Patent Application
20250090728
The invention is part of the framework of materials science and technology at the service of society. Specifically, the present invention provides a silicone material functionalized with PEG-coated copper nanoparticles with antimicrobial and antibiofilm activity, so as to reduce the bacterial load and the development of biological films or biofilms of pathogenic bacteria on the surface of products or devices made with the material in accordance with the invention.
Microorganisms accumulate naturally on a wide variety of surfaces, where they form sedentary and sessile communities. These surfaces include domestic and industrial pipes, biocompatible materials, such as contact lenses, or medical devices, such as implants or urinary catheters, as well as plant and animal tissues, among many other surfaces. The accumulation of these aggregates of microorganisms in mono- or polymicrobial form are known as biological films or biofilms and can be composed of different bacterial and fungal communities.
On the other hand, silicone-based materials are increasingly used in various industries, as they have excellent heat resistance, dielectric properties, water repellency, flexible structure, as well as being biocompatible. In the case of cooking, for example, it is possible to use food-grade silicone in baking pans, mold coatings, and utensils. In the case of the pharmaceutical industry, it is possible to use medical devices made of silicone to deliver drugs with controlled release, and pharmaceutical-grade silicones can be used in molded devices such as intravaginal rings (IVRs) or intrauterine devices (IUDs) and in medicated implants, such as stents. Another frequent use of silicone in the biomedical industry is the manufacture of tubular devices such as cannulas, probes, and catheters.
Taking into account the versatility of the new uses of silicone and the fact that it is a surface that comes into contact with food, drugs and the body itself, it is necessary to have a material that prevents the formation of biofilms after the sustained use of articles and/or devices made with this material.
In particular, in the case of medical or clinical devices, healthcare-associated infections (HAIs) increase morbidity, mortality, and health care costs. Among them, catheter-associated urinary tract infection (CAUTI) is one of the most prevalent, where the use of an indwelling urinary catheter (used for more than 24 hours) is one of the main causes of bacteriuria and bacteremia. These infections remain uncontrolled despite improved clinical practices and the development of medical devices with antibacterial properties. Therefore, these infections are a current field of research that requires new control and prevention strategies.
The ideal material for the manufacture of a urinary catheter should be biocompatible, radiopaque, resistant to fouling and colonization by microorganisms, causing low discomfort, durable and affordable at a reasonable price. However, there is no urinary catheter on the market that meets all these requirements. The most commonly used materials in the manufacture of these devices are latex, silicone, polyurethane (PU) and polyvinyl chloride (PVC). Latex and PVC catheters are cheaper, but they can be quickly embedded and have a higher incidence of allergies. PU catheters have good biocompatibility, but their increased rigidity compared to silicone causes discomfort in some patients. Currently, the use of PU is more widespread in the manufacture of central venous catheters than in urinary catheters. In contrast, silicone is more suitable for permanent probing as it has high biocompatibility, absence of allergic responses, good chemical and thermal stability, and also a long shelf life (90 days). The main disadvantage of silicone is its higher cost relative to the other materials, and that it could be colonized by microorganisms.
There are currently a variety of urinary catheters that have been designed with the goal of reducing the risk of infection. These include catheters impregnated with antibiotics (nitrofural, minocycline, and rifampicin), silver oxide, and silver alloys. However, these catheters have little effect on the adhesion of uropathogens and in clinical trials have been effective in reducing the incidence of asymptomatic bacteriuria but not of CAUTI. More recent studies have evaluated the use of copper (CuNP) and silver (NpAg) nanoparticles to develop antimicrobial materials that can be used in medical devices. For example, Sehmi et al. (2015), embedded CuNP in polyurethane and silicone and showed that these polymers have an antimicrobial effect in vitro against Staphylococcus aureus and Escherichia coli. Similarly, Ballo et al. developed catheters impregnated with CuNP and NpAg that had antimicrobial activity in vitro but failed to prevent biofilm formation in vivo.
In the previous art, various solutions are known that address the control or prevention of microbial growth and biofilm formation of pathogenic organisms in silicone materials and devices for medical use.
The patent U.S. Pat. No. 7,993,390B2 provides an implantable or insertable medical device that is resistant to microbial growth and biofilm formation, both in the device and in its environment. The devices described include a biocompatible matrix polymer region, an antimicrobial agent, and/or an inhibitor of biofilm formation. Among the alternatives developed, a polymer ureteral stent is shown where the bioactive agent is triclosan. Although it is the same technical problem, the solution used is different, as they use triclosan as an active compound that inhibits biofilm formation.
Sehmi, Sandeep K., et al. (2015) employs 2.5 nm copper nanoparticles as an antimicrobial compound in medical-grade silicone and polyurethane materials for in-hospital use. However, there is talk of encapsulation due to swelling and contraction, so in the case of silicone and considering the smaller size of copper nanoparticles, it is not the same type of material. In addition, the desired feature is to reduce microbial contamination on frequently handled surfaces in and around hospital wards, such as bed rails, tables, push plates, etc. The problem of preventing or reducing the formation of biofilm in the processed material is not addressed.
Iqbal, Zohora, et al. (2018) describes the effect of sterilization on modified silicone surfaces. It focuses on the stability of medical device biomaterials by considering all stages of preparation for surgery, including sterilization, so it explores the effect of five standard sterilization methods (autoclave, dry heat, hydrogen peroxide, ethylene oxide gas, and electron beam) on three surface coatings: PEG, pSBMA, and pMPC. The data shown suggest that autoclave and EtO treatments are suitable for PEG-silane.
Regmi, Amrit, et al. (2019) describes the formation of copper nanoparticles, made with two different concentrations of copper precursors by the coprecipitation method using NaBH4 as a reducing agent and (PEG6000) as a stabilizer. The paper evaluates the antibacterial activity of these nanoparticles. It is suggested that antimicrobial activity increases as a function of nanoparticle concentration, based on in vitro tests on plates, but does not analyze the effect on biofilms.
In summary, although there are products aimed at preventing microbial growth in catheters for medical use, they are not demanded by highly complex public hospital establishments, due to their higher cost and the low effectiveness observed in clinical trials to reduce the incidence of CAUTI. Therefore, there is currently a need for a commercial catheter with antibiofilm activity to prevent such infections, which is particularly relevant for immunocompromised and critically ill patients, as they are the most at-risk groups.
The invention corresponds to a material based on silicone and copper nanoparticles (CuNP) coated with polyethylene glycol (PEG) that exhibits antimicrobial and antibiofilm activity, i.e. it allows for the reduction of the bacterial load and the formation of biofilm. The non-stick properties of PEG and the antimicrobial activity of copper act synergistically, generating resistance to protein adsorption and bacterial adhesion, and at the same time eliminate the bacterial load, resulting in a significant reduction in biofilm formation. In the case of medical uses, the material reduces the growth of microorganisms and the development of biofilms, and thus the risk of infection.
In a mouse model of catheter-associated urinary tract infection (CAUTI), it was determined that the intravesical implantation of a silicone catheter and copper nanoparticles (cNP) coated with polyethylene glycol (PEG) showed a bacterial load in the bladder and kidney compared to a silicone catheter. In addition, scanning electron microscopy showed less biofilm formation in the silicone catheter and copper nanoparticles (CuNP) coated with polyethylene glycol (PEG) compared to the silicone catheter. Both catheters produced a mild degree of inflammation when compared to a normal bladder.
A silicone material functionalized with PEG-coated copper nanoparticles with antimicrobial and antibiofilm activity is provided, comprising:
The silicone films to be used must be clean and their surface is activated, washed and dried, prior to functionalization with a polyethylene glycol solution (PEGylating process). The excess solution is removed and the material is washed and dried (
The material, according to the invention, differs from other alternatives in that, in addition to conferring a powerful antimicrobial activity to the material granted by the presence of copper nanoparticles, it manages to prevent the adsorption of proteins on its surface, which in turn contributes to the prevention or reduction of biofilm formation. An additional benefit is that the use of copper as an active component decreases the likelihood of generating antimicrobial resistance. In addition, the materials made in accordance with the invention are not cytotoxic. The concentration of the components of the invention is such that it is the minimum that allows for obtaining the antimicrobial activity and preventing or reducing the formation of biofilm in the silicone material.
The material demonstrates a decrease in bacterial load in vivo, for example, when analyzing bladders and kidneys in the assays at 96 h post-infection. The decrease in bacterial count is an indicator of the antibacterial effect of the material according to the invention, by obtaining values of colony-forming units (CFUs) lower than those reported in the use of traditional silicone.
The material also demonstrated its antibiofilm activity, or ability to reduce bacterial colonization and the formation of a layer of bacteria on the surface of the material, when evaluated in a continuous flow system, with fluids containing bacteria and similar in composition to urine.
Although the problem initially addressed was the development of urinary catheters that prevent the formation of biofilm, the material according to the invention allows the production of various products where silicone subjected to the same type of curing can be used.
Thus, it is possible to use the material in pharmaceutical, medical and food use, among others. Devices for medical use include those described in Table 1 and, in general, any device that can be made or coated with medical-grade silicone.
In the case of kitchen utensils, it is also possible to use the silicone material functionalized with copper nanoparticles according to the invention, as it is food-safe and safe for use in the kitchen.
In the case of the pharmaceutical industry, many patients do not take their medications as prescribed and need medical devices made of silicone to deliver controlled-release medications. The silicone material functionalized with copper nanoparticles, according to the invention, makes it possible to obtain new pharmaceutical-grade silicone materials that exhibit antibacterial and antibiofilm activity.
The silicone films (diameter 20 mm, thickness 0.024″) were supplied by Interstate IPS (USA). Deionized water was used to rinse the PDMS surfaces and prepare aqueous solutions. 2-[methoxy (polyethyleneoxy)-6-9-propyl]trimethoxysilane, 3-aminopropyltrimethoxysilane, N-(6-aminohexyl)aminopropyltrimethoxysilane were acquired from Gelest SA (USA). Hydrogen peroxide, copper (II) sulfate, sodium hydroxide, L-ascorbic acid, polyethylene glycol (PM g/mol: 2000, 3000, 4000) were purchased from Sigma-Aldrich.
Six materials were developed for prototype testing (see Table 2).
The silicone films were cleaned by ethanol/water sonication (1:1, v/v) for 10 min before use. Activation of the silicone surface was carried out by treatment with HCl (30%) for 12 h at room temperature. The samples were washed 3 times in water, sonicated in water for 10 min, dried with nitrogen, and immediately immersed in the respective PEG-silane solution. Functionalization was performed using a 1:5:5 solution of PEG-silane, 0.1 M acetic acid and isopropyl alcohol, respectively, for 12 h at room temperature. Subsequently, the films were washed twice in ethanol and twice in water and then sonicated in water for 2 min to remove excess non-functionalized material. The films were dried with nitrogen and stored dry under ambient conditions.
The CuNP were obtained by chemical synthesis, using copper sulfate (CuSO4*5H2O), ascorbic acid (AA—0.01 M) as a reducing agent and PEG of different sizes (2000-3000-4000 g/mol) as a stabilizing agent.
A set of solutions was prepared by mixing 4.0 ml of CuSO4·5H2O (0.01 M) with 16.0 ml of PEG2000 at various concentrations. A stock solution of PEG2000 with a concentration of 0.5 M was prepared and various volumes of this stock solution (8, 4, 2 mL) were diluted to 16 mL with water, and it was these more dilute solutions that were added to the copper solution. In separate flasks, 0.5 ml of 0.1 M ascorbic acid was diluted in 10 ml of water and 3.0 ml of 0.5 M NaOH in 10 ml of water. Next, the ascorbic acid and sodium hydroxide solutions are mixed, and then added over the copper solution while agitating. The complete solution was kept in agitation for another 30 minutes.
After agitating, the solutions were centrifuged at 6000 rpm for 30 minutes. The precipitates were collected and redispersed in water. The obtained CuNP were centrifuged at the same rate for another 15 minutes, twice, to remove excess PEG.
The silicone surface previously functionalized with PEG-silane was incubated with synthesized CuNP for a period of 12 h to obtain the chemical interaction between the PEG-silane chains present on the silicone surface with the CuNP.
Subsequently, the physicochemical characterization of the prototype materials was carried out, which is relevant for the use of this material in the manufacture of medical devices.
The evaluation of antimicrobial capacity was carried out with the formulation of the material Filme/PEG6-9 silane/PEG2000+CuNP) with an CuNP size: 94.3±1.2 nm). Subsequently, the lowest necessary concentrations of CuNP were determined, obtaining conditions C1 and C2 (see Table 3):
Antimicrobial activity against E. coli ATCC 25922 was determined at 2 incubation times in two types of samples.
The antimicrobial activity of four films with different concentrations of CuNP was also evaluated against a clinical isolate of urinary tract infection: E. coli ATCC 25922.
In addition, the C1 formulation was evaluated for its ability to reduce bacteriuria and prevent bacteremia in a murine model of catheter-associated urinary tract infection (CFU≈1×107 of E. coli), observing that E. coli biofilms do not occur in vivo in C1 material during the course of infection.
The following table compares the percentages of bacterial load reduction obtained for C1 (CuNP 2.2±0.10 pM) and C2 (CuNP 5.6±0.28 pM) materials at each incubation time and against the four bacteria used. In general, the two films showed similar antibacterial activities, obtaining a greater reduction of the bacterial load at 6 hours of incubation. For both materials, the percentages of reduction against E. coli and K. pneumoniae were greater than 90% in all the times evaluated, and against E. coli the reduction in some cases was even 100%. This is particularly important from an epidemiological point of view given that E. coli and K. pneumoniae are the most important etiological agents of CAUTI in Chile, the USA and Europe. In the case of E. faecalis and P. aeruginosa, the percentage reduction reached values greater than 80% in some cases.
These results demonstrate the influence of surface chemistry on interaction with bacteria, which in turn has a significant effect on the antimicrobial efficacy of CuNP surfaces. In that sense, increasing the release of copper ions in the bacteria/surface contact area on the invention material leads to greater efficiency in the elimination of bacteria.
E. coli
K. pneumoniae
E. faecalis
P. aeruginosa
The biofilm formation capacity of the prototype material C1 (CuNP 2.2±0.10 pM) and C2 (CuNP 5.6±0.28 pM) was determined in a continuous flow system, using the uropathogenic E. coli strain ATCC 25922 and K. pneumoniae ATCC 700603.
Continuous flow system: A preinoculum was loaded at 37° C. of 10 mL of LB with K. pneumoniae, or of LB supplemented with Amp for E. coli ATCC 25922 (transformed with the pDiGc plasmid). The next day, the films (C1, C2, Si control) were sterilized for 30 min (15 min per side) with UV radiation under a hood. The entire flow-through system was also sterilized separately. Sterile films were duplicated in 4 chambers of the system (2 films each) under sterile conditions. A continuous flow rate of 3.3 mL/min was set for 24 h at 37° C.
Biofilm quantification: After incubation for 24 h at 37° C., the films were washed with 4 mL PBS and then fixed with 70% methanol for 7 min (7). Subsequently, the methanol was extracted, the films were left to dry and incubated with 0.5% violet crystal for 15 min. Then 3×10 mL were washed with distilled water. To quantify staining, the films were immersed in 1 mL of 33% acetic acid, vortexed for 20 s, and quantified at OD 595 nm of solution. Tests were carried out in duplicate for each prototype.
Statistical analysis: The Mann-Whitney test was used to determine significant differences in the formation of E. coli or K. pneumoniae biofilms in C1 or C2 films compared to control films (silicone).
Results: The evaluation time for biofilm formation was 24 h in AUM medium. As expected, the bacteria evaluated formed a biofilm on the control silicone surface. Notably, K. pneumoniae generated 6 times more biofilm compared to E. coli, which is similar to what has been reported in other studies evaluating biofilm production in these bacteria. Notably, C1 and C2 films significantly reduced (p<0.05) biofilm formation for both E. coli and K. pneumoniae (
No statistical differences were observed in the reduction of biofilm formation between C1 and C2. Therefore, C1 and C2 films significantly reduce the formation of biofilms generated by E. coli ATCC 25922 and K. pneumoniae ATCC 700603 compared to silicone films.
Female C57BL/6 mice weighing 26-35 g, 8-10 weeks old were divided into 2 groups, group C1 and control group (5 animals/group). A piece of 7 mm polyethylene tubing (PE10 BD cat No. 427400) was placed on a 30 G×½ sterile metal needle (0.3 mm×13 mm BD Precision Glide) followed by a 5 mm segment of C1 prototype material or the control. The needle was placed into the urethral opening and the tube was moved over the needle until the material segment of the invention or control was deposited inside the bladder. The needle and the 7 mm tube were later removed.
The mice were infected immediately after insertion of the silicone segment, where the inoculum was administered in 50 μL of 1×PBS at a rate of 10 μL/s, using a tuberculin syringe. The number of CFUs present in the inoculum corresponded to ˜ 1×107 CFU of E. coli.
The devices (C1 and silicone) retrieved from the bladders were fixed in 2.5% glutaraldehyde and prepared for scanning electron microscopy (SEM) to evaluate biofilm formation.
The control implants (silicone) of the infected mice were coated with bacteria in the lumen that are observed embedded in what appears to be an extracellular matrix (
The mice were euthanized 96 hours after infection and subsequently the bladders and kidneys were removed, homogenized and diluted in PBS and platelet in LB/ampicillin agar media in dilutions 10−1 to 10−8. The bacterial load was determined after incubation for 24 to 48 h at 37° C. and was expressed as total CFU per organ.
Statistical analysis: The Student's T test was used to determine significant differences in the number of colony-forming units (CFUs) of E. coli in bladder and kidney homogenates of mice with C1 catheter compared to controls (silicone catheter).
Results: In animals with C1 implants, E. coli bacteria reached an average titer of 1.05×103 CFU/ml at 96 hours in the bladders, which was significantly lower (P=0.432) than the bacterial titers (2×103 CFU/ml) recovered from the bladders of the control implant animals (
Inflammation evaluation. For histological analyses, the bladders were fixed in buffered formaldehyde for 2 hours and dehydrated in 70% ethanol overnight at 4° C. They were then embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) for light microscopy.
Results: The implantation of catheters in the bladder and infection with E. coli caused histological changes in the bladder at the epithelium, submucosa and muscle levels. The changes were mild (1 out of 3). Although there are no significant differences in the degrees of inflammation between the C1 implanted and control animals, it was observed that in the control animals a mild, perivascular, chronic lymphocytic to neutrophilic cystitis was observed, with mild edema and mild epithelial changes. compared to the mild, perivascular, chronic lymphocytic cystitis observed in C1 implanted animals.
The HepG2 cell line of hepatocellular carcinoma (passage 19) was used for the assays. The cells were incubated in Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and 1% antibiotic (penicillin and streptomycin). The crops were kept at 37° C. with an atmosphere humidified with 5% CO2.
The “In vitro toxicology assay, Resazurin based” kit (Sigma-Aldrich, Darmstadt, Germany; Cat No. TOX8-1KT).
Evaluation of physical, antimicrobial and antibiofilm properties of material subjected to UV sterilization and autoclave.
The samples were subjected to UV irradiation sterilization processes in the in vitro phase. As a result, the materials showed antimicrobial capacity.
| Number | Date | Country | Kind |
|---|---|---|---|
| 3572-2021 | Dec 2021 | CL | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CL2022/050126 | 12/9/2022 | WO |
