CA3208941A1 - Accelerated neutral atom beam (anab) modified polypropylene for reducing bacteria colonization without antibiotics - Google Patents

Abstract

Surfaces of a surgical implant material are modified with an accelerated neutral atom beam, surface properties of the modified material are characterized, and a reduction of wide range of bacteria colonization on such surfaces is achieved without using antibiotics.

Description

ACCELERATED NEUTRAL ATOM BEAM (ANAB) MODIFIED POLYPROPYLENE
FOR REDUCING BACTERIA COLONIZATION WITHOUT ANTIBIOTICS
CROSS REFERENCE TO RELATED APPLICATIONS
The present application claims priority to US Provisional Patent Application Number, 63/140,513 entitled Accelerated Neutral Atom Beam (ANAB) Modified Polypropylene For Reducing Bacteria Colonization Without Antibiotics, which was filed on January 22, 2021, the content of which is incorporated herein by reference in its entirety FIELD OF TECHNOLOGY
The present application is in the field of accelerated neutral atom beam technology, and more particularly related to the field of modifying the surface characteristics of orthopedic medical devices to reduce harmful contamination of the surfaces.
BACKGROUND
Since the discovery of penicillin back in 1928 by Sir Alex Flemings, hundreds of new antibiotics have been developed with the aim to fight bacterial infections, especially those related to implantable devices. However, the overuse and misuse of antibiotics trigger genetic modification in bacteria, leading to the development of one of the most disturbing health concerns that society is facing nowadays: antimicrobial resistance (AMR) to antibiotics. Currently, the Centers for Disease Control and Prevention (CDC) indicates that there are around 14 bacterial strains ranked as urgent or serious threats, meaning that there are only a few available antibiotics to treat their drug-resistant phenotypes. For instance, Staphylococcus aureus (SA), commonly found on skin, developed resistance to beta-lactam antibiotics, such as methicillin, hence leading to the appearance of Methicillin-resistant Staphylococcus aureus (MRSA), one of the most proliferative killers in the healthcare system. Currently, 2 out of 100 people carry MRSA
leading to around 94,360 invasive infections which are diagnosed annually in the U.S., with 18,650 associated deaths, more than the number of deaths by AIDS/HIV. Data from the CDC shows that 65% of all reported staphylococcus infections in the US are caused by MRSA, which represents a 300%
increase in 10 years. But healthcare problems do not rely with MRSA alone as the CDC has also predicted that by 2050 more deaths will occur from all AMR bacteria than all cancers combined, leading to one person dying every 3 seconds from AMR. Therefore, there is an urgent need to find new approaches to defeat AMR, which are far away from the current use of antibiotics.
Polypropylene is the most commonly used biomaterials for surgical hernia meshes. Ventral hernia repair is one of the most common surgical procedures worldwide. The incidence of surgical site infection (S SI) associated with wound or mesh complications has been reported to be as high as 27.7% in open surgical procedures and 10.5% laparoscopically.
SSI lead to very high costs associate with the mesh infection hospital cost as well as follow-up costs.
While the average cost of a mesh-based hernia repair without complications in the US is approximately $38,700 plus $1,400 in follow-up costs, mesh infections could raise the total charges to $82,800 in hospital costs and $63,400 in follow-up costs.
Decreasing bacterial infections of implanted mesh materials is therefore extremely important.

It would be desirable to develop improved techniques for reducing bacterial infections on implanted biomaterials. Aspects of the present disclosure describe techniques for modification of biomaterial surfaces to render the surfaces more resistant to bacteria attachment. This improves the likelihood that bacteria present on the surfaces can be cleared by the immune system of a subject patient.
Accelerated Neutral Atom Beam (ANAB) technology is a low energy accelerated particle beam gaining acceptance as a tool for easy nano-scale surface modification of implantable medical devices. ANAB is created by acceleration of neutral argon (Ar) atoms with very low energies under a vacuum which bombard a material surface, modifying it to a shallow depth of 1-3 nm.
This is a non-additive technology that results in modifications of surface topography, wettability, and surface chemistry. These modifications are understood to be important in cell-surface interactions on implantable medical devices since they change surface energy and initial protein interactions for which cells rely on to adhere. Controlling surface properties of biomaterials is vital in improving the biocompatibility of devices by enhancing tissue integration and reducing bacterial attachment.
SUMMARY
Aspects of the present disclosure utilize Accelerated Neutral Atom Beam (ANAB) technology to modify polypropylene to inhibit bacteria colonization in vitro after 24 hours without the use of drugs or antibiotics. As described herein an ANAB was designed and used to increase the surface energy of polypropylene to be closer to that of two critical proteins (mucin and casein) contained in bodily fluids that if adsorbed to a material surface can decreased bacteria colonization.
Materials as characterized using atomic force microscopy demonstrate an expected greater surface roughness and surface area for the ANAB-treated samples compared to controls. A wide range of gram- positive, gram-negative, and antibiotic resistant bacteria were tested here (including Staph. epidermidis, Staph. aureus, MRSA, multi-drug resistant E.
coli, and Pseudomonas aeruginosa) and demonstrated on average an over a 3-log reduction in bacteria after 24 hours. Further, this study confirmed a greater adsorption of mucin and casein on ANAB-treated polypropylene as the mechanism to decrease bacteria colonization.
Lastly, this study utilized an aggressive cleaning procedure and showed strong durability of the ABAN-treated surfaces. This study is important as it demonstrates a way to potentially decrease polypropylene based implant infections using ANAB modification without using antibiotics.
DETAILED DESCRIPTION
Aspects of the present disclosure provide a method for reducing bacterial colonization on a polypropylene surface of a surgical mesh implant. In an illustrative embodiment, the method includes steps of generating an accelerated neutral atom beam (ANAB) with an energy level selected to impart a predetermined increase in surface energy to a polypropylene surface of a mesh implant. The predetermined increase in surface energy is selected to alter the surface

2 energy of the polypropylene surface to approach the surface energy of mucin and/or casein proteins contained in bodily fluids that when absorbed to the polypropylene surface decrease bacteria colonization.
In the illustrative embodiment, generating the ANAB includes flowing argon gas at 200 standard cubic centimeters per minute (SCCM) through a 100 mm diameter nozzle to create weakly bonded clusters of between 100 argon atoms and 10,000 argon atoms. The weakly bonded clusters of argon atoms are then impacted with electrons to ionize the clusters to a charge of +1 or +2. The ionized clusters are then subj ected to a first electrostatic field having a field strength of 30 kilovolt configured to accelerate the ionized clusters. The accelerated clusters is then broken apart by colliding the accelerated clusters with residual argon atoms in the path of the accelerated clusters. Subsequent to breaking apart the accelerated clusters, the accelerated clusters are subjected to a second electrostatic field configured to deflect remaining ionized portions of clusters from the ANAB path and allow neutral argon atoms from the clusters to maintain a predetermined momentum along the ANAB path. In the illustrative embodiment, the surface is irradiated with an effective ANAB dose of about 2.5 x 1017 argon atoms per cm2, which modifies the surface to a depth of between 1 nanometer and 3 nanometers.
Another aspect of the present disclosure provides a method for reducing contamination of a polypropylene surface. The method includes generating an accelerated neutral atom beam (ANAB) with an energy level selected to impart a predetermined increase in surface energy to a polypropylene surface of an object to be irradiated with the ANAB, and irradiating the polypropylene surface of an object with the ANAB. The polypropolyne object can be a surgical mesh such as a hernia mesh implant, for example.
According to an aspect of the present disclosure the predetermined increase in surface energy is selected to alter the surface energy of polypropyene to approach the surface energy of one or more proteins contained in bodily fluids that when absorbed to the surface decrease bacteria colonization. Examples of such proteins include mucin and casein, for example.
According to an illustrative embodiment, the method may include accelerating neutral argon atoms with very low energies under a vacuum and bombarding the surface with the neutral argon atoms. In the illustrative embodiment the wherein the bombarding modifies the surface to a depth of between 1 nanometer and 3 nanometers.
According to another aspect of the present disclosure, the method for generating the ANAB
include flowing argon gas through a nozzle to create weakly bonded clusters of argon atoms, In an illustrative embodiment, the argon gas flow is provided at about 200 standard cubic centimeters per minute (SCCM) through a nozzle having a diameter of about 100 mm. The weakly bonded clusters consist of between 100 argon atoms and 10,000 argon atoms.
The method then includes impacting the weakly bonded clusters of argon atoms with electrons to ionize the clusters and subjecting the ionized clusters to a first electrostatic field configured to accelerate the ionized clusters. In the illustrative embodiment, impacting the weakly bonded

3 clusters of argon atoms with electrons ionize the clusters to a charge of +1 or +2. In an illustrative embodiment, the first electrostatic field has a strength of about 30 kilovolts.
In the illustrative embodiment, the method then includes breaking apart the accelerated clusters by colliding the accelerated clusters with residual argon atoms in the path of the accelerated clusters. Subsequent to breaking apart the accelerated clusters, the method includes subjecting the accelerated clusters to a second electrostatic field configured to deflect remaining iononized portions of clusters from the ANAB path and allow neutral argon atoms from the clusters to maintain a predetermined momentum along the ANAB path.
According to an aspect of the present disclosure, subsequent to forming the ANAB, the surface of the object is irradiated with an effective ANAB dose of about 2.5 x 1017 argon atoms per cm2.
Examples:
Commercial grade polypropylene sheets (0.75 mm thick; Misumi Plastics) were cut into 12 mm diameter disks and cleaned in 70% isopropanol for 30 min followed by 3 x 15 min washes in deionized H20. Polypropylene was prepared as a control or treated by ANAB
using argon (Ar) gas on an accelerated particle beam system (nAccel 100, Exogenesis Corp.) with a deflector to remove charged clusters as described in detail previously. Briefly, Ar gas was flowed at 200 SCCM through a 100 mm diameter nozzle to create weakly bonded clusters consisting of a few hundred to a few thousand Ar atoms. These clusters are then impact ionized by electron impact ionization resulting in a +1 or +2 charged cluster which is then accelerated by introducing it to a 30-kV electrostatic field. Once accelerated, the cluster is then immediately broken apart by orchestrating its collisions with residual Ar gas atoms present along the beam path in the acceleration chamber. These collisions break the weak van der Waals bonds thus releasing individual neutral atoms along with smaller, charged clusters. The remaining clusters are then pushed away with an electrostatic deflector allowing the neutral atoms to maintain their initial momentum until they reach and collide with the material surface. The effective dose of the ANAB was 2.5 x 1017 Ar atoms per cm2.
An important objective of the present study was also to assess how durable the ANAB-treated surfaces were to minimize bacteria colonization. For this, some samples were cleaned by serially soaking and sonicating in acetone and ethanol for 10 minutes each, respectively, and were re-used in the surface characterization, bacteria, and protein adsorption experiments Contact angle measurements & surface energy calculations Samples were characterized for surface energy and bacteria functions. Nine replicates were selected corresponding for each sample type and placed into 12-well plates.
The well plates containing the coupons were subsequently transferred to a clean room equipped with a Phoenix 150 Contact Angle Analyzer. A three-solvent system, i.e., deionized water, ethylene glycol, and glycerol, was adopted for evaluating the surface energies of the coupons.
Specifically, 1611.1 per solvent were dropped onto the coupon surfaces in triplicate for each of the coupon identities, and images were obtained after 2 s. Contact angles were measured using the DropSnake plugin on Fiji. The surface energy of each substrate was determined by applying the Owens/Wendt theory

4 in tandem with contact angle data and solvent surface tension values, of which the latter were obtained from the literature. The Owens/Wendt model structurally follows the mathematical formulation shown in Equation I below, where a LP and GLP are the dispersive and polar components, respectively, of the wetting liquid's surface tension, where asp and asP are the dispersive and polar components, respectively, of the substrate's surface energy, and where 0 is the contact angle that the solvent makes with the substrate surface. The goal was to use ANAB to modify the polypropylene surface until a surface energy close to the surface energy of two endogenous proteins known to reduce bacteria colonization (mucin and casein) was achieved.
CrL(COS0+1) (015)1/2 Crl. 1/2 ,TIJ 1/2 Equation I. Owens/Wendt theory 2(4)1/2 D1/2 ='S
o-L
Atomic force microscopy (AFM) AFM measurements were taken using a Park Systems XE-70 instrument in non-contact mode.
Silicon tips with a resonant frequency of ¨330 kHz and a force constant of 42 N/111 were used (PointProbeg Plus, Nanosensors). 1 1112 regions of the polypropylene were imaged and the arithmetical mean roughness (Ra) and ten-point mean roughness (Rz) was measured across this region.
Bacterial assays Colony forming units Standard colony counting procedures were implemented for determining bacterial functions on the polypropylene coupons for supporting bacterial attachment and proliferation. Staphylococcus epidermidis (ATCC 35984), methicillin-resistant Staphylococcus aureus (MRSA;
ATCC 43300), Staphylococcus aureus (ATCC 25923), multi-drug resistant Escherichia coli (E.
coli; ATCC
25922) and Pseudomonas aeruginosa (P. aeruginosa; ATCC 27853) were obtained from the American Type Culture Collection and cultured overnight in 4 ml of a 3%
Tryptic soy broth (TSB) solution.
After a minimum of 16 hours inside a shaking incubator which operated at 37 C
and 110 rpm, the bacteria were diluted in TSB (inside a sterile Class II biological safety cabinet) to a concentration ofl x 109 CFU/ml. Bacterial concentrations were measured using a SpectraMax M3 series plate reader, whereby an absorbance output reading of 0.52 at the wavelength ka = 562 nm corresponded to a bacterial density ofl x109 CFU/ml. The microbial suspensions were diluted further in TSB to a concentration of lx1 06 CFU/ml, which were used to treat the coupons inside separate 24-well plates. Surfaces were sterilized, decontaminated and cleaned using 70%
ethanol.
After the samples were inoculated with 1 ml ofl x106 CPU/ml bacteria, the 24-well plates were left, over a period of 24 hours, inside a stationary incubator with internal conditions of 37 C and

5% CO2. After 24 hours of incubation, the plates were removed, consecutively, from the controlled environment and washed gently with 1 ml of sterile phosphate buffered saline (PBS) to remove unattached and non-adherent bacteria from the sample surfaces. The coupons were carefully removed, using sterile spatulas, from the initial wash solution and immersed in 1 ml of sterile PBS, which had been pre-injected into the wells of new 24-well plates.
The coupons were washed once more with 1 ml of sterile PBS (3x washes total per sample) and distributed into polypropylene conical tubes containing 10 ml of sterile PBS. The tubes and their contents were subsequently agitated using a Branson water bath sonicator for 15 min. This facilitated the detachment of bacteria from the coupon surfaces, and the resulting suspensions were serially diluted 10 - 106x. 10 .1 of each dilution were dropped, in triplicate, onto Trypticase soy agar (TSA) plates, and left to air dry in a sterile BSC-II. After complete deposition of the bacteria onto the TSA, the plates were lidded, inverted (to disable condensate from washing away or disturbing the bacterial colonies), and placed inside a stationary incubator (37 C, 5% CO2). The plates were removed from the incubator after 15 hours, and the bacterial colonies were counted manually, with the assistance of the Cell Counter plugin on Imagek .. Live/dead and crystal violet assays For the live/dead assay, at the end of the prescribed time period, the substrates were vortexed for 60 seconds in a Tris-buffered saline (TBS) solution comprised of 42 mM Tris-HC1, 8 mM Tris Base, and 0.15 M NaCl (Sigma Aldrich). Samples were then incubated for 15 minutes with the BacLight Live/Dead solution (Life Technologies Corporation, Carlsbad, CA) dissolved in TBS
at the concentration recommended by the manufacturer. Substrates were rinsed twice with TBS
and placed into a 50% glycerol solution in TBS prior to imaging. Substrates were checked by staining with the BacLight Live/Dead staining procedure mentioned above to ensure that all of the bacteria were removed during vortexing, After it wi'ls found that all the hacteria were removed from the substrate, each vortexing solution was combined and tested for live/dead bacteria using the procedure outlined above. Similar volumes were maintained for all samples to ensure the same dilution. It has been found that when bacteria are stained via the BacLight Live/Dead stain they can still be subsequently by stained by crystal violet, adding a third way bacteria colonization was assessed in the present study. For this, bacteria were visualized and counted using a Leica DM5500 B fluorescence microscope with image analysis software captured using a Retiga 4000R camera. Using standard techniques, separate aliquots of the vortexed bacteria solution were also obtained and tested for crystal violet.
Mechanisms of bacteria colonization Protein adsorption experiments Samples were soaked in the bacteria culture media described above for 24 hours. At the end of the prescribed time period, proteins were desorbed from the surface by soaking samples in 10%
SDS for 5 minutes. All samples were checked to ensure all proteins were removed through this soaking. The protein eluant supernatant was then analyzed using ELISA assays to determine which proteins adsorbed and how much adsorbed, with a special focus on mucin, lubricin, and casein which are all proteins known to reduce bacteria attachment.
Correlation of absorbed proteins to bacteria attachment inhibition

6 Lastly, to correlate the increased adsorption of key proteins from the bacteria culture media to the samples of interest for inhibition of bacteria colonization, samples were first coated with various concentrations (from 1 microgram/ml to 100 micrograms/nil) of proteins that demonstrated an increased adsorption trend on the sample through simple soaking for 1 hour.
Proteins were purchased from Sigma. Then, the bacteria experiments mentioned in the bacteria experiments section above were conducted on the protein pre-adsorbed samples.
Since the polypropylene samples were created to have a surface energy close to that of mucin and casein, it was expected that we would measure decreased bacteria adsorption on the ANAB-treated samples that adsorbed more anti-bacterial adhesive proteins, thus, providing a mechanism of why .. the samples decreased bacteria attachment.
Statistical analysis All cell experiments were run in triplicate and repeated a minimum of three times per substrate type. Numerical data were analyzed using Analysis of Variance (ANOVA); values of p < 0.05 were considered significant. Duncan's multiple range tests were used to determine differences between means.
Contact angle measurements & surface energy calculations Following from the contact angle analyses section, the angles that 16 .1 droplets of deionized water, ethylene glycol, and glycerol formed with the solid interfaces were quantified, and the results are tabulated and summarized graphically in Table 1. The ANAB-treated sample was .. significantly 3more wettable compared to the untreated control, as designed.
Table 1. Contact angle values (degrees)for Si coupons using the three-solvent system:
deionized water (DI-H20), ethylene glycol ((CH2OH)2), and glycerol (C3H803).
Data=
mean+standard error of the mean. N= 3.
Sample DI H20 (CH2OH)2 C3Hs03 Control 87.34+/-1 .44 68.44+/-1.39 81.39+/-1.26 ANAB-treated 70.83+/-1 .36 53.00+/-0.89 76.70+/-0.71 sample The contact angle results were translated into surface energies for the tested coupons by application of the Owens/Wendt theory in linear form as described above.
Previously reported surface tensions of the solvents at room temperature were applied. For deionized water, ethylene .. glycol, and glycerol, these were amo = 72.8 mN/m (amoD =26.4 mN/m, aH2013=46.4 mN/m), G(CH2OH)2 - 47.7 mN/m, G(cF2oi-)2D¨(26.4 mN/m, a(cH2oH)2P =21 .3 mN/m), and GC3H803- 63.4 mN/m (ac3H8o3D=37.0 mN/m, ac3H8o3P=26.4 mN/m). After fitting the three-solvent results to the Owens/Wendt equation, the slope and the y-intercept of the resulting linear trendlines corresponded to the square roots of the polar and dispersive components of the coupon surface .. energies, and total surface energies were approximated by summation of asp and Gs. These values are shown in Table 2. Results showed that the ANAB-treated samples has significantly

7 greater surface energy, in fact, very close to the optimal surface energy of mucin and casein previously found to maximally inhibited bacteria colonization (40.2 mN/m).
Table 2. Surface energy values (mN/m) for the Si coupons, as determined by application of the Owens/Wendt equation, broken into polar (ifs") and di, persive (cr5,13) components.
Sample Gsifs 0-S1 (mN/m) (mN/m) Control 21.73 7.72 14.01 ANAB-treated 35.58 31.56 4.02 sample Atomic force microscopy analysis Results of the present study demonstrated significantly decreased, nanoscale surface roughness, as measured by atomic force microscopy for the ABAN-treated compared to control samples (Figures 1A-B). Specifically, the Ra values decreased from 5.29 nm 0.348 nm to 3.80 nm 0.14 nm on ANAB- treated compared to controls; respectively. Similarly, the Rz values decreased from 56.02 nm 2.78 run to 45.04 nm 5.25 run on ANAB-treated compared to coupons, respectively.
Figures 1A-B shows AFM imaging of the nanotextured surface on the ANAB-treated coupons (B) as compared to the as-received control coupons (A). Surface roughness as measured by Ra decreased from 5.29 nm 0.348 run on the control to 3.80 run 0.14 nm on the ANAB-treated samples, p<0.025; Rz decreased from 56.02 nm 2.78 run on the control to 45.04 run 5.25 nm on the treated coupons, p=0.135.
Bacterial assays Bacterial adhesion to the various polypropylene coupons was evaluated using the plating technique described previously. Impressively, all bacteria colonization decreased on the ANAB-treated samples compared to controls after 24 hours no matter if they were drug-resistant bacteria or gram positive bacteria or gram negative bacteria as shown in Figures 2, 3, and 4 for colony forming units, live/dead assays, and crystal violet staining. Specifically, for the colony forming results, counts (in 105 cells/cm 2) went from 3.1 to 0.003 for Staph epi, 5.5 to 0.0011 for Staph aureus, 0.8 to 0.0003 for MRSA, 70 to 0.015 for drug resistant E. coli, and 62 to 0.019 for Pseudomonas aeruginosa on ANAB-treated versus controls after 24 hours.
Moreover, while most of the cells were alive on both samples, there were less living cells on the ANAB than control samples, making the total number of living bacteria even less on the ANAB-treated samples.
Such results are significant since no drug or anti-biotic coating was used in the study and typically, different antibiotics are needed for gram positive versus gram negative bacteria. In this

8 study, the same nanoroughing approach via the ANAB-treatment significantly decreased both gram negative and gram positive bacteria close to values typically seen for antibiotics.
Figure 2 shows Colony counting data aper 24 h treatment by Staphylococcus epidermidis, Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus, drug resistant Escherichia coli, and Pseudomonas aeruginosa. Data = mean standard deviation: N=3,= all ANAB-treated samples were significantly less (p<0.01) than controls and the log reduction is indicated directly above the respective bacteria. Numbers indicated respective colony forming units/square cm on ANAB-treated samples.
Figure 3 shows live/dead data after 24 h treatment by Staphylococcus epidermidis, Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus, drug resistant Escherichia coli, and Pseudomonas aeruginosa. Data = mean standard deviation: N=3,= all ANAB-treated samples were significantly less (p<0.01) than controls.
Figure 4 shows crystal violet staining data after 24 h treatment by Staphylococcus epidermidis, Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus, drug resistant Escherichia coli, and Pseudomonas aeruginosa. Data = mean standard deviation: N=3; all ANAB-treated samples were significantly less (p<0.01) than controls.
SEM analysis of bacteria colonization Results of the present study also confirmed the significantly less S. aureus colonization on the ABAN-treated compared to control samples after 24 h of culture (Figures 5A-B).
Figures 5A-B shows SEM imaging (2000X magnification) of S. aureus attachment on control (A) and ANAB-treated (B) polypropylene coupons. Treatment shows a marked reduction of bacteria on the surface after 24 h. Bar represents 30 p.m.
Mechanisms of bacteria colonization Results of protein adsorption studies showed significantly greater levels of both mucin and casein adsorption of ANAB-treated samples compared to controls (0.9 pg/m1 compared to 0.1 pg/m1 for mucin and 0.4 pg/m1 compared to 0.1 pg/m1 for casein respectively).
There were no differences observed for another key protein which decreases bacteria colonization, lubricin.
Lastly, results supported significantly less bacteria colonization after 24 hours onto ANAB-treated samples coated with mucin and casein compared to controls (Figures 6 and 7).
Collectively, this provided evidence that ANAB-treated samples increased the adsorption of mucin and casein which in turn inhibited bacteria colonization. Both mucin and casein have surface energies around 40 mN/m.
Figure 6 shows colony counting data after 24 h treatment by Staphylococcus epidermidis, Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus, drug resistant Escherichia coli, and Pseudomonas aeruginosa on mucin pre-adsorbed ANAB-treated samples and control samples. Data = mean standard deviation: N=3; all AHAB-treated samples were significantly less (p<0.01) than controls.

9 Figure 7. Colony counting data after 24 h treatment by Staphylococcus epidermidis , Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus, drug resistant Escherichia coli, and Pseudomonas aeruginosa on casein pre-adsorbed ANAB-treated and control samples.
Data = mean standard deviation: N=3; all ANAB-treated samples were significantly less (p<0.01) than controls.
Lastly, as can be observed by the low standard deviations throughout this study, it was clear that the cleaning procedure employed did not significantly alter surface characterization, bacteria colonization, and the measured mechanism of action; although more studies are required, this implies a strong surability of the ABAN-treated samples.
Conclusions Results of this study showed that polypropylene can be treated by Accelerated Neutral Atom Beam (ANAB) to generate a surface energy close to the surface energy of key proteins contained in the body (mucin and casein) to in turn inhibit bacteria colonization after 24 hours, all without resorting to the use of antibiotics. Such results are significant as they were demonstrated for both gram positive, gram negative, and multi-drug resistant bacteria.
What is claimed is:

10

Claims (13)

PCT/US2022/013388

1. A method for reducing bacterial colonization on a polypropylene surface of a surgical mesh implant, the method comprising:
generating an accelerated neutral atom beam (ANAB) with an energy level selected to impart a predetermined increase in surface energy to a polypropylene surface of a mesh implant, wherein the predetermined increase in surface energy is selected to alter the surface energy of the polypropylene surface to approach the surface energy of mucin and/or casein proteins contained in bodily fluids that when absorbed to the polypropylene surface decrease bacteria colonization, wherein the generating comprises, flowing argon gas at 200 standard cubic centimeters per minute (SCCM) through a 100 mm diameter nozzle to create weakly bonded clusters of between 100 argon atoms and 10,000 argon atoms, impacting the weakly bonded clusters of argon atoms with electrons to ionize the clusters to a charge of +1 or +2, subjecting the ionized clusters to a first electrostatic field having a field strength of 30 kilovolt configured to accelerate the ionized clusters, breaking apart the accelerated clusters by colliding the accelerated clusters with residual argon atoms in the path of the accelerated clusters, and subsequent to breaking apart the accelerated clusters, subjecting the accelerated clusters to a second electrostatic field configured to deflect remaining iononized portions of clusters from the ANAB path and allow neutral argon atoms from the clusters to maintain a predetermined momentum along the ANAB path;
irradiating the at least one surface with an effective ANAB dose of about 2.5 x 10'7 argon atoms per cm2, wherein the irradiating modifies the at least one surface to a depth of between 1 nanometer and 3 nanometers.

2. A method for reducing contamination of a polypropylene surface, the method comprising:
generating an accelerated neutral atom beam (ANAB) with an energy level selected to impart a predetermined increase in surface energy to a polypropylene surface of an object to be irradiated with the ANAB;
irradiating at least one polypropylene surface of an object with the ANAB.

3. The method of claim 2, wherein the object is a surgical mesh.

4. The method of claim 3, wherein the object is a hernia mesh implant.

5. The method of claim 2, wherein the predetermined increase in surface energy is selected to alter the surface energy of polypropyene to approach the surface energy of one or more proteins contained in bodily fluids that when absorbed to the surface decrease bacteria colonization.

6. The method of claim 5, wherein the one or more proteins in the group consisting of mucin and casein.

7. The method of claim 2, comprising:
accelerating neutral argon atoms with very low energies under a vacuum;
bombarding the at least one surface with the neutral argon atoms, wherein the bombarding modifies the at least one surface to a depth of between 1 nanometer and 3 nanometers.

8. The method of claim 2, wherein generating the ANAB comprises:
flowing argon gas through a nozzle to create weakly bonded clusters of argon atoms;
impacting the weakly bonded clusters of argon atoms with electrons to ionize the clusters;
subjecting the ionized clusters to a first electrostatic field configured to accelerate the ionized clusters;
breaking apart the accelerated clusters by colliding the accelerated clusters with residual argon atoms in the path of the accelerated clusters;
subsequent to breaking apart the accelerated clusters, subjecting the accelerated clusters to a second electrostatic field configured to deflect remaining iononized portions of clusters from the ANAB path and allow neutral argon atoms from the clusters to maintain a predetermined momentum along the ANAB path.

9. The method of claim 8, comprising flowing the argon gas at 200 standard cubic centimeters per minute (SCCM), wherein the nozzle has a diameter of 100 mm.

10. The method of claim 8, wherein the weakly bonded clusters consist of between 100 argon atoms and 10,000 argon atoms.

11. The method of claim 8, wherein impacting the weakly bonded clusters of argon atoms with electrons ionize the clusters to a charge of +1 or +2.

12. The method of claim 8, wherein the first electrostatic field is a 30 kilovolt field.

13. The method of claim 2, comprising irradiating at least one surface of the object with an effective ANAB dose of about 2.5 x 1017 argon atoms per cm2.