Silver nanoparticles (AgNPs) have unlocked numerous novel disciplines in nanobiotechnological protocols due to their larger surface area-to-volume ratios, which are attributed to the marked reactivity of nanosilver, and due to their extremely small size, which enables AgNPs to enter cells, interact with organelles, and yield distinct biological effects. AgNPs are capable of bypassing immune cells, staying in the system for longer periods and with a higher distribution, reaching target tissues at higher concentrations, avoiding diffusion to adjacent tissues, releasing therapeutic agents or drugs for specific stimuli to achieve a longer duration at a specific rate, and yielding desired effects. The phytofabrication of AgNPs is a cost-effective, one-step, environmentally friendly, and easy method that harnesses sustainable resources and naturally available components of plant extracts (PEs). In addition, it processes various catalytic activities for the degradation of various organic pollutants. For the phytofabrication of AgNPs, plant products can be used in a multifunctional manner as a reducing agent, a stabilizing agent, and a functionalizing agent. In addition, they can be used to curtail the requirements for any additional stabilizing agents and to help the reaction stages subside. Azadirachta indica, a very common and prominent medicinal plant grown throughout the Indian subcontinent, possesses free radical scavenging and other pharmaceutical properties via the regulation of proinflammatory enzymes, such as COX and TOX. It also demonstrates anticancer activities through cell-signaling pathways, modulating tumor-suppressing genes such as p53 and pTEN, transcriptional factors, angiogenesis, and apoptosis via bcl2 and bax. In addition, it possesses antibacterial activities. Phytofabricated AgNPs have been applied in the areas of drug delivery, bioimaging, biosensing, cancer treatment, cosmetics, and cell biology. Such pharmaceutical and biological activities of phytofabricated AgNPs are attributed to more than 300 phytochemicals found in Azadirachta indica, and are especially abundant in flavonoids, polyphenols, diterpenoids, triterpenoids, limonoids, tannins, coumarin, nimbolide, azadirachtin, azadirone, azadiradione, and gedunin. Parts of Azadirachta indica, including the leaves in various forms, have been used for wound healing or as a repellent. This study was aimed at examining previously biosynthesized (from Azadirachta indica) AgNPs for anticancer, wound-healing, and antimicrobial actions (through MTT reduction assay, scratch assay, and microbroth dilution methods, respectively). Additionally, apoptosis in cancer cells and the antibiofilm capabilities of AgNPs were examined through caspase-3 expression, dentine block, and crystal violet methods. We found that biogenic silver nanoparticles are capable of inducing cytotoxicity in HCT-116 colon carcinoma cells (IC50 of 744.23 µg/mL, R2: 0.94), but are ineffective against MCF-7 breast cancer cells (IC50 >> 1000 µg/mL, R2: 0.86). AgNPs (IC50 value) induced a significant increase in caspase-3 expression (a 1.5-fold increase) in HCT-116, as compared with control cells. FITC-MFI was 1936 in HCT-116-treated cells, as compared to being 4551 in cisplatin and 1297 in untreated cells. AgNPs (6.26 µg/mL and 62.5 µg/mL) induced the cellular migration (40.2% and 33.23%, respectively) of V79 Chinese hamster lung fibroblasts; however, the improvement in wound healing was not significant as it was for the controls. AgNPs (MIC of 10 µg/mL) were very effective against MDR Enterococcus faecalis in the planktonic mode as well as in the biofilm mode. AgNPs (10 µg/mL and 320 µg/mL) reduced the E. faecalis biofilm by >50% and >80%, respectively. Natural products, such as Syzygium aromaticum (clove) oil (MIC of 312.5 µg/mL) and eugenol (MIC of 625 µg/mL), showed significant antimicrobial effects against A. indica. Our findings indicate that A. indica-functionalized AgNPs are effective against cancer cells and can induce apoptosis in HCT-116 colon carcinoma cells; however, the anticancer properties of AgNPs can also be upgraded through active targeting (functionalized with enzymes, antibiotics, photosensitizers, or antibodies) in immunotherapy, photothermal therapy, and photodynamic therapy. Our findings also suggest that functionalized AgNPs could be pivotal in the development of a novel, non-cytotoxic, biocompatible therapeutic agent for infected chronic wounds, ulcers, and skin lesions involving MDR pathogens via their incorporation into scaffolds, composites, patches, microgels, or formulations for microneedles, dressings, bandages, gels, or other drug-delivery systems.

Nanoparticles (NPs) have garnered a lot of interest in sectors like medicine, cosmetics, food, and pharmaceuticals for antibacterial catalytic properties, reduced toxicity, and easy production. Biological synthesis of silver nanoparticle (AgNPs) is considered as green, eco-friendly, and cost-effective approach; therefore, Azadirachta indica extracts were utilized for a dual role of fabrication and functionalization of AgNPs. Optical and physical characterizations were achieved for confirming the biosynthesized AgNPs. SEM images detected quasi-spherical AgNPs of 44.04 to 66.50 nm. Some of potent phytochemicals like flavonoids and proteins from Azadirachta indica formed a strong coating or capping on the AgNPs without affecting their secondary structure by interacting with Ag+ and NPs for the formation of AgNPs. AgNPs exhibited strong antibacterial activity (MIC 10 μg/ml) against multidrug-resistant bacteria Enterococcus faecalis; at different concentrations, no IC50 values were recorded for AgNPs as well as Azadirachta indica signifying low cytotoxicity in the exposed concentration range. The DNA degradation activity of AgNPs through the TUNEL assay revealed no significant increase in the overall FITC mean fluorescence intensity as well as a DNA fragmentation index with 5.45% DNA damage (10 μg/ml AgNPs). Drug uptake of AgNPs was also investigated through a permeability assay via Caco-2 cell lines at test concentrations where apparent permeability was detected as moderate.

Nanobiotechnology, as a novel and more specialized branch of science, has provided a number of nanostructures such as nanoparticles, by utilizing the methods, techniques, and protocols of other branches of science. Due to the unique features and physiobiological characteristics, these nanostructures or nanocarriers have provided vast methods and therapeutic techniques, against microbial infections and cancers and for tissue regeneration, tissue engineering, and immunotherapies, and for gene therapies, through drug delivery systems. However, reduced carrying capacity, abrupt and non-targeted delivery, and solubility of therapeutic agents, can affect the therapeutic applications of these biotechnological products. In this article, we explored and discussed the prominent nanobiotechnological methods and products such as nanocarriers, highlighted the features and challenges associated with these products, and attempted to conclude if available nanostructures offer any scope of improvement or enhancement. We aimed to identify and emphasize the nanobiotechnological methods and products, with greater prospect and capacity for therapeutic improvements and enhancements. We found that novel nanocarriers and nanostructures, such as nanocomposites, micelles, hydrogels, microneedles, and artificial cells, can address the associated challenges and inherited drawbacks, with help of conjugations, sustained and stimuli-responsive release, ligand binding, and targeted delivery. We recommend that nanobiotechnology, despite having few challenges and drawbacks, offers immense opportunities that can be harnessed in delivering quality therapeutics with precision and prediction. We also recommend that, by exploring the branched domains more rigorously, bottlenecks and obstacles can also be addressed and resolved in return. Graphical Abstract

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