Impact of Ligand Concentration on the Properties of Nucleic-Acid-Encapsulated MOFs and Inflammation Modulation in Prostate Cancer Cells.

The zeolitic imidazolate framework (ZIF) is one of the most explored metal-organic-framework-based systems for nucleic acid delivery to cancer cells. Current nucleic acid delivery tools exhibit several drawbacks, such as high manufacturing costs, endosomal entrapment, toxicity, and immunogenicity. However, the biomimetic mineralization of Zn-based ZIFs offers a low-cost and facile encapsulation of nucleic acids at room temperature in aqueous conditions. The efficiency of nucleic acid delivery and its subsequent impact on inflammation in cells are influenced by the physicochemical properties of the material. The imidazole content determines the formation and crystallinity of ZIF, and an optimal ratio ensures the formation of well-defined and highly crystalline structures. In this study, a series of siRNA-encapsulated ZIFs (siRNA@ZIF) were systematically prepared by varying ligand-to-metal (L/M) molar ratios. Our study demonstrates that variations in ligand concentrations influence the crystalline structures, particle size, and shape of siRNA@ZIF particles. At low L/M, two-dimensional siRNA@ZIF particles form with a size of 1 µm. As the L/M ratio increases gradually, the particle size decreases, resulting in three-dimensional particles ∼200 nm in size. We also observed better stability of siRNA@ZIF in water prepared using high L/M values and time-dependent cellular uptake by the cells. Additionally, no significant impact of the biocomposites on inflammation was found, indicating the lack of an unwanted immune response and nonimmunotoxic nature over longer periods (96 h). These findings highlight the necessity of fine-tuning ligand concentrations and synthesis chemistry in designing efficient and optimal ZIF-based systems as versatile delivery platforms for nucleic acids.

Advances in CRISPR/Cas systems-based cell and gene therapy.

Cell and gene therapy are innovative biomedical strategies aimed at addressing diseases at their genetic origins. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) systems have become a groundbreaking tool in cell and gene therapy, offering unprecedented precision and versatility in genome editing. This chapter explores the role of CRISPR in gene editing, tracing its historical development and discussing biomolecular formats such as plasmid, RNA, and protein-based approaches. Next, we discuss CRISPR delivery methods, including viral and non-viral vectors, followed by examining the various engineered CRISPR variants for their potential in gene therapy. Finally, we outline emerging clinical applications, highlighting the advancements in CRISPR for breakthrough medical treatments.

Recent Progress and Prospect of Metal-Organic Framework-Based Nanozymes in Biomedical Application.

A nanozyme is a nanoscale material having enzyme-like properties. It exhibits several superior properties, including low preparation cost, robust catalytic activity, and long-term storage at ambient temperatures. Moreover, high stability enables repetitive use in multiple catalytic reactions. Hence, it is considered a potential replacement for natural enzymes. Enormous research interest in nanozymes in the past two decades has made it imperative to look for better enzyme-mimicking materials for biomedical applications. Given this, research on metal-organic frameworks (MOFs) as a potential nanozyme material has gained momentum. MOFs are advanced hybrid materials made of inorganic metal ions and organic ligands. Their distinct composition, adaptable pore size, structural diversity, and ease in the tunability of physicochemical properties enable MOFs to mimic enzyme-like activities and act as promising nanozyme candidates. This review aims to discuss recent advances in the development of MOF-based nanozymes (MOF-NZs) and highlight their applications in the field of biomedicine. Firstly, different enzyme-mimetic activities exhibited by MOFs are discussed, and insights are given into various strategies to achieve them. Modification and functionalization strategies are deliberated to obtain MOF-NZs with enhanced catalytic activity. Subsequently, applications of MOF-NZs in the biosensing and therapeutics domain are discussed. Finally, the review is concluded by giving insights into the challenges encountered with MOF-NZs and possible directions to overcome them in the future. With this review, we aim to encourage consolidated efforts across enzyme engineering, nanotechnology, materials science, and biomedicine disciplines to inspire exciting innovations in this emerging yet promising field.

Amino Acid-Coated Zeolitic Imidazolate Framework for Delivery of Genetic Material in Prostate Cancer Cell.

Metal-organic frameworks (MOFs) are currently under progressive development as a tool for non-viral biomolecule delivery. Biomolecules such as proteins, lipids, carbohydrates, and nucleic acids can be encapsulated in MOFs for therapeutic purposes. The favorable physicochemical properties of MOFs make them an attractive choice for delivering a wide range of biomolecules including nucleic acids. Herein, a green fluorescence protein (GFP)-expressing plasmid DNA (pDNA) is used as a representative of a biomolecule to encapsulate within a Zn-based metal-organic framework (MOF) called a zeolitic imidazolate framework (ZIF). The synthesized biocomposites are coated with positively charged amino acids (AA) to understand the effect of surface functionalization on the delivery of pDNA to prostate cancer (PC-3) cells. FTIR and zeta potential confirm the successful preparation of positively charged amino acid-functionalized derivatives of pDNA@ZIF (i.e., pDNA@ZIFAA). Moreover, XRD and SEM data show that the functionalized derivates retain the pristine crystallinity and morphology of pDNA@ZIF. The coated biocomposites provide enhanced uptake of genetic material by PC-3 human prostate cancer cells. The AA-modulated fine-tuning of the surface charge of biocomposites results in better interaction with the cell membrane and enhances cellular uptake. These results suggest that pDNA@ZIFAA can be a promising alternative tool for non-viral gene delivery.

Targeting telomerase utilizing zeolitic imidazole frameworks as non-viral gene delivery agents across different cancer cell types.

Telomerase, a ribonucleoprotein coded by the hTERT gene, plays an important role in cellular immortalization and carcinogenesis. hTERT is a suitable target for cancer therapeutics as its activity is highly upregulated in most of cancer cells but absent in normal somatic cells. Here, by employing the two Metal-Organic Frameworks (MOFs), viz. ZIF-C and ZIF-8, based biomineralization we encapsulate Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 plasmid system that targets hTERT gene (CrhTERT) in cancer cells. When comparing the two biocomposites, ZIF-C shows the better loading capacity and cell viability. The loaded plasmid in ZIF-C is highly protected against enzymatic degradation. CrhTERT@ZIF-C is efficiently endocytosed by cancer cells and the subcellular release of CrhTERT leads to telomerase knockdown. The resultant inhibition of hTERT expression decreases cellular proliferation and causing cancer cell death. Furthermore, hTERT knockdown shows a significant reduction in tumour metastasis and alters protein expression. Collectively we show the high potential of ZIF-C-based biocomposites as a promising general tool for gene therapy of different types of cancers.

Advanced implications of nanotechnology in disease control and environmental perspectives.

Nanotechnology encompasses a wide range of devices derived from biology, engineering, chemistry, and physics, and this scientific field is composed of great collaboration among researchers from several fields. It has diverse implications notably smart sensing technologies, effective disease diagnosis, and sometimes used in treatment. In medical science, the implications of nanotechnology include the development of elements and devices that interact with the body at subcellular (i.e., molecular) levels exhibiting high sensitivity and specificity. There is a plethora of new chances for medical science and disease treatment to be discovered and exploited in the rapidly developing field of nanotechnology. In different sectors, nanomaterials are used just because of their special characteristics. Their large surface area of them enables higher reactivity with greater efficiency. Furthermore, special surface chemistry is displayed by nanomaterials which compare to conventional materials and facilitate the nanomaterials to decrease pollutants efficiently. Recently, nanomaterials are used in some countries to reduce the levels of contaminants in water, air, and soil. Moreover, nanomaterials are used in the cosmetics and medical industry, and it develops the drug discovery (DD) system. Among a huge number of nanomaterials, Cu, Ag, TiO2, ZnO, Fe3O4, and carbon nanotubes (CNTs) are extensively used in different industries for various purposes. This extensive review study has introduced the major scientific and technical features of nanotechnology, as well as some possible clinical applications and positive feedback in environmental waste management and drug delivery systems.

Investigation of Antimicrobial Activity and Biocompatibility of Biogenic Silver Nanoparticles Synthesized using Syzigyum cymosum Extract.

Nanotherapeutics has emerged as the most sought after approach to tackle the menace of drug-resistant pathogenic bacteria. Among others, biogenic silver nanoparticles (bAgNPs) synthesized using medicinal plant extracts demonstrate promising antibacterial propensity with excellent biocompatibility. Herein, bAgNPs were synthesized through the green chemistry approach using Syzygium cymosum leaf extract as a reducing agent at different pH values (i.e., 5, 7, 8, and 10). The average size of bAgNPs synthesized at pH 5, 7, 8, and 10 was 23.3, 21.3, 17.2, and 35.3 nm, respectively, and all the nanoparticles were negatively charged. Their antibacterial potential was investigated against Bacillus subtilis, Escherichia coli DH5α, E. coli K12, enteropathogenic E. coli, and Salmonella typhi. The highest antibacterial activity was exhibited by bAgNPs synthesized at pH 8 against all the tested bacterial strains, which can be attributed to their small size and greater surface area to volume ratio. The bAgNPs demonstrated the highest zone of inhibition (29.5 ± 0.8 mm) against B. subtilis through oxidation of membrane fatty acids that resulted in the formation of the malondialdehyde-thiobarbituric acid (MDA-TBA) adduct. However, bAgNPs demonstrated excellent hemocompatibility with rat and human red blood cells. Biogenic AgNPs synthesized at pH 8 also exhibited biocompatibility in terms of liver and kidney function biomarkers. Furthermore, hematoxylin and eosin staining of the tissue sections of vital organs (i.e., liver, kidneys, lungs, heart, spleen, and brain) also confirmed the biocompatibility of bAgNPs.

Functionalized Concave Cube Gold Nanoparticles as Potent Antimicrobial Agents against Pathogenic Bacteria.

Gold (Au) is an inert metal in a bulk state; however, it can be used for the preparation of Au nanoparticles (i.e., AuNPs) for multidimensional applications in the field of nanomedicine and nanobiotechnology. Herein, monodisperse concave cube AuNPs (CCAuNPs) were synthesized and functionalized with a natural antioxidant lipoic acid (LA) and a tripeptide glutathione (GSH) because different crystal facets of AuNPs provide binding sites for distinct ligands. There was an ∼10 nm bathochromic shift of the UV-vis spectrum when CCAuNPs were functionalized with LA, and the size of the as-synthesized monodisperse CCAu nanoparticles was 76 nm. The LA-functionalized CCAu nanoparticles (i.e., CCAuLA) showed the highest antibacterial activity against Bacillus subtilis. Both fluorescence images and scanning electron microscopy images confirm the damage of the bacterial cell wall as the mode of antibacterial activity of CCAuNPs. CCAuNPs also cause the oxidation of bacterial cell membrane fatty acids to produce reactive oxygen species, which pave the way for the death of bacteria. Both CCAu nanoparticles and their functionalized derivatives showed excellent hemocompatibility (i.e., percentage of hemolysis is <5% at 80 µg of AuNPs) to human red blood cells and very high biocompatibility to HeLa, L929, and Chinese hamster ovary-green fluorescent protein (CHO-GFP) cells. Taken together, LA and GSH enhance the antibacterial activity and biocompatibility, respectively, of CCAu nanoparticles that interact with the bacteria through Coulomb as well as hydrophobic interactions before demonstrating antibacterial propensity.

Targeted in vitro gene silencing of E2 and nsP1 genes of chikungunya virus by biocompatible zeolitic imidazolate framework.

Chikungunya fever caused by the mosquito-transmitted chikungunya virus (CHIKV) is a major public health concern in tropical, sub-tropical and temperate climatic regions. The lack of any licensed vaccine or antiviral agents against CHIKV warrants the development of effective antiviral therapies. Small interfering RNA (siRNA) mediated gene silencing of CHIKV structural and non-structural genes serves as a potential antiviral strategy. The therapeutic efficiency of siRNA can be improved by using an efficient delivery system. Metal-organic framework biocomposits have demonstrated an exceptional capability in protecting and efficiently delivering nucleic acids into cells. In the present study, carbonated ZIF called ZIF-C has been utilized to deliver siRNAs targeted against E2 and nsP1 genes of CHIKV to achieve a reduction in viral replication and infectivity. Cellular transfection studies of E2 and nsP1 genes targeting free siRNAs and ZIF-C encapsulated siRNAs in CHIKV infected Vero CCL-81 cells were performed. Our results reveal a significant reduction of infectious virus titre, viral RNA levels and percent of infected cells in cultures transfected with ZIF-C encapsulated siRNA compared to cells transfected with free siRNA. The results suggest that delivery of siRNA through ZIF-C enhances the antiviral activity of CHIKV E2 and nsP1 genes directed siRNAs.

Candidate antiviral drugs for COVID-19 and their environmental implications: a comprehensive analysis.

Emerging from Wuhan, China, SARS-CoV-2 is the new global threat that killed millions of people, and many are still suffering. This pandemic has not only affected people but also caused economic crisis throughout the world. Researchers have shown good progress in revealing the molecular insights of SARS-CoV-2 pathogenesis and developing vaccines, but effective treatment against SARS-CoV-2-infected patients are yet to be found. Several vaccines are available and used in many countries, while many others are still in clinical or preclinical studies. However, this involves a long-term process, considering the safety procedures and requirements and their long-term protection capacity and in different age groups are still questionable. Therefore, at present, the drug repurposing of the existing therapeutics previously designed against other viral diseases seems to be the only practical approach to mitigate the current situation. The safety of most of these therapeutic agents has already been tested. Recent clinical reports revealed promising therapeutic efficiency of several drugs such as remdesivir, tenofovir disoproxil fumarate, azithromycin, lopinavir/ritonavir, chloroquine, baricitinib, and cepharanthine. Besides, plasma therapies were used to treat patients and prevent fatal outcomes. Thus, in this article, we have summarized the epidemiological and clinical data from several clinical trials conducted since the beginning of the pandemic, emphasizing the efficiency of the known agents against SARS-CoV-2 and their harmful side effects on the human body as well as their environmental implications. This review shows a clear overview of the current pharmaceutical perspective on COVID-19 treatment.

Antimicrobial Peptides from Plants: A cDNA-Library Based Isolation, Purification, Characterization Approach and Elucidating Their Modes of Action.

Even in a natural ecosystem, plants are continuously threatened by various microbial diseases. To save themselves from these diverse infections, plants build a robust, multilayered immune system through their natural chemical compounds. Among the several crucial bioactive compounds possessed by plants' immune systems, antimicrobial peptides (AMPs) rank in the first tier. These AMPs are environmentally friendly, anti-pathogenic, and do not bring harm to humans. Antimicrobial peptides can be isolated in several ways, but recombinant protein production has become increasingly popular in recent years, with the Escherichia coli expression system being the most widely used. However, the efficacy of this expression system is compromised due to the difficulty of removing endotoxin from its system. Therefore, this review suggests a high-throughput cDNA library-based plant-derived AMP isolation technique using the Bacillus subtilis expression system. This method can be performed for large-scale screening of plant sources to classify unique or homologous AMPs for the agronomic and applied field of plant studies. Furthermore, this review also focuses on the efficacy of plant AMPs, which are dependent on their numerous modes of action and exceptional structural stability to function against a wide range of invaders. To conclude, the findings from this study will be useful in investigating how novel AMPs are distributed among plants and provide detailed guidelines for an effective screening strategy of AMPs.

Prospects of Exploring the Metal-Organic Framework for Combating Antimicrobial Resistance.

Infectious diseases are a major public health concern globally. Infections caused by pathogens with resistance against commonly used antimicrobial drugs or antibiotics (known as antimicrobial resistance, AMR) are becoming extremely difficult to control. AMR has thus been declared as one of the top 10 global public health threats, as it has very limited solutions. The drying pipeline of effective antibiotics has further worsened the situation. There is no absolute treatment, and the limitations of existing methods warrant further development in antimicrobials. Recent developments in the nanomaterial field present them as promising therapeutics and effective alternative to conventional antibiotics and synthetic drugs. The metal-organic framework (MOF) is a recent addition to the antimicrobial category with superior properties. The MOF exerts antimicrobial action on a wide range of species and is highly biocompatible. Additionally, their porous structures allow the incorporation of biomolecules and drugs for synergistic antimicrobial action. This review provides an inclusive summary of the molecular events responsible for resistance development and current trends in antimicrobials to combat antibiotic resistance and explores the potential role of the MOF in tackling the drug-resistant microbial species.

Synthesis of Biogenic Silver Nanoparticles Using Caesalpinia digyna and Investigation of Their Antimicrobial Activity and In Vivo Biocompatibility.

Among metallic nanoparticles, silver nanoparticles (AgNPs) have a wide spectrum of medical applications. Herein, biogenic silver nanoparticles (bAgNPs) were prepared from extracts of Caesalpinia digyna leaf as a reducing agent at different pH values (i.e., 5, 7, 8, and 10). The as-synthesized bAgNPs were characterized using UV-vis and Fourier transform infrared (FTIR) spectroscopies, scanning transmission electron microscopy, powder X-ray diffraction analysis, dynamic light scattering, and ζ-potential analysis. The sizes of bAgNPs prepared at pH 5, 7, 8, and 10 were 45.4, 11.3, 11.4, and 40.8 nm, respectively, and all of the nanoparticles were negatively charged. The antimicrobial activity of the as-prepared bAgNPs was investigated against Bacillus subtilis, Escherichia coli DH5α, E. coli K12, enteropathogenic E. coli (EPEC), and Salmonella typhi. The bAgNPs prepared at pH 8 showed the highest antibacterial propensity against all of the bacterial strains as exhibited in the zone of inhibition (ZOI) as well as the CellTox green assay, which can be due to their relatively small size, stability, and higher surface area-to-volume ratio. The bAgNPs synthesized at pH 8 showed the highest ZOI against B. subtilis, which was ∼25 mm in diameter. The lipid peroxidation assay demonstrated the formation of the malondialdehyde-thiobarbituric acid (MDA-TBA) adduct while treating the bacteria with bAgNPs due to the oxidation of fatty acids present in the membrane. The highest amount of MDA-TBA adduct was observed when Gram-positive B. subtilis was exposed to bAgNPs. On the contrary, rats treated with bAgNPs demonstrated no significant toxicity in terms of hematological and biochemical parameters. The bAgNPs also showed excellent compatibility with human red blood cells. Overall, bAgNPs synthesized at pH 8 have superior antimicrobial activity and excellent biocompatibility and, therefore, can be used as potential antibacterial agents.

Investigation of the Antibacterial Activity and in vivo Cytotoxicity of Biogenic Silver Nanoparticles as Potent Therapeutics.

Biogenic nanoparticles are the smartest weapons to deal with the multidrug-resistant "superbugs" because of their broad-spectrum antibacterial propensity as well as excellent biocompatibility. The aqueous biogenic silver nanoparticles (Aq-bAgNPs) and ethanolic biogenic silver nanoparticles (Et-bAgNPs) were synthesized using aqueous and ethanolic extracts of Andrographis paniculata stem, respectively, as reducing agents. Electron microscopic images confirmed the synthesis of almost spherical shaped biogenic silver nanoparticles (bAgNPs). The zeta potentials of the nanoparticles were negative and were -22 and -26 mV for Aq-bAgNPs and Et-bAgNPs, respectively. The antibacterial activity of bAgNPs was investigated against seven pathogenic (i.e., enteropathogenic Escherichia coli, Salmonella typhi, Staphylococcus aureus, Vibrio cholerae, Enterococcus faecalis, Hafnia alvei, Acinetobacter baumannii) and three nonpathogenic (i.e., E. coli DH5α, E. coli K12, and Bacillus subtilis) bacteria at different time points (i.e., 12, 16, 20, and 24 h) in a dose-dependent manner (i.e., 20, 40, and 60 µg) through broth dilution assay, disk diffusion assay, CellToxTM Green uptake assay, and trypan blue dye exclusion assay. The lowest minimum inhibitory concentration value for both the bAgNPs was 0.125 µg. Et-bAgNPs showed the highest antibacterial activity against S. aureus at 60 µg after 16 h and the diameter of inhibited zone was 28 mm. Lipid peroxidation assay using all the bacterial strains revealed the formation of malondialdehyde-thiobarbituric acid adduct due to the oxidation of cell membrane fatty acids by bAgNPs. The bAgNPs showed excellent hemocompatibility against human as well as rat red blood cells. Furthermore, there was no significant toxicity observed when the levels of rat serum ALT, AST, γ-GT (i.e., liver function biomarkers), and creatinine (i.e., kidney function biomarker) were determined.

Functionalization of Elongated Tetrahexahedral Au Nanoparticles and Their Antimicrobial Activity Assay.

Gold nanoparticles are inert for the human body, and therefore, they have been functionalized to provide them with antibacterial properties. Here, elongated tetrahexahedral (ETHH) Au nanoparticles were synthesized, characterized, and functionalized with lipoic acid (LA), a natural antioxidant with a terminal carboxylic acid and a dithiolane ring, to generate ETHH-LA Au nanoparticles. The antioxidant activity of Au nanoparticles was investigated in vitro, showing that LA enhances the 2,2-diphenyl-1-picrylhydrazyl free-radical scavenging and Fe3+ ion reducing activity of ETHH-LA at higher amounts. The antimicrobial propensities of the nanoparticles were investigated against Gram-positive ( Bacillus subtilis) and Gram-negative ( Escherichia coli) bacteria through propidium iodide assay as well as disk diffusion assay. ETHH-LA Au nanoparticles showed significantly higher antimicrobial activity against B. subtilis compared with E. coli. Furthermore, ETHH-LA Au nanoparticles also showed significantly better antimicrobial activity against both bacterial strains when compared with ETHH. ETHH Au nanoparticles also bring about the oxidation of bacterial cell membrane fatty acids and produce lipid peroxides. ETHH-LA showed higher lipid peroxidation potential than that of ETHH against both bacteria tested. The hemolytic potential of Au nanoparticles was investigated using human red blood cells and ETHH-LA showed reduced hemolytic activity than that of ETHH. The cytotoxicity of Au nanoparticles was investigated using human cervical cancer cells, HeLa, and ETHH-LA Au nanoparticles showed reduced cytotoxicity than that of ETHH. Taken together, LA enhances the antimicrobial activity of ETHH Au nanoparticles and Au nanoparticles interact with the bacteria through electrostatic interactions as well as hydrophobic interactions and damage the bacterial cell wall followed by oxidation of cell membrane fatty acids.