3.2. Polymeric Micelles Characterization
Curcumin-loaded and void polymeric micelles were prepared by dissolving the Pluronics in PBS at a concentration of 1.5% (
w/
w). This specific concentration was chosen in order to exceed the CMC values and to possess a minimum toxicity against normal cells, as is shown in our previous studies [
31]. The toxicity of Pluronic derivatives was demonstrated to be dependent on the cell type, chemical characteristics of the polymer, and time of incubation. The hydrophobicity of the Pluronics plays a distinct role in the micelles’ internalization pattern, thus strongly influencing the cytotoxicity. The derivative F127, with the highest HLB value in the series used in the present work (HLB = 22), is efficiently internalized in the cytoplasm, while the more hydrophobic Pluronics P123 (HLB = 8) and P84 (HLB = 14) are retained in the membrane and exhibit a slower transport into the cells [
32]. In general, the more hydrophobic Pluronics show higher cytotoxicity compared to the hydrophilic ones, against all types of normal cells at concentrations above 10
−4 M [
33]. Thus, the selection of the Pluronic derivatives to be used as micelles material must take into account all the effects of the hydrophobicity, i.e., the influence on the CMC value (and, consequently, the stability under dilution), the size of the micellar core, and the cytotoxicity of the carrier.
The size and size distribution of micelles, both void and with Curcumin encapsulated, are summarized in
Table 4.
The main value for the size of the micelles in the F127 solution was found to be 25.08 nm, a significantly higher value (
p < 0.05) compared to the ones recorded for Pluronic P84 and P123, since the last two block copolymers possess smaller molecular weights. As is expected for polymeric solutions at rather low concentrations, all samples show moderate polydispersity, according to the PdI values, ranging from 0.056 to 0.380. The size of polymeric micelles looks similar to other data reported in the literature [
30].
The encapsulation of the drug CURC into micelles results in a slight increase in the dimensions in the case of Pluronic P84 micelles. For the compounds F127 and P123, the size of CURC-loaded micelles significantly increases (p < 0.05) compared to void micelles, which suggests drug’s encapsulation mainly into the hydrophobic core of the micellar aggregates.
The values of zeta potential found for void Pluronic micelles are similar to those reported in the literature— −14.53 ± 0.32 mV for P84, −7.92 ± 1.51 mV for P123, and −13.93 ± 1.07 mV for F127, respectively—and no statistically significant changes were recorded after the encapsulation of the drug. Zeta potential values of the micelles with Curcumin encapsulated vary from −13.83 ± 0.32 mV for CURC-loaded P84 micelles to −7.07 ± 1.23 mV for CURC-loaded P123 micelles and to −12.40 ± 1.53 mV for CURC-loaded F127 micelles. The values are relatively low and explain the tendency of aggregation observed at high concentrations of polymer in solution. In
Figure 1, the DLS diagrams for size distribution and zeta potential measured for P123 micellar dispersion with CURC encapsulated are shown as representative examples.
The DLS diagram exhibits a major signal at 25.29 nm, which corresponds to the micelles with encapsulated Curcumin, while the signal at 5.8 nm corresponds to the unassociated polymeric chains. The signal located at the higher value of 261 nm is probably due to the aggregation of micelles, which is reported in other papers, in particular for Pluronic derivatives with low zeta potential values. For the two others derivatives P84 and F127, similar behavior was recorded.
In samples with all three Pluronic derivatives, the micelles are spherical in shape, as shown in
Figure 2. The solubilization of the Curcumin in micelles does not change the shape of the aggregates in the case of all three polymers P84, P123, and F127, and the TEM images of samples with drug loaded carriers does not reveal any other polymeric nanostructures such as worm-like micelles or vesicles.
A selected concentration of polymers in solutions of 1.5% was chosen to exhibit the minimum toxicity against normal mammalian cells, but at the same time, to exceed the CMC value in order to allow for the existence of micelles. Another advantage of this low concentration is that all three Pluronic derivatives produce small spherical micelles, none of them showing transitions to worm-like micelles or vesicles.
The possible interactions between drug molecules and polymer in the CURC-loaded Pluronic micelles were investigated by using FTIR spectroscopy, and the spectra are shown in
Figure 3a–c.
In the FTIR spectra of pure Curcumin (
Figure 3), a sharp peak at 3512 cm
−1 is present, which corresponds to the stretching vibration of the phenolic –OH group. The peak at 1625 cm
−1 can be associated with the –C=C– and –C=O vibrations, and the band at 1593 cm
−1 with the symmetric aromatic ring stretching vibrations –C=C–. There are also present peaks at 1502 cm
−1, corresponding to the –C=O vibration, and at 1023 cm
−1, corresponding to the C–O–C stretching vibration.
Due to similar chemical compositions, FTIR spectra of freeze-dried Pluronic micelles contain the same specific peaks. The intense band located at 1089 cm−1 is attributed to the C–O–C vibration, while the peaks at 2867 and 2978 cm−1 are related to the asymmetric and symmetric stretching vibrations of C–H.
All three spectra of Pluronic micelles loaded with CURC look similar, containing all specific peaks of Pluronic derivatives and preserving most of the bands characteristic for CURC, without significant shifting. Thus, the presence of the bands associated with the main functional groups of Curcumin confirms the encapsulation of the drug into micelles, without changes in chemical bonds. The attenuation in the intensity of the peaks at 3512 cm
−1 suggests the formation of the intermolecular hydrogen bonds between the Curcumin –OH groups and POE groups in Pluronic corona, as is reported for the behavior of CURC in other polymeric matrices containing polyoxyethylene groups [
34].
3.3. Drug Loading and Entrapment Efficiency
One of the main drawbacks of Curcumin is its low solubility in water (less than 20 µg/mL); thus, the solubilization in micellar dispersions proves to be a viable solution to increase the drug’s solubility in aqueous formulations. A significant increase in CURC’s solubility, up to 3 mg/mL was reported in the literature in Pluronics P105, P108, and F127 solutions at concentrations above 10
−2 M [
35]. Unfortunately, these concentrations are usually cytotoxic for most normal animal cells.
The presence of the block copolymer micelles leads to increased solubility of the Curcumin in aqueous solutions, as is observed from the enhancement of the fluorescence emissions of the CURC (
Figure S1).
The encapsulation of CURC in a Pluronic micellar solution at low concentrations (1% w/v) was evaluated at room temperature, and the encapsulation efficiency (EE%) was found to range from 31.2 ± 3.52 for Pluronic P123 and 43.7 ± 3.29 for Pluronic F127. The encapsulation capacity is significantly lower for P123, while the derivatives with higher HLB values, P84 and F127, show similar values of EE%: 42.7 ± 1.69 for P84 and 43.7 ± 3.29 for F127. The values for drug-loading efficiency (DL%) at the drug–polymer ratio 1:20 (w/w) were found to be 4.39 ± 0.53% for P84, very close to the value for F127 4.48 ± 0.46%, while for P123, a value of 3.71 ± 0.57% was obtained.
The drug-loading and entrapment efficiency are influenced by several factors, such as polymer concentration, the length of the hydrophobic core-forming block in the macromolecule, and the extent of the PEO corona. Various mechanisms have been proposed for the encapsulation of hydrophobic drugs, such as Curcumin, into micelles, in particular chemical conjugation or physical entrapment. The physical entrapment of drug molecules in micellar aggregates is due to the existence of intermolecular interactions: hydrophobic, van der Waals interactions, or hydrogen bonds. Curcumin’s chemical structure suggests that the formation of hydrogen bonds between the oxygen in the ether groups of PEO chains and hydroxyl groups in CURC play an important role in the drug’s encapsulation in Pluronic micelles, beyond the van der Waals interaction in the PPO core of the micelles. According to the dimension of the PPO block in the Pluronic molecule, P84 is expected to encapsulate less Curcumin than the P123 and F127 derivatives. The same PPO length in P123 and F127 suggests that the two micellar systems exhibit a similar solubilization capacity. The higher encapsulation efficiency of Pluronic F127 is due to the longer length of the PEO block, which generates a larger oxyethylene corona.
3.6. Influence of Curcumin-Loaded Pluronic Micelles on Microbial Membrane Permeability
3.6.1. Microbial Membrane Permeability
There are many events involved in the complex interaction of Pluronic compounds with multi-drug resistant (MDR) tumoral cells, such as modification in the microviscosity of the cell membranes, due to the incorporation or attachment of the polymer molecules, reduction of the adenosine-5′-triphosphate (ATP) level, inhibition of the multidrug resistance proteins, release of cytochrome C level in cytoplasm, enhancement of the pro-apoptotic signaling, or inhibition of the drug efflux transporter, in particular P–Glycoprotein (Pgp). The interesting aspect is that these effects are most evident at the concentrations below the CMC value of the block copolymer and are diminished or absent at concentrations above CMC [
38]. It was suggested that the unimers, not micelles, are responsible for the changes in tumoral cell behavior, due to their ability to penetrate the cellular membrane according to the hydrophobicity of the macromolecule. Considering the same type of mechanism of action, it is presumable that block copolymers could also have an effect in reversing antibiotic resistance in the case of microorganisms. However, very limited studies were performed on the possible influence of Pluronics on the bacterial membranes’ integrity.
To evaluate the effect of the presence of Pluronics on the membranes of bacterial and fungal cells, the accumulation of propidium iodide (PI) and 1-N-phenylnaphthylamine (NPN) as hydrophobic probes in E. coli, S. aureus, and C. albicans strains was measured, using both reference strains and resistant clinical isolates.
Propidium iodide is a fluorescent dye with affinity to DNA which is commonly used to evidence or quantify the dead cells, since it does not trespass the cell membrane and exhibits no accumulation in viable cells. Based on this behavior, it is also used as a marker of cellular membrane damage in both mammalian cells and microbial cultures. To evidence the alteration of the outer membrane of Gram-negative bacteria, 1-N-phenylnaphthylamine (NPN) was proposed as a fluorescent probe, with the intensity of the emitted fluorescence strongly dependent on the polarity of the microenvironment. When some chemicals affect the permeability of the outer membrane, NPN dye enters into the hydrophobic zone of the membrane, and thus, the intensity of the emitted fluorescence increases spectacularly.
In
Figure 7a–c, the variation in the relative fluorescence of PI in
E. coli strains (
Escherichia coli ATCC 25922 and
Escherichia coli ESBL 135, clinical isolate) in the presence of various Pluronic derivatives is presented.
In the case of standard
E. coli cultures treated with Pluronic solutions, an increase in membrane permeability was recorded only at very low concentrations for all types of polymer used in the study. For hydrophilic Pluronics P84 and F127, a slight increase (
p < 0.05) in the fluorescence relative to the control samples, considered as unity, up to 1.34 and 1.46 was recorded only at concentrations 100 times lower than CMC (denoted CMC/100), when the polymeric solution contains monomer and no micelles. At higher concentrations, for both micellar and pre-micellar, no significant changes in the fluorescence of PI is recorded. A different behavior is shown by the more hydrophobic derivative P123, when the increase in the membrane permeability is observed in an extent domain of concentrations, from very diluted (100 smaller than CMC) up to micellar concentrations (CMC). The further increase in the polymer concentration up to 10 times CMC (denoted CMC×10) results in the decrease in fluorescence at values close to untreated cells. The maximum permeation of PI in the bacterial cells seems to be located at the polymer concentration one order of magnitude lower than the CMC. Similar results were reported by Batrakova et al. [
39] for the hydrophobic Pluronics in the internalization of Rhodamine 123 as a membrane permeabilization marker, affecting normal and MDR tumoral cells. In the case of resistant
E. coli cultures, no changes in cell membrane permeability was recorded over the entire domain of concentrations investigated with all three Pluronic derivatives.
The Gram-positive bacteria
S. aureus proves to be more susceptible to the effect of the Pluronics on the cell membrane (
Figure 8a–c). The polymer–membrane interaction in Gram-positive microorganisms is governed by the ability of the PPO block of the polymeric chain to attach to or insert into the hydrophobic phospholipidic zone of the cell wall after crossing the peptidoglycan layer of the cell wall. The situation is more complex in the case of Gram-negative bacteria, which possess also an outer membrane, composed mainly from proteins, phospholipids, and lipopolysaccharides (LPSs); thus, the binding of Pluronic molecules involves others mechanisms.
As it is expected, hydrophilic block copolymer F127 show no effect at higher concentrations, with only a small increase in the relative fluorescence measured at very low concentrations (100 time smaller than CMC). Polymer F127 is considered a non-Pgp-inhibiting member of the Pluronic family, and its role in drug delivery is restricted to the carrier-forming ability. For the standard S. aureus strain, an increase in the relative fluorescence emission of PI was observed when the cultures were incubated with both unassociated molecules and micellar solutions, for Pluronic derivatives P84 and P123. The exception is Pluronic F127, which shows a slight increase in PI fluorescence in the low concentration region up to CMC. The derivative with moderate HLB, hydrophilic P84, also shows the highest effect on membrane permeability at concentrations 10 times higher than CMC, and after treatment with Curcumin-loaded micelles, too.
The most intense effect is recorded, for all block copolymers, in very diluted polymeric solutions, 100 and 10 smaller than the CMC of the respective Pluronics.
The MRSA strain shows no membrane changes when exposed to a Pluronic solution over the entire concentration range. Unexpectedly, a very small increase in the PI probe’s fluorescence was recorded in the samples incubated with micelles of P84 and Curcumin encapsulated in micelles of P84, regardless of the lack of the changes recorded in the presence of Curcumin dissolved in DMSO.
The results obtained with
C. albicans cultures show that the fungus exhibits higher sensitivity to the presence of the Pluronic polymers, and the membrane is more affected, compared to bacterial strains, as is demonstrated by the changes in the PI fluorescence (
Figure 9a–c).
In the case of Candida albicans ATCC 10231 incubated with all three Pluronics, membrane permeability was increased. For the hydrophilic derivatives P84 and F127, the increase in the relative fluorescence intensity is in the range of 1.2–1.8, at the lowest concentration of polymer in solution (100 lower than CMC values). The highest effect is observed for the polymer P84, with an HLB value of 14 and the smallest molecule among the tested block copolymer, at a concentration 100 times smaller than the CMC value. A significant increase in membrane permeability is also observed for Pluronic F127 at low concentrations (100 times and 10 times smaller than the CMC value of the polymer), as demonstrated by the values recorded for relative fluorescence intensity (1.52 and 1.66, respectively).
The more hydrophobic Pluronic P123 shows efficiency in membrane damaging on the largest domain of concentration, up to CMC. The highest increase in PI fluorescence is recorded at the lowest concentration (100 times smaller than CMC) when only monomeric species are present.
The resistant C. albicans strain is less influenced by the incubation with Pluronic solutions, for the polymers P123 and F127, with high molecular weights. As an exception, a slight effect is observed in the case of cultures treated with concentrated F127 solution (10 times higher than CMC) and the related micellar solution loaded with Curcumin. The Pluronic P84 with hydrophilic characteristics, but a smaller molecule than P123 and F127, proved to be effective over the whole domain of concentrations. An unexpected high effect was recorded for the micellar solution with Curcumin encapsulated, despite the same concentration of CURC dissolved in DMSO showing no change to the fluorescence of PI.
It is to be noted that also in the case of Candida albicans ATCC 10231, the Curcumin seems to be effective, producing a slight increase in membrane permeability (1.26 relative fluorescence intensity), while no such effect could be observed in the case of clinical isolate Candida albicans 6853.
3.6.2. Outer Membrane Permeabilization
The differences in the behavior of Gram-negative and Gram-positive bacteria in the presence of various antimicrobial agents are due to the presence of the outer membrane, consisting mainly of lipopolysaccharide (LPS) in the first case. This organelle acts as a barrier that protects Gram-negative bacteria against the aggression of antibiotics existing in the extracellular environment. For a certain chemical compound to actively interact with the bacterial membrane, it is mandatory to first trespass the outer layer of LPS. The hydrophobic NPN dye exhibits an increase in fluorescence when it is located in the phospholipid bilayer. Thus, the NPN uptake is considered a direct measurement of the outer membrane’s integrity, since an intact one prevents the penetration of the compound into the hydrophobic area of the bacterial membrane.
The strain of reference and resistant
E. coli were incubated with the Pluronic derivatives, and the results of the NPN fluorescence modification is presented in
Figure 10.
The hydrophilic Pluronic F127 with high molecules induces modification of the outer membrane of the reference E. coli ATCC 25922 only at the lowest concentration (CMC/100), while the Pluronic P84, with smaller molecules, produces the permeabilization of the outer membrane over the whole domain of concentrations, in the presence of both micelles and unassociated molecules.
For the hydrophobic derivative P123, the effect is evidenced only at concentrations near the CMC values.
In the experiments with Escherichia coli ESBL, NPN uptake was increased only when incubated with Pluronic P84, while no changes in the fluorescence intensity were recorded for samples with P123 and F127. Very peculiar behavior was registered with the Pluronic P84 solution, with the highest increase in the relative fluorescence of NPN (more than twice compared to the untreated control bacterial cultures) for the smallest concentration of polymer (CMC/100) and the highest one (10 times highest than CMC) and the corresponding micellar solution loaded with Curcumin.
The ability of CURC to induce cell membrane modifications was investigated for various microorganisms. Unfortunately, the effect on the integrity and fluidization of the bacterial membrane was investigated with various methods (TEM and SEM images, flow cytometry, confocal microscopy, spectrofluorimetry), and experimental conditions vary in a large domain; thus, the reported results are contradictory. For example, an increase of 47% for PI fluorescence intensity in
E. coli cells treated with high concentrations of CURC was determined by using flow cytometry [
40].
Furthermore, the CURC effect on membrane fluidization was found to be time dependent, and an increase of the fluorescence of 54% was measured using a concentration of 500 µg/mL (7-fold higher than the concentration used in our study), after 24 h of incubation [
41]. Oppositely, in another study performed with CURC solutions in DMSO at various concentrations 0.25–2.5 mM, with or without irradiation at 470 nm, the authors concluded that CURC does not produce membrane permeabilization [
42]. A higher increase in PI fluorescence intensity produced by 100 µg/mL of CURC was produced in
S. aureus cultures, incubated for 2 h, while the fluorescence in
E. coli cells was found to increase after 30 min of incubation, but disappear after 2 h [
43].
In conclusion, in the experimental conditions of our study, at low concentrations and short incubation times, CURC in DMSO did not influence the permeability of the microbial cell membranes in C. albicans clinical isolates and both types of S. aureus strains. A moderate effect was recorded in the fluidization of the membrane for standard C. albicans and for both standard and resistant E. coli. However, the effect of CURC on the cellular membrane was lost when encapsulated in Pluronic micelles.
Regarding the effect of the block copolymer solutions on the microviscosity of the microbial cell membrane, there are very few papers reporting the variation in the fluorescence intensity of standardized probes PI and NPN. The effect of the micelles on membrane integrity was absent in the majority of published papers, considering that only the monomeric species of surfactant produce the membrane fluidization. Experiments performed with highly concentrated Pluronic F127 on
P. aeruginosa and
E. coli suggested that polymeric micelles do not induce membrane permeability, based on tetraphenylethylene fluorescence, determined by using confocal microscopy. However, Bondar et al. [
44], in a study on the effect of novel glycerol-based trifunctional block copolymers (TBC) of propylene oxide and ethylene oxide and Pluronic L61 on the cell’s plasma membrane, suggest that these compounds are also able to produce membrane damage at increased concentrations above CMC, since the micellar aggregate may fuse with the membrane and disrupt it. A possible explanation for the results that we obtained in some microbial strains with micellar solutions is due to the ability of Pluronic aggregates to encapsulate the hydrophobic dye and increase cellular uptake, without directly damaging the membrane. Such mechanisms for penetration in the bacteria, with a first step of the attachment of the carrier to the bacterial wall, followed in a second step by the disruption of the peptidoglycan layer and incorporation of the particle inside the microorganism, were proposed for many drug delivery systems as an explanation for the increased cellular uptake. The interaction of Pluronics with cellular membranes is very complex, being different in the case of monomeric species and micellar aggregates. In addition, the complexity of the interaction must be taken into account in view of the fact that block copolymers interact with the microdomains of cell membranes in a different way, hence the very large variation in membrane fluidization effects at different concentrations, also depending on the membrane composition in various types of cells.
3.7. Photoinduced Antimicrobial Activity of Curcumin-Loaded Pluronic Micelles
As a general consideration, the CURC sample dissolved in DMSO (Curcumin control) expressed an inhibition zone toward all tested microbial strains in both conditions (darkness and 470 nm light), with higher diameters for C. albicans strains in the 470 nm light incubation condition. The lower efficiency was recorded in the growth control of E. coli in both standard and resistant strains, incubated in dark or irradiated. For the samples PM P84_CURC, enhanced antimicrobial activity can be highlighted when the incubation was performed in the presence of Gram-positive strains S. aureus, while a moderate effect was found against E. coli and C. albicans strains. For CURC encapsulated in the other two polymeric micelles P123_CURC and PM F127_CURC, the inhibition zones are in the range 8–10 nm, with a minor increase for the effect of P123_CURC on resistant E. coli and for PM F127_CURC on resistant C. albicans, under irradiation.
Instead, the results reveals that samples containing similar CURC amounts in unassociated molecule Pluronic solutions (concentrations below CMCs), namely, Premicellar P84_CURC, Premicellar P123_CURC, and Premicellar F127_CURC, expressed large inhibition zones toward S. aureus Gram-positive strains and E. coli strains, while significantly higher values are obtained for C. albicans strains when the incubation was performed in 470 nm light, compared with the darkness condition.
All the tested samples that contain Pluronic compound (considered as control samples) expressed no inhibition zone after the contact with the tested microbial strains in the same two incubation conditions (
Table S1 in Supplementary materials).
In order to demonstrate the light activation of Curcumin and to quantify the antimicrobial activity of the tested samples, following the incubation in the same two conditions (470 nm light and darkness), the MIC values have been determined and included in
Table 6.
None of the tested Pluronic derivatives (Pluronic
® P84, Pluronic
® P123, and Pluronic
® F127) exhibited antibacterial activity against all microbial strains over the concentration domain used in the study (
Table S2 in Supplementary materials).
As it is shown in
Table 6, the quantitative results confirm the qualitative results regarding the efficiency of the samples Premicellar P84_CURC, Premicellar P123_CURC, and Premicellar F127_CURC after 470 nm light incubation, the MIC values being lower compared to those obtained in darkness incubation conditions for the same microbial strains
S.aureus and
C. albicans.
In addition, the increase in the efficiency of the samples Premicellar P84_CURC, Premicellar P123_CURC, and Premicellar F127_CURC against Candida strains can be observed after incubation in 470 nm light, for which MIC values of 6.25 µM–2.5 µM were obtained, compared to the control sample of Curcumin, for which the MIC values were 25 µM.
The MIC values obtained in the case of incubation with premicellar Pluronic solutions containing CURC confirm the qualitative results of inhibition zones. Some differences were observed in the behavior of CURC-loaded polymeric micelles. In this case, the two experiments could not be compared since the MIC values could only be estimated to be higher than 100 µM, because this is the maximum concentration of CURC possible to be encapsulated in polymeric micelles. This could explain, for example, the apparent contradiction in the behavior of CURC-loaded P84 micelles, which shows a moderate radius of the inhibition zone, with various values according to the incubation conditions, while no difference between the MIC values could be observed. Additionally, the differences between the antibacterial activity reported as an inhibition zone and the minimum inhibitory concentration are due mainly to the different testing protocols for quantitative and qualitative antimicrobial measurements. In the first experiment, CURC-loaded carriers ensure contact with the microorganism grown at the surface of the solid medium, while in the second experiment, the CURC-loaded carriers interact with the microorganism in the volume of the liquid medium. Thus, it is presumable that CURC encapsulated in polymeric micelles exhibits different behavior in these two experimental conditions, due to the 2D and 3D diffusion (on the surface and in the volume of the nutrient solution). The differences were attenuated in the experiments involving free CURC in Pluronic solutions containing unassociated molecules.
All these results have been confirmed by the total number of microbial cell value (CFU/mL) determinates in the well corresponding to the MIC, using the viable cell count (VCC) method. The logarithmic representation of these values shows significant differences between the results obtained for Premicellar P84_CURC, Premicellar P123_CURC, and Premicellar F127_CURC samples in the two incubation conditions, with differences of at least two logarithmic units in the case of the same two microbial species:
S aureus strains (
Figure 11a) and
C. albicans strains (
Figure 11b).
In the case of C. albicans cultures, the photoinactivation effect produced by CURC is also correlated with the results of bacterial cell membrane permeabilization in the presence of Pluronic derivatives, especially in the premicellar area.
The lack of inhibitory activity against Gram-negative strains of
E. coli, in the condition of this experiment, was also observed after the MIC determination test and confirmed by the viable cell count method (VCC method) (
Figure 11c). Even if the block copolymer P84 produces some increase in membrane permeability in the case of
E. coli, this is not sufficient to ensure the photoinhibition effect under the conditions of using a low concentration of Cucumin.
Literature surveys show a large variety of MIC values reported for free Curcumin, usually dissolved in DMSO, against common pathogens. For example, for E. coli, values ranging from 8 to 156 µg/mL were found, probably due to the MIC dependence of the experimental conditions. In the conditions selected for our study, we obtained for a reference sample CURC dissolved in DMSO against standard bacterial cultures, 50–100 µg/mL for S. aureus and C. albicans, similar MIC values to those reported in literature.
Moreover, the comparison between the efficiency proved by CURC encapsulated in various drug delivery systems based on published data becomes more difficult [
45].
In some cases, the encapsulation of the antibiotic into micellar carriers from Pluronic enhances the antibacterial efficiency, but in other experiments, it does not produce any change in the biological effect [
46].
In the present work, samples with CURC encapsulated in polymeric micelles exhibit a reduction in the antibacterial effect compared to the efficiency of the same concentration of CURC dissolved in DMSO, for all three Pluronic derivatives. The reduction in the photoinactivation efficiency is probably due to the lower extent of free CURC molecules able to interact with the cell membrane, since most of the drug is trapped in the micellar core.
The decrease in the photoinactivation efficiency of CURC encapsulated in surfactant micelles compared to the free CURC (dissolved in DMSO) was also reported by Ryu et al. [
47]. The encapsulation of Curcumin in gemini surfactant Surfynol 465 or Tween 80 micelles leads to a reduction in photosensitizing efficiency against
Escherichia coli O157 : H7 compared to CURC in DMSO.
Similar results have been obtained by using rather close concentrations of CURC-Pluronic micelles (100 µg/mL), showing that encapsulated Curcumin produces a lower reduction for
C. albicans of 4.28 log compared to the one produced by free CURC in DMSO (4.97 log). Other studies report more effective photoinactivation of microorganism, but using very concentrated CURC drug carriers (500 µM) and long-term exposure (up to 24 h), which is not feasible for clinical applications [
48].
A very interesting point of behavior must be noted for samples of CURC in premicellar solutions of Pluronic derivatives, where a significant increase in the growth reduction of
C. albicans cultures, for both standard and clinical isolate strains, was determined. These results well correlate with the findings in the measurement on cell membrane permeability, while the increase in fluidization produced by micelles (
Figure 9) is not reflected in an enhancement of antifungal activity.
Even if the photoinhibitory efficiency of CURC in Pluronic micelles is not superior to that presented by free CURC, encapsulation in polymeric micelles brings other benefits, such as increasing stability, bioavailability, and drug release kinetics. Further research is needed to elucidate the effects that the presence of Pluronic monomers in the carrier can bring, and the possibility of increasing the amount of encapsulated drug while maintaining the lowest possible amount of micelle-forming polymer.