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Development of noncytotoxic chitosan-gold nanocomposites as efficient antibacterial materials.

Regiel-Futyra A, Kus-Liśkiewicz M, Sebastian V, Irusta S, Arruebo M, Stochel G, Kyzioł A - ACS Appl Mater Interfaces (2015)

Bottom Line: The resulting nanocomposites did not show any cytotoxicity against mammalian somatic and tumoral cells.They produced a disruptive effect on the bacteria wall while their internalization was hindered on the eukaryotic cells.This selectivity and safety make them potentially applicable as antimicrobial coatings in the biomedical field.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Chemistry, Jagiellonian University , Ingardena 3, 30-060 Kraków, Poland.

ABSTRACT
This work describes the synthesis and characterization of noncytotoxic nanocomposites either colloidal or as films exhibiting high antibacterial activity. The biocompatible and biodegradable polymer chitosan was used as reducing and stabilizing agent for the synthesis of gold nanoparticles embedded in it. Herein, for the first time, three different chitosan grades varying in the average molecular weight and deacetylation degree (DD) were used with an optimized gold precursor concentration. Several factors were analyzed in order to obtain antimicrobial but not cytotoxic nanocomposite materials. Films based on chitosan with medium molecular weight and the highest DD exhibited the highest antibacterial activity against biofilm forming strains of Staphylococcus aureus and Pseudomonas aeruginosa. The resulting nanocomposites did not show any cytotoxicity against mammalian somatic and tumoral cells. They produced a disruptive effect on the bacteria wall while their internalization was hindered on the eukaryotic cells. This selectivity and safety make them potentially applicable as antimicrobial coatings in the biomedical field.

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SEM micrographs representingthe morphology of the bacteria cellwall upon contact with chitosan and chitosan–gold nanocomposites(CS_M with 5 and 10 mM gold initial precursor) on (A) S. aureus ATTC 25923 and (B) P. aeruginosa ATCC 27853.
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fig8: SEM micrographs representingthe morphology of the bacteria cellwall upon contact with chitosan and chitosan–gold nanocomposites(CS_M with 5 and 10 mM gold initial precursor) on (A) S. aureus ATTC 25923 and (B) P. aeruginosa ATCC 27853.

Mentions: Films based on chitosanwith medium Mw and one of the uppermostgold content (M5) demonstrated the highest antibacterial effect incomparison to chitosan with low and high Mw based composites. Gram-negativebiofilm forming strains (P. aeruginosa) appeared to be more resistant than Gram-positive S. aureus at each gold nanoparticle concentration.Based on these results, CS_M was selected for the preparation of nanocompositeswith the highest AuNP content, M10. A total bactericidal effect forthose materials was obtained (Figure 7*). Themolecular weight of chitosan clearly affects the antibacterial activityof the resulting nanocomposites. The biocidal effect was reduced formaterials based on CS_H, intermediate for CS_L and finally the mosteffective antimicrobial material appeared to be CS_M. Additionally,SEM analysis was carried out in order to evaluate the morphologicalchanges in the bacterial cell wall upon contact with the bactericidalfilms (M5 and M10). Bacterial cell structural damage, induced by CS-AuNPs,was clearly observed for both tested strains (Figure 8). Multiple holes and perforations were formed on the surfaceof S. aureus after exposure to M5 andM10 films, resulting in a total cell disintegration. Similarly, P. aeruginosa cells seem to alter their form, fromelongated bacillus to ragged and irregular shapes, which confirmstheir total lysis. Results stay in agreement with the obtained CFUvalues. The presented characteristics of the prepared nanocompositesenable to analyze and understand their biological activity more accurately.Several reports concerning the mechanism of chitosan or gold nanoparticlesantibacterial activity have been presented.49,57 However, the exact mechanisms have not been elucidated yet. Otherauthors demonstrate that polycationic chitosan interacts with negativelycharged bacterial cell wall and leads to intracellular componentsleakage.67 The higher DD and amino groupsnumber, the higher positive charge enabling interactions with cellwall and finally, the better antibacterial potential of pure polymericfilms.68 Also, low molecular weight ofthe polymer facilitates cell wall penetration and interaction withintracellular components whereas high Mw enables only surface interactions.69 Here, the main bactericidal effect is a resultof the AuNPs activity, which is also an object of many scientificpapers trying to explain their mechanism. AuNPs can interact withsulfur-containing proteins in the cell membrane changing its permeability,leading to intracellular components leakage and finally cell deathor/and bind to DNA and inhibit transcription.70 As the positive charge of the polymer is greatly reduced upon AuNPsynthesis and further film formation, the antibacterial activity ofchitosan films decreases in comparison to the polymeric dispersion.Still, bacteriostatic activity of chitosan films can be observed.It has been shown that size and AuNP dispersion degree influence theirantimicrobial activity. The smaller and well-distributed gold nanoparticles,the more significant bacteria depletion occurs. Chitosan with mediumMw appears to be the best stabilizing agent for AuNP formation. Theobtained gold nanoparticles have the smallest size and the most uniformdistribution across the resulting films when using this medium Mwchitosan. Thanks to the high DD and thus the high number of aminogroups responsible for NPs formation, a high reduction rate for thegold ions is also obtained.71 Because thereduction and seed formation occur in many places at once, the smallestnanoparticles are formed compared to the other Mw chitosans tested.As the viscosity of the polymer increases, the formation of less nucleationcenters is more probable due to the hindered ion diffusion and reducingagent across the gel. The molecular weight of the polymer influencesalso further AuNP distribution in the resulting film. Low and highMw polymers do not ensure good NP distribution across the film dueto their insufficient stabilization and thus diffusion or aggregatesformation, respectively. Our results stay in agreement with previouswork of Prema et al. and Zhang et al., who presented chitosan andother polysaccharide stabilized gold nanoparticles as antibacterialagents.57,72 Bacterial cell wall morphology upon incubationwith chitosan medium based nanocomposites was further analyzed, andthe results support their bactericidal action (Figure 8). For both bacterial strains tested, significant and progressivedamage on the cell wall can be observed, which resulted in total celllysis. Another important aspect that we present is the importanceof a direct contact between materials and bacteria in order to achievebactericidal effect. Even when the XPS results showed a low gold concentrationon the surface, during the bactericidal test swelling of the polymerwould occur, allowing the contact of AuNP with the bacteria. We confirmedthe absence of AuNP detachment from the prepared nanocomposites byUV–vis spectrophotometry. Those results importantly contributeto the cytotoxicity test outcomes explanation.


Development of noncytotoxic chitosan-gold nanocomposites as efficient antibacterial materials.

Regiel-Futyra A, Kus-Liśkiewicz M, Sebastian V, Irusta S, Arruebo M, Stochel G, Kyzioł A - ACS Appl Mater Interfaces (2015)

SEM micrographs representingthe morphology of the bacteria cellwall upon contact with chitosan and chitosan–gold nanocomposites(CS_M with 5 and 10 mM gold initial precursor) on (A) S. aureus ATTC 25923 and (B) P. aeruginosa ATCC 27853.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4326049&req=5

fig8: SEM micrographs representingthe morphology of the bacteria cellwall upon contact with chitosan and chitosan–gold nanocomposites(CS_M with 5 and 10 mM gold initial precursor) on (A) S. aureus ATTC 25923 and (B) P. aeruginosa ATCC 27853.
Mentions: Films based on chitosanwith medium Mw and one of the uppermostgold content (M5) demonstrated the highest antibacterial effect incomparison to chitosan with low and high Mw based composites. Gram-negativebiofilm forming strains (P. aeruginosa) appeared to be more resistant than Gram-positive S. aureus at each gold nanoparticle concentration.Based on these results, CS_M was selected for the preparation of nanocompositeswith the highest AuNP content, M10. A total bactericidal effect forthose materials was obtained (Figure 7*). Themolecular weight of chitosan clearly affects the antibacterial activityof the resulting nanocomposites. The biocidal effect was reduced formaterials based on CS_H, intermediate for CS_L and finally the mosteffective antimicrobial material appeared to be CS_M. Additionally,SEM analysis was carried out in order to evaluate the morphologicalchanges in the bacterial cell wall upon contact with the bactericidalfilms (M5 and M10). Bacterial cell structural damage, induced by CS-AuNPs,was clearly observed for both tested strains (Figure 8). Multiple holes and perforations were formed on the surfaceof S. aureus after exposure to M5 andM10 films, resulting in a total cell disintegration. Similarly, P. aeruginosa cells seem to alter their form, fromelongated bacillus to ragged and irregular shapes, which confirmstheir total lysis. Results stay in agreement with the obtained CFUvalues. The presented characteristics of the prepared nanocompositesenable to analyze and understand their biological activity more accurately.Several reports concerning the mechanism of chitosan or gold nanoparticlesantibacterial activity have been presented.49,57 However, the exact mechanisms have not been elucidated yet. Otherauthors demonstrate that polycationic chitosan interacts with negativelycharged bacterial cell wall and leads to intracellular componentsleakage.67 The higher DD and amino groupsnumber, the higher positive charge enabling interactions with cellwall and finally, the better antibacterial potential of pure polymericfilms.68 Also, low molecular weight ofthe polymer facilitates cell wall penetration and interaction withintracellular components whereas high Mw enables only surface interactions.69 Here, the main bactericidal effect is a resultof the AuNPs activity, which is also an object of many scientificpapers trying to explain their mechanism. AuNPs can interact withsulfur-containing proteins in the cell membrane changing its permeability,leading to intracellular components leakage and finally cell deathor/and bind to DNA and inhibit transcription.70 As the positive charge of the polymer is greatly reduced upon AuNPsynthesis and further film formation, the antibacterial activity ofchitosan films decreases in comparison to the polymeric dispersion.Still, bacteriostatic activity of chitosan films can be observed.It has been shown that size and AuNP dispersion degree influence theirantimicrobial activity. The smaller and well-distributed gold nanoparticles,the more significant bacteria depletion occurs. Chitosan with mediumMw appears to be the best stabilizing agent for AuNP formation. Theobtained gold nanoparticles have the smallest size and the most uniformdistribution across the resulting films when using this medium Mwchitosan. Thanks to the high DD and thus the high number of aminogroups responsible for NPs formation, a high reduction rate for thegold ions is also obtained.71 Because thereduction and seed formation occur in many places at once, the smallestnanoparticles are formed compared to the other Mw chitosans tested.As the viscosity of the polymer increases, the formation of less nucleationcenters is more probable due to the hindered ion diffusion and reducingagent across the gel. The molecular weight of the polymer influencesalso further AuNP distribution in the resulting film. Low and highMw polymers do not ensure good NP distribution across the film dueto their insufficient stabilization and thus diffusion or aggregatesformation, respectively. Our results stay in agreement with previouswork of Prema et al. and Zhang et al., who presented chitosan andother polysaccharide stabilized gold nanoparticles as antibacterialagents.57,72 Bacterial cell wall morphology upon incubationwith chitosan medium based nanocomposites was further analyzed, andthe results support their bactericidal action (Figure 8). For both bacterial strains tested, significant and progressivedamage on the cell wall can be observed, which resulted in total celllysis. Another important aspect that we present is the importanceof a direct contact between materials and bacteria in order to achievebactericidal effect. Even when the XPS results showed a low gold concentrationon the surface, during the bactericidal test swelling of the polymerwould occur, allowing the contact of AuNP with the bacteria. We confirmedthe absence of AuNP detachment from the prepared nanocomposites byUV–vis spectrophotometry. Those results importantly contributeto the cytotoxicity test outcomes explanation.

Bottom Line: The resulting nanocomposites did not show any cytotoxicity against mammalian somatic and tumoral cells.They produced a disruptive effect on the bacteria wall while their internalization was hindered on the eukaryotic cells.This selectivity and safety make them potentially applicable as antimicrobial coatings in the biomedical field.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Chemistry, Jagiellonian University , Ingardena 3, 30-060 Kraków, Poland.

ABSTRACT
This work describes the synthesis and characterization of noncytotoxic nanocomposites either colloidal or as films exhibiting high antibacterial activity. The biocompatible and biodegradable polymer chitosan was used as reducing and stabilizing agent for the synthesis of gold nanoparticles embedded in it. Herein, for the first time, three different chitosan grades varying in the average molecular weight and deacetylation degree (DD) were used with an optimized gold precursor concentration. Several factors were analyzed in order to obtain antimicrobial but not cytotoxic nanocomposite materials. Films based on chitosan with medium molecular weight and the highest DD exhibited the highest antibacterial activity against biofilm forming strains of Staphylococcus aureus and Pseudomonas aeruginosa. The resulting nanocomposites did not show any cytotoxicity against mammalian somatic and tumoral cells. They produced a disruptive effect on the bacteria wall while their internalization was hindered on the eukaryotic cells. This selectivity and safety make them potentially applicable as antimicrobial coatings in the biomedical field.

Show MeSH
Related in: MedlinePlus