RO132576B1 - Polymeric composites for producing urinary drainage tube with antimicrobial properties and process for preparing the same - Google Patents

Description

The invention relates to polymeric composites with antimicrobial properties and to a process for obtaining the urinary drainage tube, for medical use, from them.

There are known urinary drains that are frequently colonized by uropathogenic bacteria that lead to the formation of biofilms, associated with the initiation of an infectious process of the surrounding tissues, a situation that requires removal of the medical device (Burton E., Gawande PV, Yakandawala N., LoVetri K., Zhanel GG., Romeo T., Friesen AD., Madhyastha S. 2006. Antibiofilm Activity of GlmU Enzyme Inhibitors against CatheterAssociated Uropathogens. Antimicrobial agents and chemotherapy. 50: 1835-1840). It has been estimated that the risk of acquiring an infection increases by 5% each day for an in situ medical drain (Saint S., Wiese J., Amory JK., Bernstein ML., Patel UD., Zemencuk JK., Bernstein SJ. , Lipsky BA., Fio fer TP. 2000. Are physicians aware of which of their patients have indwelling urinary catheters? Am J Med. 109: 476-480; Flumphreys H., Newcombe RG., Enstone J., Smyth ET., Mcllvenny G., Fitzpatrick F., Fry C, Spencer RC., Hospital Infection Society Steering Group. 2008. Four country healfhcare associated infection prevalence survey 2006: risk factor analysis. J Hosp Infect. 69: 249-257), and the possibility that a patient to develop a catheterization-associated infection is 35 ... 39% (Maki DG, Tambyah PA. 2001. Engineering out the risk for infection with urinary catheters. Emerg Infect Dis Mar. 7: 342-347). Urinary tract infections associated with the use of medical drains are difficult to treat because the microorganisms included in the biofilm matrix are tens, hundreds, and even up to a thousand times more resistant to antibiotics than their planktonic counterparts (Ceri H., Olson M., Stremick C. , Read RR., Morck D., Buret A. 1999. The calgary biofilm device: new technology for rapid determination of antibiotic susceptibilities of bacterial biofilms J Clin Microbiol 37: 1771-1776; Kaali P., Stromberg E., Karlsson S. 2011. Prevention of Biofilm associated Infections and Degradation of Polymeric Materials used in Biomedical Applications. Pub. In Tech Biomedical Engineering, Trends in Materials Science, Edited by Mr Anthony Laskovski, Chapter 22, pp. 513-540).

There are many known strategies for managing the infection associated with the presence of a foreign body by antibiotic therapy or combining drug therapy with surgical removal of the implanted medical device and its replacement, but these are only applicable if the tissue at the site of insertion of the device is in good condition and the pathogen involved in the infection is not part of the therapy-resistant microorganisms (Trampuz A., Andreas FW 2006, Infections associated with orthopedic implants. Current Opinion in Infectious Diseases. 19: 349-356). Removal of implantable medical devices, regardless of their type, is recommended in the event of severe sepsis, septic phlebitis, or septic shock. For example, in catheterized patients with persistent bacteremia, for 48 to 72 hours, the drain must be removed (von Eiff C, Jansen B., Kohnen W., Becker K. 2005. Infections associated with medical devices: pathogenesis , management and prophylaxis (Drugs. 65: 179-214). Prophylactic antibiotic therapy is a postoperative procedure, commonly used in surgery, to prevent microbial colonization on the implanted medical device. However, cases have been reported in which patients developed infectious complications even under antibiotic treatment (McCann MT, Gilmore BF, Gorman SP 2008. Staphylococcus epidermidis device-related infections: pathogenesis and clinical management. Journal of Pharmacy and Pharmacology. 60: 1551-1571). However, the amount of drug administered may be insufficient to achieve a prolonged antimicrobial effect. All these measures to reduce the infections associated with the use of urinary drains lead to increased morbidity and mortality, as well as to the extension of the period of 1 hospitalization of the patient (Holroyd-Leduc JM, Sen S., Bertenthal D., Sands LP, Palmer RM, Kresevic DM , Covinsky KE, Seth Landefeld C. 2007. The relationship of 3 indwelling urinary catheters to death, length of hospital stay, functional decline, and nursing home admission in hospitalized older medical patients J. Am Geriatr Soc, 55: 5

227-233; Guidelines for the Prevention of Catheter Associated Urinary Tract Infection. Published on behalf of SARI by HSE Health Protection Surveillance Center. 2011). 7

One method of reducing these drawbacks is to directly incorporate an antimicrobial agent into a polymeric matrix. Thus arose the need to develop 9 antimicrobial medical devices that inhibit microbial adhesion and thus the development of biofilms. With all the efforts made, it is very difficult to obtain a completely non-stick material, because in vivo any type of material is covered by a conditioning film (Arciola CR, Campoccia D., Gamberini S., Baldassarri L., Montanaro L. 13 2005). Prevalence of cna, fnbA and fnbB adhesin genes among Staphylococcus aureus isolates from orthopedic infections associated with different types of implant (FEMS 15 Microbiol Lett. 246: 81-86). In addition, new polymeric materials must be biocompatible, have adequate physical and mechanical properties and be sterile. 17

Numerous studies have been conducted on the design of antimicrobial materials (capable of preventing surface contamination and / or eradication of microbial biofilms) 19 by various methods (nanotechnology, coatings or surface modifications).

Composites based on inorganic antimicrobial compounds 21 (eg titanium dioxide, copper, gold, gallium, silver) and antimicrobial peptides (eg nisin, mersacidin, colicin, cecropin A, etc.) are also known (Ribeiro AMC, Barbassa L., Delo de Melo L. 23 2011. Antimicrobial Biomimetics. InTech Europe. 227-284).

Among the antimicrobial agents studied, silver is known as a metal with a broad antimicrobial spectrum compared to Gram-positive, Gram-negative, microfungal, protozoan and virus strains, and has been used for centuries to treat chronic burns and wounds. Differences between 27 bacterial species may influence behavior toward antimicrobials. The cell wall of Gram-positive bacteria contains 3 to 20 times more peptidoglycans, compared to Gram-negative bacteria. Consequently, Gram-positive bacteria are generally less sensitive to the action of silver ions than Gram-negative species 31 (Kawahara K., Tsuruda K., Morishita M., Uchida M. 2000. Antibacterial effect of silverzeolite on oral bacteria under anaerobic conditions (Dent Mater. 16: 452-5). 33 Silver nanoparticles (AgNPs) are insoluble particles smaller than 100 nm in size that can be obtained by reducing AgNO ₃ with citrate (Turkevich J., Stevenson PC, Hillier 35 J. 1951. A study of nucleation and growth processes in the synthesis of colloidal gold (Discuss Faraday Soc. 11: 55-75). Recently, silver bionanoparticles were obtained by adding plant extracts _{to the aqueous solution of AgNO 3.} Plant biomolecules induce the reduction of silver ions from AgNO ₃ to AgNPs (Sahayaraj K., Rajesh S. 2011. 39 Bionanoparticles: synthesis and antimicrobial applications. Formatex. Science against microbial pathogens: communicating current research and technological advances. 41 228-244 ). Due to their large specific surface area, silver nanoparticles can be used in low concentrations without altering the mechanical properties of the material. The release of 43 silver ions depends on the nature and concentration of silver in the nanocomposites, as well as the polymer matrix. The mechanism of action of silver on microorganisms is not yet well known, as possible mechanisms are described to induce morphological and structural changes in microbial cells (Rai M., Yadav A., Gade A. 2009. Silver nanoparticles 47 as a new generation of antimicrobials. Biotechnology Advances. 27: 76-83) by: the interaction of silver with thiol (-SH) groups in the structure of enzymes involved in cellular respiration (Klasen HJ. 2000. A historical review of the use of silver in the treatment of burns. Part I early uses (Burns, 30: 1-9); inhibiting the process of DNA replication by inducing condensation of the DNA molecule (Liau SY, Read DC, Pugh WJ, Furr JR, Russell AD 1997. Interaction of silver nitrate with readily identifiable groups: relationship to the antibacterial action of silver ions. Lett Appl Microbiol 25: 279-283); degradation of intracellular proteins and nucleic acids (Knetsch MLW., Koole LH. 2011. New Strategies in the Development of Antimicrobial Coatings: The Example of Increasing Usage of Silver and Silver Nanoparticles. Polymers. 3: 340-366), generation of reactive species of oxygen (ROS), such as oxygen (O ₂ ) or hydroxyl (HO) radicals (Danilczuk M., Lund A., Saldo J., Yamada H., Michalik J. 2006. Conduction electron spin resonance of small silver particles. Spectrochim Acta A. 63: 189-191), direct transfer of silver ions without a solvent from oxidized nanoparticles to biological targets (such as cell membrane) (Knetsch MLW., Koole LH. 2011. New Strategies in the Development of Antimicrobial Coatings: The Example of Increasing Usage of Silver and Silver Nanoparticles (Polymers. 3: 340-366).

Silver-containing polymeric composites are of major interest to academia and industry. The long-term biocidal character, thermal stability and low volatility are decisive factors in medical applications. Three techniques have been developed for the preparation of antimicrobial nanocomposites: (i) the silver nanoparticles are mixed with the polymer or are distributed in the matrix of the host polymer; (ii) the nanoparticles are synthesized during polymerization, a technique that requires the use of electronically active polymers to reduce silver salts to silver nanoparticles, and (iii) the dispersion of nanoparticles in the monomer, in which case the polymerization is initiated in the presence of nanoparticles that are simultaneously trapped in the polymer network (Bălan L., Schneider R., Lougnot DJ 2008. A new and convenient route to polyacrylate / silver nanocomposites by light-induced cross-linking polymerization. Prog Org Coat. 62: 351-357).

Treatment of polyvinyl chloride (PVC) with NaOH and AgNO ₃ resulted in a composite that completely inhibited microbial adhesion and colonization of P. aeruginosa over a period of 72 h (Balazs DJ, Triandafillu K., Wood P., Chevolot Y. , van Delden C, Harms H., Hollenstein C, Mathieu HJ 2004. Inhibition of bacterial adhesion on PVC endotracheal tubes by RF-oxygen glow discharge, sodium hydroxide and silver nitrate treatments. Biomateriah. 25: 2139-2151).

It has been shown that mixing PLA with natural polymers can improve biocompatibility properties, as they allow better attachment of human cells to the polymeric substrate. Collagen is a natural polymer, very often used in bone restoration engineering (Wahl DA and Czernuszka JT, 2006. Collagen-hydroxyapatite composites for hard tissue repair. European cells and Materials. 11: 43-56), in the manufacture of controlled release systems of drugs (Ruszczaka Z., Friessc W. 2003. Collagen as a carrier for on-site delivery of antibacterial drugs. Advanced Drug Delivery Reviews doi: 10.1016 / j.addr.2003.08.00703). The use of collagen in the production of high biocompatibility polyvinyl chloride wires is known (RO 113648 B1).

A method and composition for coating a medical device with a solution of a hydrophobic polymer, such as polysaccharides and a crosslinking agent such as aldehydes (US 7597903 B2) are known.

The disadvantage of the method of coating hydrophobic polymer matrices with antimicrobial agents is that these polymeric matrices do not have active chemical groups capable of interacting with an antimicrobial agent. After treatment of the surface of the polymeric matrix 3, the antibacterial agent cannot bind to the polymeric matrix and consequently does not provide an antimicrobial agent release effect. 5

US 7314857 B2 discloses an antimicrobial composition for inhibiting biofilm formation comprising an iron-capturing glycoprotein, a cationic polypeptide 7 and a chelating agent or an iron-capturing glycoprotein and a cationic polypeptide. In addition, surfactants and quaternary ammonium compounds can be advantageously combined with iron-containing glycoproteins in an antimicrobial composition.

U.S. Pat. No. 4,747,882 B2 discloses the use of a salt of an organic acid 11 (benzoate or sorbate) to obtain an antimicrobial coating solution of a hydrophilic urinary catheter to prevent bacterial infection. The H of the antimicrobial coating solution can be controlled with citrate or phosphate buffer.

It is also known to obtain a urinary catheter that provides both short- and long-term15 microbiocidal effects on strains of antibiotic-resistant microorganisms. This catheter consists of hydrophobic polyurethane, polyethylene vinyl acetate 17 hydrophilic and a silver salt (US 8173151 B2).

The problem solved by the invention is the presentation of a process for obtaining19 a polymeric bionanocomposite based on polylactic acid, hydrolyzed collagen and silver nanoparticles by a specific process.21

The process for obtaining the invention removes the above disadvantages in that it is processed in the melt at a temperature of 170 ° C for 7 minutes and a screw speed of 60 rpm, a mixture based on 75.6. .79.6% by weight polylactic acid, 7.56 ... 7.96% by weight plasticizer, 11.34 ... 11.94% by weight polyethylene glycol, 0 ... 5% by weight hydrolyzed collagen and 0 ... 0,5% by weight of silver nanoparticles, the resulting mixture is passed through a screw extruder, provided with a dose having a diameter φ of 4 mm 27 and a mandrel with a diameter φ of 3 mm, at a temperature of 135 ... 150 ° C, rotational speed of 30 ... 60 rpm, obtaining a urinary tube with an inner diameter of 3 ± 0.1 mm and an outer diameter 29 of 4 ± 0.1 mm, with resistance to rupture of 16 ... 32 MPa, elongation at break of 126.5 ... 216.8%, modulus of elasticity of 1891 ... 2058 MPa, has a cell proliferation of 31 88.96 ... 98.77 %, resistance to biofilm formation / fungal activity against Candida albica ns ATCC10231, stable in the short term, having a mass loss of 1.2 ... 2.9% and being 33 degradable after a longer time.

By applying the invention, as described above, the following advantages are obtained: 35

- environmentally friendly materials are used;

- biocompatible and antimicrobial materials are obtained; 37

- the process for obtaining antimicrobial composites and drainage tubes is carried out by melt processing, without high energy consumption, on equipment specific to traditional 39 polymers;

- the polymeric composites obtained have physico-mechanical characteristics, of biocompatibility and antimicrobial character adequate to the proposed use.

The present invention eliminates the disadvantages due to the use of non-biodegradable solvents and polymers 43, the technical solution being to obtain by melt processing at a temperature of 170 ° C, for 7 min and a speed of 60 rpm of a mixture based on 45 75.6 ... 79.6% by weight polylactic acid (PLA) type 2003D (NatureWorks), 7.56 ... 7.96% by weight Lapol 108 masterbatch (LAPOL, LLC, USA), 11.34 ... 11 , 94% by weight PEG 47 BioUltra 4000 (Sigma), 0 ... 5% by weight hydrolyzed collagen (Sigma-Aldrich) and 0 ... 0.5% by weight silver nanoparticles (US Research Nanomaterials, Inc. ), with a particle size of 20 nm, 99.99% Ag coated with 0.2% polyvinyl pyrrolidone (PVP). The bionanocomposites obtained in the invention have tensile strength, elongation at break, biocompatibility and antimicrobial activity, which recommends them for obtaining drainage tubes.

The process for obtaining the drainage tube according to the invention consists in extruding a single screw screw into a single extruder at a temperature of 139 ... 150 ° C and a speed of 30 ... 60 rpm 75 , 6 ... 79.6% by weight polylactic acid (PLA), 7.56 ... 7.96% by weight Lapb masterbatch 108, 11.34 ... 11.94% by weight PEG, 0 .. .5% by weight of hydrolyzed collagen and 0 ... 0.5% by weight of silver nanoparticles.

The composites obtained were characterized by the determination of physicomechanical properties, cell proliferation, behavior in biofilm formation and in vitro degradability. Drain tubes were characterized by sterility with ethylene oxide. The results are presented in tables 1 ... 3 and fig. 1 ... 3 on which the formulation of the embodiments of the invention is based. The results are discussed in comparison with the reference mixture, namely polylactic acid (80%) plasticized with Lapb 108 masterbatch (8%) and PEG 4000 (12%).

The following are 3 embodiments of the invention in connection with FIG. 1 ... 3 which represent:

- fig. 1, the morphological aspect of cell culture in contact with polymeric biocomposites; - fig. 2, evaluation of in vitro degradability for 30 days of polymeric biocomposites; - fig. 3, in vivo macroscopic evaluation of polymeric biocomposites.

Example 1

Mix in melt on a Brabender Plastograph, at 170 ° C, for 7 min and 60 rpm, 38 g PLA type 2003D, 3.8 g Lapol masterbatch 108, 5.7 g PEG with molecular weight 4000 g / mol and 2.5 g of collagen hydrolyzate. Prior to use, the polylactic acid and masterbatch are dried in an air-heated oven at 60 ° C for 24 hours. The collagen hydrolyzate is also dried in an air-cooled oven. 40 ° C for 6 h. Composite made of plasticized PLA and component for improving biocompatibility - collagen hydrolyzate has the following mass composition: 76% PLA, 7.6% Lapol masterbatch 108.11, 4% PEG and 5% hydrolyzed of collagen.

The resulting mixture is processed on a screw extruder at a temperature of 150 ° C, a rotational speed of 30 rpm. The extruder is equipped with a nozzle measuring φ = 4 mm and a mandrel measuring 3 mm, used to make a tube with an inside diameter of 3 ± 0.1 mm and an outside diameter of 4 ± 0.1 mm.

The resulting mixture has a cell viability of 88.9% - Table 1, a higher biofilm formation trend of 18821.1 CFU / ml compared to 18210.9 CFU / ml of the control sample Table 2, a tensile strength of 16 MPa, elongation at break 128% and Young mode 1970 MPa - table 3. The mixture does not degrade significantly after 30 days of maintenance in PBS, the mass loss being 8% and in the short term, it records a mass loss of 2, 9% - fig. 2. There were no macroscopic abnormalities at the 8-week evaluation of the subcutaneous tissue of the mice around the implantation site of the material - fig. 3, and the drain pipe obtained is sterile.

Example 2

Proceed according to the procedure described in Example 1 and mix 37.8 g of 2003D type PLA, 3.78 g of Lapol 108 masterbatch, 5.67 g of PEG with a molecular weight of 4000 g / mol, 2.5 g of collagen hydrolyzate and 0, 25 g of silver nanoparticles with a particle size of 20 nm are mixed in a melt on a Brabender Plastograph at 170 ° C for 7 min and 60 rpm. The resulting nanocomposite containing biocompatibility, bio-compatibility enhancing agent and antimicrobial agent - silver nanoparticles has the following mass composition: 75.6% PLA, 7.56% Lapol 108 masterbatch, 11.34% PEG, 5% hydrolyzed 3 collagen, 0.5% silver nanoparticles. The resulting mixture is processed on an extruder with a screw, at a temperature of 145 ° C, a rotational speed of 40 rpm, to obtain a tube 5 with an inner diameter of 3 ± 0.1 mm and an outer diameter of 4 ± 0.1 mm.

The resulting nanocomposite has a cell viability of 93.6% - Table 1, a lower tendency of biofilm formation of 12393.4 CFU / ml compared to 18210.9 CFU / ml of the control sample - Table 2, a tensile strength of 14.1 MPa, elongation at break 126.6% 9 and Young's mode 1891 MPa - table 3. It does not degrade significantly after 30 days of maintenance in PBS, the mass loss being 8%, and in the short term, it records a weight loss of 2.6% - fig. 2. There were no macroscopic abnormalities at the 8-week evaluation of the subcutaneous tissue of the mice around the implantation site of the material 13 fig. 3, and the drain pipe obtained is sterile.

Example 3 15

According to the procedure described in Example 1, 39.8 g of 2003D type PLA, 3.98 g of Lapol 108 masterbatch, 5.97 g of PEG with a molecular weight of 4000 g / mol and 0.25 g of 17 nanoparticles of silver with a particle size of 20 nm. The resulting nanocomposite containing both biocompatibilizer and antimicrobial agent silver nanoparticles has the following composition: 79.6% PLA, 7.96% Lapol 108 masterbatch, 11.94% PEG and 0.5% silver nanoparticles. The resulting mixture is processed on a screw extruder at a temperature of 139 ° C, a rotational speed of 60 rpm to obtain a tube with an inside diameter of 3 ± 0,1 mm and an outside diameter of 4 ± 0,1 mm. The resulting nanocomposite has a cell viability of 98.8% - Table 1, a lower tendency of biofilm formation of only 8187.9 CFU / ml compared to 18210.9 CFU / ml of the control sample - Table 2, a resistance to 25 traction of 32 MPa, elongation at break 216.8% and Young's mode 2058 MPa - table 3. It does not degrade significantly after 30 days of maintenance in PBS, the mass loss being 27 of 5%, and in the short term it records a 1.2% mass loss - fig. 2. There were no macroscopic abnormalities at the 8-week assessment of the subcutaneous tissue of mice from 29 around the site of implantation of the material fig. 3, and the drain pipe obtained is sterile.

Characterization methods 31

From the polymer mixtures obtained according to the procedures described above, plates with a size of (150 x 150 x 1) mm and films with a size of 33 (150 x 150 x 0.1) mm were obtained by pressing for physical-mechanical characterization and biological. The pressing parameters are: working pressure: 147 bar; pressing temperature: 175 ° C, for 35 preheating 3 ... 5 min; pressing time: 5 ... 10 min; cooling time: 45 ... 50 min. For comparison, a control sample containing 80 g of 2003D PLA, 8 g of Lapol 108 masterbatch and 37 g of PEG BioULTRA 4000 was used, both in the form of a plate and film and a tube, made under the same conditions as the composites above. 39

Results obtained In vitro evaluation of the cytotoxicity of polymeric biocomposites 41

In vitro biocompatibility was assessed by cytotoxicity of the studied polymeric biocomposites in accordance with SR EN ISO 10993-5: 2009, using method 43 of direct contact with the stabilized cell line obtained from mouse fibroblasts (NCTC clone L929 cell line). The viability and morphology of L929 cells treated with 45 polymeric biocomposites were evaluated.

Cell viability was assessed by MTT- (3- [4,5-dimethylthiazol-2-yl] -2,5diphenyltetrazolium bromide). The quantification of cell viability is performed by spectrophotometric method and is based on the conversion of succinate dehydrogenase of the terazolium salt of MTT- (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide) into formazan. The amount of formazan is directly proportional to the number of viable cells and is determined spectrophotometrically after dissolution in a suitable solvent.

The cell morphology was analyzed by staining the cells with Hematoxylin-Eosin, after direct contact of the cells with the sectioned samples with an area of 0.5 cm ² .

The MTT method highlighted the viability of L 929 stabilized fibroblast cells, treated for 24 h and 48 h with the resulting films, respectively. The determination of cell viability was performed in order to establish the non-cytotoxic character of the materials, based on which the biocompatibility of the materials was established. The results of cell viability are presented in Table 1.

Cell viability for NCTC clone L929 cell line treated with polymeric biocomposites compared to control cells at 24 h and 48 h

Table 1

Test code MTT 24 h,% MTT48 h,% Culture Witness (Mc) 100 100 PLA / MB / PEG (reference) 957 814 Example 1 889 845 Example 2 936 865 Example 3 988 919 Hydrogen peroxide (M +) 110 67

The cell viability of the investigated samples was over 80% compared to the culture control, which suggests that they did not exert a cytotoxic effect on fibroblast cells, so the materials are biocompatible. Cells treated with bionanocomposite PLA / MB / PEG / AgNPs had the highest percentage of cell viability of 98.8% and 91.9%, respectively, after 24 h and 48 h of contact, respectively.

The cell morphology was analyzed by staining the cells with Hematoxylin-Eosin, after direct contact of the cells with the sectioned samples with an area of 0.5 cm. The non-cytotoxic nature of the materials was also confirmed by the morphological appearance of the cells treated with the above-mentioned bionanocomposites. The morphological aspect of the cell culture, after 48 h of contact with the bionanocomposites is shown in fig. 1.

The witness of culture. The cell culture used as such in the experiment was that of mouse dermal fibroblasts, stabilized cell line - NCTC (L929 clones), whose morphological appearance is given by round and polygonal cells, circular nucleus containing 2 ... 3 nucleoli and fine intracytoplasmic granulation; at 48 h of the experiment, the NCTC cell culture was predominantly in the subconfluence phase (Fig. 1f).

The appearance of the cells in contact for 48 hours with the PLA-based samples of different compositions showed a morphology similar to the culture control, which suggests the biocompatibility of all these samples studied. However, differences in cell density were observed in culture, the proliferation of cells being influenced by the specific composition of each sample. A lower degree of proliferation compared to the culture control was observed in cells contacted with the plasticized PLA sample (Fig. 1a), while cell culture 1 was in contact with the PLA / MB / PEG / AgNPs sample (Fig. 1d). ) had a cell density close to that of the culture control. Study of the biofilm deposited on the surface of the 3 materials obtained - antifungal activity.

The development of monospecific biofilms was performed using the static model, in plates 5 with 24 wells. Samples in the form of 1 cm x 1 cm pieces (sectioned from polymer plates) were sterilized by exposure to ethylene oxide. The samples were divided into wells and then completely coated with 1.5 ml of standard density microbial inoculum prepared for the microfungal strain Candida albicans ATCC 10231 in 9 Sabouraud liquid (Merck, Germany). After preparation, the plates were incubated for 24 h at 37 ° C. After 24 h, the pre-adhesion stage, the samples were washed three times with PBS (Phosphate 11 Buffered Saline) (Biochrom), in order to remove the non-adherent cells, then they were repositioned in fresh TSB medium (Tryptic Soy Broth - a medium of general use, used for the cultivation of a wide variety of microorganisms, including fungi, aerobic bacteria, anaerobes) and incubated for another 24, 48 and 72 h. , namely determination of CFU / ml (Colony Forming Units / ml) .17

After each incubation period, 24 h, 48 h and 72 h, respectively, the samples were lightly washed with sterile PBS, so as not to destroy the biofilm and then placed in tubes19

Eppendorf 2 ml, containing 1 ml PBS each, followed by vortexing for 1 min, in order to disperse in suspension the cells of the formed biofilm. From the formed suspension, 21 successive serial microdilutions were performed, then 10: L were spotted, in triplicate, on PCA (Plate Count Agar) / Sabouraud agar medium, to determine the number of viable cells.23

The number of viable microbial cells (CFUs) is determined by the relation (1):

CFU / ml = N x 1 / D x 102 (1) 25 where:

N = average of the colonies counted at the level of the three spots, 27

10 ² = volume correction factor, D = dilution read.29

The antibiofilm activity of polymeric bionanocomposites was evaluated against the reference microfungal strain Candida albicans ATCC 10231 (density ~ 05-106 CFU / ml). Bio-31 monospecific film developed on the surface of the materials was evaluated at 24 h, 48 h and 72 h (Table 2). 33

Number of viable cells detached from the biofilm of Candida albicans ATCC 10231 developed on plasticized PLA, biocomposite and bionanocomposites at 24 h, 48 h and 72 h

Table 2

Test code 24 h 48 h 72 h PLA / MB / PEG (reference) 182109 108124 87032 Example 1 18821,1 8705.2 6615.5 Example 2 12393.4 9752.1 6305.4 Example 3 8187.9 10753.4 4025.2

The results obtained from the biofilm evaluations showed that the materials obtained have an antifungal effect, manifested both on the process of colonization of the substrate and on the development of a mature fungal biofilm (table 2). Also, in the case of these materials, it was observed that the development of the mature biofilm of 48 hours and 72 hours, respectively, is lower, a behavior proven by the small number of viable cells included in the biofilm.

Determination of mechanical traction properties

Tensile properties (tensile strength, elongation at break, Young's modulus) were determined according to ISO 527-2: 2012. The INSTRON (USA) traction machine with built-in software was used to test the 1 mm thick dumbbell (plates). The reported results represent the mean value of five specimens and the standard deviation.

The effect of hydrolyzed collagen and AgNPs on the mechanical behavior of PLA (tensile strength, tensile elongation and Young's modulus) are illustrated in Table 3.

Tensile properties of plasticized PLA samples, biocomposite and bionanocomposites obtained

Table 3

Sample code Tensile strength (MPa) Elongation at break (%) Young's module (MPa) PLA / MB / PEG (reference) 36.57 ± 2.43 245.35 ± 13.1 1837 ± 59 Example 1 16 ± 3.96 127.95 ± 7.85 1970 ± 47 Example 2 14.12 ± 2.95 126.58 ± 8.56 1891 ± 69 Example 3 32 ± 6.8 216.84 ± 45.37 2058 ± 105

Table 3 shows that the tensile strength decreases for the plasticized PLA sample compared to the reference PLA; the introduction of collagen also leads to a further decrease in tensile strength. The introduction of Ag nanoparticles does not have a significant effect on the tensile properties of the bionanocomposites obtained. However, taking into account the physical and mechanical requirements of the urinary drains (tensile strength at break must be at least 10 MPa, elongation at break must be at least 100%) it can be seen that the two types of materials (PLA / MB / PEG / HC / AgNPs and PLA / MB / PEG / AgNPs) meet the minimum traction properties requirements for use in the medical field.

In vitro evaluation of the degradability of polymeric biocomposites

The degradability assessment of polymeric biocomposites was performed by immersing (10 x 3 x 0.01) cm (obtained from film) samples in PBS, p H = 7.4, at 37 ° C, for different time intervals. Material degradation was assessed by determining the mass variation. The samples were tested in vitro after drying at 40 ° C in the MEMMERT oven for 24 hours and brought to a constant mass. The samples were also dried in an oven at 40 ° C for 24 hours before each determination of mass variation.

In fig. 2 shows the mass loss of the mixtures made at different time intervals. It is observed that the samples containing hydrolyzed collagen (PLA / MB / PEG / HC and PLA / MB / PEG / HC / AgNPs) show a mass loss of about 7% after 3 days of immersion. These samples show the highest mass loss compared to the collagen-free samples, probably due to the rapid attack of PBS in the spaces between the polymer chains, plasticizer and collagen. For short-term use (24 h) of drains, the recipe containing 3 contains plasticized PLA and antimicrobial agent has a weight loss of about 1%.

In vivo evaluation of polymeric biocomposites5

Implantation of polymeric biocomposites was performed after anesthesia of mice with ketamine / xylazine to minimize the pain and discomfort of the animals used in the tests.7

4 CD 1-Nude mice (CDl-Foxnlnu) were used; weight 20 ... 25 g / mouse (having an average weight of 22.5 g) per type of material tested. Implantation was performed dorsally, 9 by making an incision and inserting the test material. The viability check of all animals was performed at least once a day, and detailed clinical examinations were performed and recorded once a week. Body weight was measured weekly. The observation period was 8 weeks. After slaughtering the animals, the site of implantation of the biomaterial was examined to assess any obvious adverse tissue reactions to the implanted material. At 8 weeks, immediately after slaughter, the test material was carefully removed from the implant site, taking care to cause as little damage to the adjacent tissues as possible. The tissues at the site of implantation (skin, muscle) were resected and preserved in buffered formalin (approximately 4% formaldehyde solution) and then paraffined. Approximately 5 micron thick sections were prepared from each region 19 for implantation of test or control materials. The slides were stained with hematoxylin and eosin. 21

There were no effects on the implantation of biomaterials on body weight.

All animals showed normal body weight gain until slaughter. No 23 abnormal clinical signs were observed. There were no macroscopic abnormalities at the 8-week assessment. Macroscopic examination of the tissue around the implant site did not reveal the presence of hematomas at the implant site at 8 weeks, but revealed the presence of fibrotic tissue of approximately 1 mm, inseparable or adherent to the surrounding tissues, encapsulating the tested material (fig. 3). Histopathologically, no changes were observed in the implantation of biomaterials in animals. No evidence of muscle necrosis or degeneration was observed during this period.

Sterility control 31

Drain samples are seeded directly into the culture medium so that the volume of the product does not exceed 10% of the volume of the culture medium. 33 culture media (Medium I - Fluid thioglycollate medium and Medium II - Sabouraud liquid) are poured into sterile test tubes (approximately 10 ml) under aseptic conditions. The first medium is regenerated for 20 to 35 minutes at 100 ° C in a water bath and then cooled abruptly. Two test tubes from each medium are used for one sample. Aseptically open the package with the seed sample. With 37 sterile scissors, remove the sample from the package, cut a portion of about 1 cm from it and place it in the test tube with medium. The operation is repeated in the same way until the whole group of test tubes has been sown. At the end of the work, the seeds sown with the sterilized sample are incubated for 7 days, as follows: the tubes with Medium I at a temperature of 41 30 ... 35 ° C and the tubes with Medium II at a temperature of 20 ... 25 ° C. The inoculated tubes are examined daily during incubation for macroscopic evidence of microbial growth. If in all tubes seeded with the test sample the culture medium retains its initial transparency compared to the control sample throughout the 45 incubation period, then the test sample is sterile. Otherwise, the sample is sterile.

Drain tubes obtained from polymeric biocomposites were sterile. 47

demand

Claims (2)

1 Claims 3 1. Process for the production of a polymeric bionanocomposite based on polylactic acid, collagen hydrolyzate and silver nanoparticles, characterized in that it is processed in the melt at a temperature of 170 ° C for 7 minutes and a rotational speed of the screw of 60 rpm a mixture based on 75.6 ... 79.6% by weight polylactic acid, 7.56 ... 7.96% by 7 weight plasticizer, 11.34 ... 11.94% by weight polyethylene glycol , 0 ... 5% by weight hydrolyzed collagen and 0 ... 0.5% by weight silver nanoparticles, the resulting mixture is passed through 9 screw extruder, provided with a dose having a diameter φ of 4 mm and a mandrel with a diameter φ of 3 mm, at a temperature of 135 ... 150 ° C, rotational speed of 30 ... 60 rpm, obtaining 11 a urinary tube with an inner diameter of 3 ± 0.1 mm and a diameter outer of 4 ± 0.1mm, with breaking strength of 16 ... 32 MPa, elongation at break of 126.5 ... 216.8%, modulus of 13 elasticity of 1891 ... 2058MPa, has a cell proliferation of 88.96 ... 98.77%, resistance to biofilm formation / fungal activity against Candida albicans ATCC10231, stable for 15 short term, having a mass loss of 1.2 ... 2.9% and being degradable after a longer time. A polymeric bionanocomposite based on polylalkic acid, hydrolyzed collagen and silver nanoparticles obtained by the production process defined in claim 1.