proteinase, CPT releases rapidly from the nanocarrier with an accumulated release ratio of ~85% in 72 h monitored by HPLC. To demonstrate that the release is really triggered by MMP-2 proteinase digestion, MMP-2 inhibitor (Marimastat, 1 M) was added and incubated with MMP-2 and drug-containing nanoparticles together. It is found that the CPT release dramatically slows down. The inhibition of drug release in the presence of MMP-2 inhibitor verifies that the novel nanoparticle based carriers can respond to cancer specific micro-environment and achieve a controllable drug release at the tumor site.

Figure 4: Accumulative release of CPT from nanoparticles in different media: (1) PBS buffer with MMP-2 (2 μg/mL), (2) PBS buffer, (3) PBS buffer with MMP-2 and MMP-2 inhibitor Marimastat (1 mM). In Vitro Cytotoxicity Evaluation of PPDC and Drug-Loaded Nanoparticles. As a new material used for medical purpose, its biocompatibility should be taken into consideration at first place.18 Therefore, we evaluated the potential toxicity of PPDC to colorectal cancer cell line (HT-29) via standard methyl thiazolyl tetrazolium (MTT) assay. As demonstrated in Figure 5A, even incubating with polymers at the concentration of 320 μg/mL for 72 h, HT-29 cells still remain at relatively high cell



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

viability (~80%), indicating the nanocarrier used in our study has no obvious cytotoxicity. With well-established responsive property and low cytotoxicity of the carrier, we further investigated the therapeutic efficacy of the combination therapy using the prepared drug-containing nanoparticles for in vitro study. HT-29 cells that highly express the MMP-2 proteinase and negative control 3T3 cells with low level of MMP-2 expression were incubated with free drugs (CPT, sorafenib, or CPT/sorafenib mixture), CPT-loaded only, sorafenib-loaded only, and nanoparticles loaded with both drugs (containing (CPT:7 μg/mL)，(sorafenib:4.2 μg/mL)) at different concentrations for 72 h. Cells without any treatment were also used as control. Based on MTT results, dose-dependent cytotoxicity to HT-29 cells was observed after treatment with all drug-containing samples, with 50% cellular growth inhibition (IC50) value of 1.213, 0.085, 7.29, 0.012 μg/mL for CPT, sorafenib, drug mixture, nanoparticles, respectively. Moreover, it could be noticed that the combined administration of CPT and sorafenib simultaneously exhibits obvious synergistic effect (Figure 5B). As the expression level of MMP-2 is low in 3T3 cells, both sorafenib and nanoparticles give the low cytotoxicity to 3T3 cells in contrast to CPT and drug mixture (Figure 5C). All these results demonstrate that our drug delivery system could realize the responsive release of therapeutics at the tumor site while being stimulated by specific cancer micro-environment.

Figure 5. (A) cell viability rate of HT-29 cells cultured with mPEG-CPLGLAGG at different concentrations; (B) cell viability rate of HT-29 cells cultured with CPT, sorafenib, drug mixture, drug containing nanoparticles at different concentrations; (C) cell viability rate of 3T3 cells cultured with CPT, sorafenib, drug mixture, drug containing nanoparticles at different concentrations. Data are represented as average ± standard error (n = 3).


Page 8 of 31

Page 9 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


In Vitro Apoptosis-Inducing Effect of Drug-loaded Nanoparticles. To determine whether the inhibition of cancer cell proliferation by these drug-loaded nanoparticles was a consequence of CPT and sorafenib induced apoptosis, we conducted the FITC-Annexin V/PI double-staining assay in HT-29 cells.19 First, HT-29 cells were incubated with 1 μg/mL CPT, 0.6 μg/mL sorafenib, CPT/sorafenib mixture (1 μg/mL + 0.6 μg/mL), and nanoparticles containing 1 μg/mL CPT and 0.6 μg/mL sorafenib for 48 h followed with FITC-Annexin V/PI staining. At the same time, HT-29 cells without any treatment were used as control. Flow cytometry analysis indicates that the frequencies of apoptotic cells are 65.4%, 45.3%, 71.6%, 58.9% induced by CPT, sorafenib, CPT/sorafenib mixture, nanoparticles, respectively (Figure 6). The apoptosis assay indicates that nanoparticles do not exhibit the superiority over the free drugs in traditional 2D culture micro-environment. The reason for such result we speculate is that the excretion of MMP-2 is relatively low when HT-29 cells are cultured in 2D micro-environment, leading to incomplete drug release from nanocarrier.

Figure 6. Apoptosis of HT-29 cells incubated with CPT, sorafenib, drug mixture, drug containing nanoparticles for 48 h by flow cytometry analysis. Inserted numbers in the profiles present the percentage of the cells in this area. Lower left: living cells; upper left: necrotic cells; lower right: early apoptotic cells; upper right: late apoptotic cells. Each experiment group is repeated three times.

3D Tumor Spheroid Inhibition Evaluation of Drug-Loaded Nanoparticles. Wu and coworkers had reported that the expression of MMP-2 was increased when tumor cells were cultured in 3D micro-environment.20 As 3D culture model could better simulate in vivo growth environment of cancer cells, as well as to verify our



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

speculation, herein 3D culture model for HT-29 cells was constructed to evaluate the different drug formulations. After the formation of tumor spheres, the cells were incubated with aforementioned formulations (free CPT, sorafenib, CPT/sorafenib drug mixture, and drug-containing nanoparticles) used in 2D culture. The volume of tumor spheres was detected by fluorescence microscope on day 0, 3, and 7. It could be noticed that drug-containing nanoparticles exerted the highest inhibiting effect to tumor sphere when compared with other three cases, indicating that the expression level of MMP-2 was significantly improved in the 3D culture environment. When MMP-2 inhibitor (Marimastat, 1 M) was added, tumor sphere inhibiting efficacy was obviously reduced. This result further indicated that our nanoparticles were MMP-2 responsive (Figure 7A & 7B).

Figure 7. (A) Representative photos of tumor spheroids after treatment with various formulations and blank controls, the scale bar is 100 μm; (B) The ratio of volume change of HT-29 spheres volume (%) after applying different formulations. Data are represented as average ± standard error (n = 3), and the statistical significance level is * p < 0.05, **p < 0.01. Pharmacological Mechanism Evaluation of Synergistic Effect. Although the in vitro assay showed that nanoaprticles containing both CPT and sorafenib and drug mixture exhibited synergistic effect to HT-29 cells, pharmacological mechanism of synergistic effect was still not clear in this case. Sorafenib, a multi-targeted tyrosine kinase inhibitor, mainly exerts anti-angiogenic effect by blocking VEGF receptor


Page 10 of 31

Page 11 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(VEGFR). Additionally, it regulates the Raf-MEK-ERK pathway by inhibiting B-Raf kinase. Therefore, it can strongly affect tumor cell growth. Based on these knowledge, western blot analysis was carried out to identify how different drug combination could exert synergistic effect. After incubating HT-29 cells with CPT, sorafenib, drug mixture for 36 h, they were harvested and the cellular proteins were extracted in Laemmli buffer and then protein contents in the extracts were quantified using a bicinchoninic acid (BCA) protein assay kit. Equal amounts of proteins (20 μg per lane) were used for western blot analysis. The results show that the total ERK of HT-29 cells has no obvious change after incubation with sorafenib, CPT, drug mixture, respectively. The negative control shows that ERK signaling is activated in HT-29 cells, revealed by the phosphorylation of ERK. When sorafenib and drug mixture are added, the phosphorylation of ERK is decreased. However, CPT alone cannot inhibit phosphorylation of ERK. As Raf-MEk-ERK pathway could promote tumor cell proliferation and growth, inhibiting this signaling pathway could induce a higher level of apoptosis. In Figure 8, it can also be noticed that both CPT alone and sorafenib alone down-regulates the expression of anti-apoptosis protein BCL-2 while the drug mixture almost completely inhibits its expression. Based on the western blot analysis, synergistic effect induced by drug mixture can be attributed to the inhibition of Raf-MEK-ERK pathway and the down-regulation of BCL-2 expression.

Figure 8. The expression level of ERK, p-ERK, BCL-2 and MCL-1 in HT-29 cells induced by drug mixture, CPT and sorafenib at a certain concentration (CPT 2 μg/mL, sorafenib 1.2 μg/mL) for 36 h, determined by western blot analysis. Cells untreated are used as the control, and the β-actin is the loading control. Data represent three individual experiments. Each experiment group is repeated three times.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

In Vivo Photoacoustic Imaging for Detecting the Angiogenesis of HT-29 Bearing Mice. To evaluate the anti-angiogenesis effect induced by the responsive and combination nanodrug, dynamic photoacoustic (PA) imaging of CRC peripheral vasculature in the tumor bearing mice was conducted. During the imaging process, hemoglobin was used as an endogenous contrast agent, which can generate sufficient PA signals for vasculature 3D tomography.21-23 Based on this technique, the efficacy of responsive nanodrug in HT-29 tumor bearing mice can be evaluated in real-time. First, in vivo PA imaging was performed on the HT-29 xenograft tumor model to validate the potential of anti-angiogenesis. Before intravenous injection of therapeutics into nude mice, the absorption of oxyhemoglobin and deoxyhemoglobin in the near-infrared region was monitored via PA imaging. After systematic administration of PBS (200 μL), CPT (8 mg/kg), sorafenib (3.8 mg/kg), drug mixture (8 mg/kg + 3.8 mg/kg), and drug-containing nanoparticles for one week, the absorptions of oxyhemoglobin and deoxyhemoglobin were monitored again to detect the changes of PA signal. It is noticed that the intensities of PA signal increase obviously after injection of PBS and CPT, indicating that conventional chemotherapeutics cannot block the process of angiogenesis. Due to the low bioavailability of free sorafenib, there is a little enhancement of PA signals after administration of free sorafenib and drug mixture (Figure 9 & Figure S4, Supporting Information). However, when our responsive nanoparticles were used, it can significantly inhibit neovascularization and there is almost no change of PA signals for the whole week. From the PA imaging assay, we can conclude that sorafenib-loaded responsive nanoparticles really accumulate in the tumor tissue and then exert good anti-angiogenesis effect.


Page 12 of 31

Page 13 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 9. In vivo photoacoustic imaging for detecting the angiogenesis of HT-29 bearing mice after intravenous injection of different formulations and the quantitative analysis of hemoglobin intensity. Data are represented as average ± standard error (n = 4). In

Vivo

Pharmacokinetics

Investigation

and

Targeting

Capacity

of

Nanoparticles. As nanoparticles with a suitable size (< 200 nm) usually display longer retention time in the bloodstream compared to free drugs.24,25 The pharmacokinetic study was carried out by intravenous injection of free CPT, sorafenib, and nanoparticles to Sprague-Dawley (SD) rats (nearly 220 g). Figure 10A shows the time profiles of free CPT, sorafenib, and drug loaded nanoparticles (Containing CPT (880 μg/mL) and sorafenib (522 μg/mL)) in plasma, which were quantified by HPLC. It can be found that nanodrug is retained at a higher concentration in the bloodstream up to 12 h compared with free drugs. Therefore, the longer retention time of nanoparticles provides the possibility of enhanced drug accumulation in the tumor tissue. To further evaluate the passive targeting of nanoparticles, Cy5.5, a near-infrared fluorescence dye was loaded in the nanoparticles (Figure S5, Supporting Information). Then free Cy5.5 and Cy5.5-loaded nanoparticles were intravenously injected via tail vein into HT-29 tumor-bearing nude mice. Biodistribution profiles show that a large amount of Cy5.5-loaded nanoparticles accumulate in the tumor in the first 2 h. At 6 h after injection, the quantity of Cy5.5-loaded nanoparticles decreases by about 50%. However, the free Cy5.5 is almost completely eliminated at



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

the same point in time due to the fast clearance of the small molecules (Figure 10B). These data indicate that Cy5.5-loaded nanoparticles could really be accumulated in tumors by passive targeting through enhanced permeability and retention (EPR) effect. Moreover, the real-time imaging of nanoparticles in the tumor-bearing mice was monitored over the course of 8 h (Figure 10C). The near-infrared fluorescence signals of Cy5.5-loaded nanoparticles at the tumor site are observed after 2 h, and then decrease from 6 h to 8 h. In contrast, for the free Cy5.5 injection, accumulated fluorescence signals could not be observed at the tumor site from 0.5 h to 8 h, indicating that longer rentation time can promote the tumor accumulation of nanoparticles.

Figure 10. (A) Representative plasma concentration-time profiles of free CPT, sorafenib, and nanoparticles after i.v. injection into rats (a dose of CPT 8 mg/kg, sorafenib 1.5 mg/kg). The data are presented as the average ± standard error (n = 5).


Page 14 of 31

Page 15 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(B) Tissue distribution of Cy5.5 after intravenous injection of free Cy5.5 (5 mg/kg) and Cy5.5 (5 mg/kg) loaded nanoparticles, and the statistical significance level is ***p < 0.001. (C) In vivo near-infrared images of time-dependent whole body imaging of HT-29 bearing nude mice after intravenous injection of free Cy5.5 and Cy5.5 loaded nanoparticles. In Vivo Antitumor Efficacy. Based on positive results in vitro, a study of in vivo tumor growth inhibition was subsequently conducted to assess the antitumor effects of dual drug containing and responsive nanoparticles. 25 nude mice bearing HT-29 tumors were divided into five groups. As displayed in Figure 11A, after 20 days treatment, the volume of HT-29 tumor treated with PBS (200 μL) via tail vein injection increases about from 100 mm3 to 1500 mm3, demonstrating no antitumor effects. When mice were treated with CPT (8 mg/kg, tail vein injection) and sorafenib (3.8 mg/kg, tail vein injection) alone, the volume of tumor increases by 1100 mm3 and 600 mm3, respectively, suggesting a single antitumor agent in this study cannot inhibit tumor growth effectively. As a comparison, tumor volume is only raised by ~200 mm3 when administrating the drug mixture (tail vein injection), indicating that combined administration can exert remarkable synergistic effect. In contrast, mice treated with nanoparticles by tail vein injection show prominent tumor inhibition, as the volume of tumor undergoes almost no change. The body weight changes are further shown in Figure 11B. It could be noticed that injecting CPT and drug mixture (CPT combined with sorafenib) results in an obvious weight loss of tumor mice, indicating the severe toxicity of traditional chemotherapeutics. For other groups, the weights of the tumor mice do not undergo large changes. According to the tumor volume variation, tumor growth inhibition rate is calculated to quantitatively evaluate the antitumor effect. As shown in Figure 11C, our responsive nanoparticles exhibit the most superior inhibition rate compared to other groups. Finally, the efficient and synergistic apoptosis and anti-angiogenesis in the combination therapy using the responsive nanoparticles were further investigated by TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling) staining and immunofluorescence analysis. The sections of tumor



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

tissue in all groups were collected and TUNEL assay was performed using the in situ cell death detection kit. The percentage of TUNEL-stained cells was observed at a 200x magnification field. The results demonstrate that nanoparticles induce tumor cell apoptosis most effectively (Figure 12A). To detect the vascular function and density within the tumor, the excised tumors were sectioned for anti-CD31 antibody and DAPI staining. Immunofluorescence assay suggests that our responsive nanoparticles result in fewer functional blood vessels compared to other formulations, which is consistent with the PA imaging (Figure 12B). Histological examination of hematoxylin and eosin (H&E) stained tumor tissue sections were conducted for observing tissue morphology between PBS and drug-treated groups. The tumor cells treated with PBS group were observed with large nucleus in the tumor tissue, indicating a rapid tumor growth. In contrast, the tumor cellularity, as evaluated by average tumor cell numbers of each microscopic field, decreases obviously, and nucleus shrinkage and fragmentation are observed in the CPT, sorafenib, drug mixture, and nanoparticle treated groups. Meanwhile, a large necrotic area is observed in the nanoparticle group. H&E staining assay reveals significant difference of tissue morphology between PBS and treated groups, especially for the nanoparticle case (Figure 12C).


Page 16 of 31

Page 17 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 11. In vivo anticancer activity. (A) Changes of tumor volume after intravenous injection of PBS, CPT, sorafenib, drug mixture, and nanoparticles in HT-29-bearing nude mice. (B) Body weight changes of HT-29-bearing nude mice after treatment with PBS, CPT, sorafenib, drug mixture, and nanoparticles. (C) The tumor inhibition rate after treatment with PBS, CPT, sorafenib, drug mixture, and drug-containing nanoparticles in HT-29-bearing nude mice. Tumor inhibition rate (TIR) is calculated as the following equation: TIR (%) = 100 × (mean tumor weight of control group – mean tumor weight of experimental group)/mean tumor weight of control group. Data are represented as average ± standard error (n = 5), and the statistical significance level is *p < 0.05, **p < 0.01, ***p < 0.001. .



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 12. TUNEL assay, immunofluorescence, and H&E analysis of the tumor tissue sections. (Upper panel) Histological images of the tumor tissue using TUNEL assay. Green: apoptosis cells. Blue: DAPI-stained cell nuclei. Original magnification: 200×. (Middle panel) Tumor associated blood vessels (red) were stained with antibody against CD31. The cell nuclei (Blue) were stained with DAPI. Original magnification: 200×. (Bottom panel) Tumors are sectioned and stained with H&E. Original magnification: 200×. CONCLUSIONS In summary, we rationally designed an enzyme-responsive drug nanocarrier that was easy to synthesize and could be used for simultaneously deliver CPT and sorafenib to realize combination therapy. The drug-containing nanoparticles could particularly respond to the MMP-2 proteinase and achieve the smart and controlled release of agents at the tumor site. Moreover, both in vitro and in vivo studies have demonstrated that the responsive nanodrug exhibited much higher tumor inhibition effect than single antitumor agents or their mixture administrated in a traditional way. When cancer cells are treated with our smart and dual drug containing nanoparticles, synergistic effect of chemotherapy and anti-angiogenesis has been clearly validated through various characterizations and analysis. With the unique responsiveness to cancer specific micro-environments, we believe this new type of nanoparticle-based delivery system and combination antitumor strategy may be applied to clinic practice for CRC treatment in future.


Page 18 of 31

Page 19 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


EXPERIMENTAL SECTION Materials. All chemicals were purchased from Sigma-Aldrich or Adamas. Commercially available reagents were used without further purification. All chemicals were reagent grade or better. Sorafenib and camptothecin (CPT) were purchased from Meilun Biotech (Dalian, China). The protease sensitive peptide substrate (CPLGLAGG) was obtained from Ketai (Shanghai, China). MMP-2 and antibodies used for western blot analysis were obtained from Abcam. MMP-2 inhibitor (Marimastat) was obtained from Med ChemExpress. Dulbecco’s modified eagle’s medium (DMEM), fetal bovine serum (FBS) were purchased from Gibco. The Annexin V-FITC/PI apoptosis detection kit was purchased from Invitrogen and used as received. Dialysis tubes were purchased from Shanghai Lvniao Technology Corp. Clear polystyrene tissue culture treated 6-well, 12-well and 96-well plates were obtained from SangonBiotech (Shanghai, China). Measurements. Nuclear magnetic resonance (NMR) spectroscopy (Mercury plus 400 spectrometer 400 MHz, Varian, U.S.A.) was used with dimethyl sulfoxide-d6 (DMSO-d6) as solvents. Transmission electron microscopy (B-TEM, Tecnai G2 Spirit Biotwin) studies were performed to investigate the morphology and size of micelles. A drop of the micelle solution (0.5 mg/mL) was spread onto an amorphous holey-carbon film supported by a copper grid, then lyophilized by a freeze-dryer for observation. Dynamic light scattering (DLS) measurements were performed with a Malvern Zetasizer Nano S apparatus equipped with a 4.0 mW laser operating at λ = 633 nm. All samples were measured at a scattering angle of 173°. Synthesis of mPEG-COOH. Following previous literature,26 both methoxyl poly(ethylene glycol) (mPEG, Mw: 2.0 kDa) (2 g, 1 mmol) and succinic anhydride (SA) (120 mg, 1.2 mmol) with mole ratios (1:1.2) were added into a flask with pyridine as a solvent. Then the mixture was stirred by a magnetic bar in an oil-bath at 60 oC for 24 h. After pyridine was removed by reduced pressure distillation, the crude was precipitated by ice ether. After vacuum drying, the product was obtained (1.8 g, yield 84.7%) and then characterized by 1H NMR (Figure 1).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 31

Synthesis of mPEG-Peptide.27 To a solution of mPEG-COOH (1 g, 0.5 mmol) in N,N-dimethylformamide (DMF, 10 mL), N-hydroxysuccinimide (NHS, 75 mg, 0.65 mmol)

was

added

with

an

ice/water

bath.

Then

1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC, 125 mg, 0.65 mmol) was added into the mixture and stirred for overnight. The generated mPEG-NHS ester was used in the next step without further purification. Peptide (301 mg, 0.5 mmol) was suspended in DMF (2 mL), then mPEG-NHS ester (1.05g, 0.5 mmol) and triethylamine (TEA, 100 μL) were added. After 24 h reaction, DMF was removed by rotary evaporators. Then, the crude was re-suspended in the water and purified with cellulose dialysis membrane (molecular weight cut-off (MWCO): 1.0 kDa) against deionized water for removing TEA and EDC. Then, preparative chromatograph was used to remove the free peptide. After lyophilization, the product was obtained (0.76 g, yield 58%) and characterized by 1H NMR and IR. (Figure 2 and Figure S1). Preparation of CPT, Sorafenib-Loaded Nanoparticles. Briefly, Sorafenib (5 mg), CPT (5 mg), mPEG-peptide (10 mg) were suspended in the ultrapure water and sonicated for 2 min via ultrasonic cell disruptor (Xo 1200D) where the ultrasonic power was 240 W. After ultrasound, the obtained suspension was centrifuged with 3000 rpm for 10 min. The supernate was used for transmission electron microscopy (120 kV B-TEM) and dynamic light scattering (DLS, Malvern instruments) analysis. Drug Loading Content and Encapsulation Efficiency Measurement. The obtained nanoparticles were dissolved in methanol. The drug concentration was determined by HPLC (Agilent 1200). The fluorescence intensity of CPT was measured by HPLC (Agilent 1200) with methanol (V/V 60%) and water (V/V 40%) as the eluent at an excitation wavelength of 470 nm and emission wavelength of 370 nm. Sorafenib was measured via ultraviolet detector at the wavelength 266 nm with acetonitrile (V/V 49%), 0.1% TFA (V/V 20%), H2O (V/V 31%) as the eluent. The standard curve of CPT and sorafenib was displayed in Figure S2. The loading contents of CPT and sorafenib were about 960 μg and 575 μg, respectively. The mass ratio of CPT and sorafenib in the mixture group was subsequently adjusted to 1.67:1, as the


Page 21 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


same as the ratio of drug-containing nanoparticles. The encapsulation efficiency of CPT and sorafenib was 19.2% and 11.5%, respectively. DLC (wt%) = (weight of loaded drug/weight of polymer) ×100% Encapsulation Rate (ER) (%) = (weight of loaded drug/ total drug) × 100% In Vitro Drug Release from Nanoparticles. 2 mL of nanoparticles solution was added into a dialysis tube (MWCO: 3.5 kDa) 18 mL of the PBS buffer solution (containing 0.1% Tween-8) at different conditions (PBS only, MMP-2, MMP-2 + (Marimastat, 1 μM)), and gently shaken at 37 oC in a shaker at 120 rpm. At predetermined time intervals, the total buffer solution was withdrawn, followed by replacing with 18 mL of fresh buffer solution with the same condition. The fluorescence intensity of CPT released was measured by HPLC (Agilent 1200) with methanol (V/V 60%) and water (V/V 40%) as the eluent at an excitation wavelength of 470 nm and emission wavelength of 370 nm. Cell Cultures. HT-29 cells were cultured in McCoy’s 5A medium (Gibco) supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin at 37 oC, 5% CO2 and humid atmosphere. 3T3 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Hycolon) supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin at 37 oC, 5% CO2 and humid atmosphere. Three-Dimensional (3D) Tumor Sphere Construction.28 Agarose solution was prepared in serum-free DMEM (2%, w/v) by heating at 85 oC for 30 min. Normal 24-well plate was coated with 2% agarose solution to reduce the surface absorption. Then, HT-29 cells (4000 cells/400 μL per well) were suspended in McCoy’s 5A medium and seeded in 24-well plate. The cells were cultured at 37 oC in a humidified atmosphere containing 5% CO2. After 4 days, the formed tumor spheres were used for biological evaluation. In Vitro Cytotoxicity Studies. HT-29 and 3T3 cells were seeded in the 96-well plates. 12 h later, the cells were treated with mPEG-peptide, CPT, sorafenib, drug mixture, nanoparticles containing both CPT and sorafenib with different



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

concentrations and incubated for 72 h. Then, 20 μL MTT solution (5 mg/mL) was added. After 4 h incubation, the medium was replaced with DMSO. The obtained blue formazan crystals were dissolved in 200 μL per well DMSO and the absorbance was measured in a BioTek® Synergy H4 at a wavelength of 490 nm. Apoptosis Analyses with Flow Cytometry. Apoptosis of HT-29 cells was measured by FITC-Annexin V/propidium iodide (PI) method. HT-29 cells were incubated with CPT, sorafenib, CPT/sorafenib mixture, nanoparticles at a concentration (CPT 2 μg/mL, sorafenib 1.2 μg/mL). After 48 h incubation, FITC-Annexin V/PI was used to stain with the cells. The cells without any treatment were used as control. The samples were analyzed with a BD LSR Fortessa flow cytometer. Tumor Sphere Inhibition Evaluation. The tumor spheroid inhibition evaluation was conducted according to the procedure reported in the literature.29 Briefly, the serum-free McCoy’s 5A medium with different drug formulations, including CPT, sorafenib, CPT/sorafenib mixture, nanoparticles were added into the 24-well plate where HT-29 3D tumor sphere had been formed. The final concentrations of CPT and sorafenib was 2 μg/mL and 1.2 μg/mL, respectively. The HT-29 spheroids incubated in McCoy’s 5A medium without any treatment were used as blank control. After treatments, tumor spheres were observed under an inverted microscope (Leica, Germany) on day 0, 3, 7. The volumes of spheres were calculated by following formula: V = (π×dmax×dmin)/6 where dmax is the major diameter and dmin is the minor diameter of each spheroid. The growth inhibition rate was calculated with the following formula: Inhibition Rate (%) = [1-(Vday7/Vday0)]×100 Western Blot Analysis. HT-29 cells were seeded in the 6-well plates at a density of 1.0 × 106 cells in 4 mL of complete McCoy’s 5A medium and allowed to attach for 12 h. The cells were treated with CPT (2 μg/mL), sorafenib (1.2 μg/mL), drug mixture (2 μg/mL + 1.2 μg/mL) for 36 h. HT-29 cells without any treatment were used as a


Page 22 of 31

Page 23 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


negative control. After treatment, cells were harvested. The cellular proteins were extracted in Laemmli buffer and the protein content in the extracts was quantified using a bicinchoninic acid (BCA) protein assay kit (Pierce, USA). Equal amounts of proteins (20 μg/lane) were separated on sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and electrotransferred to 0.45 μm polyvinylidene fluoride (PVDF) membranes. The membranes were then blocked with 5% non-fat dry milk in TBST (Tris buffered saline supplemented with 0.1% Tween-20) and probed with antibodies against β-actin (1:1000 dilution), BCL-2 (1:1000 dilution), p-ERK (1:1000 dilution), followed

by

HRP-conjugated

(HRP:

horseradish

peroxidase)

anti-mouse

immunoglobulin-G (IgG, 1:5000 dilution). β-Actin was used as the loading control. Protein bands were visualized using Chemiluminescent HRP Substrate (Themo Scientific, USA) S13 according to the manufacture’s protocol and analyzed using the ChemiDocTM MP Imaging System (Bio-Rad, USA). In Vivo Photoacoustic Imaging. A commercial Endra Nexus 128 PA tomography system (Endra Inc., Ann Arbor, Michigan) was utilized in our experiment. The system provides a tunable nanosecond pulsed laser (7 ns pulses, 2 Hz pulse repetition frequency, 7 mJ/pulse on the animal surface, wavelength range 680-950 nm), 128 unfocused ultrasound transducers (with 5 MHz center frequency and 3 mm diameter) arranged in a hemispherical bowl filled with water, animal tray on top of the bowl, data acquisition/reconstruction console, servo motors for 3D rotation of the bowl, and a temperature monitor of the water bath. In Vivo Pharmacokinetics Investigation. SD rats (~200 g) were randomly divided into three groups, (CPT, sorafenib, nanoparticles (n = 4)). The aqueous solutions of nanoparticles and free CPT and sorafenib were intravenously injected via tail vein at a dose of CPT (8 mg/kg), sorafenib (3.8 mg/kg). The blood samples (500 μL) were taken from the eye socket at the 0.5, 1, 2, 4, 8, 12 h time points after injection. The plasma was obtained by centrifugation at 12000 rpm for 5 min and stored at -80 oC. 200 μL of plasma was treated with 250 μL of acetonitrile and methanol mixture (1:1 V/V). The fluorescence intensity of CPT was measured by HPLC (Agilent 1200) with



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

methanol (V/V 60%) and water (V/V 40%) as the eluent at an excitation wavelength of 370 nm and emission wavelength of 470 nm. Sorafenib was measured via ultraviolet detector at the wavelength 266 nm with acetonitrile (V/V 49%), 0.1% TFA (V/V 20%), H2O (V/V 31%) as the eluent. In Vivo Targeting Capacity of Nanoparticles and Biodistribution Analysis. Experiments involving animals were approved by the Animal Ethics Committee of Shanghai Jiao Tong University School of Medicine. SD rats (~220 g) and Balb/c male nude mice (4-5 weeks of age) were purchased from Chinese Academy of Science (Shanghai). The nude male mice were injected subcutaneously with 200 μL of cell suspension containing 3 × 106 HT-29 cells. The tumors were allowed to grow to about 100-150 mm3 before experiment. HT-29 bearing nude mice were randomly divided into two groups, and intravenously injected via tail vein with 200 μL Cy5.5-loaded nanoparticles and free Cy5.5 dissolved PBS solution. The fluorescence distribution was monitored at 0.5, 1, 2, 6, 8 h using an in vivo imaging system with appropriate wavelength (λex = 690 nm, λem = 700 nm). The characterization of Cy5.5-loaded nanoparticles was shown in Figure S9. To assess the tissue distribution of free Cy5.5 and Cy5.5-loaded nanoparticles, the HT-29 tumor-bearing mice were intravenously injected via tail vein with Cy5.5 and Cy5.5 nanoparticles (5 mg/kg), Mice were sacrificed by cervical vertebra dislocation at 30 min, 1 h and 6 h after drug administration (n = 3 at each time point), and the heart, liver, spleen, lung, kidney and tumor were collected. Tissue samples were rinsed in saline, blotted using paper towel, weighed and stored at -80 °C before being homogenized. Cy5.5 and Cy5.5-loaded nanoparticles were extracted from the homogenate using 2 mL of dichloromethane and methanol (4:1, v/v). The organic phases were collected and dried, and the samples were dissolved in acetonitrile for analysis. The samples of Cy5.5 and Cy5.5-loaded nanoparticles were directly examined by using fluorescence spectroscopy. In Vivo Antitumor Efficacy Assay. The HT-29 tumor-bearing mice were randomly divided into five groups. Mice in various groups were intravenously injected via the tail vein with PBS, CPT (8 mg/kg), sorafenib (3.8 mg/kg), drug mixture (8 mg/kg and


Page 24 of 31

Page 25 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3.8 mg/kg), nanoparticles (8 mg/kg) once every two days for 20 days. The length and width of tumor and the bodyweight of mice were monitored before every injection during the treatment. The tumor volume was calculated using the formula: V (mm3) = 1/2 × length (mm) ×width (mm)2 After 20 days therapy, all of mice were sacrificed and tumors were separated, weighted and photographed. Additionally, the tumors were cut into small pieces, fixed in 10% formalin and embedded in the paraffin. Then, the tissues were sectioned for histopathological analysis with H&E staining, TUNEL, and immunofluorescence assay. Statistical Analysis All data represent at least 3 separate experiments. Individual data points were compared by Student’s-test. In all cases, p < 0.05 was considered statistically significant. ASSOCIATED CONTENT Supporting Information The supporting information is available free of charge on the ACS Publications website. FTIR spectrum of mPEG-CPLGLAGG; the standard curve of CPT and sorafenib; accumulative release of CPT; 3D images of in vivo oxyhemoglobin and deoxyhemoglobin PA signal change; TEM image of Cy5.5-loaded nanoparticles. AUTHOR INFORMATION E-mail: [email protected]. E-mail: [email protected] E-mail: [email protected]. Phone: +86-21-54746215. ACKNOWLEDGMENTS. This work was financially supported by the National Basic Research Program (2015CB931801), National Natural Science Foundation of China (51473093, 21374062), the Recruitment Program of Global Experts (15Z127060012). ABBREVIATIONS CRC = Colorectal Cancer, MMP-2 = Matrix Metalloproteinase-2; CPT = Camptothecin, HPLC = High Performance Liquid Chromatograph;



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

DLS = Dynamic Light Scattering, TEM = Transmission Electron Microscope; DLC = Drug Loading Content, ER = Encapsulation Rate. REFERENCES

(1) Ferlay, J., Shin, H.-R., Bray, F., Forman, D., Mathers, C., and Parkin D. M. (2010) Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int. J. Cancer 127, 2893-2917. (2) Pilar, G. A., Enrique, G., Eduardo, P., Ruth, A., Juan, J. R., Mónica, J., Juan, M. C., Virginia, M., Cristina, Á., and Clara, M. (2014) The role of antiangiogenic agents in the treatment of patients with advanced colorectal cancer according to K-RAS status. Angiogenesis 17, 805-821. (3) Mazard, T., Causse, A., Simony, J., Leconet, W., Vezzio-Vie, N., Torro, A., Jarlier, M., Evrard, A., Del Rio, M., Assenat, E., et al. (2013) Sorafenib overcomes irinotecan resistance in colorectal cancer by inhibiting the ABCG2 drug-efflux pump. Mol. Cancer. Ther. 12, 2121-2134. (4) Hicklin, D. J., and Ellis, L. M. (2005) Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis. J. Clin. Oncol. 23, 1011-1027. (5) Awasthi, N., and Schwarz, R. E. (2015) Profile of nintedanib in the treatment of solid tumors: the evidence to date. Onco. Targets Ther. 8, 3691-3701. (6) Ma, J., and Waxman, D. J. (2008) The role of TRPV6 in breast carcinogenesis. Mol. Cancer. Ther. 7, 271-279.


Page 26 of 31

Page 27 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(7) Maeda, H., Wu, J., Sawa,T., Matsumura, Y., and Hori, K. (2000) Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review. J. Control. Release 65, 271-284. (8) Huang, S., Shao, K., Liu, Y., Kuang, Y., Li, J., An, S., Guo, Y., Mao, H., and Jiang, C. (2014) Tumor-targeting and microenvironment-responsive smart nanoparticles for combination therapy of antiangiogenesis and apoptosis. ACS Nano 7, 2860-2871. (9) Patel, K., Angelos, S., Dichtel, W. R., Coskun, A., Yang, Y. W., Zink, J. I., and Stoddart, J. F. (2008) Enzyme-responsive snap-top covered silica nanocontainers. J. Am. Chem. Soc. 130, 2382-2383. (10) Sun, Y., Zhou, Y., Li, Q., and Yang, Y. (2013) Enzyme-responsive supramolecular nanovalves crafted by mesoporous silica nanoparticles and choline-sulfonatocalix[4]arene[2] pseudorotaxanes for controlled cargo release Chem. Commun. 49, 9033-9035. (11) Wang, Y., Li, Q. Y., Liu, X. B., Zhang, C. Y., Wu, Z. M., and Guo, X. D. (2015) Mesoscale Simulations and Experimental Studies of pH-Sensitive Micelles for Controlled Drug Delivery. ACS Appl. Mater. Interfaces. 7, 25592-25600. (12) Zhang, R., Su, S., Hu, K., Shao, L., Deng, X., Sheng, W., and Wu, Y. (2015) Smart micelle@polydopamine core–shell nanoparticles for highly effective chemo–photothermal combination therapy. Nanoscale 7, 19722-19731. (13) Qiao, Z. Y., Hou, C. Y., Zhao, W. J., Zhang, D., Yang, P. P., Wang, L., and Wang, H. (2015) Synthesis of self-reporting polymeric nanoparticles for in situ



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

monitoring of endocytic microenvironmental pH. Chem. Commun. 51, 12609-12612. (14) Zhang, J., Yuan, Z.-F., Wang, Y., Chen, W.-H., Luo, G.-F., Cheng, S.-X., Zhuo, R.-X., and Zhang, X.-Z. (2013) Multifunctional envelope-type mesoporous silica nanoparticles for tumor-triggered targeting drug delivery. J. Am. Chem. Soc. 135, 5068-5073. (15) Salem, N., Kamal, I., Al-Maqhrabi, J., Abuzenadah, A., Peer-Zada, A. A., Qari, Y., Al Ahwal, M., Al-Qagtani, M., and Buhmedia, A. (2016) High expression of matrix metalloproteinases: MMP-2 and MMP-9 predicts poor survival outcome in colorectal carcinoma. Future Oncol. 12, 323-331. (16) Kessenbrock, K., Plaks, V., and Werb, Z. (2010) Matrix metalloproteinases: regulators of the tumor microenviroment. Cell 141, 52-67. (17) Vihinen, P., Ala-aho, R., and Kahari, V. M. (2005) Matrix metalloproteinases as therapeutic targets in cancer. Curr. Cancer Drug. Targets. 5, 203-220. (18) Callmann, C. E., Carback, C. V., Thompson, M. P., Hall, D. J., Mattrey, R. F., and Gianneschi, N. C. (2015) Therapeutic enzyme-responsive nanoparticles for targeted delivery and accumulation in tumors. Adv. Mater. 27, 4611-4615. (19) Zhang, T., Huang, P., Shi, L., Su, Y., Zhou, L., Zhu, X., and Yan, D. (2015) Self-assembled nanoparticles of amphiphilic twin drug from floxuridine and bendamustine for cancer therapy. Mol. Pharm. 12, 2328-2336.


Page 28 of 31

Page 29 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(20) Wu, Y. M., Tang, J., Zhao, P., Chen, Z. N., and Jiang, J. L. (2009) Enhanced expression of Hab18g/CD147 and activation of integrin pathway in HCC cells under 3-D co-culture conditions. Cell. Biol. Int. 33, 199-206. (21) Kim, C., Favazza, C., and Wang, L. H. V. (2010) In vivo photoacoustic tomography of chemicals: high-resolution functional and molecular optical imaging at new depths. Chem. Rev. 110, 2756-2782. (22) Wang, L. V., and Hu, S. (2012) Photoacoustic tomography: in vivo imaging from organelles to organs. Science 335, 1458-1462. (23) Pu, K., Mei, J., Jokerst, J. V., Hong, G., Antaris, A. L., Chattopadhyay, N., Shuhendler, A. J., Kurosawa, T., Zhou, Y., Gambhir, S. S., et al. (2015) Diketopyrrolopyrrole-based semiconducting polymer nanoparticles for in vivo photoacoustic imaging. Adv. Mater. 27, 5184-5190. (24) Barreto, J. A., OˊMmalley, W., Kubeil, M., Graham, B., Stephan, H., and Spiccia, L. (2011) Nanomaterials: applications in cancer imaging and therapy. Adv. Mater. 23, 18-40. (25) Huang, P., Wang, D., Su, Y., Huang, W., Zhou, Y., Cui, D., Zhu, X., and Yan, D. (2014) Combination of small molecule prodrug and nanodrug delivery: amphiphilic drug-drug conjugate for cancer therapy. J. Am. Chem. Soc. 136, 11748-11756. (26) Xia, Y., Wang, Y., Wang, Y., Tu, C., Qiu, F., Zhu, Li., Su, Y., Yan, D., Zhu, B., et al. (2011) A tumor pH-responsive complex: carboxyl-modified hyperbranched polyether and cis-dichlorodiammineplatinum(II). Colloid Surf. B 88, 674-681.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 31

(27) Yang, Y., Velmurugan, B., Liu, X., and Xing, B. (2013) NIR photoresponsive crosslinked upconverting nanocarriers toward selective intracellular drug release. Small 9, 2937-2944. (28) Wang, K., Zhang, X., Liu, Y., Liu, C., Jiang, B., and Jiang, Y. (2014) Tumor penetrability

and

anti-angiogenesis

using

iRGD-mediated

delivery

of

doxorubicin-polymer conjugates. Biomaterials 35, 8735-8747. (29) Jiang, X., Sha, X., Xin, H., Xu, X., Gu, J., Xia, W., Chen, S., Xie, Y., Chen, L., Chen, Y., et al. (2013) Integrin-facilitated transcytosis for enhanced penetration of advanced gliomas by poly(trimethylene carbonate)-based nanoparticles encapsulating paclitaxel. Biomaterials 34, 2969-2979.


Page 31 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


For Table of Contents use only

Matrix Metalloproteinase Responsive Nanoparticles for Synergistic Treatment of Colorectal Cancer via Simultaneous Anti-Angiogenesis and Chemotherapy Leilei Shi, Yi Hu, Ang Lin, Chuan Ma, Chuan Zhang,* Yue Su, Linzhu Zhou, Yumei Niu,* and Xinyuan Zhu*

Keywords: Colorectal cancer, synergistic therapy, nanoparticle, MMP-2


Matrix Metalloproteinase Responsive ... - ACS Publications

Recommend Documents