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Controlled Release and Delivery Systems

Extracellular Matrix Components shelled Nanoparticles as Dual Enzyme-Responsive Drug Delivery Vehicles for Cancer Therapy Juan Zhou, Mingyu Wang, Huiyan Ying, Dandan Su, Huijie Zhang, Guozhong Lu, and Jinghua Chen ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00327 • Publication Date (Web): 14 May 2018 Downloaded from http://pubs.acs.org on May 14, 2018

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ACS Biomaterials Science & Engineering

Extracellular Matrix Components shelled Nanoparticles as Dual Enzyme-Responsive Drug Delivery Vehicles for Cancer Therapy ‡

‡

Juan Zhou,a, Mingyu Wang, a, Huiyan Ying,a Dandan Su,a Huijie Zhang,a Guozhong Lub and Jinghua Chena,* a

Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Pharmaceutical Science, Jiangnan University, Wuxi, China.

b

Department of Burns & Plastic Surgery, the Third Affiliated Hospital with Nantong University, Wuxi, China.

Contact information of the corresponding author is as follows: Jinghua Chen: Jiangnan University, School of Pharmaceutical Science, No. 1800, Lihu Avenue, Wuxi 214122, China. Email: [email protected].

KEYWORDS: collagen I, hyaluronic acid, mesoporous nanoparticle, enzyme-stimulus, target drug delivery, cancer therapy.

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ABSTRACT Stimuli-responsive drug delivery systems with reduced side-effects offer promising prospects for cancer therapy. In this study, we developed an enzyme-responsive nanomedicine system based on extracellular matrix components (ECM) shelled mesoporous silica nanoparticles. The covalently conjugated ECM biomacromolecules, hyaluronic acid and collagen I, can not only enhance the biocompatibility of the particles and avoid early drug leakage, but also allow selective biodegradation triggered by hyaluronidase (HAase) and Matrix metalloproteinases 2 (MMP-2), which are over-expressed enzymes in some tumor tissues. The in vitro cytotoxicity test confirmed favourable biocompatibility of the as-prepared nanomedicine system. Moreover, this system showed distinguishing controlled release efficiency toward cancer cells induced by different levels of HAase and MMP-2. The in vivo antitumor test demonstrated the excellent efficiency of our system for tumor targeting drug delivery and tumor growth inhibition. Therefore, this dual enzyme-responsive drug delivery system provided an efficient platform for cancer therapy.

INTRODUCTION In tumor tissues, interactions between tumor microenvironment and cancer cells play critical roles in promoting tumor growth, invasion and metastasis.1-2 Most of anticancer drugs or nanodrug delivery systems (NDDS) are targeting directly on cancer cells, however, drug resistance of cancer cells have been frequently reported in literature, which might limit the effects of celltargeting drugs on cancer therapy.3 Therefore, tumor microenvironment becomes another


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penetration point for anti-cancer drug delivery. The tumor microenvironment as the replacing target, not only possess hypoxic, low pH and high pressure state,4 but also fills with abnormally over-expressed proteases and growth factors,5-6 which can be exploited for designing stimulusresponsive NDDS.7-8 The high expression of Matrix metalloproteinases 2 (MMP-2) can be found in most of tumor tissues, such as breast, cervical, ovarian cancer and so on.9 It has been demonstrated that MMP-2 can potentially promote the metastasis of cancer cells, regulate their signal pathways of growth and inhibit tumor apoptosis.10-11 In addition, MMP-2 in tumor microenvironment enables biodegradation of extracellular matrix (ECM) components, providing more space for cancer cells invasion. These features have been exploited in the designing of several different enzymeresponsive drug delivery systems.12-14 Meanwhile, tumor microenvironment is acidic owing to the large amounts of lactic acid, where hyaluronidase (HAase) has the ability to degrade hyaluronic acid. In contrast, the pH of normal tissue is usually neutral, making HAase inactivated in other biological environments.15-16 Thus, hyaluronidase is also considered as a cancer marker, and used in various cancer therapy and diagnosis applications.17-18 With these considerations in mind, we prepared hyaluronic acid and collagen I multi-shelled drug delivery system based on fluorescence doped mesoporous silica nanoparticles (FMSN-DoxC2H). On one hand, both of hyaluronic acid and collagen I are the main biomacromolecules of natural ECM, which can contribute to enhancing the biocompatibility of nanoparticles and prolonging the blood circulation.19-20 On the other hand, hyaluronic acid and collagen I shells in FMSN-Dox-C2H would block the pores of nanoparticles and stay intact in normal tissues.21-22 Moreover, they can be removed gradually due to the specific degradation induced by HAase and MMP-2 in tumor tissues. Additionally, hyaluronic acid is able to specifically interact with CD44,


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a receptor which is over-expressed on the surface of cancer cells.23 Hyaluronic acid shell can improve the targeting efficiency of FMSN-Dox-C2H. Mesoporous silica nanoparticle is one of the most commonly used carrier for drug delivery and cancer targeting therapy, owing to its high accumulation rate at the tumor site resulted from enhanced permeability and retention (EPR) effect.24-25 Moreover, the large surface area of silica nanoparticles provided enough space for different types of functionalizations readily.26-28 Therefore, in the current study, mesoporous silica nanoparticle was adopted as the carrier, and the formed FMSN-Dox-C2H system is expected to take prolonged circulation time to be accumulated at tumor tissues and respond to the enzymatic stimulus to release anticancer drug. To verify the above hypothesis, the drug release actions of FMSN-Dox-C2H toward HAase and MMP-2 were investigated, and the cytotoxicity, biodistribution and anticancer effect of FMSNDox-C2H were further studied in HeLa-tumor-bearing mice.

EXPERIMENTAL SECTION Preparations of Dox-loaded particles (FMSN-Dox). FMSN-NH2 (500 mg) were dispersed in methanol (100 mL) containing Dox (250 mg), and the mixture was stirred gently overnight at room temperature, then the solution was evaporated. After that, the particles were dispersed in methanol (100 mL) containing Dox (250 mg), the suspension was stirred overnight as previous step. After drying under vacuum rotary evaporation, ultrapure water (50 mL) was added and the particles (FMSN-Dox) were obtained by centrifugation. The supernatant was collected for measuring the drug loading amount. Preparations of FMSN-Dox-C2. Collagen I (100 mg) was dispersed in ultrapure water (100 mL), and kept in solution with pH 5.5. Then EDC (55 mg), NHS (32.5 mg) and FMSN-Dox was


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added into the solution and stirred for another 16 h. The mixture was centrifuged and washed by ultrapure water three times. After drying, the particles were conjugated with another layer of collagen I using the same method to provide FMSN-Dox-C2. Preparations of FMSN-Dox-C2H. HA (100 mg) was dissolved in ultrapure water (100 mL) and the solution was kept pH 5.5. After activation of carboxyl groups by EDC (55 mg) and NHS (32.5 mg) for 1 h, FMSN-Dox-C2 was added for another 16 h stirring. The mixture was centrifuged and washed by ultrapure water three times. FMSN-Dox-C2H was obtained after drying under vacuum. Drug release of FMSN-Dox-C2H. FMSN-Dox-C2H (2 mg) and FMSN-Dox particles (2 mg) were dispersed in PBS buffer solution (2 mL, 0.01 M, pH 7.4) respectively, and then sealed in dialysis bags. These bags were immersed in PBS buffer solution (20 mL) and incubated in shaking table at 37 °C, 100 rpm. At pre-determined time intervals, PBS medium (3 mL) was taken out and analyzed by UV absorbance spectrometer. Simultaneously, another 3 mL of fresh PBS buffer was added for supplementary. Noticeably, collagenase (1 mg/mL, 10 µL)29 and hyaluronidase (500 units) was added into FMSN-Dox-C2H dialysis bag after 3 h shaking. Cytotoxic studies of FMSN, FMSN-Dox-C2 and FMSN-Dox-C2H. FMSN, FMSN-Dox-C2 and FMSN-Dox-C2H particles were first dispersed in DMEM medium and 1640 medium at a concentration of 1 mg/mL. Cos-7, HeLa and Hep G2 cells were maintained in DMEM medium and 1640 medium, respectively, supplemented with 10% FBS and 1% penicillin-streptomycin in a humidified incubator at 37 °C with 5% CO2 and 95% air, grown in 96 well plates at 5000 cells per well overnight (100 µL). FMSN, FMSN-Dox-C2 and FMSN-Dox-C2H samples were added to make sample concentrations from 10 µg/mL to 80 µg/mL. After 24 h culturing, the medium was removed and MTT (100 µL) was added. 4 h later, DMSO (100 µL) was also added. Finally,


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the cell viabilities were measured by Thermo Scientific Microplate Reader at which an emission wavelength of 590 nm. Cellular uptake studies by confocal fluorescence microscopy. HeLa, Hep G2 and Cos-7 cells were maintained in 1640 medium and DMEM medium, respectively, supplemented with FBS (10%) and penicillin-streptomycin (1%) in a humidified incubator at 37 °C with 5% CO2 and 95% air. For intracellular localization, 105 cells per well were seeded in glass bottom cell culture dish and allowed adhering for 24 h. After incubation with 60 µg/mL of FMSN-Dox-C2 and FMSN-Dox-C2H particles for 6 h, 12 h and 24 h, the cells were washed with PBS, fixed with paraformaldehyde (4%) and observed by confocal fluorescence microscopy. Animal tumor model. Female Balb/c-nu mice (4 weeks old) feeded under SPF environment at the Laboratory Animal Care Facility of Jiangsu Province Institute of Parasitic Diseases Control (SYXK (Su) 2012-0034). The in vivo experiments were carried out under the guideline approved by the Institutional Animal Care and Use Committee (IACUC) of the Jiangsu Province Institute of Parasitic Diseases Control (2017-012). For the xenografts established from cultured cells, HeLa cells were suspended and harvested after trypsinization, and approximately 1.5 × 106 HeLa cells in 100 µL of PBS were subcutaneously injected into the right hind leg of the mice. The tumor volume (V) was determined by measuring the length (L) and width (W) at pre-determined time and was calculated as V = L × W2/2. Each treatment was conducted when the tumor volume reached approximately 100 mm3. When the tumor volume reached approximately to 100 mm3, the mice were divided into four groups (n ≥ 3) and subjected with different treatments: saline (0.9% NaCl), doxorubicin, FMSN-Dox-C2 and FMSN-Dox-C2H. The injected dose was 5 mg/Kg, the tumor volume and the body weights of the mice were measured every day.


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In vivo biodistribution studies. Female Balb/c-nu mice bearing HeLa tumors were intravenously injected with Doxorubicin or FMSN-Dox-C2H at a dose of 5 mg/Kg. At different post injection times (6 and 24 h), mice were sacrificed, and the major organs (heart, liver, spleen, lung, kidney, tumor) were harvested. The organ samples were first ground with a mortar and then homogenized with an ultrasonic cell breaker (15-25 min, 200 W). After centrifugation of the tissue homogenate, the supernatant (1 mL) was well mixed with 1 mL of Boric acid (0.2 mol/L, pH = 9), 4 mL of chloroform in methanol (chloroform: methanol = 4: 1), then the mixture was centrifuged at 3000 rpm for 20 min. Chloroform layer was retained and the upper mixture was dispersed in another 4 mL of chloroform-methanol solution. After centrifugation, the chloroform layer was combined and dried. The resulting product was dissolved in PBS and the absorbance was measured at 480 nm. Pharmacokinetics study. The mice were randomly divided into two groups, injected with DOX solution and FMSN-Dox-C2H at DOX dose of 5 mg/kg via the tail vein. 1.0 mL of blood was obtained via the eyeball at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 10 h, 16 h, 24 h, 48 h and placed at r.t for 30 min, then transferred into fridge at 4 ℃ for 2 h. After that, the samples were centrifuged at 9000 rpm for 10min to obtain serum. PBS buffer (pH=6.8, 0.5 mL) was mixed with 0.3 µL of serum, then added another 0.5 mL of methylene chloride and vortexed. After 1 min, the sample was centrifuged for 10 min at 2500 rpm. The organic layer was combined and dried by nitrogen. The resulting product was dissolved in PBS and the absorbance was measured at 480 nm. The pharmacokinetic parameters were calculated by following formula: ܶଵ/ଶ =

ln2 k

CL = kV


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AUC଴→୲ = MRT =

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‫ܥ‬଴ ݇

AUMC଴→୲ AUC଴→୲

where T1/2 is half-life, k is first order elimination rate constant, CL is total body clearance, C0 is drug initial concentration, AUC is the area under the curve, MRT is mean resident time. Statistical analysis. All the experiments were carried out at least in triplicate and the results were expressed as mean ± SD. Differences between the control and experimental groups were analyzed by two-tailed Student's t-test. A P-value less than 0.05 was considered to be statistically significant.

RESULTS AND DISCUSSION Characterization of nanomedicine delivery system. Fluorescence doped nanoparticles (FMSN) was prepared according to our previous work (see Supporting Information for details).30 As shown in Figure 1A, FITC and APTES were conjugated, together with the surfactant CTAB and silica source TEOS to form FMSN. The resulting FMSN particles displayed cylindrical shape with 140 nm length and 70 nm width (Figure 1B, Figure S1). Then, FMSN was aminolated, loaded with doxorubicin and crosslinked with collagen I. The sealing efficiency of one layer collagen I was low according to the drug release study results. To avoid drug leakage, another layer of collagen I was attached (FMSN-Dox-C2). Subsequently, hyaluronic acid was covalently anchored onto collagen I layer, yielding the final drug delivery system (FMSN-DoxC2H). Seen from Figure 1C, the shape of nanoparticle still maintained columnar, while the surface displayed uneven state, the size increased to 160 nm length and 80 nm width, due to the introduction of collagen I and hyaluronic acid. This performance was in accordance with the


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hydrodynamic diameter monitored by dynamic light scattering (DLS). As shown in Figure 1D, free particle FMSN displayed a hydrodynamic diameter of approximately 120 nm, which increased to 170 nm after doxorubicin loading and dual layer of collagen I capping. After hyaluronic acid conjugation, the size of FMSN-Dox-C2H increased to 200 nm.

Figure 1. (A) Schematic description of hyaluronic acid and collagen I functionalized mesoporous silica nanoparticles; Transmission electronic microscopy images of (B) FMSN and (C) FMSN-Dox-C2H; (D) Dynamic light scattering analysis of FMSN, FMSN-Dox-C2 and FMSN-Dox-C2H.

The Brunauer-Emmett-Teller (BET) and Barret-Joyner-Halenda (BJH) analysis were carried out for verifying the mesoporous structure of nanoparticles. Observed from Figure 2A the nitrogen adsorption-desorption curve of FMSN was presented as typical mesoporous pattern,


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whereas, FMSN-Dox-C2H was not mesostructured any longer. Moreover, particles showed significant decrease in surface area form 848 m2/g to 174 m2/g and pore volume from 0.37 to 0.15 cm3/g. These changed features were resulted from the loading of doxorubicin in pores followed by the coverage of hyaluronic acid and collagen I on surface. On the other hand, the pore size increased after modification. FMSN was of approximately 2.9 nm pore diameter, and the pore size of FMSN-Dox-C2H increased to 3.7 nm, which accounted for a small portion. Perhaps it was attributed to the porous structure came from network conjugation of collagen I or hyaluronic acid on surface (Figure 2B). The conjugation of hyaluronic acid and collagen I on particles was estimated qualitatively by zeta potential and FT-IR analysis. As shown in Figure 2C, the zeta potential of FMSN changed from negative value to positive after collagen I conjugation. Once hyaluronic acid was anchored, the charge returned to negative again. In FT-IR images (Figure S2), the peaks at 1655 and 1540 cm-1 of FMSN-Dox-C2 were corresponding to the amide I and amide II bands, respectively, indicating the presence of collagen I. Additionally, the modification of hyaluronic acid in FMSN-Dox-C2H was supported by the appearance of signal at 1160 cm-1, arising from the C-O bonds of hyaluronic acid. To obtain the modified quantity of attachments, particles were analyzed by thermo gravimetric analyzer (TGA, Figure 2D) accompanied with derivative thermogravimetric (DTG, Figure S3). An approximately 8% weight loss in the particles that conjugated with two layers of collagen I (FMSN-Dox-C2) because of the thermal evaporation of collagen I shell around 340 °C. Besides, another 6% weight decrease was resulted from thermal desorption of hyaluronic acid around 220 °C.


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Figure 2. Characterization of drug delivery system. (A) Nitrogen adsorption-desorption isotherms; (B) pore size distribution of (a) FMSN and (b) FMSN-Dox-C2H. (C) Zeta potential; (D) TGA of FMSN, FMSN-Dox-C2, FMSN-Dox-C2H.

Doxorubicin loading and release from FMSN-Dox-C2H in vitro. Before functionalization of collagen I and hyaluronic acid, FMSN was suspended and stirred in doxorubicin solution for drug loading. The amount of doxorubicin encapsulated in particles was calculated to be 110 mg per gram of particles based on the UV absorbance at 480 nm of original drug solution and the collected washing solution (Figure S4).


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The release efficiency of doxorubicin from FMSN-Dox-C2H was next evaluated before and after exposure to HAase and MMP-2. As shown in Figure 3A, no detectable doxorubicin signal was recorded when FMSN-Dox-C2H was suspended in PBS buffer (pH 7.4) in the absence of HAase and MMP-2, indicating that particles remained largely intact at this condition. When only adding HAase, the drug delivery system still remained intact under the protection of collagen layer. We next dispersed FMSN-Dox-C2H in buffer for 3 h, during which time the system did not exhibit any release. After the addition of HAase and MMP-2 into this buffered suspension, the doxorubicin signal significantly increased, thus displaying hyaluronic acid and collagen I layer degradation and drug release from nanoparticles. The degree of doxorubicin release increased to 75% within 72 h. In contrast, the control of unfunctioanlized drug-loaded nanoparticles FMSN-Dox presented doxorubicin release in both absence and presence of enzymes. Thus, these results demonstrated that FMSN-Dox-C2H showed efficient sealing properties before exposure to HAase or MMP-2, but specific release of drug upon dual enzymatic stimuli. In vitro cytotoxicity of FMSN-Dox-C2H. Since we would utilize this hyaluronic acid and collagen I functionalized nanoparticles as HAase and MMP-2 responsive drug vehicles for tumor therapy, the biological evaluation was subsequently assessed. Cytotoxicity assays were performed with three different cells: normal cell Cos-7 and liver cancer cell Hep G2 as control groups, cervical cancer cell HeLa as experimental group. Cos-7 negatively expressed HAase and MMP-2,31 Hep G2 expressed the two enzymes in low level,32-33 whereas, HeLa cell was in high expression of HAase and MMP-2.9 The three cells were treated with different concentrations of FMSN, FMSN-Dox-C2 as well as FMSN-Dox-C2H, and then cell viabilities over 24 h were recorded. No significant cytotoxicity


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was observed when Cos-7, Hep G2 and HeLa cells were incubated with FMSN for 24 h, all of the cell viabilities remained beyond 80% at nanoparticle concentrations from 10 to 80 µg/mL. In addition, the viability of Cos-7 cell incubating with different concentrations of FMSN-Dox-C2 and FMSN-Dox-C2H still kept at high level, because there were no effective HAase or MMP-2 in normal cell culture for degrading collagen I and hyaluronic acid shells, Dox in pores could not be released (Figure 3B, Figure S5). FMSN-Dox-C2H thus showed a good sealing effect in healthy cells with minimal premature drug leakage. In the case of Hep G2 cell, the viability reached to 70% in the culture of FMSN-Dox-C2 and 61% with FMSN-Dox-C2H at the highest particle concentration, respectively (Figure 3C). It demonstrated that HAase and MMP-2 in Hep G2 had the capability of breaking up protected shell and releasing Dox, but these enzymes was insufficient to degrade collagen I and hyaluronic acid in high efficiency, therefore the cytotoxicity of drug delivery system toward Hep G2 was not remarkable. However, HeLa cell was obviously affected by FMSN-Dox-C2 and FMSN-Dox-C2H. With increasing concentrations of nanoparticles, the viability of HeLa cell significantly decreased to 45% in terms of FMSNDox-C2 and 39% of FMSN-Dox-C2H, verifying the prominent degradation of collagen I and hyaluronic acid under the stimuli of HAase and MMP-2 (Figure 3D). Specially, cancer cells treated with FMSN-Dox-C2H always had lower survival ratio than that incubated with FMSNDox-C2 during the experimental periods. It can be attributed to the selective interaction of hyaluronic acid conjugated on FMSN-Dox-C2H with CD44 on cancer cell surface, leading to targeted drug delivery.


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Figure 3. (A) Release profiles of doxorubicin from FMSN-Dox, FMSN-Dox-C2H in the presence of HAase; FMSN-Dox-C2H in the absence and presence of HAase and MMP-2. Percent viability of (B) Cos-7, (C) Hep G2 and (D) HeLa cells against FMSN, FMSN-Dox-C2 and FMSN-Dox-C2H in 24h. Data as presented as mean ± SD (n = 5, ** P < 0.01, *** P < 0.001).

Cellular uptake of FMSN-Dox-C2 and FMSN-Dox-C2H. To further investigate the efficiency of enzyme responsive and cancer cell targeted drug release, the cellular uptake performances of FMSN-Dox-C2H toward Hep G2 and HeLa cells were evaluated through flow cytometry firstly. As shown in Figure 4A and 4B, the FITC fluorescence intensities of nanoparticles in both cells were similar at the beginning 6 h, indicating no significant difference in uptake efficiency. Furthermore, the intensity in HeLa cells treated with FMSN-Dox-C2H was


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1.29-fold and 2.60-fold greater than that in Hep G2 after incubation for 12h and 24 h, respectively. HeLa cells took up more nanoparticles because of richer CD44 receptor on the cell surface.34-35 Next, the intracellular distribution of FMSN-Dox-C2H in Hep G2 and HeLa cells was observed using confocal fluorescence microscopy. Overall, both cancer cells exhibited a similar trend. As shown in Figure 4C and 4D, green fluorescence from FITC was observed when Hep G2 and HeLa cell incubated with FMSN-Dox-C2H for 6 h, but the released Dox was mainly distributed in the cytoplasm. With an increase in the incubation time, more and more Dox came out and some of them were observed in cell nuclei. Until 24 h of incubation, FMSN-Dox-C2H has shown efficient taken up by Hep G2 and HeLa cells, since red fluorescence presented throughout all cells. Nevertheless, there were some differences between Hep G2 and HeLa cells have been observed. Red fluorescence from Hep G2 cell was weaker than that from HeLa cell after incubation with FMSN-Dox-C2H for 24 h. It demonstrated that particles were taken by both cells, but large part of Dox was entrapped within pores owing to low level of HAase and MMP-2 in Hep G2 cell culture. Therefore, FMSN-Dox-C2H could realize controlled drug release in different extents toward cancer cells, induced by various levels of HAase and MMP-2.


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Figure 4. Cellular uptake efficiency of FMSN-Dox-C2H in (A) Hep G2 and (B) HeLa cells; Confocal laser scanning microscopy (CLSM) images of (C) Hep G2 and (D) HeLa cells for 6h, 12 h and 24h incubation.

BSA adsorption and pharmacokinetic studies of FMSN-Dox-C2H. For assessing the biocompatibility of FMSN and FMSN-Dox-C2H in vivo, their circulation performances were tested firstly. FMSN and FMSN-Dox-C2H were suspended in bovine serum albumin (BSA) solution (0.6 mg/mL), and then the absorbance intensities of supernatant at predetermined time were collected. As shown in Figure S6, the rest of BSA in the FMSN-Dox-C2H suspended solution was lower than in FMSN, indicating that hyaluronic acid and collagen I on the surface of FMSN-Dox-C2H enabled protecting particles from protein adsorption, which was in favour of prolonged circulation in vivo. Then, the pharmacokinetic studies of free drug and FMSN-DoxC2H were investigated to evaluate their circulated behaviors in vivo. Figure S7 displayed Dox concentration versus time curve after intravenous injection of Dox and FMSN-Dox-C2H, then the major parameters were calculated and summarized in Table S1. The clearance half-time of FMSN-Dox-C2H was about 6.2 h, which was much longer than free drug Dox (T1/2: 2.2 h). Accompanying with the higher area under the curve (AUC) and mean resident time (MRT) as well as the lower clearance, it was verified that FMSN-Dox-C2H had a longer circulation time compared with Dox. This extended circulation behavior ensured the delivery, tumor retention and antitumor efficiency of FMSN-Dox-C2H. Anti-tumor activity of FMSN-Dox-C2H in vivo. Next, we investigated the in vivo behaviours of multi-shelled nanoparticles by monitoring the biodistribution of doxorubicin at 24 h postinjection of free drug and FMSN-Dox-C2H to HeLa-tumor-bearing mice. In free drug


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treated group, the doxorubicin concentration was nearly vanished at the tumor site in comparison with other normal organs (Figure 5A). In contrast, high levels of doxorubicin (0.8 mg/g) were observed at the tumor site in the FMSN-Dox-C2H treated group. This can be probably attributed to the eluded macrophage clearance by hyaluronic acid and collagen I during blood circulation, and then they were accumulated in the tumor by the targeting action of EPR effect and hyaluronic acid. Additionally, HeLa-tumor-bearing mice were divided into four groups, followed by treatments with saline, free doxorubicin, FMSN as well as FMSN-Dox-C2H to evaluate antitumor efficiency. Firstly, the mice’s body weights were monitored each day. We observed from Figure 5B that all body weights of experimental groups kept slight fluctuations in 14 days except for the free Dox group. Free Dox-treated mice displayed an obvious decrease in body weight at the second day and remained low weight at the following days, while the body weights of mice injected with FMSN-Dox-C2H remained nearly constant, in a smooth status similar to saline. The results indicated that this drug delivery vehicle had reduced side effects in comparison with free drug. Subsequently, the tumor volumes of all groups were measured in real-time. Mice treated with saline presented an unrestrained tumor growth as shown in Figure 5C, reaching about 4.3 times of the initial size. The tumors in FMSN group also presented a quick growth. Whereas, the mice treated with FMSN-Dox-C2H exhibited obviously suppressed tumor growth, holding approximately 1.3 times of the initial tumor size in 12 days, but they appeared a pickup in the last 2 days. On the other hand, the tumors treated with free Dox also grew slowly at the beginning several days owing to the high dose, meanwhile they showed a tendency to enlarge from day 10. The contrasting results from representative tumor images of mice with different treatments further confirmed the tumor growth inhibition of FMSN-Dox-C2H (Figure 5D). Thus,


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all of the above results in certain degree demonstrated that the multi-shelled nanoparticles had the advantages of prolonged circulation, targeted accumulation and enzyme-responsive drug release, producing enhanced cancer therapeutic effectiveness in vivo.

Figure 5. The anti-tumor effect of different formulations in HeLa-tumor-bearing mice. (A) The biodistribution of doxorubicin after treating mice with free doxorubicin and FMSN-Dox-C2H; (B) The profile of body weight during the treatment period; (C) changes in tumor volume. Data as presented as mean ± SD (n = 5, *** P < 0.001); (D) the images of tumors after 1 and 14 day administration of of saline, doxorubicin, FMSN and FMSN-Dox-C2H.

CONCLUSIONS In conclusion, this work described the development of multi-ECM components shelled mesoporous silica nanoparticles (FMSN-Dox-C2H) as drug delivery vehicle for tumor therapy. The introduction of collagen I and hyaluronic acid on the surface of particles was proven


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effective in sealing the pores of FMSN-Dox-C2H, avoiding the drug leakage in advance. Additionally, the controlled dual enzyme-responsive release of Dox from the drug delivery system was achieved owing to the cleavage of hyaluronic acid and collagen I under the specific degradation of HAase and MMP-2. The cytotoxicity and BSA adsorption test in vitro exhibited favourable biocompatibility and cancer cell targeting of FMSN-Dox-C2H. The in vivo antitumor experiments indicated that the drug delivery system could inhibit tumor growth effectively with reduced adverse effects on normal tissues. Therefore, this hyaluronic acid and collagen I multishelled nanoparticle can be a promising candidate as dual enzyme-responsive drug delivery system for tumor therapy.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. FT-IR images, DTG results, calibration curve of DOX, BSA absorbance of nanoparticles (PDF) AUTHOR INFORMATION Corresponding Author * Jinghua Chen. Jiangnan University, School of Pharmaceutical Science, No. 1800, Lihu Avenue, Wuxi 214122, China. Email: [email protected]. Fax: 86-0510-85329042 ORCID Juan Zhou: 0000-0001-9770-7229 Huijie Zhang: 0000-0001-9676-4264 Author Contributions


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The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of Jiangsu Province (grant number: BK20170203) and National Natural Science Foundation of China (grant number: 21574059). We also thank the Fundamental Research Funds for the Central Universities (grant number: JUSRP11748) and national first-class discipline program of Light Industry Technology and Engineering (LITE2018-20). REFERENCES 1.

Liotta, L. A.; Kohn, E. C., The microenvironment of the tumour-host interface. Nature

2001, 411 (6835), 375-379. DOI: 10.1038/35077241. 2.

Fukumura, D.; Jain, R. K., Tumor microenvironment abnormalities: Causes,

consequences, and strategies to normalize. J. Cell. Biochem. 2007, 101 (4), 937-949. DOI: 10.1002/jcb.21187. 3.

Dean, M.; Fojo, T.; Bates, S., Tumour stem cells and drug resistance. Nat. Rev. Cancer

2005, 5 (4), 275-284. DOI: 10.1038/nrc1590. 4.

Vaupel, P., The Role of Hypoxia-Induced Factors in Tumor Progression. Oncologist

2004, 9 (suppl 5), 10-17. DOI: 10.1634/theoncologist.9-90005-10. 5.

Huang, W. C.; Chen, S. H.; Chiang, W. H.; Huang, C. W.; Lo, C. L.; Chern, C. S.; Chiu,

H. C., Tumor Microenvironment-Responsive Nanoparticle Delivery of Chemotherapy for


20

Page 21 of 27 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


Enhanced Selective Cellular Uptake and Transportation within Tumor. Biomacromolecules 2016, 17 (12), 3883-3892. DOI: 10.1021/acs.biomac.6b00956. 6.

Li, H.-J.; Du, J.-Z.; Liu, J.; Du, X.-J.; Shen, S.; Zhu, Y.-H.; Wang, X.; Ye, X.; Nie, S.;

Wang, J., Smart Superstructures with Ultrahigh pH-Sensitivity for Targeting Acidic Tumor Microenvironment: Instantaneous Size Switching and Improved Tumor Penetration. Acs Nano 2016, 10 (7), 6753-6761. DOI: 10.1021/acsnano.6b02326. 7.

Tang, S.; Meng, Q.; Sun, H.; Su, J.; Yin, Q.; Zhang, Z.; Yu, H.; Chen, L.; Chen, Y.; Gu,

W.; Li, Y., Tumor-Microenvironment-Adaptive Nanoparticles Codeliver Paclitaxel and siRNA to Inhibit Growth and Lung Metastasis of Breast Cancer. Adv. Funct. Mater. 2016, 26 (33), 6033-6046. DOI: 10.1002/adfm.201601703. 8.

Gao, Z.; Hou, Y.; Zeng, J.; Chen, L.; Liu, C.; Yang, W.; Gao, M., Tumor

Microenvironment-Triggered

Aggregation

of

Antiphagocytosis

99mTc-Labeled

Fe3O4

Nanoprobes for Enhanced Tumor Imaging In Vivo. Adv. Mater. 2017, 29 (24), 1701095. DOI: 10.1002/adma.201701095. 9.

Roomi, M. W.; Monterrey, J. C.; Kalinovsky, T.; Rath, M.; Niedzwiecki, A., In vitro

modulation of MMP-2 and MMP-9 in human cervical and ovarian cancer cell lines by cytokines, inducers and inhibitors. Oncol. Rep. 2010, 23, 605-614. DOI: 10.3892/or_00000675. 10. Kessenbrock, K.; Plaks, V.; Werb, Z., Matrix Metalloproteinases: Regulators of the Tumor Microenvironment. Cell 2010, 141 (1), 52-67. DOI: 10.1016/j.cell.2010.03.015. 11. Elegbede, A. I.; Banerjee, J.; Hanson, A. J.; Tobwala, S.; Ganguli, B.; Wang, R.; Lu, X.; Srivastava, D. K.; Mallik, S., Mechanistic Studies of the Triggered Release of Liposomal


21


Page 22 of 27

Contents by Matrix Metalloproteinase-9. J. Am. Chem. Soc. 2008, 130 (32), 10633-10642. DOI: 10.1021/ja801548g. 12. Chen, W.-H.; Luo, G.-F.; Lei, Q.; Jia, H.-Z.; Hong, S.; Wang, Q.-R.; Zhuo, R.-X.; Zhang, X.-Z., MMP-2 responsive polymeric micelles for cancer-targeted intracellular drug delivery. Chem. Commun. 2015, 51 (3), 465-468. DOI: 10.1039/C4CC07563C. 13. Peng, Z.-H.; Kopeček, J., Enhancing Accumulation and Penetration of HPMA Copolymer–Doxorubicin Conjugates in 2D and 3D Prostate Cancer Cells via iRGD Conjugation with an MMP-2 Cleavable Spacer. J. Am. Chem. Soc. 2015, 137 (21), 6726-6729. DOI: 10.1021/jacs.5b00922. 14. Taghizadeh, B.; Taranejoo, S.; Monemian, S. A.; Salehi Moghaddam, Z.; Daliri, K.; Derakhshankhah, H.; Derakhshani, Z., Classification of stimuli–responsive polymers as anticancer

drug

delivery

systems.

Drug

Deliv.

2015,

22

(2),

145-155.

DOI:

10.3109/10717544.2014.887157. 15. Bertrand, P.; Girard, N.; Duval, C.; d'Anjou, J.; Chauzy, C.; Ménard, J.-F.; Delpech, B., Increased hyaluronidase levels in breast tumor metastases. Int. J. Cancer 1997, 73 (3), 327-331. DOI: 10.1002/(SICI)1097-0215(19971104)73:33.0.CO;2-1. 16. Victor, R.; Chauzy, C.; Girard, N.; Gioanni, J.; d'Anjou, J.; Stora De Novion, H.; Delpech, B., Human breast-cancer metastasis formation in a nude-mouse model: Studies of hyaluronidase, hyaluronan and hyaluronan-binding sites in metastatic cells. Int. J. Cancer 1999, 82 (1), 77-83. DOI: 10.1002/(SICI)1097-0215(19990702)82:13.0.CO;2-Q.


22

Page 23 of 27 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


17. Liu, D.; Pearlman, E.; Diaconu, E.; Guo, K.; Mori, H.; Haqqi, T.; Markowitz, S.; Willson, J.; Sy, M. S., Expression of hyaluronidase by tumor cells induces angiogenesis in vivo. Proc. Natl. Acad. Sci. 1996, 93 (15), 7832-7837. 18. Cardoso, M. J.; Caridade, S. G.; Costa, R. R.; Mano, J. F., Enzymatic Degradation of Polysaccharide-Based Layer-by-Layer Structures. Biomacromolecules 2016, 17 (4), 1347-1357. DOI: 10.1021/acs.biomac.5b01742. 19. Badylak, S. F.; Freytes, D. O.; Gilbert, T. W., Extracellular matrix as a biological scaffold material: Structure and function. Acta Biomater. 2009, 5 (1), 1-13. DOI: 10.1016/j.actbio.2008.09.013. 20. Theocharis, A. D.; Skandalis, S. S.; Gialeli, C.; Karamanos, N. K., Extracellular matrix structure. Adv. Drug Deliver. Rev. 2016, 97, 4-27. DOI: 10.1016/j.addr.2015.11.001. 21. Li, D.; He, J.; Cheng, W.; Wu, Y.; Hu, Z.; Tian, H.; Huang, Y., Redox-responsive nanoreservoirs based on collagen end-capped mesoporous hydroxyapatite nanoparticles for targeted drug delivery. J. Mater. Chem. B 2014, 2 (36), 6089-6096. DOI: 10.1039/C4TB00947A. 22. Luo, Z.; Cai, K.; Hu, Y.; Zhao, L.; Liu, P.; Duan, L.; Yang, W., Mesoporous Silica Nanoparticles End-Capped with Collagen: Redox-Responsive Nanoreservoirs for Targeted Drug Delivery. Angew. Chem. Int. Ed. 2011, 50 (3), 640-643. DOI: 10.1002/anie.201005061. 23. Yen, J.; Ying, H.; Wang, H.; Yin, L.; Uckun, F.; Cheng, J., CD44 Mediated Nonviral Gene Delivery into Human Embryonic Stem Cells via Hyaluronic-Acid-Coated Nanoparticles. ACS Biomater. Sci. Eng. 2016, 2 (3), 326-335. DOI: 10.1021/acsbiomaterials.5b00393.


23


Page 24 of 27

24. Tang, F.; Li, L.; Chen, D., Mesoporous Silica Nanoparticles: Synthesis, Biocompatibility and Drug Delivery. Adv. Mater. 2012, 24 (12), 1504-1534. DOI: 10.1002/adma.201104763. 25. Tarn, D.; Ashley, C. E.; Xue, M.; Carnes, E. C.; Zink, J. I.; Brinker, C. J., Mesoporous Silica Nanoparticle Nanocarriers: Biofunctionality and Biocompatibility. Acc. Chem. Res. 2013, 46 (3), 792-801. DOI: 10.1021/ar3000986. 26. Ahmadi, E.; Dehghannejad, N.; Hashemikia, S.; Ghasemnejad, M.; Tabebordbar, H., Synthesis and surface modification of mesoporous silica nanoparticles and its application as carriers for sustained

drug delivery. Drug Deliv. 2014,

21

(3), 164-172.

DOI:

10.3109/10717544.2013.838715. 27. Wang, S.; Liu, F.; Li, X. L., Monitoring of "on-demand" drug release using dual tumor marker mediated DNA-capped versatile mesoporous silica nanoparticles. Chem. Commun. 2017, 53 (62), 8755-8758. DOI: 10.1039/C7CC02752D. 28. Zhou, J.; Jayawardana, K. W.; Kong, N.; Ren, Y.; Hao, N.; Yan, M.; Ramström, O., Trehalose-Conjugated, Photofunctionalized Mesoporous Silica Nanoparticles for Efficient Delivery of Isoniazid into Mycobacteria. ACS Biomater. Sci. Eng. 2015, 1 (12), 1250-1255. DOI: 10.1021/acsbiomaterials.5b00274. 29. Yu, H.; Chen, J.; Liu, S.; Lu, Q.; He, J.; Zhou, Z.; Hu, Y., Enzyme sensitive, surface engineered nanoparticles for enhanced delivery of camptothecin. J. Control. Release 2015, 216, 111-120. DOI: 10.1016/j.jconrel.2015.08.021.


24

Page 25 of 27 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


30. Zhou, J.; Hao, N.; De Zoyza, T.; Yan, M.; Ramström, O., Lectin-gated, mesoporous, photofunctionalized glyconanoparticles for glutathione-responsive drug delivery. Chem. Commun. 2015, 51 (48), 9833-9836. DOI: 10.1039/C5CC02907D. 31. Knudson, W.; Bartnik, E.; Knudson, C. B., Assembly of pericellular matrices by COS-7 cells transfected with CD44 lymphocyte-homing receptor genes. Proc. Natl. Acad. Sci. 1993, 90 (9), 4003-4007. 32. Li, X.; Deng, D.; Xue, J.; Qu, L.; Achilefu, S.; Gu, Y., Quantum dots based molecular beacons for in vitro and in vivo detection of MMP-2 on tumor. Biosens. Bioelectron. 2014, 61 (Supplement C), 512-518. DOI: 10.1016/j.bios.2014.05.035. 33. Son, G.; Kim, H.; Ryu, J.; Chu, C.; Kang, D.; Park, S.; Jeong, Y.-I., Self-Assembled Polymeric Micelles Based on Hyaluronic Acid-g-Poly(d,l-lactide-co-glycolide) Copolymer for Tumor Targeting. Int. J. Mol. Sci. 2014, 15 (9), 16057. DOI: 10.3390/ijms150916057. 34. Wang, K.; Zeng, J.; Luo, L.; Yang, J.; Chen, J. I. E.; Li, B. I. N.; Shen, K., Identification of a cancer stem cell-like side population in the HeLa human cervical carcinoma cell line. Oncol. Lett. 2013, 6 (6), 1673-1680. DOI: 10.3892/ol.2013.1607. 35. Coradini, D.; Zorzet, S.; Rossin, R.; Scarlata, I.; Pellizzaro, C.; Turrin, C.; Bello, M.; Cantoni, S.; Speranza, A.; Sava, G.; Mazzi, U.; Perbellini, A., Inhibition of Hepatocellular Carcinomas in vitro and Hepatic Metastases in vivo in Mice by the Histone Deacetylase Inhibitor HA-But. Clin. Cancer Res. 2004, 10 (14), 4822-4830. DOI: 10.1158/1078-0432.ccr-04-0349.


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For Table of Contents Graphic Use Only

Extracellular Matrix Components shelled Nanoparticles as Dual EnzymeResponsive Drug Delivery Vehicles for Cancer Therapy Juan Zhou,a, ‡ Mingyu Wang, a, ‡ Huiyan Ying,a Dandan Su,a Huijie Zhang,a Guozhong Lub and Jinghua Chena,*


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Extracellular Matrix Components shelled ... - ACS Publications

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