N-Glycidyl d-tryptophan ether-based ointment with anti-infective, anti-inflammatory, and wound-healing properties

Abstract

Anti-infective hydrogel is an emerging and innovative material used as an antibacterial ointment or to coat medical devices. Here, we synthesized a novel derivative of N-glycidyl d-tryptophan ether using the d-isoform of tryptophan through a ring-opening polymerization reaction. The compound was characterized using gel permeation chromatography (GPC), HPLC, ¹H NMR, ¹³C NMR, MALDI-TOF-MS, and FTIR spectroscopy. The results demonstrated its antibacterial activity by inhibiting quorum sensing and subsequent biofilm formation. In vivo studies revealed the ability of the compound to promote wound healing by reducing inflammatory cytokine levels, such as tumor necrosis factor alpha, interleukin-1β, and IL-6. Moreover, the compound showed antioxidant activity by scavenging the DPPH radical due to the presence of polymeric hydroxyl acidic protons near the nitrogen. Since inflammation prompted ROS-initiated DNA strand breaks, it was also confirmed that the compound could reduce DNA strand break accumulation, as demonstrated through testing against bleomycin-induced DNA strand break accumulation. Therefore, the synthesized compound, which could be used as a base material for ointments, was found to be effective for antibacterial and wound healing actions by (a) inhibiting biofilm formation by bacteria, (b) reducing the expression of inflammatory cytokines, and (c) preventing the accumulation of DNA strand breaks through free-radical scavenging activity.

N-Glycidyl d-tryptophan ether derivative may be suitable for use as ointment base to reduce the inflammation, ROS, DNA damage and bacterial load over wounds.

Introduction

Ointments are used topically for protective, therapeutic, or prophylactic purposes.^1,2 In ointment therapy, the base polymer is of prime importance because, in a protective ointment, the base material should not infiltrate the human skin boundaries and must retain moisture to shield the skin from drying due to air, sunlight and other external factors.³ However, an antiseptic ointment must penetrate deeply into the infection site to deliver the loaded medications and prevent the growth of microorganisms.

Petrolatum and mineral oil, or combinations of petrolatum with waxy/fatty alcohols, are generally used to make ointment bases. The ratio of these ingredients is chosen to provide the required viscosity/spreadability of the finished product. However, these types of ointment bases do not possess direct antimicrobial or wound healing activity. A great variety of ointment bases, under four fundamental categories, hydrocarbon or oleaginous bases, absorption bases, water-removable bases, and water-soluble bases, have certain limitations. Both hydrocarbon (or oleaginous bases) and absorption bases have poor patient acceptance and cannot be removed easily with washing. Hydrocarbon bases cannot absorb water, which hinders the dissipation of skin secretions, and can absorb only limited amounts of alcoholic solutions. Absorption bases containing wool-wax or soap-type emulsifiers may be sensitizing or can have compatibility problems. Absorption bases containing water are also at a risk of microbial contamination. On the other hand, both water-removable and water-soluble bases are less emollient and may experience chemical stability problems. Water-removable bases may dry out quickly, are susceptible to microbial growth, and those with soap-like emulsifiers may have compatibility problems. Water-soluble bases can be irritating to denuded skin or mucous membranes and those having PEG-type bases may face compatibility problems.⁴ To overcome these limitations, another sort of ointment base has been developed using a low-cost lignin derivative copolymer with multifunctional advantages, such as strong antibacterial, anti-biofilm and anti-inflammatory properties.^5–7 Another kind of nanohydrogel preparation protocol, derived from the copolymer of renewable phenolic derivatives, which is easy to synthesize and highly efficient in drug delivery, has been developed for use as an ointment base.⁸ The synthesized compound described here is multifunctional and exhibits anti-inflammatory, antibacterial, and wound-healing capabilities. Tryptophan-rich peptides have been the subject of intensive research over the past few decades, aimed at improving antibacterial chemotherapeutic agents. Additional tryptophan residues in suitable positions cause the antibacterial peptide to embed profoundly into negatively charged bacterial cell membranes and repress the survivability of bacteria. Although the more commonly occurring l-form of tryptophan is generally utilized in protein synthesis, the d-form has been found to exhibit specific biological activities that differentiate it from its l-counterpart. However, the exact mechanisms through which d-tryptophan acts as an antibacterial agent may vary and are subject to ongoing research. In general, the l-isomer of tryptophan residues is routinely utilized for peptide synthesis.⁹d-Tryptophan is an effective chemical for maintaining gut health by inhibiting the biofilms of harmful bacteria.¹⁰ It has been demonstrated that d-amino acids, specifically d-Leu, d-Met, d-Trp, and d-Tyr, are also growth inhibitors. Moreover, d-Tyr was the most effective of the four d-amino acids and was a metabolic inhibitor of B. subtilis.¹¹

Here, we used the d-isomer of tryptophan to synthesize a base material, N-glycidyl tryptophan ether derivative, the starting material. The synthesized derivative is characterized through NMR, MALDI-TOF-MS and FTIR spectroscopy. It shows antibacterial activity by unequivocally repressing the in vitro quorum sensing ability of bacteria. This compound is found to reduce the expression of multiple inflammatory cytokines in mouse macrophages and raw cells, which significantly reduces the ROS-induced DNA strand break accumulation in HEK293 cells with its free radical scavenging activity, subsequently promoting the in vivo wound healing ability.

Experimental

Materials

d-Tryptophan and epichlorohydrin were purchased from Sigma Aldrich. Sodium hydroxide was purchased from Merck, India. All analytical grade solvents were purchased from Loba Chemicals, India. Fatal bovine serum, Hank's balanced salt solution (HBSS), RPMI-1640 and Dulbecco's modified Eagle's medium (DMEM)/F-12 were purchased from Mediatech, USA.

Synthesis of N-glycidyl tryptophan ether derivative (GTE)

N-Glycidyl tryptophan ether complex was synthesized by ring opening polymerization reaction according to the modified procedure by Mahata et al.¹² In brief, d-tryptophan (2.04 gm, 0.01 molar) was dissolved in 50 ml water with pH adjusted at 8.0 by the addition of 1 N NaOH at room temperature and stirred for 2 hours. Then, epichlorohydrin (2 gm, 0.02 molar) was added drop-wise in the mixture and continued for 3 hours with constant stirring. Further, 1 N NaOH solution was added in the mixture and refluxed for 24 hours at 60 °C temperature with stirring to maintain a pH of 10. A viscous reddish color solution was obtained and neutralized by the addition of dilute HCl. Then, the solution was precipitated in the cold ethanol, washed and dried under a vacuum oven over night at 60 °C. The obtained product was characterized by the FTIR, ¹HNMR and MALDI-TOF-MS for structural analysis.

Characterization of the compound

Fourier transform infrared (FTIR) spectroscopy

The FTIR spectra of the synthesized compound were taken using a Shimadzu 8400 spectrophotometer. The sample was dissolved in chloroform and placed onto a KBr pellet and dried. Absorbance spectra were collected by scanning the range from 4000 to 400 cm⁻¹ with a 4 cm⁻¹ resolution after subtracting the background spectra.

Nuclear magnetic resonance (NMR)

The ¹HNMR spectra were recorded using Bruker DPX400 (400 MHz) in D₂O solvent with tetramethylsilane (TMS) as an internal standard. About 5 mg ml⁻¹ of the concentrated solutions were prepared in an NMR tube. All signals were referenced to TMS within 0.1 ppm.

Gel permeation chromatography (GPC)

The molecular weight of the compound was determined by GPC (Agilent 1260 Infinity instrument, PLgel 10 mm MIXED-B columns) using polystyrene standard and THF as an eluent at a flow rate of 1 ml min⁻¹. The synthesized compound solution (2 mg mL⁻¹) was filtered using a 0.2 mm pore Teflon filter. An RI detector was used to record the signal.

MALDI-TOF-MS

A Voyager time-of-flight mass spectrometer (Applied Biosystem, USA) is an instrument with a 337 nm N₂ laser used for MALDI mass spectra by operating an accelerating voltage of 20 kV. The spectra were recorded in positive ion linear mode. The reproducibility of the spectrum was checked three times from the separately spotted samples.

Cell viability measurement

Cell viability was measured with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma).¹³ In brief, RAW (RAW 264.7 cells, murine macrophage cells) cells (5 × 10⁵ cells per ml) were seeded using 24-well culture plates with 500 μl medium (DMEM/F12) and treated with different concentrations of tryptophan and tryptophan derivatives. After 2 h, cells were treated with 1 μg of LPS for 24 h, 300 μl of culture medium were removed from each well, and cells were incubated with 20 μl of MTT solution (0.5 mg ml⁻¹) for 1 h. The supernatant was removed, and the formation of formazon crystals in the cells was dissolved by adding DMSO and measuring the OD at 540 nm using a microplate reader (Thermofisher Scientific, USA).

Lactate dehydrogenase assay

Lactate dehydrogenase activity (LDH) was measured in the culture medium after 24 h using a direct spectrophotometric assay with an assay kit from Sigma.

Isolation of mouse peritoneal macrophages and immunostaining

Macrophages were isolated by peritoneal lavage from mice with sterile RPMI1640 medium containing 10% FBS and an antibiotic–antimycotic mixture (Sigma), as described earlier.⁶ Raw cells and mouse primary macrophages were treated with tryptophan and tryptophan derivative; after 2 h, cells were treated with 1 μg mL⁻¹ LPS for 24 h. The immunostaining was performed following the protocol described previously.¹⁴ Semi-quantitative RT-PCR; real-time PCR; and assay of IL-1β, TNF-α and IL-6 promoter-driven reporter activity antioxidant properties following DPPH free radicals scavenging activity assay and gene-specific LA-QPCR assays¹⁵ along the primers used in this study are described in detail in the ESI† section.

Antimicrobial activity

The tryptophan derivative was dissolved in sterile distilled water to test its antimicrobial activity against two Gram-positive bacteria, S. epidermidis (MTCC435) S. aureus (MTCC1430) and two Gram-negative bacteria, P. aeruginosa (ATCC27853) and E. coli in a microtiter plate assay in triplicates following CLSI guidelines using different concentrations. MIC was determined at the lowest concentration of compound inhibiting the growth of the test strain without showing any turbidity,¹⁶ and biofilm and quorum sensing inhibitory assays were performed as described earlier.¹⁷

In vivo assay

The study was approved by the Institutional Animal Ethics Committee of the University of North Bengal, Siliguri, District-Darjeeling, West Bengal, India (Ref. No. IAEC/NBU/2018/04 dated 12.09.2018). In brief, studies were conducted using Swiss albino rats at the age of 8 weeks, weighing between 140 and 150 g on average, all of which were male, and the burn was produced in the same area on the dorsal side of the rat. Thirty rats were employed in the investigation. During the selection of the mice, both C-reactive protein (CRP) and cytokine levels were measured and confirmed that the mice were not immuno-compromised by any infection. The healthy rats were housed in a room with a temperature of 26 ± 2 °C. The cages bedded with rice husk were kept in an animal enclosure under proper conditions of humidity (55% ± 5%) and a 12 h photoperiod. Food pellets (Pranav Agro Pvt. Ltd., India) and filtered tap water (Aquaguard Eureka Forbes, India) were fed to the rats. The details of the scratch assay,¹⁷ burning procedure, and external infection in the burn-wound site are described in detail in the ESI† section.

Statistical analysis

All values are expressed as the mean ± SD of three independent experiments. Statistical differences between means were calculated by Student's t test. A p value of <0.05 (p < 0.05) was considered statistically significant.

Results

Synthesis of N-glycidyl tryptophan ether

The compound was synthesized by a reaction between tryptophan and epichlorohydrin in an alkaline medium. Tryptophan reacts with epichlorohydrin in the first step to form an epoxy derivative at the indole ring, which then undergoes ring opening polymerization to yield N-glycidyl tryptophan ether oligomer in the presence of NaOH (Scheme 1).

Scheme 1. Schematic of the synthesis of N-glycidyl tryptophan ether (GTE).

Separation of the compound

The product was further separated with reverse phase HPLC using a 300SB C-18 analytical column with solvent A: 0.1% aqueous TFA and solvent B: 0.1% TFA containing acetonitrile; flow rate was 1 ml min⁻¹. Separation was monitored at 215 nm, with a linear gradient of solvent B (0–60%). Five prominent fractions were observed, and each fraction was collected (Fig. S1†); vacuum evaporated, dried samples were redissolved in 0.1% TFA containing acetonitrile, and the antimicrobial activity was determined (Fig. S2†). These five fractions of the synthesized product indicate different dimers, oligomers or polymers. It was observed that only fraction 3 exhibited antibacterial activity, with the other fractions being inactive. Consequently, we decided to complete the whole investigation in this study using the proportion that demonstrated antibacterial activity and selected fraction 3. The GPC results of active fraction 3 exhibit an oligomeric nature with an average molecular weight of 4865 g mol⁻¹ with PDI 1.2 (Fig. S3†).

Characterization of the compound

The compound collected from fraction 3 was characterized using FT-IR, ¹H NMR and MALDI-TOF-MS analyses. The synthesized derivative is reddish in colour, odourless, melting point of 121 °C, fully soluble in water and partially soluble in organic solvents. FT-IR analysis of pure tryptophan gives a sharp absorption peak at 3396 cm⁻¹ owing to the NH stretching of the imidazole ring.¹⁸ The peak around 1660 cm⁻¹ indicates the C O stretching frequency of the acid functionality in tryptophan. Interestingly, the peak of pure tryptophan at 1573 cm⁻¹ is due to the stretching vibration of C C in the aromatic ring, which shifts to 1666 cm⁻¹ after the substitution of the polyether unit on the imidazole ring. The appearance of a new peak at 1051 cm⁻¹ for C–O–C also indicates the presence of a polyether unit in the compound. Moreover, few peaks around 1590 cm⁻¹, 3000 cm⁻¹ and 1250 cm⁻¹ correspond to –C O, aromatic –CH and ether stretching vibration, respectively (Fig. 1a and b). The ¹H NMR spectrum clearly indicates a broad signal around 3.5–3.6 ppm due to the formation of polyether units (Fig. 1c) and ¹³C NMR (Fig. S4†). Additionally, MALDI-TOF-MS analysis gives the various fragments at m/z 795, 262, and 204 of oligomer with tryptophan glycidyl ether in each unit (Fig. 2). Therefore, the results indicate that the reaction passes through the formation of a glycidyl derivative of tryptophan monomer that undergoes ring-opening polymerization.

Fig. 1. Characterization of the synthesized derivative. FTIR spectrum of the synthesized derivative (a) and the starting material d-tryptophan (b). Proton NMR spectrum of synthesized derivative (c).

Fig. 2. MALDI-TOF-MS analysis of the synthesized derivative and its fragment analysis.

Expression analysis of pro-inflammatory cytokine IL-6, IL-1β, and TNF-α

To determine the effect of tryptophan and N-glycidyl tryptophan ether derivatives on the activation of mouse macrophages, RAW cells were exposed to different concentrations of only tryptophan and their derivatives (32–96 μg mL⁻¹) for 2 h, followed by treatment with 1 μg LPS for 6 h, and RNA was extracted for semi-quantitative RT-PCR and real-time PCR (Fig. 3A and B). As expected, LPS markedly induced the mRNA expression of proinflammatory cytokines in RAW cells. It was found that tryptophan and complex dose-dependently reduced LPS-induced expression levels of IL-6, IL-1β, and TNF-α in RAW cells. It was also observed that tryptophan and the complex inhibited the LPS-induced CD11B expression level, an integrin marker of macrophages. These results were also corroborated by real-time PCR (Fig. 3B).

Fig. 3. Semiquantitative RT-PCR and quantitative real time-PCR (A and B) of the respective genes. After 18 h of treatment, iNOS, IL-6, IL-1β and total β-actin proteins were analyzed by western blot (C). The western blot bands were scanned for quantification and are represented as relative expression (D). Cells were immunostained with CD11B and IL-1β (E and F); DAPI was used to visualize the nucleus. Firefly and Renilla luciferase activities were determined using a dual luciferase kit (Promega), following the manufacturer's protocol (G). Data are presented as mean ± S.D. of three separate experiments. **p < 0.01 vs. control; and *p < 0.05 vs. treated group show an outcome that is statistically significant.

Further, the effect of tryptophan and complex on the production of IL-6, iNOS and IL-1β protein levels, in response to LPS in RAW cells was examined by western blot analysis. As illustrated in Fig. 3C and D, the complex inhibited the IL-6, iNOS and IL-1β production in RAW cells. Similarly, immunofluorescence analysis also observed that the compound markedly suppressed the LPS-induced IL-1β and CD11B production in mouse primary macrophages and RAW cells (Fig. 3E and F). Further, to test the effect of tryptophan derivative on the regulation of IL-1β, IL-6 and TNF-α transcription, mouse RAW cells were transfected with the reporter plasmid PGL3-enhancer (Promega) expressing luciferase under treatment. As evident from Fig. 3G, the compound inhibited IL-6, IL-1β and TNF-α promoter-driven luciferase activity in RAW cells in a dose-dependent manner. To rule out the possibility that cytotoxicity was responsible for this inhibitory effect, a cell viability assay was performed in the presence of tryptophan and a new compound using 32–96 μg mL⁻¹ concentration, but no significant loss of cell viability was observed by MTT assay and LDH release assay. All these results clearly demonstrate that d-tryptophan ether derivatives are potent anti-inflammatory agents.

Compound prevents DNA strand break prophylactically

To analyze the ability of tryptophan derivative to protect against ROS-induced DNA strand break accumulation and its efficient repair, we assessed the DNA strand-break levels by long amplicon quantitative PCR (LA-QPCR), as described previously.¹⁵ Because infection-/wound-related inflammatory response leads to the simultaneous production of ROS, we mimicked the situation with bleomycin treatment of HEK-293 cells in the presence of different doses of the derivative. The levels of strand-break accumulation in the HPRT and POLB genes were compared. The cells accumulated DNA strand breaks following bleomycin treatment. However, either prophylactic treatment of the cells with the derivative or treatment following bleomycin-induced strand break accumulation led to much less accumulation of DNA strand break levels, indicating the potency of the compound to either protect against DNA damage or to efficiently repair the DNA strand break (Fig. 4a). These data corroborate the antioxidant activity of the compound, as shown earlier.

Fig. 4. DNA damage recovery in a dose-dependent manner (64 to 96 μg mL⁻¹) (a). Antioxidant activity determined through the DPPH assay, showing increased activity with higher derivative concentrations (b). Derivative hydroxyl scavenging mechanism by the DPPH radical (c).

Free radical scavenging assay

l-Tryptophan is an essential amino acid for animals and human beings, also known as a precursor of melatonin and serotonin. The antioxidative properties have recently attracted much attention¹⁹ because it has been observed that melatonin and l-tryptophan protect the gastric mucosa from oxidative damage due to ischaemia and stress. This antioxidant activity is mainly due to the presence of an acidic proton in the hydroxyl group. The acidic hydrogen of hydroxyl is responsible for antioxidant properties because amino acids exist in zwitter ionic form and the acidic proton conjugates with the amine group to form ammonium ions. Thus, the proton remains unavailable for the DPPH assay; therefore, free tryptophan showed lower DPPH scavenging activity than the derivative (Fig. 4b). In the case of our synthesized derivative, the first polymeric hydroxyl proton is acidic because it is present near the nitrogen and other hydroxyl groups and scavenges by DPPH radicals to exhibit antioxidant properties by changing the reaction mixture from violet to yellow. In the first step of the reaction, we obtain a reduced form of DPPH radical and carboxylic radical of tryptophan derivative, which readily dimerizes to give peroxy byproduct. In the next step, the DPPH radical was combined with this free radical to form an ether-linked byproduct (Fig. 4c).

Antimicrobial and anti-quorum sensing activity

The antimicrobial activity of the N-glycidyl tryptophan ether derivative was tested against S. epidermidis, S. aureus and P. aeruginosa, and E. coli following both agar diffusion and microdilution methods (Fig. 5a and b). Both the l- and d-isomers of tryptophan were tested, and only the compound of d-isoform was active against the tested bacterial strains. For the anti-quorum sensing activity assay, QS-mediated violacein production in C. violaceum was analyzed after treating the bacteria with a tryptophan compound in the agar diffusion assay. A colourless translucent zone around the zone of diffusion was observed in bacteria treated with N-glycidyl tryptophan ether derivative, indicating inhibition of violacein production during the growth of C. violaceum. It was evident from the broth dilution assay that a concentration of 64 μg ml⁻¹ of derivative inhibited the production of violacein without any hindrance to bacterial growth. Inhibition of violacein pigment production without inhibiting bacterial growth is the best-studied trait in the determination of anti-quorum sensing activity (Fig. 5c). The minimum inhibitory concentration (MIC) of the derivative was determined against S. epidermidis, S. aureus, P. aeruginosa and E. coli using the microdilution method. The MIC values were found to be 125 μg mL⁻¹, 125 μg mL⁻¹, 250 μg mL⁻¹, and 250 μg mL⁻¹, respectively. Additionally, growth curve analyses for both bacterial strains, conducted in the presence and absence of the derivative, confirmed its ability to inhibit bacterial growth (Fig. 5d).

Fig. 5. Antimicrobial activity of both isomer types of tryptophan and their polymer derivatives. Images were captured from overnight culture plates of E. coli. The derivative was applied at concentrations of 50, 100, 150, 200, and 250 μg mL⁻¹ for wells l, 2, 3, 4 and 5, respectively. The centre well was treated with a chloramphenicol antibiotic disc for d-tryptophan (b) and 20 μL 4% DMSO as a control in the l-tryptophan derivative tested plate (a). Significant anti-quorum sensing activity was observed with the d-tryptophan ether derivative at concentrations of 64–70 μg mL⁻¹ (c). Killing kinetics of the derivative at half-MIC values (64 μg mL⁻¹) against S. aureus (open circle symbol, red line) and without the derivative (open square symbol, black line); similarly, against P. aeruginosa with the derivative at 125 μg mL⁻¹ (closed circle symbol, red line) and without the derivative (closed square symbol, black line) (d). Data are presented as the mean of triplicates ± S.E. Antibiofilm activity of the synthesized derivative on S. aureus (e and f) and P. aeruginosa strain (g and h). Derivative treatment reduces biofilm biomass of 48-hour grown biofilms. Biofilms were grown on glass coverslips and treated with the derivative (96 μg mL⁻¹) for 24 h, indicating a significant reduction in biomass, as confirmed with fluorescence laser scanning microscopy. Images were captured at 400X magnification from the untreated group (e and g) and treated group (f and h). Biofilms were stained with BacLight live/dead stain (Invitrogen India), where live cells are green and dead cells are red. Images reveal a marked decrease in biofilm thickness and live cells within biofilms after treatment.

In vitro and in vivo wound healing

Confluent HaCaT cells were scratched to create a linear wound to mimic wound healing in vitro and to determine whether the synthesised derivative promotes cell migration during wound closure. After 24 hours of culture, the wound margin was marked on the image (Fig. 6a), and the wound healing rate was calculated. The results showed that cell migration was greater in the presence of the compound than in the absence of the compound. The wound closure of HaCaT scratch assays was 81.67% ± 3.54% in the treated group and only around 15% in the untreated group after 24 hours. The scratch wound assay confirmed that the treated d-tryptophan derivative could significantly increase the wound healing rate in vitro when compared to the control.

Fig. 6. In vitro scratch assay against HaCaT cells using the synthesized derivative. The images were taken immediately after the scratches were made, as well as after 6 hours and 24 hours of treatment, in the presence and absence of the derivative. Images were captured at 100X magnification. The area was quantified for the control and compound-treated groups, and data are presented as the mean of triplicate individual experiments ± S.E (a). An in vivo burn was created on the dorsal side of the Swiss albino rats' skin by flame-burning, and the wound area was infected with P. aeruginosa.⁶ The infected wound was than treated with N-glycidyl tryptophan ether derivative complex (96 μg mL⁻¹) in combination with ciprofloxacin (1 μg mL⁻¹). Compared to the control wound (which received only ciprofloxacin treatment), the derivative combination-treated wound recovered quickly and completely healed, with the reappearance of fur within 20 days (Fig. 6). Images were captured at regular intervals: 0 days, 5 days, 15 days, and 20 days, using Samsung M51 mobile camera at 10X magnification (b).

An in vivo burn was created in the rat skin by the flame burning process, and the wound area was infected with P. aeruginosa. The infected wound was then treated with an N-glycidyl tryptophan ether derivative in combination with ciprofloxacin. The wound treated with PBS-1X with ciprofloxacin was only used as a control, which showed slow recovery in comparison to the derivative and was finally cured by treatment for 20 days (Fig. 6). A higher rate of reappearance of fur in the derivative-treated burn was observed, and the effectiveness of the derivative in wound repair was confirmed.

Discussion

The development of an antimicrobial ointment base with diverse activities is critical for wound infection control. Previously, we developed a series of biocompatible wound care hydrogels based on renewable polyphenolic base material, each with unique wound repair benefits.^5–8 Recent developments in the biomedical uses of antibacterial metallic, organic, and nonmetallic nanocompounds are effectively employed in wound care, and various surface treatment strategies have also been reported to combat implant-related infection.^20–22 Here, we synthesized a d-tryptophan-based smart derivative by ring opening polymerization for better wound treatment. The compound was synthesized from the d-isoform of tryptophan amino acid residue, which was more active than the l-isoform. Tryptophan-rich antimicrobial peptides are generally more active than other amino acid residues.²³

Seki et al.²⁴ reported that d-Trp suppressed microbe-induced colitis by inhibiting the growth of enteric pathogens and colitogenic pathobionts. l-Tryptophan is more rapidly metabolized than d-tryptophan because d-tryptophan oxidation activity is stronger than l-tryptophan oxidation activity in the cells grown in the medium supplemented with d-tryptophan. l-Amino acids are necessary for all forms of life because they serve as the building blocks of proteins, such as enzymes, antibodies, and hormones. d-Amino acids (d-AAs), the enantiomeric counterparts of l-amino acids, were previously assumed to be non-functional, but these d-AAs are also powerful antibacterial compounds. It is evidenced that d-AAs, such as d-Met, directly target cell wall biosynthesis, while d-Arg exhibits the greatest inhibition in terms of cellular metabolic activity.²⁵d-Trp increases the intracellular level of indoleacrylic acid (IAA), a key molecule that determines the susceptibility of enteric microbes. Moreover, IA therapy enhances the survival of mice infected with C. rodentium. Consequently, d-Trp may function as a gut environmental modulator that affects intestinal homeostasis.²⁴ These various d-amino acids have either different molecular targets or the same target. This discovery is consistent with previous studies showing that antimicrobial peptides with a maximum number of tryptophans can kill bacteria by targeting both membrane and intracellular pathways. Although the positions of the residues are also crucial for activity, the aromatic side chain of tryptophan facilitates hydrogen bond formation with membrane components and disrupts the membrane bilayer.

To date, the vast majority of studies have used the l-isomer of tryptophan, and d-isoform has rarely been reported. Following application, bacteria consume the d-amino acid and catabolyze with d-amino acid oxidase (DAAO) to produce α-keto acid, ammonia and hydrogen peroxide molecules. This reaction requires one oxygen atom, where d-amino acid oxidases serve as an active oxygen scavenger. The DAAO's antimicrobial activity, first reported in 1969,^26,27 is widely expressed in most eukaryotes, including humans; H₂O₂ resulting from oxidative deamination of d-amino acids has been identified as the antibacterial agent.²⁸ The synthesized tryptophan derivative demonstrated considerable antimicrobial activity. The derivative has quorum sensing inhibitory activity, which inhibits the formation of biofilm over the surface. Inflammation in a wound poses a significant threat as it disrupts the immune response and subsequently delays the healing process. Inflammation occurs because the attacks of pathogens and secretary inflammatory cytokine molecules are required to eliminate the infection. NF-κB, a transcription factor, is rapidly overexpressed by induction with bacterial LPS. LPS also induced activation of mouse TNFα, IL-1β and IL-6 promoter. It was found that the d-tryptophan compound efficiently reduced the level of activation TNFα, IL-1β and IL-6 levels and suppressed the NF-κB expression. The roles of NF-κB and several cytokines, such as tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β), have been well documented in the inflammation process.²⁹ All these signalling molecules are involved in platelet activation and secondary feedback circuits. TNF-α and IL-1β are the major signalling molecules related to bacterial infections, and they have a direct contribution to tissue damage.

Bleomycin can induce DNA single and double strand breaks by ROS production along these lines; thus, it is utilized as a chemotherapy medication to treat cancer.³⁰ Because the inflammatory response is known to work through ROS production, we treated HEK293 cells with bleomycin to produce a comparable impact. The bleomycin-treated cells were prophylactically treated with d-tryptophan compound to test its efficacy in limiting the DNA strand break accumulation. In wound lesions, the production of free radicals is normal, prompting oxidative damage to prolong wound repair. The synthesized derivative similarly demonstrates its viability to fix the oxidative DNA damage following bleomycin treatment and hence might be useful to keep up normal cell physiology. A few antimicrobial drugs also directly or indirectly induce oxidative DNA damage in cellular processes^31,32 to produce genotoxic effects, which may cause multiple secondary illnesses or gene mutations. Interestingly, the synthesized derivative, apart from its role as an anti-inflammatory agent, is found to have significant repair activity for the oxidative damaged DNA. The current investigation similarly foresees the efficiency of the progress of the healing of burn wounds infected with P. aeruginosa without any antibiotic treatment. The derivative effectively fixes the wound in contrast with the control group of animals flushed with sterile PBS only. The synthesized derivative-treated wound healed with a rapid recovery compared to the control wound (treated with PBS-1X only) (Fig. 6). A higher rate of fur regrowth was observed in the burns treated with the derivative than in the control. Previously, we reported another copolymer hydrogel from renewable polyphenol lignin.⁶ According to the current investigation, the d-tryptophan ether is a unique biomedical anti-infective derivative for wound care.

Conclusion

It can be concluded that the synthesized d-tryptophan-based ether derivative showed anti-quorum sensing properties and subsequently inhibited biofilm formation. The derivative can altogether decrease the level of inflammatory cytokine molecules to enhance the process of wound healing. It can protect DNA damage caused by reactive oxygen species and can also help to repair the damage in infected tissue. In this way, the developed compound has multifunctional advantages, such as antibacterial, anti-quorum sensing, antioxidant, and anti-inflammatory activities in accelerating the wound healing process.

Data availability

The datasets generated and analyzed during the current study are available from the corresponding author upon request to facilitate reproducibility and transparency.

Conflicts of interest

The author declares no conflict of interests.