In the present study, the cationic MB-loaded PLGA nanoparticles exhibited the highest phototoxicity towards bacteria, followed by anionic-MB loaded PLGA nanoparticles and free MB in both suspensions and biofilms. In suspensions, cationic nanoparticles produced approximately 1 log10 killing. In oral microcosm laboratory biofilms, they reduced bacterial viability by 48%. The average percent killings for anionic nanoparticles and free MB were approximately 60% and 40% in suspensions and biofilms, respectively. Although these data were not statistically significant (p>0.05), results exhibited the same trend in all six planktonic or biofilm experiments concerning the greater phototoxicity of cationic MB-loaded nanoparticles.
The planktonic experiments clearly demonstrated the superiority of MB-loaded nanoparticles over anionic nanoparticles and free MB. It is promising that nanoparticles were taken up by microorganisms within a short period of time and were able to release MB amounts that led to bacterial destruction following their exposure to light. Recently, cationic biodegradable PLGA nanoparticles composed of chitosan were studied as gene carriers in the nasal mucosa of mice in vivo
). The results of this study showed that PLGA nanoparticles facilitated gene delivery and subsequent expression with increased efficiency. Also, cationic eudragit containing PLGA nanoparticles showed better adhesion to Pseudomonas aeruginosa
and Staphylococcus aureus
than anionic PLGA nanoparticles (30
). The enhanced electrostatic interaction between the cationic nanoparticles and the negatively charged residues of the lipid bilayer generate nanoscale holes in the outer membrane (31
). This may explain the greater toxicity of cationic nanoparticles in the absence of light in planktonic phase compared with anionic nanoparticles and free MB. It is also possible that the use of MB-loaded nanoparticles limited the ability of microorganisms to pump the MB molecule back out. The faster release of MB by cationic nanoparticles may also have contributed to their greater phototoxicity over anionic nanocarriers. The incomplete photodestruction of dental plaque bacteria in suspension may be related to the phenotypic changes carried by these microorganisms once they were biofilm species (14
). However, it is possible that that the use of other drug and light parameters (e.g. amount of MB encapsulated in nanoparticles, incubation time, power density, energy fluence) may lead to complete bacterial destruction.
The effect of light resulted in lower reductions of microorganisms within biofilms, which was not surprising (11
). Biofilm bacteria showed resistance to PDT, with killing not exceeding 48% (for cationic MB-loaded nanoparticles) compared with dark controls. The microcosm biofilm model employed in this study originated directly from the whole-mixed natural dental plaque and showed a structure that resembled that of natural dental plaque as revealed by confocal scanning laser microscopy. This model was validated and tested previously (11
). Despite the reduced PDT bacterial destruction in biofilms compared with suspensions, the effect was much greater than seen with antibiotic therapy. In planktonic experiments the bacterial killing was two-fold greater compared with biofilms, whereas antibiotics were approximately 250-fold less effective in biofilm state (33
). The reduced susceptibility of bacteria in biofilms may be attributed to the negatively charged matrix that hinders penetration of a positively charged agent, such as MB and cationic nanoparticles, because of strong ionic interactions. However, this generalization is difficult to justify because many different factors play a role including the particular system under investigation, the chemical composition of the matrix as well as the physicochemical properties and chemical reactivity of the antimicrobial agent (34
). It has been reported that even when there is strong ionic interaction between a negatively charged matrix and a positively charged antimicrobial agent, diffusion of the agent is not hindered to a great extent and, once all of the binding sites have been filled, the matrix would not present any further barrier to diffusion (36
). It is also possible that MB penetration may have been enhanced by either passive targeting or by active targeting via the charged surface of the nanoparticle. Our efforts to study penetration and distribution of nanoparticles into the biofilms by confocal scanning laser microscopy were not successful; it was not possible to detect traces of MB fluorescence in biofilms. The reduced susceptibility of biofilms to PDT using charged nanoparticles may also be due to a failure of nanoparticles to penetrate into the interior of cell clusters by forming aggregates with other nanoparticles as well as sticking to biofilm surface. Aggregation of nanoparticles can form a mass larger than the size of a biofilm channel and therefore block or hinder the entrance of released MB completely. Finally, the increased density of bacterial clusters within biofilms results in a microenvironment with low pO2
that may be responsible for the reduced PDT effect.
Our findings suggest that cationic PLGA nanoparticles have the potential to be used as carriers of MB for photodestruction of oral biofilms. The greater PDT bacterial killing by cationic MB-loaded nanoparticles showed the ability of nanocarriers to diffuse in biofilms and release the encapsulated drug in the active form. However, it is not certain that the sufficient concentrations of MB were released in order to have the greatest possible effect in eradication of the biofilm organisms. Therefore, future studies should define the physical characteristics of nanoparticles (e.g. size, zeta potential) that are important in determining their intracellular uptake and trafficking. In addition, the optimal PDT parameters for effective elimination of biofilm species should be determined and the safety of PDT should be demonstrated by defining the therapeutic window where bacteria could be killed leaving mammalian cells intact.