Antimicrobial activity, mechanism of action, and methods for stabilisation of defensins as new therapeutic agents

Bioactive compounds, such as antimicrobial peptides (AMPs), have increasingly been used recently to counteract the rapidly increasing incidence of bacterial resistance to usual antibiotics and chemotherapeutics. In humans, endogenous AMPs are part of the immune system and act against pathogens. Defensins compose a class of AMPs that have activity against gram-positive and -negative bacteria, viruses, and fungi. For some, antitumour activity has also been reported. Such characteristics indicate that they represent a potential new class of therapeutic agents against microorganisms, including multidrug resistant pathogens. However, pH and enzymatic degradation and variable tissue distribution of these compounds limit their clinical application. New technologies and different methods have been developed to overcome these limitations and increase their half-life, such as cyclization, lipidation, design of peptidomimetics, synthesis of hybrid peptides, and use of nanocarriers. The objective of this review was to analyse current applications of defensins as antimicrobial agents and their mechanism of action. Moreover, new technologies and methods for stabilizing defensins are discussed

Biopolymeric Nanogel as a Drug Delivery System for Doxorubicin—Improved Drug Stability and Enhanced Antineoplastic Activity in Skin Cancer Cells

In this study, doxorubicin was loaded in a chitosan–albumin nanogel with the aim of improving its stability and exploring the potential of the system in the treatment of skin cancer. Infrared spectroscopy and X-ray diffraction confirmed the encapsulation of the drug. Transmission electron microscopy revealed the spherical shape of the nanogel particles. The drug-loaded nanogel was characterized with a small diameter of 29 nm, narrow polydispersity (0.223) and positive zeta potential (+34 mV). The exposure of encapsulated doxorubicin to light (including UV irradiation and daylight) did not provoke any degradation, whereas the nonencapsulated drug was significantly degraded. In vitro studies on keratinocytes (HaCaT) and epidermoid squamous skin carcinoma cells (A-431) disclosed that the encapsulated doxorubicin was more cytotoxic on both cell lines than the pure drug was. More importantly, the cytotoxic concentration of encapsulated doxorubicin in carcinoma cells was approximately two times lower than that in keratinocytes, indicating that it would not affect them. Thus, the loading of doxorubicin into the developed chitosan–albumin nanogel definitely stabilized the drug against photodegradation and increased its antineoplastic effect on the skin cancer cell line

Cytotoxicity and Microbicidal Activity of Commonly Used Organic Solvents: A Comparative Study and Application to a Standardized Extract from Vaccinium macrocarpon

The cytotoxicity and microbicidal capacity of seven organic solvents commonly applied for studying plant extracts and bioactive compounds were systematically investigated based on international standards. Four cell lines of normal (CCL-1, HaCaT) or tumor (A-375, A-431) tissue origin, seven bacterial and one fungal strain were used. The impact of the least toxic solvents in the determination of in vitro cytotoxicity was evaluated using a standardized extract from Vaccinium macrocarpon containing 54.2% v/v proanthocyanidins (CystiCran®). The solvents ethanol, methoxyethanol and polyethylene glycol were the least cytotoxic to all cell lines, with a maximum tolerated concentration (MTC) between 1 and 2% v/v. Ethanol, methanol and polyethylene glycol were mostly suitable for antimicrobial susceptibility testing, with minimum inhibitory concentrations (MICs) ≥ 25% v/v. The MTC values of the solvents dimethyl sulfoxide, dimethoxyethane and dimethylformamide varied from 0.03% to 1.09% v/v. The MICs of dimethyl sulfoxide, methoxyethanol and dimethoxyethane were in the range of 3.125–25% v/v. The cytotoxic effects of CystiCran® on eukaryotic cell lines were directly proportional to the superimposed effect of the solvents used. The results of this study can be useful for selecting the appropriate solvents for in vitro estimation of the cytotoxic and growth inhibitory effects of bioactive molecules in eukaryotic and prokaryotic cells

ANALYTICAL STUDY AND ANTIMICROBIAL ACTIVITY OF ALPHA-DEFENSIN 2 DISSOLVED IN PHARMACOPOEIA BUFFERS WITH DIFFERENT pH

Human neutrophil peptides (HNPs, alpha-defensins) are a group of six defensins being considered as new antimicrobial drugs because of their multifunctional efficiency against bacteria, viruses and fungi. Regardless of the unique biological properties alpha-defensins are unstable compounds and their activity depends on many physical and chemical factors as well as on the kind of the used cultivation media. This leads to research difficulties and obstructions in their therapeutic application. The purpose of this study was to determine antibacterial activity of alpha-defensin 2 dissolved in pharmacopeia buffers and in parallel to develop selective and accurate analytical tests for identification and assay of alpha-defensin 2 in the course of study. The antibacterial effect of alpha-defensin 2 was determined against the strain Escherichia coli (ATCC 25922). It was found that 10 mg/l of Human neutrophil peptide-2 (HNP-2, alpha-defensin 2) dissolved in pH 9.0 buffer caused 90% inhibition of the bacterial respiratory activity. This buffer was considered as a suitable environment for deploying the antibacterial activity of the alpha-defensin. А selective MS analysis method for identification of alpha-defensin 2 in sample mixtures was developed. Also HPLC method with alternative selectivity was elaborated for identification and assay of mixtures containing alpha-defensin 2 and aminoacids in Mueller Hinton broth. The procedure includes development of system suitability test determination