Interaction mechanism of chitosan oligomers in pure water with cell membrane models studied by SFG vibrational spectroscopy.

Chitosan is a versatile and biocompatible cationic antimicrobial polymer obtained from sustainable sources that is effective against a wide range of microorganisms. Although it is soluble only at low pH, chitosan oligomers (ChitO) are soluble in pure water and thus more appropriate for antibacterial applications. Although there is a vast literature on chitosan's antimicrobial activity, the molecular details of its interaction with biomembranes remain unclear. Here we investigate these molecular interactions by resorting to phospholipid Langmuir films (zwitterionic DPPC and anionic DPPG) as simplified membrane models (for mammalian and bacterial membranes, respectively), and using SFG vibrational spectroscopy to probe lipid tail conformation, headgroup dynamics and interfacial water orientation. For comparison, we also investigate the interactions of another simple cationic antimicrobial polyelectrolyte, poly(allylamine) hydrochloride - PAH. By forming the lipid films over the polyelectrolyte solutions, we found that both have only a very small interaction with DPPC, but PAH adsorption is able to invert the interfacial water orientation (membrane potential). This might explain why ChitO is compatible with mammalian cells, while PAH is toxic. In contrast, their interaction with DPPG films is much stronger, even more so for ChitO, with both insertion within the lipid film and interaction with the oppositely charged headgroups. Again, PAH adsorption inverts the membrane potential, while ChitO does not. Finally, ChitO interaction with DPPG is weaker if the antimicrobial is injected underneath a pre-assembled Langmuir film, and its interaction mode depends on the time interval between end of film compression and ChitO injection. These differences between ChitO and PAH effects on the model membranes highlight the importance of molecular structure and intermolecular interactions for their bioactivity, and therefore this study may provide insights for the rational design of more effective antimicrobial molecules.

Interaction of chitosan derivatives with cell membrane models in a biologically relevant medium.

HYPOTHESIS: The interaction of chitosan, a natural biopolymer with various biomedical applications, with lipid Langmuir films has been widely investigated as a simple model for cell membranes. However, to ensure polymer solubility, up to now only acidic subphases with pH significantly below biological fluids have been used. To increase the biological significance of these investigations, here we evaluated the effects of two chitosan derivatives (low molecular weight - CH, and positively charged - CH-P40) on phospholipid films (either zwitterionic DPPC or anionic DPPG) using phosphate buffered saline solutions (PBS) as a subphase. EXPERIMENTS: Surface pressure - area (π-A) isotherms were used to evaluate the expansion and changes in film elasticity, while Sum-Frequency Generation (SFG) vibrational spectroscopy provided information about the chain conformation of lipids. FINDINGS: It was found that chitosans caused a small expansion of the DPPC film by its insertion within the monolayer. In contrast, they distinctly expanded DPPG monolayers by both chitosan insertion within the lipid monolayer and by interacting with the anionic head group. Therefore, PBS buffer can be used as a subphase for more biologically relevant studies of chitosan interactions with Langmuir films, shedding light on why chitosan is antibacterial but not toxic to mammals, as the interaction mechanism depends on lipid headgroup charge.

Genosensor made with a self-assembled monolayer matrix to detect MGMT gene methylation in head and neck cancer cell lines.

DNA methylation is involved in the oncogenesis of head and neck squamous cell carcinoma and could be used for early detection of cancer to increase the chances of cure, but unfortunately diagnosis is usually made at late stages of the disease. In this work we developed genosensors to detect DNA methylation of the MGMT gene in head and neck cancer cell lines. The probe for MGMT promoter methylation was immobilized on gold electrodes modified with 11-mercaptoundecanoic acid (11-MUA) self-assembled monolayers (SAM). Detection was performed with electrochemical impedance spectroscopy, with clear distinction between methylated and non-methylated DNA from head and neck cell lines. The genosensor is sensitive with a low detection limit of 0.24 × 10-12 mol L-1. In addition, the cell lines FaDu, JHU28 and SCC25 for the MGMT gene, could be distinguished from the HN13 cell line which has a high degree of MGMT methylation (97%), thus confirming the selectivity. Samples with different percentages of MGMT DNA methylation could be separated in multidimensional projections using the visualization technique interactive document mapping (IDMAP). The genosensor matrix and the immobilization procedures are generic, and can be extended to other DNA methylation biomarkers.

Self-assembled amphiphilic chitosan nanoparticles for quercetin delivery to breast cancer cells.

Novel drug delivery strategies are needed to meet the complex challenges associated to cancer therapy. Biocompatible pH-sensitive drug delivery nanocarriers based on amphiphilic co-polymers seem to be promising for cancer treatment. In the present study, a drug delivery system was produced by encapsulating quercetin into novel pH-sensitive self-assembled amphiphilic chitosan nanoparticles. Up to 83% of quercetin was entrapped by the nanoparticles. The particle diameter, as measured by dynamic light scattering (DLS), ranged from ∼235 to ∼312 nm for the blank and ∼490 to ∼502 nm for the loaded carriers. The results showed that the payload release is larger at acidic pH (5.0) than at physiological pH (7.4). Fitting the data to the Korsmeyer-Peppas model indicated that anomalous diffusion is the predominant release mechanism at pH 5.0, while Fickian diffusion operates at pH 7.4. The MTT assay revealed that blank nanoparticles were non-antiproliferative for the cell tested. The results further revealed that quercetin maintains its metabolism inhibition against MCF-7 cells after encapsulation. Cellular uptake experiments showed that nanoparticles accumulated on the cell surface, whereas few were internalized. Haemocompatibility test results suggest that the nanoparticles exhibit suitable blood compatibility for biological applications. Results suggest that nanoparticles might be a promising pH-sensitive drug delivery system for applications in anticancer treatment.

Synergistic effect of quercetin and pH-responsive DEAE-chitosan carriers as drug delivery system for breast cancer treatment.

Amphiphilic chitosans, which may self-assemble in aqueous solution to form nanoaggregates with different conformations depending to the environmental pH, can be used as drug transport and delivery agents, when the target pH differs from the delivery medium pH. In this study, quercetin, a bioactive flavonoid, was encapsulated in a pH-responsive system based on amphiphilic chitosan. The hydrophilic reagent 2-chloro-N,N-diethylethylamine hydrochloride (DEAE), also known to inhibit the proliferation of cancer cells, was used as a grafting agent. Drug loading experiments (DL ∼5%) showed a quercetin entrapment efficiency of 73 and 78% for the aggregates. The sizes of blank aggregates measured by dynamic light scattering (DLS) varied from 169 to 263nm and increased to ∼410nm when loaded with quercetin. The critical aggregation concentration, zeta potential and morphology of the aggregates were determined. pH had a dominant role in the release process and Fickian diffusion was the controlling factor in drug release according to the Korsmeyer-Peppas mathematical model. In vitro studies indicated that the DEAE-modified chitosan nanoaggregates showed a synergistic effect with quercetin on the control of the viability of MCF-7 cells. Therefore, DEAE-modified chitosan nanoaggregates with pH-sensibility can be used as optimized nanocarriers in cancer therapy.

Syntheses and characterization of amphiphilic quaternary ammonium chitosan derivatives.

Amphiphilic chitosan derivatives possess improved physico-chemical properties and could be used as carriers in drug delivery systems. The aim of this study was to investigate the behaviour of an amphiphilic system involving (5-pentyl) trimethylammonium and dodecyl aldehyde-modified chitosan. Amphiphilic chitosan derivatives were synthesized and characterized by (1)HNMR and ATR-FTIR spectroscopy. Self-assembled aggregates formed in aqueous solution have hydrophobic cores that were characterized by fluorescence spectroscopy using pyrene as probe and dynamic light scattering (DLS). The critical aggregation concentration of the aggregates in water varied from 0.004 to 0.037g/L and the average size distribution was in the 230-500nm range. The ζ-potential (+15.5 to +44.8mV) confirmed that the surfaces of the aggregates were positively charged and stable in physiological-like environments. TEM images suggest that the aggregates have a spherical shape, showing good agreement with DLS results. These results suggest that the synthesized copolymers have the capability of being used as carriers for hydrophobic drugs.

Hydrophobic effect of amphiphilic derivatives of chitosan on the antifungal activity against Aspergillus flavus and Aspergillus parasiticus.

Low molecular weight amphiphilic derivatives of chitosan were synthesized, characterized and their antifungal activities against Aspergillus flavus and Aspergillus parasiticus were tested. The derivatives were synthesized using as starting material a deacetylated chitosan sample in a two step process: the reaction with propyltrimethyl-ammonium bromide (Pr), followed by reductive amination with dodecyl aldehyde. Aiming to evaluate the effect of the hydrophobic modification of the derivatives on the antifungal activity against the pathogens, the degree of substitution (DS1) by Pr groups was kept constant and the proportion of dodecyl (Dod) groups was varied from 7 to 29% (DS2). The derivatives were characterized by ¹H-NMR and FTIR and their antifungal activities against the pathogens were tested by the radial growth of the colony and minimum inhibitory concentration (MIC) methods. The derivatives substituted with only Pr groups exhibited modest inhibition against A. flavus and A. parasiticus, like that obtained with deacetylated chitosan. Results revealed that the amphiphilic derivatives grafted with Dod groups exhibited increasing inhibition indexes, depending on polymer concentration and hydrophobic content. At 0.6 g/L, all amphiphilic derivatives having from 7.0 to 29% of Dod groups completely inhibited fungal growth and the MIC values were found to decrease from 4.0 g/L for deacetylated chitosan to 0.25-0.50 g/L for the derivatives. These new derivatives open up the possibility of new applications and avenues to develop effective biofungicides based on chitosan.

Synthesis, characterization and antifungal activity of quaternary derivatives of chitosan on Aspergillus flavus.

Two series of new chitosan derivatives were synthesized by reaction of deacetylated chitosan (CH) with propyl (CH-Propyl) and pentyl (CH-Pentyl) trimethylammonium bromides to obtain derivatives with increasing degrees of substitution (DS). The derivatives were characterized by (1)H NMR and potentiometric titration techniques and their antifungal activities on the mycelial growth of Aspergillus flavus were investigated in vitro. The antifungal activities increase with DS and the more substituted derivatives of both series, CH-Propyl and CH-Pentyl, exhibited antifungal activities respectively three and six times higher than those obtained with commercial and deacetylated chitosan. The minimum inhibitory concentrations (MIC) were evaluated at 24, 48 and 72 h by varying the polymer concentration from 0.5 to 16 g/L and the results showed that the quaternary derivatives inhibited the fungus growth at polymer concentrations four times lower than that obtained with deacetylated chitosan (CH). The chitosans modified with pentyltrimethylammonium bromide exhibited higher activity and results are discussed taking into account the degree of substitution (DS).