pH-Sensitive tumor-targeted hyperbranched system based on glycogen nanoparticles for liver cancer therapy

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pH-Sensitive tumor-targeted hyperbranched system based on glycogen nanoparticles for liver cancer therapy Yuning Han, Bin Hu, Mingyu Wang, Yang Yang, Li Zhang, Juan Zhou ∗ , Jinghua Chen ∗ Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Pharmaceutical Sciences, Jiangnan University, Wuxi 214122, China

a r t i c l e

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Article history: Received 12 July 2019 Received in revised form 18 November 2019 Accepted 26 November 2019 Keywords: Glycogen nanoparticle Hyperbranch Biocompability pH-Response Liver cancer target

a b s t r a c t A multifunctionalized nanodevice based on natural hyperbranched glycogen nanoparticles had been fabricated for tumor therapy. Glycogen nanoparticles were decorated with liver cancer cell-targeted agent ␤-galactose and anticancer drug doxorubicin via schiff-base reaction. It was found that the glycogen nanocarrier could be taken by liver cancer cell selectively owing to galactose-ASGPR binding. After endocytosis, drug could be released from nanoparticles due to the cleavage of hydrazone-based bond at acidic tumor cell environment. The in vivo study demonstrated that this glycogen based drug delivery system minimized uptake and drug leakage in normal organs, enhanced accumulation and efficient drug release at tumor sites, inhibiting tumor growth with only slight retention in normal liver tissues. This strategy on exploiting glycogen nanoparticles as anticancer drug vehicle provides alternative platform for on-demand and targeted cancer therapy. © 2019 Elsevier Ltd. All rights reserved.

1. Introduction Carbohydrate functionalized nanoparticles, polymers, dendrimers and liposomes have been served as multivalent scaffolds for improving the efficiency of targeting carbohydrate binding proteins [1–4]. Through multiple receptor-ligand interactions in high affinity, these glyconanomaterials can potentially mediate various biological activities, including cell-cell communication, bacterial infections, immune response and virus invasion, which are desirable for recognition studies and therapeutic applications [5–8]. Thus, glyconanomaterials were widely explored as drug delivery vehicle for tumor therapy [9,10]. Ideally, except meeting the target capability, drug delivery system also needs to the requirements of prolonged blood circulation, controlled dissociation and release of cargo under intracellular stimuli, as well as nontoxic degradation products [11,12]. In these, biosourced polysaccharide have been extensively developed as drug carriers, such as chitin [13], hyaluronic acid [14], dextran [15], cellulose [16] and so on. They not only carry multiple copies of carbohydrate components themselves, but also possess excellent properties of hydrophilicity, nontoxicity, degradability and nonimmunogenicity [17]. While, most of these natural saccharides are liner polymers, carrying

∗ Corresponding authors. E-mail addresses: [email protected] (J. Zhou), [email protected] (J. Chen).

limited functionalized sites. Herein, glycogen, the hyperbranched polysaccharide like natural dendrimer, is concerned by us and its therapeutic applications are explored. Glycogen consists of ␣-D-(1–4) and ␣-D-(1–6) glycosidic bonds, presenting as nanoparticles in microscopic morphology [18,19]. Glycogen nanoparticles present innate characters of dendritic and hyperbranched structure without being structured by chemical synthesis [20]. Besides, the modified sites of glycogen nanoparticles are not only confined to the outer sphere but distributed in the interior space, which contribute to the modification of periodate oxidation [21], alkylation [22] and electrification [23] in high efficiency. Additionally, glycogen tends to aggregate at liver, where it is very popular to conduct metabolic activities [24]. This inherent characteristic endows glycogen the capability of “fusion targeting” as cargo vehicle. Besford et al. successfully developed lactose-functionalized glycogen, which exerts high affinity to peanut agglutinin (PNA) and interacts with galectin-1 that is overexpressed in prostate cancer cells, highlighting the targeted imaging effect to prostate cancer cells [25]. Bergkvist [23] and Caruso group [26] extended the use of glycogen nanoparticle as siRNA carrier that can bring gene silencing effect in multicellular tumor spheroids. These research broadened the applications of glycogen in drug delivery system, while we found that the delivery capabilities of the reported glycogen vehicles were not yet complete. While, the ideal drug carriers need to meet specific criteria in one system for successful cancer therapy in vivo, including prolonged blood circulation, special accumulation in tumor, release

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of drug cargo under stimuli as well as nontoxic degradation products [27]. Therefore, a strategy for glycogen modification must be devised to fully exploit all potential benefits of glycogen nanoparticles. Herein, we exploited glycogen as a smart vehicle for targeted transporting hydrophobic drug to liver cancer and releasing drug under pH response (Scheme 1). Glycogen was oxidized and conjugated with doxorubicin through schiff-base reaction, thus forming the pH responsive drug release model [28–30]. To enhance the liver targeted efficiency, we grafted glycogen with galactose derivate, which showed high sensitivity and specificity to the asialoglycoprotein receptor (ASGPR) on hepatic parenchymal cells [31]. The structure characterization and pH sensitive drug release behavior of the resulting systems were studied in detail. Their cytotoxicity and targeting activity were assessed against normal cell Cos 7 and liver cancer cell Hep G2 through intracellular drug release. Furthermore, the anticancer capability of these glycogen based drug delivery system were unambiguously investigated in vivo. We hypothesized that the evaluating intracellular uptake, drug distribution and glycogen based drug delivery system could be used as a potential platform of treating liver tumor. 2. Experimental section 2.1. Materials Glycogen was purchased from Shyuanye Biotech (Shanghai, China). Doxorubicin hydrochloride (DOX) was purchased from Melonepharma (Dalian, China). Penta-O-acetyl-␤-D-galactopyranose and 2-[2-(2-Chloroethoxy) ethoxy]ethanol was from TCI (Shanghai, China). Amberlite IR-120 H+ resin, bovine serum albumin (BSA) and ␣-amylase was from Macklin (Shanghai, China). BCA was purchased from Beyotime (Shanghai, China). All the chemicals were used as received. Cos 7 and Hep G2 cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Dulbecco’s modified eagle’s medium (DMEM), RPMI 1640 medium, fetal bovine serum (FBS) and penicillin-streptomycin solution were purchased from Gibco. Pancreatin, 4 , 6-diamidino2-phenylindole (DAPI) and thiazolyl blue tetrazolium bromide (MTT) was from Sigma-Aldrich. Balb/c-nu mice (4–6 weeks) were obtained from Model Animal Research Center of Slaccas (Shanghai, China). Dynamic light scattering (DLS) measurements was carried with a ZEN 3600 instrument (Malvern, England). Infrared spectrums (FT-IR) were obtained from a TENSOR II infrared spectrometer (Bruker, Germany). Transmission electron microscopy (TEM) images was recorded by a JEM-2100 TEM microscope (JEOL, Japan). Nuclear magnetic resonance spectrums was from an Aduance III NMR Spectroscopy (Bruker, Germany). Confocal fluorescence images (CLSM) were photographed by a Ti2-E + A1 instrument (Nikon, Japan). Flow cytometry analysis was carried using a CytoFLEX flow cytometer (Beckman Coulter, America). Single cell detection was conducted with an analyzer from Rayme (China). in vivo images were collected on IVIS imaging system (PerkinElmer, USA). 2.2. Synthesis of Gly-DOX-Gal [21] Glycogen was dissolved in water (10 mL) and mixed with an appropriate amount of sodium periodate in dark. The reaction was carried out at r.t for 1 h under stirring, then an excess of ethylene glycol (10 periodate equivalents) was added. The solution was dialyzed against pure water for two days and then lyophilized to give Gly-CHO. Gly-CHO (50 mg) was dissolved in formamide (20 mL). Then, DMSO solution (5 mL) containing DOX (37.6 mg) and TEA (30 ␮L)

was added, the mixture was allowed to stir in the dark for 48 h. After the reaction, the solution was put into dialysis tube (MWCO 3500 Da) and subjected to dialysis against DMSO for 1 d to remove unreacted DOX, then the dialysis tube was immersed into distilled water and dialyzed for another 3 days to remove impurities and organic solvents. Finally, the product Gly-DOX was lyophilized. Gly-DOX (30 mg) was dissolved in formamide (15 mL). Then, DMSO solution (2 mL) containing 1-(2-(2-(2aminoethoxy)ethoxy)ethoxy-D-galactopyranoside (20 mg) [16] and TEA (50 ␮L) was added, the mixture was allowed to stir in the dark for 48 h. After the reaction, the solution was put into dialysis tube (MWCO 3500 Da) immersed into distilled water and dialyzed for 3 d to remove impurities and organic solvents. Finally, the product Gly-DOX-Gal was lyophilized. 2.3. Determination of oxidation degree of Gly-CHO Hydroxylamine hydrochloride salt (17.375 g) was dissolved in water (100 mL). Then methylorange (0.05 % wt) were added to the solution and the pH was adjusted to 4 with NaOH (0.1 M, aq.). The solution was diluted to 1 L. Gly-CHO (0.105 g) was dispersed in the above solution (25 mL) and the mixture was stirred for 2 h followed by being titrated with 0.1 N NaOH to pH 4. The degree of oxidation was calculated as percentage by mole of oxidized glucoside units by assuming the presence of two aldehyde groups for each oxidized unit. 2.4. Determination of drug-loading rate The absorbance of Gly-DOX-Gal at 480 nm was meausred when its concentration was 0.1 mg mL−1 . Then the drug-loading amount of Gly-DOX-Gal was calculated according to the formula that transformed from calibration curve as follows: P =

A + 0.0006

(1)

4.35 × 10-3

where P is drug-loading rate (mg/g), A is the absorbance of GlyDOX-Gal at 480 nm. 2.5. pH responsive drug release of doxorubicin in Gly-DOX-Gal Gly-DOX-Gal was dispersed in PBS of pH 7.4 to the concentration of 1 mg/mL and 2 mL of the solution was added to the dialysis bag. Then the bag was respectively put into the centrifuge tube containing PBS (20 mL) of pH 5.0 and 7.4. The outer dialysis solution (2 mL) was removed at the pre-determined period (30 min, 1 h, 1.5 h, 3 h, 5 h, 8 h, 12 h, 24 h, 30 h, 36 h, 48 h, 60 h and 72 h). Finally, the ultraviolet absorption of all the samples at 480 nm was measured and the amount of released drug was calculated following Eq. (1). The release test at each pH value was repeated for 3 times to correct the errors. 2.6. In vitro BSA adsorption test Gly-DOX-Gal (2 mg) was dispersed in PBS (pH 7.4, 1 mL) followed by adding BSA standard solution (1 mL, 0.6 mg mL−1 ) to the mixture. The solution was incubated at 37 ◦ C under 200 rpm after it was well-mixed. After incubating for 0.5 h, 1 h, 2 h, 3.5 h, 6 h, 9 h, 12 h and 24 h, the mixture was centrifuged under 10,000 rpm and then the supernatant (20 ␮L) was mixed with BCA(200 ␮L) and kept in 37 ◦ C for 1 h. The absorbance of the solutions at 562 nm was measured with ultraviolet spectrophotometer and the absorption percentage was calculated using Eq. 2 as follows: AP =

m -

 A-0.1624  1.354

m

×V

× 100

(2)

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Scheme 1. Design of pH sensitive glycogen nanoparticle for targeted drug delivery to liver cancer cells.

where AP is absorption percentage (%), m is the total mass of BSA in supernatant (mg), A is the absorbance of the solution at 562 nm, V is the volume of the supernatant (mL). 2.7. Cytotoxicity assay Cos 7 and Hep G2 cells were respectively plated on 96-wells plates with a seeding density of 4 × 103 cells and 5 × 103 cells per well in 1640 supplemented with 10 % FBS and 1 % penicillinstreptomycin solution (100 ␮L) and incubated overnight at 37 ◦ C with 5 % CO2 and 95 % air. The medium was exchanged with fresh medium containing free doxorubicin, glycogen, Gly-DOX and Gly-DOX-Gal of various concentrations. The medium was removed followed by adding MTT solution (0.5 mg/mL in PBS, 100 ␮L per well). After incubating for another 4 h, DMSO (100 ␮L per well) was added too. The absorbance at 570 nm was measured after 15 min standing. The test was held in sextuplicate. 2.8. Cell uptake by flow cytometry Hep G2 cells were seeded in 6-well plates with a density of 8 × 104 cells per well. The medium was replaced by fresh medium containing Gly-DOX or Gly-DOX-Gal (50 ␮g/mL) after incubating overnight, meanwhile fresh medium was directly exchanged as control. After 6 h, 12 h and 24 h incubation, cells were trypsinized, collected and preserved with 1 % paraformaldehyde to maintain the morphology, respectively. Flow cytometry analysis was conducted during which 1 × 104 cells were tested for each example. 2.9. Drug release studies in vitro Hep G2 cells were seeded in cell culture dishes with the density of 5 × 103 cells per dish and incubated overnight. The medium was replaced by fresh medium containing Gly-DOX-Gal (50 ␮g/mL). After 6 h, 12 h and 24 h incubation, the medium was removed and DAPI (20 ␮mol/L, 400 ␮L) was added, then cells were incubated for another 30 min away from light. 4 % paraformaldehyde was added to get cells immobilized. After 15 min standing, the dishes were washed with PBS 3 times and the cell were finally preserved in PBS at −20 ◦ C. Red emission of doxorubicin and blue emission of DAPI were respectively collected by exciting at ␭ =552 nm and ␭=405 nm.

Hep G2 were seeded on 20 mm cell culture dishes in 4 × 103 per dish and cultured in 1640 cell medium for 24 h. The cells were then treated with Gly-DOX-Gal respectively for 6 h, 12 h and 24 h. The cells were then washed with PBS three times and immobilized with 4 % paraformaldehyde for 20 min. The fixed cells were washed with PBS 3 times before detecting fluorescence intensity of doxorubicin by optical fiber nanoprobes. 2.10. Lysosome escape studies Hep G2 cells with the density of 4 × 104 cells per dish were incubated with Gly-DOX-Gal (50 ␮g/mL). After 2 h, 6 h and 12 h incubation, the medium was removed and Lyso Tracker (200 times dilution with medium, 1 mL) was added, then cells were incubated for 30 min away from light. 4 % paraformaldehyde was added to immobilize cells. After 15 min standing, DAPI (20 ␮mol/L, 400 ␮L) was added and cells were incubated for another 30 min. The dishes were washed with PBS and the cells were finally preserved in PBS at −20 ◦ C. Red fluorescence of doxorubicin, blue emission of DAPI and green emission of Lyso Tracker were respectively collected by exciting at ␭ =552 nm, 405 nm and 488 nm using corresponding emission filter. 2.11. Hemolysis analysis Mouse blood sample (1 mL) was added to NaCl solution (20 mL, 0.9 %). Red blood cells (RBCs) were isolated from serum via centrifugation. After being washed with NaCl solution (0.9 %), purified RBCs were diluted to 1/50 of its volume with PBS (pH 7.4) solution. Diluted RBC suspension (0.2 mL) was mixed with PBS solution (0.8 mL) as a negative control, water (0.8 mL) as a positive control, and solution containing deferent nanoparticles (0.8 mL). All the mixtures were vortexed and kept at 37 ◦ C for another 3 h. After centrifugation, absorbance of supernatants at 541 nm was tested via UV–vis spectroscopy. Hemolysis Rate (%) =

OD (sample) − OD(PBS) OD (water) − OD(PBS)

(3)

where OD(sample), OD(PBS) and OD(water) are the supernatant absorbance of the sample, negative and the positive control, respectively.

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2.12. Pharmacokinetic studies DOX, Gly-DOX and Gly-DOX-Gal were injected into the tail vein of the rats (200 g ± 20 g) at a dosage of 5 mg/kg (n = 5). At each time point, blood was collected and adjusted pH to 5.0 for 12 h. All samples were then processed and analyzed by a validated high performance liquid chromatographic (HPLC) method. Prior to HPLC analysis, the blood samples were treated with methanol (100 ␮L) in Eppendorf tubes. These tubes were vortexed for 10 s, then centrifuged for 5 min at 10,000 rpm. The supernatant was transferred in an autosampler vial, and then each sample (20 ␮L) were injected into the chromatographic system. 2.13. In vivo anticancer efficacy Balb/c-nu mice (4–6 weeks) were fed with standard chow and all experimental protocols were approved by the Institutional Animal Care and Use Committee of the Animal Experiment Center of Jiangnan University (Wuxi, China, Animal JN.No 20190615b0500915). All mouse experimental procedures were performed in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals approved by the State Council of People’s Republic of China. Tumor-bearing mice were prepared by implanting 50 ␮L of Hep G2 cell suspension (2 × 107 cells/mL) at the right hind hip in Balb/c-nu mice. After about 10 d inoculation, the subcutaneous mouse tumor model was well built when the tumor volume reached to nearly 100 mm3 . Free DOX and DOX grafted glycogen nanoparticles were administered by tail vein injection (5 mg/kg) in above mice. After injecting 1 h, 3 h, 5 h, 7 h, 12 h and 24 h, doxorubicin fluorescence intensities in these mice were measured. Then mice were sacrificed and major organs including heart, liver, spleen, lung, kidney, and tumor tissue were excised and directly imaged (at 5 h and 24 h). The mice were then randomly divided into five groups (n = 5) and each group was intravenously injected with saline, Glycogen, free doxorubicin, Gly-DOX, Gly-DOX-Gal solution at a dose of 5 mg/kg. The tumor volumes and body weights were measured at desired time intervals for an additional 21 d. The tumor volumes of mice were measured using a vernier caliper and computed as follows: V= (length) × (width)2 /2. All of the mice were sacrificed and their body tissues including tumor, heart, liver, spleen, lung and kidney in various groups were excised, fixed, and sliced for H&E staining for the histopathological assay. 2.14. Statistical analysis The experiments were conducted at least in triplicate. The student t-test were carried out to identify the differences between the control and experimental groups. The p-value < 0.05 (*) or < 0.01 (**) was considered as statistically significant. 3. Results and discussion 3.1. Preparation and characterization of Gly-DOX-Gal Glycogen were initially oxidized by sodium periodate to provide aldehyde groups (Fig. 1A). The resulting oxidized nanoparticle Gly-CHO was sphere with an average diameter of 60 nm (Fig. 1B). The typical signals of aldehyde protons of the resulting oxidized glycogens were observed at 9.2 and 9.7 ppm by 1 H NMR (Fig. S1). Parallel experiments with various concentrations of glycogen and sodium periodate were carried out, the corresponding degrees of the resulting oxidation were also analyzed by treating the oxidized products with hydroxylamine hydrochloride and then titrating the evolved hydrochloric acid (Table S1), in which the one equipped

with most abundant aldehyde groupswas chosen for the following experiments. Subsequently, doxorubicin and ␤-D-galactoside residue (see supporting information for the synthesis, Figs. S2–S10) were conjugated onto Gly-CHO in turn by schiff-base reaction of amino and aldehyde groups, yielding the drug delivery systems GlyDOX and Gly-DOX-Gal. Both of them appeared in regular circles, about 50 nm in diameter (Fig. 1C, D). It could be seen from Fig. 1E that the hydrated size distribution of all glycogen nanoparticles was similar and concentrated, demonstrating that the modification of particles had no great effect on their morphologies. In addition, the formation of hydrazone was supported by the absorption around 1618 cm−1 in the FT-IR spectrum (Fig. 1F). Then, 1H NMR analysis was proceeded to confirm the successful conjugation of doxorubicin and ␤-D-galactoside residue. As expected, the signals of hydrogen in -CH = N- were determined at 8.0 and 8.2 ppm. The proton signals of doxorubicin were visible between 7.0–7.9 ppm and 1.4–2.3 ppm, which were assigned to hydrogen from benzene and cyclohexane fragments, respectively (Fig. S11). The amount of doxorubicin and galactose conjugated in glycogen was evaluated as 90.3 mg/g and 131.3 mg/g, respectively (Figs. 1G, S12 and Table S2). 3.2. Protein adsorption, drug release and cytotoxicity studies of Gly-DOX-Gal in vitro As we known, some drug delivery system in particular nanoparticles are exposed to proteins in the blood circulation, generating a certain amount of protein adsorption, which affects the normal movement of the particles, resulting in reduced therapeutic effect. Therefore, we firstly evaluated the adsorption capability of GlyDOX-Gal toward protein. Herein, bovine serum albumin (BSA) was chosen as the model protein [32]. Gly and Gly-DOX-Gal were suspended in BSA solution, and then the supernatant at predetermined time were collected and the absorption efficiency was calculated (Fig. S13). As shown in Fig. 2A, Gly-DOX-Gal absorbed approximately 14 % of BSA at beginning 12 h, which was about 2.5 times less than Gly nanoparticles. Then the ratio remained almost the same level in the following 12 h, and Gly-DOX-Gal. This low absorption was beneficial to the prolonged circulation of Gly-DOX-Gal in vivo. To investigate the pH-dependent releasing characteristics, the release performance of doxorubicin from Gly-DOX-Gal was monitored by measuring the UV absorbance of supernatant solution when incubating Gly-DOX-Gal in PBS buffer under pH 7.4 and 5.0, simulating normal physiological environment and tumor lysosome environment respectively. As shown in Fig. 2B, the amount of released drug increased as the pH decreased. Especially, about 13 % of DOX was spared from the system within pH 7.4 PBS buffer in 3 d. In contrast, when suspending the system in PBS buffer (pH 5.0), the DOX released efficiency raised to 56 % and it kept upward trend. These results indicated that this drug delivery system stayed nearly intact in neutral condition while rapid release of drug upon acid stimulation owing to the break of hydrazone bond. Therefore, Gly-DOX-Gal would be a potential vehicle for transporting drug to cancer cells without premature release into blood vessels and normal tissue. The purpose of this stage was to examine the cytotoxicity of glycogen based drug delivery system, Cos 7 and Hep G2 cells were incubated with different concentrations of free DOX, glycogen, Gly-DOX and Gly-DOX-Gal nanoparticles, then MTT assays were performed. At first, we tested the toxicity of free drug on both types of cells. Free DOX could enter into Cos 7 cell without resistance and produce cytotoxicity. This behavior also occurred in Hep G2 cell, indicating that free drug played killing effect on either normal cell or cancer cell without selectivity (Fig. S14). Then the two cells were incubated with glycogen nanoparticles series. As shown in Fig. 2C and D, bare glycogen nanoparticles presented no signif-

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Fig. 1. (A) Synthetic route of glycogen based, liver cancer targeted, pH responsive drug delivery system; TEM image of (B) Gly-CHO, (C) Gly-DOX and (D) Gly-DOX-Gal nanoparticles; (E) Size distribution of glycogen nanoparticles series by DLS; (F) FT-IR images of glycogen, Gly-CHO, Gly-DOX and Gly-DOX-Gal; (G) UV absorbance spectra of glycogen, Gly-CHO, Gly-DOX and Gly-DOX-Gal.

icant cytotoxicity toward both cell lines, where the cell viability was beyond 90 % at nanoparticle concentrations from 20 ␮g/mL to 100 ␮g/mL, indicating favourable biocompatibility of bare glycogen nanoparticles. Furthermore, Gly-DOX and Gly-DOX-Gal displayed low toxicities toward Cos 7 cell, since they were not able to release drug in neutral environment of normal cells. In the case of Hep G2 cell, both of Gly-DOX and Gly-DOX-Gal exhibited obvious cytotoxicity, furthermore, the damage of Gly-DOX-Gal towards cell was more powerful than Gly-DOX. 3.3. Cell uptake and distribution of Gly-DOX-Gal To exam cell uptake performance of Gly-DOX and Gly-DOXGal, Hep G2 cells were co-cultured with those nanoparticles and assessed using flow cytometry. After being cultured for 6 h, 12 h and 24 h, Hep G2 took up both of nanoparticles, indicating that glycogen based nanoparticles could be accumulated in liver cancer cells. But there were differences, the endocytosed amount of GlyDOX-Gal by cell was higher than that of Gly-DOX, and the uptake of Gly-DOX-Gal by Hep G2 cell over time (Fig. 3A). During the process, the targeting ligand of galactose accessory on the surface of Gly-DOX-Gal mediated endocytosis and were helpful for cellular

uptake of nanoparticles. The distribution of Gly-DOX-Gal in Hep G2 cell was observed by CLSM. The red fluorescence from DOX in nanoparticles was mainly observed outside nuclei after incubating Hep G2 cell with Gly-DOX-Gal for 6 h. As culturing time longer, the nuclei of Hep G2 cell gradually gave rise to red fluorescence, especially, most of cell nucleus was filled with red fluorescence at the end of 24 h (Fig. 3B). This phenomenon was confirmed by single cell nanobiochemical detecting of doxorubicin in membrane and nuclei of Hep G2 cell at 6 h, 12 h and 24 h. The fluorescence inensity of doxorubicin near membrane kept approximate level at the three time points, while its concentration in nucleus increased significantly with time, keeping twice than that in the membrane (Fig. 3C, D). We speculate that the nano-drug delivery system first accumulated near the cell membrane, and then enter into cell via ASGPR-mediated endocytosis followed by escaping from endosome and lysosome. The system would release DOX in response to acidic environment, which spontaneously trended into the nucleus. 3.4. Efficiency of escaping from lysosomes To further verify the above conjecture, we next used CLSM to examine the intracellular actiity of Gly-DOX-Gal in Hep G2 cell

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Fig. 2. (A) UV absorbance of supernatant liquor after suspending Gly, Gly-DOX-Gal in BSA solution 6 h, 12 h and 24 h. (B) Release profile of doxorubicin from Gly-DOX-Gal in pH 5.0 and 7.4 PBS buffer. Cell viabilities of (C) Cos 7 cells against glycogen, Gal-DOX and Gly-DOX-Gal nanoparticles. (D) Hep G2 cells against glycogen, Gal-DOX, Gly-DOX-Gal nanoparticles.

Fig. 3. (A) Quantitative analysis of Gly-DOX and Gly-DOX-Gal uptake in Hep G2 cell at 6 h, 12 h and 24 h through flow cytometry; (B) Confocal microscope images of Hep G2 cells incubated with Gly-DOX-Gal for 6 h, 12 h and 24 h. Blue and red fluorescence indicated DAPI and doxorubicin, respectively. The scale bar was 25 ␮m. (C) Photographs of nanoprobes used to measure the real-time amount of doxorubicin in single cell membrane and nucleus after treating Hep G2 cell with Gly-DOX-Gal for 24 h; (D) Quantitative analysis of doxorubicin in single cell membrane and nucleus after treating Hep G2 cell with Gly-DOX-Gal for 6 h, 12 h and 24 h.

(Fig. 4). Hep G2 cell were treated with lysosome probe LysoTracker (green fluorescence) and Gly-DOX-Gal (red fluorescence from doxorubicin belong to Gly-DOX-Gal), then monitored at different time points (2 h, 6 h and 12 h). The red fluorescence signal represented nanoparticles outside of the lysosome whereas the yellow fluores-

cence demonstrated nanoparticles trapped in lysosomes. As shown in Fig. 4, the yellow fluorescence doxorubicin was observed in lysotracker-labeled lysosome after 2 h culturing Hep G2 cell with Gly-DOX-Gal, indicating that the nanoparticles were trapped in lysosome. While after 6 h incubation, both of yellow fluorescence

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Fig. 4. Confocal microscope images of Hep G2 cells treated with Gly-DOX-Gal for 2 h, 6 h and 12 h. Blue, green and red colour indicated DAPI, lysotracker green and DOX, respectively. The scale bar was 25 ␮m.

and red fluorescence increased. At 12 h, red fluorescence dominated almost all cells, especially the nucleus. The results demonstrated that Gly-DOX-Gal nanoparticles could escape from lysosme and release drug, otherwise the red fluorescence would continue to overlap with green fluorescence.

3.5. Antitumor therapy in vivo Investigation of in vitro blood compatibility of Gly-DOX-Gal was especially important because biomedical applications required the nanoparticles to be delivered via intravenous injection. Thus, hemolysis assay was employed to assess the mouse red blood cell (MRBC) compatibility of nanoparticles by exposuring cells toward nanoparticles at a concentration range of 0.1–1.6 mg/mL for 3 h. As shown in Fig. 5A and B, the level of hemolysis was lower than 5 % in all samples, and these hemolytic effects were within acceptable range. The optical images of MRBCs incubated with Gly-DOX-Gal implied that nanoparticles had low interaction with erythrocytes membrane. The pharmacokinetics studies of DOX, Gly-DOX and Gly-DOXGal were evaluated by recording DOX concentrations in plasma. As observed in Figs. S15 and 5 C, DOX was rapidly cleared through systemic circulation in 6 h, while Gly-DOX and Gly-DOX-Gal presented prolonged circulation in blood until 24 h at least. In detail, DOX had a half-life of about 1.08 h. At this time point, the concentration of DOX in the blood of Gly-DOX and Gly-DOX-Gal was 4.33-fold higher than that of DOX. The half-life of Gly-DOX and Gly-DOX-Gal was close to 3.5 h. These results determined that our glycogen nanoparticles enabled reducing the clearance of DOX from bloodstream and keeping favorable stabilities in blood circulation, contributing to the enhancement of drug accumulation in tumor site. Then, the in vivo antitumor behavior of glycogen based nanoparticles were evaluated using Hep G2 tumor-bearing Balb/c-nu mice. When the tumor reached to 100 mm3 , the mice were injected with free DOX, Gly-DOX and Gly-DOX-Gal through tail vein, the biodis-

tribution of DOX in mice was detected firstly. As shown in Fig. 5D and E, the DOX injected into mice were primarily accumulated in the liver for free DOX group at 5 h postinjection, leading to a low drug concentration in tumor site. Gly-DOX also appeared in the liver due to the intrinsic targeting. Meanwhile, it was concentrated in tumor because of enhanced EPR effect, whereas the intensity of DOX was lower than that of Gly-DOX-Gal, which was attributed to the function of galactose moieties in Gly-DOX-Gal. At 24 h postinjection, the fluorescence intensity in major organs and tumor reduced for free DOX group while increased for Gly-DOX nanoparticles. However, compared to other organs, tumors treated with Gly-DOX-Gal nanoparticle group showed the strongest fluorescence, which was 3-fold higher than that of DOX. The results indicated that the presence of targeting ligand in Gly-DOX-Gal leaded to the enhancement of tumor accumulation and retention of drug. Next, the mice were randomly divided into five groups (5 each group) followed by intravenously injecting with 100 ␮L of saline, free doxorubicin, Gly, Gly-DOX as well as Gly-DOX-Gal to estimate antitumor efficiency. The mice’s body weights were monitored at specific time. We observed from Fig. 6A that all body weights of control and experimental groups kept slight fluctuations in 21 d. Subsequently, the tumor volumes of all groups were measured in real time. As shown in Fig. 6B, the tumor volume unrestrainedly increased in the groups of saline and glycogen nanoparticle without statistical difference, reaching an average tumor size approximately 8.3 times of the initial tumor size at the end of 21 d, which indicated that the glycogen nanoparticles alone had no prominent influence on tumor development. The treatment with free DOX inhibited the tumor growth in the starting 6 d due to the high drug concentration, but thereafter the tumor grew to 4.1 times the initial tumor size because of quick elimination. In contrast, the mice treated with Gly-DOX and Gly-DOX-Gal exhibited obvious tumor growth inhibition, holding approximately1.8 and 1.1 times of the initial tumor size at 21 d, respectively. More importantly, the stronger antitumor effect was achieved by Gly-DOX-Gal due to the

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Fig. 5. (A) Photographs of hemolysis of RBCs in the presence of glycogen based drug delivery systems, the presence of red hemoglobin in the supernatant indicates damaged RBCs. D.I. water (+) and PBS (-) were used as positive and negative control, respectively; (B) Percentage of hemolysis of RBCs incubated with Gly-DOX-Gal at different concentrations ranging from 0.1 to 1.6 mg/mL for 3 h; (C) Pharmacokinetic results of DOX, Gly-DOX and Gly-DOX-Gal; (D) The fluorescence images and (E) the quantified fluorescence distribution of the major organs and tumors sacrificed mice after injecting with free DOX, Gly-DOX and Gly-DOX-Gal at specific intervals.

Fig. 6. (A) Body weight of mice in different groups; (B) Relative tumor weight of mice in different groups; (C) Representative images of harvested tumors from corresponding mice after different treatments; (D) (D) H&E stained tissue sections of heart, liver, spleen, lung, kidney and tumor of the mice treated with saline, glycogen, DOX, Gly-DOX and Gly-DOX-Gal.

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targeted and preferential tumor accumulation. These tumor measured in living mice agreed with that of the excised tumor (Fig. 6C). To further evaluate the tissue damage, the histological analysis of tumor and major organs of experimental mice were carried out. Tumor slices of mice treated with saline and glycogen nanoparticles showed abundant large cell nucleus, indicating the vigorous proliferation of tumor cells. There was a fall in the number of cells in the group that received free DOX. The tumors treated with GlyDOX and Gly-DOX-Gal nanoparticles exhibited obvious decrease of cells density, accompanied with degrees of cell necrosis like nuclear condensation, shrinkage and fragmentation. Moreover, Gly-DOXGal group exhibited the largest necrosis areas, which indicated the best tumor suppression efficiency. There was no apparent difference of heart, liver, spleen, lung, and kidney of mice treated with glycogen, Gly-DOX and Gly-DOX-Gal nanoparticles compared to those treated with saline, which manifested that these drug delivery systems had no significant impact on normal tissues (Fig. 6D). 4. Conclusion In conclusion, we explored the potential applications of glycogen nanoparticles as drug transportor for liver tumor therapy. The hyperbranched glycogen nanoparticles were functionalized galactose and doxorubicin using a simple method. The resulting system showed good biocompability, effective targeting toward liver cancer cell and pH responsive drug release in vitro. Additionally, the system exhibited good anticancer effects in vivo. These results verified that glycogen, the biosourced nanoparticle, would be effective drug vehicles for treating liver tumor. Declaration of Competing Interest There are no conflicts to declare. Acknowledgements This work was supported by the Natural Science Foundation of Jiangsu Province (No. BK20170203) and National Natural Science Foundation of China (No. 21574059). We also thank Top-notch Academic Programs Project of Jiangsu Higher Education Institutions (No. PPZY2015B146), National First-class Discipline Program of Light Industry Technology and Engineering (No. LITE2018-20) and Fundamental Research Funds for the Central Universities (No. JUSRP51709A). 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.apmt.2019. 100521. References [1] M. Marradi, F. Chiodo, I. Garcia, S. Penades, Glyconanoparticles as multifunctional and multimodal carbohydrate systems, Chem. Soc. Rev. 42 (2013) 4728–4745. [2] X. Wang, O. Ramström, M. Yan, Glyconanomaterials: synthesis, characterization, and ligand presentation, Adv. Mater. 22 (2010) 1946–1953. [3] S. Cecioni, A. Imberty, S. Vidal, Glycomimetics versus multivalent glycoconjugates for the design of high affinity lectin ligands, Chem. Rev. 115 (2015) 525–561. [4] A. Bukchin, N. Kuplennik, Á.M. Carcaboso, A. Sosnik, Effect of growing glycosylation extents on the self-assembly and active targeting in vitro of branched poly(ethylene oxide)-poly(propylene oxide) block copolymers, Appl. Mater. Today 11 (2018) 57–69. [5] N.C. Reichardt, M. Martin-Lomas, S. Penades, Glyconanotechnology, Chem. Soc. Rev. 42 (2013) 4358–4376.

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Please cite this article as: Y. Han, B. Hu, M. Wang et al., pH-Sensitive tumor-targeted hyperbranched system based on glycogen nanoparticles for liver cancer therapy, Appl. Mater. Today, https://doi.org/10.1016/j.apmt.2019.100521