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.
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.
BACKGROUND: We previously developed substitutes for red blood cells (RBCs) and platelets (PLTs) for transfusion. These substitutes included hemoglobin vesicles (HbVs) and fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV, H12)-coated, adenosine diphosphate (ADP)-encapsulated liposomes [H12-(ADP)liposomes]. Here, we examined the efficacy of combination therapy using these substitutes instead of RBC and PLT transfusion in a rabbit model with traumainduced massive hemorrhage with coagulopathy. ABBREVIATIONS: ADP = adenosine diphosphate; APTT = activated partial thromboplastin time; AT = antithrombin; CR = clot rate; CT = clotting time; HbVs = hemoglobin vesicles; HSA = human serum albumin; MAP = mannitol adenine phosphate; NO = nitric oxide; NOx = NO 2 − /NO 3 ; PPP = plateletpoor plasma; PRP = platelet-rich plasma; PT = prothrombin time.From the
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.
Biogenic silver nanoparticles demonstrate excellent antibacterial activity against a broad range of bacteria. Herein, aqueous biogenic silver nanoparticles (Aq@bAgNPs) and ethanolic biogenic silver nanoparticles (Et@bAgNPs) were synthesized using aqueous as well as ethanolic extracts of Diospyros malabarica fruit, respectively. The as-prepared biogenic silver nanoparticles (bAgNPs) were characterized using UV-Vis, FTIR as well as energy dispersive X-ray (EDS) spectroscopy, electron microscopy, dynamic light scattering spectroscopy (DLS), and zetasizer. The zeta potentials of Aq@bAgNPs and Et@bAgNPs were −9.8 ± 2.6, and −12.2 ± 1.9 mV, respectively. The antibacterial activity of bAgNPs was investigated against seven bacterial strains (i.e., pathogenic and nonpathogenic) and Et@bAgNPs exhibited the highest antibacterial propensity (i.e., 20 nm in diameter) against Bacillus subtillis through disk diffusion assay. The trypan blue dye exclusion assay also confirmed the antibacterial propensity of as-prepared bAgNPs. Furthermore, both Aq@bAgNPs and Et@bAgNPs oxidize bacterial membrane fatty acids and generate lipid peroxides which eventually form complexes with thiobarbituric acid (i.e., malondialdehyde-thiobarbituric acid adduct) to bring about bacterial death. Both the nanoparticles demonstrated good hemocompatibility against human as well as rat red blood cells (RBCs). In addition, they exhibited excellent biocompatibility in vivo in terms of rat liver (i.e., serum ALT, AST, and γ-GT) and kidneys (i.e., serum creatinine) function biomarkers.
Aim The uptake pathway of liposomes into cells is mainly via endocytosis or membrane fusion; however, the relationship between the uptake pathway and the intracellular pharmacokinetics of the liposome components remains unclear. This study aimed at revealing the relationship by using cationic liposomes having similar physical properties and different uptake pathways. Materials and Methods We prepared cationic liposomes composed of amino acid-type lipids, K3C14 and K3C16, which have different uptake pathways by a hydration method, and fluorescently modified them by encapsulating FITC-dextran and surface conjugation with Alexa Fluor ® 488 (AF488). Then, we investigated their intracellular distribution in HeLa cells over time. Results The liposomes had similar physical properties and did not cause significant cell mortality after treatment for 180 min. The delivery rate and efficiency of encapsulated FITC-dextran with the fusogenic K3C16 liposomes were 3 and 1.6 times higher, respectively, than with the endocytic K3C14 liposomes. FITC-dextran molecules delivered with K3C16 liposomes were observed throughout the cytosolic space after 10 min, while those delivered with K3C14 liposomes were mainly observed as foci and took 60 min to diffuse into the cytosolic space. K3C14 lipids modified with AF488 were distributed mostly in the cytosolic space. In contrast, fluorescently labeled K3C16 lipids were colocalized with the plasma membrane of 50% of the HeLa cells after 10 min and were gradually internalized intracellularly. Conclusion Fusogenic K3C16 liposomes internalized into HeLa cells faster than endocytic K3C14 liposomes, and their components differently distributed in the cells.
Fibrinogen γ-chain peptide-coated, adenosine 5′-diphosphate (ADP)-encapsulated liposomes (H12-ADP-liposomes) are a potent haemostatic adjuvant to promote platelet thrombi. These liposomes are lipid particles coated with specific binding sites for platelet GPIIb/IIIa and encapsulating ADP. They work at bleeding sites, facilitating haemostasis by promoting aggregation of activated platelets and releasing ADP to strongly activate platelets. In this study, we investigated the therapeutic potential of H12-ADP-liposomes on post-cardiopulmonary bypass (CPB) coagulopathy in a preclinical setting. We created a post-CPB coagulopathy model using male New Zealand White rabbits (body weight, 3 kg). One hour after CPB, subject rabbits were intravenously administered H12-ADP-liposomes with platelet-rich plasma (PRP) collected from donor rabbits (H12-ADP-liposome/PRP group, n = 8) or PRP alone (PRP group, n = 8). Ear bleeding time was greatly reduced for the H12-ADP-liposome/PRP group (263 ± 111 s) compared with the PRP group (441 ± 108 s, p < 0.001). Electron microscopy showed platelet thrombus containing liposomes at the bleeding site in the H12-ADP-liposome/PRP group. However, such liposome-involved platelet thrombi were not observed in the end organs after H12-ADP-liposome administration. These findings suggest that H12-ADP-liposomes could help effectively and safely consolidate platelet haemostasis in post-CPB coagulopathy and may have potential for reducing bleeding complications after cardiovascular surgery with CPB. Platelet transfusions are broadly and commonly used for the prevention or treatment of bleeding in settings such as haematologic disorder, trauma, and surgery 1,2. Globally, the supply of blood products is currently insufficient to meet demand, although this is influenced in part by the socioeconomic background of the countries 3-5. In ageing society where the blood-donating population is decreasing and the blood-consuming population is increasing, blood shortages are anticipated to become chronic and critical 4,6. For example, in Japan, as long as current blood donation behaviour continues, the estimated shortfall of blood donations is expected to double from 2025 to 2050 7. In addition, it is extrapolated that the scarcity of platelet preparations will certainly increase in the future because of their limited shelf life and storage methods 8. Therefore, effective countermeasures
Bioactive peptides, which act as biologically active regulators, often require intracellular delivery systems to access their therapeutic targets in the cytosolic space maintaining their bioactivity. Here, we report on the delivery of a polar cell impermeable bioactive peptide, phalloidin, into living HeLa cells with cationic liposomes prepared from lysine-based lipids. Liposome/Alexa Fluor 594 phalloidin complexes were characterized regarding their size and zeta potential, which were 85 ± 38 nm and +24.5 ± 4.21 mV, respectively. The delivery of Alexa Fluor 594 phalloidin into live HeLa cells with K3C14 liposomes was evaluated using a fluorescence activated cell sorter and confocal laser scanning microscopy. The highest Alexa Fluor 594 phalloidin delivery efficiency was 92% when using 200 μg of the cationic lipid/1 × 105 cells seeded at 37 °C. The cellular uptake mechanism for the cationic liposome/Alexa Fluor 594 phalloidin complexes was investigated using various endocytosis inhibitors. We confirmed the complexes were mainly taken up through caveolae-mediated endocytosis. Incubation with bafilomycin A1, which inhibits the acidification of lysosomes, revealed that Alexa Fluor 594 phalloidin did not pass through the lysosomal pathway. Rather, Alexa Fluor 594 phalloidin was released from early endosomes or caveosomes to the cytosol to exhibit its bioactive effects including the multinucleation of HeLa cells.
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