Regular ArticleCurcumin-functionalized silk biomaterials for anti-aging utility
Graphical abstract
Introduction
Aging is a universal physiological process featured by progressive impairment in functional capacity of an organism [1]. At present, there are several assumptions concerning aging, with the most popular based on the free radical theory initially proposed by Harman [2], [3]. The excessive generation of reactive oxygen species (ROS) could cause significant senescence, apoptotic or necrotic cell death. Aging cells lose their ability to replicate, yet remain metabolically active. The cells are enlarged and express aging-related markers, including alkaline β-galactosidase, telomerase, 70 kilodalton heat shock proteins (hsp70), cyclin-dependent kinase inhibitor 2A (p16), and tumor protein p53 (p53) [4], [5].
Antioxidants can delay or prevent the oxidation of cellular substances that can be otherwise oxidized. Herbs, spices, extracts of phenolic compounds such as gallic acid, carnosic acid catechin, eugenol [6], green tea extract, polyphenol constituents (e.g., epigallocatechin gallate (EGCG)) [7] and fruit extracts (e.g., ascorbic acid (Vitamin C), α-tochopherol (Vitamin E) and β-carotene) [8] all have antioxidant activity.
Curcumin, also called curcuminoids, is a natural antioxidant that is isolated from turmeric (Curcuma longa) [9]. Curcumin has attracted attention as a food ingredient and also in drugs and nutraceuticals. Curcumin up-regulates the expression of cell proliferation-related genes, thus improving cell viability and reversing telomere shortening by increasing telomerase activity. Curcumin also inhibited the expression of cancer-related genes, such as P53 and P21 [10], reduced cell damage caused by oxidative stress, increased the expression of HSP70 [11], inhibited protein oxidation and lipid peroxidation during cell mitosis [12], induced apoptosis of cancer cells while had almost no damage to normal cells [13], and effectively alleviated oxidative damage and inflammatory reactions induced by chronic aging [14]. However, the instability of curcumin in neutral and alkaline conditions, where the compound undergoes hydrolytic degradation to feruloyl methane, ferulic acid and vanillin, hampers its utility [15]. Curcumin lost more than 90% of its anti-oxidative activity within 30 min in 0.1 M phosphate buffer and serum-free medium, while it was more stable in cell culture medium containing 10% fetal calf serum and in human blood; less than 20% of curcumin decomposed within 1 h, and approximately 50% of the curcumin remained after incubation for 8 h [16]. Therefore, curcumin in solution did not protect cells from ROS for an extended period of time. Modes to prevent curcumin from physical and chemical damage have become important issues when curcumin is to be utilized for many applications. Incorporation of curcumin in solid or semi-solid biomaterial forms is a promising approach; including encapsulation-based systems such as micro/nanoparticles [17], films [18], and hydrogels [19].
Silk is a high molecular weight structural protein derived from the Bombyx mori silkworm and has been widely used as a building block to fabricate biomaterials for tissue engineering and drug delivery. Silk materials, due to their hydrophobic nature and crystallization, are inherently more resilient against changes in temperature, moisture and pH than most other natural or synthetic polymers [20]. The entrapment and release of bioactive molecules from silk has been widely studied [21], [22], [23], [24], [25]. Curcumin has been loaded in different silk material formats, such as nanofibers [26], hydrogels [27], sponges [28], and thin films [29]. When curcumin was incorporated into methanol-treated (crystallized) silk films, and samples were stored at 37 °C in PBS at pH 7.4, more than 80% of the oxygen scavenging activity of curcumin remained after 14 days [29]. Furthermore, curcumin-incorporated silk films promoted adipogenesis from human mesenchymal stem cells (hMSCs), demonstrating retention of bioactivity upon processing into these films [30]. Since the level of curcumin released from the silk films was very low due to the strong binding of curcumin to hydrophobic silk beta-sheet domains, pro-adipogenesis was likely due surface exposed curcumin rather than the free curcumin in solution accessible to the stem cells.
In the present study, the focus was on the anti-aging effect of silk-stabilized curcumin. Two silk material forms, i.e., film and nanoparticles, were studied and compared with free curcumin, using aging-related markers, including HSP70, P16 and P53 gene expression and β-galactosidase activity [31]. Inclusion of silk-curcumin nanoparticles helped to elucidate the mechanism of anti-aging of silk-associated curcumin and also provided data to support future applications of silk-stabilized curcumin, such as oral-delivered silk/curcumin nanoparticles. Silk-curcumin nanoparticles have been previously reported in the literature [32], [33], however, the present studies focused more on the biological responses of different types of silk associated curcumin and the mechanisms underlying the responses.
Section snippets
Materials
Partially degummed silk fibers were purchased from Xiehe Silk Corporation Ltd. (Hangzhou, China). Lithium bromide (LiBr) was purchased from Aladdin (Shanghai, China). Dialysis tubing (3500 MWCO) was purchased from Thermo Fisher (Shanghai, China). Curcumin (Cat#C7727, >80% pure) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich (Shanghai, China). Ethanol, polyethylene glycol 1000, 400 and other reagents were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai,
Silk nanoparticles
Curcumin loaded nanoparticles (silk/cur NPs) had a size range of 150–700 nm, smaller than that of plain silk (no curcumin) nanoparticles (700–1200 nm) (Fig. 1A). This was consistent with the SEM observations, which showed a relatively homogeneous size distribution for the silk-curcumin materials (Fig. 1B). When observed under larger magnification, silk/cur NPs showed non-spherical shapes with some aggregates (Fig. 1B inset). The secondary structures of silk in silk/cur NP were analyzed by FTIR.
Discussion
Curcumin has been incorporated into silk films and its long-term stability and release profiles have been studied previously [29]. For example, in our prior studies, the adipogenic differentiation of human mesenchymal stem cells (hMSCs) cultured on curcumin-incorporated silk films was reported [30]. Compared to the curcumin that was added directly to the cell culture medium, the silk film-associated curcumin significantly promoted adipogenic differentiation of hMSCs, suggesting the curcumin on
Conclusions
Curcumin has been successfully incorporated into silk films and silk nanoparticles without significantly affecting the structure and morphology of the materials. The strong association of curcumin with silk, likely binding to the hydrophobic beta-sheet domains on silk, retarded its release while preserving free radical scavenging activity. Long-time exposures of functional curcumin on silk surfaces helped maintain the viability of stem cells that were cultured on silk/cur films, as well as silk
Conflict of interest
The Authors declare that there is no conflict of interest.
Acknowledgements
This work was supported by Natural Science Foundation of China grant (project no. 51273138, GZ1094 and 81301844), Start-up Fund of Soochow University (project no. 14317432), Natural Science Foundation of Suzhou City Jiangsu Province, China (Grants No SYN201403), and The Natural Science Foundation of Jiangsu Province, China (Grants No BK20150371).
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The first two authors contributed equally to this paper.