Structural, Mechanical, and Barrier Properties of the Polyvinylidene Fluoride-Bacterial Nanocellulose-Based Hybrid Composite.

This study presents an analysis of films which consist of two layers; one layer is PVDF as the matrix, along with fillers BaTiO3 (BT), and the second is one bacterial nanocellulose (BNC) filled with Fe3O4. The mass fraction of BT in PVDF was 5%, and the samples were differentiated based on the duration of the mechanical activation of BT. This innovative PVDF laminate polymer with environmentally friendly fillers aligns with the concept of circular usage, resulting in a reduction in plastic content and potential improvement of the piezoelectric properties of the entire composite. This work presents new, multifunctional "green" packaging materials that potentially could be a good alternative to specific popular materials used for this purpose. The synthesis of the films was carried out using the hot press method. Tensile tests, water vapor permeability examination, and structural analyses using SEM-EDS and FTIR have been conducted. The sample PVDF/BT20/BNC/Fe3O4 exhibited the best barrier properties (impermeability to water vapor), while the highest tensile strength and toughness were exhibited by the PVDF/BT5/BNC/Fe3O4 sample.

Reduction in Pathogenic Biofilms by the Photoactive Composite of Bacterial Cellulose and Nanochitosan Dots under Blue and Green Light.

In this study, nanochitosan dots (ChiDs) were synthesized using gamma rays and encapsulated in bacterial cellulose (BC) polymer matrix for antibiofilm potential in photodynamic therapy. The composites were analyzed for structural changes using SEM, AFM, FTIR, XRD, EPR, and porosity measurements. Additionally, ChiD release was assessed. The results showed that the chemical composition remained unaltered, but ChiD agglomerates embedded in BC changed shape (1.5-2.5 µm). Bacterial cellulose fibers became deformed and interconnected, with increased surface roughness and porosity and decreased crystallinity. No singlet oxygen formation was observed, and the total amount of released ChiD was up to 16.10%. Antibiofilm activity was higher under green light, with reductions ranging from 48 to 57% under blue light and 78 to 85% under green light. Methicillin-resistant Staphylococcus aureus was the most sensitive strain. The new photoactive composite hydrogels show promising potential for combating biofilm-related infections.

Effects of Synthesis Parameters on Structure and Antimicrobial Properties of Bacterial Cellulose/Hydroxyapatite/TiO2 Polymer-Ceramic Composite Material.

Bacterial cellulose (BC) is a highly pure polysaccharide biopolymer that can be produced by various bacterial genera. Even though BC lacks functional properties, its porosity, three-dimensional network, and high specific surface area make it a suitable carrier for functional composite materials. In the present study, BC-producing bacteria were isolated from kombucha beverage and identified using a molecular method. Two sets of the BC hydrogels were produced in static conditions after four and seven days. Afterwards, two different synthesis pathways were applied for BC functionalization. The first method implied the incorporation of previously synthesized HAp/TiO2 nanocomposite using an immersion technique, while the second method included the functionalization of BC during the synthesis of HAp/TiO2 nanocomposite in the reaction mixture. The primary goal was to find the best method to obtain the functionalized material. Physicochemical and microstructural properties were analyzed by SEM, EDS, FTIR, and XRD methods. Further properties were examined by tensile test and thermogravimetric analysis, and antimicrobial activity was assessed by a total plate count assay. The results showed that HAp/TiO2 was successfully incorporated into the produced BC hydrogels using both methods. The applied methods of incorporation influenced the differences in morphology, phase distribution, mechanical and thermal properties, and antimicrobial activity against Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Proteus mirabilis (ATCC 12453), and Candida albicans (ATCC 10231). Composite material can be recommended for further development and application in environments that are suitable for diseases spreading.

Dielectric and Structural Properties of the Hybrid Material Polyvinylidene Fluoride-Bacterial Nanocellulose-Based Composite.

In the search for environmentally friendly materials with a wide range of properties, polymer composites have emerged as a promising alternative due to their multifunctional properties. This study focuses on the synthesis of composite materials consisting of four components: bacterial nanocellulose (BNC) modified with magnetic Fe3O4, and a mixture of BaTiO3 (BT) and polyvinylidene fluoride (PVDF). The BT powder was mechanically activated prior to mixing with PVDF. The influence of BT mechanical activation and BNC with magnetic particles on the PVDF matrix was investigated. The obtained composite films' structural characteristics, morphology, and dielectric properties are presented. This research provides insights into the relationship between mechanical activation of the filler and structural and dielectric properties in the PVDF/BT/BNC/Fe3O4 system, creating the way for the development of materials with a wide range of diverse properties that support the concept of green technologies.

Anticancer effect of novel luteolin capped gold nanoparticles selectively cytotoxic towards human cervical adenocarcinoma HeLa cells: An in vitro approach.

BACKGROUND: Although luteolin has been confirmed as potent anticancer agent, its potential application as therapeutic is limited by its water solubility. To overcome this shortcoming nanoparticle technology approach was applied. Owing to their proven low toxicity and the possibility to be easily functionalized gold nanoparticles (AuNP) were the nanosystem of choice used in this study. Novel luteolin capped gold nanoparticles (AuNPL) were synthesized and their anticancer effect towards human cervical adenocarcinoma HeLa cells was investigated in vitro. METHODS: AuNPL were synthesized by reducing chloroauric acid by trisodium citrate with subsequent addition of luteoline during synthesis and their physicochemical characterization was done. AuNPL cytotoxicity against HeLa, human malignant melanoma A375, and normal human keratinocytes HaCaT cells was tested by MTT cell survival assay, and their IC50 values were determined. The capability of AuNPL to induce cell cycle arrest and apoptosis in HeLa cells were demonstrated by flow cytometry. The antioxidant activity of AuNPL was assessed by DPPH· and ABTS·+ scavenging assays. Cytoprotective properties of AuNPL towards HaCaT cells were examined by measuring the physiological and H2O2 induced intracellular reactive oxygen species (ROS) levels using flow cytometry. Also, genotoxicity of AuNPL in HaCaT cells was investigated by the single cell alkaline comet assay. RESULTS: Spherical AuNPL, stable in aqueous solution up to six months at 4 °C were obtained in the synthesis. The selectivity in the cytotoxic action of AuNPL on HeLa and A375 cancer cells compared with their cytotoxicity on normal keratinocytes HaCaT was observed. AuNPL exerted their cytotoxic activity against HeLa cells through accumulation of the cells in the subG1 phase of the cell cycle, inducing the apoptotic cell death mediated by the activation of caspase-3 - 8, and - 9. AuNPL antioxidative potential was confirmed by DPPH· and ABTS·+ scavenging assays. IC50 concentration of AuNPL exerted cytoprotective effect against HaCaT cells by the significant reduction of the physiological intracellular ROS level. Additionally, AuNPL were shown as more cytoprotective towards HaCaT cells then luteolin due to the more successful elimination of H2O2 induced intracellular ROS. Moreover, nontoxic concentrations of AuNPL did not cause considerable DNA damage of HaCaT cells, indicating low genotoxicity of the nanoparticles. CONCLUSION: Synthesized AuNPL showed selective cytotoxic activity against HeLa cells, while being nontoxic and cytoprotective against HaCaT cells. The observed findings encourage further investigation of AuNPL as a promising novel anticancer agent.

Employing Gamma-Ray-Modified Carbon Quantum Dots to Combat a Wide Range of Bacteria.

Nowadays, it is a great challenge to develop new medicines for treating various infectious diseases. The treatment of these diseases is of utmost interest to further prevent the development of multi-drug resistance in different pathogens. Carbon quantum dots, as a new member of the carbon nanomaterials family, can potentially be used as a highly promising visible-light-triggered antibacterial agent. In this work, the results of antibacterial and cytotoxic activities of gamma-ray-irradiated carbon quantum dots are presented. Carbon quantum dots (CQDs) were synthesized from citric acid by a pyrolysis procedure and irradiated by gamma rays at different doses (25, 50, 100 and 200 kGy). Structure, chemical composition and optical properties were investigated by atomic force microscopy, transmission electron microscopy, X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, Raman spectroscopy, UV-Vis spectrometry and photoluminescence. Structural analysis showed that CQDs have a spherical-like shape and dose-dependent average diameters and heights. Antibacterial tests showed that all irradiated dots had antibacterial activity but CQDs irradiated with dose of 100 kGy had antibacterial activity against all seven pathogen-reference bacterial strains. Gamma-ray-modified CQDs did not show any cytotoxicity toward human fetal-originated MRC-5 cells. Moreover, fluorescence microscopy showed excellent cellular uptake of CQDs irradiated with doses of 25 and 200 kGy into MRC-5 cells.

Structural, optical, and bioimaging characterization of carbon quantum dots solvothermally synthesized from o-phenylenediamine.

Carbon quantum dots as a novel type of carbon nanomaterials have attracted the attention of many researchers because of their unique optical, antibacterial, and anticancer properties as well as their biocompatibility. In this study, for the first time, carbon quantum dots were prepared from o-phenylenediamine dissolved in toluene by a solvothermal route. Subsequently, the prepared carbon quantum dots were encapsulated into polyurethane films by a swelling-encapsulation-shrink method. Analyses of the results obtained by different characterization methods (AFM, TEM, EDS, FTIR, photoluminescence, and EPR) indicate the significant influence of the precursor on structural, chemical, and optical properties. Antibacterial and cytotoxicity tests showed that these dots did not have any antibacterial potential, because of the low extent of reactive oxygen species production, and showed low dark cytotoxicity. By investigating the cellular uptake, it was established that these dots penetrated the HeLa cells and could be used as probes for bioimaging.

Goat milk proteins enriched with Agaricus blazei Murrill ss. Heinem extracts: Electrophoretic, FTIR, DLS and microstructure characterization.

This study aimed to characterize mixtures of goat milk proteins and Agaricus blazei Murrill (ABM) extracts (aqueous, AE and polysaccharides, PE). The mixtures showed stable particles with ζ-potential more negative than -41.1 mV. The addition of AMB extracts to goat milk did not result in a significant particle size change, whereas their addition to heated goat milk significantly increased mean particle diameter (from 194 nm to 225 nm). Fourier Transform Infrared Spectroscopy (FTIR) showed that ABM extracts provoked changes in the secondary structure of goat milk proteins and interactions between polysaccharides and milk proteins predominantly via hydrogen and/or glycoside bonds and hydrophobic interactions. The milk protein profiles revealed proteolytic activity in mixtures with AE resulting in the formation of five new polypeptides. The different microstructures of mixtures with AE and PE were found by Scanning Electron Microscopy (SEM). A schematic representation of possible milk proteins-ABM extracts interactions has been given.

Structural Characterization of Nanocellulose/Fe3O4 Hybrid Nanomaterials.

The rise of innovation in the electrical industry is driven by the controlled design of new materials. The hybrid materials based on magnetite/nanocellulose are highly interesting due to their various applications in medicine, ecology, catalysis and electronics. In this study, the structure and morphology of nanocellulose/magnetite hybrid nanomaterials were investigated. The effect of nanocellulose loading on the crystal structure of synthesized composites was investigated by XRD and FTIR methods. The presented study reveals that the interaction between the cellulose and magnetic nanoparticles depends on the nanocellulose content. Further, a transition from cellulose II to cellulose I allomorph is observed. SEM and EDS are employed to determine the variation in morphology with changes in component concentrations. By the calculation of magnetic interactions between adjacent Fe3+ and Fe2+ ions within composites, it is determined that ferromagnetic coupling predominates.

Freeze vs. Spray Drying for Dry Wild Thyme (Thymus serpyllum L.) Extract Formulations: The Impact of Gelatin as a Coating Material.

Freeze drying was compared with spray drying regarding feasibility to process wild thyme drugs in order to obtain dry formulations at laboratory scale starting from liquid extracts produced by different extraction methods: maceration and heat-, ultrasound-, and microwave-assisted extractions. Higher total powder yield (based on the dry weight prior to extraction) was achieved by freeze than spray drying and lower loss of total polyphenol content (TPC) and total flavonoid content (TFC) due to the drying process. Gelatin as a coating agent (5% w/w) provided better TPC recovery by 70% in case of lyophilization and higher total powder yield in case of spray drying by diminishing material deposition on the wall of the drying chamber. The resulting gelatin-free and gelatin-containing powders carried polyphenols in amount ~190 and 53-75 mg gallic acid equivalents GAE/g of powder, respectively. Microwave-assisted extract formulation was distinguished from the others by a higher content of polyphenols, proteins and sugars, higher bulk density and lower solubility. The type of the drying process mainly affected the position of the gelatin-derived -OH and amide bands in FTIR spectra. Spray-dried formulations compared to freeze-dried expressed higher thermal stability as confirmed by differential scanning calorimetry analysis and a higher diffusion coefficient; the last feature can be associated with the lower specific surface area of irregularly shaped freeze-dried particles (151-223 µm) compared to small microspheres (~8 µm) in spray-dried powder.

Photoactive and antioxidant nanochitosan dots/biocellulose hydrogels for wound healing treatment.

Bacterial infection and their resistance to known antibiotics delays wound healing. In this study, nanochitosan dots (nChiD) produced by gamma irradiation have been encapsulated in bacterial cellulose (BC) polymer matrix to study the antibacterial potentials of these nanocomposites and their possible usage in wound healing treatment (scratch assay). Detailed analyses show that nChiDs have disc-like shape and average diameter in the range of 40 to 60 nm depending of the applied dose. All nChiDs as well as BC-nChiD nanocomposites emit green photoluminescence independently on the excitation wavelengths. The new designed nanocomposites do not have a cytotoxic effect; antioxidant analysis shows their moderate radical scavenging activity whereas antibacterial properties show significant growth inhibition of strains mostly found in difficult-to-heal wounds. The obtained results confirm that new designed BC-nChiD nanocomposites might be potential agent in wound healing treatment.

Bactericidal and antioxidant bacterial cellulose hydrogels doped with chitosan as potential urinary tract infection biomedical agent.

Therapy of bacterial urinary tract infections (UTIs) and catheter associated urinary tract infections (CAUTIs) is still a great challenge because of the resistance of bacteria to nowadays used antibiotics and encrustation of catheters. Bacterial cellulose (BC) as a biocompatible material with a high porosity allows incorporation of different materials in its three dimensional network structure. In this work a low molecular weight chitosan (Chi) polymer is incorporated in BC with different concentrations. Different characterization techniques are used to investigate structural and optical properties of these composites. Radical scavenging activity test shows moderate antioxidant activity of these biocompatible composites whereas in vitro release test shows that 13.3% of chitosan is released after 72 h. Antibacterial testing of BC-Chi composites conducted on Gram-positive and Gram-negative bacteria causing UTIs and CAUTIs (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae) and encrustation (Proteus mirabilis) show bactericidal effect. The morphology analysis of bacteria after the application of BC-Chi shows that they are flattened with a rough surface, with a tendency to agglomerate and with decreased length and width. All obtained results show that BC-Chi composites might be considered as potential biomedical agents in treatment of UTIs and CAUTIs and as a urinary catheter coating in encrustation prevention.

Altered organization of collagen fibers in the uninvolved human colon mucosa 10 cm and 20 cm away from the malignant tumor.

Remodelling of collagen fibers has been described during every phase of cancer genesis and progression. Changes in morphology and organization of collagen fibers contribute to the formation of microenvironment that favors cancer progression and development of metastasis. However, there are only few data about remodelling of collagen fibers in healthy looking mucosa distant from the cancer. Using SHG imaging, electron microscopy and specialized softwares (CT-FIRE, CurveAlign and FiberFit), we objectively visualized and quantified changes in morphology and organization of collagen fibers and investigated possible causes of collagen remodelling (change in syntheses, degradation and collagen cross-linking) in the colon mucosa 10 cm and 20 cm away from the cancer in comparison with healthy mucosa. We showed that in the lamina propria this far from the colon cancer, there were changes in collagen architecture (width, straightness, alignment of collagen fibers and collagen molecules inside fibers), increased representation of myofibroblasts and increase expression of collagen-remodelling enzymes (LOX and MMP2). Thus, the changes in organization of collagen fibers, which were already described in the cancer microenvironment, also exist in the mucosa far from the cancer, but smaller in magnitude.

Graphene quantum dots as singlet oxygen producer or radical quencher - The matter of functionalization with urea/thiourea.

Due to their low cost and possible green synthesis, high stability and resistance to photobleaching, graphene quantum dots (GQDs) can be considered as one of the class of carbon nanomaterials which may have great potential as an agent for photosensitized oxygen activation. In such a way, GQDs can be used as a theranostic agent in photodynamic therapy. In this work pristine GQDs, GQDs irradiated with gamma rays and GQDs doped with N and N, S atoms are produced using a simple, green approach. By using different techniques (AFM, HR-TEM, SEM-EDS, FTIR, XRD, PL and UV-Vis) we investigated structural and optical properties of the new types of GQDs. We showed that GQDs functionalized with thiourea (GQDs-TU) completely lost the ability to produce singlet oxygen (1O2) upon photoexcitation while functionalization with urea (GQDs-U) improves the capability of GQDs to produce 1O2 upon the same conditions. Thus, presented GQDs modification with urea seems like a promising approach for the production of the efficient photosensitizer. On the opposite, GQDs-TU are efficient OH quencher. Due to high singlet oxygen production and low cytotoxicity below 100 µg/mL against HeLa cells, GQDs-U is a good candidate as an agent in photodynamic therapy at this concentration.

Rheology and Microstructures of Rennet Gels From Differently Heated Goat Milk.

Rennet coagulation of goat milk heated to 65 °C/30 min (Gc), 80 °C/5 min (G8) and 90 °C/5 min (G9) was studied. A rheometer equipped with a vane geometry tool was used to measure milk coagulation parameters and viscoelastic properties of rennet gels. Yield parameters: curd yield, laboratory curd yield and curd yield efficiency were measured and calculated. Scanning electron microscopy of rennet gels was conducted. Storage moduli (G') of gels at the moment of cutting were 19.9 ± 1.71 Pa (Gc), 11.9 ± 1.96 Pa (G8) and 7.3 ± 1.46 Pa (G9). Aggregation rate and curd firmness decreased with the increase of milk heating temperature, while coagulation time did not change significantly. High heat treatment of goat milk had a significant effect on both laboratory curd yield and curd yield. However, laboratory curd yield (27.7 ± 1.84%) of the G9 treatment was unreasonably high compared to curd yield (15.4 ± 0.60%). The microstructure of G9 was notably different compared to Gc and G8, with a denser and more compact microstructure, smaller paracasein micelles and void spaces in a form of cracks indicating weaker cross links. The findings of this study might serve as the bases for the development of different cheese types produced from high-heat-treated goat milk.

Graphene oxide size and structure pro-oxidant and antioxidant activity and photoinduced cytotoxicity relation on three cancer cell lines.

Photoactive materials called photosensitizers can be used for treatment of different types of cancer in combination with light source. In this paper, we have investigated pro-oxidant and antioxidant potentials of four graphene based nanomaterials (graphene oxide-GO, graphene quantum dots-GQDs, carbon quantum dots-CQDs and N-doped carbon quantum dots-N-CQDs) depending on the presence/absence of visible light source. Structural and optical properties of these materials and their potentials for reactive oxygen species generation/quenching are investigated by applying different microscopy and spectroscopy techniques (transmission electron microscopy, FTIR, UV-Vis, photoluminescence, electron paramagnetic resonance). Results show that all types of quantum dots has pro-oxidant and antioxidant potentials whereas GO demonstrated only moderate antioxidant effect. The best free radical scavenger is CQDs sample in the absence of light. CQDs are the best singlet oxygen generator under blue light irradiation as well. To check photo-cytotoxicity of these materials, photo-cytotoxic concentrations of the GO, GQDs, CQDs and N-CQDs were determined for three cellular lines: human rhabdomyosarcoma (RD), cell line derived from human cervix carcinoma Hep2c (HeLa) and fibroblast cell line from murine (L2OB). Cytotoxicity test has indicated that all samples are much less photocytotoxic than cis-diamminedichloroplatinum (cis-DPP). The production method and doping of quantum dots affect the photodynamic activity of tested samples very much.

Controllable synthesis of Fe3O4-wollastonite adsorbents for efficient heavy metal ions/oxyanions removal.

Iron oxide, in the form of magnetite (MG)-functionalized porous wollastonite (WL), was used as an adsorbent for heavy metal ions (cadmium and nickel) and oxyanions (chromate and phosphate) removal from water. The porous WL was synthesized from calcium carbonate and siloxane by controlled sintering process using low molecular weight submicrosized poly(methyl methacrylate) as a pore-forming agent. The precipitation of MG nanoparticles was carried out directly by a polyol-medium solvothermal method or via branched amino/carboxylic acid cross-linker by solvent/nonsolvent method producing WL/MG and WL-γ-APS/MG adsorbents, respectively. The structure/properties of MG functionalized WL was confirmed by applying FTIR, Raman, XRD, Mössbauer, and SEM analysis. Higher adsorption capacities of 73.126, 66.144, 64.168, and 63.456 mg g-1 for WL-γ-APS/MG in relation to WL/MG of 55.450, 52.019, 48.132, and 47.382 mg g-1 for Cd2+, Ni2+, phosphate, and chromate, respectively, were obtained using nonlinear Langmuir model fitting. Adsorption phenomena were analyzed using monolayer statistical physics model for single adsorption with one energy. Kinetic study showed exceptionally higher pseudo-second-order rate constants for WL-γ-APS/MG, e.g., 1.17-13.4 times, with respect to WL/MG indicating importance of both WL surface modification and controllable precipitation of MG on WL-γ-APS.

Photo-induced antibacterial activity of four graphene based nanomaterials on a wide range of bacteria.

Due to controversial reports concerning antibacterial activity of different graphene based materials it is very important to investigate their antibacterial action on a wide range of Gram-positive and Gram-negative bacteria. In this paper we have investigated the structure induced phototoxic antibacterial activity of four types of graphene based materials: graphene oxide (GO), graphene quantum dots (GQDs), carbon quantum dots (CQDs) and nitrogen doped carbon quantum dots (N-CQDs). Antibacterial activity was tested on 19 types of bacteria. It is found that nanometer-size CQDs and N-CQDs are the most potent agents whereas micrometer-size GO has very poor antibacterial activity. Electron paramagnetic resonance measurements confirmed photodynamic production of singlet oxygen for all types of used quantum dots. Detailed analysis has shown that N-CQDs are an excellent photodynamic antibacterial agent for treatment of bacterial infections induced by Enterobacter aerogenes (E. aerogenes), Proteus mirabilis (P. mirabilis), Staphylococcus saprophyticus (S. saprophyticus), Listeria monocytogenes (L. monocytogenes), Salmonella typhimurium (S. typhimurium) and Klebsiella pneumoniae.

Effects of different carrier materials on physicochemical properties of microencapsulated grape skin extract.

The goal of this study was to investigate the characteristics of grape skin extract (GSE) spray dried with different carriers: maltodextrin (MD), gum Arabic (GA) and skim milk powder (SMP). The grape skin extract was obtained from winery by-product of red grape variety Prokupac (Vitis vinifera L.). The morphology of the powders, their thermal, chemical and physical properties (water activity, bulk and tapped densities, solubility), as well as release studies in different pH conditions were analyzed. Total anthocyanin content and total phenolic content were determined by spectrophotometric methods. MD and GA-based microparticles were non-porous and spherical, while SMP-based ones were irregularly shaped. The process of spray drying Prokupac GSE using these three carriers produced powders with low water activity (0.24-0.28), good powder characteristics, high yields, and solubility higher than 90%. The obtained dissolution/release profiles indicated prolonged release of anthocyanins and phenolic compounds in different mediums, especially from GSE/GA microparticles. These results have shown that grape skin as the main by-product of wine production could be used as a source of natural colorants and bioactive compounds, and microencapsulation as a promising technique for the protection of these compounds, their stabilization in longer periods and prolonged release.

Antibacterial potential of electrochemically exfoliated graphene sheets.

Electrochemically exfoliated graphene is functionalized graphene with potential application in biomedicine. Two most relevant biological features of this material are its electrical conductivity and excellent water dispersibility. In this study we have tried to establish the correlation between graphene structure and its antibacterial properties. The exfoliation process was performed in a two electrode-highly oriented pyrolytic graphite electrochemical cell. Solution of ammonium persulfate was used as an electrolyte. Exfoliated graphene sheets were dispersed in aqueous media and characterized by atomic force microscopy, scanning electron microscopy, Raman spectroscopy, Fourier transform infrared spectroscopy, X photoelectron spectroscopy, X-ray diffraction, electron paramagnetic resonance, zeta potential, contact angle measurements and surface energy. Antibacterial assays have shown lack of the significant antibacterial activity. Major effect on bacteria was slight change of bacteria morphology. Membrane remained intact despite significant change of chemical content of membrane components.