Current Pharmaceutical Biotechnology - Volume 18, Issue 2, 2017

Antimicrobial peptides (AMPs) and proteins are produced by a wide range of organisms as important elements of their defense mechanisms, forming a large number of antimicrobial compounds that can be used to treat several human infections. The potential for the use of AMPs and antimicrobial proteins in therapeutics is growing, but their application is often limited, due to their poor physical and/or chemical properties. In recent years, several drug delivery systems have been proposed to carry such molecules, in an attempt to overcome the difficulties regarding their properties. However, no review has yet systematized the most relevant information on this subject. Therefore, this review summarizes the work that has been conducted to develop delivery systems for the transport and protection of AMPs and antimicrobial proteins, including their description and potential applications, while highlighting the opportunities for future research in this field.

Background: Pullulan is a microbial polysaccharide of low energy value, which can component of low-calorie foods and in dietary snacks for diabetics. The objective of the study was to determine the effect of pullulan on the growth and fermentation activity of selected human intestinal bacteria. Methods: Commercial pullulan purchased from Focubase (China) of a molecular weight of 100,000 Da constituted as experimental material. Food grade pullulan 99% purity. Two control media were prepared: the first standard RCM composed of (g/100 ml): 0.5 glucose, 0.1 soluble starch, 1.0 peptone, 1.0 meat extract, 0.3 yeast extract, 0.3 sodium acetate, 0.05 cysteine hydrochloride, 0.5 NaCl, pH 6.8; and the second modified RCM, wherein the soluble starch was replaced by increased glucose concentration to 2.0% (RCM+G). Experimental medium was the modified RCM medium, wherein the soluble starch and glucose were replaced by pullulan at a concentration of 2.0% (RCM+P). Stool suspensions were prepared from fresh stool samples (1 g) in peptone water (9 g), which were previously homogenized. Then, suspensions at a volume of 300 μl were transferred to the media (RCM, RCM+G, and RCM+P). After mixing, flasks were placed in anaerobic tubes with AnaeroGenTM 2.5 l sachets. Incubation of samples was carried out at 37°C for 48 h. Results: The number of Bifidobacterium, Lactobacillus, and Escherichia coli bacteria, as well as pH and total acidity of the culture during 0, 24, and 48 h were measured. It was found that the numbers of bacteria of the Bifidobacterium and Lactobacillus genus in medium with pullulan were one logarithmic cycle lower in comparison to their numbers in the control media. Higher total acidity (1.48 g/100 ml) of pullulan culture in comparison to the control media was obtained (1.10 and 0.60 g/100 ml), and lower pH values than RCM medium, particularly 4.15 and 4.70, respectively. Pullulan exhibited selective effect on the natural microflora of the colon. Increase in the fermentation activity of bacteria in medium with pullulan favorably influenced modification of the composition of gut microbiota. Conclusion: In summary, pullulan exhibited a selective effect on the natural microflora of the infants’ colon. Although no stimulating effect of pullulan on the growth of Bifidobacterium and Lactobacillus was observed, their increased acidifying activity, which probably was the cause of reduction in the number of E. coli bacteria, was confirmed.

Background: Cancer patients treated with alkylating agents and radiotherapy are exposed to high level of reactive oxygen species (ROS) in tissues. ROS can involve superoxide free radicals, peroxynitrite, singlet oxygen, nitric oxide and hydrogen peroxide. It is well documented that increased exposure to oxygen through a high metabolic rate could lead to a shortened life span. Ionizing radiation, use of drugs and the development of cancer can lead to cancer-induced anemia. Recombinant human erythropoietin (Epo) supplementation is one of the methods for treating anemia. Erythropoietin through an increase in the number of erythrocytes, improves oxygenation tissue. The aim of this work was to study the effect of Epo on colon adenocarcinoma cells (DLD-1) given alone or in combination with hydrogen peroxide (H2O2). Cell proliferation and number were measured. Methods: Expression of EpoR, Bcl-2 and Akt1 protein was assessed by RT-PCR, Western blot, and confocal microscopy. Results: The results show that the coadministration of Epo and H2O2 indicates antitumor action, which occurs via a dose-dependent inhibition of DLD-1 cell growth and proliferation. Moreover, the coadministration of Epo and H2O2 resulted in a decrease of cell numbers, as well as Bcl-2 expression. The incubation of DLD-1 cells with those agents led to a decrease in EpoR and phosphorylated EpoR expression and an increase in Akt1 and phosphorylated Akt expression. The addition of Epo to H2O2 intensified the cytotoxic effect of the latter. Conclusion: These preclinical results suggest that Epo during chemotherapy or radiotherapy may possess potential benefits in colon cancer patients.

Background: Gram positive bacteria produce peptides, defined bacteriocins which exhibit good antibacterial activity. Objective: We evaluated the ability of L. fermentum to produce bacteriocins having therefore, good probiotic features and finally, be safe towards microglial cells. Method: Eight wild strains, identified using molecular techniques, were investigated for the evaluation of resistance to bile salts, low pH, H2O2 production, biofilm formation, antibacterial activity and safety on microglia cells (BV2). Results: The determination of the susceptibility/resistance profile showed that the strains are sensitive to the antibiotics tested. All strains showed a good tolerability to extremely low pH as well as resisting in presence of bile salts. In addition, the strains showed excellent activity against pathogens and one of them (LAC 42) showed activity also against Pseudomonas aeruginosa and Klebsiella pneumoniae. Finally, LAC 42 and its active compound did not change microglia cell viability following 24h exposure. Our data on this antibacterial molecule suggest that it is a compound with low molecular weight and with highly hydrophilic component. Conclusion: These results describe the characteristics of Lactobacillus strains and provide evidences for their possible use as new potential probiotic. In addition, other studies are now warranted to exploit the antibacterial activity of the supernatant LAC 42 and for its complete chemical characterization.

Background: In this study, the effect of low concentrations of poly(amido)amine dendrimers (G2-G4) on human lymphocytes was studied. Some works revealed that PAMAMs can adversely affect the morphology of blood components and mitochondria functions. In this context, the present report aimed to investigate the in vitro cationic dendrimers’ effect on mitochondrial respiration and cell morphology in lymphocytes isolated from human blood. Methods: To monitor the mitochondrial changes, the high-resolution respirometer was used, whereas the cell morphology was analyzed using a flow cytometer and fluorescence microscopy. Results:The concentration-dependent dendrimers’ influence on lymphocytes morphology was shown. Changes in mitochondrial respiration revealed the concentration- and generation-dependent differences between dendrimer activity. There were no alterations in the routine respiration and in the state of the inner mitochondrial membrane (L/E), but decreased ADP- and FCCP-stimulated respirations were detected after treatment with G3 and G4 dendrimers. The markers of mitochondrial membrane integrity (RCR) and OXPHOS efficiency (P/E) significantly decreased regardless of the dendrimer generation used. Conclusion: Based on these in vitro evaluations, we state that cationic PAMAM dendrimers can impair both the morphology and the bioenergetics of human lymphocytes, even when used at low concentrations and in a short time (up to 1 h). However, these results do not imply that similar findings could be possible for in vivo observations.

Background: Phosvitin (PV) is a highly-phosphorylated metal-binding protein in egg yolk. Phosphoserine clusters make PV resistant to enzymatic digestion, which might be nutritionally undesirable. Objective: This study was designed to determine the effects of high hydrostatic pressure and enzymatic hydrolysis (HHP-EH) on the antioxidant and anti-inflammatory properties of PV hydrolysates (PVHs). Methods: PV was hydrolyzed by alcalase, elastase, savinase, thermolysin, and trypsin at 0.1, 50, and 100 MPa pressure levels. PVHs were evaluated for degree of hydrolysis, molecular weight distribution patterns, antioxidant and anti-inflammatory properties in chemical and cellular models. The effect of PVH on gene expression of pro-inflammatory cytokines (TNF-α and IL-1β) was also evaluated using real time-PCR. The hydrolysate with most potent antioxidant and anti-inflammatory properties was subjected to LC-MS/MS analysis to identify the peptide sequence. Results: Hydrolysates produced at 100 MPa exhibited higher degree of hydrolysis and greater reducing power and free radical scavenging activity compared to those obtained at atmospheric pressure. After adjusting the phosphate content, alcalase- and trypsin-digested PVHs showed superior iron chelation capacity (69-73%), regardless of pressure. Both alcalase- and trypsin-digested PVHs significantly inhibited nitric oxide production by RAW264.7 macrophage cells. LPS-stimulated up-regulation of proinflammatory cytokines was also suppressed by alcalase-digested PVH. Conclusion: The HHP-EH method could play a promising role in the production of bioactive peptides from hydrolysis-resistant proteins. HHP-assisted PVH may be useful in preparing a potential pharmaceutical with antioxidant and anti-inflammatory properties.

Background: Biologically synthetized silver nanoparticles are promising antimicrobial agent. Flow cytometry, well diffusion methods, colony-forming units (CFU) and spectroscopic approach are commonly used in antimicrobial study. The aim of this study was investigation of effectiveness of Bio- AgNPs synthesized by Actinomycetes HGG16n using fluorescence flow cytometry method as an alternative to the standard ones (well and disc diffusion method). Methods: Flow cytometry technique was applied to monitor the antibacterial effect of biocolloidal silver and its combination with various commercial antibiotics against selected pathogens. The observed effectiveness was confirmed by fluorescence micrographs. Result: Silver nanoparticles synthesized by Actinomycetes HGG 16n strain were as effective antimicrobial agent as the tested commercial antibiotics. Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Salmonella infantis and Bacillus subtilis strains are not able to create the resistant mechanisms under treatment of biocolloidal silver. Furthermore, the flow cytometry technique was more sensitive than disc and well diffusion and confirmed the effectiveness of BioAgNPs against all tested bacterial cells. Precipitation and limited diffusion of biocolloidal silver was observed by using well diffusion method. Conclusion: Specificity and selection of antimicrobial approach are related with different nature of lowmolecular compounds (e.g. antibiotic) compared with biocolloids. An alternative method, flow cytometry was designed for antimicrobial study of biocolloidal silver nanoparticles and compared to the classical microbial techniques. Moreover, this work highlights the development of novel and inexpensive antimicrobial agent for most oral and skin infections caused by Gram (+) and Gram (-) bacteria.

Background: Streptococcus agalactiae (group B Streptococcus - GBS) remains a leading cause of neonatal infections and an important cause of invasive infections in adults with underlying conditions. Methods: This study evaluated for the first time the effect of an oleoresin collected from Copaifera multijuga Hayne (copaiba oil) alone or in combination with silver nanoparticles produced by green synthesis using Fusarium oxysporum (AgNPbio) against planktonic and sessile cells of GBS isolated from colonized women. Results: Copaiba oil showed a dose-dependent bactericidal activity against planktonic GBS strains, including those resistant to erythromycin and/or clindamycin. Scanning and transmission electron microscopy of GBS treated with copaiba oil revealed morphological and ultrastructural alterations, displaying disruption of the cell wall and decreased electron density due to leakage of cytoplasmic content. Copaiba oil also exhibited antibacterial activity against biofilms of GBS strains, inhibiting their formation as well as the viability of mature biofilms. In addition, the combination of copaiba oil with AgNPbio resulted in a synergistic effect against planktonic cells and biofilm formation, reducing the minimal inhibitory concentration values of both compounds. No hemolytic activity was detected for both compounds. Conclusion: These results indicate the potential of copaiba oil, alone or in combination with AgNPbio, for the development of new alternative strategies for controlling GBS infections.