Hydrolytic Degradability, Cell Tolerance and On-Demand Antibacterial Effect of Electrospun Photodynamically Active Fibres

Photodynamically active fibres (PAFs) are a novel class of stimulus-sensitive systems capable of triggering antibiotic-free antibacterial effect on-demand when exposed to light. Despite their relevance in infection control, however, the broad clinical applicability of PAFs has not yet been fully realised due to the limited control in fibrous microstructure, cell tolerance and antibacterial activity in the physiologic environment. We addressed this challenge by creating semicrystalline electrospun fibres with varying content of poly[(l-lactide)-co-(glycolide)] (PLGA), poly(ε-caprolactone) (PCL) and methylene blue (MB), whereby the effect of polymer morphology, fibre composition and photosensitiser (PS) uptake on wet state fibre behaviour and functions was studied. The presence of crystalline domains and PS–polymer secondary interactions proved key to accomplishing long-lasting fibrous microstructure, controlled mass loss and controlled MB release profiles (37 °C, pH 7.4, 8 weeks). PAFs with equivalent PLGA:PCL weight ratio successfully promoted attachment and proliferation of L929 cells over a 7-day culture with and without light activation, while triggering up to 2.5 and 4 log reduction in E. coli and S. mutans viability, respectively. These results support the therapeutic applicability of PAFs for frequently encountered bacterial infections, opening up new opportunities in photodynamic fibrous systems with integrated wound healing and infection control capabilities.


Introduction
Antibiotic resistance poses a risk to mankind, threatening not just our health and food safety but also our environment [1]. If not treated in a timely manner, bacterial contamination causes infection and detrimentally affects the performance of medical devices implanted in vivo as well as the outcome of surgical therapies. With the growing trends in antibiotic resistance, timely management of infection is therefore at risk, especially in the context of chronic ulcers [2], oral wounds [3] and bone fractures [4], which frequently cause significant pain, death risks and economic burden. Surgical wound infections alone account for one-quarter of all healthcare-associated infections, causing an estimated 2.5-fold increase in hospital stay and additional healthcare costs [5]. There is therefore a critical need for antimicrobial strategies that minimise antibiotic reliance and that treat antibiotic-resistance infections when activated on-demand via safe external stimuli.
Photodynamic therapy (PDT) is a promising antibiotic-free antimicrobial treatment, which is based on the application of light to a photosensitiser (PS) in the presence of molecular oxygen. The light is absorbed by the PS, enabling the transfer of an electron from a ground to an excited state. The consequent reaction of the excited PS molecule with molecular oxygen generates toxic reactive oxygen species (ROS) [6]. Due to the high reactivity of ROS, PDT can be conveniently used to eradicate fungi, viruses [7] and bacteria [8], regardless of resistant strains and with minimal risks of inducing antimicrobial resistance. Consequently, the application of PS and light for PDT should therefore be tailored to accomplish antibacterial photodynamic effect, on the one hand, and cellular viability and low dark cytotoxicity (i,e., in the absence of light), on the other hand.
Variation of PS dosage and light intensity can be exploited to control the extent of photodynamic effect; however, accomplishing selective antimicrobial activity is still a challenge due to the concentration-dependent aggregation of PS molecules [9], which can lead to limited ROS generation [10], water solubility [6,11] and visible light absorption [12]. These limitations restrict the applicability of PDT in the clinical management of infectious diseases [13], at a time when the need for new antimicrobial strategies has never been more critical. Although the synthesis and testing of new PSs to address the above-mentioned challenges has been reported [10,11,14], encapsulation of PSs already in use in the clinic in polymer carriers represents a promising route to minimise PS aggregation, ensure a prolonged therapeutic window and comply with current regulatory framework [15][16][17][18]. With this strategy, control of the (i) release profile of PS from the carrier, (ii) PS uptake in both cells and bacteria and (iii) selectivity between the bactericidal effect and sparing effect upon host cells are key to successful performance.
Electrospun meshes are frequently used for the development of wound [19] and oral [20] dressings, whereby the fibre diameter and pore size can be tuned to accommodate drugs and support soft tissue repair and infection control [21][22][23][24][25]. By selecting appropriate fibre-forming building blocks, bespoke molecular configurations [26,27] and drug-polymer nanoscale interactions [28], the electrospinning process can be developed to enhance hydrolytic fibre stability and the retention of both drugs and fibres in wet environment. Aliphatic polyesters, e.g., PLGA, have been used for the fabrication of electrospun biomedical fibres [29,30], as they can be used in FDA-approved devices, e.g., surgical sutures [31]; are biodegradable; and can release degradation products that support wound repair [32,33]. In light of their superior processability, these polymers can be encapsulated with soluble factors during fibre formation, so that factor release can be achieved through either fibre erosion, diffusion or swelling [34].
In this work, we describe the simple and effective fabrication of long-lasting electrospun PAFs that induce bactericidal effect on both Gram-negative E. coli and Gram-positive S. mutans when irradiated with visible light, while preserving the viability of mammalian cells. Our strategy to deliver on this was to leverage both the photodynamic capability of single PSs and the semicrystalline morphology of fibre-forming aliphatic polyesters, so that PAFs with controlled photodynamic effect, controlled PS release capability and retained wet state fibrous architecture could be successfully accomplished, while avoiding multistep synthetic routes of polymer functionalisation and post-spinning fibre crosslinking. In solution, methylene blue (MB) displayed higher uptake in either aforementioned bacteria, i.e. E. coli and S. mutans, or L929 mouse fibroblasts with respect to erythrosine (ER), and was therefore selected as a suitable PS for the creation of PAFs. Electrospinning of MB-loaded solutions containing varying PLGA:PCL weight ratios generated submicron fibres with ~100 wt.% encapsulation efficiency and up to ~1.3 MPa of averaged Young's modulus. The hydrolytic degradation and MB release profiles of PAFs were strongly affected by the polymer morphology and chemical composition, so that PAFs could successfully support attachment and proliferation of L929 mouse fibroblasts over a 7-day cell culture and still trigger up to 4 log reduction in bacteria viability. The knowledge gained with these electrospun PAFs could be useful to accomplish photodynamic systems with integrated anti-infection and wound healing functionalities for the management of chronic tissue states.

Hydrolytic Degradation Tests
Dry electrospun samples (n = 3) were cut into 1 cm 2 squares and weighed prior The mass loss was calculated via Equation (1): whereby m t and m d represent the dry weights of both the sample collected at the selected time point t and the fresh electrospun sample, respectively.

Methylene Blue (MB) Release Tests
Dry samples (~20 mg) were incubated in PBS (

Cytotoxicity Tests
Dry electrospun samples were cut into squares (~20 mg) and exposed to 15 min of UV light on each side. Resulting samples were then employed for both extract and contract cytotoxicity tests, as reported below.

Extract Cytotoxicity Tests
Extract sample solutions were prepared by incubating previously accomplished UV-disinfected samples in 5 mL of PBS at 37 °C for 0, 2 and 24 h. In parallel, L929 murine fibroblasts were seeded onto an opaque walled 96-well plate at a density of 5 × 10 3 cells per well with 100 µL of complete α-MEM and incubated for 24 h (37 °C, 5% CO 2 ) to allow for cell attachment. The cell culture medium was then removed and each well washed with fresh PBS, prior to addition of the extract solutions.
Control wells contained fresh PBS with cells (negative control) and 10 vol.% OPDEGE solution in PBS (positive control). Following a 10-min incubation, the well plates were either exposed to light for 60 min or wrapped with aluminium foil and exposed to light for the same period of time (dark control). Following light treatment, an ATP luminescence assay (Promega) was carried out to quantify cell viability, according to manufacturer instructions. Briefly, 100 µL of CellTiter-Glo ® 2.0 solution was added to each well, and the well plate covered with aluminium foil and shaken at 200 RPM for 5 min, prior to 25-min equilibration at room temperature. On each plate, luminosity readings of each tested sample group were compared to the averaged luminosity of the negative dark control (fresh PBS with cells and dark light exposure) to determine the percentage of killed cells, according to Equation (2): whereby Lum test and Lum control represent the luminosity of the test and control wells, respectively. onto an opaque walled 96-well plate and immediately exposed to light for 60 min, or wrapped with aluminium foil and exposed to light for the same period of time (dark control). Following light treatment, the solutions were serially diluted and 100 µL aliquots were spread onto fresh agar plates. The plates were then incubated at 37 °C for 24 h. The following day, manual counting of colonies was performed and the number of CFU for each solution was calculated and compared to the initial inoculation. The experiment was performed in triplicate.

Contact Photodynamic Tests
Lawns of bacteria were spread by using a sterile swab dipped in bacterial broth and streaking across a fresh agar plate. Sterile tweezers were used to place UV-disinfected electrospun discs (Ø 10 mm, n = 3) onto the agar plate inoculated with bacteria in triplicate. The plates were incubated for 60 min to allow for MB release, before being exposed to light for 30 or 60 min (or wrapped in foil as the dark control). Agar plates were then incubated at 37 °C overnight to allow for bacterial growth. The following day, images were taken of each plate. Zones of inhibition were calculated using the straight-line tool on ImageJ software, to count the number of pixels in a known section of the image to produce a scale, and then to measure the zone size for each image.

Statistical Analysis
Significant differences were evaluated using an unpaired Student's t-test (p < 0.05). All data were collected in triplicate and presented as mean ± standard deviation.
Despite these initial advances, however, the biocompatibility of PAFs in both inert and antimicrobial states has only partially been addressed [24,33,35], which is critical to enable applicability in medical devices. Furthermore, electrospun fibres often suffer from instability and macroscopic shrinking in aqueous environments due to water-induced fibre swelling and merging [16,27,35], which can lead to uncontrolled release of PS and fast hydrolytic degradation [34]. To address these Gram-negative and Gram-positive bacteria models, given their occurrence in chronic wounds [38] and periodontal diseases [39], respectively. These preliminary investigations informed the selection of the PS for fibre encapsulation, so that PAFs with varied chemical composition (Table 1) were characterised with respect to their hydrolytic stability, release capability, bactericidal effect and cytotoxicity in dark and following light exposure.

PS Uptake of Mammalian and Bacterial Cells
To assess the effectiveness of the PSs in vitro, uptake and photodynamic tests  The increased cellular uptake observed with MB-with respect to ER-supplemented solutions can be attributed to the different electrostatic charge and decreased molecular weight of the phenothiazine, compared to the xanthene, dye. The cationic charge of MB has been shown to mediate electrostatic interactions and binding with the bacterial wall and cellular membrane [6,9,42], while the decreased molecular weight facilitates passive diffusion of the PS in the cell, ultimately leading to increased PS uptake [43]. On the other hand, ER displays a dianionic configuration in the neutral pH of the PBS solution [44], whereby the presence of the two negative charges decreases the interaction of ER with the cell membrane, therefore hindering the uptake by bacteria.

PS Impact on L929 Cells, E. coli and S. Mutans
Following the uptake tests, the impact of PS solutions on cellular and bacterial activity was investigated via an ATP luminescence assay, in line with its employability to assess the metabolic activity of cells [45] and bacteria [46], and its  Overall, these results confirmed the photodynamic activity of both PSs against selected bacteria, in line with previous reports [47]. The photodynamic effect of selected ER-supplemented solutions proved to be lower than previous antibacterial results with S. mutans (≥ 2 log reduction) [44], a result that is attributed to the   Figure S1A, Supplementary   Information). This observation was hypothesised to be attributed to the poor miscibility of PLGA and PCL [50], which was expected to be decreased when equivalent weight fractions of both polymers were applied. The poor miscibility of selected fibre-forming polymers was supported by DSC measurements (Figure 3D-F).
Unlike fibres made with increased PLGA content, however, a different thermal behaviour was observed in PCL-rich PAFs MB20-PLGA20-CL80 and respective MB-free variant. While the former sample revealed only the PCL-related melting transition, both PCL-and PLGA-related melting transitions were detected in the fibre control ( Figure 3D). The most likely explanation for this observation is that the MB molecules encapsulated in the fibre are more compatible with the PLGA than with the PCL phase. The excess of carbonyl groups in the repeat unit of the former compared to the latter polymer may promote increased electrostatic interactions with MB, so that PLGA crystallisation is hindered in MB-encapsulated fibres, similarly to other polyester fibres loaded with low molecular weight additives [37,49].
This observation is supported by the constantly decreased PLGA-related melting enthalpy (∆H m2 ) measured in PAFs compared to the corresponding fibre controls (Table 2). In order to further elucidate the effect of either MB encapsulation or PLGA:PCL weight ratio on the macroscopic behaviour of respective fibres, tensile tests were  Table 2).
Other than that, MB-free fibres made with increased PLGA content revealed a gradual increase of stress at break ( Figure 4B) and Young's modulus ( Figure 4C), a result that is in agreement with the inherently higher mechanical properties of PLGA with respect to PCL fibres and the non-miscibility of the two polymers [49,[51][52][53].

Fibre Behaviour in Physiological Environment
Additionally to microstructure and thermal analysis, the attention moved to the characterisation of PAFs in an aqueous environment, aiming to accomplish fibres with controlled degradability, retained fibrous architecture and sustained release of MB. The equilibrium water uptake of both MB-encapsulated and MB-free samples was initially measured, as the first event in the hydrolytic degradation process of aliphatic polyesters [36,54,55]. No detectable macroscopic changes (e.g., shrinking) were observed in the retrieved 24-h incubated fibres, while a significantly increased water uptake was measured in PAFs (WU = 301 ± 2-348 ± 25 wt.%) with respect to MB-free controls (WU = 62 ± 6-250 ± 19 wt.%, p < 0.05) ( Figure 6A). MB-free fibres made with the highest PLGA content also displayed a significant increase in water uptake with respect to the other two sample groups, while a counterintuitive opposite trend was observed with respective MB-encapsulated samples ( Figure 6A). Polymer crystallinity is known to play a critical role on the diffusion of water in aliphatic polyesters [36,54,55], as the presence of ordered domains limits water diffusion, water uptake and volumetric swelling of fibres. The semicrystalline morphology of both PAFs and fibre controls therefore explains the dimensional stability of these samples following incubation with water, in contrast to the prompt macroscopic shrinking observed when amorphous fibrous architectures were tested [16,27,35]. The increased values of water uptake measured in PAFs compared to the corresponding fibre controls therefore reflects aforementioned considerations, as decreased melting enthalpies, suggesting lower crystallinity [56], were constantly measured in the former compared to the latter groups (Table 2).
Together with the polymer morphology, the hydrophilicity and secondary interaction capability of the fibre-forming polymer are also key in explaining the lowest and highest water uptake of samples MB-PLGA80-CL20 and PLGA80-CL20 among each sample group, respectively. PLGA is known to be more hydrophilic compared to both PLA and PCL [36,55], given the absence of methyl groups and reduced number of methylene bridges in respective repeat unit. Consequently, the higher water uptake measured in samples PLGA80-CL20 compared to both samples PLGA50-CL50 and PLGA20-CL80 is attributed to the excess of PLGA content and respective increase in fibre hydrophilicity in the former with respect to the two latter groups. The presence of PLGA is also likely to explain the counterintuitively lower water uptake of sample MB20-PLGA80-CL20 with respect to the other two samples of PAF ( Figure 6A), due to the presence of PLGA-MB secondary interactions ( Figure 3D).
Following the water uptake measurements, an 8-week hydrolytic degradation study was carried out, whereby the sample mass loss ( Figure 6B) and microstructure ( Figure 7) were assessed at specific time points.

(G) (H) (I)
MB20-PLGA20-PCL80 and PLGA20-PCL80 exhibited the lowest change in weight ( Figure 6B). These trends in mass loss were also supported by the changes in fibrous architectures observed in retrieved samples over the course of hydrolytic degradation. Although samples MB20-PLGA20-CL80 ( Figure 7A-C) and MB20-PLGA50-CL50 ( Figure 7D-F) retained their fibrous configuration for the whole incubation period, PAFs with the highest content of PLGA displayed significant fibre merging after 6 weeks of degradation ( Figure 7H) and complete loss of mechanical integrity after 8 weeks. Both trends in mass loss and fibrous microstructure therefore reflect previous considerations regarding the higher hydrolytic reactivity of PLGA with respect to PCL [36].
The observed water uptake and degradation characteristics of PAFs were then considered to study respective MB release capability. In contrast to the complete release of MB described by sample MB20-PLGA20-CL80 within 3 h ( Figure 8A), fibres with increased PLGA content revealed a significantly increased retention of MB, whereby samples MB20-PLGA80-CL20 and MB20-PLGA50-CL50 released 95 wt.% of MB only after 3 weeks ( Figure 8B). Selected PLGA-rich fibres also indicated a slower release with respect to Indocyanine green-encapsulated PLA fibres [33], whereby ~65 wt.% release was observed within 1 week in PBS despite only 1 wt.% mass loss being recorded. In contrast, an even slower release than the one reported in this study was reported with curcumin-encapsulated polyurethane fibres with low hydrolytic degradability (≤ 5 wt.%, 2.5 weeks) [24], a result that was attributed to the hydrophobic interactions between curcumin and the fibre-forming polymer. The aforementioned release profiles therefore suggest a diffusion-rather than degradation-led mechanism of release, whereby the PLGA-rich fibres displaying ≤ 40 wt.% mass loss over three weeks described the highest retention of MB, while significant burst release was measured with the PCL-rich samples MB20-PLGA20-CL80 displaying just 5 wt.% mass loss ( Figure 6B). The most likely explanation for this observation is attributed to the role played by the PLGA in mediating secondary interactions with MB, again supported by previous DSC results ( Figure 3D-F, Table 2), so that diffusion of MB out of the fibres is hindered in fibres with increased PLGA content. Conversely, an excess of the PCL phase in the PAFs is demonstrated to ensure superior hydrolytic stability ( Figure 6B) and wet state retained fibrous microstructure (Figure 7), in agreement with the semicrystalline morphology and hydrophobicity of this polymer.
In the following, the cytotoxicity and antibacterial testing in vitro will be presented, whereby PAFs made with equivalent PLGA:PCL weight fraction will be selected due to their controlled release and long-lasting MB retention capabilities in aqueous environment.

Contact and Extract Cytotoxicity Tests
The morphology of cells cultured on to PAFs and MB-free controls was inspected via SEM after 1-and 7-day cell culture, whereby either an initial 60-min light exposure or 60-min dark incubation (dark controls) was applied to cells. Clear cellular proliferation with evidence of ECM deposition was observed with MB-free fibres PLGA50-CL50, with cell spreading depicted already after 24 h-culture ( Figure   9A and Figure S2A, Supplementary Information). At day 7, the surface of both the light-treated sample ( Figure 10A) and dark control ( Figure S3A, Supplementary Information) appeared completely coated by cells, confirming the high cellular tolerability of selected MB-free fibres, in line with previous reports on the biocompatibility of polyester fibres in vitro [33,37,50,57].   [58][59][60]. After 7 days of culture, cell growth was spread over the sample surface, whereby a layer of ECM appeared to have deposited and round cells were no longer present (Figures 10D and S3D). These SEM images therefore indicate high cell tolerance of these fibres, a result that agrees with the low concentration (~0.6 and ~1.5 µg·100 µL −1 ) of MB expected to be released at this time point ( Figure   8). Unlike previous reports demonstrating the lack of dark cytotoxicity in vitro [24,33,35], these data confirm high cell viability even following light irradiation.
The cell tolerance of PAFs with decreased MB content were further confirmed by quantitative ATP assays on L929 cells cultured with 0-24-h fibre extracts ( Figure   11). The extracts of sample MB2-PLGA50-CL50 displayed the highest degree of cell viability, whereby an averaged cell killing of 35% and 4% was measured following 60-min light activation and 60-min dark incubation, respectively. Although cell tolerance (≤ 20% ATP reduction) was also revealed in dark with the extracts of the other two fibre groups, significant cytotoxic effects (≥ 70% ATP reduction) were measured in both cases following activation with light, providing first indication of fibre photodynamic activity.

Antibacterial Photodynamic Capability of PAFs
Once the cytotoxicity of PAFs was investigated, the attention moved to the quantification of the antibacterial photodynamic capability of PAFs. Fibres electrospun with varied MB content were initially cultured in contact with agar plates inoculated with either E. coli or S. mutans, so that the zone of inhibition (ZOI) following light irradiation (for either 30 or 60 min) and dark incubation was measured  Other than E. coli, significant antibacterial effects were also observed against S. mutans ( Figure 12B), whereby variation in fibre MB dosage, rather than the irradiation time, seemed to play a significant role. ZOIs in the range of 14.8 ± 0.7-18.7 ± 0.9 mm were measured on the 60-min light-treated samples, which proved to be similar to the one recorded following dark incubation (Ø = 10.7 ± 0.7-18.5 ± 1.0 mm). This observation is in agreement with previous photodynamic data, whereby similar viability reduction was expressed by S. mutans after 60-min incubation with MB-supplemented solutions in dark ( Figure 2C) and under light irradiation ( Figure   2F).
To confirm that the photodynamic effect was governed by the release of MB out of the PAFs, 0-24-h extracts of fibre MB2-PLGA50-CL50 were tested and bacteria counts measured ( Figure 13). In contrast to the results obtained in dark (≤ 0.1 log reduction) and with the PBS negative control, a significant reduction (2-2.5 log) in E. coli viability was again recorded following light treatment, whereby an extraction time of at least two hours was needed to enable detectable effects on E. coli. When the same test was carried out with S. mutans, a significant reduction (> 1 log) was still observed following dark incubation with both 2 and 24-h fibre extract, a result that is again in agreement with the decreased tolerance of this bacteria to native MB ( Figure 2C). These values were further increased (> 2 log reduction) when light was applied, whereby even the 0-h extract was proven to play a significant antibacterial effect. Overall these data confirm the key role played by the release capability of PAFs in triggering photodynamic effect on-demand and ensuring bacterial tolerance when no light exposure is applied. In this regard, the release profile of the PS should also be customised according to the target bacteria, given aforementioned difference in photodynamic susceptibility according to the bacterial structure.

Conclusions
Electrospun PAFs with varying PLGA:PCL weight fraction and MB content were studied to accomplish retained wet-state fibrous microstructure, controlled degradability and controlled PS release profile, as a means to deliver long-lasting fibres with integrated cellular tolerance and antibacterial photodynamic effect.