3.1. Preparation of RuPLA Nanoconjugates
was conjugated to FDA-approved polylactide via the drug-initiated ROP of LA to give RuPLA
conjugates with different microstructures and chain lengths (i.e., 2000, 4000 and 7000 g/mol), as previously reported (Table 1
]. These conjugates were prepared either from a racemic mixture of d
-lactide or from the enantiopure d
-lactide or l
-lactide. Polymers derived from d
-lactide yielded an amorphous polymer as a result of the random sequence of d
-units along the polymer backbone, whereas polymers derived from the enantiopure monomers yielded semi-crystalline isotactic polymers PLLA and PDLA with a melting temperature Tm
around 140 °C. Mixing two isotactic polylactides of opposite configurations (PLLA and PDLA) at an equimolar ratio allowed the formation of a stereocomplex characterized by superior physical properties, in particular thermal properties, with a Tm
60 °C higher than that of the respective homochiral polymers, in accordance with what has already been reported in the literature [37
]. These conjugates were then formulated into reproducible and narrowly dispersed NPs with an intensity-average diameter Dz lower than 300 nm and a polydispersity index (PdI) around 0.2. The reproducibility of the preparation with respect to particle size was investigated by analyzing three independent batches (Figures S1–S4
). The surface charge of NPs was also investigated by zeta-potential (ζ, mV) measurements. Depending on the microstructure of the RuPLA
nanoconjugates, the zeta potential was different. While stereocomplex NPs (namely, NPs3
) possess a zeta potential below 10 mV, atactic NPs (namely, NPs1
) have a positive zeta potential ca. 30 mV, suggesting that RuOH
might be present on the surface of the atactic NPs.
The ruthenium loading % RuOH was determined by 1H NMR spectroscopy and confirmed after formulation by UV-vis spectrophotometry using the absorption peak of RuOH at 450 nm.
3.2. Biological Evaluation of RuPLA Nanoconjugates
The photobactericidal activity of RuOH and NPs was established through biological assays against four bacterial strains, two Gram-positive strains (S. aureus and S. epidermidis) and two Gram-negative strains (E. coli, and P. aeruginosa), by determining their minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC). While MIC assays determine the lowest concentration of an antimicrobial agent that prevents visible growth of a microorganism, MBC assays determine the lowest concentration that reduces the viability of the initial bacterial inoculum by ≥99.9% which represents a three-logarithmic decrease. Each experiment was realized in triplicate and repeated three times.
RuOH and all NPs were ineffective against both Gram-negative strains at 50 μM with or without light irradiation, indicating that their MIC and MBC are higher than 50 μM.
On Gram-positive strains, RuOH
and all NPs were ineffective under dark conditions at 50 µM. However, after light irradiation with LED visible light (4.83 mW/cm2
) for a total fluence of 25 J/cm2
, NPs1, 3
showed a photobactericidal activity against S. aureus
and S. epidermidis
). The most active NPs are NPs3
, the two stereocomplex ones, with a photobactericidal activity at 12.5 µM. We can therefore conclude that NPs increase the photobactericidal capacity of RuOH
. Indeed, this enhancement is probably related to the increased hydrophobicity rendered possible by the PLA chain and the microstructure of the NPs.
The use of high fluence rates of the exciting light can cause oxygen depletion and PS photobleaching. Therefore, low fluence rate is more clinically relevant. To observe the influence of light dose on the survival rate of bacteria, S. aureus
and S. epidermidis
were directly put in contact with the most active NPs (NPs 1
) in PBS (Figure 1
). After an incubation period of 4 h in the dark in the different solutions, the washed bacteria were irradiated by the same device used previously. Bacterial concentration and survival rate were monitored following the increasing light dose. For each strain, three independent experiments were performed.
NPs3 and 5 showed the best activity against these two bacterial strains since a low light dose (6.25 J/cm2) is sufficient to induce a reduction of up to three log of the survival rate. For NPs 1, a light dose of 18.75 J/cm2 is necessary to induce a complete eradication of bacterial strains.
To understand more the results observed from the bacteria viability assays, flow cytometry experiments were performed to compare the interaction of NPs with bacteria (Figure 2
). The two Gram-positive strains and one Gram-negative strain (P. aeruginosa
) were incubated with the solutions of NPs (NPs1
) at 10 µM. After 4 h of incubation in the dark at 37 °C, the bacteria were washed with PBS by centrifugation and the washed bacteria were resuspended with 500 µL of PBS. Fluorescence emissions were analyzed with a BD FACS Aria III cell sorter. Histograms were used to determine the percentage of labeled bacteria with NPs. These percentages were compared with the percentage obtained with the untreated bacteria used as a reference. NPs1
showed weak to no interaction with bacteria. These results are consistent with the low or lack of photobactericidal activity of NPs1
observed above. Although NPs3
showed a photobactericidal activity, a weak interaction is observed with these NPs. Concerning NPs5
, a remarkable fluorescence is obtained after incubation of these NPs with bacteria. This high fluorescence indicates a strong interaction of NPs with bacteria. We can observe a difference between the two types of bacteria. A lower interaction is obtained with the Gram-negative strain. However, it must be pointed out that flow cytometry only gives an idea of possible interaction between NPs and bacteria but does not give information about the uptake. This difference of interaction was highlighted in numerous studies that discuss the fundamental difference in susceptibility to aPDT between Gram-positive and Gram-negative due to the difference of the structure of the bacterial cell walls [40
]. Gram-positive bacteria possess a thick cell wall (20–80 nm) of peptidoglycan as outer shell of the cell. In contrast, Gram-negative bacteria have a relatively thin (<10 nm) layer of cell wall composed of peptidoglycan but harbor an additional outer membrane made of layer of lipopolysaccharide [43
]. These differences in the cell envelope confer different properties to the cell. One would have expected NPs1
to have the best interaction with bacteria, and hence photobacterial activity, because of their positive zeta potentials, especially NPs1
which, in addition, is more hydrophobic. Indeed, the presence of a positive zeta potential should favor the electrostatic interaction between the particles and Gram-negative and Gram-positive bacteria whose membranes are negatively charged. However, the microstructure of RuPLA
nanoconjugates seems to play an important role in their interaction with bacteria, and hence in their photobactericidal activity. More work needs to be done to have a complete understanding of the role of polymer microstructure on the interaction with bacteria.