Free and Nanoencapsulated Tobramycin: Effects on Planktonic and Biofilm Forms of Pseudomonas

Cystic fibrosis (CF) is a genetic disorder in which frequent pulmonary infections develop secondarily. One of the major pulmonary pathogens colonizing the respiratory tract of CF patients and causing chronic airway infections is Pseudomonas aeruginosa. Although tobramycin was initially effective against P. aeruginosa, tobramycin-resistant strains have emerged. Among the strategies for overcoming resistance to tobramycin and other antibiotics is encapsulation of the drugs in nanoparticles. In this study, we explored the antimicrobial activity of nanoencapsulated tobramycin, both in solid lipid nanoparticles (SLN) and in nanostructured lipid carriers (NLC), against clinical isolates of P. aeruginosa obtained from CF patients. We also investigated the efficacy of these formulations in biofilm eradication. In both experiments, the activities of SLN and NLC were compared with that of free tobramycin. The susceptibility of planktonic bacteria was determined using the broth microdilution method and by plotting bacterial growth. The minimal biofilm eradication concentration (MBEC) was determined to assess the efficacy of the different tobramycin formulations against biofilms. The activity of tobramycin-loaded SLN was less than that of either tobramycin-loaded NLC or free tobramycin. The minimum inhibitory concentration (MIC) and MBEC of nanoencapsulated tobramycin were slightly lower (1–2 logs) than the corresponding values of the free drug when determined in tobramycin-susceptible isolates. However, in tobramycin-resistant strains, the MIC and MBEC did not differ between either encapsulated form and free tobramycin. Our results demonstrate the efficacy of nanoencapsulated formulations in killing susceptible P. aeruginosa from CF and from other patients.


Introduction
Cystic fibrosis (CF) is the most common genetic disorder in the Caucasian population and it is characterized by a high morbidity and mortality. The CF lung is compromised by the production of viscous mucus secretions, resulting in a debilitated mucociliary clearance that promotes bacterial infection and inflammation [1]. Pseudomonas aeruginosa is the predominant opportunistic pathogen infecting the respiratory tract of CF patients. Once chronic lung colonization occurs, P. aeruginosa changes phenotypically to produce alginate, which allows the bacterium to become established within nanoparticles spread homogenously through the lung and there is no migration of lipid nanoparticles to other organs, such as liver, spleen, or kidneys [21].

Bacterial Isolates
The 34 clinical isolates of P. aeruginosa (17 non-mucoid and 17 mucoid) included in this study were obtained from the sputum samples and pharyngeal exudates of CF patients seen at the University Hospital Vall d'Hebrón and University Hospital Sant Joan de Déu (Barcelona, Spain) between January and April 2012. The patients (59% female, 41% male) ranged in age from 9 to 50 years (mean: 27 years). P. aeruginosa strains ATCC 27853 and PAO1 served as the control strains in the drug susceptibility assays and biofilm studies, respectively. Two CF clinical isolates, P. aeruginosa strain 362VH (tobramycin-resistant) and strain 056SJD (tobramycin-susceptible), were used to evaluate the ability of free and nanoencapsulated tobramycin to inhibit bacterial growth and eradicate bacterial biofilms. Table 1 summarizes the isolates used and their main characteristics.  S  S  S  S  S  S  S  S  S  2881M SJD  13  Male  +  -S  I  R  R  R  S  S  S  S  R  610M SJD  13  Male  +  -S  S  R  R  R  S  S  S  S  R  610 SJD  13  Male  -ß  S  S  R  I  R  S  S  S  S  R  805 SJD  15  Female  -ß  S  S  S  S  S  R  S  S  S  S  555.1 SJD  7  Female  +  -S  S  S  S  S  S  S  S  S  S  PA 417 VH  17  Female  -ß  R  R  R  S  S  S  S  S  S  R  PA 362 VH  36  Male  +  ß  S  S  S  S  S  S  R  S  S  S  PA 684 VH  32  Male  --S  S  I  R  R  S  S  I  S  I  PA 103 VH  29  Female  +  -R  S  S  S  S  R  S  R  S  S  023 VH  15  Male  +  -S  R  I  R  R  R  R  S  S  852 VH  17  Male  --S  S  S  S  S  S  S  S  S  S  153 VH  17  Female  +  -S  S  S  S  S  S  S  S  S  R  516 VH  20  Female  +  -S  S  S  S  S  S  S  S  S  S  547 VH  15 Male

Preparation of Lipid Nanoparticles
Tobramycin-loaded nanoparticles were prepared as described. Briefly, two loaded formulations were elaborated, namely solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) [21]. An emulsion solvent evaporation technique was chosen for the preparation of SLN. Briefly, 10 mg of antibiotic (Sigma-Aldrich, St. Louis, MO, USA) were mixed with a 5% (w/v) Precirol ® ATO 5 (Gattefossé, Madrid, Spain) dichloromethane solution. Then, the organic phase and an aqueous surfactant containing solution (Poloxamer 188 at 1% w/v and Polysorbate 80 at 1% w/v) were mixed and emulsified by sonication at 20 W for 30 s (Branson Sonifier 250, Danbury, CT, USA). The solvent was allowed to evaporate by magnetic stirring for 2 h at room temperature. Subsequently, the resulting SLNs were washed by centrifugation in Amicon ® centrifugal filtration units (100,000 MWCO, Merck Millipore, Billerica, MA, USA) at 2500 rpm for 15 min three times. For the NLC elaboration, a hot melt homogenization technique was selected. In brief, Precirol ® ATO 5 and Miglyol ® 812 (Sasol, Johannesburg, South Africa) were selected as the lipid core. Those lipids were mixed with the API and heated above the melting temperature of the solid lipid. The surfactant solution consisted of 1.3% (w/v) of Polysorbate 80 and 0.6% (w/v) of Poloxamer 188. The lipid and aqueous solutions were heated to the same temperature and then emulsified by sonication for 15 s at 20 W. Nanoparticles were stored at 4 • C overnight to allow lipid re-crystallization and particle formation. Then, a washing step was undergone by centrifugation at 2500 rpm in Amicon ® centrifugal filtration units (100,000 MWCO) three times. All the nanoparticles prepared were freeze-dried with two different cryoprotectants, either D-mannitol or trehalose (15%). In SLN formulations, emulsifiers constituted the aqueous phase of the emulsions, stabilizing the lipid dispersion of the nanoparticles and preventing their agglomeration [22]. Thus, the influence of the emulsifier on the bioactivity of the lipid nanoparticles was examined in two different types of SLN. SLN-tobramycin nanoparticles were prepared using the emulsifiers poloxamer 188 and polysorbate 80, each at 1% w/v. SLN-SDS-tobramycin nanoparticles were prepared using 2% sodium dodecyl sulfate (SDS) as the co-emulsifier. NLCs loaded with tobramycin (NLC-tobramycin) were prepared using a hot melt homogenization technique, following the method described by Pastor et al. [21].
All three types of nanoparticles used in this work (SLN-tobramycin, SLN-SDS-tobramycin, and NLC-tobramycin) were stabilized by trehalose, since in previous research we determined that it was a better cryoprotectant than mannitol [20]. Solid Lipid Nanoparticles and Nanostructured lipid carriers were characterized for size, polidispersity index (PDI) and Z-potential by means of Zetaseiser Nano ZS (Malvern Instruments, Worcestershire, UK). Measurements were based on Dynamic Light Scattering (DLS). Atomic force microscopy images were obtained by using a XE-70 atomic force microscope (Park Systems, Suwon, Korea).

Drug Susceptibility Assay in Planktonic Bacteria
Susceptibility to free tobramycin and to the three formulations of nanoencapsulated tobramycin was determined using the broth microdilution method in accordance with the Clinical Laboratory Standards Institute [23]. Briefly, the isolates were grown overnight at 37 • C in MHBCA, after which 2 mL of the culture was used to inoculate 20 mL of fresh MHBCA medium. After 2 h at 37 • C and 200 rpm, the bacterial cultures were adjusted to an optical density at 625 nm (OD 625nm ) of 0.08-0.1 and diluted 1:1000 in fresh MHBCA medium. Five µL of each diluted suspension was added to the wells (10 4 UFC/well) of 96-well microtiter plates previously filled with MHBCA and serially diluted antibiotic (free and nanoencapsulated). The plates were incubated at 37 • C for 24 h, after which the minimal inhibitory concentration (MIC) was determined macroscopically, based on the visually assessed turbidity of the wells. All experiments were performed in triplicate with three technical replicates.

Effect of Free and Nanoencapsulated Tobramycin on P. aeruginosa Growth
Two P. aeruginosa CF isolates, tobramycin-susceptible strain 056SJD and tobramycin-resistant strain 362VH, were used to examine the effect of free and nanoencapsulated (SLN and NLC) tobramycin. The antimicrobials were added to exponentially growing liquid cultures (1 × 10 8 CFU/mL, in MHBCA) at concentrations above and below the MIC. Samples were taken aseptically at 0, 1, 2, 3, 4, and 5 h from bacterial cultures incubated at 37 • C with shaking (250 rpm). Bacterial growth was measured optically to determine the OD 625nm . All measurements were carried out in triplicate.

Antimicrobial Susceptibility of Sessile Bacteria
The minimal biofilm eradication concentration (MBEC), defined in this study as the minimal antibiotic concentration required to eliminated >90% of the non-treated biofilm, was determined as described by Moskowitz et al. [24], with modifications. Briefly, the formation of bacterial biofilms was promoted as follows: the pegs of a modified polystyrene microtiter lid (catalog No. 445497; Nunc TSP system) were immersed into 96-well microtiter plates containing inoculated (10 4 UFC/well) 200 µL MHBCA/well. The modified plates were left undisturbed at 37 • C for 24 h. The pegs were then gently rinsed in 0.9% NaCl and the bacterial biofilms exposed to different concentrations of free and nanoencapsulated tobramycin for 24 h at 37 • C in MHBCA. The pegs were then rinsed again with 0.9% NaCl and the biofilms removed by 10 min sonication and centrifugation (2000 rpm, 10 min) in a BioSan Laboratory Centrifuge LMC-3000. Bacteria recovered from the biofilms were incubated for 24 h at 37 • C. Pegs were again rinsed with 0.9% NaCl solution and biofilms removed by 10 min sonication. Recovered bacteria were incubated for 24 h at 37 • C. Optical densities at 620 nm were measured in order to determine MBEC values. All experiments were performed in triplicate on at least three occasions.

Statistical Analysis
The antimicrobial susceptibilities of the tested P. aeruginosa strains to free and nanoencapsulated tobramycin were statistically analyzed using Cochran's Q test. A p-value < 0.05 was considered to indicate statistical significance.

Nanoparticle Characterization
Main characterization data of nanoparticles are shown in Table 2. AFM imaging and size measurements of particles are presented in Figure 1.

Antimicrobial Activity of Free and Nanoencapsulated Tobramycin
Nearly all the isolates tested in this study resulted to be susceptible (MIC ≤ 4 µg/mL) to both the free and nanoencapsulated tobramycin formulations ( Figure 1). The MIC of free tobramycin tested against the isolates was 0.5 µg /mL, whereas that of NLC-tobramycin was slightly lower (between 0.25 µg /mL and 0.5 µg /mL) and was also lower than the MICs of SLN-and SLN-SDS-tobramycin (between 1 and 4 µg /mL and 0.5 µg /mL, respectively) type.
In addition, NLCs were much more active than either of the SLN preparations, as evidenced by MIC values of 0.5 and 1-4 µg /mL (p < 0.05), respectively. Among the two types of SLN, the formulation prepared without SDS lost antimicrobial activity (up to eight-fold higher MICs) ( Figure  1). Thus, further experiments were conducted using NLC and SLN-SDS.
The efficient antibacterial activity of lipid nanoparticles loaded with tobramycin may be due to their small size and physic-chemical properties, which facilitates diffusion of the drug into the bacterial cell [25]. Similar results were reported by Ghaffari et al. [19] in their study of P. aeruginosa clinical isolates obtained from CF patients. The authors showed that tobramycin loaded in lipid

Antimicrobial Activity of Free and Nanoencapsulated Tobramycin
Nearly all the isolates tested in this study resulted to be susceptible (MIC ≤ 4 µg/mL) to both the free and nanoencapsulated tobramycin formulations ( Figure 1). The MIC of free tobramycin tested against the isolates was 0.5 µg /mL, whereas that of NLC-tobramycin was slightly lower (between 0.25 µg /mL and 0.5 µg /mL) and was also lower than the MICs of SLN-and SLN-SDS-tobramycin (between 1 and 4 µg /mL and 0.5 µg /mL, respectively) type.
In addition, NLCs were much more active than either of the SLN preparations, as evidenced by MIC values of 0.5 and 1-4 µg /mL (p < 0.05), respectively. Among the two types of SLN, the formulation prepared without SDS lost antimicrobial activity (up to eight-fold higher MICs) ( Figure 1). Thus, further experiments were conducted using NLC and SLN-SDS.
The efficient antibacterial activity of lipid nanoparticles loaded with tobramycin may be due to their small size and physic-chemical properties, which facilitates diffusion of the drug into the bacterial cell [25]. Similar results were reported by Ghaffari et al. [19] in their study of P. aeruginosa clinical isolates obtained from CF patients. The authors showed that tobramycin loaded in lipid nanoparticles had the same or higher antimicrobial activity than the free form of the drug. The slightly higher bioactivity of tobramycin-loaded NLC than SLN can be attributed to the higher drug-loading capacity of these nanoparticles and the avoidance of drug loss during storage [14,26]. As demonstrated by Moreno-Sastre et al. [27], second-generation NLC are more stable than first-generation SLN and they can be stored at a wider range of temperatures without relevant modifications of their antimicrobial activity.
The improved antibacterial activity of SLN-SDS vs. the SLN particles suggests that SDS, when used as a co-emulsifier, confers improved drug stability and release. SDS may also facilitate contact between the lipid nanoparticles and water, resulting in a better distribution equilibrium of the drug. Of relevance to our findings is the major challenge posed by ensuring drug stability in the development of colloidal drug carriers, which offer a high surface area and short diffusion pathways [28]. Figure 2b shows the data separated for mucoid and non-mucoid strains of P. aeruginosa.
The improved antibacterial activity of SLN-SDS vs. the SLN particles suggests that SDS, when used as a co-emulsifier, confers improved drug stability and release. SDS may also facilitate contact between the lipid nanoparticles and water, resulting in a better distribution equilibrium of the drug. Of relevance to our findings is the major challenge posed by ensuring drug stability in the development of colloidal drug carriers, which offer a high surface area and short diffusion pathways [28]. Figure 2b shows the data separated for mucoid and non-mucoid strains of P. aeruginosa.

Effect of Free and Nanoaencapsulated Tobramycin on Bacterial Growth
The susceptibilities of non-mucoid, susceptible (isolate 056SJD) and mucoid, resistant (isolate 362VH) P. aeruginosa to free and nanoencapsulated tobramycin were similar at all concentrations of the antibiotic tested (Figure 3). At sub-inhibitory concentrations (1/2 × MIC), the effect of the tobramycin-loaded lipid formulations on the growth kinetics of susceptible isolate was slightly lower than that of the free drug (Figure 3a) whereas the response of the resistant isolate did not differ (Figure 3d). At the MIC, greater inhibition of the susceptible isolate was achieved, since after 5 h of antimicrobial exposure none of the formulations was able to fully inhibit the growth of the resistant isolate (Figure 3b,e). At concentrations above the MIC, the growth of the susceptible isolate was inhibited immediately after the addition of the antimicrobial (Figure 3c), but, again, none of the formulations fully inhibited the growth of the resistant isolate (Figure 3f). Empty lipid nanoparticles had no antibacterial activity in either isolate (data not shown).
Taken together, our results demonstrate that the loading of tobramycin into lipid nanoparticles does not adversely affect the antimicrobial activity of the drug against planktonic P. aeruginosa. The preserved potency of lipid nanoparticles containing tobramycin may be due to their facilitated diffusion across the bacterial cell membranes. Mugabe et al. [29] showed that the effective antimicrobial activity of gentamicin loaded into liposomes involved fusion of the particles with the bacterial membrane, leading to its deformation. Further experiments are needed to better understand the interactions between the lipids in nanoformulations and the cellular membrane of microorganisms that promote drug diffusion.
The slower killing of the mucoid, resistant strain of P. aeruginosa than of the non-mucoid, susceptible strain by free as well as nanoencapsulated tobramycin can be explained by the additional time needed for outer membrane permeabilization by the drug, regardless of its method of preparation, and the subsequent delay in its reaching its intracellular target.
A previous study showed an immediate effect of tobramycin against most of the susceptible populations tested but not against the resistant population [30].
tobramycin-loaded lipid formulations on the growth kinetics of susceptible isolate was slightly lower than that of the free drug (Figure 3a) whereas the response of the resistant isolate did not differ (Figure 3d). At the MIC, greater inhibition of the susceptible isolate was achieved, since after 5 h of antimicrobial exposure none of the formulations was able to fully inhibit the growth of the resistant isolate (Figure 3b,e). At concentrations above the MIC, the growth of the susceptible isolate was inhibited immediately after the addition of the antimicrobial (Figure 3c), but, again, none of the formulations fully inhibited the growth of the resistant isolate (Figure 3f). Empty lipid nanoparticles had no antibacterial activity in either isolate (data not shown).
Taken together, our results demonstrate that the loading of tobramycin into lipid nanoparticles does not adversely affect the antimicrobial activity of the drug against planktonic P. aeruginosa. The preserved potency of lipid nanoparticles containing tobramycin may be due to their facilitated diffusion across the bacterial cell membranes. Mugabe et al. [29] showed that the effective antimicrobial activity of gentamicin loaded into liposomes involved fusion of the particles with the bacterial membrane, leading to its deformation. Further experiments are needed to better understand the interactions between the lipids in nanoformulations and the cellular membrane of microorganisms that promote drug diffusion.
The slower killing of the mucoid, resistant strain of P. aeruginosa than of the non-mucoid, susceptible strain by free as well as nanoencapsulated tobramycin can be explained by the additional time needed for outer membrane permeabilization by the drug, regardless of its method of preparation, and the subsequent delay in its reaching its intracellular target.
A previous study showed an immediate effect of tobramycin against most of the susceptible populations tested but not against the resistant population [30].

Anti-Biofilm Efficacy of Free and Nanoencapsulated Tobramycin
To test the influence of the lipid nanoparticles on tobramycin's ability to kill sessile bacteria, biofilms of four P. aeruginosa strains were exposed to free and nanoencapsulated (SLN and NLC)  Figure 3d). At the MIC, greater inhibition of the susceptible isolate was achieved, since after 5 h of antimicrobial exposure none of the formulations was able to fully inhibit the growth of the resistant isolate (Figure 3b,e). At concentrations above the MIC, the growth of the susceptible isolate was inhibited immediately after the addition of the antimicrobial (Figure 3c), but, again, none of the formulations fully inhibited the growth of the resistant isolate (Figure 3f). Empty lipid nanoparticles had no antibacterial activity in either isolate (data not shown).
Taken together, our results demonstrate that the loading of tobramycin into lipid nanoparticles does not adversely affect the antimicrobial activity of the drug against planktonic P. aeruginosa. The preserved potency of lipid nanoparticles containing tobramycin may be due to their facilitated diffusion across the bacterial cell membranes. Mugabe et al. [29] showed that the effective antimicrobial activity of gentamicin loaded into liposomes involved fusion of the particles with the bacterial membrane, leading to its deformation. Further experiments are needed to better understand the interactions between the lipids in nanoformulations and the cellular membrane of microorganisms that promote drug diffusion.
The slower killing of the mucoid, resistant strain of P. aeruginosa than of the non-mucoid, susceptible strain by free as well as nanoencapsulated tobramycin can be explained by the additional time needed for outer membrane permeabilization by the drug, regardless of its method of preparation, and the subsequent delay in its reaching its intracellular target.
A previous study showed an immediate effect of tobramycin against most of the susceptible populations tested but not against the resistant population [30].

Anti-Biofilm Efficacy of Free and Nanoencapsulated Tobramycin
To test the influence of the lipid nanoparticles on tobramycin's ability to kill sessile bacteria, biofilms of four P. aeruginosa strains were exposed to free and nanoencapsulated (SLN and NLC) ); Free tobramycin ( (Figure 3d). At the MIC, greater inhibition of the susceptible isolate was achieved, since after 5 h of antimicrobial exposure none of the formulations was able to fully inhibit the growth of the resistant isolate (Figure 3b,e). At concentrations above the MIC, the growth of the susceptible isolate was inhibited immediately after the addition of the antimicrobial (Figure 3c), but, again, none of the formulations fully inhibited the growth of the resistant isolate (Figure 3f). Empty lipid nanoparticles had no antibacterial activity in either isolate (data not shown).
Taken together, our results demonstrate that the loading of tobramycin into lipid nanoparticles does not adversely affect the antimicrobial activity of the drug against planktonic P. aeruginosa. The preserved potency of lipid nanoparticles containing tobramycin may be due to their facilitated diffusion across the bacterial cell membranes. Mugabe et al. [29] showed that the effective antimicrobial activity of gentamicin loaded into liposomes involved fusion of the particles with the bacterial membrane, leading to its deformation. Further experiments are needed to better understand the interactions between the lipids in nanoformulations and the cellular membrane of microorganisms that promote drug diffusion.
The slower killing of the mucoid, resistant strain of P. aeruginosa than of the non-mucoid, susceptible strain by free as well as nanoencapsulated tobramycin can be explained by the additional time needed for outer membrane permeabilization by the drug, regardless of its method of preparation, and the subsequent delay in its reaching its intracellular target.
A previous study showed an immediate effect of tobramycin against most of the susceptible populations tested but not against the resistant population [30].

Anti-Biofilm Efficacy of Free and Nanoencapsulated Tobramycin
To test the influence of the lipid nanoparticles on tobramycin's ability to kill sessile bacteria, biofilms of four P. aeruginosa strains were exposed to free and nanoencapsulated (SLN and NLC) ); SLN-Tobramycin ( (Figure 3d). At the MIC, greater inhibition of the susceptible isolate was achieved, since after 5 h of antimicrobial exposure none of the formulations was able to fully inhibit the growth of the resistant isolate (Figure 3b,e). At concentrations above the MIC, the growth of the susceptible isolate was inhibited immediately after the addition of the antimicrobial (Figure 3c), but, again, none of the formulations fully inhibited the growth of the resistant isolate (Figure 3f). Empty lipid nanoparticles had no antibacterial activity in either isolate (data not shown).
Taken together, our results demonstrate that the loading of tobramycin into lipid nanoparticles does not adversely affect the antimicrobial activity of the drug against planktonic P. aeruginosa. The preserved potency of lipid nanoparticles containing tobramycin may be due to their facilitated diffusion across the bacterial cell membranes. Mugabe et al. [29] showed that the effective antimicrobial activity of gentamicin loaded into liposomes involved fusion of the particles with the bacterial membrane, leading to its deformation. Further experiments are needed to better understand the interactions between the lipids in nanoformulations and the cellular membrane of microorganisms that promote drug diffusion.
The slower killing of the mucoid, resistant strain of P. aeruginosa than of the non-mucoid, susceptible strain by free as well as nanoencapsulated tobramycin can be explained by the additional time needed for outer membrane permeabilization by the drug, regardless of its method of preparation, and the subsequent delay in its reaching its intracellular target.
A previous study showed an immediate effect of tobramycin against most of the susceptible populations tested but not against the resistant population [30].

Anti-Biofilm Efficacy of Free and Nanoencapsulated Tobramycin
To test the influence of the lipid nanoparticles on tobramycin's ability to kill sessile bacteria, biofilms of four P. aeruginosa strains were exposed to free and nanoencapsulated (SLN and NLC) ); NLC-Tobramycin ( (Figure 3d). At the MIC, greater inhibition of the susceptible isolate was achieved, since after 5 h of antimicrobial exposure none of the formulations was able to fully inhibit the growth of the resistant isolate (Figure 3b,e). At concentrations above the MIC, the growth of the susceptible isolate was inhibited immediately after the addition of the antimicrobial (Figure 3c), but, again, none of the formulations fully inhibited the growth of the resistant isolate (Figure 3f). Empty lipid nanoparticles had no antibacterial activity in either isolate (data not shown).
Taken together, our results demonstrate that the loading of tobramycin into lipid nanoparticles does not adversely affect the antimicrobial activity of the drug against planktonic P. aeruginosa. The preserved potency of lipid nanoparticles containing tobramycin may be due to their facilitated diffusion across the bacterial cell membranes. Mugabe et al. [29] showed that the effective antimicrobial activity of gentamicin loaded into liposomes involved fusion of the particles with the bacterial membrane, leading to its deformation. Further experiments are needed to better understand the interactions between the lipids in nanoformulations and the cellular membrane of microorganisms that promote drug diffusion.
The slower killing of the mucoid, resistant strain of P. aeruginosa than of the non-mucoid, susceptible strain by free as well as nanoencapsulated tobramycin can be explained by the additional time needed for outer membrane permeabilization by the drug, regardless of its method of preparation, and the subsequent delay in its reaching its intracellular target.
A previous study showed an immediate effect of tobramycin against most of the susceptible populations tested but not against the resistant population [30].

Anti-Biofilm Efficacy of Free and Nanoencapsulated Tobramycin
To test the influence of the lipid nanoparticles on tobramycin's ability to kill sessile bacteria, biofilms of four P. aeruginosa strains were exposed to free and nanoencapsulated (SLN and NLC) ).

Anti-Biofilm Efficacy of Free and Nanoencapsulated Tobramycin
To test the influence of the lipid nanoparticles on tobramycin's ability to kill sessile bacteria, biofilms of four P. aeruginosa strains were exposed to free and nanoencapsulated (SLN and NLC) tobramycin at antibiotic concentrations between 0 and 256 µg/mL. ATCC strain 27,853 and strain PAO1 were used as controls, and strains 056SJD (non-mucoid, tobramycin-susceptible) and 362VH (mucoid, tobramycin-resistant) as the P. aeruginosa CF isolates. All P. aeruginosa strains used in this experiment formed adequate biofilms (data not shown). The MIC and MBEC values of the four strains are shown in Table 3. Among the isolates susceptible to tobramycin, the MIC and MBEC values of the nanoencapsulated drug were slightly lower (1-2 logs) than those of the free drug. However, for the clinical isolate resistant to tobramycin, there were no differences in the MIC and MBEC values obtained with the nanoparticles and free tobramycin. The exception was NLC-tobramycin, in which the MBEC was slightly lower than the value obtained with the free form. The much higher MBEC vs. MIC values of both free and nanoencapsulated tobramycin likely reflected the interaction between the anionic mucopolysaccharide of the biofilm and the cationic aminoglycoside, such that the amount of free tobramycin available to act against the resident bacteria was limited [31]. Among the two types of nanoparticles (SLN and NLC), NLC were slightly more active than SLN (1 log) for all strains tested. Specifically, the concentrations of free tobramycin needed to completely eradicate the P. aeruginosa biofilm were 8-16 µg/mL (tobramycin-susceptible strains) and 32 µg/mL (tobramycin-resistant strain), but the effective NLC-tobramycin concentration was lower (2-4 µg/mL and 16 µg/mL, respectively). The better results obtained with the NLC formulation of tobramycin were in agreement with our previously published results showing that colistin-loaded NLCs were highly effective in biofilm eradication [20]. A modification of MBEC assay was performed to test the efficacy of NLC-tobramycin to prevent the biofilm formation [32]. For all the isolates tested, BPC (biofilm prevention concentration) values of NLC-tobramycin were identical to values for free tobramycin. Thus, whereas NLC-tobramycin was more effective than its free form in eradicating biofilms, both free and nanoencapsulated tobramycin did not show any differences on the prevention on biofilm formation. Although the mechanisms underlying the efficacy of tobramycin in NLC are not fully understood, a role for charge distribution seems likely. Tobramycin loaded into NLC has a negative net charge because of the negatively charged nanoparticles, in contrast to the positive net charge of free tobramycin. The superior mucus penetration of negatively charged nanoparticles has been reported [33] and suggests the greater ability of NLC-tobramycin to penetrate the exopolyssacharide matrix surrounding the biofilm structure. Increased penetration would better allow tobramycin to reach its cellular target, in contrast to its free form. Alternatively, the fast-antimicrobial release reported by Pastor et al. [21] would ensure an initial antimicrobial concentration that is high enough to inhibit the biofilm growth of P. aeruginosa. Moreover, a sustained antimicrobial concentration higher than the MIC value would enable the eradication of surviving cells. However, further experiments are needed to determine which, if any, of our hypotheses is the correct one.

Conclusions
New antimicrobial formulations, such as lipid nanoparticles, can improve the transfer of antimicrobials to their sites of action, potentially allowing a dose reduction and therefore the avoidance of adverse side effects. Our study of planktonic cultures and biofilms of P. aeruginosa demonstrated that antimicrobial activity of tobramycin was not affected by nanoencapsulation. Thus, we found that nanoencapsulation of tobramycin did not improve its efficacy against planktonic P. aeruginosa. However, nanoencapsulation did improve its ability to eradicate P. aeruginosa biofilms. Given the key role of biofilms in respiratory infections of P. aeruginosa in CF patients, the results obtained in this study, and especially with NLC-tobramycin, may provide new options in the treatment of these infections, particularly taking into account the better distribution of antibiotics when inhaled as nanoparticles.