Antimicrobial Coating Compositions and Formulations Thereof

FIELD OF INVENTION

The present invention relates to antimicrobial coating and formulation thereof. More particularly the invention relates to an antimicrobial coating and formulation thereof that can be used to coat different materials such as silicone, silicon resins, polyvinyl chloride, polyvinyl chloride resins, polypropylene, polypropylene resins, polyethylene, polyethylene resins, latex rubber, glass, glass resins, stainless steel, ceramic, certain polyurethane etc. Even particularly the present invention relates to antimicrobial coating to coat various surfaces, such as medical devices, including urinary catheters, vascular catheters, oxygen tubing, endotracheal and nasogastric tubes, hemodialysis tubing, stents, non-invasive ventilation masks etc.

BACKGROUND OF INVENTION

Medical devices are being used widely in the critical sector of hospital settings and therefore considered as a lifesaving invention in the medical field. However, the use of these commonly used conventional medical devices such as urinary catheters, vascular catheters, oxygen tubing, endotracheal and nasogastric tubes, hemodialysis tubing, stents, non-invasive ventilation masks etc. is responsible for device-associated nosocomial infections. The colonization of bacteria on the inner and outer surfaces of the indwelling medical devices lead to both infection and malfunction of the medical device which make them life-threatening instead of life saving in most of the critically ill patients. Literature reports suggest that in population of critically ill patients, urinary tract infections are 95% associated with catheter, bloodstream infections are 87% due to an indwelling vascular catheter, and pneumonia is 86% associated with mechanical ventilation. Apart from bacteria as a major cause of device-associated infections, fungi and viruses are also occasionally contributing to the device-associated nosocomial infections. These hospital acquired infections result in patient incompliance, reduced quality of life, increased length of hospital stays, extra financial burden etc. To solve this serious problem, currently available antimicrobial coating solutions have several drawbacks such as non-compatibility of the coating solution with various substrate surfaces, weak adherence of the film formed, non-uniformity of the coating layer, use of the commercially available antibiotics contributing to a global crisis of bacterial resistance, lack of the long-term antimicrobial potential of the coating layer etc. Therefore, to address this challenging problem there is an urgent need to develop antimicrobial coating formulation which forms a uniform, well adhering and long-term functional antimicrobial coatings having no bacterial resistance on both inner and outer surfaces of the medical devices.

Many prior studies have explored the development of antimicrobial coatings using different materials such as prepolymers, polymers, chain extenders, and various cross-linkers, including photo-initiators. Some of these coatings rely on a UV light curing process, while others use thermal curing to create a covalent bond within the polymer network. In certain previous research, antimicrobial coatings were developed using polyurethaneurea, which is synthesized using NCO- terminated polyurethane prepolymers and bifunctional amines as chain extenders or cross-linkers. These coatings often included various antimicrobial agents like antibiotics, metal particles (alloy), silver salts, silver ions and silver nanoparticles. However, these previous studies primarily focused on short-term antimicrobial performance and often lacked uniformity, durability, stability, and long-term functionality in their coatings. Additionally, using polyurethaneurea or polyurethane with a chemical reagent as a cross-linker resulted in covalently bonded coatings that could potentially cause a loss of their antimicrobial activity due to the disrupted release of antimicrobial agents from these tightly bound coatings. This combination of polyurethane polymer and crosslinkers also had a short pot life and often led to significant composition wastage due to the polymer initiating irreversible curing in the coating solution itself. Conversely, using polyurethane dispersions without cross-linkers could result in less durable and easily removable antimicrobial coatings upon contact with water.

Considering these challenges with polyurethane as a supporting polymer in antimicrobial coating development, there is a need to find a suitable cross-linker that forms an optimized polyurethane network through ion-pairing (ionic bond) rather than covalent bonding. Additionally, optimizing the concentrations of the coating composition ingredients is essential to produce uniform, durable, and long-term functional antimicrobial coatings on various substrate surfaces, including latex, silicone, polyvinyl chloride, and polyurethane.

OBJECTIVES OF THE INVENTION The primary object of the present invention is to provide an antimicrobial coating composition for prevention of device-associated infection/ hospital-acquired infections.

Another object of the present invention is to provide an antimicrobial coating composition for uniform, durable and long term functional antimicrobial coatings.

Yet another object of the present invention is to provide an antimicrobial coating composition having polyurethane as a supporting polymer, ethylenediamine as a cross linker, colloidal silver solution as an antimicrobial agent, polyvinyl pyrrolidone (K90) as a film former and glycerol as a humectant or wetting agent.

Still another object of the present invention is to provide an antimicrobial coating composition restricting bacterial resistance towards the colloidal silver nanoparticles used in the composition and effectively killing the existing resistant bacterial strains.

Still another object of the present invention is to provide an antimicrobial coating composition suitable for various coating methods such as dip coating, spray coating, roll coating, spin coating, brushing, flow coating etc.

Still another object of the present invention is to provide an antimicrobial coating composition with an extended pot-life or shelf life at room temperature.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides an antimicrobial coating formulation for having the appropriate combination and quantities of the added components which are synergistically important for achieving the desired properties of antimicrobial coating formed on the surfaces of various medical devices. While there have been reports of using NCO-terminated polyurethane prepolymers as supporting polymers and amino functional reagents as cross-linkers or chain extenders to create durable coatings on medical devices, the combination of a water-based polyurethane with no -NCO terminal as a supporting polymer and ethylenediamine as a cross- linker to form an optimized polyurethane network through ion-pairing (ionic bond) instead of a covalent bond for creating desired antimicrobial coatings is being reported for the first time in this invention. The present invention, the composition of polyurethane as a supporting polymer, ethylenediamine as a cross linker, colloidal silver solution as an antimicrobial agent, polyvinyl pyrrolidone (K90) as a film former and glycerol as a humectant or wetting agent are combined in appropriate quantities which results into synergistic effect by formulating uniform, durable and long term functional antimicrobial coatings on the surfaces of medical devices.

The antimicrobial coating composition of the present invention is a composition containing a supporting polymer, a cross linker, an alcohol-water mixture as a solvent, an ammonium methacrylate copolymer stabilized inorganic antimicrobial agent, a film forming polymer and a humectant or wetting agent. The substrate surfaces coated with antimicrobial coating composition when dipped in to water showed no removal of the coating layer or film and indicated a long-term antibacterial potential performed using zone of inhibition assay for up to 15 days.

DETAILED DESCRIPTION OF THE INVENTION

The antimicrobial composition of the present invention comprises of a supporting polymer, an antimicrobial agent, a film forming polymer, a humectant or wetting agent, a cross linker and a solvent

In the present invention, antimicrobial coating composition comprises polyurethane as a supporting polymer. The selection of a suitable polyurethane polymer having appropriate functional groups available for cross linking (ion-pairing) is crucial for achieving desired antimicrobial coatings on the various substrate surfaces. Also, an appropriate or optimized concentration of polyurethane in the composition is important to hold the film forming polymer along with colloidal silver nanoparticles without affecting their long-term functionality. Higher concentration of it in the composition could result into loss of antimicrobial property and stripping of the film formed on the substrate. Whereas, lower concentration of it in the composition could lead to weak networking and bonding with the substrate surface holding colloidal silver nanoparticles, this could further result into quick removal of a film formed upon contact with water. In the present invention, the polyurethane employed is water based aromatic hybrid N- Methyl-2-Pyrrolidone (NMP) free polyurethane dispersion having pendant carboxylic acid functional groups available for ion-pairing (cross linking). The carboxylate (COO ) ions from the pendant carboxylic acid functional groups present in the structure of polyurethane polymer form stable and desired polymer network through ion-pairing with the ammonium (NH3 ⁺ ) ions of cross linker used. This network securely anchors the colloidal silver nanoparticles to the substrate surface for an extended duration.

In the present invention related to an antimicrobial coating composition, purpose of colloidal silver solution is as an antimicrobial agent. The colloidal silver solution used in the composition is novel and developed by the inventors of the present invention, An Indian patent (Patent number: 347091) has been already granted for this invention under the title “A composition of silver nanoparticles and formulation thereof’. This developed aqueous solution of colloidal silver was slightly modified using an ammonium methacrylate copolymer and used further in an antimicrobial coating composition for achieving a long-term antimicrobial activity of the coating films formed on various substrates. In the present invention related to an antimicrobial coating composition, purpose of polyvinylpyrrolidone (K90) is as a film forming polymer. Considering the need of non-ionic film forming polymer in the formulation of antimicrobial coating composition, polyvinylpyrrolidone (K90) was selected as a film former. Apart from the charges present on the polymer molecules, an optimized concentration of polyvinylpyrrolidone (K90) in the composition is important to achieve uniform distribution of colloidal silver nanoparticles in the antimicrobial coatings. Higher concentration of it in the composition could mask the functional groups of supporting polymer and cross linker, this could lead to formation of a weak cross-linked networking of supporting polymer, this could in turn result into removal of an antimicrobial film formed upon contact with water. Whereas, lower concentration of it in the composition could lead to non-uniform distribution of the colloidal silver nanoparticles in an antimicrobial film formed on the substrate surface. In the present invention, a non-ionic nature and an appropriate concentration of the selected film forming polymer in the antimicrobial coating composition resulted into formation of long term functional antimicrobial films on various substrate surfaces.

In the present invention related to an antimicrobial coating composition, purpose of glycerol is as a humectant or wetting agent. The curing step of antimicrobial coating films on substrate surface involves the heating in the range of 60 to 80 °C for 30 min. An over drying of the antimicrobial films on substrate surface during curing process could lead to breaking and stripping of the films formed. Therefore, to avoid this over drying by retaining moisture in the antimicrobial films formed, glycerol was used as a humectant or wetting agent. An optimized concentration of glycerol in the composition is important to achieve the non-sticky and intact antimicrobial coatings. Higher concentration of it in the composition could result into formation of sticky antimicrobial films, whereas lower concentration of it in the composition could lead to breaking and stripping of the films formed during their handling. In the present invention, use of an appropriate concentration of the selected humectant or wetting agent in the antimicrobial coating composition resulted into formation of the non-sticky and intact antimicrobial coatings.

The selection of a cross linker having appropriate functional groups available for ionic cross linking (ionic bonding) with carboxylic acid functional groups of polyurethane (supporting polymer) is an important step for achieving desired antimicrobial coatings. In the present invention, the purpose of ethylenediamine is acting as a cross linker. The chain length of the selected bifunctional amine, base strength, solubility in water and alcohol, boiling point and its affinity towards carboxylic acid functional groups of polyurethane are equally important in achieving the optimized cross-linking network of polyurethane for effectively holding the film former and colloidal silver nanoparticles on the substrate surface. An appropriate concentration of ethylenediamine was used in the composition based on the mole equivalent ratio of carboxylic acid and amine functional groups available for cross-linking. In the present invention, use of an appropriate concentration of the selected cross-linker in the antimicrobial coating composition resulted into formation of long-term functional antimicrobial films on various substrate surfaces.

The developed antimicrobial coating composition involves the use of alcohol to make the alcoholdistilled water mixture as a solvent. Though all the composition ingredients are freely soluble in water, the use of completely water-based composition was avoided to get the desired antimicrobial films on substrate surface. The completely water-based antimicrobial composition produces non- uniform films on the substrate surfaces after drying due to high boiling point and slow evaporation rate of water at around 70 °C. The examples of the alcohols used for making alcohol-water solvent system includes ethanol, isopropyl alcohol, methanol etc. An appropriate concentration of alcohol used in the antimicrobial composition of the present invention produced uniform antimicrobial films/coatings on various substrate surfaces by accelerating the evaporation rate of the solvent system used at around 70 °C.

The developed antimicrobial coating composition involves the use of distilled water for making alcohol-water mixture as a solvent. As polyurethane, a supporting polymer is soluble in plain water or alcohol-water mixture, the use of completely alcohol-based composition was not preferred to avoid the precipitation of polyurethane in alcohol and to get the desired antimicrobial films on substrate surface. An appropriate concentration of water used in the antimicrobial composition of the present invention produced uniform antimicrobial films/coatings on various substrate surfaces by keeping polyurethane uniformly dispersed in the formulated composition.

Example 1

Table 1. Optimization of an antimicrobial coating composition using various composition ingredients Optimization of antimicrobial coating compositions

Composition Compo 1 Compo 2 Compo 3 Compo 4 Compo 5 Compo 6 Compo 7 Compo 8 Compo 9 ingredients

(14 ml) (14 ml) (14 ml) (14 ml) (14 ml) (14 ml) (I L) (14 ml) (I L)

Amount (% v/v)

Polyurethane 7.142 7.142 10.714 10.714 10.714 7.142 7.2 10.714 10.714

Distilled water 35.714 35.714 17.857 17.857 17.857 7.142 7.2 7.142 7.142

Colloidal silver 28.571 21.428 solution

Ethylenediamine 28.571 10.714 15.714 10.714 10.71 16.071 16.071

Jeffamine D230 15.714 methylenediamine solution

Methanol 25 35.714 35.714 35.714 39.285 39.28

Mixture of 3 % 18.071 18.071 18.071 w/v ofPVP K90 and 2 % w/v of glycerol in colloidal silver solution

Mixture of 3 % 35.714 35.71 w/v ofPVP K90 and 5 % w/v of glycerol in ammonium methacrylate copolymer modified colloidal silver solution.

Mixture of 4 % 35.714 35.714 w/v ofPVP K90 and 5 % w/v of glycerol in ammonium methacrylate copolymer modified colloidal silver solution.

Ethanol 30.357 30.357

The various compositions were tested during the optimization process of an antimicrobial coating solution, as shown in Table 1. In Compo 1, not using methanol and PVP K90 resulted in the formation of uneven coating films on the substrate surfaces. Compo 2, which added methanol along with distilled water to accelerate drying, still produced non-uniform and hard coatings with cracks in the films after turning and twisting the substrates. On the other hand, Compo 3, 4, and 5, which used PVP K90, glycerol, and methanol together, produced uniform coatings without cracks and exhibited antibacterial activity (zone of inhibition) for up to 24 hours

Among these, only Compo 3 created well-adhered coatings due to a stable cross-linking between polyurethane and ethylenediamine. Compo 4 and 5 failed to produce strongly adhered coating films because of weak or no cross-linking between polyurethane and jeffamine D230 in Compo 4 and polyurethane and 4,4 methylenediamine in Compo 5. Although all three screened cross-linkers were bifunctional amines, only ethylenediamine could form the optimized cross-linking network of polyurethane to effectively hold the film former and colloidal silver nanoparticles on the substrate surface. This highlights the importance of selecting a suitable cross-linker, considering factors like its base strength, solubility in water and alcohol, chain length, boiling point, and affinity towards carboxylate (COO ) ions of polyurethane. This choice not only determines the durability of the coating films containing silver nanoparticles but also allowing a sustained release of silver ions for long-term functionality. Compo 6 successfully produced uniform, durable, and long-term functional coating films on the substrate surfaces using an ammonium methacrylate copolymer modified colloidal silver solution. Therefore, Compo 6 was considered the optimized coating composition and scaled up to 1 L (Compo 7). Similarly, Compo 8 also created uniform, durable, and long-term functional coating films on the substrate surfaces using an ammonium methacrylate copolymer modified colloidal silver solution. However, Compo 8 used ethanol as a solvent instead of methanol. Therefore, it was also considered an optimized coating composition and scaled up to 1 L (Compo 9) for use on various substrates such as latex rubber, silicone, polyvinyl chloride, and polyurethane.

In an embodiment of an optimized antimicrobial coating composition of the present invention can be used to coat the uncoated latex Foley balloon catheters.

In an embodiment of an optimized antimicrobial coating composition of the present invention can be used to coat the uncoated silicone Foley balloon catheters.

In an embodiment of an optimized antimicrobial coating composition of the present invention can be used to coat the uncoated endotracheal tubing made up of polyvinyl chloride.

In an embodiment of an optimized antimicrobial coating composition of the present invention can be used to coat the uncoated hemodialysis tubing and central venous catheter made up of polyurethane.

Example 2

Preparation method of an ammonium methacrylate copolymer modified colloidal silver solution.

The present invention discloses a preparation method of an ammonium methacrylate copolymer modified colloidal silver solution. The preparation method comprises the following steps

• Preparing the 1 % w/v ethanolic solution of an ammonium methacrylate copolymer (Solution A).

• Preparing the 1 % w/v aqueous solution of an ascorbic acid (Solution B).

• Mixing of 40 ml of solution A and 16 ml of solution B using magnetic stirrer for 5 min (Solution C).

• Preparing the 2.4 % w/v solution of glycerol using patented (Patent number: 347091) colloidal silver solution (Solution D). Slowly adding 56 ml of solution C to 400 ml of solution D using magnetic stirrer for 10 to 15 min to finally produce an ammonium methacrylate copolymer modified colloidal silver solution.

Example 3

Coating and curing process

The present invention also discloses a coating and curing method utilizing an optimized antimicrobial coating composition of the present invention. The coating and curing process comprises the following steps

• Cleaning the substrate surfaces (latex rubber, silicone, polyvinyl chloride and polyurethane) using isopropyl alcohol and drying in a hot air oven at 60-80 °C for 5 min.

• Dipping the cleaned substrates into primer solution for 1 to 2 min at room temperature.

• After 1 to 2 min of dipping, removing all the substrates from the primer solution tank and immediately transferring into the hot air oven maintained at 60-80 °C for 30 min.

• After 30 min of drying of a primer layer, dipping all the substrates into antimicrobial coating solution for 10 min at room temperature.

• After 10 min of dipping, removing all the substrates from the antimicrobial solution tank and immediately transferring into the hot air oven maintained at 60-80 °C for 30 min.

• After 30 min of drying, dipping all the substrates into distilled water for 30 sec and transferring into the hot air oven maintained at 60-80 °C for 3 to 10 min.

• After complete drying, removing all the substrates from the hot air oven and testing individually for antimicrobial activity using zone of inhibition assay. Example 4

Coating of various medical devices

We coated various uncoated medical devices, including latex Foley balloon catheters, silicone Foley balloon catheters, endotracheal tubing, hemodialysis tubing, and central venous catheters, using an optimized antimicrobial coating compositions and the method described in Example 3. The optimized antimicrobial coating composition had a density of 0.919 gm/ml and a viscosity of 30-40 cps. The latex Foley balloon catheters had an outer diameter of 5.33 mm (16 Fr), an inner diameter of 2.3 mm, and a length of 40 cm. The silicone Foley balloon catheters had the same specifications. The endotracheal tubing had an outer diameter of 9.3 mm and an inner diameter of 7 mm. The hemodialysis tubing was 13.5 cm long and had a 2-lumen indwelling catheter measuring 11.5 Fr x 5-2/5". The central venous catheter was 16 cm long and had an indwelling catheter measuring 7 Fr x 6-2/5". After coating these devices, the coated latex Foley balloon catheter and coated silicone Foley balloon catheters were further utilized for the in-depth evaluation of coatings.

Example 5

Antimicrobial coating film/layer morphology (Figure 2 and 3)

The morphology analysis was performed to determine the structural uniformity of the coated antimicrobial film on the silicone and the latex Foley catheter surfaces. We used scanning electron microscopy to analyze the structure of the antimicrobial coating films on the surfaces of silicone and latex Foley catheters. In Fig. 2, SEM images of the coated silicone Foley catheter show a continuous, uniform, and porous antimicrobial coating without any uncovered areas. The pore sizes in this film ranged from 0.1 to 1.8 pm (Fig. 2)(b). The images also clearly reveal the boundary between the coated and uncoated sections of the silicone Foley catheter (Fig. 2)(c). In Fig. 2, SEM images of the coated latex Foley catheter similarly show a continuous and uniform antimicrobial coating layer with no uncovered areas. However, the coating on the latex surface was found to be less porous compared to the silicone catheter (Fig. 3)(b). SEM analysis also clearly distinguishes between the coated and uncoated sections of the latex Foley catheter (Fig. 3)(c).

Example 6 Antimicrobial coating film thickness (Fig 4 and 5)

An antimicrobial coating film thickness analysis was performed to measure the thickness and uniformity of the inner and outer antimicrobial coating films on both silicone and latex Foley catheter surfaces using the scanning electron microscopy. SEM images in Fig. 4 and Fig. 5 confirmed that there are continuous and uniform antimicrobial coating films along the periphery of both silicone (Fig. 4)(a) and latex (Fig. 5)(a) Foley catheters. The SEM analysis also showed that there is uniform antimicrobial coating on the inner surfaces of silicone (Fig. 4)(b) and latex (Fig. 5)(b) Foley catheters, with thicknesses of about 1.5 pm and 1 pm, respectively. We also examined the uniformity of the outer coating towards the upper and lower portions of the catheters. For silicone Foley catheters, the thickness of the outer coating film was consistent, measuring between 3-5 pm (Fig. 4)(c) and (d). Similarly, for latex Foley catheters, the outer coating film had a consistent thickness in the range of 4-6 pm (Fig. 5)(c) and (d).

In summary, these results confirm that the antimicrobial coating films, produced using our optimized composition, are uniform and have consistent thicknesses on both silicone and latex surfaces.

Example 7

Distribution of silver nanoparticles in the cured coating films (Fig 6)

As depicted in Fig. 6, SEM images of the antimicrobial coating film verified the presence of an ammonium methacrylate copolymer modified silver nanoparticles distributed within the crosslinked polyurethane network. These nanoparticles appeared spherical and measured between 120 to 230 nm in size (Fig. 6). The particle size was also confirmed through dynamic light scattering experiments, which yielded a consistent result of 148 nm, aligning with the SEM analysis.

Table 2.

Silver content values measured in both the upper and lower halves of the coated silicone and latex Foley catheters using atomic absorption spectroscopy. Substrate coated Silver content found in the cured coating films (ppm)

Upper half portion Lower half portion

Coated silicone catheter 39.36 36.64

Coated latex catheter 43.52 45.58

The silver content values were measured in both the upper and lower halves of the coated silicone and latex Foley catheters to check how evenly the antimicrobial coating films were distributed. We separately digested and analyzed the upper and lower halves of the coated catheters using atomic absorption spectroscopy to determine the silver concentration in each section. For the coated silicone catheter, we found silver content values of 39.36 ppm in the upper half and 36.64 ppm in the lower half (Table 2). These similar silver concentrations in both sections suggest that the silver nanoparticles were evenly distributed throughout the length of the silicone catheter due to the uniform coating film formation. Similarly, for the coated latex catheter, we observed silver content values of 43.52 ppm in the upper half and 45.58 ppm in the lower half (Table 2). Once again, the similar silver concentrations in both sections indicate uniform distribution of silver nanoparticles, likely due to uniform coating film formation. The slightly higher silver concentration in the latex catheter compared to the silicone catheter may be attributed to slightly more hydrophilic and porous nature of the latex rubber. However, it's important to note that the silver content in the coating films can be easily adjusted by varying the concentration of silver nanoparticles during the formulation of the antimicrobial coating composition.

Therefore, the results discussed in examples 5, 6, and 7 collectively validate the uniformity of antimicrobial coating films produced using an optimized antimicrobial coating formula on silicone and latex surfaces.

Example 8

Durability of antimicrobial coatings

The durability of the coatings was determined by making slight modifications to the previously reported percent weight loss method. To investigate how well the antimicrobial coating films held up in an aqueous environment, we cut the coated silicone and latex Foley catheter samples into 4 cm lengths and weighed them individually to determine their initial mass using an analytical weighing balance. After weighing, we immersed both sets of samples in 30 ml of artificial urine and placed them in an incubator at 100 rpm for up to 20 days at 37 °C. At specific time intervals, we removed the samples from the artificial urine, dried them at 60 °C, and weighed them to check for any weight loss compared to their initial masses.

Additionally, to assess their durability under shear stress, we cut the samples into 2 cm lengths and weighed them individually to determine their initial mass using an analytical weighing balance. After weighing, we placed all the samples separately in 2 ml eppendorf tubes containing 1.5 ml of artificial urine. Each set of samples was then spun at 1500, 3000, and 6000 rpm for 2 min, and we monitored any weight loss.

The percentage of weight lost by the antimicrobial coating films after being incubated in artificial urine at 100 rpm for 20 days.

The percentage of weight lost by the antimicrobial coating films after being spun in artificial urine for 2 min at 1500, 3000, and 6000 rpm.

The purpose of the durability testing was to evaluate how effectively the antimicrobial coating films adhered to the surfaces of silicone and latex Foley catheters when exposed to an aqueous system. To do this, an artificial urine was utilized as a representative aqueous system, considering the commercial application of these catheters in assisting urination. After 20 days of incubation in artificial urine, the coated films on both silicone and latex Foley catheters exhibited weight losses of 1.8 % and 2 %, respectively. Additionally, we performed durability tests to evaluate the ability of coating films to maintain adherence under shear stress. The coating films on silicone and latex Foley catheters showed minimal weight losses of 0.6 % and 0.2 % at 3000 rpm and the highest weight losses of 1.8 % and 3.2 % at 6000 rpm, respectively These minimal weight losses in the coating films after 20 days in artificial urine and exposure to shear stress confirm the robust and stable bonding between the cross-linked polyurethane network and the substrate surfaces, which holds silver nanoparticles in place for long-term antimicrobial effectiveness. In summary, these findings further highlight the durability of the antimicrobial coating films on silicone and latex Foley catheters.

Example 9

Adhesion test of coating films (Fig 9) An adhesion test was performed on the antimicrobial coating films applied to silicone and latex Foley catheters. This test involved slight modifications to an X-cut method described in standard guideline (ASTM D3359-09). A Medigrip adhesive tape USP was utilized to measure the force required to peel off the coating films. Briefly, one end of the Medigrip tape was fixed firmly to the intersection of the X-shaped cut on the coating films and the other end of the tape was attached to a universal testing machine. Subsequently within 90 sec of applying the tape, we rapidly removed it by pulling the machine up at an angle of approximately 180°. At the end, the X-cut area on the coating films was inspected to check if any of the coatings had been removed from the catheter surfaces.

The purpose of adhesion test was to determine how effectively the antimicrobial coating films adhered to the surfaces of the silicone and latex Foley catheters when subjected to the applied peel- off force. The results revealed that there was no removal or peel-off of the coating films from the surfaces of the silicone and latex Foley catheters, even when subjected to forces of 60 and 50 gm/cm, respectively.

These findings suggest that the coatings are durable because they demonstrate strong adhesion between the cross-linked polyurethane network and the substrate surfaces, which holds silver nanoparticles in place for long-term antimicrobial effectiveness.

Example 10

Durability of inner side coating (Fig 7)

Considering the extended potential for commercial use of silicone Foley catheters in aiding urination over the long term, it becomes crucial to assess the durability of the inner side coating on these coated silicone catheters. The durability testing of the inner side coating of the silicone Foley catheter was performed by making slight modifications to the in-vitro bladder model previously described by Wang et al. in 2019. Briefly, the coated silicone Foley catheter was fixed within an in-vitro bladder model filled with artificial urine. The inner side coating was then exposed to a steady flow of artificial urine at a rate of 0.5 ml/min for 40 days. Throughout this period, we regularly replenished the bladder with fresh artificial urine to maintain a continuous flow through the inner side of the coated catheter. After completing the experiment, the coated catheter was removed from the in-vitro bladder model and dried it at 60 °C for 1 h. Following the drying process, the catheter was sliced longitudinally to examine the inner side coating for any changes using a scanning electron microscope.

The purpose of conducting the durability test on the inner side coating was to assess how effectively an antimicrobial coating film adhered to the inner side surface of silicone Foley catheter when exposed to a continuous flow of artificial urine for a period of 40 days. SEM image confirmed the presence of a fully intact antimicrobial coating on the inner side of the silicone catheter, even after enduring a continuous urine flow for 40 days. However, we noticed that the observed coating had a somewhat less porous polymer structure compared to the coating before exposure to artificial urine. The decreased porosity in the 40-days treated coating film could be attributed to the retention of salts from the artificial urine. Nevertheless, the prolonged adhesion of the coating film to the inner surface of the silicone catheter after a continuous 40-days urine flow confirms the durability of the inner side coating.

Therefore, the results discussed in examples 8, 9, and 10 collectively validate the durability of antimicrobial coating films produced using an optimized antimicrobial coating formula on silicone and latex surfaces.

Example 11

Mechanism of bonding of coating films with the substrate surfaces (Fig 10)

There have been a few reports on the use of NCO-terminated polyurethane prepolymer along with amino functional cross linkers forming a covalently bonded network to create durable coatings on medical devices. However, this invention introduces a novel approach, utilizing water -based polyurethane without -NCO terminals as the supporting polymer and ethylenediamine as the crosslinker. This method results in an optimized polyurethane network formed through ion-pairing (ionic bonding) instead of covalent bonding to produce the desired antimicrobial coatings. In this process, the carboxylate (COO ) ions from the pendant carboxylic acid functional groups within the polyurethane polymer form a stable and desired polymer network through ion-pairing with the ammonium (NH3 ⁺ ) ions from the cross-linker. The ionic bonding between the carboxylate (COO ) ions and the ammonium (NH3 ⁺ ) ions was confirmed using FT-IR spectroscopic analysis of the ingredients in the optimized antimicrobial coating composition.

The actual ionic cross-linking process occurs during the curing step of an antimicrobial coating method. In this step, a coating composition applied to substrate surfaces is heated to approximately 60 to 80 °C for 30 min. The polyurethane dispersion within the coating composition is a base salt of triethylamine, formed through the interaction between the carboxylic acid group of polyurethane and the amine group of monofunctional triethylamine. This results in having one monofunctional and one bifunctional amine (ethylenediamine) available in the coating composition during the curing process. When the coating composition is heated during curing, triethylamine dissociates from the carboxylic groups and evaporates due to its lower boiling point (89.28 °C) compared to ethylenediamine (116 °C). Meanwhile, the higher boiling point of ethylenediamine causes minimal loss through evaporation, allowing it to remain available for ion pairing with the dissociated carboxylic groups. This forms an optimized cross-linked polyurethane network. A similar ionpairing reaction between the carboxylic group of polyurethane and hexamethoxymethyl melamine has been reported previously to demonstrate the effect of the neutralizing amines on the curing of polyurethane dispersions (Mequanint et al., 2003).

The bonding of the coating films occurs in a series of steps on the substrate surfaces. First, a preliminary primer layer is applied, which attaches to the substrate surfaces and leaves the outer side epoxy functional group accessible on the surface after the curing step. Second, the secondary antimicrobial coating layer is applied, creating an optimized cross-linked network through a combination of both covalent and ionic bonding. This happens by covalently linking one end of ethylenediamine's terminal amine group to the epoxy functional group provided by the primer and simultaneously ion pairing the other terminal amine group of ethylenediamine with the pendant carboxylic groups of polyurethane. Lastly, the pendant carboxylic groups of polyurethane can further create an additional polyurethane network by ion pairing with both terminal amine groups of ethylenediamine.

This unique combination of covalent and ionic bonding led to stable coatings on the substrate surfaces. These coatings securely hold silver nanoparticles in position, enabling their gradual release for long-term antimicrobial effectiveness. Furthermore, when combining polyurethane dispersion having no -NCO terminal and bifunctional amines as a cross linker, it resulted in an antimicrobial coating composition with a long pot life. This is because there are no covalently reactive functional groups in the composition that would cause it to cure at room temperature and thus avoids significant loss.

Example 12

Antimicrobial activity tests

(a) Antibacterial activity by zone of inhibition assay (Fig 1)

We conducted a zone of inhibition assay to assess the antibacterial effectiveness of antimicrobial- coated substrates against multi-drug resistant E. coli. Since this bacterium is a common cause of urinary tract infections, we used a clinical isolate of multi-drug resistant E. coli in the assay. In this assay, we vertically placed approximately 1.5 cm long pieces of both the antimicrobial-coated and plain (uncoated) substrates in sterile petri plates containing Mueller-Hinton agar and E. coli. These plates were then incubated at 37 °C for 18 h. After this incubation period, the coated samples of latex Foley catheter, silicone Foley catheter, endotracheal tube (polyvinyl chloride), and central venous catheter (polyurethane) displayed substantial zones of inhibition, measuring 13, 9, 13, and 6 mm, respectively. In contrast, the uncoated substrates showed no zones of inhibition after the 18 h incubation. These results confirm that the coatings effectively release silver nanoparticles, which in turn eliminate multi-drug resistant bacteria.

(b) Long-term antibacterial activity testing by zone of inhibition assay:

Zone of inhibition measurements for an antimicrobial coated latex Foley catheter that was immersed in water for a period of 15 days.

We conducted a long-term antibacterial test on the coated latex Foley catheter to assess its sustained antibacterial effectiveness against multi-drug resistant E. coli, a common cause of urinary tract infections. We used a zone of inhibition assay for this assessment. In this assay, we took 1.5 cm long pieces of both the coated and uncoated latex Foley catheter and immersed them in distilled water for up to 15 days at room temperature. At predetermined intervals, we removed the samples from the water, dried them at 60 °C for 15 min, and then placed them vertically in sterile petri plates containing Mueller-Hinton agar and E. coli. These plates were incubated at 37 °C for 18 h. The results showed that the coated latex catheter samples, which had been immersed in distilled water for 1, 2, 3, 7, and 15 days, displayed prominent zones of inhibition measuring 11.5, 11, 11.5, 11, and 9.5 mm, respectively. Conversely, the uncoated catheter samples showed no zones of inhibition throughout the entire experiment. These findings confirm that the coatings are capable of retaining ammonium methacrylate copolymer-modified silver nanoparticles for an extended period when exposed to water. These results also validate the long-term antibacterial potential of the coatings on the latex Foley catheter against multi-drug resistant bacteria. This sustained antibacterial activity can be attributed to the strongly bonded coatings with the substrate surfaces, which keeps the ammonium methacrylate copolymer-modified silver nanoparticles in place and allows for the gradual release of silver ions through the water swelled ammonium methacrylate copolymer when in contact with water.

(c) Long-term antibacterial activity testing by biofilm extraction method (Figure 8)

Table 3. The data showing number of colonies and the percentage reduction in bacterial load after 40 days of bacterial exposure to the inner surfaces of both coated and uncoated silicone catheters.

Samples tested Dilution Number of CFU/ml Reduction factor bacterial (%) colonies

Uncoated silicone 1 (f 9 9 x 10 catheter

Antimicrobial 10 ⁶ 22.5 2.25 x 10 ⁸ 75 % coated silicone catheter

Considering the extended potential for commercial use of silicone Foley catheters in assisting urination over the long term and the significant issue of catheter-associated urinary tract infections, it becomes important to evaluate the long-term antibacterial activity of the inner side coating on these coated silicone catheters. The long-term antibacterial testing of the inner side coating of the silicone Foley catheter was performed by making slight modifications to the in-vitro bladder model previously described by Wang et al. in 2019. In detail, the coated and uncoated silicone Foley catheter were fixed separately within the in-vitro bladder models filled with artificial urine. The inner side coating was then exposed to a steady flow of non-sterile artificial urine at a rate of 0.5 ml/min for 40 days. Non-sterile work environment and artificial urine were used intentionally in the experiment to induce contamination as no bacterial culture was added to the in-vitro bladder model. After completing the experiment, we removed both catheters from the in-vitro bladder model and cut 1 cm pieces from each catheter. These pieces were washed gently with sterile IX PBS and transferred to fresh tubes containing 1 ml of solution of IX PBS with Tween 20 (0.01% w/v). We then vortexed the tubes for 1 min each to extract the cells attached to the catheters in the form of biofilms. The suspensions were serially diluted using IX PBS, and dilutions of 10 ⁶ and 10 ⁷ for the coated and uncoated samples, respectively were plated on sterile nutrient agar plates in duplicates. These plates were incubated for 48 h at 37°C. Additionally, we stained the bacterial cells extracted from the coated and uncoated samples using gram staining to visualize any biofilm formation on the inner side surfaces under an optical microscope.

After 40 days of exposure to bacteria in a non-sterile environment, the number of colonies obtained indicated a higher bacterial load on the inner side surface of the uncoated silicone catheter (9 x 10 ⁸ CFU/ml) compared to the coated silicone catheter (2.25 x 10 ⁸ CFU/ml) (Table 3). This confirms a reduction of 75% in bacterial growth on the inner side surface of the coated silicone catheter due to the antimicrobial coating, in comparison to the uncoated silicone catheter. Furthermore, optical microscope images of stained samples captured at 400X magnification supported the quantitative results for both samples. In the uncoated sample, there were continuous and highly concentrated violet-colored regions, suggesting the presence of biofilm on the inner side layer. In contrast, the coated sample displayed very few violet-colored regions, indicating the absence of biofilm formation and minimal bacterial presence on the inner side coating.

These findings affirm the long-term antibacterial potential and the ability to prevent biofilm formation in the inner side coatings of silicone Foley catheters.

(d) Time kill assay of the coated latex and silicone catheters:

Table 4. The time kill assay data showing colony forming units (CFU/ml) at various time intervals for the various samples tested.

Samples tested Colony forming units (CFU/ml) 0 min I h 3 h 6 h 24 h

Coated silicone catheter 6 x 10 ⁴ 0 0 0 0

Coated latex catheter 7 x 10 ⁴ 1 x 10 ⁴ 0 0 0

Positive control silicone 1 x 10 ⁵ TNTC TNTC TNTC TNTC catheter

Positive control latex 1.2 x 10 ⁵ 5 x 10 ⁴ 1 x 10 ⁴ l x 10 ⁴ 0 catheter

Uncoated silicone catheter 3 x 10 ⁵ l.l x lO ⁵ TNTC TNTC TNTC

Uncoated latex catheter 3 x 10 ⁴ 2.6 x 10^ TNTC TNTC TNTC

Culture control 9 x 10 ⁴ 8 x 10 ⁴ 2.27 x 10 ⁶ TNTC TNTC

Note: TNTC = Too numerous to count

We performed a time kill assay on coated silicone and latex Foley catheter samples to determine how long it takes to release the minimum bactericidal concentration of silver from the coated films. In this assay, we compared these coated samples to commercially available positive control coated samples of silicone and latex Foley catheters, and uncoated (plain) samples of silicone and latex Foley catheters as negative controls. We used clinical isolates of multi-drug resistant E. coli, a common cause of urinary tract infections, for the testing. The time kill assay for all the samples was performed using 0.5 % Mueller-Hinton broth (MHB). The samples were cut into 2 cm pieces and sterilized them using UV light. Each catheter piece was placed in an eppendorf tube containing 1 ml of MHB inoculated with an overnight-grown bacterial culture to achieve a final concentration of approximately 10 ⁵ CFU/ml. The tubes were then incubated at 37 °C with periodic sampling at intervals of 0, 1, 3, 6, and 24 h. Appropriate dilutions were spread plated on sterile Luria Bertini agar plates and incubated at 37 °C for 24 h to obtain the colony forming unit (CFU/ml).

After incubating all the samples for 24 h, as indicated in Table 4, only the coated silicone catheter showed no bacterial growth at all the tested time intervals (1, 3, 6, and 24 h). In contrast, the coated latex catheter had 1 x 10 ⁴ CFU/ml after 1 h, but it did not show any bacterial growth during the remaining testing periods (3, 6, and 24 h). Among the other samples, the commercially available positive control latex catheter exhibited no bacterial growth only after completing 24 h. In summary, the data in Table 4 confirms the following outcomes: (i) The coated silicone catheter released a minimum bactericidal concentration of silver, exhibiting bactericidal activity from 1 h onwards up to 24 h. (ii) The coated latex catheter released a silver concentration that initially managed bacteriostatic activity up to 1 h, followed by a release of the minimum bactericidal concentration of silver, exhibiting bactericidal activity from 3 h onwards up to 24 h. (iii) the commercially available positive control latex catheter showed bacteriostatic activity from 1 h onwards up to 6 h and exhibited bactericidal activity only after completing 24 h. (iv) All other samples, including the uncoated silicone catheter, uncoated latex catheter and the commercially available positive control silicone catheter, did not demonstrate antibacterial activity at any of the tested time intervals.

These findings confirm that the antimicrobial coatings developed in this invention are effective at preventing bacterial growth when compared to both the uncoated samples and the infection prevention coating samples available in the market.

Therefore, the results discussed in examples 12a, 12b, 12c and 12d collectively validate the longterm functionality of antimicrobial coating films produced using an optimized antimicrobial coating formula on silicone and latex surfaces.

Brief Description of the drawings

Figure 1 describes Zone of inhibition measurements for an antimicrobial coated latex Foley catheter that was immersed in water for a period of 15 days.

Figure 2 describes Scanning electron microscopic (SEM) images of uncoated surface of silicone Foley catheter.

Figure 3 shows Scanning electron microscopic (SEM) images of uncoated surface of latex Foley catheter

Figure 4 shows Scanning electron microscopic (SEM) images showing curvature of uniformly coated surface for coating film thickness Figure 5 shows Scanning electron microscopic (SEM) images showing curvature of uniformly coated surface.

Figure 6 shows scanning electron microscopic (SEM) images showing distribution of an ammonium methacrylate copolymer modified silver nanoparticles within the crosslinked network of polyurethane of antimicrobial coating film.

Figure 7 Scanning electron microscopic (SEM) image of the inner side coating of silicone Foley catheter exposed to a continuous flow of artificial urine for 40 days.

Figure 8 shows biofilm formation was examined using gram staining and microscopic observation for both uncoated (a and b) and coated (d and e) samples, and quantifying the bacterial colonies by counting for both uncoated (c) and coated (f) samples.

Figure 9 shows Optical microscopic images of the antimicrobial coatings on the surfaces of silicone (a) and latex (b) Foley catheters after they were turned and twisted.

Figure 10 shows FT-IR spectra of plain polyurethane dispersion (a), plain ethylenediamine (Yang et al., 2017) (b), physical mixture (c) and cured antimicrobial coating film (d).

Detailed Description of Drawings

Figure 1 describes after this incubation period, the coated samples of latex Foley catheter, silicone Foley catheter, endotracheal tube (polyvinyl chloride), and central venous catheter (polyurethane) displayed substantial zones of inhibition, measuring 13, 9, 13, and 6 mm, respectively. In contrast, the uncoated substrates showed no zones of inhibition after the 18 hours incubation. These results confirm that the coatings effectively release silver nanoparticles, which in turn eliminate multidrug resistant bacteria.

Figure 2 further shows structural uniformity of the coated antimicrobial film on the silicone and the latex Foley catheter surfaces. SEM images of the coated silicone Foley catheter show a continuous, uniform, and porous antimicrobial coating without any uncovered areas. The pore sizes in this film ranged from 0.1 to 1.8 pm (Fig.2)(b). The images also clearly reveal the boundary between the coated and uncoated sections of the silicone Foley catheter (Fig.2)(c). Figure 3 further explains SEM images of the coated latex Foley catheter similarly show a continuous and uniform antimicrobial coating layer with no uncovered areas. However, the coating on the latex surface was found to be less porous compared to the silicone catheter (Fig.3)(b). SEM analysis also clearly distinguishes between the coated and uncoated sections of the latex Foley catheter (Fig 3)(c).

Figure 4 and Figure 5 further explains an antimicrobial coating film thickness analysis was performed to measure the thickness and uniformity of the inner and outer antimicrobial coating films on both silicone and latex Foley catheter surfaces using the scanning electron microscopy. SEM images in Fig. 4 and Fig. 5 confirmed that there are continuous and uniform antimicrobial coating films along the periphery of both silicone (Fig. 4)(a) and latex (Fig. 5)(a) Foley catheters. The SEM analysis also showed that there is uniform antimicrobial coating on the inner surfaces of silicone (Fig 4)(b) and latex (Fig 4)(b) Foley catheters, with thicknesses of about 1.5 pm and 1 pm, respectively. The uniformity of the outer coating towards the upper and lower portions of the catheters was also examined. For silicone Foley catheters, the thickness of the outer coating film was consistent, measuring between 3-5 pm (Fig 4)(c) (d). Similarly, for latex Foley catheters, the outer coating film had a consistent thickness in the range of 4-6 pm (Fig 5)(c)(d).

Figure 6 talks about SEM images of the antimicrobial coating film verified the presence of an ammonium methacrylate copolymer modified silver nanoparticles distributed within the crosslinked polyurethane network. These nanoparticles appeared spherical and measured between 120 to 230 nm in size. The particle size was also confirmed through dynamic light scattering experiments, which yielded a consistent result of 148 nm, aligning with the SEM analysis.

Figure 7 further explains the purpose of conducting the durability test on the inner side coating was to assess how effectively an antimicrobial coating film adhered to the inner side surface of silicone Foley catheter when exposed to a continuous flow of artificial urine for a period of 40 days. SEM image confirmed the presence of a fully intact antimicrobial coating on the inner side of the silicone catheter, even after enduring a continuous urine flow for 40 days.

Figure 8 further describes the coated and uncoated silicone Foley catheter were fixed separately within the in-vitro bladder models filled with artificial urine. The inner side coating was then exposed to a steady flow of non-sterile artificial urine at a rate of 0.5 ml/min for 40 days. Non- sterile work environment and artificial urine were used intentionally in the experiment to induce contamination as no bacterial culture was added to the in-vitro bladder model.

After 40 days of exposure to bacteria in a non-sterile environment, the number of colonies obtained indicated a higher bacterial load on the inner side surface of the uncoated silicone catheter (9 x 10 ⁸ CFU/ml) compared to the coated silicone catheter (2.25 x 10 ⁸ CFU/ml). This confirms a reduction of 75% in bacterial growth on the inner side surface of the coated silicone catheter (Fig.8)(f) due to the antimicrobial coating, in comparison to the uncoated silicone catheter (Fig.8)(c). Furthermore, optical microscope images of stained samples captured at 400X magnification supported the quantitative results for both samples. In the uncoated sample, there were continuous and highly concentrated violet-colored regions, suggesting the presence of biofilm on the inner side layer (Fig.8) (a) and (b). In contrast, the coated sample displayed very few violet-colored regions, indicating the absence of biofilm formation and minimal bacterial presence on the inner side coating (Fig.8) (d) and (e).

Figure 9 further describes in addition to the standard adhesion test, we used a non-standard qualitative method to assess how well the coatings adhered to the surfaces of silicone and latex Foley catheters. In this approach, we turned and twisted the coated samples, then observed them under an optical microscope to look for any signs of microcracking or the removal of coating films. As shown in Fig. 9, the optical microscope images confirmed that there were no microcracks or removed coating films on the surfaces of the silicone and latex Foley catheters.

Figure 10 further explains FT-IR spectrum of the plain polyurethane dispersion displayed a carbonyl stretching peak from the pendant carboxylic group at 1725 cm ¹ . In contrast, the FT-IR spectrum of plain ethylenediamine exhibited absorption peaks at 3352 and 3276 cm ¹ , corresponding to the asymmetric and symmetric stretching of NH2 groups, respectively. The FT- IR spectrum of the physical mixture of polyurethane dispersion, ethylenediamine, PVP K90, and glycerol showed combined peaks at 1725 cm' ¹ (carbonyl stretching of the pendant carboxylic group from polyurethane dispersion) and 3356 cm' ¹ (asymmetric stretching of NH2 groups) and 3293 cm' ¹ (symmetric stretching of NH2 groups) from ethylenediamine. Nevertheless, in the FT- IR spectrum of the cured antimicrobial coating film (Fig. lOd), a notable absorption peak at 1725 cm' ¹ was observed, while there were no absorption peaks at 3356 and 3293 cm ¹ . This suggests that the NH2 groups of ethylenediamine formed ionic bonds with the pendant carboxylic groups of polyurethane, resulting in the creation of carboxylate (COO ) ions and ammonium (NH3 ⁺ ) ions through ion-pairing. Therefore, the characteristic absorption peak observed at 1725 cm' ¹ in the FT- IR spectrum of the cured antimicrobial coating film corresponds to the carbonyl stretching of the carboxylate (COO ) ions in the ion pair formed.

Advantages of the present invention

1. The antimicrobial coating formulations of the present invention keeps the medical devices surfaces antimicrobial once coated with the formulation and hence reduces the deviceassociated infections or hospital acquired infections.

2. The reduced rate of device-associated infections or hospital acquired infections results in reduced morbidity and mortality, improved patient compliance, improved quality of life.

3. The use of any medical device coated with the antimicrobial coating formulation of the present invention eliminates the additional hospital stays and extra financial burden on patients.

4. Apart from this, the appropriate combination and quantities of the selected components in the antimicrobial coating composition provides the uniform, long term functional antimicrobial coatings on various medical devices.

5. Also, as compared to the commercially available antimicrobial coatings, the developed antimicrobial coating composition involves the use of novel inorganic colloidal silver nanoparticles instead of commercially available antibiotics which avoids the global problem of bacterial resistance and effectively killing the existing resistant bacterial strains.

6. Also, the developed antimicrobial coating composition is suitable for any type of coating method and does not involve any harsh conditions in the curing process.

7. Also, the developed antimicrobial coating composition has long pot life and therefore can be utilized up to 1 month for effective antimicrobial coating on medical devices.