A dermatan sulfate-functionalized biomimetic nanocarrier for melanoma targeted chemotherapy

Carbohydrate Polymers 235 (2020) 115983

Contents lists available at ScienceDirect

Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol

A dermatan sulfate-functionalized biomimetic nanocarrier for melanoma targeted chemotherapy

T

Shanshan Lia,1, Fuzhong Zhangb,1, Yang Yua, Qixiong Zhangc,* a

Department of Pharmaceutical Engineering, College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China Chengdu Institute of Chinese Herbal Medicine, Chengdu 610000, China c Department of Pharmaceutics, Army Medical University, Chongqing 400038, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Dermatan sulfate Biomimetic Melanoma Targeted therapy

Melanoma is a malignant tumor of melanocytes that is a serious threat to human health. Dermatan sulfate (DS) is a natural glycosaminoglycan. Inspired by the origin of DS, we report a DS-functionalized biomimetic chitosan nanocarrier (DCNP) for melanoma targeted chemotherapy. DS can anchor to the surface of the chitosan nanocarrier (CNP) by forming amide bond. The SN38/DCNP can rapidly release the anti-tumor drug under acidic conditions. The functionalization of DS not only promoted the specific uptake behavior of melanoma cells, but also up-regulated cleaved caspase-3 and PARP promote tumor cell apoptosis. In vivo model, DCNP reduced the non-specific distribution of SN38 in the circulation and other tissues, while shows superior tumor targeting ability. SN38/DCNP significantly inhibit tumor growth and improved the survival rate. Moreover, SN38/DCNP has a milder myelosuppressive effect. The above results indicated that DS could be used as an excellent targeting unit for the treatment of melanoma.

1. Introduction Melanoma is a malignant tumor that develops on the skin. Although the best choice is surgical resection, the prevalence of metastatic lesions makes surgical treatment very limited (Streit & Detmar, 2003). Chemotherapy is one of the commonly used methods for treating primary or metastatic melanoma in clinical practice (Lee, Betticher, & Thatcher, 1995). The camptothecin chemotherapeutics, represented by SN38, have attracted widespread attention from medicinal chemists because they inhibit DNA replication by inhibiting topoisomerase. However, SN38 is highly toxic, strong side effects, and have low bioavailability due to the low water solubility, which makes them have many limitations in cancer treatment (Payne, James, & Weiss, 2006; Staff, Grisold, Grisold, & Windebank, 2017; Weiss & Poster, 1982). These disadvantages are mainly due to the low selectivity of systemically administered chemotherapeutic drugs (Lee, Dees, & Wang, 2017). In order to enhance the selectivity of anti-cancer drugs, targeted drug delivery systems (targeted DDS) have become a hot topic in current research (Oliveira Pinho, Matias, & Gaspar, 2019). targeted DDS could enhance many chemotherapeutic drugs for targeting tumor cells, and effectively reduce their damage to normal cells (Garnacho, 2016; Srinivasarao & Low, 2017). targeted DDS has carrier forms such as liposomes,

microspheres, and nanoparticles (Zhu, Zhu, Pan, Chen, & Zhu, 2019). Among them, long-chain polymers, as a common carrier material, have the advantages of prolonging the circulation time of drugs and continuously releasing drugs, improving drug efficacy, etc., and have received widespread attention and research in recent years (Han, Thurecht, Whittaker, & Smith, 2016). For example, polyetherimide (PEI) is an artificial cationic polymer that is often used as a carrier for nucleic acid drugs (Pandey & Sawant, 2016; Pinnapireddy, Duse, Strehlow, Schafer, & Bakowsky, 2017), but strong hydrophilicity and cytotoxicity limit its application to chemotherapeutic drugs. However, polysaccharide compounds have a long-chain structure and have good potential as pharmaceutical carriers (Debele, Mekuria, & Tsai, 2016; Ganguly, Chaturvedi, More, Nadagouda, & Aminabhavi, 2014). Chitosan (CS) is a natural cationic polysaccharide prepared from chitin deacetylation and is widely found in nature (Younes & Rinaudo, 2015). CS is cheap, biocompatibility and degradability. Since CS has a positive charge, it can be ion-crosslinked with some polyanions such as tripolyphosphate (TPP) to form nanoparticles (Bhattarai, Gunn, & Zhang, 2010; Sawtarie, Cai, & Lapitsky, 2017). The CS-based nanocarriers have the advantages of sustained drug release and increased drug absorption (Hu, Sun, & Wu, 2013; Prabaharan, 2015). Meanwhile, another natural polysaccharide, dermatan sulfate (DS)

⁎

Corresponding author. E-mail address: [email protected] (Q. Zhang). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.carbpol.2020.115983 Received 7 December 2019; Received in revised form 21 January 2020; Accepted 10 February 2020 Available online 15 February 2020 0144-8617/ © 2020 Elsevier Ltd. All rights reserved.

Carbohydrate Polymers 235 (2020) 115983

S. Li, et al.

2. Materials and methods

is widely found in connective tissue and is the most important glycosaminoglycan in the skin (Sugahara et al., 2003). DS is hydrophilic and has a negative charge to prevent the phagocytic system from clearing it too quickly, so that it maintains good stability and biocompatibility in the circulatory system. These properties provide a good basis for the functionalization of the nanocarrier surface. There are a variety of proteoglycans containing DS chains (DSPGs) in the human body, such as biglycan, decorin, versican and syndecan (Trowbridge & Gallo, 2002; Wight, 2002; Yu et al., 2017). The complex structural sequence information of the DS chain makes DSPGs not only the basic structural components of human tissues, but also has many important physiological functions, such as participating in cell signal transduction, regulating cell adhesion, maintaining cell growth. Among them, DS plays an extremely important role and is a cofactor for many cell behaviors (Mizumoto, Yamada, & Sugahara, 2015). In view of the function of DS, it is considered to have a certain therapeutic application potential (Yamada & Sugahara, 2008). In the field of tumor research, it has been reported that decorin composed of DS can down-regulate the activity of the oncoprotein erb-B2, and induce the up-regulation of the tumor suppressor gene P21, thereby blocking the cell cycle (Goldoni et al., 2008; Reed et al., 2005; Santra, Eichstetter, & Iozzo, 2000). In addition, the Yoon Yeo group found that PEI/DS complexes could binding to CD146 expressed on the surface of cells (Kim et al., 2017). However, the PEI/DS system prepared via weaker electrostatic interactions may case cargo leakage. Moreover, PEI/DS is highly water-soluble and is not suitable for a wide range of hydrophobic chemotherapy drugs. Herein, we designed a dermatan sulfate-functionalized chitosan nanocarrier (DCNP), which mimic the CD146-bonding characteristics of proteoglycans, effectively achieve specific targeting to melanoma (Fig. 1). Briefly, CS and TPP can form drug-loaded nanocarriers (SN38/ CNP) together with the hydrophobic topoisomerase inhibitor SN38 by ion crosslinking (Xu et al., 2012). Under the guidance of EDC/NHS, DSfunctionalized was performed to obtain SN38/DCNP. After injection into mice via tail vein, SN38/DCNP can be distributed to the tumor by the EPR effect and binding to highly expressed CD146 on the surface of B16F10 cells. Due to the pH-triggered degradability of CS, SN38/DCNP can hydrolyze and release drugs in the acidic organelles, and enhance the delivery efficacy of SN38 to the nucleus of tumor cells.

2.1. Materials Dermatan sulfate (DS, average Mw 30 kDa, preserves the sulfated sequences, 2-O-sulfation of the iduronic acid units and/or 4 or 6 positions of glucosamine N acetyl, #263301, low molecular weight chitosan CS, 50−190 kDa, viscosity 20–300 cps referred to 1 wt.% in 1% acetic acid at 25℃, deacetylation degree 75–85 %, #448869, sodium tripolyphosphate TPP, N-3-Dimethylaminopropyl-N’-ethylcarbodiimide hydrochloride EDC and N-Hydroxysuccinimide NHS were purchased from Sigma-Aldrich U.S.A. Dulbecco's Modified Eagle’s Medium DMEM, fetal bovine serum FBS, and trypsin for cell culture were purchased from Gibco U.S.A. Purified anti-mouse CD146 antibody #134701, FITC-labeled anti-mouse CD146 antibody #134705 and FITC Annexin V Apoptosis Detection Kits with Propidium Iodide #640914 was purchased from Biolegend U.S.A, while topoisomerase I inhibitor SN38 and Cy5 NHS ester was obtained from MedChemExpress MCE, U.S.A. Alexa Fluor 488-labeled rabbit anti-mouse topoisomerase I monoclonal antibodies #ab197505 were supplied by Abcam U.S.A. PE-labeled rabbit anti-mouse cleaved caspase-3 monoclonal antibodies #9978 and PElabeled rabbit anti-mouse cleaved polyADP-ribose polymerase monoclonal antibodies #67495 were purchased from Cell Signaling Technology, Inc. U.S.A. Cytofix/Cytoperm Plus Fixation/ Permeabilization Kit with BD GolgiStop was purchased from BD Biosciences U.S.A. MTT Cell Proliferation and Cytotoxicity Assay Kit was supplied by Beyotime Biotechnology Co. Ltd. China. All the other reagents are commercially available and used as received. 2.2. Preparation and characterization of dermatan sulfate-functionalized chitosan NP According to the ion crosslinking method (Gierszewska & Ostrowska-Czubenko, 2016), an acetic acid (98 %) solution of CS (2.5 mg/mL, 3.6 mL) was taken and added to a solution of SN38 in absolute ethanol (1 mg/mL, 5 mL) with stirring (Xuan, Zhang, & Ahmad, 2006. The pH of this solution is about 2.57. 1 mL of TPP aqueous solution 2 mg/mL was slowly added dropwise under stirring, and reacted at room temperature for 30 min. It was then centrifuged 12,000 r/min, 40 min and the precipitate was washed with deionized water and lyophilized to give SN38/CNP. The preparation method of SN38/DCNP is similar.

Fig. 1. Schematic representation of the dermatan sulfate-functionalized SN38-loaded chitosan nanocarrier and the acid-triggered drug release in tumor tissue. The CNP were constructed by chitosan and tripolyphosphate (TPP) via ionic cross-linking. The dermatan sulfate was utilized to functionalize the CNP. After SN38-loaded DCNP were delivered to tumor tissue through EPR effects, sufficient cellular uptake mediated by melanoma-specific expression of CD146. The DCNP allow SN38 to be successfully endocytosed while the acid induced chitosan dissolution ensures adequate hydrolysis and release of SN38. 2

Carbohydrate Polymers 235 (2020) 115983

S. Li, et al.

BioLegend, Cat#134701 followed by 20 μg/mL Cy5/DCNP for 1 h and analyzed with Accuri C6 or a confocal microscope. For confocal microscopy, cells were fixed with 4% paraformaldehyde for 15 min at room temperature and stained with DAPI. B16F10 cells were cultured overnight on a tissue culture plate. Then, different concentration (0, 5, 10, 20, 40, 80 μg/mL) of Cy5/CNP and Cy5/DCNP was added into the culture medium. After incubation for 2 h, B16F10 cells were washed with PBS thrice to remove excess NPs. Intracellular Cy5 fluorescence intensity (FI) was measured by flow cytometry.

Briefly, 90 mg DS was dissolved in 30 mL of 4 ℃ deionized water and 180 mg of EDC was added. After stirring for 30 min, 195 mg of NHS was further added, and the pH was adjusted to about 7.4 with a 0.1 M NaOH solution. After reacting at 4 °C for 2 h, 30 mg of SN38/CNP was added to the above solution, magnetically stirred for 24 h. Then, the reaction solution was centrifuged at 4 °C 36,000 r/min, 60 min. The precipitate was washed with deionized water, and lyophilized to obtain SN38/ DCNP. Follow the same procedure and replace SN38 with Cy5, we can also obtain two Cy5-loaded nanoparticles: Cy5/CNP and Cy5/DCNP. Since DS has the uronic acid structure which can produce a product having ultraviolet absorption at 530 nm by reacting with carbazole (Bitter & Muir, 1962). The amount of non-binding DS is determined by quantifying the structure of the uronic acid. Then, DS binding rate (BR) in DCNP was calculated by the following formula (Mt refers total mass of DS, Mf refers mass of non-binding DS).

2.5. Cytotoxicity evaluation by MTT assay

The particle size, monodispersity and zeta potential of nanocarriers were determined by dynamic light scattering (DLS) using a multi-angle particle sizing method (90 Plus/BI-MAS) or DLS under an electric field (Zeta Plus analyzer). Each nanocarrier was dispersed in water at an appropriate concentration, dropped onto a copper mesh film. After airdrying, the morphological observation was performed through a transmission electron microscope (TEM, JEM-1400 microscope, JEOL, Japan) and analyzed.

B16F10 cells were cultured in DMEM medium supplemented with 10 % FBS. For the methyl thiazolyl tetrazolium (MTT) assay, cells were planted at 10,000 cells/well in 96-well plates for 24 h. Subsequently, cells were treated with the medium containing SN38/DCNP or SN38/ CNP or free SN38 at various concentrations (varying from 0, 0.1, 0.5, 1, 2, 4, 8, 16, 32 μM) for 12 h or 24 h. After the incubation, the medium was replaced with fresh medium. The MTT reagent and stop/solubilization solution were sequentially added with a 3 h interval. The formazan concentration was measured at 550 nm. The cell viability was normalized to the absorbance of PBS-treated control cells. The half inhibition concentration (IC50) was calculated based on the results obtained.

2.3. Release properties of SN38-loaded NP

2.6. In vitro anti-tumor activity of SN38/DCNP

According to the literature (Palakurthi, 2015), SN38 can maintain the structure of the lactone ring in an acidic environment. For the quantification of SN38, 10 mg SN38/CNP or SN38/DCNP was completely dissolved in 1% HCl. The concentration of SN38 was measured by an UV/Vis spectrophotometer at 372 nm (Zhang et al., 2019). The SN38 drug loading content (DLC) and encapsulation efficiency (EE) were calculated by the following formula.

Apoptosis analysis was conducted using FITC Annexin V Apoptosis Detection Kit with Propidium Iodide according to the manufacture’s protocol. Specifically, B16F10 cells were seeded in a 6-well plate at 5 × 106 cells/well and incubated overnight. The medium was then replaced with fresh growth medium containing SN38/DCNP or SN38/CNP or SN38 (calculated with 8 μM SN38). After 12 h of incubation, cells were washed with cold BioLegend’s cell staining buffer, digested with 0.25 wt % trypsin, and collected by centrifugation. After the cells were resuspended in 100 μL of Annexin V binding buffer with 5 μL of Annexin V and 10 μL of PI viability staining solution at 2.5 × 106 cells/mL, they were vortexed gently and incubated in a dark room for 15 min. Finally, 400 μL of Annexin V binding buffer was added for analysis by flow cytometry (Accuri C6, BD). The effects of SN38/DCNP treatment on the expression of topoisomerase I, cleaved caspase-3, and cleaved PARP in B16F10 cells were also measured by flow cytometry. B16F10 cells were seed in 6-well plates at 5 × 106 cells/well and incubated overnight. After the cells have undergone the same treatment as described above, cells were washed, digested, collected, and then resuspended in 1 mL of PBS containing 250 μL of fixation/permeabilization solution at 4 °C for 20 min. Subsequently, cells were washed two times with 1 mL of BD Perm/ Wash buffer, and resuspended in 200 μL of BD Perm/Wash buffer. After cells were stained with Alexa Fluor 488-labeled topoisomerase I antibody at 22 °C for 30 min, analysis was performed by flow cytometry. Similarly, the expression levels of cleaved caspase-3 and cleaved PARP were quantified by corresponding antibody.

BR= ( Mtds− Mfds)/Mt×100%

DLC= Msn/Mn×100% EE= ( Mtsn− Mfsn)/Mt× 100% Here Msn refers mass of SN38, Mn refers mass of nanocarriers, Mtsn refers total mass of SN38, Mfsn refers mass of free SN38. The drug release profiles of NPs were determined using a dialysis method. In brief, SN38/CNP and SN38/DCNP were suspended in PBS and transferred into dialysis bag with molecular weight cut off of 10,000 Da. Thereafter, the bag was put into a centrifuge tube (50 mL) and immersed in PBS release medium with pH 4.8 or 7.4. The tube was kept in a shaking water bath at 120 rpm at 37 °C. Fixed amount of outer release solution was collected at various time points and replaced with fresh medium. Finally, the released SN38 in the outer solution was determined using the UV spectrophotometer at 372 nm. Similarly, DLC and EE of Cy5 were quantified by UV–vis spectrophotometry (detection wavelength was 646 nm). 2.4. Cell culture and intracellular uptake of NPs B16F10 mouse melanoma cells and RAW264.7 mouse macrophages were obtained from Chinese Academy of Sciences. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 100 IU/mL penicillin, 100 μg/mL streptomycin, and 10 % FBS. Cells were maintained in a 5% CO2 humidified atmosphere at 37 °C. Firstly, the expression of CD146 on the surface of B16F10 cells and macrophages RAW264.7 was detected by flow cytometry (BD, Accuri C6) using FITC-labeled CD146 antibody BioLegend, Cat#134705. For competition assay, 1 × 105 cells/well B16F10 cells were seed in 24 well plate with or without 35 mm glass-bottom cell culture dishes. After overnight incubation, cells incubated with purified CD146 antibody

2.7. Animals All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals proposed by the National Institutes of Health. All procedures and protocols were approved by the Animal Ethics Committee at Southwest University. Six- to eight-week-old female BALB/c nude mice were obtained from the Chongqing Academy of Chinese Materia Medica. Animals were housed in standard mouse cages under conditions of optimum light, temperature, and humidity, with ad libitum access to water and food. Before further experiments were performed, all mice were acclimatized for at 3

Carbohydrate Polymers 235 (2020) 115983

S. Li, et al.

Fig. 2. Characterization of chitosan nanocarrier (CNP) and dermatan sulfate-functionalized chitosan nanocarrier (DCNP). A–B, The TEM photograph of CNP (A) and DCNP (B). Scale bar represents 100 nm. C–D, The particle size distribution of CNP (C) and DCNP (D). E–F, The hydrodynamic diameter (E) and ζpotential (F) of CNP and DCNP. All data are presented as mean ± SD (n = 3).

volume, but also observes the apoptosis of the tumor tissue. Apoptotic cells were detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay using the in situ cell death detection kit (Roche Diagnostics, U.S.A). Images were acquired using an Olympus fluorescence microscope equipped with a Hamamatsu ORCA03 G digital camera. Similarly, in order to investigate the effect of SN38/DCNP on the survival rate of tumor-bearing mice, the treatment time was extended to 54 days. The treatment plan is the same as before. For the survival test, 10 mice were in each group.

least 7 days. 2.8. Establishment of a mouse model of melanoma xenograft Based on previously reported methods (Kim et al., 2017), we established a mouse model of melanoma xenograft. 1 × 106 B16F10 cells were suspended in 100 μL RPMI 1640 medium and subcutaneously injected to the left flank of 6–8 week old male BALB/c nude mice (6week old). Tumor size was measured every other day with a digital caliper. The volume was calculated as LW2/2, where L was the length (the longest tumor diameter) and W was the width (the shortest tumor diameter) measured in millimeters. Mice were randomly assigned groups (n = 6 per group).

2.11. Detection of bone marrow suppression in melanoma-bearing mice Myelosuppression is a common side effect of SN38, and we evaluated the degree of myelosuppression of tumor-bearing mice with different SN38 formulations treatment. After the 42-day course of treatment, the surviving mice in all groups were sacrificed, the unilateral femurs were collected, washed with PBS and collected the rinse. The number of bone marrow cells was determined by a hematology analyzer.

2.9. In vivo pharmacokinetic study After the first treatment, the blood of the mice was taken out periodically, and the amount of SN38 in the blood was measured. Briefly, at predefined time points (0.15, 0.5, 1, 2, 4, 8, 24, 48 and 72 h), blood samples and major organ were collected. The whole blood samples were immediately centrifuged at 12,000 g for 2 min to obtain plasma. Then 800 μL of ice-cold methanol was added into 200 μL of plasma to precipitate protein. After centrifugation at 12,000 g for 2 min, the supernatant was purged with nitrogen, reconstituted with methanol, and detected by high-performance liquid chromatography (HPLC) (Rivory & Robert, 1994). The mobile phase was consisted of acetonitrile and ammonium acetate buffer (75 mM, pH 6.4) containing 5 mM tetrabutylammonium phosphate at 22:78 (v/v). Reversed-phase C18 columns were used, and detection wavelength was 370 nm. To determine drug biodistribution in major tissues, they were homogenized and centrifuged. SN38 in the supernatant was extracted with acetonitrile. Quantification was also performed by HPLC.

2.12. Statistical analysis Data are expressed as mean ± standard deviation (SD). Statistical analysis was assessed using one-way ANOVA test. A value of p < 0.05 was considered statistically significant. 3. Results and discussion 3.1. Preparation of dermatan sulfate-functionalized chitosan nanocarrier Based on a large number of free positively charged primary amino groups of hydrophobic chitosan (CS), an ion crosslinking reaction method was used to crosslink the negatively charged TPP to form nanocarriers, named CNP. Furthermore, under the help of EDC/NHS, the carboxyl group of dermatan sulfate (DS) reacts with the amino group of CS to form an amide bond. Thereby, DS-functionalized chitosan nanocarrier was obtained, referred to as DCNP. From the transmission electron microscope (TEM) images (Fig. 2A–B), both CNP and DCNP have a uniform spherical morphology. The representative PDI of CNP and DCNP measured by dynamic light scattering (DLS) were 0.119 and 0.245, respectively (Fig. 2C–D). It’s indicated that the above two

2.10. In vivo anti-tumor efficacies of SN38/DCNP in melanoma-bearing mice According to the modeling method mentioned above. About 12th day, mice were administered with PBS (100 μL) or treatments - SN38 (3 mg/kg), equal amount of SN38/DCNP and SN38/CNP suspended in 100 μL PBS, via tail vein injection every three days. Tumor volume was monitored for 30 days after the first treatment. The anti-tumor effect not only monitors the body weight of the mouse as well as the tumor 4

Carbohydrate Polymers 235 (2020) 115983

S. Li, et al.

negatively ζ-potential exhibited stronger cellular uptake results. This may be due to the functionalization of DS. In order to demonstrate the relationship between enhanced cell uptake and CD146, a competitive experiment was conducted. We found that after pre-incubation of CD146 antibodies (αCD146) with B16F10 cells, the uptake of Cy5/ DCNP was reduced. This also indicated that the uptake process is CD146 mediated. Consistent with the results observed with confocal laser scanning microscope, this phenomenon was also quantified by flow cytometry. Compared to Cy5/CNP, the fluorescence intensity of Cy5 increased to about 1.8-fold after incubation with Cy5/DCNP (Fig. 3C). When the αCD146 was involved in the incubation system, the intracellular fluorescence intensity is also significantly reduced. In fact, for CD146-negative MALME-3M cells (Luo et al., 2012) and RAW264.7 cells, Cy5/DCNP will not be more easily endocytosed than Cy5/CNP (Fig. S2), this also revealed the selectivity of DCNP for CD146+ cells. In dose-dependent experiments (Fig. 3D), flow cytometry analysis showed enhanced endocytosis of Cy5/DCNP in B16F10 cells with increased dose. It is worth noting that there is no occurrence of uptake saturation as the concentration of nanocarriers increases, which ensures sufficient intracellular delivery capability of DCNP. All of the above results showed that the uptake of DCNP by B16F10 cells is promoted by binding of DS to CD146 expressed in melanoma cells.

nanocarriers have a narrow size distribution, which is also consistent with the TEM images. Fig. 2E reflects the average particle size of the two nanocarriers. When the hydrodynamic diameter of the CNP is 98.5 ± 2.2 nm, the DCNP is 103.3 ± 2.3 nm. We know that particle sizes below 200 nm ensure maximum “enhanced permeability and retention effect” in hematological malignancies (Deshantri et al., 2018). Unlike the similar properties of the particle size, due to the negative charge of DS, the ζ-potential of DCNP (−12.45 ± 4.25 mV) was significantly decrease, compared to 0.457 ± 3.25 mV of CNP (Fig. 2F). It is also due to the CS, TPP, DS all have different charges, so the ratio of the three can be controlled to regulate the surface charge of the nanocarrier. We know that the negative of the DS-functionalized can avoid the attachment of too many biomacromolecules (such as HSA) on the surface of the nanocarriers, prolong the cycle time, and avoid the elimination of the phagocytic system in the body (Hu et al., 2018). Further, according to the measurement of the content of uronic acid, the binding ratio of DS was measured to be about 32 % (data not shown). These results indicated that we had successfully prepared DSfunctionalized chitosan nanocarriers. 3.2. Cellular uptake profiles of DCNP by CD146+ melanoma cell Flow cytometry was used to demonstrate that DS functionalization enhances the uptake of nanocarriers by CD146+ tumor cells. Basically, it was demonstrated that the CD146 expression level of B16F10 melanoma cells was approximately 39 times that of macrophage RAW264.7 cells (Fig. 3A). The CD146-specificity of B16F10 cells provides an effective target for delivery systems. For cytotoxicity experiments, ∼1000 μg/mL CNP and DCNP didn’t cause significant effect on the cell viability of B16F10 cells or RAW264.7 cells, indicating that the nanocarriers are well biocompatible (Fig. S1). Furthermore, the uptake behavior of B16F10 cells was studied using fluorescein Cy5-labeled CNP or DCNP. After incubation with 20 μg/mL of Cy5/DCNP for 2 h, the intracellular Cy5 fluorescence was observed by confocal laser scanning microscope (Fig. 3B). Interesting, contrary to what is generally thought to be positively correlated with surface positive charge and endocytosis, DCNP with more

3.3. Preparation, characterization and release properties of SN38-loaded NP It is well known that irinotecan is an inhibitor of topoisomerase I, which can significantly kill a variety of tumors including melanoma (Dumez et al., 2006). As an active metabolite of irinotecan, 7-ethyl-10hydroxy camptothecin (SN38) has an anti-tumor activity in the range of several hundred to one thousand times that of irinotecan (Kawato, Aonuma, Hirota, Kuga, & Sato, 1991). However, SN38 is extremely poorly water soluble and insoluble in any biocompatible reagent, making it unusable for clinical use. Moreover, there are significant individual differences in the therapeutic effect of SN38, and the predictability is poor, and it is easy to produce serious side effects such as delayed diarrhea and bone marrow suppression (Bala, Rao, Boyd, &

Fig. 3. Cellular uptake profiles of NPs by B16F10 melanoma cells. A, Typical flow cytometry (left) and quantitative analysis (right) of CD146 expression levels in B16F10 cells and RAW264.7 macrophages. CD146 levels were tested by flow cytometry using FITC-labeled CD146 monoclonal antibody. B, The CLSM images of Cy5labeled NP intercellular uptake. C, Representative flow cytometry plot (left) and histograms (right) of the fluorescence intensities in B16F10 cells treated with Cy5labeled NPs for 1 h. Receptor neutralization was performed using a CD146 monoclonal antibody (αCD146) to demonstrate the specificity of uptake pathway. D, Representative flow cytometry curves (left) and quantitative data (right) illustrating concentration-dependent internalization of Cy5/DCNP in B16F10 cells for 1 h. All data are presented as mean ± SD (n = 6). 5

Carbohydrate Polymers 235 (2020) 115983

S. Li, et al.

Fig. 4. Characterization and release properties of SN38-loaded NPs. A-B, The TEM photos of SN38/CNP (A) and SN38/DCNP (B). Scale bar represents 200 nm. C-D, The size distribution of SN38/CNP (C) and SN38/DCNP (D). E, The drug loading content and encapsulation efficiency of SN38-loaded NPs. F-G, The SN38 release properties of NPs in PBS with pH 7.4 (F) or pH 4.8 (G). All data are presented as mean ± SD (n = 6).

faster than CNP, which should be attributed to the hydrophilic DS covalently linked to CS. In previous reports (Dmour & Taha, 2017), TPP and CS also formed phosphoramide bond under the action of EDC. Under acidic conditions, CS-TPP-DS could change from hydrophobic to hydrophilic more quickly. These results also demonstrated that SN38 can be fully embedded in the hydrophobic core of CNP and DCNP. The prepared SN38/DCNP was capable of changing the solubility under conditions close to the tumor acidic microenvironment, resulting in release of the drug.

Prestidge, 2013; Friberg, Henningsson, Maas, Nguyen, & Karlsson, 2002). Therefore, we chose SN38 as a model drug. Generally, large molecular weight CS has poor water solubility, and low molecular weight CS (1000−1500 Da) obtained through degradation can be dissolved in water (Muanprasat & Chatsudthipong, 2017). With its own hydrophobicity, SN38 can be easily embedded in the hydrophobic core of chitosan nanocarriers (Desbrieres, Martinez, & Rinaudo, 1996). Fig. 4A–B are the TEM images of SN38/CNP and SN38/DCNP, respectively. Similar to the blank nanocarriers, the two nanocarriers loaded with SN38 maintain a regular spherical appearance with a relatively uniform particle size. From DLS results in Fig. 4C–D, the hydrodynamic particle sizes of SN38/CNP and SN38/DCNP are 101.2 nm and 102.5 nm, respectively. Both PDI were lower than 0.3, indicating a narrow particle size distribution. The maximum absorption wavelength (λmax) of SN38 measured by UV spectrometer was 372 nm (Fig. S3A). According to the standard curve and regression equation (R2 = 0.995) of SN38 (Fig. S3B), the encapsulation efficiency and drug loading content of the two drug-loaded nanoparticles were measured (Fig. 4E). It can be seen that the drug loadings of SN38/CNP and SN38/ DCNP are 8.7 ± 0.6 % and 11.2 ± 1.2 %, respectively. The encapsulation efficiency is above 85 %. This shows that CNP and DCNP have excellent encapsulation ability for SN38. In addition, we also investigated the drug release of NPs in PBS at different pH values. It can be clearly seen from Fig. 4F that the release profile of SN38/DCNP is approximately the same as CNP in the neutral environment of pH 7.4, even after 24 h, the cumulative release is about only 20 %. In the environment of pH 4.8, the cumulative release of two NPs was obviously increased, and even the cumulative release of SN38/DCNP is over 80 % (Fig. 4G). This is due to the positive charge of CS itself, which remains relatively hydrophobicity in a neutral environment and doesn’t cause hydrolysis of the NPs. With the decrease of pH, the imine of chitosan can easily form the imine salt, the solubility of CS increases, which means the destruction of the structure of the NPs, thereby releasing the drug (He, Lou, Wang, & Zhao, 2015). As the release rate of DCNP is

3.4. Bioactivity of SN38/DCNP in CD146+ melanoma cell To verify that SN38/DCNP produce greater cytotoxicity against CD146-positive melanoma cells that SN38 or SN38/CNP, the MTT assay and flow cytometry were assessed. Fig. 5A–B are the experimental results measured by the MTT method, showing the cell viability of the two SN38-loaded NPs after incubation with B16F10 cells for 12 h or 24 h. In all case, significant dose-dependent cytotoxicity was observed for B16F10 cells. Moreover, the cytotoxicity of SN38/DCNP was stronger than SN38 and SN38/CNP at any concentration. From Fig. 5C, after 12 h incubation, the calculated half-inhibitory concentration (IC50) of SN38, SN38/CNP and SN38/DCNP on B16F10 cells was 19.87 μM, 16.15 μM and 8.48 μM respectively. As the incubation time was extended to 24 h (Fig. 5D), the IC50 was also reduced to 18.47 μM, 13.15 μM and 7.46 μM, respectively. Notably, the desirable antitumor activity observed herein should be largely attributed to the more internalization, because the IC50 of SN38/DCNP is only 40 % of free SN38. In addition, we also tested other CD146-positive cells, A375SM cells (Jean et al., 1998) and PC-3 cells (Wu et al., 2001), which also showed sensitivity to SN38/DCNP (Fig. S4A–B). But, for CD146-negative MALME-3 M melanoma cells and macrophage RAW264.7 (Fig. S4C–D), SN38/ DCNP did not show stronger cytotoxicity than SN38/CNP. This also shows that the stronger cytotoxicity of SN38/DCNP depends on CD146mediated more internalization. 6

Carbohydrate Polymers 235 (2020) 115983

S. Li, et al.

Fig. 5. In vitro anti-tumor effects of SN38, SN38/CNP and SN38/DCNP in B16F10 cells. A-D, Relative cell viability of B16F10 cells (A, B) and calculated IC50 values (C, D) after incubation with different concentrations of SN38/CNP or SN38/DCNP or SN38 for 12 h or 24 h. E–F, Flow cytometry (E) and quantification (F) of topoisomerase I expression in B16F10 cells. After 12 h of incubation, intracellular topoisomerase I was stained with Alexa Fluor 488-labeled rabbit anti-mouse topoisomerase I monoclonal antibodies. G–H, Flow cytometric profiles (left) and quantitative analysis (right) illustrating the levels of cleaved caspase-3 (G) and cleaved PARP (H) in B16F10 cells. I–J, The apoptotic profiles (I) and quantitative analysis (J) of B16F10 cells treated with SN38 or SN38/CNP or SN38/DCNP (SN38, 8 μM) were obtained by flow cytometry. All data are presented the mean ± SD (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001.

(calculated as 8 μM SN38) induced 46.9 % of necrotic cells and 4.58 % of early apoptotic cells. Of note, necrotic cells and early apoptotic cells are considered to be apoptotic cells (Renvoize, Biola, Pallardy, & Breard, 1998). It can be seen from Fig. 5J that cells incubated with SN38/DCNP showed significant high apoptosis, compared to those treated with SN38/CNP or free SN38. Consequently, this result demonstrated that SN38/DCNP also exerted its antitumor activity by inhibiting topoisomerase I, increase cleaved caspase-3 and cleaved PARP expression level, resulting in a significant increase in CD146+ cell apoptosis.

It is well known that SN38 affect the spatial structure of topoisomerase I, the expression of topoisomerase I in B16F10 cells were quantified by flow cytometry (Fig. 5E). As expected, treatment with SN38/DCNP most effectively inhibited topoisomerase I expression, among all the SN38 formulations (Fig. 5F). As well demonstrated, cleaved caspase-3 plays an extremely important role in the process of apoptosis caused by SN38. On the other hand, cleaved caspase-3 would induce the cleavage of PARP. Therefore, cleaved caspase-3 and cleaved PARP are also considered to be related proteins of camptothecin-induced cytotoxicity. SN38/DCNP increased the levels of cleaved caspase3 and PARP to 3.2 and 2.1 times higher than those of untreated cells, respectively (Fig. 5G–H). Moreover, significant differences were observed between the free SN38 and SN38/DCNP groups. In parallel experiments, apoptosis of B16F10 cells was measured by flow cytometry using FITC Annexin V Apoptosis Detection Kit with PI. In line with the inhibited formation of DNA spatial structure, treatment with SN38 resulted in significant apoptosis of B16F10 cells (Fig. 5I). SN38/DCNP

3.5. Pharmacokinetic study and tissue distribution of different SN38 formulations Subsequently, pharmacokinetic and biodistribution profiles of SN38, SN38/CNP and SN38/DCNP in melanoma-bearing mice were compared. Firstly, we verified the feasibility of SN38 detection by 7

Carbohydrate Polymers 235 (2020) 115983

S. Li, et al.

Fig. 6. In vivo pharmacokinetic study. A, Protocol for B16F10 melanoma xenograft model and monitor process. B-C, Relative drug content in blood (B) and biodistribution (C) of SN38, SN38/CNP or SN38/DCNP treatment group in melanoma-bearing mice. All data are presented the mean ± SD (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001 and ns means no significance.

3.6. In vivo effects of SN38/DCNP in melanoma-bearing mice

HPLC. The HPLC chromatogram (Fig. S5A–B) and regression equation (Fig. S5C, R2 = 0.999) have fully proved that this method can detect trace SN38. From Fig. 6A, a single vail injection of different SN38 formulations was performed by subcutaneous injection of 1 × 106 B16F10 cells in BALB/C nude mice, approximately on 12th day (tumor size approximately 50 mm3). For the next 3 days, mouse blood was collected at predetermined time points, and concentration of SN38 was performed by HPLC. It can be seen from the relative SN38 content reflected in Fig. 6B, the free SN38 group has the largest drug content at the beginning and rapidly decreases, which may be due to the rapid metabolism elimination. Compared to free SN38, the initial content of SN38/DCNP and SN38/CNP group is lower because the drug is embedded in the core of the nanocarrier. It is worth noting that the content of SN38/DCNP in blood is always lower than that of SN38/CNP, which may also be due to the tumor targeting of SN38/DCNP. Compared to metabolism in normal mice (Wu et al., 2019), SN38 has a shorter halftime (t1/2 = 0.78 h) in tumor-bearing mice. The prolonged half-life of SN38/CNP (t1/2 = 1.1 h) is may due to the slow-release effect of CNP. The shortest half-life of SN38/DCNP (t1/2 = 0.6 h) may be due to the tumor-specific targeting and drug release effect of DCNP, which significantly reduced distribution in blood, and slightly affected the t1/2 of SN38. By calculation, the area under the drug-time curve (AUC) of SN38/ DCNP decreased by 55 % and 49 % compared with SN38 and SN38/ CNP, respectively (Fig. S6A). From Fig. 6C, after 72 h, although the drug concentration of SN38, SN38/CNP and SN38/DCNP showed no significant differences in the liver and spleen, SN38/DCNP dramatically reduced the drug distribution in the lungs and kidneys. Excitingly, SN38/DCNP has more accumulation of drug in tumors than in SN38 (2.23-fold) or SN38/CNP (1.60-fold) group (Fig. S6B). Taken together, SN38/DCNP can avoid undesired leakage and release in the circulation and targets tumor sites via binding to CD146 positive cell, like B16F10, pericytes (Chen et al., 2017) and so on, resulting in reduced the nonspecific distribution of SN38 and achieved targeted delivery to melanoma.

Anti-tumor effects of SN38/DCNP were investigated in melanomabearing mice (Fig. 7A). Model mice were treated with PBS in the control group, while the model mice received i.v. administration of different SN38 formulations every 3 days, respectively. Mouse body weight and tumor volume were recorded every 6 days during the 30-day treatment period. From Fig. 7B, the body weight of the untreated mice was remained stable, while the mice received treatment showed a slight decrease. The most obvious weight change of the mice was founded in SN38-treatment group (about 5% decreased), which maybe induced by the side effects from chemotherapeutic drugs. While SN38/CNP attenuated the trend of weight loss, the body weight of mice treated with SN38/DCNP only decreased by about 1%. In the most intuitive comparison of tumor volume, untreated BALB/C nude mice showed significant tumor growth, reaching nearly 252 mm3 till day 42 (Fig. 7C). Different SN38 formulations decreased tumor volume to different extents. When the tumor volume of SN38/CNP and SN38/DCNP treatment groups decreased to 134 ± 17 mm3 and 91 ± 16 mm3, the inhibition effect of free SN38 was the worst, only 179 ± 26 mm3. Meanwhile, TUNEL assay showed that SN38/DCNP remarkably increased the apoptosis of tumor cells (Fig. 7D). In Fig. 8A, the effect of each treatment regimen on survival in mice were discussed. In addition to continuing to prolong the treatment period, other treatment regimens are consistent with the aforementioned. From Fig. 8B, untreated nude mice all died on day 51, while mice given SN38, SN38/CNP and SN38/DCNP had survival rates of 30 %, 40 % and 60 %, respectively. Meanwhile, in another parallel experiment shown in Fig. 8C, chemotherapy-induced myelotoxicity was also assessed at 30 day after treatment. Compared with untreated mice, free SN38 treatments resulted in only 37.3 % bone marrow cell (BMC) counts, whereas the BMC of the SN38/DCNP and SN38/CNP treatment groups were 48.7 % and 78.2 %, respectively. It is noteworthy that SN38/DCNP has a milder myelotoxicity than SN38/CNP. This indicated that SN38/DCNP can effectively alleviate the myelosuppressive side effects caused by the non-specific distribution of SN38, and also contributes to the improvement of mouse survival rate. 8

Carbohydrate Polymers 235 (2020) 115983

S. Li, et al.

Fig. 7. In vivo anti-tumor efficacies of SN38/DCNP in melanoma-bearing mice. A, Protocol for B16F10 melanoma xenograft model and treatment process. B-C, Changes in the body weight (B) and tumor volume (C) of tumor-bearing mice during 42 days. D, Analysis of tumor cell apoptosis by TUNEL assay. All data are presented the mean ± SD (n = 6). *p < 0.05 and **p < 0.01.

Fig. 8. SN38/DCNP alleviated tumor mortality and the degree of bone marrow suppression of chemotherapy. A, Protocol for B16F10 melanoma xenograft model and survival experiment. B, The survival rate of tumor-bearing mice received different treatments. SN38 or equivalent SN38-loaded NPs were intravenous administered every 3 days from 12th day to 54th day. (n = 10 in each group). C, At the 42 day, the mouse bone marrow cell (BMC) was counted to evaluated myelosuppression (n = 3). All data are presented the mean ± SD. *p < 0.05.

9

Carbohydrate Polymers 235 (2020) 115983

S. Li, et al.

4. Conclusion

localized drug delivery. Advanced Drug Delivery Reviews, 62(1), 83–99. Bitter, T., & Muir, H. M. (1962). A modified uronic acid carbazole reaction. Analytical Biochemistry, 4, 330–334. Chen, J., Luo, Y., Hui, H., Cai, T., Huang, H., Yang, F., ... Yan, X. (2017). CD146 coordinates brain endothelial cell-pericyte communication for blood-brain barrier development. Proceedings of the National Academy of Sciences of the United States of America, 114(36), E7622–E7631. Debele, T. A., Mekuria, S. L., & Tsai, H. C. (2016). Polysaccharide based nanogels in the drug delivery system: Application as the carrier of pharmaceutical agents. Materials Science & Engineering C, Materials for Biological Applications, 68, 964–981. Desbrieres, J., Martinez, C., & Rinaudo, M. (1996). Hydrophobic derivatives of chitosan: Characterization and rheological behaviour. International Journal of Biological Macromolecules, 19(1), 21–28. Deshantri, A. K., Varela Moreira, A., Ecker, V., Mandhane, S. N., Schiffelers, R. M., Buchner, M., ... Fens, M. (2018). Nanomedicines for the treatment of hematological malignancies. Journal of Controlled Release, 287, 194–215. Dmour, I., & Taha, M. O. (2017). Novel nanoparticles based on chitosan-dicarboxylate conjugates via tandem ionotropic/covalent crosslinking with tripolyphosphate and subsequent evaluation as drug delivery vehicles. International Journal of Pharmaceutics, 529(1–2), 15–31. Dumez, H., Awada, A., Piccart, M., Assadourian, S., Semiond, D., Guetens, G., & van Oosterom, A. (2006). A phase I dose-finding clinical pharmacokinetic study of an oral formulation of irinotecan (CPT-11) administered for 5 days every 3 weeks in patients with advanced solid tumours. Annals of Oncology, 17(7), 1158–1165. Friberg, L. E., Henningsson, A., Maas, H., Nguyen, L., & Karlsson, M. O. (2002). Model of chemotherapy-induced myelosuppression with parameter consistency across drugs. Journal of Clinical Oncology, 20(24), 4713–4721. Ganguly, K., Chaturvedi, K., More, U. A., Nadagouda, M. N., & Aminabhavi, T. M. (2014). Polysaccharide-based micro/nanohydrogels for delivering macromolecular therapeutics. Journal of Controlled Release, 193, 162–173. Garnacho, C. (2016). Intracellular drug delivery: Mechanisms for cell entry. Current Pharmaceutical Design, 22(9), 1210–1226. Gierszewska, M., & Ostrowska-Czubenko, J. (2016). Chitosan-based membranes with different ionic crosslinking density for pharmaceutical and industrial applications. Carbohydrate Polymers, 153, 501–511. Goldoni, S., Seidler, D. G., Heath, J., Fassan, M., Baffa, R., Thakur, M. L., ... Iozzo, R. V. (2008). An antimetastatic role for decorin in breast cancer. The American Journal of Pathology, 173(3), 844–855. Han, F. Y., Thurecht, K. J., Whittaker, A. K., & Smith, M. T. (2016). Bioerodable PLGAbased microparticles for producing sustained-release drug formulations and strategies for improving drug loading. Frontiers in Pharmacology, 7, 185. He, K., Lou, T., Wang, X., & Zhao, W. (2015). Preparation of lignosulfonate-acrylamidechitosan ternary graft copolymer and its flocculation performance. International Journal of Biological Macromolecules, 81, 1053–1058. Hu, J., Sheng, Y., Shi, J., Yu, B., Yu, Z., & Liao, G. (2018). Long circulating polymeric nanoparticles for gene/drug delivery. Current Drug Metabolism, 19(9), 723–738. Hu, L., Sun, Y., & Wu, Y. (2013). Advances in chitosan-based drug delivery vehicles. Nanoscale, 5(8), 3103–3111. Jean, D., Gershenwald, J. E., Huang, S., Luca, M., Hudson, M. J., Tainsky, M. A., ... BarEli, M. (1998). Loss of AP-2 results in up-regulation of MCAM/MUC18 and an increase in tumor growth and metastasis of human melanoma cells. The Journal of Biological Chemistry, 273(26), 16501–16508. Kawato, Y., Aonuma, M., Hirota, Y., Kuga, H., & Sato, K. (1991). Intracellular roles of SN38, a metabolite of the camptothecin derivative CPT-11, in the antitumor effect of CPT-11. Cancer Research, 51(16), 4187–4191. Kim, B. K., Kim, D., Kwak, G., Yhee, J. Y., Kwon, I. C., Kim, S. H., ... Yeo, Y. (2017). Polyethylenimine-dermatan sulfate complex, a bioactive biomaterial with unique toxicity to CD146-positive cancer cells. ACS Biomaterials Science & Engineering, 3(6), 990–999. Lee, M. S., Dees, E. C., & Wang, A. Z. (2017). Nanoparticle-delivered chemotherapy: Old drugs in new packages. Oncology (Williston Park), 31(3), 198–208. Lee, S. M., Betticher, D. C., & Thatcher, N. (1995). Melanoma: Chemotherapy. British Medical Bulletin, 51(3), 609–630. Luo, Y., Zheng, C., Zhang, J., Lu, D., Zhuang, J., Xing, S., ... Yan, X. (2012). Recognition of CD146 as an ERM-binding protein offers novel mechanisms for melanoma cell migration. Oncogene, 31(3), 306–321. Mizumoto, S., Yamada, S., & Sugahara, K. (2015). Molecular interactions between chondroitin-dermatan sulfate and growth factors/receptors/matrix proteins. Current Opinion in Structural Biology, 34, 35–42. Muanprasat, C., & Chatsudthipong, V. (2017). Chitosan oligosaccharide: Biological activities and potential therapeutic applications. Pharmacology & Therapeutics, 170, 80–97. Oliveira Pinho, J., Matias, M., & Gaspar, M. M. (2019). Emergent nanotechnological strategies for systemic chemotherapy against melanoma. Nanomaterials (Basel), 9(10). Palakurthi, S. (2015). Challenges in SN38 drug delivery: Current success and future directions. Expert Opinion on Drug Delivery, 12(12), 1911–1921. Pandey, A. P., & Sawant, K. K. (2016). Polyethylenimine: A versatile, multifunctional nonviral vector for nucleic acid delivery. Materials Science & Engineering C, Materials for Biological Applications, 68, 904–918. Payne, A. S., James, W. D., & Weiss, R. B. (2006). Dermatologic toxicity of chemotherapeutic agents. Seminars in Oncology, 33(1), 86–97. Pinnapireddy, S. R., Duse, L., Strehlow, B., Schafer, J., & Bakowsky, U. (2017). Composite liposome-PEI/nucleic acid lipopolyplexes for safe and efficient gene delivery and gene knockdown. Colloids and Surfaces B, Biointerfaces, 158, 93–101. Prabaharan, M. (2015). Chitosan-based nanoparticles for tumor-targeted drug delivery.

To date, chemotherapy is still an important method for the treatment of melanoma, especially in the treatment of metastatic lesions, chemotherapy is more effective than surgery. But its non-specific distribution limits the long-term application. Drug delivery systems, especially targeted DDS, provide an excellent platform to avoid these disadvantages of chemotherapy. As a CD146 receptor, dermatan sulfate has been reported to bind effectively to melanoma cells with high CD146 expression. In this experiment, DCNP was prepared as a drug carrier for the targeted treatment of melanoma. The preparation process is simple, the morphology of DCNP is uniform and the drug loading and encapsulation rate of SN38 are high. It is worth noting that the dissolution of chitosan will promote the release of SN38 in the acidic tumor environment. In addition, we demonstrated that functionalization of DS can promote the uptake of CNP by CD146+ cells like B16F10. Meanwhile, compared with SN38/CNP, SN38/DCNP better inhibited topoisomerase I and up-regulated the expression of cleaved caspase-3 and cleaved PARP, resulting in enhance the apoptosis of B16F10 cells. For B16F10 cells transplanted nude mouse melanoma model, SN38/ DCNP significantly increased the targeted enrichment in tumor sites and exhibited a better inhibitory effect on tumor volume. Furthermore, SN38/DCNP reduced the content of chemotherapy drugs in blood circulation, lungs and kidneys. DCNP avoids the side effects caused by the non-specific distribution of SN38, reduces the bone marrow suppression degree, improves the safety of SN38 and the survival rate of melanomabearing mice. These results indicated that the DS modification of the surface provides a new reference for targeted nanotherapy for melanoma. Author statement Qixiong Zhang and Shanshan Li designed the experiments. Shanshan Li and Fuzhong Zhang performed the experiments. The manuscript was prepared by Qixiong Zhang, Shanshan Li and Yang Yu. Data availability The authors declare that all data supporting the findings of this study are available within the paper and its supplementary information, source data in this study are available from the online version. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments The work was supported by the Fundamental Research Funds for the Central Universities [SWU118123], National Natural Science Foundation of China [81603047] and Postdoctoral Science Special Foundation of Chongqing [Xm2017018]. 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.2020.115983. References Bala, V., Rao, S., Boyd, B. J., & Prestidge, C. A. (2013). Prodrug and nanomedicine approaches for the delivery of the camptothecin analogue SN38. Journal of Controlled Release, 172(1), 48–61. Bhattarai, N., Gunn, J., & Zhang, M. (2010). Chitosan-based hydrogels for controlled,

10

Carbohydrate Polymers 235 (2020) 115983

S. Li, et al.

Cancer Treatment Reviews, 9(1), 37–56. Wight, T. N. (2002). Versican: A versatile extracellular matrix proteoglycan in cell biology. Current Opinion in Cell Biology, 14(5), 617–623. Wu, C., Zhang, Y., Yang, D., Zhang, J., Ma, J., Cheng, D., ... Deng, L. (2019). Novel SN38 derivative-based liposome as anticancer prodrug: An in vitro and in vivo study. International Journal of Nanomedicine, 14, 75–85. Wu, G. J., Varma, V. A., Wu, M. W., Wang, S. W., Qu, P., Yang, H., ... Amin, M. B. (2001). Expression of a human cell adhesion molecule, MUC18, in prostate cancer cell lines and tissues. Prostate, 48(4), 305–315. Xu, Q., Guo, L., Gu, X., Zhang, B., Hu, X., Zhang, J., ... Wang, S. (2012). Prevention of colorectal cancer liver metastasis by exploiting liver immunity via chitosan-TPP/ nanoparticles formulated with IL-12. Biomaterials, 33(15), 3909–3918. Xuan, T., Zhang, J. A., & Ahmad, I. (2006). HPLC method for determination of SN-38 content and SN-38 entrapment efficiency in a novel liposome-based formulation, LESN38. Journal of Pharmaceutical and Biomedical Analysis, 41(2), 582–588. Yamada, S., & Sugahara, K. (2008). Potential therapeutic application of chondroitin sulfate/dermatan sulfate. Current Drug Discovery Technologies, 5(4), 289–301. Younes, I., & Rinaudo, M. (2015). Chitin and chitosan preparation from marine sources. Structure, properties and applications. Marine Drugs, 13(3), 1133–1174. Yu, Y., Duan, J., Leach, F. E., 3rd, Toida, T., Higashi, K., Zhang, H., & Linhardt, R. J. (2017). Sequencing the dermatan sulfate chain of decorin. Journal of the American Chemical Society, 139(46), 16986–16995. Zhang, Q., Zhang, F., Li, S., Liu, R., Jin, T., Dou, Y., ... Zhang, J. (2019). A multifunctional nanotherapy for targeted treatment of colon cancer by simultaneously regulating tumor microenvironment. Theranostics, 9(13), 3732–3753. Zhu, C., Zhu, Y., Pan, H., Chen, Z., & Zhu, Q. (2019). Current progresses of functional nanomaterials for imaging diagnosis and treatment of melanoma. Current Topics in Medicinal Chemistry, 19(27), 2494–2506.

International Journal of Biological Macromolecules, 72, 1313–1322. Reed, C. C., Waterhouse, A., Kirby, S., Kay, P., Owens, R. T., McQuillan, D. J., ... Iozzo, R. V. (2005). Decorin prevents metastatic spreading of breast cancer. Oncogene, 24(6), 1104–1110. Renvoize, C., Biola, A., Pallardy, M., & Breard, J. (1998). Apoptosis: Identification of dying cells. Cell Biology and Toxicology, 14(2), 111–120. Rivory, L. P., & Robert, J. (1994). Reversed-phase high-performance liquid chromatographic method for the simultaneous quantitation of the carboxylate and lactone forms of the camptothecin derivative irinotecan, CPT-11, and its metabolite SN-38 in plasma. Journal of Chromatography B, Biomedical Applications, 661(1), 133–141. Santra, M., Eichstetter, I., & Iozzo, R. V. (2000). An anti-oncogenic role for decorin. Down-regulation of ErbB2 leads to growth suppression and cytodifferentiation of mammary carcinoma cells. The Journal of Biological Chemistry, 275(45), 35153–35161. Sawtarie, N., Cai, Y., & Lapitsky, Y. (2017). Preparation of chitosan/tripolyphosphate nanoparticles with highly tunable size and low polydispersity. Colloids and Surfaces B, Biointerfaces, 157, 110–117. Srinivasarao, M., & Low, P. S. (2017). Ligand-targeted drug delivery. Chemical Reviews, 117(19), 12133–12164. Staff, N. P., Grisold, A., Grisold, W., & Windebank, A. J. (2017). Chemotherapy-induced peripheral neuropathy: A current review. Annals of Neurology, 81(6), 772–781. Streit, M., & Detmar, M. (2003). Angiogenesis, lymphangiogenesis, and melanoma metastasis. Oncogene, 22(20), 3172–3179. Sugahara, K., Mikami, T., Uyama, T., Mizuguchi, S., Nomura, K., & Kitagawa, H. (2003). Recent advances in the structural biology of chondroitin sulfate and dermatan sulfate. Current Opinion in Structural Biology, 13(5), 612–620. Trowbridge, J. M., & Gallo, R. L. (2002). Dermatan sulfate: New functions from an old glycosaminoglycan. Glycobiology, 12(9), 117R–125R. Weiss, R. B., & Poster, D. S. (1982). The renal toxicity of cancer chemotherapeutic agents.

11