Anionic liposome formulation for oral delivery of thuricin CD, a potential antimicrobial peptide therapeutic.

Thuricin CD is a two-peptide antimicrobial produced by Bacillus thuringiensis. Unlike previous antibiotics, it has shown narrow spectrum activity against Clostridioides difficile, a bacterium capable of causing infectious disease in the colon. However, peptide antibiotics have stability, solubility, and permeability problems that can affect their performance in vivo. This work focuses on the bioactivity and bioavailability of thuricin CD with a view to developing a formulation for delivery of active thuricin CD peptides through the gastrointestinal tract (GIT) for local delivery in the colon. The results indicate that thuricin CD is active at low concentrations only when both peptides are present. While thuricin CD was degraded by proteases and was unstable and poorly soluble in gastric fluid, it showed increased solubility in intestinal fluid, probably due to micelle encapsulation. Based on this, thuricin CD was encapsulated in anionic liposomes, which showed increased activity compared to the free peptide, maintained activity after exposure to pepsin in gastric fluid and intestinal fluid, was stable in suspension for over 21 days at room temperature and for 60 days at 4 °C, and exhibited no toxicity to epithelial intestinal cells. These findings suggest that an anionic lipid-based nano formulation may be a promising approach for local oral delivery of thuricin CD.

Pre-formulation of an additive combination of two antimicrobial agents, clofazimine and nisin A, to boost antimicrobial activity.

According to the World Health Organization, antimicrobial resistance is one of the top ten issues that pose a major threat to humanity. The lack of investment by the pharmaceutical industry has meant fewer novel antimicrobial agents are in development, exacerbating the problem. Emerging drug design strategies are exploring the repurposing of existing drugs and the utilization of novel drug candidates, like antimicrobial peptides, to combat drug resistance. This proactive approach is crucial in fighting global health threats. In this study, an additive combination of a repurposed anti-leprosy drug, clofazimine, and an antimicrobial peptide, nisin A, are preformulated using liquid antisolvent precipitation to generate a stable amorphous, ionized nanoparticle system to boost antimicrobial activity. The nanotechnology aims to improve the physicochemical properties of the inherently poorly water-soluble clofazimine molecules while also harnessing the previously unreported additive effect of clofazimine and nisin A. The approach transformed clofazimine into a more water-soluble salt, yielding amorphous nanoparticles stabilized by the antimicrobial peptide; and combined the two drugs into a more soluble and more active formulation. Blending pre-formulation strategies like amorphization, salt formation, and nanosizing to improve the inherent low aqueous solubility of drugs can open many new possibilities for the design of new antimicrobial agents. This fusion of pre-formulation technologies in combination with the multi-hurdle approach of selecting drugs with different effects on microbes could be key in the design platform of new antibiotics in the fight against antimicrobial resistance.

The development of a solid lipid nanoparticle (SLN)-based lacticin 3147 hydrogel for the treatment of wound infections.

Chronic wounds affect millions of people globally. This number is set to rise with the increasing incidence of antimicrobial-resistant bacterial infections, such as methicillin-resistant Staphylococcus aureus (MRSA), which impair the healing of chronic wounds. Lacticin 3147 is a two-peptide chain bacteriocin produced by Lactococcus lactis that is active against S. aureus including MRSA strains. Previously, poor physicochemical properties of the peptides were overcome by the encapsulation of lacticin 3147 into solid lipid nanoparticles. Here, a lacticin 3147 solid lipid nanoparticle gel is proposed as a topical treatment for S. aureus and MRSA wound infections. Initially, lacticin 3147's antimicrobial activity against S. aureus was determined before encapsulation into solid lipid nanoparticles. An optimised gel formulation with the desired physicochemical properties for topical application was developed, and the lacticin-loaded solid lipid nanoparticles and free lacticin 3147 aqueous solution were incorporated into separate gels. The release of lacticin 3147 from both the solid lipid nanoparticle and free lacticin gels was measured where the solid lipid nanoparticle gel exhibited increased activity for a longer period (11 days) compared to the free lacticin gel (9 days). Both gels displayed potent activity ex vivo against S. aureus-infected pig skin with significant bacterial eradication (> 75%) after 1 h. Thus, a long-acting potent lacticin 3147 solid lipid nanoparticle gel with the required physicochemical properties for topical delivery of lacticin 3147 to the skin for the potential treatment of S. aureus-infected chronic wounds was developed.

Intracellular Bacterial Targeting by a Thiazolyl Benzenesulfonamide and Octaarginine Peptide Complex.

A brominated thiazolyl benzenesulfonamide (BTB) derivative is conjugated with the cell-penetrating peptide octaarginine (R8) in an effort to construct innovative antibacterial products. The noncovalent complex of BTB and R8 is characterized by Fourier transform infrared (FTIR) spectroscopy, which indicates hydrogen bonding between the two constituents. Attachment of the peptide moiety renders aqueous solubility to the hydrophobic benzenesulfonamide drug and bestows bactericidal activity. Confocal imaging in conjunction with dye probes shows successful clearance of intracellular Staphylococcus aureus bacteria by the BTB-R8 complex. Scanning electron micrographs and studies with a set of fluorescent dyes suggest active disruption of the bacterial cell membrane by the BTB-R8 complex. In contrast, the complex of BTB with octalysine (K8) fails to cause membrane damage and displays a modest antibacterial effect. A complex of BTB with the water-soluble hydrophilic polymer poly(vinylpyrrolidone) (PVP) does not display any antibacterial effect, indicating the distinctive role of the cell-penetrating peptide (CPP) R8 in the cognate complex. The leakage of the encapsulated dye from giant unilamellar vesicles upon interaction with the BTB-R8 complex further highlights the membrane activity of the complex, which cannot be accomplished by bare sulfonamide alone. This work broadens the scope of use of CPPs with respect to eliciting antibacterial activity and potentially expands the limited arsenal of membrane-targeting antibiotics.

Emergent antibacterial activity of N-(thiazol-2-yl)benzenesulfonamides in conjunction with cell-penetrating octaarginine.

Hybrid antimicrobials that combine the effect of two or more agents represent a promising antibacterial therapeutic strategy. In this work, we have synthesized N-(4-(4-(methylsulfonyl)phenyl)-5-phenylthiazol-2-yl)benzenesulfonamide derivatives that combine thiazole and sulfonamide, groups with known antibacterial activity. These molecules are investigated for their antibacterial activity, in isolation and in complex with the cell-penetrating peptide octaarginine. Several of the synthesized compounds display potent antibacterial activity against both Gram-negative and Gram-positive bacteria. Compounds with 4-tert-butyl and 4-isopropyl substitutions exhibit attractive antibacterial activity against multiple strains. The isopropyl substituted derivative displays low MIC of 3.9 µg mL-1 against S. aureus and A. xylosoxidans. The comparative antibacterial behaviour of drug-peptide complex, drug alone and peptide alone indicates a distinctive mode of action of the drug-peptide complex, that is not the simple sum total of its constituent components. Specificity of the drug-peptide complex is evident from comparison of antibacterial behaviour with a synthetic intermediate-peptide complex. The octaarginine-drug complex displays faster killing-kinetics towards bacterial cells, creates pores in the bacterial cell membranes and shows negligible haemolytic activity towards human RBCs. Our results demonstrate that mere attachment of a hydrophobic moiety to a cell penetrating peptide does not impart antibacterial activity to the resultant complex. Conversely, the work suggests distinctive modes of antibiotic activity of small molecules when used in conjunction with a cell penetrating peptide.

Enhancing Aqueous Solubility and Antibacterial Activity of Curcumin by Complexing with Cell-Penetrating Octaarginine.

Bacterial resistance to antimicrobial drugs is one of the biggest threats to human health and novel drugs, and strategies are needed to obviate this resistance crisis. An innovative strategy for designing novel antimicrobial drugs is based on the hybridization of an antimicrobial agent with a second functional entity. Here, we use a cell-penetrating peptide-octaarginine (R8) as the second functional entity and develop a complex or hybrid of R8 and curcumin that possibly targets the bacterial cell membrane. Minimum inhibitory concentration assays show that the antibacterial activity of the complex is enhanced in a synergistic manner and rapid killing kinetics are obtained, emphasizing a bactericidal mode of action. In addition, electron microscopy images reveal bacterial membrane disruption by the complex. The R8-curcumin complex also displays activity against HeLa cells.

Reusable nanobiocatalysts for the efficient extraction of pigments from orange peel.

Nagpur mandarin orange (Citrus reticulata) peels were subjected to treatment with nanobiocatalysts in the form of cellulase and pectinase immobilized magnetic nanoparticles (MNPs). MNPs (Fe3O4) with average diameter in range of 40-90 nm were immobilized with cellulase and pectinase through APTES and glutaraldehyde. Treatment followed by extraction into organic solvents resulted in 8-9 fold increase in extraction of carotenoidic pigments compared to use of free enzymes. Optimum pH and temperature for the process were determined to be 5.0 and 50 °C, respectively. The nanobiocatalysts could be reused across three cycles with only 15 % drop in yield per cycle. Dinitrosalicylic acid assays showed that superior peel hydrolysis also led to greatest extent of pigment extraction.