enzyme sensitive chondroitin sulfate-based self-assembled nanoparticles loading docetaxel for the inhibition of metastasis and growth of melanoma

Carbohydrate Polymers 184 (2018) 82–93

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Redox/enzyme sensitive chondroitin sulfate-based self-assembled nanoparticles loading docetaxel for the inhibition of metastasis and growth of melanoma Mengrui Liu, Hongliang Du, Abdur Rauf Khan, Jianbo Ji, Aihua Yu, Guangxi Zhai

T

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Department of Pharmaceutics, College of Pharmacy, Shandong University, Jinan 250012, China

A R T I C L E I N F O

A B S T R A C T

Keywords: Amphiphilic self-assembled nanoparticles Redox/enzyme sensitiveness Melanoma Metastasis Chondroitin sulfate Smart drug delivery

In this report, redox/enzyme responsive chondroitin sulfate-ss-deoxycholic acid (CSCD) conjugates were synthesized using cystamine as the linkage which could self-assemble to form self-assembled nanoparticles (175.6 + 5.2 nm) in the aqueous environment. Docetaxel (DTX) was loaded in nanoparticles with desired loading efficiency for the inhibition of tumor growth and metastasis of melanoma. Interestingly, nanoparticles were demonstrated to respond to hyaluronidase-1 (Hyal-1) which could degrade chondroitin sulfate (CS) backbones. In this case, we designed dual-sensitive nanoparticles with enhanced drug release pattern under the presence of glutathione (GSH)/Hyal-1. Compared with Taxotere®, CSCD nanoparticles significantly improved the DTX distribution in tumors and lungs with about 4.4-fold higher area-under-the-curve (AUC) value. In situ tumor volume and pulmonary metastatic formation were reduced upon the administration of DTX-loaded CSCD nanoparticles via DTX-induced apoptosis and decreased metastasis-promotion protein expression. With only minor cytotoxicity, CSCD nanoparticles could be promising nano-drug delivery systems for successful management of melanoma.

1. Introduction Melanoma is a lethal malignancy with aggressive properties and its incidence has been rising steadily for the past few decades especially in white populations worldwide (Bombelli, Webster, Moncrieff, & Sherwood, 2014). Metastasis and the invasion of primary melanocytes to distant tissues severely hinder the therapy of malignant melanoma (Afshar, David, Fuehner, Gottlieb, & Gutzmer, 2016; Wurth et al., 2016) which is frequently accompanied with lung metastasis (Davies et al., 2011). Once melanoma has developed to the late-stage metastatic disease, it would become difficult to treat, resulting in high mortality. Therefore, 3-year overall survival of patients is less than 15% with conventional treatments such as surgery and chemotherapy (Balch et al., 2009). Despite advances in immunotherapy and gene therapy, options for the treatment of melanoma and metastasis are seriously limited especially due to the resistance to chemotherapeutics (Ferreira et al., 2016b). Owning to the impressive progress in material science, a large number of nanocarriers appearing recently offered the potential to improve therapeutic effects of melanoma by improving drug solubility and stability, prolonging drug circulation and minimizing off-target effects due to their small size and enhanced permeability and retention

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(EPR) effects. These nanocarriers include dendrimers, micelles, polymeric nanoparticles, inorganic nanoparticles, gold or metal oxide frameworks (Miao, Guo, Lin, Liu, & Huang, 2017). In addition, surface functionalization methodologies can endow nanocarriers with the ability to control their biodistribution and achieve active targeting property (Jurj et al., 2017). For example, Fan et al. recently developed ternary nanoparticles for melanoma therapy using poly (ethylene glycol) (PEG)-coating to reduce the protein adsorption and particle aggregation (Fan et al., 2017). Recently, although nanomedicine has made tremendous progress, only a few applicable nanodrugs achieve delightful therapeutic effects. This may be, to a certain extent, a consequence of difficulties in the control of the rapid release of the drug from targeted nanocarriers in the tumor microenvironment (TME) (Mura, Nicolas, & Couvreur, 2013). To enhance the delivery of nanocarriers into tumor and metastasis sites and improve the TME responsive drug release, stimuli-sensitive nanocarriers have been developed in response to internal triggers such as low pH values and redox potentials (Li, Xiao, Zhu, Deng, & Lam, 2014), facilitating drug release at desired sites with an “on-demand” (also termed ‘switch on/off’) strategy (Karimi et al., 2016; Mura et al., 2013). The unique and intriguing properties of these nanocarriers have

Corresponding author: Department of Pharmaceutics, School of Pharmaceutical Sciences, Shandong University, 44 Wenhua Xilu, Jinan 250012, China. E-mail address: [email protected] (G. Zhai).

https://doi.org/10.1016/j.carbpol.2017.12.047 Received 29 July 2017; Received in revised form 4 December 2017; Accepted 16 December 2017 Available online 21 December 2017 0144-8617/ © 2017 Published by Elsevier Ltd.

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2. Materials and methods

given hope for achieving tailored drug release profile with excellent spatial, temporal and dosage control (Zhu et al., 2015). Among these nanocarriers, various stimuli-responsive polymeric nanoparticles have been actively developed owing to their relative biocompatibility, multifunctionalization with targeting ligands or sensitive linkages, and enhanced stability (Cheng, Meng, Deng, Klok, & Zhong, 2013; Nicolas, Mura, Brambilla, Mackiewicz, & Couvreur, 2013). For example, smart pH/redox responsive folic acid-PEG-coated polymeric lipid vesicles were constructed for tumor targeted drug delivery (Cheng, Kumar, Zhang, & Liu, 2014; Wang et al., 2014). M., Giver Ahtoniraj et al. recently designed chitosan-cystamine-mPEG based redox responsive polymeric nanoparticles, which were allowed to swell and disrupt with rapid drug release under a reduced condition in the TME (G.A., Ayyavu, S., & Kandasamy, 2017). In this work, in order to treat melanoma by inhibiting the growth and metastasis of tumors, smart redox/enzyme responsive chondroitin sulfate-ss-deoxycholic acid (CSCD) self-assembled nanoparticles were designed by conjugating deoxycholic acid (DOCA) to CS. The redoxsensitive cystamine (CYS) was utilized as the linkage with disulfide bonds. Meanwhile, adipic dihydrazide (ADH) was applied to be another linker to form non-redox-sensitive chondroitin sulfate-ADH-deoxycholic acid (CSAD) conjugates as the control group. As a main component of bile acid, DOCA is a widely used hydrophobic segment to modify the hydrophilic polymer for the construction of stable self-assembled nanoparticles (Lee et al., 2012; Park et al., 2006). Docetaxel (DTX), which is an excellent and widely used chemotherapeutic agent especially against melanoma, was encapsulated into CSCD nanoparticles. CD44, a family of single-span transmembrane glycoproteins overexpressing in various tumors including ovarian, melanoma and breast cancers, has been proved to function as receptors of hyaluronic acid (HA) (Mattheolabakis, Milane, Singh, & Amiji, 2015; OrianRousseau & Ponta, 2015; Zhang et al., 2016), therefore, numerous HAbased nanocarriers have been developed to actively target drug to CD44 + tumors via the interaction between HA and CD44 (Qi, Fan, He, & Wu, 2015; Stefanello et al., 2017; Sun, Deng, Meng, Zhang, & Zhong, 2016; H. Zhang et al., 2017; L. Zhang et al., 2017). 27 Hyal-1 as one of HA family members is a lysosomal enzyme. It is overexpressed in various tumor cells and associated with the growth, metastasis and angiogenesis of tumors (Morera et al., 2017). The reason why HA is widely accepted as the drug delivery polymer is that HA could be degraded to low molecular weight components by Hyal-1 (Feng et al., 2016; Ji et al., 2016) after being taken up by cancer cells through receptor-mediated endocytosis (Zhang et al., 2016). On this basis, notably, increasing CS-based nanocarriers have been developed so far with the ability to target CD44 + tumors due to the structural similarity between CS and HA (Liang et al., 2015; Y.S. Liu Liu, Chiu, Chen, Chen, & Wang, 2014). However, most of them exhibit inadequate drug release efficacy in demanded sites. Although the existence of chondroitinase in mammals is still not established, it is believed that CS is also degraded by the Hyal-1 (Honda, Kaneiwa, Mizumoto, Sugahara, & Yamada, 2012), giving hope for the initial design of Hyal-1-responsive CS-based nanoparticles. Therefore, we hypothesized that our CSCD nanoparticles could not only enhance active targeted drug delivery efficiency in tumors, but also facilitate controlled release by synergetic effects of Hyal1 induced CS degradation and GSH induced disulfide bonds cleavage (as shown in Scheme ). The synthesis of novel CSCD conjugates was illustrated and clearly characterized. Tests of redox/enzyme responsive dissociation and drug release behaviors were conducted. Non-redoxsensitive CSAD nanoparticles were also constructed as a control group. Furthermore, we investigated not only in vivo anticancer effects but also antimetastasis effects due to the high propensity of melanoma to metastasize, and explored the possible mechanism. These results significantly verified the Hyal-1 responsive disassociation of CSCD nanoparticles and indicated the promising application in DTX delivery for the inhibition of the growth and metastasis of tumors.

2.1. Materials CS (Mw = 10 kDa) was provided by the Institute of Biochemical and Biotechnological Drugs of Shandong University. DOCA was purchased from Shanhai Sangon Biological Engineering Technology Services Co., Ltd. (Shanghai, China). CYS and ADH were obtained from Aladdin (China). 1-ethyl-3- (3-dimethylaminopropyl) -cabodiimide hydrochloride (EDC), N - hyodroxy succinimide (NHS), N,N,N’,N’ - tetramethylethylenediamine (TEMED), Sulforhodamine B (SRB) and fluorescein isothiocyanate (FITC) were acquired from Sigma – Aldrich (USA). DTX was purchased from Zelang Pharmaceutical Co. (Nanjing, China). Fetal bovine serum (FBS), RPMI-1640 medium were obtained from Gibco BRL (Gaithersberg, MD, USA). All reagents were of analytical grade and used without further purification. Mice melanoma cells (B16F10) were obtained from the Biochemical and microbial research institute and grown in RPMI 1640 medium containing 10% FBS, 100 U/mL penicillin G and 100 mg/mL streptomycin. The cells were cultured in a 37 °C incubator with 5% CO2. 2.2. Synthesis of CS-ss-DOCA and CS-DOCA conjugates 2.2.1. Synthesis of cystamine modified CS (CS-CYS) and adipic dihydrazide modified CS (CS-ADH) To provide amino groups on CS, CS-CYS was synthesized by coupling the amine terminals of CYS into the carboxyl groups of CS backbones via amidation reaction (Li et al., 2012). Firstly, 0.2 g CS (0.50 mmol) was dissolved in deionized water to get a concentration of 2 mg/mL, followed by addition of 1.54 g CYS (10.0 mmol) under a stirring condition. After the pH value was adjusted to 6–7 by 0.1 M hydrochloric acid, 0.8 g EDC (4.0 mmol) and 0.117 g NHS (1.0 mmol) were added to this solution. The reaction was carried out for 8 h at room temperature, and the pH of the reacting solution was then altered to neutral value by 0.1 M sodium hydroxide to quench this reaction. The resulting mixture was dialyzed (MWCO 3500) exhaustively against 0.1 M sodium chloride and then deionized water to remove the trace of CYS, EDC and NHS coupling agents. Finally, these conjugates were isolated by lyophilization. CS-ADH was synthesized as our previously reported literature (Liu, Du, & Zhai, 2016). 2.2.2. Synthesis of CSCD and CSAD conjugates DOCA was chemically grafted into CS-CYS and CS-ADH via amide bonds to prepare amphiphilic CSCD and CSAD conjugates, respectively (Wei, Dong, & Liu, 2015). In brief, DOCA was dissolved in N,N- dimethyl formamide (DMF) and equal amount (2 equiv. / DOCA) of EDC and NHS were added simultaneously to activate the carboxyl groups of DOCA. The activation was allowed for 40 min and TEMED (2 equiv. / DOCA) was then added to adjust the pH, finally acquiring NHS ester of DOCA ((DOCA-NHS)). CS-CYS and CS-ADH were dissolved in 20 mL mixed solvent (deionized water: DMF (1:1, v/v)), respectively, with the concentration of 5 mg/mL. Then, the (DOCA-NHS) dissolved in 3 mL DMF was drop-wise to the solution and the feeding molar ratio of (DOCA-NHS) to CS ranged from 0.4 to 1.0. The resulting solution was dialyzed (MWCO 3500) against excess amount of water/ethanol (1:1, v/v) for 24 h and then deionized water for 48 h. These conjugates were subsequently isolated by lyophilization. The synthesis process of CSCD and CSAD conjugates were shown in Fig. 1A and Fig. S1, respectively. 2.3. Characterization of amphiphilic CSCD and CSAD conjugates The chemical structures of polymers were characterized by 1H NMR spectrometer (AvanceTM DPX-300). CS was dissolved in D2O, while CSCD and CSAD were dissolved in D2O / DMSO-d6 (1:1, v/v). FT-IR spectra of polymers were analyzed via FT-IR spectrometer (Nicolet 6700, USA) by KBr plates. 83

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Scheme 1. Illustration of the formation, tumor-targeted accumulation and redox/ enzyme-responsive drug release of self-assembled DTX-CSCD nanoparticles. EPR effect meant enhanced permeation and retention effect.

sulfuric acid (65/50, v/v)), respectively. The reaction was conducted for 15 min at 70 °C under agitation condition. When conjugate solutions were cooled to room temperature, the UV absorbance was measured at 378 nm against the free solvent as the blank group. The DS was calculated via the following equation:

The degree of substitution of DOCA (DS, mol%, the number of DOCA molecules per 100 sugar residues of CS) was quantified using UV/vis spectrophotometer (Yu, Li, Qiu, & Jin, 2009). Accurately weighted CSCD and CSAD conjugates were dissolved in 10 mL mixed solution (0.5 mL DMSO, 0.5 mL aqueous acetic acid, 9.0 mL water/

Fig. 1. The synthesis and characterization of CSCD copolymer. (A) Synthesis route of CSCD. 1H NMR (B) and FT-IR (C) spectra of CS, CS-CYS, CSAD and CSCD copolymers.

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m /M DS = ⎛ 1 DOCA ⎞ ⎝ (m -m1)/MCS ⎠ ⎜

mL Hyal-1 (Feng et al., 2016). After being incubated in a shaking bed at 37 °C with a rotation speed of 100 rpm, these solutions were extracted at each time point. After the exposure to triggers, nanoparticles were purified by centrifugation (3300 rpm, 20 min) to remove residues of GSH and Hyal-1 for size measurement by DLS and TEM. Notably, CSCD and CSAD nanoparticles incubated without triggers were observed to confirm the self-assembled ability and morphology as control groups. Meanwhile, the morphology change of CSAD nanoparticles exposed to triggers was monitored.

⎟

(1)

where m1 is the amount of DOCA in CS polymer; m is the amount of CSCD or CSAD; MDOCA is the molecular weight of DOCA residues; MCS is the molecular weight of CS units. 2.4. Preparation and characterization of self-assembled nanoparticles 2.4.1. Preparation of self-assembled nanoparticles CSCD self-assembled nanoparticles were prepared via modified sonication-dialysis method (Liu et al., 2016). Briefly, 4 mg conjugates were dissolved in 2 mL deionized water under stirring condition, and then treated with sonication for 4 min (4 s on, 2 s off) by a probe sonicator at 120 W in an ice bath. In order to guarantee good dispersion of conjugates, the sonication was repeated three times. The resulting nanoparticle solution was filtrated through 0.45 μm syringe filter. CSAD nanoparticles were prepared by the same protocol.

2.6. Preparation of DTX-loaded CSCD (DTX-CSCD) nanoparticles and DTX-loaded CSAD (DTX-CSAD) nanoparticles DTX, an insoluble chemotherapeutic agent in aqueous solution, was encapsulated to nanoparticles by modified ultrasound-dialysis method (H. Zhang et al., 2017; L. Zhang et al., 2017). In brief, CSCD and CSAD nanoparticles were completely dissolved in deionized water and different amount of DTX dissolved in methanol was added to the solution under the drastic stirring condition. After being stirred for 4 h, the suspension was sonicated with a probe sonicator and then dialyzed for 24 h against deionized water to remove methanol. The suspension was subsequently centrifuged at 4000 rpm for 20 min to discard precipitate (unencapsulated DTX) followed by being filtered through a 0.45 μm syringe filter. The drug loading (DL) and entrapment efficiency (EE) were calculated by following equations:

2.4.2. Characterization of self-assembled nanoparticles The particle size distribution and zeta potential of CSCD and CSAD nanoparticles were measured on a Malvern Zetasizer Nano-ZS90 dynamic light scattering (DLS) (Malvern instruments, UK) (n = 3). Moreover, the morphology of nanoparticles was observed using transmission electron microscopy (TEM, JEM-100CX II, Hitachi, Japan). The samples were prepared by dropping 1 mg/mL nanoparticle solution on the copper grid, before being stained with 2% phosphotungstic acid. The self-aggregation behavior was evaluated via the measurement of critical micelle concentration (CMC) value using pyrene as a hydrophobic fluorescence probe by a fluorospectrophotometer. Pyrene solution in acetone with the concentration of 6.0 × 10−5 M was added to a series of brown volume flask, after which the acetone was evaporated capitalizing on nitrogen flow. Then, CSCD and CSAD conjugates solutions (the concentration ranged from 1.0 × 10-3 to 0.5 mg/mL) were added to volume flask, respectively, getting a final pyrene concentration of 6.0 × 10-7 M. Subsequently, these conjugates solutions were treated with ultrasonic incubation for 40 min and then kept overnight at 37 °C. Finally, the spectrophotometer (F-7000, Hitachi, Japan) was used to scan the emission spectra of pyrene from 350 nm to 450 nm at a fixed excitation wavelength of 334 nm. The CMC values of CSCD and CSAD nanoparticles were determined by the cross point of acquired curves when plotting the intensity ratios of I373/I384 against the logarithm of conjugate concentration.

DL% =

Weight of DTX in nanoparticles Weight of the feeding conjugates and weight of DTX in nanoparticles × 100%

EE% =

Weight of DTX in nanoparticles × 100% Weight of the feeding DTX

(3)

(4)

DTX concentration was measured using high performance liquid chromatography (HPLC) equipped with a Hypersil-ODS2 C18 column (5 μm particle size, 250mm × 4.6 mm). The mobile phase was the mixed solution of H2O: CH3CN (45:55, v/v) and the flow rate was 1.0 mL/min at UV wavelength of 230 nm. DTX-loaded nanoparticles were also characterized including particle size and zeta potential as mentioned in 2.4.2. 2.7. In vitro drug release from self-assembled nanoparticles

2.4.3. Hemolytic test The hemolytic test was conducted to evaluate the safety of nanoparticles (Guo et al., 2013). Briefly, 2.5 mL of CSCD nanoparticles solutions with different concentrations were mixed with 2.5 mL of erythrocytes suspension in nature saline (NS) (2%, v/v). All samples were incubated for 1 h at 37 °C and subsequently centrifuged at 3000 rpm for 10 min to remove intact erythrocytes. The absorption of collected supernatant was analyzed by a UV/vis spectrophotometer at 541 nm. The hemolysis rate was calculated by the following equation:

Hemolysis (%) =

Asample - Anegative × 100% Apositive - Anegative

The release profile of DTX from CSCD and CSAD nanoparticles was evaluated by dialysis method. Briefly, DTX-CSCD nanoparticles were placed in dialysis tubes (MWCO 3500) which were immersed in the release medium (PBS buffer, pH 7.4, 0.1 M), containing 0 mM GSH, 10 mM GSH, 150 U/mL Hyal-1 or 10 mM GSH and 150 U/mL Hyal-1 together, respectively. These samples were gently shaken at 37 °C in a water bath at 100 rpm. At desired time intervals, certain medium was extracted followed by supplement of the same amount of fresh medium to retain the consistent release medium. Drug release from DTX solution, DTX-CSAD in free PBS and PBS with 10 mM GSH and 150 U/mL Hyal-1 was conducted, respectively. The released DTX content was measured by HPLC analysis and all samples were conducted in triplicate.

(2)

where Asample, Apositive and Anegative are the absorbance of sample, a solution which is 100% hemolysis and 0% hemolysis, respectively. The sample solution without erythrocytes was used as a blank control to compensate the turbidity of conjugate samples.

2.8. Intracellular uptake and endocytosis inhibition assay To trace uptake capacities of nanoparticles in B16F10 cells, FITClabeled CSCD and CSAD conjugates were synthesized (Liu et al., 2016). Cells were seeded in a 12-well plate (1.5 × 103 cells/well) and allowed to adhere overnight. The medium was then replaced by the fresh medium containing FITC-CSCD or FITC-CSAD nanoparticles to treat

2.5. Disassembly of nanoparticles triggered by GSH and Hyal-1 To investigate the disassembly pattern of CSCD nanoparticles responding to GSH and Hyal-1, nanoparticles were dissolved in PBS (pH 7.4, 0.1 M) (2 mg/mL) containing 10 mM GSH with or without 150 U/ 85

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accurately and mixed with extraction buffer, before being homogenized using FJ-200 high-speed shearing machine (Jincheng Guosheng Experimental Instrument Factory, Jintan, China). Then, tissue homogenates were conducted as the protocol of plasma samples mentioned above in 2.10. Finally, the concentration of DTX was measured by HPLC. Data were normalized to the tissue weight.

cells for further 0.5 h and 4 h, respectively. Then, cells were washed by PBS followed by being collected and the mean fluorescence intensity (MFI) of cells was measured by a flow cytometer (FCM) to quantitatively measure the cellular uptake capacity of nanoparticles. Meanwhile, fluorescence microscope was used to monitor the endocytosis of nanoparticles using the same protocol mentioned above. Cells were washed by PBS and incubated with Hoechst 33,342 to stain nucleus for 1 h before observation. To investigate the internalization mechanism of nanoparticles, various inhibitors were used to per-incubate cells for 2 h including free CS (10 mg/mL), chlorpromazine (CPZ, 10 μg/mL), sucrose (0.45 M), genistein (10 mg/mL), Cytochalasin D (3.5 μg/mL) and sodium amide (NaN3, 25 M) (Y. Liu et al., 2014). Following procedures were processed as mentioned above.

2.12. In vivo antitumor and antimetastasis efficacy Before experiments, tumor-bearing mice were achieved as described above. When the tumor volume grew to a volume of 100–150 mm3, mice were randomly divided into four groups (n = 6) which were intravenously administrated with DTX-CSCD and DTX-CSAD nanoparticles, Taxotere® and NS for three times (interval 4 days), respectively. Body weight was recorded and tumor volume was measured by the formula: V = (W2 × L) /2 (Width (W) is the shortest diameter while Length (L) is the longest diameter perpendicular to width). After 18 days, mice were sacrificed and major organs were removed for histological examination by H&E staining. Among these organs, tumors and lungs (main metastatic tissues) were further collected for photo imaging, counting the macroscopic metastasis nodules in lungs.

2.9. In vitro cytotoxicity and apoptosis studies Cell killing abilities of DTX-CSCD, DTX-CSAD nanoparticles and DTX solution were evaluated by SRB assay as described by Showkat et al. (Ganie et al., 2016). In brief, cells were seeded in 96-well plate at the density of 5 × 103 cells/well and incubated by 100 μL medium. Fresh medium containing a series concentration of DTX-loaded nanoparticles and DTX solution were added to replace previous medium after cell adhesion. Cells were further treated for 24 h, 48 h and 72 h, respectively, before being fixed with 10% TCA (w/v) and then stained by SRB solution (100 μL, 0.4% in 1% acetic acid). The unbound SRB was washed by 1% acetic acid and the bound dye was extracted with 10 mM Tris-hydrochloric acid buffer (100 μL, 0.01 M, pH 10.4) for determination of optical density (OD) values at 570 nm. Cells without drug treatment were conducted by the same protocol and each sample was conducted in triplicate. The percentage of cell viability was calculated by the following formula:

2.13. TUNEL analysis and immunohistochemical assay of tumor tissues To further evaluate the tumor apoptosis of tumor sections in 2.12, the TdT-mediated dUPT Nick-End Labeling (TUNEL) assay was conducted by manufacturer’s guidelines (Roche, Basel, Switzerland). Moreover, the immunohistochemical (IHC) staining assay of tumor sections was proceed. Briefly, tumor sections were initially incubated with anti-COX-2 goat antibody (Santa Cruz Biotechnology, Dallas, USA) overnight at 4 °C followed by incubation of secondary antibody (Dako, Glostruo, Denmark) for 1 h at room temperature. Sections were then stained with diaminobenzidine (DAB) solutions for about 5 min, which were finally counterstained with hematoxylin.

Cell viability (%) = (A570 sample-A570 blank) / (A570 positive control-A570 blank) × 100%

2.14. Statistical analysis

Qualitative cell apoptosis was assessed by nuclear staining. Cells were treated with different DTX formulations, before being stained by Hoechst 33,342 for 30 min in darkness at room temperature. Eventually, cells were washed three times by PBS and observed under a fluorescence microscope (BX40, Olympus).

Statistical analysis was carried out using the SPSS (version 15.0) (SPSS Inc., Chicago, IL) and Origin 6 (MicroCal Inc., Northampton, MA) software followed by the evaluation with one-way ANOVA and the Bonferroni’s t-test. All data were expressed as mean values together with the corresponding standard deviation (Mean + SD). The statistically significance was confirmed when the acquired P value was less than 0.05.

2.10. Pharmacokinetics of self-assembled CSCD and CSAD nanoparticles Wistar rats were fasted for 12 h before the experiment and randomly divided into three groups (n = 4), which were intravenously administrated by DTX-CSCD and DTX-CSAD nanoparticles and Taxotere®, respectively, with the DTX dosage of 15 mg/kg. At predetermined time point, 0.5 mL of blood was extracted from sinuses jugularis of rats into heparinized tubes followed by further centrifugation at 4000 rpm for 10 min to retain plasma. Samples were stored at − 20 °C until further measurement. At the next step, 200 μL of plasma was mixed with 2 mL of methyl-tert-butyl ether, and the mixture was then centrifuged at 4000 rpm for 15 min to collect supernatant. The methyl-tert-butyl ether was evaporated by nitrogen flow, before adding acetonitrile to dissolve DTX for the measurement of drug concentration by HPLC.

3. Results and discussion 3.1. Synthesis of conjugates and characterization CS-CYS and CS-ADH were primarily synthesized by amide-reactive coupling. Carboxyl groups on DOCA were activated by EDC and NHS, followed by coupling with amino groups on CS-CYS or CS-ADH. Therefore, the linkage was the only difference between redox-sensitive CSCD conjugates and non-redox-sensitive CSAD conjugates (Liu et al., 2016). The chemical structures of CSCD conjugates were demonstrated by 1 H NMR and FT-IR, as shown in Fig. 1B–C. The signals at 2.01 ppm and 3.23–4.65 ppm were the characteristic peaks of CS polymer. The cystamine exhibited peaks at the range of 2.80–2.90 ppm and 2.93–3.10 ppm in CS-CYS with a little shift in CSCD result, in addition, the DOCA was founded at 0.54–1.56 ppm. The amount of cystamine and DOCA could be quantitatively characterized via the integration ratio between the methylene in cystamine (δ = 2.85 ppm [4H, -CH2-S]), or the methyl group in DOCA (δ = 0.77 ppm [3H, -CH3]) and the Nacetyl group in CS (δ = 2.01 ppm [3H, -CH3]), respectively. In FT-IR spectrum, CS polymer showed its characteristic peaks in

2.11. In vivo biodistribution In vivo biodistribution of DTX formulations was investigated in B16F10-bearing C57 mice with the initial subcutaneous oxter inoculation of 5 × 105 cells/each mouse. Then, mice were randomly divided into three groups which were intravenously injected with DTX-CSCD and DTX-CSAD nanoparticles and Taxotere®, respectively. At predetermined time point, mice (n = 3 of each group) were sacrificed and autopsied with major organs excision. Isolated tissues were weighted 86

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and their low CMC values helped them form stable self-assembled nanostructures easily and retain their spherical morphology in high dilution aqueous environment. Thereafter, CSCD and CSAD nanoparticles could be designed as the ideal entrepot for lipophilic agents. CSCD3 and CSAD3 were chosen as the nanoparticles studied in the following experiments. The result of the hemolysis ratio of CSCD nanoparticles was depicted in the Fig. S2, and the hemolysis ratio was 3.6% + 0.25 even at a DTX concentration as high as 3 mg/mL. In addition, the hemolysis ratio of CSAD nanoparticles in our previous literature indicated 4.5% + 0.43 at 3 mg/mL (Liu et al., 2016). These results demonstrated the good blood compatibility of CSCD and CSAD nanoparticles, suggesting the suitable abilities of these nanoparticles for intravenous administration.

Table 1 Characterization of self-assembled nanoparticles. Values are presented as Mean + SD, n = 3. sample

DS (%)

Diameter (nm)

Polydispersity index (PDI)

Zeta potential (mV)

CMC (mg/ mL)

CSCD1 CSCD2 CSCD3 CSAD3

4.5 7.2 10.1 7.0

169.5 + 0.9 157.6 + 6.5 139.7 + 6.1 165.2 + 4.3

0.168 + 0.5 0.130 + 0.5 0.175 + 0.1 0.234 + 0.0

−18.7 + 1.8 −20.5 + 0.7 −21.5 + 1.1 −20.5 + 0.8

0.048 0.031 0.022 0.027

3441.54 cm−1 for the eOH stretch vibration and eNH symmetrical vibration, 2931 cm−1 for CeH stretch vibration, 1632.76 cm−1 for carbonyl (eC]O) stretch vibration, 1422.41 cm−1 for eOH and eCH deformation (ring), 1383.77 cm−1 for eCH3 deformation (bend) vibration, 1230.65 cm-1 for S]O stretching vibrations, 1040.33 cm−1 for eCeO stretch vibration. In the spectra of CSAD and CSCD conjugates, there were carbonyl (eC]O) stretch vibration for amide moieties in1658.52 cm−1 for CSCD and 1651.36 cm−1 for CSAD, respectively. The new peaks for those tow conjugates appearing at 1564.08 cm−1 were corresponded to the amide band II absorption (NeH bond bending vibration) due to the formation of amide linkages. Other chemical bonds also appeared with a little shift from the peak positions of those shown in the CS spectrum. Different amount of DOCA were grafted to CS backbones and the highest DS of DOCA in CSCD conjugates could be 10.1% while the figure for CSAD was 7.0%. The characteristics were shown in Table 1.

3.3. The stimuli sensitive disassembly of self-assembled nanoparticles To investigate the responsive disassembly of nanoparticles, they were incubated with 10 mM GSH or (/ and) 150 U/mL Hyal-1 (Agrahari et al., 2016; Z. Chen Chen, Li, Yin, Ren, & Qu, 2013; Huang et al., 2015; Lin et al., 2016; Toole, 2004), after which the size distribution was quantitatively measured with DLS (Fig. S3) and the morphology was observed under TEM (Fig. 2A). The size distribution of CSCD3 was stable after 24 h incubation without GSH, changing from 144.1 nm to 146.1 nm for 24 h (Fig. S3a-b). Being treated with Hyal-1 for 24 h, the size of some particles reduced which might be attributed to the degradation of CS backbones (Fig. S3c). When incubated with 10 mM GSH for 24 h, the unimodal peak of size distribution changed to two peaks and the size of some nanoparticles increased to 440.2 nm while some remained about 100 nm (Fig. S3d), which were observed in Fig. 2Ab. It was mainly because that these redox-sensitive particles became slack and thereby swelled via the cleavage of disulfide linkages, leading to the detachment of CS shells from micellar nanoparticles and the hydrophilic-hydrophobic imbalance (Li et al., 2012). In addition, the size distribution was shifted to multimodal peaks and the size of a large number of particles was larger than 1000 nm or smaller than 40 nm when these particles were incubated with GSH and Hyal-1 for 24 h (Fig. S3e, Fig. 2Ac). These results indicated the synergetic effects of double triggers in the contribution to the rapid disassembly of nanoparticles. As shown in Fig. 2A, CSCD3 (a) and CSAD3 (d) nanoparticles showed the spherical state and uniform distribution without any trigger incubation. The mean diameters of CSAD3 nanoparticles remained steady even in the existence of GSH (e). However, these CSAD3 nanoparticles swelled and performed aggregation when treated with Hyal-1 (f), demonstrating the enzyme responsive property of CS.

3.2. Preparation and characterization of self-assembled nanoparticles The self-assembled CSCD and CSAD nanoparticles were prepared with a sonication method and the mean diameter of CSCD nanoparticles measured by DLS ranged from 139 to 170 nm with low PDI, achieving homogeneously core-shell nano-structured particles in aqueous medium. When the DS increased, the particle size of CSCD nanoparticles decreased, which indicated the formation of more dense hydrophobic cores via the enhanced hydrophobic interaction between DOCA moieties. Particle size plays an important role in pharmacokinetics and the internalization of drug formulations (Yousefpour, Atyabi, Vasheghani-Farahani, Movahedi, & Dinarvand, 2011). It has been reported that nanoparticles with high size distribution tend to be scavenged and cleared by macrophages, and if the diameter is below 300 nm, particles could avoid the complement system activation and blood clearance. In particular, particles with about 150 nm in size could perform satisfied cellular uptake efficiency (He, Hu, Yin, Tang, & Yin, 2010). The zeta potential of CSCD nanoparticles was about − 21 mV and the result revealed the favorable stability of colloidal systems. These properties endowed the nanoparticles with compact structures, enhanced stability and good biocompatibility in blood circulation. In addition, the TEM results of CSCD nanoparticles in Fig. 2Aa revealed the homogeneous spheres morphology, indicating the self-assembled ability of CSCD conjugates. CSAD nanoparticles (b) exhibited similar size distribution, zeta potential and spherical shape to CSCD. Obviously, the size of particles under TEM was slightly smaller than that determined by DLS, it was mainly ascribed to the particle shrink when samples were dried before observation under TEM, while samples were conducted in hydrated state for DLS measurement (Jiang, Quan, Liao, & Wang, 2006). The CMC, determined using pyrene as a hydrophobic fluorescence probe, plays a pivotal role in the formation and stability of nanoparticles. The CMC of CSCD decreased from 0.048 mg/mL to 0.022 mg/ mL with increased DS of DOCA, suggesting that the formation of compact core was derived from the hydrophobic interaction between increased DOCA moieties. CSCD3 nanoparticles exhibited 0.022 mg/mL in CMC while the figure for CSAD3 nanoparticles was 0.027 mg/mL,

3.4. The preparation and characteristics of DTX-loaded self-assembled nanoparticles The chemotherapeutic drug, DTX, was encapsulated into CSCD3 and CSAD3 nanoparticles through an ultrasound-dialysis method and the characteristics were shown in Table 2. Increasing the feed ratio of PTX to conjugates from 1:10 to 5:10, the DL of CSCD3 nanoparticles sharply increased from 3.6% to 20.7%. However, the EE primarily showed a remarkable increase from 37.4% to 61.8% when the feed ratio rose from 1:10 to 3:10, but it decreased to 52.32% when the feed ratio further increased to 5:10. The possible reason was that enhanced hydrophobic interaction between drug and conjugates facilitated the upward trend in EE, but further increase of DTX amount induced the unbalance between hydrophilic and hydrophobic sections, leading to more precipitation of unloaded DTX and sharply declined EE. For size distribution, the figure for DTX-CSCD3 nanoparticles showed a slight increase with increased DTX ratio, but not an obvious change compared to blank CSCD3 nanoparticles. It demonstrated the conjugates could provide adequate spaces for drug storage without dramatic size increase. In addition, the zeta potential results revealed the stability of drug loaded nanoparticles. 87

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Fig. 2. In vitro characteristics of nanoparticles. (A) The TEM of CSCD nanoparticles in free medium (a) or treated by 10 mM GSH after 12 h (b) or treated by 10 mM GSH and 150 U/mL Hyal-1 after 12 h (c). The TEM of CSAD nanoparticles in free medium (d) or treated by 10 mM GSH after 12 h (e) or treated by 10 mM GSH and 150 U/mL Hyal-1 after 12 h (f). (B) Plot of the intensities ratio I384/I373 from pyrene excitation spectra as a function of the logarithms of concentration of CSCD and CSAD conjugates. (C) The cumulative release profile of DTX solution and DTX-loaded CSAD3 and CSCD3 nanoparticles under different conditions at 37 °C (n = 3). (D) The plasma DTX concentration-time profiles in rats following i.v. administration of Taxotere®, DTX-CSCD3 and DTX-CSCD3 nanoparticles (n = 4).

induced by Hyal-1 due to the specific enzymolysis of outer CS. Therefore, the above results indicated redox- and enzyme- sensitive drug release behaviors of CSCD3 nanoparticles.

3.5. In vitro drug release triggered by glutathione and enzyme As shown in Fig. 2C, DTX released from CSCD3 nanoparticles without GSH and Hyal-1 in the release medium was about 15.2% at 6 h and about 67.1% at 48 h, respectively. Accelerated drug release could be observed with single trigger, for instance, DTX release reached 32.1% in the presence of 10 mM GSH and 37.8% with 150 U/mL Hyal-1 at 6 h, respectively. When CSCD3 nanoparticles were incubated with GSH and Hyal-1 simultaneously, DTX release even reached 65.5% with a sharp release rate at 6 h. However, it took around 48 h for DTX to be released from CSCD3 without triggers. Non-redox-sensitive CSAD3 nanoparticles displayed a relatively steady rise pattern in DTX release in the presence of 10 mM GSH, which was similar to the trend of nontriggers induced DTX release from CSCD3 nanoparticles. This trend was consistent with that of CSAD3 nanoparticles without triggers reported in our previous study (Liu et al., 2016). Notably, there was a similarly accelerated release pattern between CSAD3 and CSCD3 nanoparticles

3.6. Cellular uptake and internalization mechanism The internalization of FITC labeled CSCD3 and CSAD3 nanoparticles was quantitatively analyzed by flow cytometry and the MFI was used to denote uptake capacity. As shown in Fig. 3Aa, the fluorescent intensity in B16F10 cells was weak at 0.5 h and enhanced at 4 h, demonstrating the time-dependent internalization pattern. As presented in Fig 3Ab, the MFI of CSAD3 increased from 37.93 (0.5 h) to 118.02 (4 h), while the figure for CSCD3 ranged in 62.42–190.95. In addition, the MFI of CSCD3 showed a 1.70- and 1.62-fold higher than that of CSAD3 nanoparticles at 0.5 h and 4 h, respectively, manifesting the advantageous uptake efficiency of CSCD3 compared with that of CSAD3 nanoparticles.

Table 2 The characterization of DTX-loaded self-assemble nanoparticles. Values are presented as mean + SD, n = 3. sample

drug / carrier (DTX:polymer)

DL(%)

EE(%)

Diameter (nm)

Zeta potential (mV)

CSCD3

1:10 2:10 3: 10 5:10 3: 10

3.6 + 0.2 8.0 + 1.2 15.6 + 0.9 20.7 + 0.2 11.8 + 1.2

37.4 + 2.5 43.3 + 2.1 61.8 + 4.1 52.3 + 0.6 44.8 + 5.0

143.0 + 4.8 159.3 + 4.0 175.6 + 5.2 191.5 + 5.6 178.6 + 4.2

−20.9 + 1.4 −21.8 + 0.7 −23.6 + 1.1 −25.4 + 0.9 −26.0 + 0.6

CSAD3

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Fig. 3. (A) Cellular uptake of CSAD3 and CSCD3 nanoparticles and internalization mechanism of CSCD3 nanoparticles by flow cytometer. (a) FCM histograms for CSAD3 and CSCD3 in B16F10 cells at different times. (b) The quantitative results of CSAD3 and CSCD3 uptake in B16F10 cells at different times. Indicated values were mean + SD (n = 3), *p < .05, **p < .01, significant difference between CSAD3 and CSCD3 groups. (c) FCM histograms for CSCD3 in B16F10 cells after 4 h incubation in the presence of endocytic inhibitors. (d) The quantitative results of the effect of different endocytic inhibitors in cellular uptake of CSCD3 nanoaprticles in B16F10 cells. Indicated values were mean + SD (n = 3), *p < . 05, **p < .01, vs. control. (B) Fluorescence microscopy images of B16F10 cells after 0.5 h and 4 h with CSCD3, CSAD3 nanoparticles, CSCD3 nanoparticles with free CS, respectively.

viability exceeded 95% even when the concentration of nanoparticles reached 10 mg/mL. As shown in Fig. S4a-c, the cytotoxicity of DTXCSCD3 nanoparticles was higher than that of DTX-CSAD3 nanoparticles at DTX concentration of 0.01 μg/mL at all incubation time points. Table S1 summarized the IC50 values of DTX-loaded nanoparticles, DTX solution at different incubation times. After 24 h, a lower IC50 value of DTX solution was observed compared to that of DTX-CSAD3 nanoparticles. The observation commonly depended on the fact that DTX solution was able to diffuse into cytosol rapidly via passive diffusion without energy support. DTX-loaded nanoparticles could be internalized via receptor-mediated uptake especially CD44 mediated endocytosis capitalizing on energy, which might delay the intracellular drug release process and reduce cell inhibition efficiency (Li et al., 2012). However, GSH/Hyal-1 responsive DTX-CSCD3 nanoparticles could achieve rapid disassembly and drug release in cytosol and exhibited enhanced cell killing ability, suggesting the potent of DTXCSCD3 nanoparticles for intracellular delivery of DTX. After 24 h incubation of DTX formulations, the morphology of cells observed in Fig. S5 also revealed the highest apoptosis percentage of DTX-CSCD3 nanoparticles followed by DTX-CSAD3 nanoparticles, which were both higher than that of DTX solution.

To further investigate the internalization pathway of CSCD3 nanoparticles, the MFI was quantified by flow cytometry with pre-incubation of various inhibitors (Fig. 3Ac-d). When cells were pretreated with free CS before the treatment of CSCD3 nanoparticles, the MFI was several times lower than that of untreated one (60.45% for control). It could be attributed to the competitive bind between free CS and CD44, which blocked the CD44 receptor-mediated endocytosis of CSCD3 nanoparticles. Notably, the chemical modification of CS with DOCA did not affect the receptor-mediated internalization of CS because DOCA moieties were located in the core while CS backbones formed the hydrophilic surface. Treatment of NaN3 could obviously reduce the cellular uptake of CSCD3, which demonstrated the energy-dependent internalization. CPZ and Sucrose induced about 21.47% and 25.00% reduction of MFI values, respectively, indicating the slight effect of clathrin- and osmotic pressure-mediated pathway in CSCD3 internalization. By contrast, weak inhibition by cytochalasin D revealed the negligible effect of macropinocytosis in the uptake process. Interestingly, the uptake of CSCD3 saw an increase after the incubation with genistein which is an inhibitory agent of caveolin-mediated pathway. 3.7. Cytotoxicity and apoptosis

3.8. Pharmacokinetic study

To construct biocompatible drug delivery systems, the cytotoxicity of DTX-CSCD3, DTX-CSAD3 and free DTX was evaluated in B16F10 cells by SRB assay. As shown in Fig. S4d, free CSCD3 and CSAD3 nanoparticles had a neglect impact on the cell growth and the cell

The mean plasma concentration level of DTX during 48 h was determined after a single tail intravenous injection of DTX-CSCD3, DTX89

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occurred after the release from nanoparticles, and the nano-formed DTX was able to improve the retention in blood circulation. Both the known “EPR effect” (passive targeting) and CS receptor-mediated endocytosis (active targeting) may benefit nanoparticles to accumulate into tumor sites and therefore have the potential to improve antitumor effects of nanoparticles.

Table 3 Mean pharmacokinetic parameters of DTX after intravenous (i.v.) administration of DTX solution, DTX-CSAD3 and DTX-CSCD3 self-assembled nanoparticles to rats (n = 4). Parameter

Taxotere®

DTX-CSAD3

DTX-CSCD3

AUC0-48 a(mg/Lh) AUC0-∞ (mg/Lh) MRT0-48 b(h) MRT0-∞ (h) t1/2z c(h) CL d[L/(hkg)] Cmax e(mg/L)

6.17 + 0.82 6.28 + 0.82 1.50 + 0.09 1.66 + 0.09 1.46 + 0.02 2.42 + 0.33 4.47 + 0.66

27.39 + 1.78 27.41 + 1.78 5.64 + 0.09 5.73 + 0.10 5.04 + 0.31 0.55 + 0.04 9.88 + 1.57

27.57 + 3.62 27.65 + 3.63 4.68 + 0.43 4.82 + 0.45 6.32 + 0.24 0.55 + 0.07 13.00 + 1.54

a b c d e

3.9. In vivo tissue distribution In vivo distribution via tail vein injection can be affected by drug accumulation and elimination rate, which is of great importance to the therapeutic efficiency as well as side effects. After the administration of DTX-loaded nanoparticles and Taxotere® at drug dose of 10 mg/kg, the distribution of DTX in major tissues was determined in B16F10-bearing C57 mice metastasis tumor models. As shown in Fig. 4, DTX-loaded CSCD3 and CSAD3 nanoparticles showed an obviously increased drug accumulation in lung (the metastasis organ) (Ernsting et al., 2012; Ferreira et al., 2016a) and tumor compared with that of Taxotere®. There were about 4.40- and 3.61-fold of DTX amount from DTX-loaded CSCD3 and CSAD3 nanoparticles in lungs and tumors at 2 h, respectively, higher than that from Taxotere®. In addition, Taxotere® could be rapidly cleared in these major tissues and DTX could be hardly detected at 6 h while DTX-CSAD3 nanoparticles could be still detected in tumors even at 12 h. When DTX was loaded in nanoparticles, it could be released from nanoparticles via permeation as well as the dissociation of nanoparticle skeletons. In this case, compared with free drug, DTXloaded nanoparticles enabled drug to delay the release rate, prolong the retention time in tissues and thereby improve antitumor therapeutic effects. In addition, the expected excellent tumor targeting ability of prepared nanoparticles could be attributed to the following three reasons. Initially, these nanoparticles were able to target tumors because of the EPR effect based on their favorable size distribution (about 175.6 nm) and the receptor-ligand interaction. Secondly, the slow drug release rate contributed to less drug leakage and clearance in blood circulation. Moreover, the size and the hydrophilic surface allowed nanoparticles to avoid kidney infiltration. The advantageous tumor selectivity and high lung distribution offered DTX-loaded nanoparticles great potentials to play therapeutic effects of DTX at pathological sites. In case of nanoparticles, the DTX content from DTX-CSCD3 nanoparticles was 1.92-fold higher than that from DTX-CSAD3 nanoparticles at 2 h in tumor tissues, but it showed a rapid decrease after 6 h. As for the explanation, it may be easier for redox-sensitive CSCD3 nanoparticles to dissociate followed by prompt drug release in the abundant existence of GSH inside tumor cells. This meant that the DTX separated from nanoparticles would encounter rapid elimination and thereby induced lower residual after a period of time. Given the fact that fast

Area-under-the-curve. Mean residence time. Half-life time. Total clearance. Maximum concentration.

CSAD3 nanoparticles and Taxotere® at a drug dose of 15 mg/kg, which was shown in Fig. 2D. DTX-loaded nanoparticles showed significantly higher initial concentration compared to that of Taxotere®. Moreover, DTX-loaded nanoparticles could be detected at 48 h, while free DTX was rapidly cleared away from the blood circulation within 8 h. These results may have a positive influence on the therapeutic effect that nanoparticles prompted DTX to potentially present a long circulation time and improve the accumulation in targeting tissues. The mean pharmacokinetic parameters were shown in Table 3. DTX-CSCD3 and DTX-CSAD3 nanoparticles contributed to higher Cmax and MRT compared with Taxotere®. Compared with the short half-life time of Taxotere® (1.46 h), the parameters of DTX-CSCD3 and DTXCSAD3 nanoparticles rose by 4.3 times and 3.5 times, respectively. The AUC0-∞ was a key therapeutic index of DTX and the AUC0-∞ values for DTX-CSCD3 and DTX-CSAD3 nanoparticles were about 4.40- and 4.36fold, higher than that of Taxotere®, respectively. In addition, the CL of DTX-loaded nanoparticles showed a significant decline compared with that of Taxotere®, indicating the longer circulation time of these selfassembled nanoparticles. Different in vivo behaviors of DTX between Taxotere® and DTXloaded nanoparticles may contribute to the different pharmacokinetic behaviors of DTX formulations mentioned above (Guo et al., 2014). DTX, a small hydrophobic molecule with lower molecular weight, could be easily removed from the circulation system and filtrated through the glomerular of kidney followed by the rapid elimination. By contrast, the hydrophobic microdomain of self-assembled nanoparticles could serve as reservoirs for DTX while the hydrophilic CS backbones prompted the DTX to extend the circulation time and avoid the interaction between DTX and blood components. It is noticeable that the clearance of DTX

Fig. 4. Quantitative in vivo biodistribution analysis of DTX concentration in the main tissues of B16F10 tumor-bearing mice at different times after injections of Taxotere®, DTX-CSAD3 and DTX-CSCD3 nanoparticles through tail veins (n = 3).

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Fig. 5. Antitumor effects (A), body weights (B) of B16F10 tumor-bearing mice treat with NS, Taxotere®, DTX-CSAD3 and DTX-CSCD3 nanoparticles after a schedule of multiple doses. Indicated values were mean + SD (n = 5), *p < . 05, ***p < .001, vs. DTX-CSCD3 group. H&E staining analysis (C) and TUNEL apoptosis (D) of tumors from B16F10 tumor-bearing mice treat with different formulations. (E) H&E staining analysis of lung from B16F10 tumor-bearing mice treat with different formulations. Circles indicate the lung metastases. (F) Quantitative analysis of pulmonary metastatic modules in the B16F10 tumor-bearing mice, ***p < .001, vs. DTX-CSCD3 group. (G) IHC staining analysis of the expression of COX-2 protein in murine original tumors. Scale bar: 100 μm.

et al., 2015; Wang et al., 2011; Yuan, Chen, Zhang, & Zheng, 2010). The preferential liver internalization could be due to the nonspecific elimination of nanoparticles by the mononuclear phagocyte system (MPS) (Polomska, Gauthier, & Leroux, 2017).

release ability in targeting sites could insure adequate drug concentration and improve the therapeutic effect, the internal stimuli such as redox and enzyme have been widely applied in the design of intelligent nanocarriers (Hu et al., 2017; Zhang et al., 2016; Zhao et al., 2016). DTX amount from CSCD3 and CSAD3 nanoparticles was 1.99- and 1.87-fold higher than that of Taxotere® in liver at 0.5 h, respectively, which was consistent with other reports (C. Chen et al., 2013; P. Xu

3.10. Antitumor and antimetastasis efficiency Antitumor and antimetastasis abilities were evaluated on B16F1091

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pulmonary metastasis results, which may due to the nano-scale drug delivery system and GSH/hyal-1 responsive antitumor therapy in tumor site against COX-2. The admirable antitumor and antimetastasis abilities of intelligent CSCD3 nanoparticles compared with DTX made them more potential for clinical transform as anticancer candidates.

bearing C57 mice metastasis tumor models. As shown in Fig. 5A, DTXloaded CSCD3, CSAD3 nanoparticles and Taxotere® showed about 85% (*** p < .001), 62% and 37% of tumor inhibition rate than that of NS control group, respectively. These results demonstrated the predominant tumor targeting and accumulation capacity of nanoparticles, which was in accordance with biodistribution results. It was noticeable that DTX-CSCD3 nanoparticles were superior to DTX-CSAD3 nanoparticles in the anti-proliferation efficiency, indicating the strengths of synergetic effect of GSH/Hyal-1 in triggering burst disintegration of nanoparticles and accelerating drug release. In comparison, drug release could only be accelerated by the single Hyal-1 contribution to CSAD skeletons. These results were coincident with in vitro stimuli-responsive disassembly results. Applying stimuli-responsive nanodrugs may be an intriguing approach to achieve better anticancer management than those without stimuli responsiveness, and numerous intelligent drug delivery systems have been reported in anticancer advantages (X.D. Xu et al., 2015; Zhu et al., 2015). As for the body weight, these four groups did not see a drastic fluctuation in experiment duration (as shown in Fig. 5B), and the curves for mice in DTX-loaded CSCD3 and CSAD3 groups increased slightly. However, the mice for NS and Taxotere® groups became skinny although the curves for these mice were steady due to their growing tumor volume. The pathological H&E staining of tumor sections were shown in Fig. 5C, demonstrating the highest degree of cell necrosis and fibrosis with hemorrhage treated by DTX-CSCD3 nanoparticles. The histological analysis of tumor apoptosis was conducted via TUNEL assay (Fig. 5D), and normal cell nuclei were stained to be blue while pale brown presented TUNEL positive areas. DTX-CSCD3 nanoparticles group exhibited the largest TUNEL positive areas followed by DTX-CSAD3 nanoparticles group, by contrast, small area was observed for Taxotere® treated group while NS group resulted in almost no positive region. These results indicated the most effective chemotherapeutic efficiency of DTX-CSCD3 nanoparticles which showed slight enhancement compared with that of DTX-CSAD3 nanoparticles. It may own to the responsive fast drug release in tumor cells via the effect of synergetic triggers (GSH/Hyal-1) of CSCD3 nanoparticles compared with single enzymatic effect of CSAD3 nanoparticles. As shown in Fig. S6, no obvious destruction was found, although a large amount of DTX-loaded nanoparticles were accumulated in heart, liver and brain. These expected results demonstrated the biocompatibility and safety of CSCD3 and CSAD3 nanoparticles for DTX delivery. At the end of the treatment, all pulmonary metastasis nodules were counted (Fig. 5E-G). The largest number of nodules was observed in NS group, showing the severe lung metastasis of melanoma. After drug treatments, metastasis nodules reduced and the figures for DTX-CSCD3, DTX-CSAD3 nanoparticles and Taxotere® groups accounted for 19.19%, 28.04% and 51.84% of that of NS group, respectively. The pathological H&E staining results of lungs were shown in Fig. 5E, and lung sections administrated by DTX-CSCD3 and DTX-CSAD3 nanoparticles showed overwhelming normalization while most of the pulmonary alveoli disappeared with severe fibrillation in NS group. These results were coincident with nodules results, demonstrating the potential antimetastasis efficacy of DTX-loaded nanoparticles. It was reported that the tumor invasion and metastasis could be prompted by the rapid tumor cell proliferation (Koontongkaew, 2013), and therefore the dramatic antitumor efficiency of nanoparticles could contribute to the inhibited metastasis ability. The inflammatory factor COX-2 could enhanced the invasion and metastasis of tumor (Singh, Vaid, Katiyar, Sharma, & Katiyar, 2011), and its expression in tumor sections was measured to explore the possible antimetastasis ability against B16F10 based melanoma of DTX-loaded nanoparticles (Fig. 5G). The COX-2 expression of tumor in NS group showed the highest percentage in tumor sections while it dropped in DTX-loaded nanoparticles groups. Compared to the COX-2 percentage of tumor incubated with Taxotere®, the figure for DTX-CSCD3 groups saw a dramatic suppression followed by DTX-CSAD3 nanoparticles. This trend was coincident with

4. Conclusion In this article, we initially described smart redox/enzyme responsive self-assembled nanoparticles formed by CSCD conjugates. The high level of Hyal-1 was firstly found to degrade CS backbone, and the high concentration of GSH caused the selective breakage of disulfide bonds. Therefore, CSCD nanoparticles exhibited rapid drug release with the synergetic redox/enzyme effect at tumor cells and produced an anticancer activity. In addition, CS-based nanoparticles were demonstrated to be internalized by tumor cells via receptor-mediated uptake especially CD44. In our experiment, the lung metastasis was observed in tumor-bearing mice models due to the metastasis property of melanoma. Interestingly, the in vivo biodistribution study indicated the high tumor and lung accumulation of nanoparticles compared to that of the free drug. These nanoparticles therefore showed super-duper tumor cell-killing activity and tumor growth inhibition, further reducing the lung metastasis via metastasis induced protein expression inhibition. Our work presented valuable candidates for smart drug delivery systems that could specifically aggregate in tumor tissues, selectively release drug via dual internal triggers, and display good antitumor and antimetastasis effects. This report could not only pave the path to the design of triggers-sensitive drug delivery systems based on polysaccharides, but also bring hope for the melanoma therapy. More deep research especially the mechanism and toxicity should be investigated to further improve cancer management before the application in clinic. Acknowledgements This work was supported by the Natural Science Foundation of Shandong Province, China (No.ZR2015HM032). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.carbpol.2017.12.047 References Afshar, K., David, S., Fuehner, T., Gottlieb, J., & Gutzmer, R. (2016). BRAF inhibition in a lung transplant recipient with metastatic melanoma. JAMA Dermatology, 152(2), 228–230. Agrahari, V., Meng, J., Ezoulin, M. J., Youm, I., Dim, D. C., Molteni, A., et al. (2016). Stimuli-sensitive thiolated hyaluronic acid based nanofibers: Synthesis, preclinical safety and in vitro anti-HIV activity. Nanomedicine (London), 11(22), 2935–2958. Balch, C. M., Gershenwald, J. E., Soong, S. J., Thompson, J. F., Atkins, M. B., Byrd, D. R., et al. (2009). Final version of 2009 AJCC melanoma staging and classification. Journal Of Clinical Oncology, 27(36), 6199–6206. Bombelli, F. B., Webster, C. A., Moncrieff, M., & Sherwood, V. (2014). The scope of nanoparticle therapies for future metastatic melanoma treatment. Lancet Oncology, 15(1), e22–e32. Chen, C., Zhang, H., Hou, L., Shi, J., Wang, L., Zhang, C., et al. (2013). Single-walled carbon nanotubes mediated neovascularity targeted antitumor drug delivery system. Journal of Pharmacy and Pharmaceutical Sciences, 16(1), 40–51. Chen, Z., Li, Z., Lin, Y., Yin, M., Ren, J., & Qu, X. (2013). Bioresponsive hyaluronic acidcapped mesoporous silica nanoparticles for targeted drug delivery. Chemistry, 19(5), 1778–1783. Cheng, R., Meng, F., Deng, C., Klok, H. A., & Zhong, Z. (2013). Dual and multi-stimuli responsive polymeric nanoparticles for programmed site-specific drug delivery. Biomaterials, 34(14), 3647–3657. Cheng, W., Kumar, J. N., Zhang, Y., & Liu, Y. (2014). pH- and redox-responsive poly (ethylene glycol) and cholesterol-conjugated poly(amido amine)s based micelles for controlled drug delivery. Macromolecular Bioscience, 14(3), 347–358. Davies, M. A., Liu, P., McIntyre, S., Kim, K. B., Papadopoulos, N., Hwu, W. J., et al. (2011). Prognostic factors for survival in melanoma patients with brain metastases. Cancer, 117(8), 1687–1696. Ernsting, M. J., Murakami, M., Undzys, E., Aman, A., Press, B., & Li, S. D. (2012). A

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