Levodopa (biological precursor of dopamine) is sometimes used instead of dopamine for synthesis of highly adhesive polycatecholamine coatings on different materials. However, in comparison of polydopamine, little is known about biological safety of poly(levodopa) coatings and their efficacy in binding of therapeutically active substances.
Carbohydrate Polymers 272 (2021) 118485 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Poly(levodopa)-modified β-glucan as a candidate for wound dressings ´ ska d, Anna Belcarz a, * Anna Michalicha a, Agata Roguska b, Agata Przekora c, Barbara Budzyn a Chair and Department of Biochemistry and Biotechnology, Medical University of Lublin, Chodzki 1, 20-093 Lublin, Poland Institute of Physical Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, Poland c Independent Unit of Tissue Engineering and Regenerative Medicine, Chair of Biomedical Sciences, Medical University of Lublin, Chodzki 1, 20-093 Lublin, Poland d Independent Laboratory of Behavioral Studies, Medical University of Lublin, Chodzki 4a, 20-093 Lublin, Poland b A R T I C L E I N F O A B S T R A C T Keywords: β-Glucan Poly(levodopa)-based modification Fibroblasts Danio rerio Antibacterial activity Levodopa (biological precursor of dopamine) is sometimes used instead of dopamine for synthesis of highly adhesive polycatecholamine coatings on different materials However, in comparison of polydopamine, little is known about biological safety of poly(levodopa) coatings and their efficacy in binding of therapeutically active substances Therefore, thermally polymerized curdlan hydrogel was modified via two different modes using levodopa instead of commonly used dopamine and then coupled with gentamicin – aminoglycoside antibiotic Physicochemical properties, biological safety and antibacterial potential of the hydrogels were evaluated Poly (levodopa) deposited on curdlan exhibited high stability in wide pH range and blood or plasma, were nontoxic in zebrafish model and favored blood clot formation Simultaneously, one of hydrogel modification modes allowed to observe high gentamicin binding capacity, antibacterial activity, relatively high nontoxicity for fibroblasts and was unfavorable for fibroblasts adhesion Such modified poly(levodopa)-modified curdlan showed therefore high potential as wound dressing biomaterial Introduction Polydopamine (PDA) is a major pigment which occurs in natural melanin (eumelanin) (Simon & Peles, 2010) It also mimics the specialized adhesive foot protein (Mytilus edulis foot protein-5) in mus sels (Lee et al., 2007) Based on this phenomenon, the method of bio mimetic approach for the functionalization of a wide range of materials has been developed and proposed in 2007 (Lee et al., 2007) Since then, formation of PDA coating became very popular as a strategy of solid substrate functionalization for a variety of technical, environmentprotecting and medical purposes PDA coatings were used for example for modification of graphene nanosheets (Wang et al., 2013; Xu et al., 2010), Fe3O4 nanoparticles (for drug delivery, for catalyst support, ad sorbents and sensors) (Liu et al., 2013; Wang et al., 2013; Zhou et al., 2010), silica nanoparticles (Zhu et al., 2019), polymers (Hu & Mi, 2013; Murphy et al., 2010), titanium (Steeves et al., 2016), and many other matrices Moreover, melanin-like coatings enable the secondary coupling reactions with different organic molecules, due to the presence of catechol domains in their structure which can react with thiols and amines via Michael addition or Schiff base reactions (Burzio & Waite, 2000; LaVoie et al., 2005) Therefore, the number of scientific reports concerning this topic grows rapidly, indicating the enormous interest in this useful technique Most scientific reports state that melanin-like coatings are formed from dopamine Very rarely levodopa is used for this purpose instead of dopamine, forming poly(L-DOPA) Both dopamine and levodopa belong to catecholamine family Dopamine (3-hydroxytyramine; 2-(3,4-dihy droxyphenyl)ethylamine; 4-(2-Aminoethyl)-1,2-benzenediol) contains both catechol and amino groups in its molecule Levodopa (L-DOPA; DOPA; 3,4-Dihydroxy-L-phenylalanine; L-3-Hydroxytyrosine) is closely related to dopamine (as its precursor in catecholamine synthesis pathway) and structurally differs from this compound by the presence of carboxyl group in aminoethyl moiety Both dopamine and L-DOPA polymerize in the presence of oxidants (as O2 or Cu2+ ions) and in slightly alkaline buffers (e.g in 10–50 mM Tris pH 8.5), although Bernsmann et al (Bernsmann et al., 2011) reported that O2 as oxidant may be not effective in case of L-DOPA In fact, although dopamine is used much more frequently as a monomer for polycatecholamine coat ings formation, both these compounds are commonly called “dopamine” in numerous scientific reports In comparison with PDA, little is known about poly(levodopa) (poly(L-DOPA)) properties, both in relation to its biological safety and potential in coupling with attractive biological * Corresponding author E-mail address: anna.belcarz@umlub.pl (A Belcarz) https://doi.org/10.1016/j.carbpol.2021.118485 Received June 2021; Received in revised form 12 July 2021; Accepted 22 July 2021 Available online 24 July 2021 0144-8617/© 2021 The Authors Published by Elsevier Ltd This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) A Michalicha et al Carbohydrate Polymers 272 (2021) 118485 molecules However, carboxyl group in L-DOPA, which is absent in dopamine molecule, may affect its polymerization to poly(L-DOPA) and also the polymer properties For example, presence of carboxyl group (of approx 2–3 pKa value) is responsible for negative charge of L-DOPA which increases the poly(L-DOPA) dispersity in water (Hashemi-Mog haddam et al., 2018) Polyolefin membranes coated with poly(L-DOPA) showed notable content of free –COOH groups on their surfaces and also an increased hydrophilicity, although the latter was higher for polydopamine-coated membranes (Xi et al., 2009) Poly(L-DOPA) films deposited on polypropylene, nylon and poly(vinylidenefluoride) sub strates showed significantly higher stability in strong acidic conditions than analogous polydopamine coatings (Wei et al., 2013) L-DOPA, due to –COOH content, was also used for the synthesis of PDE-DOPA4 monomer blocks which were further oxidized (with assistance of NaIO4) to form quickly setting adhesive hydrogel (Burke et al., 2007) Importantly, free carboxyl group in poly(L-DOPA) molecule could be an additional site for interactions between poly(L-DOPA) coatings and different ions/particles/molecules As suggested by Bernsmann et al (Bernsmann et al., 2011), L-DOPA during its polymerization to poly(LDOPA) is turned to 5,6-dihydroxyindole-2-carboxylic acid – an inter mediate which is unable to undergo a 2,2′ -branching Thus, this carboxyl group in 5,6-dihydroxyindole-2-carboxylic acid units is prob ably not engaged in the formation of covalent bonds in poly(L-DOPA) coatings Poly(L-DOPA) was already effectively used to attach paclitaxel to core-shell Fe3O4@poly(DOPA) nanoparticles (Hashemi-Moghaddam et al., 2018) although the exact role of free carboxyl groups in these nanoparticles was not explained In nitrogen-doped graphene quantum dots obtained with assistance of L-DOPA, free surface carboxyl groups were prone to coordinate with Fe3+ ions, thus facilitating the electron transport between ions and dots and in consequence enabling the for mation of Fe3O4-dots hybrids (Shi et al., 2016) Thus, poly(L-DOPA) coatings may exert other properties and allow different applications than PDA coatings Recently, our group synthesized PDA-modified high-set curdlan hydrogels using thermal polymerization method Curdlan (β-1,3-glucan) is a polysaccharide of specific gelling properties, high water sorption capacity, significant elasticity and relatively high mechanical resistance (Chen & Wang, 2020) It exhibits therefore high potential for design of wound dressing materials However, due to the presence of exclusively hydroxyl groups in repeating glucose units of curdlan backbone, it is biologically inert and relatively insusceptible to modifications improving its biological properties and capability to bind therapeuti cally active molecules (Cai & Zhang, 2017) PDA coating could be therefore an excellent method to introduce modifiable domains for biological improvement of curdlan We have demonstrated that PDAmodified hydrogels showed the ability to bind molecules containing free amino groups (as proved for gentamicin and peroxidase) and exhibited unchanged mechanical stability, increased soaking capacity, prolonged antibacterial properties and Fickian-type mechanism of drug release (Michalicha et al., 2021) These results suggested that PDA-modified curdlan hydrogel may serve as a carrier of free amino groupscontaining molecules and be used for different purposes, e.g antibac terial hydrogels for wound dressings In view of this, we hypothesized that also levodopa may effectively form the biologically safe deposits on polysaccharide matrix and may be used for fabrication of wound dressings Therefore, in this paper we report the fabrication of poly(levodopa)-modified curdlan hydrogels The hydrogels were first characterized for stability as well as biological safety of the deposits during contact with blood, fibroblast cell line, fibroblast primary culture and zebrafish eggs and larvae, in relation to their possible application as wound dressings Second, the modified hydrogels were coupled with gentamicin and their antibacterial activity was evaluated Materials and methods 2.1 Synthesis of poly(L-DOPA)-modified curdlan hydrogels Curdlan powder (from Alcaligenes faecalis; cat No 281–80,531; DP 6790; average Mw 1100 kDa; specific rotation [A]20/D: +30 to +35), Cl− content < 0.5%, heavy metals content (including Pb) < 0.002%, was provided by Wako Chemicals (Japan); Tris (2-Amino-2-(hydroxymethyl) propane-1,3-diol) and L-DOPA (3,4-Dihydroxy-L-phenylalanine) by Sigma-Aldrich (USA) Control and poly(L-DOPA)-modified hydrogels were synthesized according to procedures described elsewhere (Michalicha et al., 2021), briefly: 2.1.1 With L-DOPA monomer added to curdlan suspension Before thermal Gelling (BG) Suspension of 0.4 g curdlan in ml 10 mM Tris/HCl buffer pH 8.5 was combined with 10 mg (2-LD-BG) or 20 mg (4-LD-BG) of L-DOPA, stirred 10 until L-DOPA was completely dissolved, transferred into glass tubes (ø 13 mm) and polymerized at 93 ◦ C for 15 After cooling, hydrogel was cut into mm slices and incubated in air (air oxygen as an oxidant) for 24 h at 25 ◦ C to allow L-DOPA polymerization Then slices were washed 10 times in 100 ml DI H2O, frozen and lyophilized (SRK, System Technik GMBK, Germany) 2.1.2 With L-DOPA monomer added to curdlan suspension After thermal Gelling (AG) Suspension of 0.4 g curdlan in ml 10 mM Tris/HCl buffer pH 8.5 was transferred into glass tubes (ø 13 mm) and polymerized at 93 ◦ C for 15 Cooled hydrogel was cut into mm slices, immersed in ml 10 mM Tris/HCl buffer pH 8.5 containing 10 mg (2-LD-AG) of L-DOPA and incubated in orbital shaker for 24 h at 25 ◦ C with the access to air (air oxygen as an oxidant), to allow L-DOPA polymerization Then slices were washed 10 times in 100 ml DI H2O, frozen and lyophilized 2.1.3 Control curdlan hydrogels were prepared as in Section 2.1.2 without the immersion in L-DOPA solution and further incubation Prior to cell cultures, zebrafish, drug release and antibacterial ac tivity experiments, all curdlan hydrogels were sterilized by ethylene oxide method in paper/plastic peel pouch (sterilization for h at 55 ◦ C, aeration for 20 h) 2.2 Gentamicin immobilization and quantitative analysis Immobilization of gentamicin into modified curdlan samples was performed by incubation of lyophilized slices in mg/ml gentamicin (Sigma-Aldrich, USA) in Britton-Robinson buffer pH 8.5, in proportion 33.3 ml of antibiotic solution/1 g lyophilized curdlan hydrogel slices, using DTS-4 shaker (100 rpm), 24 h at 25 ◦ C, followed by 24 h at ◦ C Then slices were washed twice in 50 ml DI water, frozen and lyophilized In case of pilot experiment, EDC/NHS activation of poly(L-DOPA) carboxyl groups was used for binding with gentamicin Hydrogel sliced were first soaked in 0,1 M MES buffer pH 6,5 and then incubated in mixture of 0.1 M EDC (N-(3-Dimethylaminopropyl)-N-ethyl carbodiimide hydrochloride; Sigma-Aldrich, USA) and 0.2 M NHS (Nhydroxy succinimide; Sigma-Aldrich, USA) in 0,1 M MES buffer pH 6,5 for h, at 25 ◦ C, on plate shaker DTS-4 (ELMI, USA), 100 rpm After wards, the samples were washed twice (10 min.) in distilled water and immersed in mg/ml gentamicin (Sigma-Aldrich, USA) in 0,05 M NaHCO3 (pH 8.5), in proportion 33.3 ml of antibiotic solution/1 g lyophilized curdlan hydrogel slices, using DTS-4 shaker (100 rpm), 24 h at 25 ◦ C Gentamicin concentration in solutions before and after incubation was evaluated according to Ginalska et al (Ginalska et al., 2004), based on gentamicin derivatization by phthaldialdehyde (Sigma-Aldrich, USA) Amount of immobilized gentamicin was calculated from formula (1): A Michalicha et al Ci(µg/g d.w.) [ ] Cb(µg/ml) –Ca(µg/ml) x V(ml) = Mg Carbohydrate Polymers 272 (2021) 118485 appropriate calibration curve (using 96-well plates and Synergy H4 hybrid microplate reader, Biotek, USA) and were 136 mg% and 0.18 mg/ml, respectively For hemolysis test, lyophilized hydrogel slices (30 mg ± mg) were immersed in ml of blood 100× diluted in PBS pH 7.4 without Ca2+ and Mg2+ ions Positive control contained 0.1% Triton X-100 while negative one: 30 mg ± mg of HDPE (high density polyethylene, Sigma-Aldrich, USA) Then samples were incubated h at 37 ◦ C in Innova 42 incubator shaker (New Brunswick Scientific, USA), 150 rpm Erythrocytes-released hemoglobin was estimated using reac tion with Drabkin reagent, as above For blood clot formation test, 100 μl of whole 10 mM CaCl2-activated blood was placed onto 30 mg ± mg lyophilized hydrogel slices or pieces of HDPE (negative control) Nonactivated Ca2+-free whole blood (100 μl) served as positive control Then samples were incubated 15 min., 30 or 45 at 37 ◦ C, without shaking (controls were performed individually per each time point) Then all samples were incubated with 2.5 ml of distilled water for Finally the hemoglobin content in solution was estimated using reaction with Drabkin reagent, as described above Each experiment was performed in triplicate Statistically significant differences between negative control and various samples were considered at p < 0.0001, according to a One-way ANOVA with post-hoc Dunnett’s test (GraphPad Prism 8.0.0 Software, San Diego, CA) (1) where: Ci(μg/g d.w.) – amount of gentamicin immobilized on samples (μg/g of dry hydrogel weight) Cb(μg/ml) – concentration of gentamicin in solution before incubation with samples (μg/ml) Ca(μg/ml) – concentration of gentamicin in solution after incubation with samples (μg/ml) V(ml) – volume of solution incubated with samples (ml) Mg – dry weight of samples (g) 2.3 Characterization of modified curdlan hydrogels FTIR-ATR spectra were collected using Vertex 70 IRspectrophotometer (4000–400 cm− 1, 64 scans, cm− spectral resolu tion; Bruker, USA) and analysed using OPUS 7.0 software (Bruker, USA) X-ray photoelectron spectroscopy (XPS) measurements were made on a Microlab 350 (Thermo Electron) spectrometer with nonmonochromatic AlKα (hν = 1486.6 eV, power 300 W, voltage 15 kV) radiation as an X-ray excitation source A lateral resolution was about 0.2 cm2 The high-resolution XPS spectra were acquired using the following parameters: pass energy 40 eV, energy step size 0.1 eV A Smart function of background subtraction was used to obtain the XPS signal intensity The all collected XPS peaks were fitted using an asymmetric Gaussian/Lorentzian mixed function The measured binding energies were calibrated with respect to the energy of C 1s at 285.0 eV Avantage-based data system software (Version 5.9911, Thermo Fisher Scientific) was used for data processing For evaluation of soaking capacity, lyophilized curdlan hydrogels were incubated in ml 0.9% NaCl at 37 ◦ C for up to 72 h In defined time points, the samples were withdrawn from the liquid, drained on What man paper to remove the excess of the liquid and weighed (XS205, Mettler-Toledo, Switzerland) The obtained data were normalized (to 100% of initial dry weight of samples) Experiment was performed in triplicate Statistically significant differences between non-activated and activated sample in each modification mode were considered at p < 0.05, according to Student’s t-test (GraphPad Prism Software, San Diego, CA) 2.6 Cell culture experiments Cell culture tests were conducted using human skin fibroblasts: normal human skin fibroblast cell line (BJ cell line, ATCC-LGC Stan dards) and primary human dermal fibroblasts (HDFs, ATCC-LGC Stan dards) Fibroblasts were cultured at 37 ◦ C in a humidified atmosphere of 5% CO2 and 95% air BJ fibroblasts were maintained in EMEM medium supplemented with 10% fetal bovine serum (FBS, Pan-Biotech), and 100 U/ml/100 μg/ml penicillin/streptomycin mixture (Sigma-Aldrich Chemicals) HDFs were maintained in a Fibroblast Basal Medium sup plemented with the components of Fibroblast Growth Kit-Low Serum (both purchased from ATCC-LGC Standards) 2.6.1 Cytotoxicity tests according to ISO 10993-5 standard Fibroblast suspension with a concentration of × 105 cells/ml was seeded in 100 μl into the wells of 96-multiwell plates After 24-h incu bation, the culture medium was discarded and the monolayer of cells was exposed to the extracts of the tested samples The extracts of the materials were prepared according to ISO 10993-12 standard by placing 0.1 g sample in ml of complete culture medium followed by 24-h in cubation at 37 ◦ C Non-toxic extract serving as a negative control of cytotoxicity was prepared by the incubation of complete culture me dium in a polystyrene vessel for 24 h at 37 ◦ C without any biomaterial (extract marked as PS control) Fibroblasts were maintained in the ex tracts for 48 h and then cytotoxicity of the samples was determined by evaluation of cell metabolism using MTT assay (Sigma-Aldrich Chem icals) and cell number using total LDH test (Sigma-Aldrich Chemicals) The MTT assay was carried out based on the procedure described earlier (Przekora et al., 2014) Total LDH test was performed after cell lysis according to the manufacturer instructions Results of MTT and total LDH tests were presented as the percentage of negative control of cytotoxicity (100% viability in terms of cell metabolism and cell num ber) Three independent experiments were conducted for both cyto toxicity tests Statistically significant differences between negative control (PS control) and various samples were considered at p < 0.05, according to a One-way ANOVA with post-hoc Dunnett’s test (GraphPad Prism 8.0.0 Software, San Diego, CA) 2.4 Stability of poly(L-DOPA) deposits Poly(L-DOPA)-modified hydrogel slices were extensively washed in distilled water (10 times in 0.5 l; first washes for h, second washes for 12 h; RM 5-30 V shaker (CAT M Zipperer Gmbh, Germany), 30 rpm.) Incubation in human serum (kindly donated by Regional Center of Blood Donation and Blood Treatment in Lublin) and in human blood (collected after approval of Bioethics Committee at the Medical University of Lublin, no KE-0254/258/2020) was performed (2 ml per 30 mg of dry hydrogel weight) on plate shaker DTS-4 (ELMI, USA), 100 rpm, at 37 ◦ C, for up to 96 h; then washed once in distilled water Effect of pH was tested using 0.1 M Britton-Robinson buffers pH 2, 4, 6, 8, 10 and 12 (the same liquid-to-biomaterial proportion as for human serum and blood), for at 37 ◦ C, for days Macro photography of plasma-, blood- and buffers-incubated hydrogels and post-incubation buffers was performed using E-520 digital camera (Olympus, Japan) 2.5 Hemolysis and blood clot formation test 2.6.2 Cell proliferation Fibroblast suspension with a concentration of 1.5 × 104 cells/ml was seeded in 100 μl into the wells of 96-multiwell plates After 24-hour incubation, total LDH test was conducted to determine cell number at starting point (time = h – before addition of the extracts) The exact Human blood was collected on citrate from healthy volunteer on approval of Bioethics Committee at the Medical University of Lublin, no KE-0254/258/2020 Its total hemoglobin and plasma hemoglobin con centration were estimated on basis of reaction with Drabkin reagent and A Michalicha et al Carbohydrate Polymers 272 (2021) 118485 number of fibroblasts was calculated using calibration curve prepared for known concentrations of BJ cells and HDFs Then, extracts of the biomaterials (prepared as described in section 2.6.1) were added to the cells which were further incubated for days Cell number was deter mined after 24- and 72-hour exposure to the extracts Three independent proliferation tests were performed Statistically significant diffrences between PS control (non-toxic control extract prepared by incubation of culture medium in a polystyrene vessel) and various samples were considered at p < 0.05, according to a One-way ANOVA with post-hoc Dunnett’s test (GraphPad Prism 8.0.0 Software; San Diego, CA) ZEISS, Germany) Incidence of morphological and physiological abnor malities e.g a lack of somite formation, scoliosis, or the pericardial oedema were observed and compared to the control embryos Image analysis was performed to determine the percentage of malformed embryos 2.7.3 Locomotor activity assay Test was performed on day post fertilization (dpf) larvae One larva per well was placed in 96 multi-well plate To each well, either 200 μl extract of control or poly(L-DOPA)-modified hydrogels or the same volume of pure E3 medium was added (each group of n = 20) and the zebrafish larvae were incubated in the extracts for 30 before the test Then, EthoVision XT 15 video tracking software (Noldus Informa tion Technology b.v., The Netherlands) was used for evaluation of lo comotor activity The distance moved in 10 period was calculated in cm, in a light condition The results were processed by the one-way ANOVA analysis with Dunnett’s post hoc test A p value