PL235824B1 - Medical use of an antibacterial composite in dressing materials, in particular for prevention or treatment of local infections - Google Patents

Abstract

The subject of the application is an antibacterial composite containing bismuth and chlorhexidine - ligand x and fluoroquinolone ligand y in a molar ratio of 1:1:1-3, respectively, with a complex structure composed of ligands x and y covalently linked to a bismuth(III) ion for use in dressing and/or medicinal materials.

Description

Description of the invention

The subject of the invention is the medical use of an antibacterial composite in dressing materials, in particular for the prophylaxis or treatment of local infections.

It is estimated that in 2015 nearly 6.5 million people worldwide were affected by the problem of chronic wounds, of which around 500,000 in Poland. Due to the aging of the society and the increase in the incidence of civilization diseases (diabetes, obesity, diseases of the cardiovascular system), this number will keep growing [Rusak. A., Rybak Z., Polim. Med., 2013, 43 (3), 199-204; Dreifke M.B. and co-authors, Mater. Sci Eng C Mater Biol Appl, 2015, 48, 651-662].

Chronic wounds are those whose healing process is disturbed and takes more than three months. They are characterized by long-term inflammation, most of which is caused by a bacterial infection caused by more than one strain. The most common microorganisms in the infected wound are Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli coagulase-negative staphylococci (including Staphylococcus epidermidis). It is very likely that these bacteria are present in chronic wounds in the form of a complex structure called biofilm, the presence of which makes treatment difficult and prolongs its duration. Treatment of chronic wound infections with the use of popular antibiotics and antiseptics is difficult due to the increasing number of strains resistant to the currently used and available preparations. The most common species of resistant bacteria in wounds are P. aeruginosa and S. aureus [Nunan R. et al., Dis. Model Mech., 2014, 7. 1205-1213; Davies C.E. et al. Rep Reg, 2001.9, 332-340; Gj0dsb0l K. et al., Int Wound J, 2006, 3 (3), 225-231].

The specificity of chronic wounds treatment requiring the use of appropriate dressings, often associated with temporary inability to work and the need for third party care, places a heavy burden on the health and social care system, generating huge expenses; while the decreasing number of possible antibacterial preparations makes it still a great therapeutic challenge for modern medicine and science, making it a global problem [Sen C.K. et al., Wound Repair Regen., 2009, 17, 763-771].

The current research focuses on searching for a more effective method of wound treatment by including substances with antibacterial, analgesic and healing properties in the structure of dressings, and thus aim to replace systemic drug administration with topical administration. Inhibition and / or elimination of bacterial infection can be achieved through the use of antibiotics, chemotherapeutic agents and antiseptics, and more and more commonly - with the use of substances of natural origin: essential oils, extracts, plants, enzymes or peptides.

Ciprofloxacin is a commonly used chemotherapeutic agent in dressing prototypes. The drug is incorporated into the structure of chitosan-alginate dressings [Han F. et al. Appl Surf Sci, 2014, 311,626-634], chitosan-gelatin [Hima B. TVL. et al., International Journal of Drug Delivery, 2010, 2, 173-182] to obtain film dressings having antibacterial activity for a long period of time (up to 28 days).

Another method is the production of polyurethane-dextran microfibers using the electrospinning technique, in which the structure includes a drug [Unnithan A.R. et al., Carbohydr Polym, 2012, 90, 1786-1793].

In order to better control the release of the chemotherapeutic agent, ciprofloxacin montmorillonite complexes are also formed and then enclosed in gelatin, which results in slower and longer release of the drug [Kevadiya B.D. et al., Colloids Surf B Biointerfaces, 2014, 122, 175-183].

Antiseptics are used instead of antibiotics in the case of a minor infection of the wound or to prevent the development of an infection. The most commonly used in the development of potential dressing materials are biguanide derivatives and iodine compounds.

Chlorhexidine has been used in several types of dressings due to its disinfecting properties. As in the case of ciprofloxacin, it was complexed with montmorillonite and incorporated into the structure of the chitosan dressing, which slowed down the release of the active substance. Studies of biological activity have shown antiseptic activity in preventing the formation of biofilm in the wound [Ambrogi V. et al., J Colloid Interface Sci 2017, 491,265-272].

PL 235 824 Β1

The discussed methods of obtaining antibacterial dressings concern compounds with proven antibacterial properties which are routinely used in medicine, but they are no longer effective due to the increasing number of cases of multi-drug resistance of microorganism strains.

The essence of the invention relates to the use of a composite containing bismuth and chlorhexidine (as ligand x) and fluoroquinolone (as ligands y) in a molar ratio of 1: 1: 1-3, respectively, in dressing materials, especially for the prophylaxis or treatment of local infections, especially dermatological, oral, urological . The composite has a complex structure composed of ligands x and y covalently linked to a bismuth (III) ion, unlike the combination known in the art.

The composite to be used as an antimicrobial and potentially healing agent in dressing materials can be in the form of a powder, solution, suspension, hydrogel, capsules (macro, micro or nanocapsules), granules, films (patch, film). The composite is preferably for use in chitosan-alginate, chitosan-gelatin-alginate, chitosan-keratin matrix.

The composite is preferably incorporated into the matrix of ready-made dressings, catheters or other biomaterials intended for the treatment of local infections.

The aim of the invention is to use a composite with the composition described above for the preparation of antibacterial and potentially therapeutic dressing forms, biocompatible with human tissues, where the composite is incorporated into a matrix with biocompatible properties, non-toxic and enabling its successive release, preferably chitosan-alginate, chitosan-gelatin-alginate , chitosan-keratin. The use of a composite in the form of a dressing allows for a prolonged antibacterial effect thanks to the gradual release of active substances. Moreover, it is possible to reduce the cytotoxicity of the composite incorporated into the matrix of the wound dressing and / or the medicinal form, especially after being encapsulated in macro-, micro- or nano-particles by slowing down its distribution.

The structure, spectrum of antimicrobial activity and toxicity to mammalian cells for a composite containing bismuth, chlorhexidine and ciprofloxacin as a fluoroquinolone (CIP-Bi-CHX), selected for the preparation of dressings for the prevention or treatment of local infections, were determined, respectively, on the basis of instrumental analysis of NMR, tests microbiological and cytotoxicity tests.

The structure of the CIP-Bi-CHX composite:

Summary formula: C39H47B1CI4FN13O3

Molar mass: 1115.67 g / mol

Melting point: 294.7 ° C

PL 235 824 B1

NMR spectra of the CIP-Bi-CHX composite indicate the presence of ciprofloxacin and chlorhexidine, as they contain signals characteristic for these substances. Comparative analysis of the NMR spectra also shows the binding of CIP and CHX via the bismuth atom due to the appearance of significant changes in the signals in the spectrum of the composite compared to the same signals in the spectra of the parent substances. The changes concern differences in band multiplicity (additional peak or peak disappearance), band extension, difference in band intensity (mainly signal reduction), significant band shifts as well as band disappearance. The changes indicate a decisive interference in the molecules of substances unrelated to the possibility of creating a composite or even hybrid structure (creating atomic and coordinating bonds).

In the 1H NMR spectrum of the composite there are proton bands characteristic for ciprofloxacin: 1.19, 1.30-1.33, 3.84-3.85, 7.55-8.66, while the proton band of the carboxyl group: 15.14 disappears probably due to the substitution of the hydrogen atom of the carboxyl group with the bismuth atom.

The spectrum ¹ H NMR present composite band characteristic proton chlorhexidine 1.29, 1.46, 3.04-3.08, 7.31-7.43. The proton bands, especially of the chlorhexidine biguanide moiety in the range 7.26-7.39, underwent significant shifts after its incorporation into the composite structure.

The changes in the ¹³ C NMR spectra of the composite in relation to the spectra of the parent substances are similar to those observed in the 1 H NMR spectrum which confirms the binding of these substances with the possibility of composite formation.

¹ H NMR spectrum (d6-DMSO, ppm)

CIP HCl: 1.19, 1.20, 1.32, 1.34, 3.57, 3.85, 3.87, 3.88, 7.60, 7.63, 7.93, 7.98, 8.68, 9.43, 15.14 (-COOH)

CHX 2CH3COOH: 1.26, 1.44, 1.72 (-CH3 in acetate), 3.04, 3.06, 3.09, 7.26, 7.28, 7.29, 7.39

CIP-Bi-CHX: 1.19, 1.29, 1.30, 1.33, 1.46, 3.04, 3.08, 3.84, 3.85, 7.31, 7.34, 7.41, 7.43, 7.55, 7.57, 7.88, 7.92, 8.66

¹³ C NMR spectrum (d6-DMSO, ppm)

CIP HCl: 8.09, 36.47, 42.95, 46.78, 46.84, 107.29, 107.41, 111.50, 111.81, 119.76 119.87, 139.55, 144.54, 144.68, 148.70, 151.70, 155.01, 166.33,176.84, 176.87

CHX 2CH3COOH: 25.29 (-CH3 in acetate), 26.37, 122.47, 126.18, 128.69 160.07, 176.39 (-COOH in acetate)

CIP-Bi-CHX: 8.05,26.39,36.37,44.89,49.79,106.81,107.23,111.30,111.61,119.08,119.18, 122.16, 126.73, 128.93, 138.28, 138.76, 139.66, 145.64, 145.77, 148.48, 151.82, 155.13, 155.38, 160.74, 166.45, 176.81

Microbiological testing

The composite (CIP-Bi-CHX), synthesized with the chemotherapeutic agent ciprofloxacin (CIP) and the antiseptic chlorhexidine (CHX), was assessed for antimicrobial activity against selected microbial strains:

Gram-positive aerobes:

- Staphylococcus aureus ATCC 25923

- Staphylococcus epidermidis ATCC 12228 Gram-negative aerobes:

- Escherichia coli ATCC 25992

- Pseudomonas aeruginosa ATCC 27853

Assessment of zones of inhibition of bacterial growth using the screening test consisting in diffusion of the active substance into Mueller-Hinton solid medium.

Experiment - the following were prepared successively: solutions or suspensions of the test substances at a concentration of 10 mg / ml in DMSO; Mueller-Hinton agar, which was poured approximately 20 ml into Petry dishes and allowed to set at room temperature; 0.5 McFarland bacterial inoculum using the bacteria strains listed.

The suspensions of individual bacteria were inoculated on the dried plates with a spatula, and then the previously prepared solutions of the test compounds with the final concentration of 100 µg were applied to the marked appropriate places on the plate. The finished plates were incubated for 24 hours at 37 ° C under aerobic conditions. After the incubation period, the zones of inhibition of bacterial growth produced around the test compounds were measured with a microbiological ruler. The results of the experiment are presented in the table below:

PL 235 824 Β1

Test compound Growth inhibition zones [mm] Gram bacteria Gram + bacteria E. coli P. aeruginosa 5 '. aureus S. epidermidis Ciprofloxacin (CIP), as hydrochloride 42 28 40 38 Chlorhexidine (CHX) as the diacetate 12 10 19 18 Composite (CIP-Bi-CHX) 44 40 42 38

The presented numerical data indicate the intensification of the antibacterial activity of the obtained composite in relation to the compounds used in its synthesis against bacteria E. coli, P. aeruginosa and S. aureus, among which Staphylococcus aureus and Pseudomonas aeruginosa are considered the most common etiological factor causing wound infections. , including infections of diabetic foot ulcers and burns (Arkadiusz Jawień et al. Guidelines for local and general management of wounds with infection. Wound treatment 2012, 9 (3), 59-75). The tested composite showed the largest zone of inhibition of growth in relation to E. coli, and the greatest intensification of antibacterial activity was observed in relation to P. aeruginosa.

Assessment of the sensitivity of the tested bacterial cells

In order to determine the degree of sensitivity of the tested bacterial cells in relation to the composite, the MIC (quantitative sensitivity assessment) and MBC values were determined:

• when MBC <4 x MIC, bactericides against a given pathogen • when MBC> 4 x MIC, bacteriostatic agents against a given pathogen

In order to determine the antimicrobial efficacy of the composite, the MBC / MIC value was determined (the lower the MBC / MIC value, the more effective the agent).

Assessment of the minimum bacterial growth inhibitory concentration (MIC). The MIC value (µg / ml) of the composite was determined using the serial microdilution method in 96-well plates in the appropriate bacterial media. For this purpose, solutions of the test substances were prepared at a concentration of 50 mg / ml in a suitable solvent that did not show any inhibitory effect on the test bacteria (2.5% acetic acid) and a bacterial inoculum at a concentration of 0.5 McFarland of the tested microorganisms (S. aureus, S. epidermidis, E. coli, P. aeruginosa). 392 µΙ was measured to the first well in the plate and to the remaining 200 µΙ of Mueller broth. Then 8.16 µΙ of the test substance solution was added to the first well and the final concentrations were obtained by double microdilution: 1000; 500; 250; 125; 62.5; 31.25; 15.62; 7.81; 3.91; 1.96; 0.976; 0.488; 0.244; 0.122; 0.061; 0.0305 μg / ml. In the next stage, each well was inoculated with the given bacterium in the amount of 2 μΙ. A positive control (i.e. broth inoculated with a given bacterium) and a negative control (pure broth) were also prepared in each plate in order to assess the correctness and sterility of the experiment. The finished plates were incubated under appropriate conditions (temperature 37 ° C, 24 hours, aerobic conditions). The reading was taken using a BioTek plate reader at 600 nm. The determined values of the minimum bacterial growth inhibitory concentration (MIG) for the tested compounds against aerobic Gram negative and Gram positive bacteria are presented in the table below.

Test compound MIC values [µg / ml] Gram bacteria Gram + bacteria E. coli P. aeruginosa S. aureus 5. epidermidis Ciprofloxacin (CIP), as hydrochloride 0.122 0.122 0.0305 0.122 Chlorhexidine (CHX) as the diacetate 0.488 7.800 0.488 0.244 Composite (CIP-Bi-CHX) 0.122 0.244 0.488 0.122

PL 235 824 Β1

The results show that the obtained composite is more active than chlorhexidine against S. epidermidis, E. coli and P. aeruginosa, especially against P. aeruginosa. In the other considered cases, its activity is equal or slightly lower than that of chlorhexidine and ciprofloxacin as compounds used in its synthesis.

Assessment of the minimum bactericidal concentration (MBC) at which 99.9% of bacteria die. The MBC ^ g / ml value) was determined against the composite using 96-well plates, in which the MIC values for specific compounds were previously determined, against selected bacterial strains (S. aureus, S. epidermidis, E, coli, P. aeruginosa). From the wells in which no bacterial growth was noted, 10 µΙ of medium containing declining concentration of the test compound was removed and transferred to clean Petry plates with a suitable agar medium. (Mueller-Hinton for aerobic bacteria or BHI for microaerobic bacteria). The plates were then incubated for 24 hours at 37 ° C under aerobic conditions. After incubation, possible bacterial growth was assessed. Plates containing the medium with the active substance concentration where no colonies appeared were taken as the bactericidal concentration (MBC). The determined values of the minimum bactericidal concentration (MBC) for the tested compounds against aerobic Gram negative and Gram positive bacteria are presented in the table below:

Test compound MBC values [gg / ml] Gram bacteria Grani + bacteria E. coli P. aeruginosa S. aureus S. epidermidis Ciprofloxacin (CIP), as hydrochloride 0.244 0.244 0.061 0.244 Chlorhexidine (CHX) as the diacetate 1.953 62,500 0.976 0.488 Composite (CIP-Bi-CHX) 0.244 0.488 1.953 0.976

Calculated Values; The MBC / MIC efficacy of the tested compounds against aerobic Gram-negative and Gram-positive bacteria is presented in the table below:

Test compound MBC / MIC values Gram bacteria gram-l- bacteria E. coli P. aeruginosa S. aureus S. epidermidis Ciprofloxacin (CIP), as hydrochloride 2 2 2 2 Chlorhexidine (CHX) as the diacetate 4 8 2 2 Composite (CIP-Bi-CHX) 2 2 4 8

The results indicate that the composite is bactericidal in nature against the bacteria tested, with the exception of S. epidermidis, where the MBC / MIC ratio of 8 indicates bacteriostatic activity and is more effective against Gram- than against Gram + bacteria. Chlorhexidine is bacteriostatic against P. aeruginosa and bactericidal against the rest of the bacteria tested. Ciprofloxacin is bactericidal against all tested bacteria.

PL 235 824 Β1

Proposal:

The CIP-Bi-CHX composite composed of ciprofloxacin and chlorhexidine bound via the bismuth atom exhibits bactericidal activity against the bacteria that most commonly cause wound infections: Staphylococcus aureus and Pseudomonas aeruginosa, similar to ciprofloxacin. Due to the fact that it is a novel compound, it may be more effective than ciprofloxacin against which bacteria have developed resistance mechanisms.

Cytotoxicity tests

For the proper assessment of cytotoxicity, several cell lines were used as cell lines of different origins may show different sensitivity to the same substance (Gruber B, Anuszewska E. Assessment of the cytotoxic effect of new anthracycline derivatives in a system of different cell lines, IL Bulletin, 2001, 45 (3 ), 569-578).

The FaDu cell line (ATCC - HTB-43) derived from human laryngeal squamous cell carcinoma was used in the study. RAW 264.7 (ATCC-TIB-71) derived from murine macrophages and Vero (ECACC - 84113001) derived from African green monkey kidney. Minimum Essential Medium (MEM) was used to cultivate FaDu cells, while Dulbecco's Modified Eagle Medium (DMEM) was used to culture RAW 264.7 and Vero cells. The media was supplemented with 10% serum (FBS) and 100 U / ml penicillin and 100 µg / ml streptomycin.

The test substances were dissolved in 10% citric acid to a final concentration of 1 mg / ml. Cell cultures were carried out in 96-well plates, into which 100 μΙ of cell suspensions were poured with a density of 2 × 10 ⁴ (FaDu), 4 × 10 ⁴ (RAW 264.7) and 1.5 × 10 ⁴ cells, respectively, per well prepared in a culture medium with the addition of 10% serum. After 24 h of incubation at 37 ° C, the culture medium was removed and the solutions of tested extractions were added in the culture fluid with the addition of 12% serum. The final substance concentrations were 0.25; 0.5; 1; 2; 3.9; 7.8; 15.6; 31.3; 62.5; 125 μg / ml. For the cell culture control, only the culture fluid with 2% serum was added. Cells treated with the test substances were incubated at 37 ° C in the presence of 5% CO2 for 48 h.

The cytotoxicity of substances was quantified using the formazan assay, MTT (Rajtar B. et al., Journal of Pre-Clinical and Clinical Research, 2013, 7, 104-106) using a plate reader at 540 and 620 nm.

On the basis of the measurements, the IC50 value was determined, i.e. the concentration of the test compound that caused a decrease in the activity of the used population of cells by 50% compared to the control. The obtained results are presented in the table below:

Test compound Cell line FaDu RAW 264.7 Yero IC ₅ o (pg / mL) ± SD Composite (CIP-Bi-CHX) 3.86 ± 0.59 1.90 ± 0.06 29.4 ± 2.36 Ciprofloxacin (CIP), as hydrochloride 13.74 ± 1.96 25.48 ± 1.93 35.95 3.89 Chlorhexidine (CHX) as the diacetate 1.87 ± 0.19 0.92 ± 0.08 11.65 ± 1.29 Citric acid (solvent) 16.15 ± 2.71 27.00 ± 2.69 33.5 ± 7.89

The CIP-Bi-CHX composite de novo synthesized according to the above-mentioned examples and subjected to cytotoxicity assessment using all the used cell lines showed a toxicity more than 2 times lower than the chlorhexidine used for its synthesis. The composite and the substances used for its synthesis showed the lowest toxicity in relation to the lines

The cell lineage of the green monkey kidney, which lineage appears to most closely correspond to human cells. Compared to this line, the composite was significantly less toxic (2.5 times lower) to the chlorhexidine used for its synthesis and slightly more toxic to ciprofloxacin, also used for its synthesis.

From the point of view of cytotoxicity, it is advantageous to use chlorhexidine in the form of the proposed composite due to the significant reduction of its cytotoxicity while maintaining the spectrum of action with the simultaneous intensification of the antibacterial effectiveness.

Composite application forms

Below are examples of the use of the composite and the obtained forms of dressing and / or therapeutic materials containing a composite of bismuth, chlorhexidine and ciprofloxacin as fluoroquinolone (CIP-Bi-CHX), used in a molar ratio of 1: 1: 1.

P r z k ł a d 1

Film dressing material: 25 ml of 1.5% chitosan dissolved in 1% acetic acid was mixed with 19 ml of 1.5% aqueous gelatin solution, and then 1 ml of glycerin as a plasticizer and 5 ml of the composite dissolved in citrate buffer were added. , pH 4.3, at a concentration of 0.1 mg / ml. In order to obtain a higher concentration of the composite, it is preferable to dissolve it in citric acid with the addition of polyethylene glycol. The mixture was mixed, degassed using an ultrasonic bath, then poured into a mold and allowed to dry.

P r z k ł a d 2

Granule (ball) dressing material: Mix 87 ml of a 2% aqueous solution of sodium alginate with 8 ml of a 1.5% aqueous solution of gelatin by adding 5 ml of the composite solution dissolved in citrate buffer, pH 4.3, at a concentration of 0.1 mg / ml. In order to obtain a higher concentration of the composite, it is advantageous to dissolve the composite in citric acid with the addition of polyethylene glycol. The resulting mixture was slowly added dropwise to 500 ml of a solution formed by mixing 3% chitosan in 1% acetic acid by volume 1: 1 with a 5% chloride solution; calcium with constant stirring. After 30 minutes, the obtained granules were filtered, rinsed with water and then dried.

P r z k ł a d 3

The granulate obtained according to example 2 was coated with silver particles by reducing Ag + to Ag ^o . The granulate was placed in the silver nitrate solution with the addition of a surfactant, and the ascorbic acid solution was added while stirring, resulting in a dark brown color of the surface.

P r z k ł a d 4

Capsule dressing material: 70 ml of 1.5% chitosan solution in 1% acetic acid was mixed with 10 ml of 1.5% aqueous gelatin solution. 15 ml of a 5% calcium chloride solution and 5 ml of the composite solution dissolved in citrate buffer, pH 4.3, at a concentration of 0.1 mg / ml. In order to obtain a higher concentration of the composite, it is preferable to dissolve the composite in citric acid with the addition of polyethylene glycol. The resulting mixture was added dropwise to 100 ml of a 0.25% sodium alginate solution. The capsules obtained were then filtered and transferred to a 2.5% calcium chloride solution for 30 minutes, then drained again and rinsed with water. They were placed in molds and dried.

P r z k ł a d 5

Ready-made, commercially available polyurethane foam wound dressings (0.5 x 0.5 cm fragments) were activated in 0.25% ethanolic iodine or 0.1% ethanolic bromine for 1 h at 37 ° C. It was taken out and drained on gauze, and then 0.25 ml of the hydrogel containing the composite was applied dropwise to each fragment of the matrix (25 mg of the composite was dissolved in 15 ml of 10% citric acid, 15 mg of chitosan was added by dissolving with ultrasound, then 0.5 ml of 5% glutaraldehyde) and 0.2 ml of polyethylene glycol solution) and dried for 24 h at 37 ° C.

P r z k ł a d 6

Ready-made, commercially available wound dressings were soaked with the composite-containing hydrogel solution or the composite solution and dried. The hydrogel was prepared by dissolving a hydrophilic polymer (preferably chitosan or chitosan and sodium alginate) in a water phase, hot or cold (depending on the type of polymer), and then introducing the composite by dissolving it in a citric acid solution and adding it to the aqueous phase before forming hydrogel, or by mixing a composite solution with a hydrogel base, or by grinding the composite powder with a hydrogel base.

PL 235 824 Β1

Example 7

Dressing material in the form of a polymer chitosan-alginate-pectin powder containing a composite, obtained by spray drying.

Example 8

Hydrogel dressing material with antimicrobial activity for dental use: the composite in the form of a powder, solution or encapsulated in micro / or nanocapsules is incorporated into a polymeric substrate prepared in an aqueous phase using known procedures. The polymer used is, for example, chitosan, sodium alginate, xanthan gum, sodium carboxymethyl cellulose, hydroxyethyl cellulose or a polymer composition.

Example 9

Antimicrobial catheter for treatment of catheter-related infections: the finished (commercially available latex catheter) is soaked in a composite solution after surface activation with an ethanolic iodine or bromine solution.

Assessment of microbiological activity of selected dressing materials

The samples of chitosan-gelatin membranes, alginate granules and ready-made polyurethane dressings containing the composite obtained according to the above examples, which were assessed for antibacterial properties against Escherichia coli and Staphylococcus aureus bacteria, showed zones of inhibition of bacterial turf growth under and around the tested samples on Mueller-Hinton solid substrate, in contrast to the control :

Composite release profiles from selected materials using the HPLC method

The chitosan gelatin membranes of Example 1 and the alginate granules of Example 2 containing the composite and comparatively containing the physical mixture of ciprofloxacin and chlorhexidine were subjected to release tests into the phosphate buffer at 37 ° C with gentle shaking of 50 rpm.

Films containing 60 μg of active substance were put into test tubes and the amounts of granules corresponding to 100 μg of active substance were weighed. The tubes were placed in the incubator. 2 ml of buffer was poured into each tube and replaced with a new one every 10 minutes. The buffer samples, after appropriate dilution, were subjected to chromatographic analysis and based on the obtained data, the release profiles of active substances were determined, which are presented in the figure below.

PL 235 824 Β1

Membranes

Granules (balls)

□ Released substance

10

3 4 S 6 7 8

The release of the active substance from the prepared dressings, membranes and granules is gradual, in smaller amounts, with a tendency to stabilize, allowing long-term antimicrobial protection, the release from the membranes being more intensive than from the granules.

The release of active substances from dressings, membranes and granules containing a physical mixture of ciprofloxacin and chlorhexidine (substances not complexed) is much faster compared to the release of the composite. A more intense release can be seen in the films when compared to the granules.

Claims (4)

1. Antimicrobial composite containing bismuth and chlorhexidine - ligand x and fluoroquinolone ligand y in a molar ratio of 1: 1: 1-3, respectively, with the structure of a complex composed of ligands x and y covalently linked with the bismuth ion (III) for use in dressing and / or therapeutic materials, especially for the prevention or treatment of local infections. 2. A composite according to claim 1 for use in a matrix with biocompatible, non-toxic and successive release properties, preferably chitosan-alginate, chitosan-gelatin-alginate, chitosan-keratin. 3. A composite according to claim 1 to be used as a dressing material in the form of a powder, solution, suspension, hydrogel, capsules (macro, micro or nanocapsules), granules, films (patch, film). 4. A composite according to claim 1 for incorporation into the matrix of ready-made dressings, catheters or other biomaterials intended for the treatment of local infections.