Development of Antimicrobial Laser-Induced Photodynamic Therapy Based on Ethylcellulose/Chitosan Nanocomposite with 5,10,15,20-Tetrakis(m-Hydroxyphenyl)porphyrin

The development of new antimicrobial strategies that act more efficiently than traditional antibiotics is becoming a necessity to combat multidrug-resistant pathogens. Here we report the efficacy of laser-light-irradiated 5,10,15,20-tetrakis(m-hydroxyphenyl)porphyrin (mTHPP) loaded onto an ethylcellulose (EC)/chitosan (Chs) nanocomposite in eradicating multi-drug resistant Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans. Surface loading of the ethylcelllose/chitosan composite with mTHPP was carried out and the resulting nanocomposite was fully characterized. The results indicate that the prepared nanocomposite incorporates mTHPP inside, and that the composite acquired an overall positive charge. The incorporation of mTHPP into the nanocomposite enhanced the photo- and thermal stability. Different laser wavelengths (458; 476; 488; 515; 635 nm), powers (5–70 mW), and exposure times (15–45 min) were investigated in the antimicrobial photodynamic therapy (aPDT) experiments, with the best inhibition observed using 635 nm with the mTHPP EC/Chs nanocomposite for C. albicans (59 ± 0.21%), P. aeruginosa (71.7 ± 1.72%), and S. aureus (74.2 ± 1.26%) with illumination of only 15 min. Utilization of higher doses (70 mW) for longer periods achieved more eradication of microbial growth.


Introduction
The rapidly growing resistance of microbial pathogens against antibiotics must be considered as one of the most significant clinical challenges facing the world nowadays [1,2]. The development of microbial resistance against most of the known classes of antibiotics has become an acute problem, particularly in hospitals [3,4]. Likewise, in the last few decades, advances in the search for new antibiotics have not kept pace with the growing number of resistant bacterial strains [1]. For example, many reports showed that extendedspectrum β-lactamase-producing Enterobacteriaceae (ESBL-PE), multidrug-resistant (MDR) Pseudomonas aeruginosa, carbapenem-resistant Enterobacteriaceae (CRE), Acinetobacter baumannii, and methicillin-resistant Staphylococcus aureus (MRSA) are increasingly identified as the predominant causative pathogens in patients, due to antibiotic misuse [1,5] Therefore, to counteract this emerging public health problem there is an urgent need to develop and The focus of the present study is the surface loading of EC/Chs nanocomposite with mTHPP, yielding the mTHPP EC/Chs nanocomposite. Characterizations of the resulting nanocomposite were performed with FT-IR, X-ray diffraction (XRD), and scanning electron microscopy (SEM). Screening of different continuous laser systems with different powers allowed the investigation of the effects of different laser wavelengths to select the most effective option for the development of using this nanocomposite for aPDT against multidrug resistant microbial cells.

Preparation of mTHPP-Loaded Nanocomposite
The antimicrobial photodynamic therapy (aPDT) of mTHPP-loaded EC/Chs nanocomposite was investigated for three pathogens. In the present study, the mTHPP photosensitizer was chosen due to its absorption capacity at several laser light wavelengths in the blue and red region, which allows for the investigation of the effect of different excitation wavelengths. The synthesis of mTHPP was achieved using the standard procedure ( Figure 1) and was followed by loading it into EC/Chs nanocomposite to overcome its solubility drawbacks and enhance drug delivery [39]. The resultant mTHPP EC/Chs nanocomposite possesses a green color, which indicates that the system acquired an overall positive charge. Chitosan was suggested to be the source of the positive charge, and this is compatible with the literature [39]. Zeta measurements in Table 1 proved an overall positive charge for the nanocomposite.

Polarized Light Microscopy
Polarized light microscopy was used to evaluate the light activity of the mTHPPloaded nanocomposite by applying different filters, as shown in Figure 2. The light-  Polarized light microscopy was used to evaluate the light activity of the mTHPPloaded nanocomposite by applying different filters, as shown in Figure 2. The lightreflection pattern of mTHPP was assigned in daylight with a cold blue filter, which indicated incorporation of mTHPP into the nanocomposite structure.

FT-IR Spectroscopy
The FT-IR spectra of the parent materials, free composite, and mTHPP EC/Chs nanocomposite are shown in Figure 3. The mTHPP spectrum displays a specific region of mTHPP between 1700-500 cm −1 . The characteristic bands of mTHPP at 3386, 3021, 2928, 1596, 1441, 978, 740, and 559 cm −1 are associated with NH stretching, CH (phenyl) stretching, CH (pyrrole) stretching, the vibration of C-C-C in phenyl rings, CH rocking on phenyl, in-plane δ and out-of-plane δ N-H, and hydrogen atom motion (NH), respectively [40,41]. The EC spectrum revealed specific bands that are characterized for the cellulose derivatives. The bands at 3453, 2976, 2481, 1363, and 1051 cm −1 are associated with OH group stretching, H group stretching, H-C-H asymmetric stretching, symmetric stretching of terminal CH3 of a primary ethyl group, C-H bending, and C-O-C stretching, respectively [42,43]. Additionally, the FT-IR spectrum of chitosan revealed bands at 3471, 2948, 2840, 1644, 1514, and 1025 cm −1 , indicative of N-H stretching, symmetric CH3 and asymmetric CH2 stretching, CH stretching, C=O stretching (amide I), -NH stretching (amide II), and the free amino group (-NH2) at the C2 position of glucosamine, respectively [44]. On the other hand, the free composite was assigned a major band at 3411 cm −1 , which corresponds to overlapping hydroxyl groups of the composite. Additionally, the intensity of the band at 2935 cm −1 was decreased and the position shifted to lower frequency. In addition, a peak at 1640 cm −1 became sharper. Moreover, upon the loading of mTHPP into

FT-IR Spectroscopy
The FT-IR spectra of the parent materials, free composite, and mTHPP EC/Chs nanocomposite are shown in Figure 3. The mTHPP spectrum displays a specific region of mTHPP between 1700-500 cm −1 . The characteristic bands of mTHPP at 3386, 3021, 2928, 1596, 1441, 978, 740, and 559 cm −1 are associated with NH stretching, CH (phenyl) stretching, CH (pyrrole) stretching, the vibration of C-C-C in phenyl rings, CH rocking on phenyl, in-plane δ and out-of-plane δ N-H, and hydrogen atom motion (NH), respectively [40,41]. The EC spectrum revealed specific bands that are characterized for the cellulose derivatives. The bands at 3453, 2976, 2481, 1363, and 1051 cm −1 are associated with OH group stretching, H group stretching, H-C-H asymmetric stretching, symmetric stretching of terminal CH 3 of a primary ethyl group, C-H bending, and C-O-C stretching, respectively [42,43]. Additionally, the FT-IR spectrum of chitosan revealed bands at 3471, 2948, 2840, 1644, 1514, and 1025 cm −1 , indicative of N-H stretching, symmetric CH 3 and asymmetric CH 2 stretching, CH stretching, C=O stretching (amide I), -NH stretching (amide II), and the free amino group (-NH 2 ) at the C 2 position of glucosamine, respectively [44]. On the other hand, the free composite was assigned a major band at 3411 cm −1 , which corresponds to overlapping hydroxyl groups of the composite. Additionally, the intensity of the band at 2935 cm −1 was decreased and the position shifted to lower frequency. In addition, a peak at 1640 cm −1 became sharper. Moreover, upon the loading of mTHPP into the composite, a significant change was that the band of the hydroxyl groups was shifted to lower frequency, and a band at 1381 cm −1 was shifted to the lower frequency resulting from terminal CH 3 groups. Moreover, a new band at 1086 cm −1 was observed.

XRD Analysis
The XRD patterns of native materials, as well as free and loaded nanocomposites, are shown in Figure 4. The mTHPP exhibits a typical diffraction pattern, as reported by Kang et al. [44]. Likewise, the polysaccharide derivatives exhibited a pattern characteristic for polysaccharides. Herein, the EC sample exhibited broad peaks at around 7.80° and a sharp band at 20.6°, which refers to a d-spacing at 10.8 and 4.33 Å respectively, like all cellulose and cellulosic derivatives [45]. In addition, the Chs sample showed a typical chitosan XRD pattern, which indicated two sharp peaks at 11 and 20° with d-spacing at 8.3 and 4.4 Å,

XRD Analysis
The XRD patterns of native materials, as well as free and loaded nanocomposites, are shown in Figure 4. The mTHPP exhibits a typical diffraction pattern, as reported by Kang et al. [44]. Likewise, the polysaccharide derivatives exhibited a pattern characteristic for polysaccharides. Herein, the EC sample exhibited broad peaks at around 7.80 • and a sharp band at 20.6 • , which refers to a d-spacing at 10.8 and 4.33 Å respectively, like all cellulose and cellulosic derivatives [45]. In addition, the Chs sample showed a typical chitosan XRD pattern, which indicated two sharp peaks at 11 and 20 • with d-spacing at 8.3 and 4.4 Å, respectively [46,47]. On the other hand, the peaks at 44 and 74 • of the mTHPP crystallographic isotropic phase were observed in the loaded nanocomposite as they had been observed in the magnified load nanocomposite pattern, which may be indicative of the inclusion of the mTHPP particle into the composite network.   Figure 5 illustrates the topographical studies of mTHPP, the free composite, and the mTHPP-loaded nanocomposite. The mTHPP surface in Figure 5a,b shows a random surface appearance with EDX chart continent from C, N, and O. In contrast, the free composite appearance in low and high magnification in Figure 5d,e appears as a collapsed network with layer-layer constriction. On the other hand, the mTHPP-loaded  Figure 5 illustrates the topographical studies of mTHPP, the free composite, and the mTHPP-loaded nanocomposite. The mTHPP surface in Figure 5a,b shows a random surface appearance with EDX chart continent from C, N, and O. In contrast, the free composite appearance in low and high magnification in Figure 5d,e appears as a collapsed network with layer-layer constriction. On the other hand, the mTHPP-loaded nanocomposite topography clearly shows a uniform network structure including mTHPP particles in nanorange (>200 nm). Moreover, the imaging process of the high magnification SEM obtained to image the average particle size of mTHPP and mTHPP EC/Chs nanocomposite was recorded at about 120 nm. This confirms that the prepared carrier system is of nano scale.

Particle Size and Zeta Potential Measurements
The dynamic light scattering (DLS) results, polydispersity index (PDI), and mean particle size determinations are listed in Table 1. The particle distribution was measured as a dispersion in aqueous solution and gave values of 175, 292, and 595 nm for the j)

Particle Size and Zeta Potential Measurements
The dynamic light scattering (DLS) results, polydispersity index (PDI), and mean particle size determinations are listed in Table 1. The particle distribution was measured as a dispersion in aqueous solution and gave values of 175, 292, and 595 nm for the mTHPP free composite, and mTHPP-loaded nanocomposite, respectively. These particle sizes indicate that the free composite is of nanoscale dimension. However, the loading process resulted in more particle aggregation where the PDI values of mTHPP EC/Chs nanocomposite was increased twofold. On the other hand, the zeta potential was calculated from the three main measured factors (cell current, phase shift, and mobility) that depend on the charge over particle surface. Zeta measurements of mTHPP, free nanocomposite, and mTHPP EC/Chs nanocomposite are tabulated in Table 1. Neat mTHPP had a negative zeta charge, which presented an average zeta potential of -20.55 mV. For the free composite, a highly positive zeta charge was recorded and determined to be 35.5 mV. This is the result of chitosan, which plays a vital role in the net charge of nanocomposite; whereas EC was charge-neutral [48,49]. In addition, the mTHPP-loaded nanocomposite system had a decreased positive charge of 23.84 mV.

Thermal Stability and UV/Vis Absorption of mTHPP and Its Nanocomposite
The UV/Vis absorption of porphyrins and materials derived thereof is one of the main analytical tools in this area [50]. In addition, the thermal stability of the porphyrin and the nanocomposite were studied to assure their stability under biological conditions. The temperature of mTHPP and mTHPP-loaded nanocomposite solutions dissolved in DMSO was varied gradually from 37 to 44 • C. The absorption spectra of the porphyrin and its nanocomposite at different temperatures exhibit five absorption bands as shown in Figure 6a,b. The first one, P 1 at 419 nm, represents the Soret or B band due to the transition from the ground state (S 0 ) to the second excited state (S 2 ), which ranges from 380 to 500 nm depending on whether the porphyrin is βor meso substituted. The other bands are P 2 at 515 nm, P 3 at 550 nm, P 4 at 591 nm, and P 5 at 647 nm. These represent the Q bands, which range from 500 to 750 nm due to the weak transitions from S 0 to the first excited state (S 1 ) [51]. The relative absorbances for the porphyrin were 0.0064, 0.0112, 0.014, 0.0952, and 0.096; while the porphyrin nanocomposite had relative absorbances of 0.0078, 0.0133, 0.0155, 0.098, and 0.0082 for the laser wavelengths used-458, 476, 488, 515, and 637 nm, respectively. Accordingly, the descending arrangement of the laser wavelengths according to their absorption by the porphyrin and its nanocomposite is 515 nm > 488 nm > 476 nm > 635 nm > 458 nm. The porphyrin nanocomposite has different absorbance enhancement ratios for P1, P2, P3, and P4, such as 22%, 18%, 11%, and 3%, respectively for the laser wavelengths used, while the absorbance of P5 is quenched by 15%. The inset of Figure 6a,b shows that the porphyrin absorption bands are unaffected by the temperature except for P 1 at 418.90 nm, which exhibits an absorbance decrease from 0.513 to 0.432 upon the slight temperature increase. The porphyrin nanocomposite enhanced the thermal stability of the porphyrin absorption P 1 .

Photodynamic Inactivation of Microbial Strains
The current study aimed to determine the degree to which photodynamic therapy using mTHPP-loaded nanocomposites could be effective against multidrug-resistant pathogens in vitro. The microwell dilution method based on turbidometry and the plate counting method (CFU method) were used to prescreen the antimicrobial efficacy of mTHPP and the mTHPP-loaded nanocomposite in the presence (irradiation with 635 nm laser light) and in the absence of laser light.
As shown in Table 2, both the mTHPP and mTHPP-loaded nanocomposite significantly suppressed the growth of bacterial (P. aeruginosa, and S. aureus) and yeast cells (C. albicans) when exposed to laser light, as compared to the absence of laser light. In the presence of mTHPP and mTHPP-loaded nanocomposite alone in the dark, the inhibiting efficiencies were less than 7% and 3%, respectively. These preliminary results showed significant photocytotoxicity efficacy of the EC/cellulose/mTHPP nanocomposite at (635 nm) and low cytotoxicity in the dark against three multi-resistant pathogens, which represented that the designed nanocomposite could be introduced as a good candidate photosensitizer for aPDT application. The current study aimed to determine the degree to which photodynamic therapy using mTHPP-loaded nanocomposites could be effective against multidrug-resistant  Accordingly, in more in-depth studies, the laser light was applied only and in combination with the mTHPP or mTHPP-loaded nanocomposite against the microbial pathogens at 50 µg/mL (dissolved in dimethyl sulfoxide (DMSO) at different wavelengths (458, 476, 488, 515, 635 nm), at the power of 5 mW/cm 2 , and at different exposure times (15, 30, 45 min).
As can be seen in Table 3, mTHPP and the mTHPP-loaded nanocomposite provided a potential photodynamic effect when excited with laser light. In the case of irradiation alone (without mTHPP or mTHPP-loaded nanocomposite), it was evident that the microbial survival rate decreased close to the blue irradiation region (458-488 nm). e.g., the P. aeruginosa, and S. aureus survival rates were reduced with 32.8 ± 0.94% and 27.7 ± 1.16% at 476 nm and 488 nm (exposure for 30 min), respectively. Notable photoinactivation of C. albicans was observed as 23.6 ± 1.45% at 458 nm and irradiation for 30 min. Upon using longer wavelengths, the reduction of microbial survival was decreased to 7.33 ± 0.94%, 10.5 ± 0.62%, and 7.8 ± 0.32% at 635 nm (red-light region) for C. albicans, P. aeruginosa, and S. aureus, respectively. These results screened the efficacy of laser illumination only on the three organisms for 15-30 min at different wavelengths between 458-635 nm. The study showed that the blue light (458, 476, and 488 nm) significantly reduces the microbial survival rate of the three organisms. However, the red light showed threefold less effectiveness than the blue light, indicating that the red light is safe and does not affect the endogenous chromophores.
The irradiation group of mTHPP with different laser light wavelengths for each microorganism indicated the combined effect of illumination and porphyrin. The degree of photoinactivation in the blue-light region was more pronounced than under red light illumination. Obviously, P. aeruginosa was strongly photoinactivated under blue light irradiation for 15 min with growth inhibition of 53.1 ± 2.3% and 65.8 ± 2.23% at 476 nm and 488 nm, respectively. The photoinactivation rate of S. aureus reached 54.06 ± 0.82% and 47.2 ± 0.55% at 476 nm and 488 nm, respectively. Indeed, the action of mTHPP supplemented with laser light for 15 min significantly suppressed bacterial growth (65.8 ± 2.23% for P. aeruginosa at 488 nm, and 54.06 ± 0.82% for S. aureus at 476 nm) about three-and eightfold as compared to the irradiation group only (irradiated for 15 min). Furthermore, the maximum inhibition of C. albicans by the irradiated mTHPP was achieved at 458 nm with 44.03 ± 0.75%, which showed a threefold efficiency as compared to the irradiation group only (13.3 ± 0.5%). Herein, we screened the efficacy of mTHPP when illuminated at different wavelengths (458-635), which showed a potential reduction in bacterial growth for the blue-laser-light region and to a lesser extent for red laser light. Irradiation of mTHPP-loaded nanocomposite had a lesser effect than mTHPP on bacterial growth under blue laser light irradiation at 476 nm for 15 min (37.9 ± 2.07% for P. aeruginosa, and 30.8 ± 1.04% for S. aureus), and at 488 nm for 15 min (41.7 ± 1.02% for P. aeruginosa, and 42.2 ± 2.17% for S. aureus). While a lower survival reduction of C. albicans with 21.8 ± 1.02%, 21.9 ± 0.89%, and 17.5 ± 1.23% was emphasized upon exposure at 458 nm, 476 nm, and 488 nm for 15 min. Notably, there was lower effect of irradiation of mTHPP-loaded nanocomposite on C. albicans survival, which resulted in 27.2 ± 1.39 growth reduction as compared to 23.6 ± 1.45% for the irradiated group alone. Surprisingly, irradiation of the loaded nanocomposite in the red region (635 nm for 15 min) more efficiently suppressed the microbial survival than in the blue-light region (458-488 nm) with 71.7 ± 1.72%, 74.2 ± 1.26%, and 59.1 ± 0.21% for P. aeruginosa, S. aureus, and C. albicans, respectively. However, irradiation of mTHPP under red laser light gave less photoinactivation than that achieved by the nanocomposite at the same exposure time (15 min), whereas the reduction of P. aeruginosa, S. aureus, and C. albicans survival at 635 nm was not more than 36.06 ± 0.82%, 52.3 ± 0.49%, and 35.06 ± 2.2%, respectively. In general, the photoinactivation of irradiated mTHPP was found to be more pronounced on the microbial survival inhibition than irradiated mTHPP-loaded nanocomposite in the blue-light region. Conversely, the irradiation of the mTHPP-loaded nanocomposite under the red-light region was more efficient than the irradiated mTHPP under the same light region. In other words, introduction of the nanocomposite dramatically reduced the bacterial survival rate in the therapeutic window of the electromagnetic spectrum (600-800) in only 15 min (Table 3).
Our findings show that both mTHPP and the mTHPP-loaded nanocomposite exert significant antimicrobial susceptibility only with irradiation by laser light; and the microbial eradication efficiencies of the composite are low in the absence of light. The synergistic effect of photodynamic inactivation and antimicrobial activity of the composite material was most effective upon irradiation with blue light. The UV/Vis absorption ( Figure 6) showed no differences between the absorption bands of mTHPP and mTHPP-loaded nanocomposite, but clear differences were found in the photoinactivation performance of mTHPP and mTHPP-loaded nanocomposite between irradiation with blue (458-486 nm) and red (635 nm) light. This variation may be attributed to the fact that the red light had more penetration into ethylcellulose/chitosan (mimicking biological tissue) than the blue light [6,52]. The efficient distribution of mTHPP in the nanocomposite network (Figure 1) increases the surface area of photoactive pigments, hence making the system more active.
There are several explanations why blue light irradiation is more effective than red light on microbial cells. Blue light may activate endogenous photosensitizers in the microbial cells resulting in the generation of reactive oxygen species (ROS) [5]. Secondly, irradiation with blue light may increase the free radical release [5,14]. Following our findings, irradiation of Escherichia coli at 415 nm in the absence of photosensitizers resulted in a 30% reduction in survival [53]. Similarly, exposure of S. aureus (Gram-positive), E. coli (Gram-negative), and C. albicans (yeast-like fungi) to laser irradiation in blue region at 405 and 445 nm for 45 min inhibited their growth by more than 50% without supplementation of exogenous photosensitizer [54].
Even though blue light irradiation reduces microbial survival without an exogenous photosensitizer, the inclusion of an exogenous photosensitizer in our design offers several advantages. Without an exogenous photosensitizer, long irradiation times are required (45 min) to reach at least 50% inhibition. Light penetration of blue light is lower than that of red light, and photodynamic therapy with red light diminishes reactions with human cells and the activation of endogenous photosensitizers.
Next, we optimized the laser power for each preferred wavelength to enhance the microbial photoinactivation of the mTHPP-loaded nanocomposite. In this regard, a suitable wavelength for each microorganism was used with different powers (10, 20, 40, 70 mW/cm 2 ) at a fixed concentration of mTHPP and the mTHPP-loaded nanocomposite (50 µg/mL). As shown in Table 4, the irradiation power makes a significant difference in the reduction rate of microbial survival in the blue-light region, particularly in the case of C. albicans. The photoinactivation efficiency for C. albicans at 458 nm for 15 min was increased upon an increase in irradiation power and reached up to maximum values 83.66 ± 1.24 and 56.13 ± 0.69% at 70 mW/cm 2 for mTHPP and mTHPP-loaded nanocomposite, respectively. In addition, the photoinactivation of P. aeruginosa was notably enhanced upon a raise in irradiation power (83.4 ± 0.53% for mTHPP and 59.2 ± 0.90% for mTHPP-loaded nanocomposite at 70 mW/cm 2 ). Similar results were obtained for the photoinactivation of S. aureus.
Note that extending the exposure time of the appropriate light wavelength was found to be more efficient for microbial cell eradication ( Table 4). The count of viable microbial colonies was significantly decreased with longer times, with bacterial survival irradiated in the presence of mTHPP for longer exposure time (45 min) reaching 92% and 94.7% for P. aeruginosa (at 476 nm and 70 mW/cm 2 ) and S. aureus (at 488 nm and 70 mW/cm 2 ), respectively. Likewise, with longer exposure times the mTHPP-loaded nanocomposite somewhat increased photoinactivation, especially under red light. Apparently, the effect of higher energy power on C. albicans suggests that higher doses may be necessary to promote further absorption, which is known to be faster in prokaryotic than in eukaryotic cells [54]. Overall, the Gram-positive bacterium was more sensitive to photosensitizers at lower doses than Gram-negative bacteria. Many photosensitizers effective for the eradication of microbes carry or are conjugated with functional groups that give it a positive charge. For example, it has been reported that cationic porphyrins provide effective photoinactivation of both Gram-positive and Gram-negative bacteria by promoting photosensitizer attachment to the microbial cell wall and ROS production [4,6,14]. Likewise, many studies reported more effective photoinactivation by forming porphyrin-conjugates. E.g., a porphyrin conjugate with mannose-hPG provided eradication of S. aureus under red light (655 nm) [55]. Similarly, it has been reported that the cationic porphyrin-polymyxin B conjugate has higher bacterial absorption than neutral porphyrins [56]. In agreement, conjugation of mTHPP and ethylcellulose/chitosan and its overall cationic nature gave significant antimicrobial activity under red light irradiation.

Minimal Inhibitory Concentration and Minimal Bactericidal Concentration
To determine the minimal inhibition concentration (MIC) and minimal bactericidal inhibition (MBC), various concentrations (25-400 µg/ mL) of mTHPP and mTHPP-loaded nanocomposite were examined under an appropriate wavelength and power for each microorganism using exposure times of 30 min. Irradiation of mTHPP under blue light gave of MIC at 50 and 25 µg/mL, and MBC of 100 and 200 µg/mL for P. aeruginosa and S. aureus, respectively. On the other hand, the mTHPP-loaded nanocomposite gave MIC of 50, and 100 µg/mL, and MBC of 100 and 200 µg/mL for P. aeruginosa and S. aureus, respectively. In addition, the MIC of C. albicans was determined to be 100 and 50 µg/mL for irradiated mTHPP under blue light and mTHPP-loaded nanocomposite under red light, while the MBC value of the irradiated mTHPP and mTHPP-loaded nanocomposite was found to be 200 µg/mL.

Materials
Microbial pathogens were kindly donated by the Microbiology and Immunology Department, Faculty of Medicine (boys), Al-Azhar University (Cairo, Egypt) (Ethylcellulose and chitosan used in this study were purchased from Sigma Aldrich (St. Louis, MO, USA), molecular weight 650,000, viscosity, 275.9 cps, and degree of deacetylation, 85.5%). Nutrient agar and potato dextrose agar media were purchased from Conda Lab (Madrid, Spain).

Composite Preparation
Chitosan (0.2 g) was dissolved in 50 mL (1% acetic acid solution). Ethylcellulose (0.3 g) was dissolved in 50 mL ethanol. The two solutions were miscible under vigorous stirring at room temperature for one hour. The temperature was raised to 70 • C and the mixture was stirred at 1500 rpm overnight. The prepared composite was ultrasonicated for 5 min and stored in a refrigerator (10 • C) for further observations. 3.2.3. Loading of mTHPP onto the Nanocomposite mTHPP (0.05 g, 0.074 mmol) was dissolved in 5 mL ethanol. The mTHPP solution was added dropwise to the previously prepared composite (100 mL) under stirring at 1500 rpm overnight.

Characterizations and Instrumentation
mTHPP and mTHPP-loaded nanocomposites were characterized via several techniques using the following instruments: polarized microscope, Leica Microsystems (Switzerland) Ltd. (NRC, Giza, Egypt) (Leica DM750P); FT-IR spectrometer (NRC, Giza, Egypt) (Nicolet Impact-400 FT-IR spectrophotometer) in the range of 400-4000 cm −1 . A topographical study was carried out using scanning electron microscopy (SEM) with energy dispersive electron spectroscopy (EDX) (JSM 6360 L V, JEOL/Noran, Tokyo, Japan). For surface morphology imaging, different samples were recorded using an accelerating voltage of 10-15 kV. Additionally, the obtained images were processed via image J free software. The X-ray diffraction (XRD) (NRC, Giza, Egypt) of samples was investigated on a Diano X-ray diffractometer using a CuKα radiation source energized at 45 kV and a Philips X-ray diffractometer (PW 1930 generator, PW 1820 goniometer) with CuKα radiation source (λ = 0.15418 nm). The XRD patterns were recorded in a diffraction angle range of 2θ from 10 • to 80 • in reflection mode. The particle size distribution and zeta potential of the composite, mTHPP, and mTHPP-loaded nanocomposite were measured using Nicomp TM 380 ZLS size analyzer, Entegris, Billerica, MA, USA. Laser light scattering was used at 170 • in the case of particle size detection, whereas zeta potential was measured at 18 • .
UV/Vis absorption spectra of the porphyrin and composite solutions samples were measured using an Ocean Optics spectrometer (HR4000 UV-NIR) (Cairo University, Giza, Egypt) with a spectral resolution of 1 nm and equipped with an optical fiber (SMA 905). The absorption spectra ranged from 200 to 1100 nm. The liquid sample was set in a quartz cuvette in a dark chamber thermo-plate holder. The thermo-plate temperature was varied from 37 to 44°C using a circulating water bath controller (MGW, Lauda M3, USA).
Laser irradiation experiments were carried out using two different continuous laser systems to investigate the effects of the laser wavelengths. A continuous argon laser system (Spectra-Physics, Model:183-C0201) with multi-wavelengths (458, 476, 488, and 515 nm) and multiple powers (from 10 to 70 mW), and a continuous diode laser (LAP Laser Applikationen, Model: LAP 5MDL-63) with a wavelength of 635 nm and power 5 mW were used.

Microorganisms and Culture Conditions
The microorganisms used in the study were methicillin-resistant Staphylococcus aureus (MRSA), multidrug resistant Pseudomonas aeruginosa (MDR-PA), and Candida albicans ATCC 10231. Before each experiment, the strains were cultivated aerobically in 20 mL of nutrient broth (S. aureus, P. aeruginosa) at 37 • C and potato dextrose broth (C. albicans) at 28 • C for 24 h while shaken. All the experimental procedures were performed under aseptic conditions [59,60]. S. aureus MRSA was susceptible to vancomycin but completely resistant to the other antimicrobial drugs. Pseudomonas aeruginosa (MDR-PA) was moderately susceptible to cefepime and highly resistant to other antibiotics tested.

Photodynamic Inactivation of Microbial Strains
The suspensions of bacteria (10 6 CFU/mL) and yeast (10 8 CFU/mL) were tested in 96-well microplates (GAMA GROUP, Czech Republic) by transferring each sample to Muller Hinton broth consisting of (g/L), beef extract (2); acid hydrolysate of casein (17.5); starch (1.5). The samples were divided into four test groups: (1) light only: microbial samples were exposed to various wavelengths of light in the absence of the PSs and composite. (2) The mTHPP or mTHPP-loaded nanocomposite only: microbial samples were treated in the presence of mTHPP or mTHPP-loaded nanocomposite and not irradiated.
(3) Both mTHPP or mTHPP-loaded nanocomposite and light: microbial samples had mTHPP or mTHPP-loaded nanocomposite as in group 2 but were subsequently treated using light of an appropriate wavelength as in group 1 for different times (15-45 min). (4) Controls using microbial samples not treated by either laser light, or mTHPP or mTHPPloaded nanocomposite.
The mTHPP and mTHPP-loaded nanocomposite were first added to the Muller Hinton medium with constant concentration (50 µg/mL). The microplates were initially irradiated with different wavelengths of light as described above for 15-45 min. The efficacy of mTHPP and mTHPP-loaded nanocomposite to induce the photoinactivation mechanism for the tested pathogens was evaluated by the turbidometry method and by the CFU plate method [53]. The experiments were repeated at least three times. The control samples were both microbial cells growing in the absence of each treatment and those exposed to light only (groups 1 and 4).

Determination of Microbial Survival
The survival of microbial cells following illumination was determined by counting their viable number (i.e., the CFU plate method) after 15 to 45 min exposure of the suspended bacteria and yeast to light. Microbial cultures grown under the same conditions but without light exposure served as controls. The absorbance of the suspension at 630 nm was monitored at predetermined periods to determine growth curves. The percent of growth inhibition and cell survival in the treated and untreated cells that were monitored via the turbidometry method was calculated using the following equation: where A and B are the absorbances of the untreated and treated samples, respectively. On the other hand, microbial survival in CFU/mL was calculated according to the following formula: CFU/mL = Number of colonies in plate × Dilution factor/Amount transferred to plate (mL) The decrease in survival in all groups was calculated as a percentage, as shown below: All experiments were repeated at least three times.

Determination of Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC)
The MIC and MBC were determined by the microdilution method in 96-well microplates with modifications. Several concentrations of mTHPP and mTHPP-loaded nanocomposite (0.025-0.400 mg) were added in wells with Mueller Hinton broth (MHB) and the suspension with microorganisms. The positive control was a well with the bacterial suspension and MHB, while the negative control contained only MHB. A control with DMSO was also performed to discard the diluent activity. Three plates were produced in the same way; one plate was exposed for 20 min to red light irradiation and another plate was incubated under dark conditions (without any light exposure). Afterward, the plates were incubated for 24 h at 37 • C. The assay was revealed with CFU/mL of the microbial growth. The lowest concentration that did not show colony formation in the plate after treatment was considered as MIC. To determine the MBC, an aliquot of 10 µL was taken of each well, seeded on nutrient agar or a PDA plate, and incubated for 24 h. After the colonies were identified, the lowest concentration that did not demonstrate microbial growth was considered as the MBC [61].
Cytotoxicity of mTHPP and mTHPP-loaded nanocomposite was determined in vitro using Vero tissue culture: the MTT protocol [62,63] with minor modification. The Vero (normal fibroblast cell line) was obtained from the American-type culture collection (ATCC). The cell quantity and the percentage of the viable cell were calculated by the following formula:

Cytotoxicity
The cytotoxicity of mTHPP and the mTHPP-loaded nanocomposite against normal cell lines in vitro was considered as the first step to detect the safety of these products as presented in Figure 7. It is well-known that the mTHPP has low cytotoxicity as reported in the literature [64]. Otherwise, the addition of biopolymer to the drug carrier system could decrease the cytotoxicity dose as predicted. Herein, the obtained results affirmed that the mTHPP has a low cytotoxicity effect with IC 50 of about 315 µg/mL and this was reduced to 475 µg/mL after loading into nanocomposite drug carrier system.

Conclusions
In this article, we designed and prepared mTHPP EC/Chs nanocomposite for laserinduced aPDT against multidrug resistant pathogens C. albicans, P. aeruginosa, and S. aureus. The surface conjugation of the ethylcellulose/chitosan nanocomposite with the

Conclusions
In this article, we designed and prepared mTHPP EC/Chs nanocomposite for laserinduced aPDT against multidrug resistant pathogens C. albicans, P. aeruginosa, and S. aureus. The surface conjugation of the ethylcellulose/chitosan nanocomposite with the mTHPP was proven and characterized by FT-IR, SEM, EDX, XRD, DLS, and UV-NIR absorption. The study of free mTHPP when irradiated with laser light in the blue region (458, 488, and 476 nm) at a power of 70 mW for only 15 min revealed a significant reduction in the microbial survival rates of C. albicans, P. aeruginosa, and S. aureus (83.66 ± 1.24%, 83.4 ± 0.53%, 87.06 ± 0.87%, respectively). However, the mTHPP EC/Chs nanocomposite reduced the microbial survival rates to a lesser extent (56.13 ± 0.69%, 59.2 ± 0.90%, 55.56 ± 0.80%, respectively) compared to mTHPP under the same conditions. Conversely, introduction of ethylcellulose and chitosan biopolymer not only retained the activity of mTHPP but also enhanced its physicochemical properties in the therapeutic red region (635 nm) of the electromagnetic spectrum. The new material mTHPP EC/Chs nanocomposite showed a significant reduction in the microbial survival rates of C. albicans, P. aeruginosa, and S. aureus (59.1 ± 0.21%, 71.7 ± 1.72%, 74.2 ± 1.26%, respectively) upon illumination with 635 nm laser light for 15 min at 5 mW only. We conclude that the new material mTHPP EC/Chs nanocomposite enhances the physicochemical and photo-killing properties of mTHPP by utilizing the thermal and penetration properties of the therapeutic region of the electromagnetic spectrum. Note that increasing the power dose from 5 mW to higher clinical doses is expected to enhance the photokilling of the mTHPP EC/Chs nanocomposite. The development of new porphyrin-based photosensitizer formulations for aPDT mTHPP nanocomposites is a potential route for use in biological systems. Next, we will study the variation of clinical laser power, illumination time, and dose in the near-infrared region and will extend the biological samples to include other hospital-borne multi-drug resistant pathogens.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.