Evaluation of new polymer-iodine complexes for the fabrication of medical devices

Povidone-iodine has been a true success story in the fight against infections by harnessing the antimicrobial and antiviral properties of elemental iodine for water-based applications. However, to date there has been little success in implementing iodine attributes in water-insoluble engineering plastics. Here, we describe the first development of biocidal active polyamide- and polyurethane-iodine complexes at laboratory and commercially relevant scales. These polymer-iodine materials are active against a broad range of microorganisms, including bacteria, yeast and fungi, and can be used as base materials for medical devices. The use of new polymer-iodine complexes for infection prevention in medical devices, such as sutures, catheters and drains, or wound care is expected to have significant positive effects at reducing healthcare-acquired infections. In addition, the materials are expected to find significant applications in other fields, such as air handling with the production of biocidal face masks and air filters to control spread of pathogens.


Introduction
Healthcare-acquired infections (HAI's) continue to be a source of countless unneeded deaths and excessive hospital costs. The main sources of HAI's are pathogenic microorganisms that have entered the body due to contamination and infection, or due to imbalances in the normal microbiota (i.e. dysbiosis) as a consequence of specific treatment conditions or weakened immune defences. Often the HAI forms at the site of a medical device (e.g. a central line catheter) that causes introduction of unwanted microorganisms at a specific body site or becomes the scaffold for unwanted adhesion of bacteria already carried by the patient. In fact, medical devices are a significant source of HAI's and represent a serious concern in many clinical settings [1][2][3] . Microbial infections caused by the use of medical devices, such as sutures, catheters, central lines and prostheses result in secondary infections that are often difficult to eradicate and compromise the well-being of the patient. Medical devices provide a perfect nidus for microbial adherence and subsequent formation of biofilms, which are very difficult to eradicate. For these reasons, there is an urgent need to develop and evaluate new materials with biocidal properties in order to reduce the risk of medical device contamination and microbial attachment to said medical devices.
Severe infections may result in removal of medical devices, tissue debridement or, at worse, patient death. Such HAI's not only worsen patient health, but also increase healthcare costs significantly. In particular, catheters are the most common implants worldwide, with 5 million central venous catheters (CVC) and 30 million urinary catheters being implanted per year, in the USA alone. Both catheter types have been identified as two of the leading origins of HAI's.
The Centers for Disease Control (CDC) reported that approximately 250,000 CVC-related bloodstream infections (BSI) occur each year in the USA at an additional hospitalization cost of ca. $34,500-$56,000 per each infection. Not only is there a huge economic cost, but the CDC estimates that 4% of patients in the USA will contract a HAI during hospitalization, which will result in 1.7 million infections and 99,000 associated deaths.
Further compromising the treatment of HAI's is the development of "superbugs", bacteria that have developed resistance to common antibiotics and medications. Not only are healthcare institutions the major source of such "superbugs", but the people they treat are very young, immunocompromised, elderly and sick, making them the most vulnerable group. The World Health Organization (WHO) has identified antimicrobial resistance as one of the global health threats of the 21 st century with estimated casualties amounting to 10 million people/year at a cost of $100 trillion by 2050 if no developments on controlling this health crisis are found.
The water-soluble iodophor complex, povidone-iodine (PVP-I), is a potent broad-spectrum antimicrobial active with excellent antiseptic properties against bacteria, including methicillinresistant Staphylococcus aureus (MRSA) and mycobacteria [4] , fungi [5] , protozoa [6] and both enveloped and non-enveloped viruses, such as the poliovirus, immunodeficiency viruses [6,7] , influenza viruses [8,9] and coronaviruses: SARS [10] , MERS [11] and SARS-CoV-2 [12] . Topical formulations of PVP-I have been widely used over the years for disinfection, wound care or treatment of burns [8] and, despite its common use for more than 60 years, no microbial resistance to PVP-I has ever been reported [5] . However, the PVP-I complex is water soluble and thus its applications in healthcare are generally limited to surface and skin disinfection to minimize the risk of infections.
The research question we addressed in the present study was whether it would be possible to develop water-insoluble iodine-polymer complexes that have similar antimicrobial properties as PVP-I, are relatively simple to manufacture, and can be used for actual medical device fabrication to reduce the risk of infections. More importantly, such new water-insoluble iodinepolymer complexes with biocidal activity could play an important role in reducing the risks of HAI's in medical devices, equipment and articles by reducing the risk of undesirable microbial contamination and inhibition of microbial adhesion leading to biofilm formation. Therefore, iodine complexes of two polymer families, polyamides (PA) and polyurethanes (PU), were manufactured and the biocidal properties of tubes and foams of PA-and PU-iodine complexes were tested against some of the most commonly found and clinically relevant microorganisms.
In brief, we show that the described polymer-iodine complexes can be simply produced by utilizing existing equipment and processes already used to make the corresponding non-iodine polymer materials, and that they have strong bactericidal and fungicidal activity.

Production of polymer-iodine complexes
Polyamide-Iodine (PA-I) thermoplastic complexes were produced by direct extrusion of the base PA polymer with a suitable iodine source in a twin-screw extruder. The extrusion conditions were dependent on the base polyamide used and the ease that the iodine source is dissolved into the molten PA matrix to generate the PA-I complex. General extrusion temperatures were in the 150-240 °C range, resulting in PA-I complexes that were homogeneous and stable.
Polyurethane-Iodine (PU-I) thermoplastic complexes were produced by direct extrusion of the base PU with a suitable iodine source in a twin extruder. Extruding temperatures of <200 °C were generally effective to generate homogeneous and stable PU-I thermoplastics. PU-I thermosets (cross-linked polymeric matrixes) were produced by first dissolving the suitable iodine source in one of the raw materials for PU fabrication, primarily the polyol or diol raw material. The PU reaction was then conducted as usual to produce the PU-I thermoset.
Suitable iodine sources used for the complexation reactions included elemental iodine (I) and povidone-iodine (PVP-I).
The resultant extruded thermoplastic PA-and PU-I tubes were directly tested for antimicrobial activity. The PU-I thermosets (foams) were allowed to cure and then cut into the desired shape for subsequent antimicrobial testing.

Inhibitory effects of thermoplastic PA-and PU-I (P-I) tubes on the growth of various microorganisms
To evaluate the potential growth inhibitory effects of P-I thermoplastic complexes on microorganisms, tubes composed of PA or PU extruded with PVP, PVP-I and/or iodine at various iodine concentrations ( Table 1) were tested. Eight of the tested tubes (i.e., numbers 3, 4, 6, 14, 15, 16, 17 and 19) showed large clearing inhibition zones for S. aureus (Figure 1).
Tubes 1, 2, 11 and 18 containing lower iodine concentrations showed more localized biocidal activity, with the clearing zones mainly apparent at the point of direct tube contact.   Based on the test results in Figure 1, two main observations can me made: (1) the size of inhibition zones is related to the total iodine loading. Generally, higher iodine loading will lead to increased activity. However, by observing and comparing tubes 6, 7, 8 and 9 that all possess an iodine loading of 5% a 2 nd observation can be made, (2) the compositional makeup of the PA-I complex is also important to determining biocidal activity.

Inhibitory effects of PA-I tubes following sterilization
To evaluate the possibilities for re-use of the different materials, a potential decrease of the inhibitory effect following sterilization was tested. For both strains of S. aureus (MRSA and MSSA), the formation of halos reflecting growth inhibition was still observed upon autoclaving (121 O C, 15 psi, 15 min) (Figure 3). This observation also strongly supports the view that antibacterial activity for the iodine containing materials will be observed over an extended time period, which is an important factor for medical devices that need to remain in contact with the body for extended periods of time (e.g. wound dressings, urinary catheters, drains, etc.).

Inhibitory effects of PU-I foams on the growth of various microorganisms
To   Based on the positive results from various lab-produced PU-I foams, new development foams, both reticulated and non-reticulated, were produced at pilot relevant scale (39-43, Table 1) and further evaluated against A. fumigatus (Figure 6). Due to the lower iodine loading in these foams, the fungal inhibition halo under the foams was evaluated to determine activity.
Noteworthy is the observation that the fungicidal activity reflected by the clearing zones seems less related to the iodine levels, but more to the actual contact of the foams with A. fumigatus

Inhibitory effect of PU-I foams on saliva samples from healthy volunteers
Due to the current great need of face masks, the developed foams were further evaluated for inhibition of oral microorganism. Freshly prepared and identical autoclaved foams 39 to 43 ( Table 1) were incubated with saliva from healthy volunteers for 1 or 7 days at room temperature. Subsequently, the foams were replica-plated by pressing onto blood agar plates.
Foams 40 to 43 showed a clear decrease in the microbial load compared to the control foam (39) after 1-day incubation of the saliva-inoculated material (Figure 7A-B). After 7 days incubation, the inhibitory effect was nearly 100% for the non-autoclaved foams (Figure 7C-D). Upon autoclaving, the foams retained their biocidal activity to varying extents.

Inhibitory effect of commercially produced PU-I face masks on microbial growth
Based on the above positive results, a commercial batch of reticulated foam 44 was produced 14 at a batch size of 1500 kg in order to show the technology was scalable and commercially viable.
The resultant high-quality foam was tested as a potential component for the development of face masks with microbial inhibitory properties. These face masks were worn for 3 h by healthy volunteers. Subsequently, the side of the foam that was in contact with the volunteer's face was pressed on a blood agar plate to assess the bacterial load. As shown in Figure 8, the microbial load of face masks with foam 44 was significantly decreased in comparison with the control 39 (reticulated commercial foam with no biocidal activity).

Discussion
Considering the impact of HAI's in many clinical settings, developing medical devices with inherent properties against bacterial contamination and biofilm formation is of great importance. Many studies are focused on the evaluation of coating agents, such as polymer coatings, antimicrobial coatings or nanostructured coatings, to antagonize the first adherence of microorganisms and, thus, inhibit or slow down biofilm formation [13] . From such studies, the use of antiseptic-based coatings was shown to be more effective when compared to the use of antibiotics-based coatings [13,14] . Moreover, the local delivery of antiseptics was successful when implemented in coatings for catheter-based applications [15,16] . However, antiseptics often pose an unknown risk once placed in the body due to their synthetic and toxic nature.
Iodine is a naturally occurring element shown to have broad antimicrobial and antiviral activity, but it is also an essential mineral nutrient. Iodide salts are often added to foods and supplements to ensure good human health. Elemental iodine, itself, shows poor water solubility, is corrosive and readily sublimes (unstable physical form), which has limited its use as a biocide until the discovery of the povidone-iodine complex, the active material in Betadine Ò . The povidoneiodine complex is: stable, water-soluble, highly active against a broad range of microorganisms and viruses, and safe for human use. As such, the povidone-iodine material has become an essential tool for the prevention of infections with broad use in both institutional and public healthcare. Though povidone-iodine represents a true success story for harnessing the antimicrobial and antiviral properties of iodine for water-based applications, there has been relatively limited success in harnessing said iodine attributes in water-insoluble engineering plastics that could be used for the fabrication of various medical devices, and healthcare and consumer products.
In the present study, we show that the excellent antiseptic properties of water-soluble povidoneiodine, can be similarly realized in water-insoluble, engineering plastics, specifically PA-and suggesting that the polymer-iodine systems can be further customized to meet specific requirements for a particular medical need. Moreover, we show that the biocidal properties of the polymer-iodine complexes included in this study are largely maintained upon sterilization, which strongly supports that the materials can be re-used or will maintain their biocidal activity for an extended period of time. Thus, these polymers have the extra advantage of a low environmental footprint, since they can be recycled or allowed to remain in use for longer periods of time.
In the case of PU-I foams, we show that the technology is readily scalable and that commercial production of both non-reticulated and reticulated foams with biocidal activity is viable. In order for the PU-I foams to have broad-use applications, it is important that both non-reticulated and reticulated foams can be produced. In non-reticulated PU foams, the gas bubbles entrapped in the foam are completely closed and thus air or liquid cannot pass through the foam structure.
Such non-reticulated foams are generally used for their cushioning/insulation properties, but cannot be used in filters. To make the PU foam acceptable for filter applications, the foam must be reticulated, which is the process of treating the corresponding non-reticulated foam with a combustible gas or chemical to bring about the controlled degradation of the gas bubbles to produce an open and highly porous foam structure. Once the PU foam is reticulated, the foam can be used for the production of efficient filters. The present research shows that the biocidal activity observed in the non-reticulated PU-I foam is maintained after reticulation. Finally, we developed a prototype face mask with a commercially produced PU-I reticulated foam that shows strong biocidal activity against oral bacterial flora. Such reticulated foams can be implemented in both face masks and filters of air handling systems to reduce the spread of airborne pathogens.

Conclusion
In conclusion, the beneficial antimicrobial attributes of the water soluble PVP-I can be similarly  Tubes or foams with different concentrations of nylon, PVP and iodine were placed on top of the plates, and the plates were then incubated at 37 O C. Growth inhibition was inspected upon 24 and 48 h of incubation.

Microbial growth inhibition by PA-I tubes upon sterilization
To evaluate a possible decrease in the microbial growth inhibitory efficacy of the investigated materials upon standard sterilization by autoclaving, the tubes were washed once with 70 % ethanol, rinsed with distilled H2O, dried and autoclaved at 121 O C (15 min, 15 psi).

Growth-inhibiting effects of PU-I foams on microbe-containing saliva samples from healthy volunteers
To evaluate the effects of PVP-I on oral microorganisms, 1 mL saliva samples from healthy volunteers were incubated with various foams (materials 39 to 43, Table 1). After 1 or 7 days of incubation at room temperature, the foams were replica plated by gently pressing them onto Blood Agar plates (BA) for 10 s. Subsequently, the plates were incubated for 24 h at 37 O C. To ensure that all the microorganisms present in the foams were detected, the materials incubated with saliva for 7 days were also plated and kept on top of the BA plates during incubation for 24 h at 37 O C. Evaluation of the growth inhibitory effects of 1x autoclaved foams was carried out in parallel.

Microbial growth-inhibiting effects of PU-I foam mouth masks
Prototype face masks were developed with commercial PU foams containing iodine (sample 44) and without iodine (sample 39) to evaluate the activity of these foam materials on microorganisms commonly transmitted from the face and breath to face masks. Mouth masks were worn for 3 h by volunteers. Afterwards, the inside of the mask was gently pressed for 10 s on BA plates. The plates were subsequently incubated for 24 h at 37 O C. After pressing the inside and outside of the mask on BA plates, a piece of the mask was cut and incubated on top of a BA plate at 37 O C for 24 h.

Ethical approval
Studies with healthy volunteers wearing PU-I foam face masks were performed based on written informed consent with approval of the Medical Ethics Review Board of the University Medical Center Groningen (UMCG; approval no. Metc2012-375), and with adherence to the Helsinki Guidelines.