5.1 Conclusion
Nanotechnology has emerged as a substantial engineering field in recent years, and the nanoscale silver-related study is one of the most promising topics that draw profound attention by scientists due to its antimicrobial properties. Microbial infection still remains a disturbing issue in health care and medical applications. As we know, many pathogens have evolved as drug-resistant microbial strains, and thus infection issue is pretty serious in actual clinical environment, in spite that various antibiotics have been developed.
Therefore, it is urgent to develop novel antibiotics that possess superior biocompatibility but do not have drug-resistance property. Among diverse antimicrobial agents, silver is the promising candidate because of its intrinsic properties of high thermal stability, versatility, low toxicity to mammalian cells, and long-term activity. However, the further applications of AgNPs have been limited owing to their potential toxicity for human body.
Fortunately, the emergence of nanotechnology may offer a novel strategy to produce various sizes of AgNPs with modifications or incorporations, which give a greater opportunity to fight against microbes but simultaneously have lower toxicity for human.
The synthesis of AgNPs or silver-based antimicrobial nano-composites can be well established with miscellaneous methods. The chemical synthesis commonly employs AgNO3 as a starting material to yield AgNPs by chemical reduction with some natural reductants such as chitosan, dextran, sodium citrate, ascorbate, and other reducing biomolecules like polypeptides, flavones, and alkaloids. Chitosan, one kind of polysaccharides derived from chitin, is a positively charged biopolymer which exhibits various biological properties such as inherent antimicrobial activity, chelating activity, biocompatibility, and biodegradability. Chitosan possesses outstanding potential for
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applications in several fields of medicine, pharmaceutics, and agriculture. In the present study, we have established two synthetic systems to produce a novel combinative form of AgNPs and chitosan as composite spheres, Ag@chitosan, by using the pump-driven syringe method and the microfluidic chip method.
In the pump-driven syringe method, a novel approach for one-step synthesis of AgNPs–embedded chitosan particles is proposed. In this approach, we can simultaneously obtain and stabilize AgNPs in chitosan polymer matrix in situ, and the products are called silver nanoparticles–chitosan composite particles (Ag@chitosan). The diameters of the synthesized Ag@chitosan spheres ranges from 1.7 to 2.5 mm, and the embedded AgNPs are measured to be 15±3.3 nm. These products were applied to evaluate the antifungal effect, and it was interesting that they exhibited selective inhibition for the growth of Cordyceps militaris (Cm) but not for Antrodia cinnamomea (Ac). It is widely known that both chitosan and Ag@chitosan can inhibit many kinds of microorganisms, and our current finding suggests that this product may improve the cultivation of Ac by avoiding the contamination of other microbes.
In the microfluidic chip method, we have invented a microfluidic system to fabricate Ag@chitosan particles on a micrometer scale. The diameters of the synthesized Ag@chitosan particles can be controlled by adjusting the concentrations of chitosan and NaOH, and the flow rates of continuous and dispersed phases. To test the biocompatibility of Ag@chitosan, we used the MTT assay for NIH-3T3 and MCF-7 cell lines to evaluate their toxicity. The results showed that our products were highly safe and without significant toxicity even with a concentration of Ag@chitosan up to 1000 μg/mL. The antibacterial ability of the Ag@chitosan spheres was evaluated also, and it appeared that E. coli were inhibited in a dose-dependent manner with Ag@chitosan concentrations.
In order to satisfy diverse requirements, two synthetic platforms have been developed and established one after another, and either of them can be successfully utilized to
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fabricate homogeneous and stable Ag@chitosan spheres with a millimeter or micrometer scale, respectively. The diameters of the Ag@chitosan spheres synthesized by the syringe method range from 1.7 to 2.5 mm, and those by the microfluidic method range from 262 to 558 μm in our experiments. The characteristics of the synthesized Ag@chitosan spheres were evaluated by scanning electron microscopy, UV-Vis spectroscopy, Fourier transform infrared spectroscopy, and X-ray diffraction. With all those tools, we confirmed that these microspheres contained AgNPs unequivocally.
In summary, there are several remarkable highlights in this research. First, we have successfully fabricated the Ag@chitosan spheres that are found to be uniform and to have antimicrobial ability. In addition, the sizes of the Ag@chitosan spheres fabricated in the present study are smaller than those in previous literature. Second, we have invented two novel methods available for syntheses of the Ag@chitosan spheres, including the pump-driven syringe method and the microfluidic chip method. Third, in the two methods, only “one-step synthesis” is needed to fabricate the Ag@chitosan spheres, indicating that the two approaches are faster and more effective than other methods in literature. Fourth, one important finding is that the Ag@chitosan particles have the antifungal potentiality.
This phenomenon is scarcely mentioned in literature before, suggesting that our finding may provide a new direction of the development for antifungal applications. Furthermore, with the unveiling of antifungal ability of both chitosan and Ag@chitosan, it may offer a novel option apart from conventional antibiotics, and also a great opportunity to develop next-generation antibiotics.
5.2 Future Work
In the present research, we have fabricated uniform Ag@chitosan spheres successfully by both the pump-driven syringe method and the microfluidic chip method. Their stabilities, antibacterial effects and antifungal effects are further evaluated. The most
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attractive finding is the antifungal effect of the Ag@chitosan products that is relatively rarely reported in literatures. Also, it is found that the synthesized Ag@chitosan spheres can inhibit the growth of Cordyceps militaris (Cm) but not Antrodia cinnamomea (Ac). Ac is an important cash crop with highly economic value in Taiwan, and our products might be an option to become a beneficial additive for cultivating Ac by inhibiting other fungi and bacteria. How to ameliorate the product to make it feasible for Ac cultivation is one of our future works.
Excepting of Ac, whether the Ag@chitosan particles possess the ability of inhibiting other fungi strains remains unclear. However, more investigations related to the antifungal effects of the Ag@chitosan spheres should be done before they can really be applicable for fungal suppression in the future.
It is also an interesting issue about whether the Ag@chitosan spheres can inhibit virus growth. Therefore, further experiments for evaluating their antivirus potentiality will be another promising research work.
The current research only offers preliminary results associated with the Ag@chitosan spheres. To develop and fabricate a new and real antimicrobial reagent via such spheres requires further investigations.
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