pH sensitive doxorubicin-loaded nanoparticle based on Radix pseudostellariae protein-polysaccharide conjugate and its improvement on HepG2 cellular uptake of doxorubicin

Journal Pre-proof pH sensitive doxorubicin-loaded nanoparticle based on Radix pseudostellariae protein-polysaccharide conjugate and its improvement on HepG2 cellular uptake of doxorubicin Xixi Cai, Qian Yang, Qingxia Weng, Shaoyun Wang PII:

S0278-6915(19)30889-0

DOI:

https://doi.org/10.1016/j.fct.2019.111099

Reference:

FCT 111099

To appear in:

Food and Chemical Toxicology

Received Date: 16 October 2019 Revised Date:

12 December 2019

Accepted Date: 25 December 2019

Please cite this article as: Cai, X., Yang, Q., Weng, Q., Wang, S., pH sensitive doxorubicin-loaded nanoparticle based on Radix pseudostellariae protein-polysaccharide conjugate and its improvement on HepG2 cellular uptake of doxorubicin, Food and Chemical Toxicology (2020), doi: https:// doi.org/10.1016/j.fct.2019.111099. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

Author Contributions Xixi

Cai:

Conceptualization,

Methodology,

Investigation,

Formal

analysis,

Data Curation, Writing-Original Draft preparation; Qian Yang: Methodology, Formal analysis; Qingxia Weng: Investigation, Formal analysis; Shaoyun Wang: Conceptualization, Resources, Writing - Review & Editing, Funding Acquisition.

pH sensitive doxorubicin-loaded nanoparticle based on Radix pseudostellariae protein-polysaccharide conjugate and its improvement on HepG2 cellular uptake of doxorubicin

Xixi Cai,1,2 Qian Yang,2 Qingxia Weng,2 Shaoyun Wang*,2 1

College of Chemical Engineering, Fuzhou University, Fuzhou 350108, China

2

College of Biological Science and Technology, Fuzhou University, Fuzhou 350108,

China

*Corresponding Author E-mail address: [email protected]

ABSTRACT Nanoparticles based on Radix pseudostellariae protein-polysaccharide conjugates were self-assembled via pH adjustment and thermal treatment. The fabricated nanoparticles (CP3) were spherical with narrow size distribution of 125.0 nm in diameter. The doxorubicin (DOX) -loaded CP3 nanoparticles exhibited pH-sensitive release behavior and accelerated the release of DOX under the acidic pH simulating tumor microenvironment and endosomal pH. In HepG2 uptake studies, CP3-DOX nanoparticles notably improved the internalization of DOX, which was 1.56-fold compared with free DOX. CP3-DOX nanoparticles could serve as P-glycoprotein efflux pump inhibitor and be internalized into HepG2 cells via clathrin-dependent endocytosis. Moreover, the cytotoxicity effect of DOX on HepG2 cells was elevated after the encapsulation by CP3, with a lower IC50 value of 0.25 µg/mL. The findings suggested that the pH-sensitive CP3-DOX nanoparticles has a great potential in facilitating the efficacy of DOX in cancer cells, and the obtained CP3 could be a good candidate as nanocarrier for the encapsulation and delivery of functional compounds.

Keywords: Radix pseudostellariae; Protein-polysaccharide conjugate; Doxorubicin; Nanoparticle; pH-sensitive release; Cellular uptake

1. Introduction As the fast-development of nano technology, nanoparticle-based drug delivery systems provide favorable platforms for cancer therapy (La-Beck et al., 2019; Shi et al., 2011; Xing et al., 2019). Nanocarriers could increase drug hydrophilic, enhance stability, alleviate side effects and targeting deliver drug to tumor sites. Doxorubicin (DOX), belongs to anthracyclines, is an effective chemotherapeutic agent widely used for cancer therapy, including breast, lung, ovarian, stomach, liver cancer, acute leukemia, etc. DOX can diffuse into cells, interact with nucleus DNA and lead to DNA denaturation, finally resulting in cell death (Varela-López et al., 2019). However, the poor pharmacokinetics, non-target specificity and systemic distribution of DOX bring about severe toxic side effects in normal tissues, which limit its clinical use (dos Santos Arruda et al., 2019). Besides, the long-term exposure of cancer cells to DOX develops drug resistance, with high P-glycoprotein (P-gp) expression, which shows high affinity to DOX and will cause drug efflux out of cancer cells (Varma et al., 2003). Various nanocarrier strategies have been propounded to attenuate toxicity and facilitate the efficacy of DOX, among which, protein-based self-assembled nanoparticles have attracted more and more attention due to their excellent biocompatibility and degradability (Golla et al., 2013; Hu et al., 2018). The amphipathicity and structural diversity of proteins provide multiple binding sites to both hydrophilic and hydrophobic molecules through electrostatic interactions, hydrophobic interaction, hydrogen bonding, van der Waals force, etc., which make 1

proteins nanoparticles easily engineered to exert multiple functions (Lv et al., 2019; Ping et al., 2017). However, the pure protein-based nanoparticle could be influenced by environmental factors, including pH, ions, temperature, etc, resulting in aggregation and precipitation. Moreover, the precipitated nanoparticles in the organisms could cause inflammatory response and even acute toxicity (Saei et al., 2017). The use of cross-linking agent or surface modification of protein nanoparticles to provide steric or electrostatic resistance to improve nanoparticle stability have been widely studied (Ma et al., 2017; Ping et al., 2017). The grafting of the polysaccharide has been proved to be an effective method to enhance the protein solubility and stability (Ding et al., 2019; Fan et al., 2018; Zhang et al., 2019). Maillard reaction is a well-known process to produce proteins and polysaccharides conjugates (Liu et al., 2014; Oh et al., 2015; Wei et al., 2018). Nanoparticles fabricated by Maillard conjugates were reported to exhibit superior stability to high temperature, pH and ionic strength, which was attributed to the strong steric repulsion of polysaccharide (Feng et al., 2016; Yi et al., 2014). Such stable glycated protein nanoparticles have also been found to be self-assembled in more than 80 kinds of herbal medicine decoctions and the studies showed that they could easily transport through the Caco-2 cells monolayer, playing key roles in the delivery of functional compounds (Zhuang et al., 2008). Radix pseudostellariae (RP), recorded as a tonic traditional Chinese medicine in the Chinese Pharmacopoeia with multiple effects, such as improve immunity and

2

appetite, nourishing vitality and moistening lung (PCCn, 2015), is widely distributed in Asia area and used for health foods. The application of RP in the field of health care is increasingly widespread (Yang et al., 2019). Protein and polysaccharide are abundant in RP, accounting for about 17 % and 20 % (dry weight), respectively, which make RP a potential raw material candidate for the development of protein nanoparticles. Our group has reported the formation conditions of the protein nanoparticles from RP via water bath heat treatment and pH control and investigated the encapsulation and improvement on curcumin stability (Weng et al., 2019). However, the constructed protein nanoparticles still had shortage in pH and ionic strength stability, which limited their practical applications. Therefore, in this study, the homologous protein and polysaccharide from RP were used to prepared conjugate with certain glycated degree through dry heating and easily self-assembled into polysaccharide-stabilized protein nanoparticles via thermal treatment. Meanwhile, the release behavior, cellular uptake and cytotoxicity effect of DOX-loaded nanoparticles were also investigated.

2. Material and methods 2.1. Materials and chemicals The raw Radix pseudostellariae (RP) were collected from the authorized planting base in Zherong, Fujian, China. DOX·HCl (98.0 % purity), thiazolyl blue tetrazolium bromide (MTT), Cyclosporine-A (CysA), chlorpromazine, indomethacin, colchicine

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and quercetin were the product of Shanghai Makclin Biochemical Co., Ltd. (Shanghai, China). Fetal bovine serum was purchased from Gibco (Beijing, China). Dulbecco's modified Eagle's medium (DMEM)-high glucose, Roswell Park Memorial Institute (RPMI) 1640 medium, phosphate-buffered saline (PBS), trypsin and 100X penicillin and streptomycin were obtained from Hyclone (Shanghai, China). Hoechst 33342 and 1,1'-dioctadecyl-3,3’-dioctadecyloxacarbocyanine perchlorate (DIO) membrane dye were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). BCA protein kit was the product of Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Annexin V-APC/7-AAD apoptosis detection kit was the product of Elabscience Biotechnology Co., Ltd (Wuhan, China). All other chemicals and regents were of analytical grade and commercially available. Human normal liver L-02 cells, human hepatoma HepG2 cells, and colon cancer Caco-2 cells were obtained from BeNa Culture Collection (Kunshan, China). L-02 cells and HepG2 cells were cultured in RPMI 1640 medium supplemented with 10 % (v/v) fetal bovine serum, 100 U/mL penicillin and 100 µg/mL streptomycin. Caco-2 cells were cultured in DMEM-high glucose medium supplemented with 15 % (v/v) fetal bovine serum, 1 % (v/v) non-essential amino acids, 100 U/mL penicillin and 100 µg/mL streptomycin.

2.2. Preparation and characterization of glycation conjugates 2.2.1. Preparation of RP protein and polysaccharide The raw RP samples (100 g) were homogenized with 500 mL phosphate buffer 4

(PBS, 10 mM, pH 7.0) and stirred at 4 °C for 12 h. The homogenate was filtered through four layers gauze and then centrifuged at 10,000 rpm, 4 °C for 15 min. The protein (pro) in the supernatant was preliminary purified by ammonium sulfate precipitation between 20-80% saturation, and then dialyzed against deionized water and lyophilized for further use. The RP residue after protein extract was resuspended in 1 L deionized water and extracted under continuous shaking at 90 °C for twice. The combined filtrates were evaporated in vacuum and then precipitated by 90 % ethanol. The precipitated polysaccharide (ps) was dialyzed against deionized water and then lyophilized for further use. 2.2.2. Preparation of glycation conjugates Solutions containing 1 % RP protein were mixed with 1 %, 3 % and 5 % polysaccharide in deionized water, respectively, and hydrated overnight. The mixture were adjusted to pH 7.0 and then freeze-dried. The powders were incubated under 60 °C and 79 % relative humidity for 72 h to produce glycation conjugates. 2.2.3. SDS-PAGE The raw RP protein and glycation conjugates were dissolved with loading buffer and subjected to SDS-PAGE. SDS-PAGE was performed with a 12.5 % separating gel and 5 % stacking gel as described (Laemmli and Favre, 1973). Proteins were then stained with 0.1 % Coomassie brilliant blue R-250 solution. 2.2.4. Glycation degree analysis

5

The glycation degrees of conjugates were determined by the reduction in free amino groups using o-phthaldialdehyde (OPA) assay (Feng et al., 2016). The resultant OPA reagent (2.7 mL) was mixed with 0.1 mL RP protein or glycation conjugates solutions and incubated at room temperature for 3 min. The absorbance at 340 nm was measured immediately. L-leucine (0-6.0 mM) was used as a standard amino-group-containing compound for calibration curve construction. 2.3. Self-assembly and characterization of nanoparticles 2.3.1. Fabrication of nanoparticles RP protein-polysaccharide conjugates were dispersed in PBS (10 mM, pH 7.0) to 0.5 mg pro/mL and adjusted to desired pH. Nanoparticles were fabricated under 100 °C water bath for 30 min. For the preparation of DOX-loaded nanoparticles, DOX (2 mg/mL in normal saline) was added dropwise to the conjugate nanoparticle solution at pH 7.4 to a ratio of 20 µg/mL DOX in 0.5 mg/ mL protein and magnetically stirred at room temperature in dark for 2 h. 2.3.2. Size distribution Particle size (mean Z-average diameter, d. nm) and polydispersity index (PDI) of the samples were analyzed by dynamic light scattering (DLS) on a Zetasizer Nano ZS instrument (Malvern Instruments Ltd, Worcestershire, UK). 2.3.3. Transmission electron microscopy (TEM) Morphology of the nanoparticles was observed using a Hitachi H-700 transmission electron microscope (Hitachi Ltd., Tokyo, Japan) as described in our 6

previous work (Weng, et al., 2019) 2.3.4. X-ray diffraction (XRD) The crystal states of DOX, DOX-free nanoparticle and DOX-loaded nanoparticle powders were analyzed using X’pert3 and Empyrean diffractometer (Panalytical, Almelo, Netherlands). Samples were scanned continuously over a 2θ range of 5-90 ° at a speed of 4 °/min. 2.4. In vitro DOX release The in vitro release of DOX from DOX-loaded nanoparticles were investigated in PBS at pH values of 7.4, 6.8 and 5.5 through dynamic dialysis. Briefly, dialysis bags containing 5 mL of the nanoparticle solutions were immersed in 50 mL release media and magnetically stirred under 100 rpm at 37 °C. At different time intervals, 1 mL of release media was collected for the DOX release analysis. And then fresh release media of equal volume was added. The DOX fluorescence intensities were measured on a microplate reader (SpectraMax iD3, Molecular Devices LLC., CA, USA) at an excitation wavelength of 480 nm and an emission wavelength of 595 nm, and the concentration was calculated by a free DOX generated standard curve. 2.5. Cellular uptake of DOX-loaded nanoparticles on HepG2 cells

2.5.1. Cellular uptake studies HepG2 cells were seeded in 6-well cell culture plate at a density of 2×104 cells/well in a 5 % CO2 humidified atmosphere at 37 °C for 24 h. The cells were

7

washed with PBS thrice and then fresh cultured medium containing free DOX or DOX loaded nanoparticles with different DOX concentration were added. After 0.5 h or 1 h incubation, the supernatants were discarded. The cells were washed with PBS thrice and then lysed with 1 % Triton x-100. The DOX concentrations in the cell lysates were determined via fluorescence intensity and the protein contents were measured by a BCA protein kit. Besides, for the investigation of the effect of P-gp on DOX cellular uptake, free DOX, DOX mixed with CysA and DOX-loaded nanoparticles were added to HepG2 cells, respectively. After incubation for 1 h, the cellular uptakes of DOX were measured as mentioned above. HepG2 cells were seeded in glass bottom dishes at a density of 2×104 cells/well and cultured for 24 h, then, the cells were treated with free DOX and DOX-loaded nanoparticles for 0.5 h or 1 h. After that, the culture medium were discarded and the cells were washed with PBS thrice. The cell nuclei and membrane were stained with Hoechst 33342 and DIO for 20 min, respectively. After washed thrice, the cells were observed by an inverted fluorescent microscope (Ts2-FL, Nikon instruments co. LTD, Shanghai, China). 2.5.2. Endocytosis pathway of DOX-loaded nanoparticles To study the endocytosis pathway of DOX-loaded nanoparticles, endocytosis inhibitors (chlorpromazine, colchicine, quercetin and indomethacin) were employed. HepG2 cells were preincubated with 20 µg/mL endocytosis inhibitors for 1 h. Subsequently, the cells were treated with DOX-loaded nanoparticles at DOX

8

concentration of 15 µg/mL in the presence of corresponding inhibitors for another 1 h. Then, the cellular uptake of DOX were analyzed via fluorescence intensity as mentioned in section 2.5.1.

2.6. Cytotoxicity analysis The cytotoxicity of samples was evaluated by MTT assay. Briefly, the cells were seeded in 96-well plate at a density of 5 ×103 cells/well and cultured for 24 h. Then, the samples at different concentrations were added to the cells. After incubated for 24 h, MTT reagent was added and the cells were incubated for further 4 h. The culture medium was then discarded and 150 µL of DMSO was added. The absorbance of each well was measured at 570 nm on a microplate reader and the cell survival rate was calculated as compared with the negative control.

2.7. Cell apoptosis detection by flow cytometry HepG2 cells were seeded in 6-well cell culture plate at a density of 5×105 cells/well. After incubated with CP3, DOX and CP3-DOX for 24 h, respectively, the cells were harvested and washed with cold PBS. The cells were stained with Annexin V-APC/7-AAD according to the introduction of the kit and then the cell apoptosis was detected by a BD Accuri C6 Plus flow cytometer (BD Life Sciences, San Jose, USA).

2.8. Statistical analysis All data are presented as means ± standard deviation (SD) of three independent experiments. Statistical evaluation was carried out with IBM SPSS 22.0 software by 9

one-way analysis of variance (ANOVA) with Duncan’s multiple range test. A confidence lecel of p < 0.05 was considered as statistical significance.

3. Results and discussion 3.1. Fabrication and characterization of RP protein-polysaccharide conjugates nanoparticles

3.1.1. Fabrication of RP protein-polysaccharide conjugates nanoparticles The RP protein-polysaccharide conjugates were characterized by SDS PAGE. As shown in Fig.1A, obvious protein bands corresponding to raw RP protein distributed below 116 kDa. After glycation with RP polysaccharide for 72 h, the smeared zones between 44.3 kDa and 200 kDa and obvious bands at about 200 kDa were observed, while the intensity of raw RP protein bands below 44.3 kDa became significantly weaker, indicating the formation of RP protein-polysaccharide conjugates with higher molecular weight by dry-heating Maillard reaction. Dextran-glycated β-lactoglobulin and the glycation of ovalbumin with dextran showed similar results (Feng et al., 2016; Yi et al., 2014). However, there was no obvious difference in the intensity of smeared zones of the conjugates prepared at different protein and polysaccharide ratios. The OPA assay showed that the glycation degree increased by 46.6 % after heating for 72 h at protein and polysaccharide weight ratio of 1:1. Consistent with the results of SDS PAGE, increasing the ratio of polysaccharides has no effect on the glycation degree of RP protein (Fig. 1A).

10

RP protein-polysaccharide conjugates nanoparticles were fabricated by coacervation via thermal treatment. Previous studies showed that protein nanoparticles with superior colloidal stability could easily self-assemble near the pI (Feng et al., 2015). Therefore, the impact of pH on the fabrication of conjugates nanoparticles was evaluated by heating the conjugates at 100 °C water bath for 30 min at the pH range of 5.5-7.0. The characteristics of the fabricated nanoparticles were analyzed by DLS (Fig. S1). Based on the results shown in Fig. S1, the optimal self-assembly conditions for the three conjugates were listed in Table 1. The conjugate prepared by RP protein and polysaccharide at weight ratio of 1:1 and 1:3 could be self-assembled into relatively monodisperse nanoparticles at pH 5.75 (CP1) and pH 5.5 (CP3), respectively. However, as the protein and polysaccharide ratio reached 1:5, the nanoparticles were polydisperse at the test pH range. Hydrophobic interaction is considered to be the major force involved in the heat-induced assembly of protein near the pI, since the electrostatic repulsion can be negligible at the pI of protein (Feng et al., 2015). The introduction of polysaccharide could provide steric repulsion to stabilize the protein nanoparticles, however, the existence of excessive hydrophilic polysaccharides might disturb the nanoparticle assembly, resulting in the formation of nanoparticles with large size and high PDI (CP5) (Feng et al., 2016; Li et al., 2008). Based on the size distribution shown in Fig. 1C, CP3 with nanoparticle size of 125.0 d.nm and a narrow distribution (PDI<0.3) was subsequently used for the study of drug encapsulation and delivery.

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3.1.2. Biocompatibility of CP3 The biocompatibility of drug-free self-assembly is a crucial indicator for the potential use as drug carrier material in living organisms. Moreover, as a kind of Maillard reaction product, the cytotoxicity of CP3 also need to be evaluated. Therefore, the biocompatibility of CP3 was investigated using human normal liver L-02 cells, human hepatoma HepG2 cells and colon cancer Caco-2 cells by MTT assay. As shown in Fig. 2, raw RP protein showed obvious cytotoxicity towards L-02 and Caco-2 cells, which might attribute to the large number of cytotoxic defense proteins such as lectin and trypsin inhibitors in the plant protein extract (Cai et al., 2018; Wang and Ng, 2001). After the drying heating and self-assembly process, CP3 showed no inhibition on the three cell lines even if the protein concentration reached 2.5 mg/mL, suggesting that the active sites of protein for cytotoxicity were changed or blocked during the conjugation and self-assembly of RP protein and polysaccharide. The results indicated that the biocompatibility of CP3 as drug nanocarrier is appropriate.

3.2. DOX encapsulation by CP3 Investigation on the nanoparticle size distribution of CP3-DOX confirmed that the characteristics of CP3 was not significantly affected after DOX loading. CP3-DOX was narrow distributed with nanoparticle size about 125.5 d.nm determined by DLS (Fig. 3A). TEM observation showed that CP3-DOX were in spherical shape (Fig. 3B). XRD was applied to study the crystal states of DOX in CP3 12

nanoparticles. As shown in Fig. 3C, the XRD pattern of CP3 did not show obvious diffraction peaks, indicating that it was in amorphous states. On the contrary, there were strong diffraction peaks of free DOX between 5-40 °, indicating that DOX had a highly crystal structure. However, the DOX diffraction peaks were almost completely disappeared in CP3-DOX, suggesting that DOX was well embedded into CP3 nanoparticles as amorphous states. 3.3. In vitro release behavior of CP3-DOX The in vitro DOX releases from CP3-DOX nanoparticles were monitored in PBS at different pH values simulating human physiological pH (pH 7.4), tumor microenvironment pH (pH 6.8) and intracellular endo/lysosomal acidic environment (pH 5.5). As shown in Fig. 4, the in vitro releases of DOX from CP3-DOX were pH sensitive. As the pH dropped from 7.4 to 5.5, the release of DOX was significantly accelerated. After a 12 h release experiment, the release rates of DOX from CP3-DOX was 63.6 % at pH 5.5, 30.0% at pH 6.8, and only 21.2% at pH 7.4. On account of the protonation and increased hydrophilicity of DOX under acidic conditions, the encapsulated DOX was more easily diffused into the release medium from CP3. Besides, since CP3 gradually approached its isoelectric point and the surface potential tended to zero as the pH decreased to 5.5, the electrostatic attraction of CP3 and DOX became weak, thereby triggering release of DOX from CP3. Recently, stimuli-sensitive nanocarriers attract much attention since they could respond to the stimuli, such as pH, temperature, metal ions, enzyme, light, etc., and controllably 13

release the drugs from nanocarriers at the target site (Ping et al., 2015; Uthaman et al., 2018). The results manifested that CP3-DOX could retain most of the DOX in natural physiological pH, such as blood circulation, while in acidic tumor pH environment and endosome, it exhibited better DOX release. The pH-sensitive drug release behavior of CP3-DOX would contribute to the tumor sites-specific DOX release and thereby, alleviate the side effects. 3.4. Cellular uptake of CP3-DOX nanoparticles on HepG2 cells Given that DOX is a kind of anticancer agents targeting in nuclear DNA, the efficient cell internalization and intracellular release are of crucial importance. The effect of CP3 on the cellular uptake of DOX was investigated in human hepatoma HepG2 cells. The fluorescence microscopy images are shown in Fig. 5. With the incubation time prolonged, the cells treated with DOX and CP3-DOX both exhibited enhanced intracellular DOX fluorescence. After internalized by HepG2 cells, free DOX can diffused through nuclear membrane rapidly and located in the nuclei. Compared with DOX, CP3-DOX with nanoparticle size of 125.5 d. nm could not directly pass through the nuclear membrane, therefore, the red fluorescence in the cytoplasm was stronger than free DOX treated cells. It is generally considered that nanoparticles enter the cell through endocytosis and then are internalized by endosome. The existence of obvious DOX fluorescence in CP3-DOX treated cells nuclei at 1 h suggesting that DOX could be fast released from CP3-DOX nanoparticles by response to the acidic endosomal pH and binding to nuclei DNA, and 14

this procedure was rapidly. The cellular uptake of DOX was quantitative through fluorescence intensity analysis. As shown in Fig. 6A, the internalization of free DOX and CP3-DOX was time- and dose-dependently. In addition, at the same incubation time and DOX concentration, DOX in the form of CP3-DOX had higher cellular uptake efficiency, indicating that CP3 could promote the internalization of DOX by HepG2 cells. Tumor cells are likely to develop drug resistance after long-term exposure to a type of drug due to the activation of a series of transport proteins in the drug efflux system, which can actively pump the drug out of the cells. P-gp efflux pump is the most well studied drug resistance-related proteins resulting in the low absorption rate of DOX (Varma et al., 2003; Wang et al., 2015). Therefore, P-gp inhibitor CysA was used to explore the inhibition of CP3-DOX on P-gp activity. As shown in Fig. 6B, the co-incubation of DOX and CysA enhanced the HepG2 cellular uptake of DOX remarkably, and the uptake amount was almost the same as CP3-DOX, which was 1.56-folds of free DOX, suggesting that CP3 plays a similar role to P-gp inhibitor in the cellular internalization of DOX. The results indicated that DOX encapsulated in CP3 could improve cellular uptake of DOX through eliminating the inhibitory effect of P-gp efflux pump. Meanwhile, the results of MTT assay of free DOX and CP3-DOX on HepG2 showed that the IC50 values of DOX and CP3-DOX were approximately 1.40 and 0.25 µg/mL DOX, respectively (Fig. 6C). The obvious lower IC50 value indicating that CP3-DOX exhibited stronger antiproliferative effects on

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HepG2 cells, which was considered to be related to the higher cellular uptake of CP3-DOX. Nanoparticles are generally considered to be internalized into cells through endocytosis, and the endocytosis pathways are classified into macropinocytosis, clathrin-dependent

endocytosis,

caveolin-dependent

endocytosis

and

clathrin-/caveolin-independent pathways (Wang et al., 2015). To investigate the pathway by which CP3 entered HepG2 cells, chlorpromazine (clathrin-dependent endocytosis inhibitor), indomethacin (caveolin-dependent endocytosis inhibitor), colchicine (macropinocytosis inhibitor) and quercetin (clathrin-/caveolin-independent endocytosis inhibitor) were applied to HepG2. Results showed that the cellular uptake of CP3-DOX was significantly inhibited in the presence of chlorpromazine, while the effects of the other three inhibitors were not obvious, indicating that the internalization of CP3-DOX in HepG2 cells might be a process mainly involving clathrin-dependent endocytosis. 3.5 Cytotoxicity effect of CP3-DOX nanoparticles on HepG2 cells

The antiproliferative effects of DOX and CP3-DOX on HepG2 cells were further investigated by Annexin V-APC/7-AAD staining and measured through flow cytometry. The results of cell apoptosis after different treatment were shown in Fig. 7. CP3 alone almost didn’t induce HepG2 cells apoptosis or necrosis at 250 µg/mL, further confirming the superior biocompatibility. The treatment of DOX and CP3-DOX at 0.25 µg/mL DOX remarkably increased the HepG2 cells apoptosis to 16

82.7 % and 90.5 % of control respectively, which was in accordance with previous reports showing that DOX intercalated to DNA and induced tumor cells apoptosis (Vijay et al., 2018). Moreover, consistent with the results of MTT assay (Fig. 6C), the CP3-DOX treated group exhibited more notably decreased normal cells and increased early-stage apoptosis cells than free DOX. The results further suggested that the encapsulation of DOX by CP3 could potentiate DOX-induced cytotoxicity.

4. Conclusion In summary, biocompatible nanoparticles (CP3) based on Radix pseudostellariae protein-polysaccharide conjugate were developed for the encapsulation and delivery of DOX in this study. CP3-DOX nanoparticles exhibited pH sensitivity and accelerated DOX release under acidic pH simulating tumor microenvironment and endosomes. DOX encapsulated in CP3 showed higher cellular uptake efficiency and stronger cytotoxicity effects on HepG2 cells. Moreover, CP3-DOX nanoparticles could be internalized into HepG2 cells via clathrin-dependent endocytosis. The results suggest that CP3 could potentially be a good candidate as nanocarrier for improvement on the cellular uptake of anticancer agents.

Acknowledgement This work was supported by National Key R&D Program of China (No. 2016YFD0400202), National Natural Science Foundation of China (No. 31901639).

Declaration of competing interest 17

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.

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Figure captions Fig.1 Preparation of RP protein-polysaccharide conjugates and the self-assembled particles. (A) SDS-PAGE of RP protein-polysaccharide conjugates; (B) Degree of protein glycation detected by OPA method; (C) Size distribution of self-assembled conjugate particles determined by DLS.

Fig. 2 Biocompatibility of CP3 nanoparticles evaluated by MTT assay. Data represented as mean ± SD (n=3).

Fig. 3 Encapsulation of DOX by CP3 nanoparticles. (A) Particle size distribution of CP3-DOX nanoparticles determined by DLS; (B) The morphology of CP3-DOX nanoparticles observed by the TEM; (C) XRD patterns of DOX and CP3-DOX nanoparticles.

Fig. 4 In vitro DOX release profiles of CP3-DOX nanoparticles in PBS at pH 7.4, 6.8 and 5.5, respectively. Data represented as mean ± SD (n=3).

Fig. 5 Fluorescence microscopy images of HepG2 cells treated with DOX and CP3-DOX nanoparticles for 0.5 and 1 h, respectively. The concentration of DOX was 15 µg/mL. The nuclei were stained by Hoechst 33342 (blue) and the cell membrane was stained by DIO (green). DOX exhibited red 24

fluorescence.

Fig. 6 Cellular uptake and cytotoxicity effect of DOX and CP3-DOX nanoparticles on HepG2 cells. (A) Effects of different concentration of DOX on HepG2 cells uptake of DOX and CP3-DOX nanoparticles. (B) Cellular uptake of DOX, DOX with CysA and CP3-DOX nanoparticles at equal DOX concentration of 15 µg/mL. The concentration of CysA was 20 µg/mL. (C) Cytotoxicity effect of DOX and CP3-DOX nanoparticles on HepG2 cells detected by MTT assay. (D) Cellular uptake of CP3-DOX nanoparticles in the presence of 20 µg/mL endocytosis inhibitors. Data represented as mean ± SD (n=3). * represented as significant difference at p<0.05.

Fig. 7 Flow cytometry analysis of apoptosis in HepG2 cells treated with DOX and CP3-DOX nanoparticles. The concentration of CP3 was 250 µg/mL. The concentration of DOX and CP3-DOX were 0.25 µg/mL DOX. Cells were stained with Annexin V-APC/7-AAD. In each panel, Q1 quadrant represented Annexin V-APC–/7-AAD+ cells (necrosis), Q2 quadrant represented Annexin V-APC+/7-AAD+ cells (late-stage apoptosis), Q3 quadrant represented Annexin V-APC–/7-AAD– cells (normal) and Q4 quadrant represented Annexin V-APC+/7-AAD– cells (early-stage apoptosis).

25

Table 1 Fabrication conditions and properties of RP protein-polysaccharide conjugates particles Maillard reaction conditions Particle

pro:ps

Reaction time

(w/w)

(h)

CP1

1:1

CP3 CP5

Particle fabrication conditions

Size (d. nm)

PDI

Buffer

pH

Heating

72

PBS, 10 mM

5.75

100 °C, 30 min

69.0±0.7

1:3

72

PBS, 10 mM

5.5

100 °C, 30 min

125.0±1.8 0.289±0.038

1:5

72

PBS, 10 mM

6.0

100 °C, 30 min

123.9±2.4 0.815±0.077

Data represented as mean ± SD (n=3).

26

0.411±0.058

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Fig. 6

Fig. 7

Highlights Biocompatible nanoparticles (CP3) were self-assembled based on RP protein-polysaccharide conjugate. DOX-loaded CP3 nanoparticles exhibited pH-sensitive DOX release behavior. CP3-DOX significantly improved DOX uptake by HepG2 cells. CP3-DOX could serve as P-gp inhibitor and be internalized into HepG2 cells via clathrin-dependent endocytosis. The cytotoxicity effect of DOX on HepG2 cells was elevated by CP3 encapsulation.

Declaration of interests ☒ 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. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: