A SHOE

Cross Reference to Related Applications

This application claims the benefit of U.S. Provisional Applications No. 63/075,122, filed September 5, 2020, entitled WEARABLE PROTECTIVE LAYER TO PROTECT SOLE OF FOOT, SOCKS WHEN WORN OR SOLE OF A SHOE, No. 63/112,006, filed November 10, 2020, entitled WEARABLE PROTECTIVE LAYER TO PROTECT SOLE OF FOOT, SOCKS WHEN WORN OR SOLE OF A SHOE and No. 63/260,357, filed August 18, 2021, entitled WEARABLE PROTECTIVE LAYER TO PROTECT SOLE OF FOOT, SOCKS WHEN WORN OR SOLE OF A SHOE, the contents of the entirety of which are explicitly incorporated herein by reference and relied upon to define features for which protection may be sought hereby as it is believed that the entirety thereof contributes to solving the technical problem underlying the invention, some features that may be mentioned hereunder being of particular importance.

Identification of parties concerned

The Applicant of the present intellectual property matter is Wearable Shoe Trees, LLC of Texas, United States. The inventor(s) of the invention described in this patent documentation is/are Paul Siragusa of Texas, United States. At the time of filing, John B. Moetteli and the firm Da Vinci Partners LLC of Switzerland represent the Applicant.

Copyright & Legal Notice

A portion of the disclosure of this patent document contains material which is subject to copyright protection. The Applicant has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever. Further, no references to third party patents or articles made herein is to be construed as an admission that the present invention is not entitled to antedate such material by virtue of prior invention.

Background of the Invention

For example, at the security checks on airports, one is typically asked by the security staff to take off its shoes before passing through a security check device. Thus, one has to walk a certain distance without wearing shoes, i.e. walking that distance either barefoot or in socks. As a consequences, the socks or even the sole of foot is exposed to whatever dirt, germs, bacteria or the like is on the floor. One embodiment of the present invention provides a solution to that problem in that socks or soles of foot may be wrapped easily with a protective layer.

A similar problem constitutes when one is wearing shoes and must walks through an area where the floor is contaminated by whatever dirt, germs, bacteria orthe like and the sole of the shoe must be protected

SUBSTITUTE SHEET (RULE 26) against such dirt/germs/bacteria/.- pick-up. Another embodiment of the present invention provides a solution to that problem in that soles of a shoe may be wrapped easily with a protective layer.

Summary of the Invention

A Personal Protective Equipment (PPE) system and method and apparatus is provided which Includes a sole-shaped anti-microbial sheet made up of at least one layer having at least one adhesive backing for releasably attaching to the sole of a foot, socks or booties, or the sole of a shoe, protected with a peel-away protective layer. The sheet may advantageously include at least one tab which is not adhesively backed to facilitate removal of the sheet by gripping using the thumb and a finger and pulling away from the sole of a foot, socks or booties, or the sole of a shoe. A wearer using the sheet to protect a sole from contact with a floor surface is able to prevent dirt, germs, bacteria, etc from being picked-up by the sole of a foot, socks, or the sole of a shoe. When the user has completed their trajectory along a potentially unsanitary path, such as through the security line at an airport, they peel the sheet away, optionally returning to a bag provided with the packaging. The bag may optionally be the same bag, such as a ZIPLOK™ baggie, that the protective sheet of the Invention was contained in at the time of purchase.

Brief Description of the Drawings

The attached drawings represent by way of example, different embodiments of the subject of the invention.

FIG. 1A shows a top view of a foot placed on an embodiment of the Invention.

FIG. 1B shows a top view of an embodiment of the invention.

FIG. 2 shows a top view of an alternative embodiment of the invention.

FIG. 3A shows a perspective view of an auto shoe cover machine.

FIG. 3B shows a perspective view of wearer's feet having an embodiment of the invention applied e.g. by an auto shoe cover machine.

FIG. 4A shows a perspective view of wearer's feet having an embodiment of the invention applied e.g. by an auto shoe cover machine.

FIG. 4B shows a perspective view of wearer's feet having an embodiment of the invention applied e.g. by an auto shoe cover machine.

FIG.5 is a diagram showing a thermostability curve of ZnO and ZnOc particles optionally used in the invention.

FIG. 6 Is a diagram showing X-ray diffraction pattern of ZnO and ZnOc particles optionally used in the invention..

FIG. 7 is a diagram showing FT-IR spectrum of ZnO and ZnOc powder optionally used in the invention.

FIGs.8A to 8D are SEM micrographs of ZnO (FIGs.8A and 8C) and ZnOc powders (FIGs. SB and

SUBSTITUTE SHEET (RULE 26) 8D) optionally used in the invention.

FIG. 9 is a diagram showing WAXD spectrum of iPP and iPP / ZnOc composites optionally used in the invention.

FIG. 10 is a diagram showing UV-Vis spectrum of iPP and iPP / ZnOc samples optionally used in the invention.

FIGs. 11A to 11F are micrographs of iPP (FIGs. 11A and 11D), iPP/2%ZnOc (FIGs. 11B and HE) and iPP/5%ZnOc (FIGs. 11C and 11F) pellets fractured in liquid nitrogen optionally used in the invention.

FIG. 12 is a diagram showing thermostability curve of iPP and iPP / ZnOc composites optionally used in the invention.

FIG. 13 is a diagram showing stress-strain curves of iPP and iPP / ZnOc films optionally used in the invention.

FIG. 14 is a diagram showing the effect of time and filler content on the antibacterial activity of iPP and iPP / 2%-5% ZnOc composites optionally used in the invention.

FIG. 15 is a flow chart showing the processes of data acquisition in certain performance tests of the invention.

FIG. 16 is a chart showing the results of the performance tests of the invention.

FIG. 17 shows the floor area used in the performance test of the invention.

FIG. 18A and 18B show the shoes used in the performance tests of the invention.

FIG. 19 shows the incubation of the performance tests of the invention.

FIG. 20 is a flow chart showing the processes of data acquisition in the performance tests of the invention.

FIG. 21 is a printed protective film of the invention with instructions printed thereon.

Those skilled in the art will appreciate that elements in the Figures are illustrated for simplicity and clarity and have not necessarily been drawn to scale. For example, dimensions may be exaggerated relative to other elements to help improve understanding of the invention and its embodiments. Furthermore, when the terms 'first', 'second', and the like are used herein, their use is intended for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. Moreover, relative terms like 'front', 'back', 'top ^' and ^' bottom ^* , and the like in the Description and/or in the claims are not necessarily used for describing exclusive relative position. Those skilled in the art will therefore understand that such terms may be interchangeable with other terms, and that the embodiments described herein are capable of operating in other orientations than those explicitly illustrated or otherwise described.

Detailed Description of the Preferred Embodiment The following description is not intended to limit the scope of the invention in any way as it is exemplary in nature, serving to describe the best mode of the invention known to the inventors as of the

SUBSTITUTE SHEET (RULE 26) filing date hereof Consequently, changes may be made in the arrangement and/or function of any of the elements described in the exemplary embodiments disclosed herein without departing from the spirit and scope of the invention.

There are situations when one walks barefoot or in socks and the floor, whereat this floor might be contaminated with dirt, germs, bacteria or the like. This is typically the case at security check points at airports. For example, at a security check point, one is typically asked by the security staff to take off its shoes before passing through a security check device. Thus, one has to walk a certain distance without wearing shoes, i.e. walking that distance either barefoot or in socks. As a consequence, the socks or even the sole of foot is exposed to whatever dirt, germs, bacteria or the like is on the floor. One embodiment of the present invention provides a solution to that problem in that socks or soles of foot may be wrapped easily with a protective layer. Such layer 112 may be made of a material selected from the group of plastic materials consisting of polyethelyne (PE), Acrylonitrile butadiene styrene (ABS), General purpose polystyrene (GPPS), High impact polystyrene (HIPS), Melamine formaldehyde (MF), Polycaprolactam/Nylon (PA6), Polyarylamide (PARA), Poiybutylene terephthalate (PBT), Polycarbonate (PC), Polyether ether ketone (PEEK), Polyethermide (PEI), Polyethylene naphthalate (PEN), Polyethylene terephthaiate (PET), Poly(methyl methacrylate) (PMMA), Polyoxymethylene (POM), Polystyrene (PS), Poiysulfone (PSU), Polytetrafluoroethylene (PTFE), Styrene acrylonitrile (SAN), Polystyrene butadiene styrene (SBS), Urea formaldehyde (UF) , Epoxy, Chlorinated polyethylene (CPE), ethylene propylene diene monomer (EPDM), High-density polyethylene (HOPE), Low-density polyethylene (LDPE), Linear low-density polyethylene (LLDPE), Medium-density polyethylene (MDPE), Poluchloroprene/Neoprene (PCP), Polypropylene (PP), Polyurethane (PU), Polyvinyl chloride (PVC), Thermoplastic polyurethane (TPU), Silicone, and Rubber.

The invention offers the ability to:

1. Easy and quickly wrap a protective layer around socks or the sole of foot;

2. Provide optionally breathability and airflow though the protective layer via micro pores;

3. Where the protection layer is made up of several protective layers laminated together via a releasable adhesive backing allowing peeling away of sheets, optionally with each layer having a separate tab, the exposed protective layer can be removed via the tab while exposing a fresh layer. Note that each tab can be separately numbered to ensure each successive layer is removed.

Referring now to FIGs. 1A and 1B, a top view of a naked foot placed on an embodiment of a Personal Protection Equipment (PPE) device of the invention is shown. A naked foot 102 is placed on a first PPE device 104 according to the present invention. The first device 104 comprises a protective layer 106, at least one tab 110, and an adhesive backing 212 applied on at least one face of the protective layer 106 adapted to enable releasable adherence to the sock, bootie, foot, or shoe sole and/or sidewall. When referred

SUBSTITUTE SHEET (RULE 26) to throughout this specification, the adhesive backing 212 is not a permanent adhesive but rather it is a tacky substance such as a Pressure Sensitive Acrylate (PSA) used in a POST-IT* note that sticks to surfaces but is easily releasable or removable. Such PSA often employ 2-Ethyl hexyl acrylate, n-Butyl acrylate, or Iso-octyl acrylate and are considered soft adhesives. Note that the adhesive backing 212 may be applied via a double stick backing such as 3M 350 Series (3M 9485, 3M 9675, 3M 9731), 3M 200 Series (3M 467, 3M 468), 3M 300LSE Series (3M 9472LE, 3M 9490LE), 3M 300 Series (3M 6038, 3M 9472), 3M VHB, 100 series (3M 9473), Adchem Double Coat (254M, 256M) and Stockwell DP-1001 (Silicone double coat on polyimide film). The adhesive backing 212 may also use a same or adapted formulation of adhesive used in the aforementioned double stick backings-

In some embodiments, one adhesive backing 212, on one side of the at least one layer 106 has an adhesiveness which is lower than that applied to the other side of the layer. ^" The outwardly facing adhesive backing has a tackiness which allows non-slip and the pickup of germs and other microbes from the floor, yet is not so tacky that one cannot walk about without the device being removed from the shoe (due to being stuck to the floor), or without the wearer not being able to walk (not being able to lift and reposition his foot, shifting his weight from foot to foot). If the adhesiveness of the inwardly facing adhesive backing is lower than the outwardly facing portion, then upon walking, the device will simply pull away from the shoe, sock, bootie, or foot, which would clearly not be desirable. The outwardly facing backing then should have very low tensile resistance (even zero Is acceptable so long as it exhibits high friction) when the wearer attempts to remove his foot from the floor, yet have a high sheer resistance (or high friction when weighted by the wearer's body or leg) when the wearer tries to slide his foot on the floor.

Adhesiveness (‘Stickiness' or "tackiness") is most commonly measured with a cylinder probe, which is pressed (application of compression), onto the surface of the sample after which the force to pul! the probe off it is measured. The higher the force to separate these surfaces, the more adhesive is the adhesive used. Consequently, the tackiness of the outwardly fating adhesive backing cannot be higher than that of the inwardly fating adhesive backing, while at the same time, the tackiness of the outwardly facing backing should not result in too much adhesion when a wearer places their entire weight (or half of it, as the wearer typically has two feet, shoes, socks or booties) on the device. Nevertheless, the friction being high, there may be zero adhesion but significant friction generated when weight is on the covered shoe, sock, bootie or foot. The tensile force (upward lifting force) required to remove the device and shoe/bootie/sock or foot combination should therefore be very low, even zero. Consequently, typically, the outwardly fating backing is merely tacky and does not "stick" or adhere to the floor and thus it is at least high friction surface, but tacky enough to pick up what is on the floor in a similar manner as scotch or masking tape which is inverted and used to clean lint off of a wool sweater (albeit, in the case of the invention, the high force due to the weight of the wearer must be considered in determining the right formulation of the adhesive. As for

SUBSTITUTE SHEET (RULE 26) the inwardly facing backing, it should always allow easy peel away from the shoe, bootie, foot or sock without damaging the same, and of course have more adherence than the outwardly facing backing. Within these parameters, the selection of the appropriate range of tackiness is within the capability of the person of ordinary skill in the art as emulsions or thinners of tackifiers can be added to the formulation of the adhesive backing to yield the desired tackiness.

Note that due to the antimicrobial agents in the adhesive backing or the layer itself, when the device comes into contact with the floor, these antimicrobial agents come into contact with germs, bacteria and viruses, which kill them.

The adhesive backing 212 is preferably covered by a removable foil or film 122 which is pulled away so as to lay open the adhesive backing 212 for application against a surface. Preferably, the at least one tab 110 is an integral part of the protective layer 106, i.e. made of the same material and an extension of the main surface of the protective layer. The outer contour of the first device, optionally comprising the at least one tab 110, is preferably made by a punching process. The surface of the at least one tab 110 or the entire surface may be structured, on one or on both sides, to provide a better grip. This structure may be applied to the protective layer 106 in the area of the at least one tab 110 during the punching process. Optionally, the protective layer 106 may be perforated 114 for to provide the functionality of breathability and airflow in order to prevent the foot from sweating. The perforations 114 are located in the protective layer 106, preferably at locations where the foot or the sock is placed when worn. The perforations 114 may be applied to the protective layer 106 during the punching process. The perforations 114 may be micro pores. The perforations 114 are an optional feature and may or may not be present on the final product. ^" The device 104 may be in slight Foot- or "V-shape" 116 for easier alignment and less material wa$te{as shown in FIG. 1B}. The surface of the protective layer 106 may be on one or on both sides and be adapted to stop sliding. The protective layer 106 of the invention may have a textured or non-slip bottom to prevent slipping on slick surfaces, such as blood and wet floors. This structure may be applied to the surface of the protective layer 106 during the punching process. The protective layer 106 may comprise a pre-cut or preperforated slitting line 120. This slitting iine 120 may be applied to the protective layer 106 during the punching process.

The protective layer 106 according to the invention is made from a special plastic that is extremely strong, stretchy, durable and made with an anti-microbial material. A suitable antimicrobial film can be found on: www.silverdefender.com or www.3M.com. or the website of other suitable adhesive suppliers The protective layer 106 has a built-in antimicrobial protection. Such an antimicrobial protection can, for example, be based on silver ions that react with and affect multiple sites in bacterial cells on the surface of the layer, such to keep the layer clean. The protective layer 106 is suitable for use in high traffic areas. The protective layer may be made in form of a tape, the tape being optionally perforated, so as to facilitate

SUBSTITUTE SHEET (RULE 26) tearing away a sheet of appropriate length. Further, the protective layer may be perforated such that, after it is installed on an object such as a shoe, a foot or an insole, said perforation serves to facilitate removal of the protective layer from the object (e.g. after it has ended its useful lifespan or is no longer needed).

However, the anti-microbial effect is optional as benefits can be gained by use of the invention without anti-microbial treatment, when the device acts as a simple protective barrier between the user and a dirty surface.

In a variant of the above embodiment, the material of the protective layer 106 is manufactured with anti-microbial properties all the way through it As a result, while walking, when the sole scrapes against the ground with each step, any plastic worn off will only reveal a new layer of anti-microbial surface.

In one embodiment, the PPE device 104 according to the present invention may be installed, i.e. partially wrapped over the sole and upwardly along the sides of a naked foot 102 or a foot wearing a sock (not shown). The method for installation may be described as follows:

1. Hold the front tab 110 by its "non-stick tab" and peel around half of the backing off and secure front portion to top of the toe area.

2. Line up the PPE device 104 substantially in the middle of your foot and pull the removable foil or film part all the way down your foot and up your heel and secure it.

3. Optionally, if wearing Sandler's shoe types, stick directly to bottoms of feet and tear pre- cut or pre-perforated slitting line 120 in between big toe and pointer toe.

4. Optionally, once the invention is installed, the wearer may place its foot into a shoe.

In another embodiment, the PPE device 104 according to the present invention is made in a thin plastic anti-microbial shoe or boot cover with a non-slip bottom and/or an elastic, the elastic being affixed to the PPE device 104 such that the elastic is situated around the ankle of a wearer when the PPE device 104 is installed. So, like a shoe cover, one may slip a shoe into such a PPE device 104 such that the PPE device 104 covers the shoe and may be worn while walking. The device 104 worn as described in the present paragraph has, for example, the effect to help stop the spread of Covid when entering venues.

In a preferred embodiment, the first device 104 is put on top of and affixed to a second device 204. The second device 204 comprising the features of the first PPE device 104, whereas the second device 204 is preferably of equal size/dimensions than the first PPE device 104, or more preferably of larger site/dimensions than the first PPE device 104 (such as exemplary shown in FIG. 1A).

In a variant of the above embodiment, the material of the first PPE device 104, and also of the second PPE device 204, will be manufactured with anti-microbial properties all the way through it. As an effect, while walking, when the sole scrapes against the ground on each step, any plastic grinded off it will only reveal a new layer of anti-microbial surface.

The PPE devices 104, 204 according to the above-described embodiments may be installed, i.e.

SUBSTITUTE SHEET (RULE 26) wrapped around a naked foot 102 or a foot wearing a sock (not shown). The method for installation may be described as follows:

1. Hold the front tab 210 by its "non-stick tab" and peei around half of the removable foil or film off and secure front portion to top of the toe area.

2. Line up the device 204 substantially in the middle of your foot and pull the removable foil or film all the way down your foot and up your heel and secure it.

3. Optionally, if wearing Sandler's shoe types, stick directly to bottoms of feet and tear precut or pre-perforated slitting line 120 in between big toe and pointer toe.

Typically for a second use of the embodiment (e.g. on a "round trip" or in a one-day scenario when one is taking the shoes off on a plane):

4. The second PPE device 204 may be separated from the first PPE device 104 by means of peeling the second PPE device 204 off and peeling the removable foil or film to secure first PPE device 104 to the foot as described.

Referring now to FIG. 2, a top view of an alternative embodiment of the invention is shown. A naked foot 202 is placed on a PPE device 204 according to the present invention. The device 204 comprises a protective layer 206 spread substantially over the whole surface of the PPE device 204. An adhesive backing 212, applied on the entire surface of the layer 206, adheres to the sock, foot sole or shoe sole. The adhesive backing 212 is preferably covered by a removable paper, foil or film 254, 254'. Optionally, the removable paper, foil or film 254, 254' may be separated in individual elements by separation gaps 256. One variant is shown in Fig. 2. The removable film 254, 254', 254" may be removed separately from one another, allowing a central panel 254 of removable film to be removed first while leaving a frame structure made up of at least the lateral removable films 254'. However, any other arrangement of removable paper, foil or film 254, 254* and separation gaps 256 are conceivable. Optionally, the protective layer 206 may be perforated to provide the functionality of breathability and airflow in order to prevent the foot from sweating. The perforations are located in the protective layer 206, preferably at locations where the foot or the sock is placed when worn. The perforations may be applied to the protective layer 206 during the punching process. The perforations may be micro pores so as to prevent bacteria from passing therethrough. The perforations are an optional feature and may or may not be present on the final product. The device 204 may be in a slight foot- or "V-shape" for easier installation (as exemplarily shown in FIG. 1B). The surface of the protective iayer 206 may be on one or on both sides and be adapted to stop sliding. The protective Iayer 206 of the invention may have a textured or non-slip bottom to prevent slipping on slick surfaces, such as wet floors (e.g. when biood is on the floor). This structure may be applied to the surface of the protective Iayer 206 during the punching process. The protective Iayer 206 may comprise a pre-cut or preperforated slitting line (as exemplarily shown in FIG. 1B). This slitting line may be applied to the

SUBSTITUTE SHEET (RULE 26) protective layer 206 during the punching process. The protective layer 206 may comprise an extended length 332 in the front area 334 to allow one to pull it over the front of the toe box of the shoe as well as the "laces area". This will protect the bottom and top of a wearer's footwear that are prone to bacteria and airborne particles falling on them. The PPE sole shield can remain on the bottom and extend over the top of the wearer's shoe, e.g. until one leaves the hospital, airport or other potentially infected location. Then the wearer can dispose of them being confident that the wearer's feet hasn't transported any harmful germs, bacteria, viruses or the like on the bottom or the top.

The protective layer 206 according to the invention is made from a special plastic that is extremely strong, stretchy, durable and made with an anti-microbial material. A suitable antimicrobial film can be found on: www.silverdefender.com or www.3M.com. or the website of other suitable adhesive suppliers. However, the anti-microbial effect is optional as benefits can be gained by use of the invention without anti-microbial treatment, when the device acts as a simple protective barrier between the user and a dirty surface. The protective layer 306 has built in antimicrobial protection. Such an antimicrobial protection can, for example, be based on silver ions that react with and affect multiple sites in bacterial cells on the surface of the layer, such to keep the layer clean. The protective layer 206 is suitable for use in high traffic areas. The layer may be made in form of a tape, the tape being optionally perforated, so as to facilitate tearing away a sheet of appropriate length. Further, the layer may be perforated such that, after it is installed on an object such as a shoe or foot, said perforation serves to facilitate removal of the layer from the object (e.g. after it has ended its useful lifespan or is no longer needed).

In a variant of the above embodiment, the material of the protective layer 206 will be manufactured with anti-microbial properties all the way through it. As a result, while walking, when the sole scrapes against the ground on each step, any plastic ground or worn off it will only reveal a new layer of anti-microbial surface.

In one embodiment, the PPE device 204 according to the present invention may be installed, i.e. partially wrapped over the sole and upwardly along the sides of a naked foot 202 or a foot wearing a sock (not shown). The method for installation may be described as follows:

1. Peel the center of the removable paper, foil or film 254 backing off of the adhesive backing 212. The sides, front and rear removable paper, foil or film 254' may have a thin border used to keep the flimsy protective layer 206 in a "flat position" for easy installation.

2. The removable paper, foil or film 254' will be peeled off after the user places his foot 202 in the center of the exposed adhesive backing 212 of the protective layer 206. The boarders of removable paper, foil or film 254' left on the edges of the protective layer 206 wiii act as borders that keep the very thin protective layer 206 straight and prevent it from folding on its self and sticking to its seif.

3. After the user places his foot 202, preferably directly in the center of the adhesive backing 212, they

SUBSTITUTE SHEET (RULE 26) will then remove the removable paper, foil or film 254' by peeling them off the same way as they did the center piece 254 that originally exposed the adhesive backing 212. Once the removable paper, foil or film 254' are peeled off, the so laid open areas of the adhesive backing 212, can be stuck or adhered to the sides of your foot 202 and top of toes and up the heal of the foot 202.

4. Optionally, once the invention is installed, the wearer may place his foot into a shoe.

In another embodiment, the PPE devices 104, 204, 304 according to the present invention are made in a thin plastic anti-microbia! shoe or boot cover with a non-slip bottom and/or an elastic, the elastic being affixed to the PPE devices 104, 204 such that the elastic is situated around the ankle of a wearer when the PPE devices 104, 204 are instalied. So, like a shoe cover, one may slip a shoe into such PPE devices 104, 204 such that the PPE devices 104, 204 cover the shoe and may be worn while walking. The PPE devices 104, 204 worn as described in the present paragraph have, for example, the effect to help stop the spread of Covid when entering venues.

In another preferred embodiment, a third PPE device (not shown) is affixed to the second PPE device 204, such that the embodiment comprises three devices. The third PPE device is affixed to the second PPE device in the same manner as the second PPE device 204 is affixed to the first PPE device 104. Further preferred embodiments, adding even further devices in the same manner, are within the scope of the invention.

In a variant of the above embodiment, the material of all the PPE devices will be manufactured with anti-microbial properties all the way through it As an effect, while walking, when the sole scrapes against the ground on each step, any plastic worn off will only reveai a new anti-microbial surface.

The embodiments as described above may be affixed to the bottom of a shoe, preferably of a medical shoe, instead of a naked foot 102 or a foot wearing sock{s) as provided above. One of the herein-described embodiments may preferably be affixed to the bottom of a medical shoe when you walk into a hospital. The embodiments can e.g. be made disposable when entering or leaving a hospital.

The present invention is suitable for various types of footwear. From sneakers/shoes, medical shoes to sandals or flip flops.

The embodiments, when installed on a foot, on a foot wearing sock(s), or on a shoe may last for 24 hours or any other time.

In another variant of the above embodiments, the material of the PPE devices may be perforated through out to be able to easily tear at certain lengths (small , medium and large) for a one size fits all feature and benefit.

In another variant of the above embodiments, they may be scented for a fresh smell at time of install.

In another variant of the above embodiments, the embodiment is specifically designed for the sole of footwear. The layer of this embodiment has a permanent and waterproof adhesive backing and consists of

SUBSTITUTE SHEET (RULE 26) a thicker anti-microbial protective material with a textured non-slip bottom. When you wear these permanent PPE sole protectors, the anti-microbial layer will easily wipe of germs, bacteria and the like when the user wipes his feet on a door mat A mat more suited for this type of wiping may be designed but will work on any regular door mat. The user would wipe off their feet on the door mat and the anti- microbial layer will easily transfer germs from the protective layer to the door mat leaving most of the germs on the mat, and not dragging them inside of the home for instance. This anti-microbial feature would last for up to 90 days, it Is possible to remove the permanent PPE sole protector by exposing it to heat from a heat gun or hot hair dryer. By applying heat it will break down the glue and make it easily removable.

The embodiments, when installed on a foot wearing sock(s), may comprise at least one front tab to avoid pulling socks off.

A detailed description on how the invention works is now provided. The invention may be of a rectangular shape or art to a dedicated shape as exemplary provided in the figures. They will be wider and longer than the actual projected surface of a foot or shoe so they may be wrapped around the foot or shoe to create an air tight fit. Logos, of sports teams, for example, may be printed on the PPE device of the invention. A user simply peels off the removable foil or film and carefully applies the adhesive backing to the foot, sock or shoe. It may be flattened out with the user's fingers so there are substantially no air bubbles in it Then the excess part of the layer is wrapped around the foot, sock or shoe. For sandals or flip flops there isa perforated slitting line 120 in between the big toe and pointertoe to allow for the piece of the sandal that is disposed in between those specific toes. This may be pre made at the factory or the consumer may simply cut the invention to accommodate for that piece of the sandal.

The user peels the removable foil or film from the invention so as to lay open the adhesive backing and applies the invention to the foot, sock or shoe they desire to protect.

As mentioned, a suitable anti-microbial film can be found on: www.siiverdefender.com or www.3M.com. or the website of other suitable adhesive suppliers The protective layer may optionally have built in antimicrobial protection. Such an antimicrobial protection can, for example, be based on silver ions that react with and affect multiple sites in bacterial cells on the surface of the layer, so as to keep the layer clean. The protective layer is suitable for use in high traffic areas. The layer may be a tape, the tape optionally being perforated, so as to facilitate tearing away a sheet of appropriate length. Further, the layer may be perforated such that, after it is installed on an object such as a shoe or foot, said perforation serves to facilitate removal of the layer from the object (e.g. after the device has served its useful purpose).

When socks or the sole of the foot are exposed to the dirt germs, bacteria, viruses or the like is on the floor, these germs will then contaminate a damp sweaty sock and become embedded into the sock and pass through the sock, and into the open pores on the sole of the foot leaving the person at a great risk of being infected. After walking through airport security screening, the person will have to then put their shoes back

SUBSTITUTE SHEET (RULE 26) on and their socks will now contaminate the inside of their shoes. The heat from one's feet will create an incubator atmosphere for the germs, bacteria, viruses etc. to stay alive and get one sick by entering the open pores on one's sweaty feet. When a foot sweats in the sock, one's pores remain open and this creates an entrance into your bloodstream for bacteria, germs, viruses or the like, to enter into the body and so ultimately, he/she gets contaminated.

A further advantage provided by this invention is to keep the bottoms of socks free from bacteria so when one finally arrives at home and takes off one's shoes, one does not spread the bacteria and germs that have been incubating in the socks all day onto the carpet or floor. There is the risk that young children may be playing on it when they crawl on the contaminated floor thereby becoming exposed to these germs just tracked in. Then, by touching the germs on the floor and then touching their eyes or mouth will leave them at great risk for becoming infected. This can be avoided when wearing the PPE device according to the invention as described herein.

It should be appreciated that the particular implementations shown and herein described are representative of the invention and its best mode and are not intended to limit the scope of the present invention in any way.

In further detail, in one embodiment, the protective layer 106 is infused with anti-microbial that has at least one adhesively-backed side 112, 212 that will adhere to the sole of a person's footwear.

Optionally, the protective layer 106 may be cut at the end of the front of the shoe or extended approximately 3 to 10 inches past the front of the footwear to be able to pull the access film back towards you and over the laces to clean them and shield them from germs, viruses, bacteria and Covid droplets.

The protective layer 106 is preferably wider and longer then then the sole to which it is sized to make sure it covers the entire sole as well as covering a portion of the sides and front and rear of the wearer's footwear.

Optionally, in another embodiment, an adhesive backing 112 may be applied to both sides of the protective layer 106, thereby providing a tacky non-slip surface (optionally covering only a portion of the surface in a zig-zag or show shoe grip pattern). The film's tackiness serves as a non-slip feature ensuring the user does not slip after application of the product when walking. The exteriorly disposed tacky (i.e. adhesive) layer on the protective layer 106 has the added benefit of picking up the bacteria and germs up off of the floor and sticking them to the tacky side 112, 212 of the protective layer 106. The longer the germs stay on the film, the more die. Still further, visually, the adhesively-backed sidell2, 212 will become soiled and so show the user what amount of dirt and germs the protective layer 106 protects him or her against.

As already described, the protective layer 106 may optionally be die cut into sheets that have at least one side attached to a removable release liner 254, 254' that is removed prior to adhering to sole of footwear.

SUBSTITUTE SHEET (RULE 26) The release liner 254, 254' is designed to prevent drying out of the adhesive backing 212 and to prevent sticking before the protective layer 106 is ready for adhering to the foot, sock or shoe.

Note that the composition of the adhesive backing directed toward the sole, sock or foot may have a different composition than that of the adhesive backing directed outwardly. This is because the function of each respective side is different. The inwardly directed adhesive backing is intended to attach, in an easily removable manner, the layer to the shoe, sock or foot. The outwardly directed adhesive backing is designed to provide slip-resistance and to clean the floor of germs. Consequently, the level of adhesion and the amount of anti-microbial to be impregnated in the layer varies.

In this and all other embodiments herein described, the antimicrobial lining of the layer may be bonded to a conventional, clear polyethylene plastic typically used to vacuum-package foods. This anti-microbial lining can optionally be a pullulan-based biopolymer produced from starch syrup during a fermentation process, which is already approved for use in foods. Pu!iulan, a water-soluble "polysaccharide," is essentially a chain of sugar, glycerin and cellulose molecules linked together. The adhesive backing applied thereon can be a low-strength adhesive or even an adhesive backing inducing static electricity such as found in 3M POST-IT* note products. A Pressure Sensitive Acrylate (PSA) provides the ability to stick and re-stick, adhering to most surfaces, and then to be easily removed without being torn apart and without leaving residues of adhesive on the substrate. Because of the different natures of the anti-microbial layer and the adhesive backing, these may be applied to different portions of the surfaces of one or both sides of the layer so as to allow dual functionality.

Still further, when the layer is removed from the sole, or sock or foot, the anti-microbial will have killed bacteria and germs on the sole, sock or foot, and the sticky adhesive will also pull off most of the germs on the bottoms of your shoes, socks or feet, leaving your soles, socks or feet cleaner than they were before the layer of the invention was installed.

Referring now to FIGs.3A and 3B, in another embodiment 401 of the invention, the antimicrobial layer 106 need not have an adhesive backing on either side but instead may be manufactured on a roll and dispensed from an auto shoe cover machine such as BOOTIE-BUTLER™, described on www.uline.com/BL_1208/Bootie-Butler?pricode=WA3825, and shown in FIG. 3A. In this embodiment of the invention, an anti-microbial, adhesive backing, optionally structured for grip, is advantageously applied to the bottom of each bootie 401 (having an integrated elastic band 403 that holds the bootie around the shoe or foot) dispensed, with a release layer optionally placed therebetween.

The invention may be applied to a wearer'sfoot or shoe as follows: stepafoot into the auto shoe cover machine and pull the foot back, whereby covers are automatically applied to a wearer's foot, shown in FIG. 3B. Such a machine may be used anywhere and may work with or without electricity. The machine and/or this embodiment of the invention optionally has anti-static properties, i.e. is made of e.g. polypropylene,

SUBSTITUTE SHEET (RULE 26) and may have a lightweight polypropylene with a sewn-in Electro Static Discharge (ESD) strip. The machine and/or this embodiment of the invention is preferably waterproof, i.e. is made of e.g. polyethylene and/or polypropylene, and may have an extra tough, water proof bottom surface.

As for the adhesive backing, this is typically applied as a double-stick tape with a release strip, which is run along the layer and pressed against the layer to adhere thereto, leaving the release trip attached so that it can be later removed at the point of application. The adhesive used is typically available from 3M corporation and is similar to that used on POST-IT™ notes. Natural rubber adhesives can optionally be used. A key ingredient in the formulation of natural rubber-based adhesives is a tackifying resin. In the case of a solvent-based adhesive, the use of a tackifying resin serves not only to improve the tackiness associated with a pressure sensitive adhesive (PSA), but also to improve the other properties of the adhesive. This improvement comes from the fact that the tackifying resin usually has a glass transition temperature that is higher than that of rubber. Mixing the two increases the Tg of the resulting adhesive and improves its stiffness. The adhesive can remain tacky as long as a certain amount of solvent is present, but it loses its tackiness when all the solvent is gone. Plasticizers are often used when the rubber is not heavily chewed. Examples of plasticizers are lanolin and liquid poly(butene). Various fillers and reinforcing agents are also used to increase the adhesive's bond strength. In this case, carbon black is often used. Reinforcement can also be achieved by crosslinking. A special type of crosslinking agent is poly(isocyanate). In the case of natural rubber, antioxidants must be used to prevent oxidation and embrittlement of the adhesive.

Referring now to FIGs. 4A and 4B, another embodiment 402 is shown as applied to a wearer's foot 404 using an automatic shoe cover machine 406. This particular embodiment is in more compact form, the layer of the invention can be dispensed using, for example, the Quen automatic shoe cover machine 406 described on www.quen-techs.com/news/why-choose-quen-automatic-shoe-cover - machine-22734285.html. Optionally both sides of the layer can have an anti-microbial adhesive backing applied, and a release layer(s) may be optional. Shown in FIG, 4A, the device 410 is preferably applied to a wearer's foot by using the principle of heat shrinkage to increase the holding capacity of shoe cover. Further, an automatic shoe cover machine such as the Quen automatic shoe cover machine may be configured to store consumables (i.e. units of at least one of the embodiments of the present invention) in the form of rolled film. By taking advantage of the principle of heat shrinkage, a large number of shoe covers can be applied to shoes or feet. One roll of layer can be used to make from 1000 to 1600 shoe covers. The automatic shoe cover machine 406 such as the Quen automatic shoe cover machine may be designed with ergonomics as its design concept, using computer micro-control technology and an automatic error-correcting electronic display system.

A variant 402 which does not require heat-shrink capabilities is shown in FIG.4B. Here, the device of the invention is dispensed under the foot and over a thick, spongy layer. The foot is pressed against the

SUBSTITUTE SHEET (RULE 26) device and into the spongy layer and in this manner, the sides of the shoes, booties, socks or foot are covered.

The anti-microbial layer of the invention typically uses a plastic material based on polyvinylidene chloride (PVC) or is made of iow density polyethylene (LORE). These coatings are optionally impregnated with a Zinc or Silver oxide, even small copper particles, thereby created an impregnated anti-microbial film. Alternatively, such a plastic layer is coated with a separate antimicrobial film. A suitable antimicrobial film-forming composition may consist of not more than 10 weight percent polyvinyl alcohol, 0.05 to 15 weight percent polyhexamethylene biguanide, 0.001 to 10 weight percent of a quaternary ammonium compound, and water or a water-based solvent The film-forming composition may form a water-soluble, biocidal, antimicrobial film containing no more than 98 weight percent polyvinyl alcohol and 1 to 15 weight percent each of polyhexamethylene biguanide and the quaternary ammonium compound. The film-forming composition is applied to the plastic film to form the antimicrobiai film, and may optionally contain an indicator dye to make the antimicrobial film more visible to the user. Service provider "BiOCOTE"* Ltd of the UK helps with specifying an appropriate antimicrobial additive for any particular application. See www.biocote.com/make-my-product-antimicrobial/.

Clara Silvestre et al. describe in their article the "Development of Antibacterial Composite Films Based on Isotactic Polypropylene and Coated ZnO Particles for Active Food Packaging", also attached in the Appendix A hereto. In one embodiment of the present invention, such antibacterial composite films are applied to the layer of the PPE device.

Referring now to FIG. 5 and, for context, the attached Appendix A, a diagram shows a thermostability curve of ZnO and ZnOc particles optionally used in the invention. "Before blending the ZnOc particles with iPP, an investigation of properties of the ZnO and ZnOc particles has been performed, to assess the amount of stearic acid present on the ZnO particles and the influence of the coating process on the structure, morphology, and thermal stability of the zinc oxide particles. The content of stearic acid present on the surface of the coated particies was evaluated through thermogravimetric analysis. FIG. 5 reports the thermogravimetric curves of ZnO and ZnOc particles recorded during the heating rate of 20 "C/min in air from room temperature to 700 ^e C. In the entire T range, for ZnO particles, no weight loss was observed. In the case of ZnOc, after the thermal treatment, a weight reduction of about 9% is found. Considering that ZnO does not undergo degradation, it can be concluded that the weight reduction percentage observed for ZnOc corresponds probably to the percentage of stearic acid present on the surface of the particles."

Referring now to FIG. 6 and, for context, the attached Appendix A, a diagram shows X-ray diffraction pattern of ZnO and ZnOc particles optionally used in the invention. To study the influence of the stearic add on the crystalline structure of the ZnO particles, "the spectra of X-ray diffraction at high angles were recorded. As shown in FIG.6, the spectrum of the partides of ZnOc presents the same peaks

SUBSTITUTE SHEET (RULE 26) as those of ZnO, suggesting that the coating does not after the crystalline structure of the partides {zincite). In order to recognize the functional groups present, the interaction between the stearic acid and the ZnO partides and to obtain information on the shape of the partides, FTIR analysis was been performed."

Referring now to FIG.7 and, for context, the attached Appendix A, a diagram shows FT-IR spectrum of ZnO and ZnOc powder optionally used in the invention. "Different bands can be observed, in particular at wavelengths of around 2916 cm' ¹ , 2848 cm ^*1 , 1460 cm ^-1 , at 1540 cm' ¹ , 1384 cm ¹ and at 454 cm ⁴ ."

Referring now to FIGs.8A to 8D and, for context, the attached Appendix A, SEM micrographs of ZnO (FIGs. 8A and 8C) and ZnOc powders (FIGs.8B and 8D) optionally used in the invention are shown. "The morphology of the ZnO and ZnOc partides was studied using scanning electron microscopy (SEM). FIGs.

8A to 8D show SEM micrographs of ZnO and ZnOc respectively. From the micrographs, it is clear that ZnO particles are characterized by a hexagonal crystal structure, as already emerged from FTIR analysis, with a smooth surface. Moreover, the ZnO particles seem to have a strong tendency to form agglomerates. Contrary ZnOc particles, more homogeneously dispersed in the matrix, have a spherical shape. Comparing the dimensions of the kinds of partides, the size of the ZnO partides ranges between 250 to 500 nm while that of the partides of ZnOc varies between Ito 1.2 micrometers."

Referring now to FIG.9 and, for context, the attached Appendix A, a diagram shows WAXD spectrum of iPP and iPP / ZnOc composites optionally used in the invention. "All samples show the presence of foe peak at 2Θ = 18 ^* -19 ^* characteristic of a form of iPP [39,40]. The sample iPP/5%Zn0c is characterized also by a small percentage of the form β, highlighted by the presence of the peak at 28 = 16°."

Referring now to FIG. 10 and, for context, the attached Appendix A, a diagram shows UV-Vis spectrum of iPP and IPP / ZnOc samples optionally used in the invention. "For ail samples, an absorption band at 280 nm is observed, probably due to the presence of stabilizer added to commercial iPP. For the samples containing ZnOc, an absorption band is also observed in the region around 385 nm, as indicated by arrows. This band is due to the inherent capacity of the ZnO particles to absorb the UV light."

Referring now to FIGs. 11A to 11F and, for context, the attached Appendix A, micrographs of iPP (FIGs. 11A and 11D), iPP/2%ZnOc (FIGs. 11B and HE) and iPP/5%ZnOc (FIGs. 11C and 11F) pellets fractured in liquid nitrogen optionally used in the invention are shown. "Comparing the results with those obtained for the system iPP/ZnO and iPP/PPgMA/ZnO at a given composition, it seems that foe coating of the ZnO with stearic add favors a better dispersion and distribution of the partides in the iPP matrix and prevents the formation of agglomerates."

Referring now to FIG. 12 and, for context, the attached Appendix A, a diagram shows the thermostability curve of IPP and IPP / ZnOc composites optionally used in the invention. "In particular, the % weight loss of the samples as function of temperature for iPP and iPP/ZnOc samples is shown." Referring now to FIG.13 and, for context, foe attached Appendix A is a diagram showing stress-strain

SUBSTITUTE SHEET (RULE 26) curves of iPP and !PP / ZnOc films optionally used in the invention. "It can be seen that all the samples have the typical behavior of a semi-crystalline polyolefin, with the phenomenon of yield strength, cold drawing, fiber elongation and final break of the fibers."

Referring now to FIG. 14 and, for context, the attached Appendix A, a diagram showing the effect of time and filler content on the antibacterial activity of iPP and iPP / 2%-5% ZnOc composites optionally used in the invention. "The antimicrobial effect against £ coii is presented as a function of time for the different composites. Without ZnO particles, the reference concentration of the microorganism is measured to be ~2 x 10 ⁶ . After 1 h, no change in the concentration was observed for all samples. By increasing the time, a decrease in the E.coli concentration is observed for the composites. The effect is more evident for the iPP/5%ZnOc composite. Significant variations in concentration are observed increasing the contact time and ZnOc content. After 24 h, the concentration of £ colt decreases to 8.8 x 10 ⁵ for iPP/2%ZnOc and 1.7 x 10 ⁵ for iPP/5%ZnOc. After 48 h, the bacterial concentration was significantly decreased for the ?PP/5%ZnOc sample (2 x 10 ³ CFU/mL). The sample iPP/2%ZnOc reaches similar values after five days. The values of percentage reduction (%R) of £ coli for all samples at different contact times are reported in Table 5. Neat iPP exhibits no bactericidal activity, and R was observed to be zero up to day 10. The iPP/5%ZnOc composite exhibited maximum reduction, 99.9%, after 48 h."

Referring now to FIG. 15 is a flow chart showing the processes of data acquisition in certain performance tests of the invention.

Referring now to FIG. 16 is a chart showing the results of the performance tests of the invention. Referring now to FIG. 17 shows the floor area used in the performance test of the invention. Referring now to FIG. 18A and 18B show the shoes used in the performance tests of the invention.

Referring now to FIG. 19 shows the incubation of the performance tests of the invention. Referring now to FIG. 20 is a flow chart showing the processes of data acquisition in the performance tests of the invention.

Referring now to FIG.21, a printed protective film 2100 of the invention has instructions printed thereon, it adheres to the layer 112 of the invention, and is peeled away at application to the shoe, sock, bootie or foot and discarded, optionally, in the zip-lock type baggie in which it was purchased. Product Test Results:

A study entitled "Test of floor interaction with shoes in reference to acquired and deposited bacteria" is attached in the Appendix B hereto. The study concludes that there are copious amounts of bacteria on a hospital floor.

A study entitled "Test of shoe interaction with "SAFE STEPS"™ in reference to acquired and deposited bacteria" is attached in the Appendix C hereto. The study concludes that bacteria from the soles of shoes are transferred from the sole of the shoe to the floors you walk on (car, home & work). These

SUBSTITUTE SHEET (RULE 26) studies also prove that a person wearing 2 shoes walking through supermarket or a mall, one shoe protected with the device of the invention and the other not, the shoe not protected by the device of the invention picks up a considerable amount of bacteria, while the shoe with the device of the invention, when the device is removed from the shoe, the sole of the shoe was even cleaner than it was before the device of the invention was applied.

In an advantage, a wearer using the sheet to protect a sole from contact with a floor surface is able to prevent dirt, germs, bacteria, etc. from being picked-up by the sole of a foot, socks, or the sole of a shoe.

Benefits, other advantages and solutions mentioned herein are not to be construed as critical, required or essentia! features or components of any or all the claims, herein incorporated by reference.

As will be appreciated by skilled artisans, the present invention may be embodied as a system, a device, or a method.

Moreover, the system contemplates the use, sale and/or distribution of any goods, services or information having similar functionality described herein.

The specification and figures should be considered in an illustrative manner, rather than a restrictive one and all modifications described herein are intended to be included within the scope of the invention claimed. Accordingly, the scope of the invention should be determined by the appended claims (as they currently exist or as later amended or added, and their legal equivalents) rather than by merely the examples described above. Steps recited in any method or process claims, unless otherwise expressly stated, may be executed in any order and are not limited to the specific order presented in any claim. Further, the elements and/or components recited in apparatus claims may be assembled or otherwise functionally configured in a variety of permutations to produce substantially the same result as the present invention. Consequently, the invention should not be interpreted as being limited to the specific configuration recited in the claims.

As used herein, the terms "comprises", "comprising", or variations thereof, are intended to refer to a non-exdusive listing of elements, such that any apparatus, process, method, article, or composition of the invention that comprises a list of elements, that does not include only those elements recited. Unless otherwise explicitly stated, the use of the term "consisting" or "consisting of" or "consisting essentially of" is not intended to limit the scope of the invention to the enumerated elements named thereafter, unless otherwise indicated. Other combinations and/or modifications of the above- described elements, materials or structures used in the practice of the present invention may be varied or adapted by the skilled artisan to other designs without departing from the general principles of the invention.

For example, a thicker, puncture resistant (e.g., using nyion, Kevlar or other suitable materia! layer), reinforced, and hypoallergenic yet flexible version may be provided to protect a barefoot user when the invention is directly attached to the user's foot. When one walks barefoot, one is extremely susceptible to

SUBSTITUTE SHEET (RULE 26) contamination by poisons, viruses or bacteria found on the floor or on the ground where people sometimes discharge body fluids, fluids which may contain dangerous pathogens.

The patents and articles mentioned above are hereby incorporated by reference herein, unless otherwise noted, to the extent that the same are not inconsistent with this disclosure. Other characteristics and modes of execution of the invention are described in the appended claims.

Further, the invention should be considered as comprising all possible combinations of every feature described in the instant specification, appended claims, and/or drawing figures which may be considered new, inventive and industrially applicable.

Additional features and functionality of the invention are described in the claims appended hereto and/or in the abstract. Such claims and/or abstract are hereby incorporated in their entirety by reference thereto in this specification and should be considered as part of the application as filed.

Multiple variations and modifications are possible in the embodiments of the invention described here. Although certain illustrative embodiments of the invention have been shown and described here, a wide range of changes, modifications, and substitutions is contemplated in the foregoing disclosure. While the above description contains many specific details, these should not be construed as limitations on the scope of the invention, but rather exemplify one or another preferred embodiment thereof. In some instances, some features of the present invention may be employed without a corresponding use of the other features. Accordingly, it is appropriate that the foregoing description be construed broadly and understood as being illustrative only, the spirit and scope of the invention being limited only by the claims which ultimately Issue in this application.

SUBSTITUTE SHEET (RULE 26) Appendix A

Development of Antibacterial Composite Films Based on Isotactic Polypropylene and Coated ZnO Particles for Active Food Packaging Clara Silvestre Donatella Duraccio ² , Antoneila Marra ¹ , Valentina Strongone ¹ and Sossio Cimmino ¹

Received: 11 December 2015; Accepted: 15 January 2016; Published: 22 January 2016 Academic Editor: Stefano Farris 1 Institute of Polymers, Composites and Biomaterials, Consiglio Nazionale delle Ricerche

(IPCB/CNR) Via

Campi Flegrei 34, Naples 80078, Italy; antonella.marra@ipcb.cnr.it (A.M.); valentina.strongone@outlook.it (V.S.); sossio.cimmino@cnr.it (S.C.)

2 Istituto per le Macchine Agricole e Movimento Terra, Consiglio Nazionale delle Ricerche (IMAMOTER-CNR)

Strada delle Cacce 73, Torino 10135, Italy; donatella.duracdo@cnr.it * Correspondence: clara.silvestre@cnr.it; Tel.: +39-081-867-5067; Fax: +39-081-867-5230 Abstract: This study was aimed at developing new films based on isotactic polypropylene (iPP) for food packaging applications using zinc oxide {ZnO} with submicron dimension particles obtained by spray pyrolysis. To improve compatibility with IPP, the ZnO particles were coated with stearic acid (ZnOc). Composites based on iPP with 2 wt% and 5 wt% of ZnOc were prepared in a twin-screw extruder and then filmed by a calendar. The effect of ZnOc on the properties of iPP were assessed and compared with those obtained in previous study on iPP/ZnO and IPP/iPPgMA/ZnO. For all composites, a homogeneous distribution and dispersion of ZnOc was obtained indicating that the coating with stearic add of the ZnO particles reduces the surface polarity mismatch between iPP and ZnO. The iPP/ZnOc composite films have relevant antibacterial properties with respect to E.coli, higher thermal stability and improved mechanical and impact properties than the pure polymer and the composites iPP/ZnO and iPP/iPP-g- MA/ZnO. This study demonstrated that iPP/ZnOc films are suitable materials for potential application in the active packaging field.

Keywords: isotactic polypropylene; zinc oxide; properties; active packaging; composites 1. Introduction

It is extensively reported that the nano/microparticules of zinc oxide (ZnO) exhibit antibacterial activity against gram-positive and gram-negative bacteria. This activity does not require the presence of UV light (unlike TiO ₂ ), being stimulated by visible light, and it is inversely dependent on particle size [1-5]. The mechanisms responsible for the antibacterial activity are not fully understood. Distinctive

SUBSTITUTE SHEET (RULE 26) mechanisms that have been put forward in the literature are listed as the following: direct contact of ZnO with ceil wails, resulting in destructing bacterial cell integrity (6-8], liberation of antimicrobial ions, mainly Zn ₂₊ ions [8], and reactive oxygen species formation [9-11].

There has been a great deal of interest in the antimicrobial property of ZnO for food packaging applications, as a viable solution for stopping infectious diseases [12-14]. Due also to low cost, ZnO partides (with sub-micro and nano-dimensions) are therefore ideal fillers for polymers to be applied in the field of active food packaging. Thus, ZnO particles have been incorporated into a number of different polymers used in food packaging (15,16], such as low-density polyethylene (LDPE) [17,18], isotactic polypropylene (iPP) [19-24], polyamide (PA) [18,25], and polylactic add (PLA) [26].

Recently, the influence of ZnO particles, obtained by spray pyrolysis with submicron dimensions [22,27,28], on the structure, morphology, thermal stability, photo stability, and mechanical and antibacterial properties of (iPP)/ZnO composites was investigated [22,24]. The addition of ZnO particles imparts improvements on the photodegradation resistance of iPP to ultraviolet irradiation and the composites exhibit significant antibacterial activity against Escherichia coli. This activity is dependent on exposure time and composition.

On the other hand, it was noticed that due to the surface polarity mismatch between iPP and ZnO, agglomeration phenomena of the ZnO particles occur and that these phenomena cause a decrease in the mechanical and other functional properties of iPP/ZnO composites with respect to plain iPP [22-24].

The main problem to be solved in adding ZnO nano/micropartides to an iPP matrix seems therefore related to the formation of agglomerated domains that occur because of the strong intermolecular interactions among the ZnO partides in combination with their high surface area. This prevents transfer of their superior properties to the composite. Good dispersion has been reported for some polar polymers [18,25], but ZnO dispersion in non-polar polymers, such as IPP, during melt processing remains a challenge.

A largely proposed strategy, to improve dispersion consists in adding a compatibilizer, containing groups suitable for interaction with the two components [29-33]. Following this strategy in a previous paper, polypropylene grafted with maleic anhydride (PPgMA) [24] was selected as the most promising candidate as a compatibiiizer between iPP and ZnO. In particular, the influence of three PPgMA (with different MW and MA% content) added to iPP/ZnO 98/2 wt% on the structure, morphology, mechanical, thermal, barrier properties and antibacterial activity against E, coli was investigated with the aim to verify if the compatibiiizer PPgMA could be beneficial in order to increase the dispersion of ZnO in an iPP matrix in order to have films with improved properties, it was found that the presence of this compatibiiizer improves the dispersion of the particles in the matrix, but, at the same time, does not cause any enhancement in the barrier and mechanical properties and indeed reduces the antibacterial activity with respect to iPP/ZnO. An important aspect found in this study is that the more the ZnO are well embedded in polymer material, the

SUBSTITUTE SHEET (RULE 26) more the antibacterial activity decreases, probably because the surface of the particle available for contact with the solution decreases.

An alternative methodology to improve the dispersion consists of modifying the particles' surfaces with groups suitable for interaction with the matrix.

The main purpose of this paper is to develop new films based on iPP for applications in the food industry as active packaging using coated ZnO particles to improve compatibility between the organic phase and inorganic one. In particular, the surface of the ZnO particles, obtained by spray pyrolysis, was coated with stearic acid (ZnOc). Objective of the paper is also to assess the influence of the coating process on the structure, morphology, and thermal stability of the zinc oxide particles.

2. Experimental

2.1. Materials and Sample Preparation

The materials used in this work are: (1) isotactic polypropylene (iPP, Moplen X30S), in pellets, kindly supplied by Basell (Ferrara, Italy), with melt flow index = 9 dg-min ^-1 (2.16 kg, 230 ^a C), M _w = 3.5 x 10 ^s and M _n = 4.7 x 10 ⁴ ; (2) zinc oxide coated with stearic acid (ZnOc) (white powder). The ZnO particles were synthesized using a preindustrial spray scale pyrolysis platform at the Pylote in Toulouse-France and then coated with stearic acid. This technique [22,27,28] provides many advantages compared to other techniques of preparation: the simplicity of the process, high purity of the powders obtained, more uniform chemical composition, narrow size distribution, better regularity in shape and the ability to synthesize muiticomponent materials. The coating of the ZnO particles was performed by preparing a solution of stearic acid and ZnO (1:10) in isopropanol under stirring for 12 h. The powder was recovered by centrifuge and dried in an oven at a temperature of 70 °C.

2.2. Composite Preparation

The powders of ZnOc were mixed with iPP at two different compositions: 2% and 5% (see Table 1), in a twin screw extruder Collin ZK 25 (D = 25 mm and L/D- 24).

Table 1. Isotactic polypropylene/Zinc oxide coated with stearic add (iPP/ZnOc) mixtures. The temperature setting of the extruder from the hopper to the die was: 180/195/195/190/180 "C. The screw speed of the dispenser was 20 rpm while the speed of the extruder screws was 25 rpm.

Films of iPP and iPP/ZnOc were obtained by compression molding in a press at 210 ”C and 100 bars. The films had a thickness of about 110-120 μm.

SUBSTITUTE SHEET (RULE 26) 2.3. Characterization

The following technologies were used to determine the properties of the films:

1) FT-IR Spectroscopy

Infrared spectra of the compression molded films were recorded with a PerkinEimer FT- IR spectrometer, mode! Paragon 500 equipment (PerkinEimer, Boston, MA, USA). The IR spectra were recorded in the range 4000-800 cm ^-1 with 4 cm ^-1 resolution and 20 scans.

2) Wide-Angle X-ray Diffraction

Wide-angle X-ray diffraction (WAXD) measurements were conducted using a Philips XPW diffractometer (Philips, Almelo, The Netherlands) with CuKa radiation (1.542 A) filtered by nickel. The scanning rate was 0.02° / s and the scanning angle was from 5° to 45°. The ratio of the area under the crystalline peaks and the total area multiplied by 100 was taken as the crystalline percentage degree.

3) Thermogravimetric Analysis

The thermal stability of the blends was examined by thermogravimetric analysis (TGA), using a PerkinElmer-Pyris Diamond apparatus (PerkinEimer, Boston, MA, USA) with a heating rate of 10 "C/min in air. Two measurements were performed for each sample.

4) Scanning Electron Microscopy

The surface analysis was performed using SEM, Fei Quanta 200 FEG (Fei, Hillsboro, OR, USA), on particles of the powders and on cryogenicaliy fractured surfaces of composites. Before the observation, samples were coated with an Au/Pd alloy using an E5 150 SEM coating unit.

5) Tensile Tests

Dumbbell-shaped specimens were cut from the compression molded films and used for the tensile measurements. Stress-strain curves were obtained using an Instron machine, Model 4505 (Instron, Torino, Italy) at room temperature (25 °C) at a crosshead speed of 5 mm/min. Ten tests were performed for each composition.

6) Charpy Impact Test

The impact test allows for determining the degree of toughness of a polymer. Charpy impact tests were performed by using a pendulum CEAST (CEAST, Torino, Italy) with appropriate software for processing the data. The tests were carried out at room temperature on slabs obtained by compression moulding. Rectangular samples, with width of 3 mm, thickness of about

4 mm and length of 5 cm were used. The samples were cut from slabs obtained by using the same conditions adopted for the preparation of films.

7) Analysis UV-Visible Spectrometric

SUBSTITUTE SHEET (RULE 26) The UV-Visible spectrometry is useful for evaluating the ability of a material to minimize radiation potentially dangerous to the packaged food. The instrument used is a spectrometer Shimadzu UV-2101PC (Shimadzu, Columbia, MD, USA). UV-Vis spectra were recorded in transmission In the range 200-850 nm.

8) Antibacterial Test

The antimicrobial activity of the IPP/ZnOc composites was evaluated using E.coli DSM 498T (DSMZ, Braunschweig, Germany) as test microorganisms. The evaluation was performed using the ASTM Standard Test Method E 2149-10 [34]. The preparation of the bacterial inoculum required to grow a fresh 18-h shake culture of E. coli DSM 498T in a sterile nutrient broth (LB composition for 1 L: 10 g of triptone, 5 g of yeast extract and 10 g of sodium chloride) The colonies were maintained according to good microbiological practice and examined for purity by creating a streak plate. The bacterial inoculum was diluted using a sterile buffer solution (composition for one litre: 0.150 g of potassium chloride, 2.25 g of sodium chloride, 0.05 g of sodium bicarbonate, 0.12 g of CaCl ₂ -6H ₂ O and pH = 7) until the solution reached an absorbance of 0.3 ± 0.01 at 600 nm, as measured spectrophotometrically. This solution, which had a concentration of 1.5 x 10 ⁸ - 3.0 x 10 ⁸ colony forming units/ml (CFUs/mL), was diluted with the buffer solution to obtain a final concentration of 1.5 x 10 ₆ -3 x 10 ⁶ CFUs/mL, that was the working bacterial dilution.

The experiments were performed in 50 mL sterilized flasks. One gram of the film was maintained in contact with 10 mL of the working bacterial dilution. After 2 min, 100 mL of the working bacterial dilution was transferred to a test tube, which was followed by serial dilution and plating out on Petri dishes (10 mm x 90 mm) in which the culture media was previously poured. The Petri dishes were incubated at 35 ^° C for 24 h. These dishes represented the To contact time. The flasks were then placed on a wrist-action shaker for 1 h, 24 h, 48 h, 5 days and 10 days. The bacterial concentration in the solutions at these time points was evaluated by again performing serial dilutions and standard plate counting techniques. Three experiments were performed for each composition. The number of colonies in the Petri dish after incubation was converted into the number of colonies that form a unit per millilitre (CFUs/m) of buffer solution in the flask. The percentage reduction (R%) was calculated using the following formula:

R% (CFU/mL) = [(B-A)/ B] x 100, (1) where A - CFUs/mL for the flask containing the sample after the specific contact time and B = CFUs/mL at To.

3. Results and Discussion

3.1. Analysis of ZnO and ZnOc Particles

Before blending the ZnOc particles with iPP, an investigation of properties of the ZnO and ZnOc particles has been performed, to assess the amount of stearic acid present on the ZnO particles and the influence of the coating process on the structure, morphology, and thermal stability of the zinc oxide

SUBSTITUTE SHEET (RULE 26) particles. The content of stearic acid present on the surface of the coated particles was evaluated through thermogravimetric analysis. Figure 5 reports the thermogravimetric curves of ZnO and ZnOc particles recorded during the heating rate of 20 ^° C/min in air from room temperature to 700 X,

In the entire T range, for ZnO particles, no weight ioss was observed. In the case of ZnOc, after the thermal treatment, a weight reduction of about 9% is found. Considering that ZnO does not undergo degradation, it can be concluded that the weight reduction percentage observed for ZnOc corresponds probably to the percentage of stearic acid present on the surface of the particles.

To study the influence of the stearic acid on the crystalline structure of the ZnO particles, the spectra of X-ray diffraction at high angles were recorded. As shown in Figure 6, the spectrum of the particles of ZnOc presents the same peaks as those of ZnO, suggesting that the coating does not alter the crystalline structure of the particles (zincite) [35].

In order to recognize the functional groups present, the interaction between the stearic acid and the ZnO particles and to obtain information on the shape of the particles, FTIR analysis was been performed.

Figure 7 reports the FTIR spectrum of ZnOc and ZnO. For the coated sample, different bands can be observed, in particular according to literature [35-38]:

• at 2916 and 2848 cm ^-1 : these vibration bands can be assigned to the "stretching” of the symmetric and asymmetric aliphatic group CH ₂ ;

• at 1460 cm ^-1 : this band is assigned to the vibration of "bending" of aliphatic groups CHa and CH3 of stearic acid;

• at 1540 and 1384 cm ^-1 : these bands are assigned to the asymmetric and symmetric vibrations of the carboxylate group of the stearic acid;

• at around 454 cm-: ¹ These bands give information about the shape of the particles. It is interesting to go deeper into the bands at points 3 and 4.

In particuiar, the bands at 1540 and 1384 cm ^-1 (region of absorption of the carbonyl C=0) can be related to the coordination behavior of the carboxylate group when it forms complexes with metais. These bands can give important information on the nature of the link between ZnO and the carboxylate group (COO-) of stearic acid.

According to literature, the carboxylate group has versatile coordination behavior, when it forms coordination complexes with metais. It can be ionic, monodentate, bidentate chelating or bridging. Measuring the frequency of asymmetric, (u _as (COO-)) and symmetric bands (u _as .(COO-) and Ua(COO-)} and the magnitude of their separation, Δ, (Δ = u _as (COO- ) - u _s (COO-)), the mode of the carboxylate binding with ZnO can be determined [36-38]. Generally, depending on the value of Δ, the following order is proposed for the coordination of carboxylates of divalent metais: Δ(chelating) < Δ(bridge) < Δ(ionic) < Δ(monodentate).

SUBSTITUTE SHEET (RULE 26) Finally, the assignment of the type of link is done comparing the Δ{ experimental) with that of the corresponding sodium salt {Δ(sodium salt)) with the following rules:

• if Δ( experimental) « Δ( sodium salt) is bidentate chelating coordination;

• if Δ(experimental) ≤ Δ( sodium salt) is bidentate coordination to bridge;

• if (experimental) > Δ( sodium salt) coordination is monodentate.

From the figure, Δ(experimental) = (1540 - 1384) cm ^-1 = 156 cm ^-1 . According to the criterion above, and taking into account that from literature data, the sodium stearate as Δ equal to 138 cm ¹ [35], it can be concluded that the coordination is monodentate.

The band at 454 cm ¹ can give information about the shape of the particles. Using the theory of dielectric media [34,36], the single band in ZnOc indicates particles with a spherical shape. It is interesting to make a comparison with the spectrum of the uncoated particles where the two bands indicate the presence of a structure with a mainiy prismatic shape.

The morphology of the ZnO and ZnOc particles was studied using scanning electron microscopy (SEM). Figures 8A to 8D show SEM micrographs of ZnO and ZnOc respectively. From the micrographs, it is clear that ZnO particles are characterized by a hexagonal crystal structure, as already emerged from FTIR analysis, with a smooth surface. Moreover, the ZnO particles seem to have a strong tendency to form agglomerates. Contrary ZnOc particles, more homogeneously dispersed in the matrix, have a spherical shape. Comparing the dimensions of the kinds of particles, the size of the ZnO particles ranges between 250 to 500 nm while that of the particles of ZnOc varies between 1 to 1.2 μm.

3.2. Analysis of the IPP/ZnOc Composites 3.2.1. Structure and Morphology

The WAXD patterns of iPP and iPP/ZnOc composites are reported in Figure 9. All samples show the presence of the peak at 2Θ = 18°-19° characteristic of a form of iPP [39,40]. The sample iPP/5%Zn0c is characterized also by a small percentage of the form β, highlighted by the presence of the peak at 2Θ = 16°.

UV-Vis spectra are reported in Figure 10. For all samples, an absorption band at 280 nm is observed, probably due to the presence of stabilizer added to commercial IPP. For the samples containing ZnOc, an absorption band is also observed in the region around 385 nm, as indicated by arrows. This band is due to the inherent capacity of the ZnO particles to absorb the UV light [29,30,35,36],

Figures 11A to 11F show SEM micrographs of the fractured surface of iPP (Figures 11A and 11D), iPP/2%ZnOc (Figures 11B and HE), iPP/5%ZnOc (Figures 11C and 11E). It is possible to note that the particles are fairly distributed within the polymer matrix. Only a few aggregates with a size of about 5 pm can be observed.

SUBSTITUTE SHEET (RULE 26) Comparing the results with those obtained for the system iPP/ZnO and iPP/PPgMA/ZnO [24] at a given composition, it seems that the coating of the ZnO with stearic acid favors a better dispersion and distribution of the particles in the iPP matrix and prevents the formation of agglomerates.

3.2.2. Thermostability

Figure 12 shows the % weight loss of the samples as function of temperature for iPP and iPP/ZnOc samples, whereas Table 2 reports the values of the temperature at the inflection point of the curve of Figure 12 detected at the maximum of the peak of the first derivative and which corresponds to the maximum rate of the degradation of the sample.

Table 2. Thermal degradation temperature at which the degradation rate is maximum

(Tmax).

For the two composites, a delay in the temperature of starting degradation, compared to «ΡΡ, and a consistent increase of the Tmax are observed. Taking into account that the presence of uncoated ZnO at the same composition did not have consistent influence on the thermostability of iPP, as reported in the paper at reference [22], the increase in thermal stability should be attributed to the stearic acid that coats the ZnO particles. As it was reported in a previous section (see Figure 5), the degradation of the stearic acid starts before the degradation of iPP. The degradation products of the stearic acid probably act as a barrier for the degradation of the matrix also slowing the diffusion of the degradation products of «ΡΡ in the sample causing an increase of the thermal stability of the iPP/ZnOc composites.

3.2.3. Mechanical and Impact Properties

Figure 13 shows the stress-strain curves of iPP and iPP/ZnOc composites, whereas Table 3 reports the values of mechanical parameters, (Young modulus (E), stress and strain at the yield point (σy, εy), and at break (σb, εb)).

It can be seen that ail the samples has the typical behavior of a semi-crystalline polyolefin, with the phenomenon of yield strength, cold drawing, fiber elongation and final break of the fibers.

From the values shown in the Table 3, it can be observed that: (1) the two composite films have similar values of Young modulus and strain at the yield point but higher than those of plain iPP; (2) the elongation at yield and the stress at break point can be considered similar for the three samples (the differences are inside the experimental error), whereas the elongation at break decreases with the addition of ZnOc. Comparing these results with those reported in reference [22], where ZnO not coated was used, it can be observed that the composites with ZnOc present improved mechanical properties.

SUBSTITUTE SHEET (RULE 26)

Table 3. Stress-strain parameters of iPP and iPP/ZnOc composites.

Table 4 shows the values of the impact test, in particular the values of the force (F) that the pendulum lost on impact with the sample, the energy (U) absorbed by the samples at the break and the toughness (T). The results demonstrate that the presence of ZnOc increases the toughness; in fact, the toughness of iPP/5%ZnOc sample is 26% higher than that of iPP.

Table 4. Impact tests values for iPP and iPP/ZnOc composites. 3.2.4. Antibacterial Properties

In Figure 14, the antimicrobial effect against £. coli is presented as a function of time for the different composites. Without ZnO particles, the reference concentration of the microorganism is measured to be ~2 x 10 ⁶ . After 1 h, no change in the concentration was observed for all samples. By increasing the time, a decrease in the E.coli concentration is observed for the composites. The effect is more evident for the iPP/5%ZnOc composite. Significant variations in concentration are observed increasing the contact time and ZnOc content. After 24 h, the concentration of £. coli decreases to 8.8 x 10 ⁵ for iPP/2%ZnOc and 1.7 x 10 ⁵ for iPP/5%ZnOc. After 48 h, the bacterial concentration was significantly decreased for the iPP/5%ZnOc sample (2 x 10 ³ CFU/mL). The sample iPP/2%ZnOc reaches similar values after five days.

The values of percentage reduction (%R) off. coli for all samples at different contact times are reported in Table 5. Neat iPP exhibits no bactericidal activity, and R was observed to be zero up to day 10. The iPP/5%ZnOc composite exhibited maximum reduction, 99.9%, after 48 h.

Table 5. Percent reduction of E.coli at different times of contact with films of iPP and iPP/2%-5% ZnOc.

In Table 6 a comparison of the bacterial activity of the three systems, iPP/ZnO, iPP/ZnOc and IPP/PPgMA/ZnO, at the same ZnO content (2%) is reported. From this table, it is clearly confirmed that;

SUBSTITUTE SHEET (RULE 26) • in the «ΡΡ /ZnOc system the ZnOc particles maintain their antibacterial properties against £. colt, with respect to the uncoated particles;

« in the system iPP/PPgMA/ZnO, the ZnO particles that are linked to the maleic anhydride groups of PPgMA [24], do not display similar antibacterial activity at least up to 48 h. Probably, the PP chains of the PPgMA, due to the link between MA and ZnO, cover the ZnO particles and hinder the antibacterial activity.

Table 6. Percent reduction of f. coli at 48 hand 5 days of contact for iPP and different films at 2% ZnO, (adapted from Table 5 of this paper and references [22,24]).

The results obtained from the analysis of antibacterial properties allow us to conclude that the particles of ZnO with stearic acid have relevant antibacterial property against E. coli, similar to that of ZnO and that the coating of the particles does not have a negative influence as the coating of the particles with PPgMA [24],

4. Conclusions

This work had as its final objective the preparation of the film based on the isotactic polypropylene matrix intended for packaging food, with improved properties by the addition of ZnO particles coated with stearic acid (ZnOc). The latter has the function of compatibilization between the inorganic metal oxide particles phase and the organic matrix of the isotactic polypropylene. The samples were prepared in a twin-screw extruder and then filmed by a compression molding.

It was observed that the stearic acid coating on the ZnO particles reduces the surface polarity mismatch between iPP and ZnO and allows the formation of a composite with fair distribution of particles.

The principal achievement of the novel composites is the strong antibacterial activity against f. coli : the bacterial concentration decreases with increasing concentration of ZnOc and the contact time between the film and the bacterial solution. After 48 h, the bacterial reduction was significantly decreased for the sample containing 5% of ZnOc (R = 99.99%); for the sample iPP/2% ZnOc, it reaches these values after five days.

Moreover, the iPP/ZnOc composites present improvement of the thermal stability, tensile parameters (Young modulus and stress at the yield point) and impact properties with respect to neat iPP,

SUBSTITUTE SHEET (RULE 26) iPP/ZnO, and iPP / PPgMA/ZnO at least for samples containing 2% ZnO [22,24]. It also has to be underlined that the films containing ZnOc show an absorption in the region around 385 nm, confirming that the ZnOc particles also have a shielding effect to UV radiation as those of ZnO as reported in the literature.

On the base of the results obtained, it can be stated that the methodology proposed (using of novel ZnO particles obtained by spray pyrolysis; coating the ZnO with stearic acid and optimization of the processing conditions) is innovative, because no literature is available (at our knowledge) on the properties of such an iPP/ZnOc system prepared directly by melt mixing. Moreover, it is also very efficient in preventing formation of agglomerated domains and in providing a system with improved properties.

In conclusion, the composites iPP/5% ZnOc films have relevant antibacterial property against E. coli, higher thermal stability and improved mechanical and impact properties than the pure «ΡΡ film so that they are suitable for application in the food industry as active packaging films.

Acknowledgments; The research described herein was partially supported by the European Community's Seventh Framework Programme (ERA-Net Susfood-CEREAL "Improved and resource efficiency throughout the post-harvest chain of fresh-cut fruits and vegetable"), Italian Health Ministry (Progetti Ricerca Corrente/ 2013 "impiego di nanopackaging innovativo ne!la filiera della carne: valutazione deH'efficacia antibatterica e della sicurezza d'uso"), and Italian Ministry of Foreign Affair (Bilateral Project Italy / Quebec 2014-2016 "Sviluppo di nanomateriali ecosostenibiii per l'lmballaggio alimentare adatti alia sterilizzazione per radiaztone"). Jannette Dexpert-Ghys and Marc Vereist from The Centre d'Elaboration de Mat6riaux et d'Etudes Structures (CEMES-CNRS), Toulouse-France, are kindly thanked for supplying ZnO particles obtained by spray-pyrolysis. Ida Romano of the Istituto di Chirnica Biomoiecoiare (CNR), Pozzuoli (NA) Italy is kindly thanked for performing the antimicrobial tests.

Author Contributions; Clara Silvestre and Sossio Cimmino supervised the research program. Clara Silvestre, Sossio Cimmino and Donatella Duraccio designed the setup. Antonei!a Marra, Valentina Strongone and Donatella Duraccio performed the experiments. All authors contributed to the analysis of the presented experiments and correlation of the different means of investigations. Antoneila Marra and Clara Silvestre wrote the initial draft. Clara Silvestre and Sossio Cimmino coordinated the revisions of the draft in the final form.

Conflicts of Interest; The authors declare no conflict of interest.

References, incorporated by reference to this patent application and relied upon;

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© 2016 by the authors; licensee MDPi, Basel, Switzerland. Modified to remove graphics. This article is an open access article distributed under the terms and conditions of the Creative Commons by Attribution (CC-BY) license (http:/ /creativecommons.org/licenses/by/4.0/).

SUBSTITUTE SHEET (RULE 26) Appendix B

Study Title

Test of floor interaction with shoes in reference to acquired and deposited bacteria. Test Method Utilizing Ensure Touch meter to gauge bacteria! activity on surfaces.

Test Location

Baylor Scott & White Health 4700 Alliance Blvd Piano, TX 75093

Test Dates: August 19-21, 2021 Abstract

This study was constructed in order to determine if shoes pick up, carry and deposit pathogens, specifically bacteria. Shoes were cleaned and worn through a busy hospital for 20 minutes. Directly afterwards, the shoes walked in a freshly cleaned floor area for 10 minutes. "SAFE STEPS"™ films were then applied to determine if they removed bacteria from the floor area. Based on the averaged results from 6 tests, the floor area CFU count went up 107% after walking with dirty shoes for 20 minutes, aka. deposition, then dropped by 46% after "SAFE STEPS"™ walked in the area for 10 minutes. Hygiena Ensure Touch Meter

This meter was chosen for 2 major advantages. 1. It is a commonly used meter for measuring cleanliness of surfaces in the food and beverage industry, health care and education. 2. It is the most sensitive instrument in its class. We are using the "ULTRASNAP"™ swabs to test for ATP, or any organic material which includes bacteria, and any other organic matter that could eventually be bacteria food. The ATP test is to check the cleanliness of the floor and shoes prior to running the study. We are using the "MICROSNAP"™ Total Viable Count swabs to test for the total bacteria colony count. The bacteria tests require an extra incubation step before results can be seen.

Visual Data Acquisition Summary

The procedure of data acquisition according to the study is shown in Figure 15.

Data Acquisition Summary

1. A 2'x3' Floor Area (FA) was selected in a hospital corridor and was taped off with blue masking tape.

2. Camera was placed on a tripod and video recorded the entire proceedings.

3. The FA was cleaned multiple times with 70% IPA and paper towels to achieve an ATP count of 100 or

SUBSTITUTE SHEET (RULE 26) less. (For healthcare public areas, 100 is the maximum allowable limit for waiting room furniture, handrails, doors and restrooms.)

4. FA was swabbed in multiple areas for bacteria.

5. The shoe soles were cleaned with 70% IPA and paper towels. Shoes were ATP tested to be under the limit of 100.

6. Shoes were swabbed for bacteria.

7. Dr. Josh Lemon put on the shoes, and walked throughout the hospital for 20 minutes.

8. Upon return, Dr. Josh Lemon walked in the clean taped area for 10 minutes.

9. FA was divided in 2, F11 and FA2.

10. FA1 was tested in multiple areas for bacteria,

11. Shoes were swabbed for bacteria.

12. Paul Siragusa walked in FA2 with the "SAFE STEPS""* film applied to the shoe soles for

10 minutes

13. “SAFE STEPS"™ films on the shoes were swabbed for bacteria.

14. FA2 was tested in multiple areas for bacteria.

15. "SAFE STEPS"™ films were removed from the shoes.

16. Shoes were swabbed for bacteria.

17. All samples were marked and stored in a cooler below 40f.

Incubation Summary

1. Samples were removed from the cooier and allowed to return to room temperature for

10 minutes.

2. Samples were put into order.

3. Sample tops were snapped and the bulbs were squeezed, before being placed in the Hygiena Dry Block

Incubators,

4. Samples were allowed to incubate for 7 hours at 86f.

5. The reagent tubes were taken out of the cooler and allowed to return to room temperature for 10 minutes.

6. Samples were removed from the incubator; tube was squeezed a few times to get the mix into the bulb.

7. Sample tube liquid was squeezed into reagent tube.

8. Reagent tube bulb was snapped and squeezed,

9. Tube was shaken for a count of 10.

10. Reading was taken.

SUBSTITUTE SHEET (RULE 26) Data Analysis Summary

1. 6 floor samples were taken for each reading.

2. Due to the imperfect nature of the study, the 2 outliers were removed from each data set. Outliers were classified as any reading that diverged from the mean CFU count by over 100 CPU's.

3. The remaining 4 sample readings were averaged into each data point,

4. Each data set has the following 3 data points: Floor before walking, Floor after walking, Floor after

"SAFE steps"TM.

5. All the 6 data sets were compiled into line and bar graphs.

6. Average % change and average CFU for all 6 data sets were included for reference.

Results of "SAFE STEPS"™ Hospital Floor Study - Data

The following tables 7 to 9 show the results of the study. In particular, table 7 shows the average CFU for each of the 6 data sets, table 8 shows the average % changes of the 6 data sets and table 9 shows the average CFO of the 6 data sets,

SUBSTITUTE SHEET (RULE 26)

The results of the study are shown in the chart as shown in Figure 16. Figure 17 shows the floor area of the study.

Figures 18A and 18B show the shoes of the study.

Figure 19 shows the incubation of the study.

SUBSTITUTE SHEET (RULE 26) Appendix C

Study Title

Test of shoe Interaction with "SAFE STEPS"™ in reference to acquired and deposited bacteria. Test Method

Utilizing Ensure Touch meter to gauge bacterial activity on surfaces.

Test Locations Food Court in Mall Monmouth County, NJ Supermarket Monmouth County, NJ

Test Date: August 29, 2021 Abstract

This study was constructed in order to determine if "SAFE STEPS"™ protects shoes from shoes picking up, carrying and depositing pathogens, specifically bacteria. Shoes were cleaned before testing and tested for bacteria. "SAFE STEPS"™ was applied to the bottom of the right shoe. The left shoe was not modified. The shoes were worn through a busy food court for 30 minutes, with some time spent in the bathroom. Directly afterwards, the "SAFE STEPS"™ film was removed and the shoes were tested for bacteria. This was repeated at a busy supermarket.

Hygiena Ensure Touch Meter

This meter was chosen for 2 major advantages. 1. it is a commonly used meter for measuring cleanliness of surfaces in the food and beverage industry, health care and education. 2. It is the most sensitive instrument in its class. We are using the Ultrasnap swabs to test for ATP, or any organic material which includes bacteria, and any other organic matter that could eventually be bacteria food. The ATP test is to check the cleanliness of the floor and shoes prior to running the study. We are using the "MICROSNAP"™ Total Viable Count swabs to test for the total bacteria colony count. The bacteria tests require an extra incubation step before results can be seen.

Visual Data Acquisition Summary

The procedure of data acquisition according to the study is shown in Figure 20.

SUBSTITUTE SHEET (RULE 26) Data Acquisition Summary

1. Flat shoes were selected so the shoe soie surface will be similar to the film.

2. Shoes were disinfected for 10 minutes with 12% H202 and then cleaned with 90% IPA.

3. "SAFE STEPS"™ was applied to one shoe.

4. Since this strictly an indoor surface test, the shoes were hand carried to the entrance of the food court and supermarket, then put on.

5. Walked in the facility wearing the shoes for 30 minutes total, including the bathrooms.

6. Shoes were tested for bacteria.

7. Shoes were tested for ATP. (This was the secondary test, so bacteria was done first over the same 2 areas of the foot: toe and heel.) incubation Summary

1. Samples were removed from the cooler and allowed to return to room temperature for

10 minutes.

2. Samples were put into order.

3. Sample tops were snapped and the bulbs were squeezed, before being placed in the Hygiena Dry Block incubators.

4. Samples were allowed to incubate for 7 hours at 86f.

5. The reagent tubes were taken out of the cooler and allowed to return to room temperature for 10 minutes.

6. Samples were removed from the incubator; tube was squeezed a few times to get the mix into the bulb.

7. Sample tube liquid was squeezed into reagent tube.

8. Reagent tube bulb was snapped and squeezed.

9. Tube was shaken for a count of 10,

10. Reading was taken. Data Analysis Summary

1. 2 shoe samples, toe and heel, were taken per reading and averaged. The results are shown in the tables 10, 11, 12 and 13. In particular, table 10 shows "MICROSNAP"™ Total Viable Count Swabs (Total Bacteria), table 11 shows Ultrasnap Swaps (Total ATP), tables 12 shows "MICROSNAP"™ Total Viable

SUBSTITUTE SHEET (RULE 26) Count Snap (Total Bacteria) including the indication in % Change and table 13 shows Ultrasnap Swaps (Total ATP) including the indication in % Change.

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