Combination therapeutics of doxorubicin with Fe₃O₄@chitosan@phytic acid nanoparticles for multi-responsive drug delivery

Abstract

Functional multi-responsive drug delivery vehicles have drawn considerable attention owing to their merits of low toxicity and improved antitumor efficiency. In this article, novel Fe₃O₄@chitosan@phytic acid (FCP) magnetic pH-sensitive nanoscaled microspheres with narrow size distribution and high drug loading efficiency for doxorubicin (DOX) of up to 94.2% have been successfully prepared in a facile one-step hydrothermal method utilized in controlled drug releasing and combination therapy. The constructed nanocarriers exhibited high saturation of 24.83 emu g⁻¹, excellent pH-response in weak acidic regions like tumor tissues while drug releasing and obvious anticancer efficiency, which was verified by MTT test in the MG-63 cancer cell line in vitro, leading to notable inhibition of tumor growth without negative consequences. Therefore, the as-prepared FCP nanoparticles show promising potential as drug carriers to improve antitumor therapeutic efficacy.

---

Introduction

Current cancer chemotherapies face long-standing challenging obstacles. Direct administration of conventional therapeutic regimes to patients is inclined to be hampered by poor physiological stability, low cell-membrane permeability, nonspecific targeting and insufficient cellular drug uptake, resulting in severe adverse effects on normal tissues and limitation of therapeutic effectiveness.^1–3 Therefore, nanoscaled drug carriers have emerged as promising candidates utilized in the biomedical field.^4–6 To achieve more efficient targeting delivery and controllable drug release, functional multi-responsive nanomaterials as drug delivery vehicles have been explored, among which magnetic nanocomposites have drawn more attention owing to their high loading capacity and good biocompatibility.^7,8 There are some investigations in the literature on magnetic nanocarriers utilized for cancer diagnosis and treatment. For instance, Li's group has designed magnetic carboxymethylcellolose (Fe₃O₄-CMC) nanocarriers for pH-responsive drug delivery.⁹ He et al. have developed multifunctional Fe₃O₄@carbon@zeolitic imidazolate framework-8 hybrid nanoparticles as pH-sensitive drug delivery vehicles for tumor therapy in vivo.¹⁰ Hassan et al. have published regarding biodegradable polymeric vesicles containing magnetic nanoscaled quantum dots and anticancer drugs for controllable drug release and ultrasound imaging.¹¹ However, there are few reports on combination therapy of nanocarriers and anticancer agents for cancer treatment while keeping multi-responsive drug release to our best knowledge.

Phytic acid (PA), a plant ingredient constituting 1–5% of most cereals, legumes, nuts and spores, serves as a natural antioxidant in the preservation of seeds and addition to foods inhibiting lipid peroxidation and concomitant oxidative spoilage.^12,13 PA also exists in erythrocytes of animals to promote the release of oxygen, improve the function of erythrocytes and prolong their survival time.^13,14 In addition, PA, which hydrolyzes into inositol and phospholipid in the human body, of which the former may slow aging down and the latter is an important component of cells, is beneficial to human nutrition.¹⁴ By the same mechanism, Graf et al. pointed out that PA might lower the incidence of colonic cancer and protect against other inflammatory bowel diseases and that dietary phytic acid (phytate) could weaken the incidence and growth rate of tumors.^15,16 Subsequently, Fedarko et al. verified that phytic acid showed inhibition in hepatoma cells.¹⁷ Shamsuddin et al. demonstrated that phytic acid was rapidly absorbed and metabolized by murine and human malignant cells to inhibit fiber metastatic tumor cells in vitro.¹⁸ Inspired by those works, we propose that PA can also be an active constituent combined with anti-cancer drugs participating in cancer therapy.

Here, biodegradable magnetic Fe₃O₄@CS nanomaterials as the precursors were prepared by binding chitosan (CS) onto the surface of magnetic Fe₃O₄ nanoparticles through a facile one-step hydrothermal method, after which the precursors were encapsulated by PA via electrostatic interaction, developing novel Fe₃O₄@CS@PA (FCP) nanocarriers with a narrow size distribution. Wherein chitosan (CS), a well-known natural polymer with abundant carboxyl groups, was chosen as the surfactant to modify magnetic Fe₃O₄ nanospheres, making the precursors easily encapsulated by PA.^19–22 The FCP nanoparticals as drug delivery vehicles exhibited outstanding pH-controlled and magnetism triggered properties, meanwhile, they may be combined with doxorubicin (DOX) easily loaded to the as-prepared carriers via electrostatic interaction with high drug loading efficiency for tumor therapy to improve the efficiency that anti-cancer drugs work only. The corresponding cell cytotoxicity of the composite was detected by MTT test with MG-63 cancer cell line in vitro.

Experimental

Materials

CS was purchased from Shandong Xiya Reagent Co., Ltd., China. Ferric chloride hexahydrate (FeCl₃·6H₂O), ammonium acetate (NH₄Ac), ethylene glycol and phytic acid (PA) in this study were obtained from Sinopharm Chemical Reagent Co., Ltd, China. Doxorubicin (DOX) was obtained from Shanghai Yuanye Biotech Co., Ltd., China. The MG-63 cancer cell line and the sigma used for the experiment were obtained from Hyclone Laboratories, Inc. (HyClone, Logan, UT, USA) and Heilongjiang Tumor Treatment and Prevention Institute, respectively. All chemicals were directly used with no further purification.

Synthesis of Fe₃O₄@CS@PA nanoparticles

The Fe₃O₄@CS nanospheres were synthesized via a simple modified solvothermal approach. For a typical preparation procedure, 0.675 g of FeCl₃·6H₂O (2.5 mmol) was dissolved in 35 mL of ethylene glycol by magnetic stirring, then 1.925 g of NH₄Ac (25 mmol) and 0.763 g (0.5 mmol) of CS were added to the above solution under stirring. After a homogeneous suspension had formed, continuous stirring was carried out for 30 min. Subsequently, the obtained solution was transferred into a Teflon-lined stainless steel autoclave (50 mL capacity), sealed and heated at 200 °C for 12 h, after which the autoclave was cooled down to room temperature. The brown precipitate was collected by magnetic separation, washed with absolute ethanol and deionized water several times, and finally re-dispersed in 5 mmol L⁻¹ phytic acid solution. The mixture was stirred in a water bath (60 °C) for 30 min. Again the product was separated with a magnet and washed with absolute ethanol and deionized water several times before being dried in a vacuum at 60 °C for 24 h. The modified process mentioned above could be repeated to increase the thickness of the PA shell.

Characterization

The morphology and structure of the as-synthesized products were observed by a field emission scanning electron microscope (SEM, Hitachi S4800) and transmission electron microscope 50 (TEM, JEOL, JEM-2010). The element composition was analysed by an energy-dispersive X-ray spectroscopy (EDX) analyser. The size distribution of the FCP nanoparticles was measured by Nano Particle Size and Zeta potential analyser (Malvern, Zetasizer Nano ZS90). The phase purity of the products was characterized by powder X-ray diffraction (XRD) taken in a Rigaku D/max-IIIB diffractometer (Tokyo) using nickel-filtered Cu Kα radiation at 5540 kV, 150 mA. The Fourier transform infrared (FTIR) spectra of the nanocarriers were obtained via an AVATAR 360 FTIR spectrophotometer in the 400–4000 cm⁻¹ region using the KBr-disk method to investigate the surface functional groups of the products. Thermogravimetric (TG) analysis was performed on a NetzschSTA409 thermo-analyser (Shimadzu Co.) from room temperature to 800 °C with a heating rate of 10 °C min⁻¹ under a nitrogen atmosphere. The DOX concentration was examined by UV-vis (Shimadzu UV 1700) spectrophotometer.

Doxorubicin loading and release test

DOX (5 mg), an anti-cancer drug selected as a model drug, was loaded onto the FCP nanocarriers (40 mg), which were dispersed into 5 mL of phosphate-buffered saline (PBS) solution (pH = 7.4) containing DOX (1.0 mg mL⁻¹). To facilitate the loading of DOX molecules, the mixture was shaken slowly for 24 h utilizing an oscillator. The obtained solution was separated by centrifugation at 6000 rpm for 5 min with the fresh PBS solution (pH = 7.4) with a constant volume (5 mL) replenished in the centrifugal tube. The process was repeated several times until the supernatant solution stayed colorless, collecting the supernatant solutions for UV-vis analysis and the FCP@DOX for further use. The drug loading capacity and efficiency (DLC & DLE%) were calculated using the formulae given below:

DLE (%) = DLC × 100% (2)

The drug releasing experiment was carried out at 37 °C. A certain amount FCP@DOX was dispersed into 5 mL PBS solution of different pH values (5.0 and 7.4) in centrifugal tubes shaken at 150 rpm with an oscillator under dark conditions. At a definite time, the tubes were centrifuged at 6000 rpm for 5 minutes and the obtained supernatant was stored at 4 °C in the dark to determine the release efficiency of DOX-loaded nanocarriers using a UV-vis spectrophotometer at 480 nm.

Cell cytotoxicity of Fe₃O₄@CS@PA nanocarriers

Typically, the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell assay using the MG63 cell line was carried out to examine the cell viabilities of the nanocarriers, DOX-loaded nanocarriers and free DOX.^29,30 The cells were seeded into a 96-well plate, providing a density of 8000 cells per well, which was then incubated in DMEM medium 75 (0.1 mL) for 24 h to enable attachment of the cells to wells at 37 °C in 5% CO₂ atmosphere. The equivalent doses of bare Fe₃O₄@CS, FCP nanocarriers, DOX-loaded FCP nanoparticles and free DOX were respectively sterilized by ultraviolet irradiation for 2 h and pipetted into each well with different concentrations of 6.25, 12.5, 25, 50 and 100 μg mL⁻¹, meanwhile, the culture wells without nanocarriers were served as the control groups. Then the whole plate was incubated for another 24 h under the same conditions. Twenty microliters of 0.5% MTT reagent in PBS buffer mixed with 100 μL of clear cell medium was transferred to each well. Subsequently, the plate was covered with aluminium foil and protected against light at 37 °C after another 4 h incubation. Then, 100 μL of dimethyl sulfoxide (DMSO) solution was added to each well to completely dissolve the formazan viable cells facilitated via shaking for 5 min at 150 rpm. The absorbance was read at 480 nm using a Molecular Devices Spectramax M5 plate reader.^31–33 The cell viability was calculated with the following formula:

The above MTT test should be measured at 570 nm again to illustrate the cell viabilities further.³⁸

Results and discussion

Synthesis and characterization of Fe₃O₄@CS@PA nanoparticles

As illustrated in Scheme 1, Fe₃O₄@CS nanoparticles were prepared in a facile one-step hydrothermal method.³⁹ In this procedure, CS as the stabilizing agent modified the chemical functional groups and charge on the surface of Fe₃O₄ nanoparticles, making it easy to induce PA growing on the surfaces of the Fe₃O₄@CS nanospheres to form the FCP nanocarriers. Subsequently, the drug molecules were efficiently loaded onto the surface of carriers by electrostatic interaction and hydrogen bonding, after which the drug-loaded carriers could release drugs triggered by physical and chemical signals, including an external magnetic field and an acidic pH, resulting in efficient release and combined action between carriers and drugs in tumor cells.

Scheme 1 Schematic representation of synthetic procedure of the FCP nanoparticles, loading and multi-responsive releasing of DOX and the FCP nanocarriers combining with DOX acting in MG-63 cells.

The information about the morphologies of FCP nanocarriers is illustrated by the SEM and TEM images below. The as-prepared nanoparticles present a spherical shape (Fig. 1a and c–f). Among them, Fig. 1f shows that there is a layer of pellicles on the surfaces of the precursors compared to the Fe₃O₄@CS nanoparticles (Fig. 1e), which indicates that PA was successfully attached. The FCP nanospheres with uniform distribution at the mean size of 243 nm and good dispersion are illustrated in Fig. 1a and b, the polydispersity index of which is 0.158. Thus, the FCP nanoparticles present excellent spherical shape with rough surface, narrow size distribution and good dispersion corresponding to the requirements of ideal drug delivery vehicles.³⁴

Fig. 1 SEM images of the Fe₃O₄@CS nanoparticles (c) and FCP nanocarriers (a and d), TEM images of the Fe₃O₄@CS nanoparticles (e) and FCP nanocarriers (f), and particle size distribution of FCP nanocarriers (b).

To demonstrate the loading of PA further, the as-synthesized carriers were also analysed by energy-dispersive X-ray (EDX) spectroscopy. The information regarding the co-existence of C, N, O, Fe and P elements is shown in Fig. 2, and the inset in Fig. 2 provides the corresponding elemental percentage in FCP nanoparticles.

Fig. 2 EDX spectroscopy of the FCP nanoparticles.

Fig. 3 indicates that the new absorption band at 1652 cm⁻¹ was ascribed to the overlap of the peaks of carbonyl group of the CS from Fe₃O₄@CS and the HPO₄²⁻ of PA from the FCP nanoparticles compared with that of pure PA (Fig. 3c).^23,24 The peak at 3468 cm⁻¹ belongs to the absorption band of OH⁻ of PA and those at 1009 cm⁻¹ and 1177 cm⁻¹ belong to absorption band of PO₄³⁻ marked in Fig. 3b.²⁵ Meanwhile, the absorption band at around 598 cm⁻¹ of FCP nanoparticles is the same as the peak at 598 cm⁻¹ of the Fe₃O₄@CS nanospheres OWING to the stretching of Fe–O bonds. The above analyses confirm that nanocarriers are successfully modified by PA without changing the chemical structure of the magnetic substrate. It is also demonstrated that the surfaces of the nanomaterials are abundant in phosphoric acid groups, which can improve the drug-loading efficiency.

Fig. 3 FTIR spectra of (a) Fe₃O₄@CS nanoparticles, (b) FCP nanocarriers and (c) pure PA.

The XRD analysis (Fig. 4) was performed to display a clear crystalline pattern to identify the existence of Fe₃O₄ (JCPDS no. 65-3107)²⁶ and the influences CS and PA have in the crystal structure of the products. The diffraction peaks intensity of Fe₃O₄@CS nanospheres and FCP nanocarriers is weaker than those of bare Fe₃O₄ owing to the introduction of CS and PA, however, the peak values of which still correspond to those of the naked Fe₃O₄. The results are consistent with the FTIR spectra analysis above, suggesting that the precursors were successfully modified by PA without changing the chemical structure of the magnetic substrate further.

Fig. 4 XRD patterns of (a) pure Fe₃O₄ and (b) Fe₃O₄@CS nanoparticles and (c) FCP nanocarriers.

The room temperature magnetization curves of the as-prepared samples are shown in Fig. 5. The saturation magnetization values of the Fe₃O₄@CS nanoparticles and the FCP nanocarriers are 53.30 and 24.83 emu g⁻¹, respectively, providing further evidence of the successful modification of the matrix with PA. Additionally, there is no obvious remanence or coercivity observed, indicating that the superparamagnetic character is attributed to the magnetic core consisting of primary nanocrystals without any modification,²⁷ which is consistent with the characterization by TEM and XRD above. The separation and re-dispersion of the products can be easily operated because of the excellent magnetic property by applying an external magnet. Therefore, the carriers with the superparamagnetic property and high magnetization can be triggered by external magnetic field, bring about controllable drug release to reach a higher local concentration.³⁵

Fig. 5 Magnetic hysteresis curves of (a) Fe₃O₄@CS nanoparticles and (b) FCP nanocarriers.

In addition, TG analysis was carried out under nitrogen flow to investigate the thermal stability of the as-prepared nanomaterials by comparison. As shown in Fig. 6, the initial weight loss of Fe₃O₄@CS nanoparticles (4.6%) in the range of 0–110 °C corresponds to the evaporation of the different types of absorbed water molecules. Then the obvious weight loss (22.1%) at around 240 °C is mainly owing to other introduced organics during the hydrothermal process²⁸ and the decomposition of CS that was adhered to the surface of the Fe₃O₄ nanoparticles. Besides, there are two large weight losses of FCP nanocarriers in the range of 100–164 °C and 230–350 °C. One loss is ascribed to the evaporation of the different types of absorbed water molecules and the decomposition of PA anchored onto the Fe₃O₄@CS nanoparticles. The other is attributed to the same reason as the weight loss of the Fe₃O₄@CS nanoparticles at around 240 °C. The results confirm that the thermal stability of the products can absolutely meet the requirement of biomedical applications and further identify the fact that PA was successfully anchored onto the Fe₃O₄@CS nanoparticles. In addition, the weight loss of FCP nanoparticles is more than that of Fe₃O₄@CS nanoparticles, which indicates that there are more organics in FCP nanocarriers compared with Fe₃O₄@CS nanoparticles because of the PA molecule.

Fig. 6 TG curves of Fe₃O₄@CS nanoparticles and FCP nanocarriers.

Drug loading and pH-responsive release of FCP nanocarriers

The DOX-loaded FCP nanocarriers, bare FCP nanocarriers and pure DOX were characterized by UV-vis spectroscopy. Fig. 7A shows that the absorption band of the DOX-loaded FCP nanocarriers corresponds to that of pure DOX at 480 nm, which clarifies the successful loading of drugs. The high loading efficiency and capacity of DOX were 94.2% and 117.8 mg g⁻¹, which could be attributed to the electrostatic interaction between positively charged DOX and negatively charged phosphoric acid groups, and the coordination bonding of PA-DOX.

Fig. 7 (A) Absorbance spectra of bare Fe₃O₄@CS nanocarriers (a), DOX-loaded FCP nanocarriers (b) and free DOX (c). (B) Release of DOX from DOX-loaded FCP nanoparticles in buffer solutions at pH 7.4 and 5.0.

To investigate the drug release behavior of the FCP nanocarriers, the DOX-loaded FCP carriers were respectively immersed in PBS at pH values of 7.4 and 5.0, chosen as the physiological environment and endosomal/lysosomal environment inside tumor issues to study the release rate of DOX from the DOX-loaded FCP nanocarriers at 37 °C in vitro. As Fig. 7B shows, DOX was released in a very slow fashion and the cumulative release of DOX was only about 32.7% after 48 h in neutral PBS (pH = 7.4). On the other hand, 83.9% of DOX was released at a faster drug release rate after 48 h at pH = 5.0. Moreover, the rate of DOX releasing in acid PBS (pH = 5.0) is higher than that in neutral PBS, the reason for which is that the intermolecular force between PA and DOX would become weaker in acid conditions.³⁶ Consequently, the pH-driven drug release FCP system is beneficial for utilizing as a drug delivery vehicle to target cancerous tissues owing to the lower extracellular pH of tumors than that of normal tissues, contributing to accelerating drug release and increasing local drug concentration.³⁷

Cytotoxicity assay

The standard MTT assay was performed on MG-63 cells read at 480 nm and 570 nm to characterize the cytotoxicity of bare Fe₃O₄@CS, FCP nanocarriers, DOX-loaded FCP nanoparticles and free DOX shown in Fig. 8 and 9, respectively. On the one hand, Fig. 8A shows that the cell viability of bare Fe₃O₄@CS slightly fluctuates above and below 100%, which indicates that the bare Fe₃O₄@CS nanoparticles are not very toxic. Compared with bare Fe₃O₄@CS nanospheres, on the other hand, Fig. 8A reveals that free DOX and DOX-loaded FCP nanoparticles exhibit a similar increasing trend of cytotoxicity with increasing concentrations of samples within the MG-63 cells. Less than 65% cell viability is observed at the concentration of 100 μg mL⁻¹ of bare FCP nanocarriers after incubation for 52 h (total incubation time mentioned in materials and methods section, 24 h, 24 h and 4 h included), indicating that the as-prepared materials can kill tumor cells to some extent. As shown in Fig. 8B, a contrast test was conducted to demonstrate the effect of DOX-loaded FCP nanoparticles on tumor cells by decreasing the concentrations of free DOX and DOX-loaded FCP nanoparticles, which suggests that DOX-loaded FCP nanoparticles present higher cytotoxicity to tumor cells compared with free DOX when the ratio of free DOX to DOX-loaded FCP nanoparticles is around 1 : 4. The results for DOX-loaded FCP nanoparticles are better than those for DOX with the same content of FCP nanocarriers loading (117.8 mg g⁻¹) working on tumor cells only, mainly because the PA of the nanocarriers can inhibit the growth of tumor cells and combine with DOX taking effect on the tumor cells together. Additionally, Fig. 9 shows a similar trend to Fig. 8, which demonstrates that PA can be an active constituent combined with anti-cancer drugs, further participating in cancer therapy. These data confirm that the as-prepared FCP nanocarriers can inhibit the growth of cancer cells to combine with antitumor drugs to treat cancer and act as promising drug carriers applied in the biomedical field to improve the antitumor therapeutic efficacy.

Fig. 8 Cell viabilities of (A) pure DOX, FCP–DOX, bare FCP and Fe₃O₄@CS nanocarriers with the same concentration and (B) pure DOX and FCP–DOX nanoparticles with different concentrations to MG-63 cells examined by MTT assay read at 480 nm.

Fig. 9 Cell viabilities of (A) pure DOX, FCP–DOX, bare FCP and Fe₃O₄@CS nanocarriers with the same concentration and (B) pure DOX and FCP–DOX nanoparticles with different concentrations to MG-63 cells examined by MTT assay read at 570 nm.

Conclusion

In summary, an ideal FCP nanocarrier based on Fe₃O₄@CS nanospheres has been successfully developed. The as-prepared carriers with a mean size of 243 nm exhibited high magnetic saturation of 53.30 emu g⁻¹ and remarkable pH response in weak acidic areas like tumor tissues while drug releasing. In addition, the obvious inhibition of tumor cells by FCP nanocarriers was verified by MTT test in the MG-63 cancer cell line in vitro. It is worth noting the drug loading efficiency and capacity for loading DOX of the constructed nanocarriers are up to 94.2% and 117.8 mg g⁻¹, respectively. As a consequence, the FCP nanoparticles can be utilized not only in controlled drug release to improve local drug concentrations but also in combination therapy by introducing anticancer agents that work together to enhance antitumor therapeutic efficacy and inhibit the regeneration of tumor cells. Overall, FCP nanomaterials show promising potential as drug carriers for biomedical applications.

Acknowledgements

The above work was supported by the National Natural Science Foundation of China (NSFC 51402065), the Fundamental Research Funds of the Central University (HEUCFZ), the Natural Science Foundation of Heilongjiang Province (B201404), the International Science & Technology Cooperation Program of China (2015DFR50050) and the Magor Project of Science and Technology of Heilongjiang Province (GA14A101).

Notes and references

1. K. Cho, X. Wang, S. Nie, Z. G. Chen and D. M. Shin, Clin. Cancer Res., 2008, 14, 1310–1316.
2. O. Tredan, C. M. Galmarini, K. Patel and I. F. Tannock, J. Natl. Cancer Inst., 2007, 99, 1441–1454.
3. A. J. Primeau, A. Rendon, D. Hedley, L. Lilge and I. F. Tannock, Clin. Cancer Res., 2005, 11, 8782–8788.
4. X. Ma, H. Tao, K. Yang, L. Feng, L. Cheng, X. Shi, Y. Li, L. Guo and Z. Liu, Nano Res., 2012, 5, 199–212.
5. J. Liu, S. Z. Qiao, Q. H. Hu and G. Q. Lu, Small, 2011, 7, 425–443.
6. S. Xuan, F. Wang, J. M. Lai, K. W. Sham, Y. X. Wang, S. F. Lee, J. C. Yu, C. H. Cheng and K. C. Leung, ACS Appl. Mater. Interfaces, 2011, 3, 237–244.
7. F. J. Hernandez, L. I. Hernandez, A. Pinto, T. Schafer and V. C. Ozalp, Chem. Commun., 2013, 49, 1285–1287.
8. F. J. Hernandez, L. I. Hernandez, M. Kavruk, Y. M. Arica, G. Bayramoglu, B. A. Borsa, H. A. Oktem, T. Schafer and V. C. Ozalp, Chem. Commun., 2014, 50, 9489–9492.
9. X. Guo, L. Xue, W. Lv, Q. Liu, R. Li, Z. Li and J. Wang, New J. Chem., 2015, 39, 7340–7347.
10. M. He, J. Zhou, J. Chen, F. Zheng, D. Wang, R. Shi, Z. Guo, H. Wang and Q. Chen, J. Mater. Chem. B, 2015, 3, 9033–9042.
11. F. Ye, A. Barrefelt, H. Asem, M. Abedi-Valugerdi, I. El-Serafi, M. Saghafian, K. Abu-Salah, S. Alrokayan, M. Muhammed and M. Hassan, Biomaterials, 2014, 35, 3885–3894.
12. C. H. Fox and M. Eberl, Compl. Ther. Med., 2002, 10, 229–234.
13. J. R. Zhou and J. W. Erdman Jr, Crit. Rev. Food Sci. Nutr., 1995, 35, 495–508.
14. V. Kumar, A. K. Sinha, H. P. S. Makkar and K. Becker, Food Chem., 2010, 120, 945–959.
15. E. Graf and J. W. Eaton, Free Radical Biol. Med., 1990, 8, 61–69.
16. E. Graf and J. W. Eaton, Cancer, 1985, 56, 717–718.
17. N. S. Fedarko, M. Ishihara and H. E. Conrad, J. Cell. Physiol., 1989, 139, 287–294.
18. A. M. Shamsuddin, Int. J. Food Sci. Technol., 2002, 37, 769–782.
19. D. H. K. Reddy and S.-M. Lee, Adv. Colloid Interface Sci., 2013, 201–202, 68–93.
20. A. Lopez-Cruz, C. Barrera, V. L. Calero-DdelC and C. Rinaldi, J. Mater. Chem., 2009, 19, 6870–6876.
21. M. Thi Thu Trang, H. Phuong Thu, P. Hong Nam, L. Thi Thu Huong, P. Hoai Linh, P. Thi Bich Hoa, T. Dai Lam and N. Xuan Phuc, Adv. Nat. Sci.: Nanosci. Nanotechnol., 2012, 3, 015006.
22. L. D. Tran, H. V. Tran, H. D. Vu, T. T. Mai, D. G. Pham, T. P. Ha, P. X. Nguyen, B. H. Nguyen and J. K. Park, Journal of Chitin and Chitosan Science, 2001, 16, 7–14.
23. P. B. Shete, R. M. Patil, N. D. Thorat, A. Prasad, R. S. Ningthoujam, S. J. Ghosh and S. H. Pawar, Appl. Surf. Sci., 2014, 288, 149–157.
24. X. Cui, Y. Li, Q. Li, G. Jin, M. Ding and F. Wang, Mater. Chem. Phys., 2008, 111, 503–507.
25. A. Tarafdar, A. B. Panda, N. C. Pradhan and P. Pramanik, Microporous Mesoporous Mater., 2006, 95, 360–365.
26. Y. Zeng, R. Hao, B. Xing, Y. Hou and Z. Xu, Chem. Commun., 2010, 46, 3920–3922.
27. M. Mahmoudi, S. Sant, B. Wang, S. Laurent and T. Sen, Adv. Drug Delivery Rev., 2011, 63, 24–46.
28. S. Zhang, Y. Zhou, W. Nie and L. Song, Cellulose, 2012, 19, 2081–2091.
29. L. Chen, K. Jiang, H. Jiang and P. Wei, Exp. Ther. Med., 2014, 8, 527–532.
30. J. Huang, J. Ni, K. Liu, Y. Yu, M. Xie, R. Kang, P. Vernon, L. Cao and D. Tang, Cancer Res., 2012, 72, 230–238.
31. D. Yu, S. Xiao, C. Tong, L. Chen and X. Liu, Chin. Sci. Bull., 2007, 52, 2913–2918.
32. K. S. Kim, S.-J. Park, M.-Y. Lee, K.-G. Lim and S. K. Hahn, Macromol. Res., 2012, 20, 277–282.
33. L. Wu, M. Wu, Y. Zeng, D. Zhang, A. Zheng, X. Liu and J. Liu, Nanotechnology, 2015, 26, 025102.
34. Y. Geng, P. Dalhaimer, S. Cai, R. Tsai, M. Tewari, T. Minko and D. E. Discher, Nat. Nanotechnol., 2007, 2, 249–255.
35. C. Sun, J. S. Lee and M. Zhang, Adv. Drug Delivery Rev., 2008, 60, 1252–1265.
36. R. K. Tekade, T. Dutta, V. Gajbhiye and N. K. Jain, J. Microencapsulation, 2009, 26, 287–296.
37. D. Schmaljohann, Adv. Drug Delivery Rev., 2006, 58, 1655–1670.
38. H. Wu, X. Li, W. Liu, T. Chen, Y. Li, W. Zheng, C. W. Y. Man, M. K. Wong and K. H. Wong, J. Mater. Chem., 2012, 22, 9602–9610.
39. G. Zhao, X. Peng, H. Li, J. Wang, L. Zhou, T. Zhao, Z. Huang and H. Jiang, Chem. Commun., 2015, 51(35), 7489–7492.

---