Chitosan-Based Nanogel Enhances Chemotherapeutic Efficacy of 10-Hydroxycamptothecin against Human Breast Cancer Cells

Chitosan (CS), the second most abundant polysaccharide in nature, has been widely developed as a nanoscopic drug delivery vehicle due to its intriguing characteristics. In this work, a positively charged CS-based nanogel was designed and synthesized to inhibit the proliferation of breast cancer cell lines. The model drug of 10-hydroxycamptothecin (HCPT) was entrapped into the core via a facile diffusion to form CS/HCPT. The characteristics of CS/HCPT were evaluated by assessing particle size, drug loading content, and drug loading efficiency. Furthermore, cell internalization, cytotoxicity, and apoptosis of CS/HCPT were also investigated in vitro. The present investigation indicated that the positively charged CS-based nanogel could be potentially used as a promising drug delivery system.


Introduction
Breast cancer is well known to be the most common malignant disease in women worldwide and is the second leading cause of death among US women [1].The main subtypes of breast cancer are identified based on the expression of hormone receptors (HR), namely, estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) [2].HR-positive breast cancer is one of the most common subtypes, which accounts for approximately 70-75% of all cases [3].The prognosis of HR-positive breast cancer is better than that of HR-negative breast cancer [4].Breast cancer is becoming a considerable public health problem because of its high morbidity and mortality.There are a variety of available treatment methods for breast cancer, including surgery, radiotherapy, chemotherapy, and hormone therapy [5].It is proven that surgery is an efficient treatment for primary breast cancer.However, cancer cells may spread to distant sites in the body before the primary lesion is found and resected [6].Chemotherapy, as a supplement to surgery, has become standard of treatment for locally advanced or poor-prognosis early-stage disease.Furthermore, neoadjuvant chemotherapy has been increasingly administered to reduce the size of primary tumor [7].
Although chemotherapy plays an important role in clinical application, it is still hindered by several unfavourable factors, such as rapid systemic elimination, low water solubility, and severe systemic toxicity [8].
To overcome these disadvantages, nanotechnology has been developed over the past few decades and offers promising perspectives for improving the therapeutic potency of chemotherapy [9].Chitosan (CS), a biopolymer derived from chitin, is known as the second most abundant polysaccharide in nature [10].CS has been widely developed as a nanoscopic drug delivery vehicle due to its intriguing characteristics, such as biocompatibility, biodegradability, antibacterial, nontoxicity, and low cost [11][12][13].In addition to these advantages, the existence of primary amino (NH 2 ) and hydroxyl (OH) groups in the CS chain facilitates its surface engineering chemical reactions [14].
On the basis of these considerations, a positively charged CS-based nanogel was designed and synthesized as a drug delivery system to inhibit the proliferation of breast cancer cell lines.The model drug of 10-hydroxycamptothecin (HCPT) is a derivative of camptothecin (CPT) [15].HCPT is an inhibitor of topoisomerase I, which can effectively inhibit DNA replication and RNA transcription in breast cancer cells [16,17].HCPT was entrapped into the CS via a facile diffusion to form CS/HCPT.The characteristics of CS/HCPT were evaluated by assessing the particle size, drug loading content, and drug loading efficiency.Furthermore, cell internalization, cytotoxicity, and apoptosis of CS/HCPT were also investigated in vitro.Ltd. (Shanghai, China).Dimethylformamide (DMF) was sourced from Sigma-Aldrich (Shanghai, China) and dried over calcium hydride (CaH 2 ) at room temperature before vacuum distillation.The purified deionized water was prepared by the Milli-Q plus system (Millipore Co., Billerica, MA, USA).

Preparation of the HCPT-Loaded
Nanogel.The CS/HCPT was prepared through a facile diffusion and dialysis method as described previously [18].Briefly, CS (50.0 mg) was dissolved in 20.0 mL of acetic acid solution (pH 3.0), and HCPT (25.0 mg) was dissolved in 20.0 mL of DMF by vortex and sonication.Then, 20.0 mL of HCPT solution was dropped slowly into the CS solution while stirring.The mixed solution was then stirred at 30 rpm for 12 h at room temperature and then dialyzed against deionized water for 24 h.The dialysis medium was refreshed five times to completely remove the free HCPT and DMF.The whole procedure was performed in darkness.Finally, the solution was filtered and followed by lyophilization to obtain CS/HCPT.

Determination of Drug Loading Content and Drug
Loading Efficiency.The drug loading content (DLC) and drug loading efficiency (DLE) were calculated as described in our previous work with slight modification [19].In short, the freeze-dried CS/HCPT was accurately weighed and dissolved in acetic acid solution (pH 3.0).In CS/HCPT, the content of HCPT was then assayed by ultraviolet-visible (UV-vis) spectrophotometry at 365 nm.The standard curve method was used.The DLC and DLE of CS/HCPT were calculated according to equations ( 1) and ( 2 2.5.Cell Uptake and Intracellular Drug Release.The cell uptake and intracellular HCPT release behavior of CS/HCPT by 4T1 cells were detected by confocal laser scanning microscopy (CLSM).Simply, 4T1 cells were suspended at a density of 0 8 × 10 5 cells mL -1 and taken 2.0 mL into a 6-well plate and incubated at 37 °C in 5% (V/V) carbon dioxide (CO 2 ) atmosphere.After 24 h of incubation, the culture medium was replaced with free HCPT or CS/HCPT at a certain HCPT concentration of 1.25 μg mL -1 .The untreated cells incubated with phosphate-buffered saline (PBS) were used as a control.The cells were incubated for 2 h or 6 h and then rinsed with PBS for three times.The obtained cells were fixed with 4% paraformaldehyde for 15 min at room temperature.The cells were washed repeatedly with PBS.The cell uptake and intracellular drug release behavior of CS/HCPT were observed by CLSM.
2.6.In Vitro Cytotoxicity Assay.In vitro cytotoxicity of CS/HCPT, including free HCPT, was evaluated in 4T1 cells by a standard MTT assay.Briefly, 1 0 × 10 4 viable cells dispersed in 200.0 μL DMEM medium were seeded in 96-well plates and incubated overnight at 37 °C.After that, the prepared free HCPT and CS/HCPT solution were added in cells and incubated for 48 h or 72 h.The concentrations of HCPT were ranged from 0 to 20.0 μg mL -1 .At predetermined time intervals, 20.0 μL of MTT solution was added to each well and the 96-well plates were incubated at 37 °C for approximately 4 h.The obtained MTT products were measured with a Bio-Rad 680 microplate reader (Bio-Rad Laboratories, Hercules, CA, USA) at the absorbance of 490 nm.All experimental samples were performed for three times.
The cell viability (%) was calculated as follows: In equation ( 3), A sample and A control represented the absorbance of sample well and control well, respectively.

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International Journal of Polymer Science 2.7.Cell Apoptosis Analysis.The apoptosis percentage of 4T1 cells induced by CS/HCPT was detected by flow cytometry (FCM) analysis.4T1 cells were incubated in 6-well plates at a density of 3 × 10 5 cells per well for 24 h.The incubation medium was then replaced with 2.0 mL of complete DMEM containing different HCPT formulations with a HCPT concentration of 0.1 μg mL -1 .The untreated cells were used as a control.The cells were incubated for additional 24 h at 37 °C and then harvested by trypsinization, washed, and resuspended in 0.5 mL 1x Annexin V binding buffer.Before analyzing, the samples were stained with 5.0 μL of Annexin V-FITC and propidium iodide (PI) for 15 min in the dark.Finally, the cells were analyzed using FCM.

Results and Discussion
3.1.Preparations and Characterizations of CS/HCPT.The hydrophobic antitumor drug of HCPT was encapsulated into CS through facile diffusion (Scheme 1).Electrostatic interaction is the main reason for drug encapsulation [20].The preparation method was simple and straightforward.The DLC and DLE of CS/HCPT were at high levels of 31.7 and 92.8 wt.%, respectively (Table 1).The resulting CS/HCPT exhibited an average diameter of 55 8 ± 1 4 nm, which was detected by DLS (Figure 1).The R h of CS/HCPT was less than 200 nm, showing the maximum enhanced permeability and retention (EPR) effect, which was important for tumor targeting within the biomedical applications of nanomedicine [21,22].Furthermore, CS/HCPT has a positive surface charge with zeta potential of 10 6 ± 2 2 mV (Table 1).The zeta potential is important for evaluating the stability and dispersion of nanoparticles [23].In addition, positively charged particles have a great efficiency in cell membrane infiltration and internalization [24].Obviously, CS/HCPT is an excellent platform for drug delivery.

Cell Uptake and Intracellular Drug
Release.The cell uptake and intracellular HCPT release behavior of CS/HCPT by 4T1 cells were monitored with CLSM.As depicted in Figure 2(a), after 2 h of treatment, the HCPT fluorescence signal was observed in the cells treated with free HCPT or CS/HCPT.The HCPT fluorescence was a little stronger in the cells incubated with free HCPT than in the cells with CS/HCPT.However, after 6 h of incubation, the CS/HCPTtreated cells exhibited much higher HCPT fluorescence intensity than those of the free HCPT treatment group (Figure 2(b)).The results indicated that CS/HCPT could be preferentially internalized via endocytosis pathway, which had a temperate efficiency at the outset and then revealed the enhanced fluorescence signal after effective drug release [25].On the contrary, free HCPT was transported into cells through passive diffusion [26,27].It was also reported that endocytosis of nanoparticles is often more efficient than passive diffusion of drug molecules [28].Similar results have been reported in our previous work [29].In that work, a positively charged polypeptide nanogel was synthesized mainly to enhance the mucoadhesiveness of HCPT for intravesical chemotherapy of bladder cancer.In this work, the positively charged nanogel was based on CS, which was the second most abundant polysaccharide in nature with good biocompatibility and biodegradability.The results of Figure 2 demonstrated that CS could increase the uptake and endocytosis of HCPT by 4T1 cells.3 International Journal of Polymer Science 3.3.In Vitro Cytotoxicity Study of CS/HCPT.4T1 is a representative cell line in breast cancer, and its growth and metastasis in BALB/c mice are very similar to that of human breast cancer.The kinetics of 4T1-induced tumors is similar in both postoperative and nonsurgical conditions, which can be used as a postoperative and nonsurgical model [30].To investigate the enhanced therapeutic efficacy of CS/HCPT against the 4T1 cell line, a standard MTT assay was applied.As shown in Figure 3(a), after 48 h of exposure to either CS/HCPT or free HCPT, the metabolic activity of 4T1 cells decreased in a concentration-dependent manner.The CS/HCPT displayed an apparently higher cytotoxicity than free HCPT at any given equivalent concentration, indicating that the drug delivery platform can indeed enhance anticancer efficiency of HCPT.The enhanced cytotoxicity of CS/HCPT was probably due to the nonspecifically internalized into cells via endocytosis, phagocytosis, or pinocytosis after accumulating on the surface of the cells, while free HCPT was transported by passive diffusion [31,32].The similar results were obtained when the incubation time was expanded to 72 h (Figure 3(b)).In addition, the half maximal inhibitory concentration (IC 50 ) value of CS/HCPT was 8.7 μg mL −1 and 4.4 μg mL −1 at 48 h and 72 h, respectively.On the contrary, the IC 50 value of free HCPT was 10.3 μg mL −1 and 7.5 μg mL −1 at 48 h and 72 h, respectively.The results indicated that the proliferation rate was time dependent.

Cell Apoptosis
Analysis.The apoptotic activities of 4T1 cells induced by CS/HCPT were assessed by FCM analyses.The cells were incubated with free HCPT or CS/HCPT at a certain HCPT concentration of 0.1 μg mL -1 for 24 h and then double labeled for viability and apoptosis.As depicted in Figure 4, CS/HCPT remarkably decreased the percentage of normal cells and significantly increased the percentage of

Conclusions
In summary, a cationic CS-based nanogel was successfully synthesized to inhibit the proliferation of breast cancer cells.The hydrophobic antitumor drug of HCPT was entrapped into the core via facile diffusion.The resulting CS/HCPT exhibited an average diameter of 55 8 ± 1 4 nm.The positive surface charge of CS/HCPT promoted intracellular drug internalization via endocytosis pathway.The cytotoxicity assay suggested excellent biocompatibility of CS/HCPT and also proved better cytotoxicity in comparison to free HCPT.The present investigation indicated that the positively charged CS-based nanogel could be potentially used as a promising drug delivery system.

Scheme 1 :
Scheme 1: Schematic illustration for the preparation of CS/HCPT and enhanced cell uptake by 4T1 cells.

Figure 2 :Figure 3 :
Figure 2: Representative CLSM microimages of 4T1 cells incubated with free HCPT and CS/HCPT for 2 (a) and 6 h (b).For each panel, the microimages from left to right showed HCPT fluorescence in cells (blue), differential interference contrast (DIC) image, and the overlay of two images.The scale bar represented 20 μm.

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International Journal of Polymer Science necrotic/late apoptotic cells.The results were attributed to the positive charge of CS/HCPT, which increased the uptake of CS/HCPT by 4T1 cells and further induced enhanced cell apoptosis.

Figure 4 :
Figure 4: Apoptotic cell populations were determined by FCM analyses with Annexin V-FITC and PI staining after coincubating 4T1 cells with PBS, free HCPT, or CS/HCPT for 24 h.The lower left (Q3), lower right (Q4), upper right (Q1), and upper left (Q2) quadrants in each panel indicated the populations of normal, early and late apoptotic, and apoptotic necrotic cells, respectively.
), respectively.Particle Size and Zeta Potential Measurements.The hydrodynamic radius (R h ) of CS/HCPT was measured by dynamic light scattering (DLS) measurements in a WyattQELS instrument with a vertically polarized He−Ne laser (DAWN EOS, Wyatt Technology Co., Santa Barbara, CA, USA).The scattering angle was fixed at 90 °.The samples were prepared in aqueous solution at a concentration of 100.0 μg mL −1 .Before measurements, the solution was filtered through a 0.45 μm Millipore filter.The zeta potential of CS/HCPT was determined by a Zeta Potential/BI-90Plus Particle Size Analyzer (Brookhaven, USA).The sample was adjusted to a concentration of 100.0 μg mL −1 in aqueous solution as recommended.Both the particle size and zeta potential measurements were performed in triplicate, and the results were presented as means values ± standard deviation (SD).