INHIBITING BACTERIA COLONIZATION WITHOUT ANTIBIOTICS

Abstract

A device such as a medical device and a method for making same provides a device surfaces modified by beam irradiation, such as a gas cluster ion beams or a neutral beam, to inhibit or delay attachment or activation or clotting of platelets or to match surface energy of the device to that of a protein with the property of inhibition of bacterial colonization that can coat the all or part of the device surface to effect such inhibition.

Claims

1. A method of modifying a surface of an object so as to inhibit attachment of platelets thereto, the method comprising: forming a Neutral beam of monomers derived from a gas-cluster ion-beam which is accelerated in a reduced-pressure chamber; introducing an object into the reduced-pressure chamber treated for dissociation to establish monomer beams content by separating charged particles and clusters therefrom introducing an object into the reduced pressers chambers; and irradiating at least a portion of the surface of said object with the neutral beam to inhibit attachment of platelets thereto.

2. The method of claim 1, wherein the at least a portion of the surface modified to inhibit the attachment of platelets thereto is adapted to promote the attachment or proliferation of non-platelet cells.

3. The method of claim 2, wherein the non-platelet cells are endothelial cells.

4. The method of claim 1, wherein the object is a medical device intended for surgical implant into a subject.

5. The method of claim 1, wherein the at least a portion of the surface comprises a metal, a ceramic, a polymer, or a glass an oxide, a metal alloy, a plastic, a polymer, a copolymer, a solid resin, a glass, quartz, a ceramic, sapphire, a glassy material, titanium, titania, an alloy of titanium, a cobalt-chrome alloy, a cobalt-chrome-molybdenum alloy, tantalum, or a tantalum alloy.

6. An object comprising of Neutral Beam implant, having a surface modified by beam irradiation to inhibit or delay attachment or activation or clotting of platelets or inhibition of bacterial colonization.

7. The object of claim 6, wherein the object surface comprises any of a metal, a ceramic, a polymer, or a glass an oxide, a metal alloy, a plastic, a polymer, a copolymer, a solid resin, a glass, quartz, a ceramic, sapphire, a glassy material, titanium, titania, an alloy of titanium, a cobalt-chrome alloy, a cobalt-chrome-molybdenum alloy, tantalum, or a tantalum alloy.

8. The object of claim 7, wherein the surface comprises titanium, titania or a titanium alloy.

9. The object of claim 7, wherein the surface comprises polypropylene.

10. The object of claim 7, wherein the surface comprises PVC.

11. The object of claim 7, wherein the surface comprises PEEK.

12. A method of modifying a surface of an object which is a medical device or natural or synthetic body part so as to inhibit bacterial on a surface of the object when placed in bacteria exposed environment, comprising: a) forming a Neutral Beam of monomers derived from a gas-cluster ion-beam which is accelerated in a reduced-pressure chamber treated for dissociation to establish monomers content by separating charged particles and clusters therefrom; b) introducing an object into the reduced-pressure chamber; c) and irradiating at least a portion of the surface of said object with the Neutral Beam to inhibit bacterial colonization.

13. The method of claim 12 wherein the surface object comprises a metal, a ceramic, a polymer, or a glass an oxide, a metal alloy, a plastic, a polymer, a copolymer, a solid resin, a glass, quartz, a ceramic, sapphire, a glassy material, titanium, titania, an alloy of titanium, a cobalt-chrome alloy, a cobalt-chrome-molybdenum alloy, tantalum, or a tantalum alloy.

14. The method of claim 13 wherein processing conditions of neutral beam irradiation produce an object surface energy substantially matching surface energy of a desired protein with microbacterial colonization inhibition properties, which protein is inherently available in the object's usage environment or from an external source.

15. The method of claim 14 wherein the protein to be surface energy matched is present in a mammalian environment in which such objects are used.

16. The method of claim 15 wherein the protein is derived from outside a mammalian host and administered to proximity to the object.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0061] FIG. 1 is a schematic illustrating elements of a GCIB processing apparatus 1100 for processing a workpiece using a GCIB;

[0062] FIG. 2 is a schematic illustrating elements of another GCIB processing apparatus 1200 for workpiece processing using a GCIB, wherein scanning of the ion beam and manipulation of the workpiece is employed;

[0063] FIG. 3 is a schematic of a Neutral Beam processing apparatus 1300, which uses electrostatic deflection plates to separate the charged and uncharged beams;

[0064] FIG. 4 is a schematic of a Neutral Beam processing apparatus 1400 using a thermal sensor for Neutral Beam measurement;

[0065] FIG. 5 is a typical SEM image 100 of a surface of a nitinol coupon from an untreated control group showing substantial progression toward platelet attachment, activation, and clot formation;

[0066] FIG. 6 is a typical SEM image 150 of a surface of a nitinol coupon from a Neutral-Beam-treated group showing noticeable inhibition of platelet attachment, activation, and clot formation, according to an embodiment of the invention;

[0067] FIG. 7 is a typical SEM image 170 of a surface of a nitinol coupon from a GCIB-treated group showing substantial inhibition of platelet attachment, activation, and clot formation, according to an embodiment of the invention.

[0068] FIGS. 8a and 8b provide AFM imaging showing 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 nm on the control to 3.80 nm 0.14 nm on the ANAB-treated samples, p<0.025; Rz decreased from 56.02 nm±2.78 nm on the control to 45.04 nm±5.25 nm on the treated coupons, p=0.135;

[0069] FIG. 9 shows colony counting 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 and the log reduction is indicated directly above the respective bacteria.

[0070] FIGS. 10 and 10a show_live/dead data after 24 h treatment by Staphylococcus epidermidis, Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus, drug resistant Escherichia coli, and Pseudomonas aeruginosa;

[0071] FIG. 11 shows results after crystal violet staining after 24 h treatment by Staphylococcus epidermidis, Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus, drug resistant Escherichia coli, and Pseudomonas aeruginosa;

[0072] FIG. 12 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.

[0073] FIG. 13. 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 casein pre-adsorbed ANAB-treated and control samples.

DETAILED DESCRIPTION OF EMBODIMENTS

[0074] In the following description, for simplification, item numbers from earlier-described figures may appear in subsequently-described figures without discussion. Likewise, items discussed in relation to earlier figures may appear in subsequent figures without item numbers or additional description. In such cases items with like numbers are like items and have the previously-described features and functions, and illustration of items without item numbers shown in the present figure refer to like items having the same functions as the like items illustrated in earlier-discussed numbered figures.

[0075] Reference is now made to FIG. 1, which shows a schematic configuration for a GCIB processing apparatus 1100. A low-pressure vessel 1102 has three fluidly connected chambers: a nozzle chamber 1104, an ionization/acceleration chamber 1106, and a processing chamber 1108. The three chambers are evacuated by vacuum pumps 1146a, 1146b, and 1146c, respectively. A pressurized condensable source gas 1112 (for example argon) stored in a gas storage cylinder 1111 flows through a gas metering valve 1113 and a feed tube 1114 into a stagnation chamber 1116. Pressure (typically a few atmospheres) in the stagnation chamber 1116 results in ejection of gas into the substantially lower pressure vacuum through a nozzle 1110, resulting in formation of a supersonic gas jet 1118. Cooling, resulting from the expansion in the jet, causes a portion of the gas jet 1118 to condense into clusters, each consisting of from several to several thousand weakly bound atoms or molecules. A gas skimmer aperture 1120 is employed to control flow of gas into the downstream chambers by partially separating gas molecules that have not condensed into a cluster jet from the cluster jet. Excessive pressure in the downstream chambers can be detrimental by interfering with the transport of gas-cluster ions and by interfering with management of the high voltages that may be employed for beam formation and transport. Suitable condensable source gases 1112 include, but are not limited to argon and other condensable noble gases, nitrogen, carbon dioxide, oxygen, and many other gases and/or gas mixtures. After formation of the gas clusters in the supersonic gas jet 1118, at least a portion of the gas clusters are ionized in an ionizer 1122 that is typically an electron impact ionizer that produces electrons by thermal emission from one or more incandescent filaments 1124 (or from other suitable electron sources) and accelerates and directs the electrons, enabling them to collide with gas clusters in the gas jet 1118. Electron impacts with gas clusters eject electrons from some portion of the gas clusters, causing those clusters to become positively ionized. Some clusters may have more than one electron ejected and may become multiply ionized. Control of the number of electrons and their energies after acceleration typically influences the number of ionizations that may occur and the ratio between multiple and single ionizations of the gas clusters. A suppressor electrode 1142, and grounded electrode 1144 extract the cluster ions from the ionizer exit aperture 1126, accelerate them to a desired energy (typically with acceleration potentials of from several hundred V to several tens of kV), and focuses them to form a GCIB 1128. The region that the GCIB 1128 traverses between the ionizer exit aperture 126 and the suppressor electrode 1142 is referred to as the extraction region. The axis (determined at the nozzle 1110), of the supersonic gas jet 1118 containing gas clusters is substantially the same as the axis 1154 of the GCIB 1128. Filament power supply 1136 provides filament voltage V.sub.f to heat the ionizer filament 1124. Anode power supply 1134 provides anode voltage VA to accelerate thermoelectrons emitted from filament 1124 to cause the thermoelectrons to irradiate the cluster-containing gas jet 1118 to produce cluster ions. A suppression power supply 1138 supplies suppression voltage Vs (on the order of several hundred to a few thousand volts) to bias suppressor electrode 1142. Accelerator power supply 1140 supplies acceleration voltage V.sub.Acc to bias the ionizer 1122 with respect to suppressor electrode 1142 and grounded electrode 1144 so as to result in a total GCIB acceleration potential equal to V.sub.Acc. Suppressor electrode 1142 serves to extract ions from the ionizer exit aperture 1126 of ionizer 1122 and to prevent undesired electrons from entering the ionizer 1122 from downstream, and to form a focused GCIB 1128.

[0076] A workpiece 1160, which may (for example) be a medical device, a semiconductor material, an optical element, or other workpiece to be processed by GCIB processing, is held on a workpiece holder 1162, which disposes the workpiece in the path of the GCIB 1128. The workpiece holder is attached to but electrically insulated from the processing chamber 1108 by an electrical insulator 1164. Thus, GCIB 1128 striking the workpiece 1160 and the workpiece holder 1162 flows through an electrical lead 1168 to a dose processor 1170. A beam gate 1172 controls transmission of the GCIB 1128 along axis 1154 to the workpiece 1160. The beam gate 1172 typically has an open state and a closed state that is controlled by a linkage 1174 that may be (for example) electrical, mechanical, or electromechanical. Dose processor 1170 controls the open/closed state of the beam gate 1172 to manage the GCIB dose received by the workpiece 1160 and the workpiece holder 1162. In operation, the dose processor 1170 opens the beam gate 1172 to initiate GCIB irradiation of the workpiece 1160. Dose processor 1170 typically integrates GCIB electrical current arriving at the workpiece 1160 and workpiece holder 1162 to calculate an accumulated GCIB irradiation dose. At a predetermined dose, the dose processor 1170 closes the beam gate 1172, terminating processing when the predetermined dose has been achieved.

[0077] FIG. 2 shows a schematic illustrating elements of another GCIB processing apparatus 1200 for workpiece processing using a GCIB, wherein scanning of the ion beam and manipulation of the workpiece is employed. A workpiece 1160 to be processed by the GCIB processing apparatus 1200 is held on a workpiece holder 1202, disposed in the path of the GCIB 1128. In order to accomplish uniform processing of the workpiece 1160, the workpiece holder 1202 is designed to manipulate workpiece 1160, as may be required for uniform processing.

[0078] Any workpiece surfaces that are non-planar, for example, spherical or cup-like, rounded, irregular, or other un-flat configuration, may be oriented within a range of angles with respect to the beam incidence to obtain optimal GCIB processing of the workpiece surfaces. The workpiece holder 1202 can be fully articulated for orienting all non-planar surfaces to be processed in suitable alignment with the GCIB 1128 to provide processing optimization and uniformity. More specifically, when the workpiece 1160 being processed is non-planar, the workpiece holder 1202 may be rotated in a rotary motion 1210 and articulated in articulation motion 1212 by an articulation/rotation mechanism 1204. The articulation/rotation mechanism 1204 may permit 360 degrees of device rotation about longitudinal axis 1206 (which is coaxial with the axis 1154 of the GCIB 1128) and sufficient articulation about an axis 1208 perpendicular to axis 1206 to maintain the workpiece surface to within a desired range of beam incidence.

[0079] Under certain conditions, depending upon the size of the workpiece 1160, a scanning system may be desirable to produce uniform irradiation of a large workpiece. Although often not necessary for GCIB processing, two pairs of orthogonally oriented electrostatic scan plates 1130 and 1132 may be utilized to produce a raster or other scanning pattern over an extended processing area. When such beam scanning is performed, a scan generator 1156 provides X-axis scanning signal voltages to the pair of scan plates 1132 through lead pair 1159 and Y-axis scanning signal voltages to the pair of scan plates 1130 through lead pair 1158. The scanning signal voltages are commonly triangular waves of different frequencies that cause the GCIB 1128 to be converted into a scanned GCIB 1148, which scans the entire surface of the workpiece 1160. A scanned beam-defining aperture 1214 defines a scanned area. The scanned beam-defining aperture 1214 is electrically conductive and is electrically connected to the low-pressure vessel 1102 wall and supported by support member 1220. The workpiece holder 1202 is electrically connected via a flexible electrical lead 1222 to a faraday cup 1216 that surrounds the workpiece 1160 and the workpiece holder 1202 and collects all the current passing through the defining aperture 1214. The workpiece holder 1202 is electrically isolated from the articulation/rotation mechanism 1204 and the faraday cup 1216 is electrically isolated from and mounted to the low-pressure vessel 1102 by insulators 1218. Accordingly, all current from the scanned GCIB 1148, which passes through the scanned beam-defining aperture 1214 is collected in the faraday cup 1216 and flows through electrical lead 1224 to the dose processor 1170. In operation, the dose processor 1170 opens the beam gate 1172 to initiate GCIB irradiation of the workpiece 1160. The dose processor 1170 typically integrates GCIB electrical current arriving at the workpiece 1160 and workpiece holder 1202 and faraday cup 1216 to calculate an accumulated GCIB irradiation dose per unit area. At a predetermined dose, the dose processor 1170 closes the beam gate 1172, terminating processing when the predetermined dose has been achieved. During the accumulation of the predetermined dose, the workpiece 1160 may be manipulated by the articulation/rotation mechanism 1204 to ensure processing of all desired surfaces.

[0080] FIG. 3 is a schematic of a Neutral Beam processing apparatus 1300 of an exemplary type that may be employed for Neutral Beam processing according to embodiments of the invention. It uses electrostatic deflection plates to separate the charged and uncharged portions of a GCIB. A beamline chamber 1107 encloses the ionizer and accelerator regions and the workpiece processing regions. The beamline chamber 1107 has high conductance and so the pressure is substantially uniform throughout. A vacuum pump 1146b evacuates the beamline chamber 1107. Gas flows into the beamline chamber 1107 in the form of clustered and unclustered gas transported by the gas jet 1118 and in the form of additional unclustered gas that leaks through the gas skimmer aperture 1120. A pressure sensor 1330 transmits pressure data from the beamline chamber 1107 through an electrical cable 1332 to a pressure sensor controller 1334, which measures and displays pressure in the beamline chamber 1107. The pressure in the beamline chamber 1107 depends on the balance of gas flow into the beamline chamber 1107 and the pumping speed of the vacuum pump 1146b. By selection of the diameter of the gas skimmer aperture 1120, the flow of source gas 1112 through the nozzle 1110, and the pumping speed of the vacuum pump 1146b, the pressure in the beamline chamber 1107 equilibrates at a pressure, PB, determined by design and by nozzle flow. The beam flight path from grounded electrode 1144 to workpiece holder 162, is for example, 100 cm. By design and adjustment PB may be approximately 6×10.sup.−5 torr (8×10.sup.−3 pascal). Thus the product of pressure and beam path length is approximately 6×10.sup.−3 torr-cm (0.8 pascal-cm) and the gas target thickness for the beam is approximately 1.94×10.sup.14 gas molecules per cm.sup.2, which is observed to be effective for dissociating the gas-cluster ions in the GCIB 1128. V.sub.Acc may be for example 30 kV and the GCIB 1128 is accelerated by that potential. A pair of deflection plates (1302 and 1304) is disposed about the axis 1154 of the GCIB 1128. A deflector power supply 1306 provides a positive deflection voltage V.sub.D to deflection plate 1302 via electrical lead 1308. Deflection plate 1304 is connected to electrical ground by electrical lead 1312 and through current sensor/display 1310. Deflector power supply 1306 is manually controllable. V.sub.D may be adjusted from zero to a voltage sufficient to completely deflect the ionized portion 1316 of the GCIB 1128 onto the deflection plate 1304 (for example a few thousand volts). When the ionized portion 1316 of the GCIB 1128 is deflected onto the deflection plate 1304, the resulting current, I.sub.D flows through electrical lead 1312 and current sensor/display 1310 for indication. When V.sub.D is zero, the GCIB 1128 is undeflected and travels to the workpiece 1160 and the workpiece holder 1162. The GCIB beam current I.sub.B is collected on the workpiece 1160 and the workpiece holder 1162 and flows through electrical lead 1168 and current sensor/display 1320 to electrical ground. I.sub.B is indicated on the current sensor/display 1320. A beam gate 1172 is controlled through a linkage 1338 by beam gate controller 1336. Beam gate controller 1336 may be manual or may be electrically or mechanically timed by a preset value to open the beam gate 1172 for a predetermined interval. In use, V.sub.D is set to zero and the beam current, I.sub.B, striking the workpiece holder is measured. Based on previous experience for a given GCIB process recipe, an initial irradiation time for a given process is determined based on the measured current, I.sub.B. V.sub.D is increased until all measured beam current is transferred from I.sub.B to I.sub.D and I.sub.D no longer increases with increasing V.sub.D. At this point a Neutral Beam 1314 comprising energetic dissociated components of the initial GCIB 1128 irradiates the workpiece holder 1162. The beam gate 1172 is then closed and the workpiece 1160 placed onto the workpiece holder 1162 by conventional workpiece loading means (not shown). The beam gate 1172 is opened for the predetermined initial radiation time. After the irradiation interval, the workpiece may be examined and the processing time adjusted as necessary to calibrate the duration of Neutral Beam processing based on the measured GCIB beam current I.sub.B. Following such a calibration process, additional workpieces may be processed using the calibrated exposure duration.

[0081] The Neutral Beam 1314 contains a repeatable fraction of the initial energy of the accelerated GCIB 1128. The remaining ionized portion 1316 of the original GCIB 1128 has been removed from the Neutral Beam 1314 and is collected by the grounded deflection plate 1304. The ionized portion 1316 that is removed from the Neutral Beam 1314 may include monomer ions and gas-cluster ions including intermediate size gas-cluster ions. Because of the monomer evaporation mechanisms due to cluster heating during the ionization process, intra-beam collisions, background gas collisions, and other causes (all of which result in erosion of clusters) the Neutral Beam substantially consists of neutral monomers, while the separated charged particles are predominately cluster ions. The inventors have confirmed this by suitable measurements that include re-ionizing the Neutral Beam and measuring the charge to mass ratio of the resulting ions. As will be shown below, certain superior process results are obtained by processing workpieces using this Neutral Beam.

[0082] FIG. 4 is a schematic of a Neutral Beam processing apparatus 1400 as may, for example, be used in generating Neutral Beams as may be employed in embodiments of the invention. It uses a thermal sensor for Neutral Beam measurement. A thermal sensor 1402 attaches via low thermal conductivity attachment 1404 to a rotating support arm 1410 attached to a pivot 1412. Actuator 1408 moves thermal sensor 1402 via a reversible rotary motion 1416 between positions that intercept the Neutral Beam 1314 or GCIB 1128 and a parked position indicated by 1414 where the thermal sensor 1402 does not intercept any beam. When thermal sensor 1402 is in the parked position (indicated by 1414) the GCIB 1128 or Neutral Beam 1314 continues along path 1406 for irradiation of the workpiece 1160 and/or workpiece holder 1162. A thermal sensor controller 1420 controls positioning of the thermal sensor 1402 and performs processing of the signal generated by thermal sensor 1402. Thermal sensor 1402 communicates with the thermal sensor controller 1420 through an electrical cable 1418. Thermal sensor controller 1420 communicates with a dosimetry controller 1432 through an electrical cable 1428. A beam current measurement device 1424 measures beam current I.sub.B flowing in electrical lead 1168 when the GCIB 1128 strikes the workpiece 1160 and/or the workpiece holder 1162. Beam current measurement device 1424 communicates a beam current measurement signal to dosimetry controller 1432 via electrical cable 1426. Dosimetry controller 1432 controls setting of open and closed states for beam gate 1172 by control signals transmitted via linkage 1434. Dosimetry controller 1432 controls deflector power supply 1440 via electrical cable 1442 and can control the deflection voltage V.sub.D between voltages of zero and a positive voltage adequate to completely deflect the ionized portion 1316 of the GCIB 1128 to the deflection plate 1304. When the ionized portion 1316 of the GCIB 1128 strikes deflection plate 1304, the resulting current I.sub.D is measured by current sensor 1422 and communicated to the dosimetry controller 1432 via electrical cable 1430. In operation dosimetry controller 1432 sets the thermal sensor 1402 to the parked position 1414, opens beam gate 1172, and sets V.sub.D to zero so that the full GCIB 1128 strikes the workpiece holder 1162 and/or workpiece 1160. The dosimetry controller 1432 records the beam current I.sub.B transmitted from beam current measurement device 1424. The dosimetry controller 1432 then moves the thermal sensor 1402 from the parked position 1414 to intercept the GCIB 1128 by commands relayed through thermal sensor controller 1420. Thermal sensor controller 1420 measures the beam energy flux of GCIB 1128 by calculation based on the heat capacity of the sensor and measured rate of temperature rise of the thermal sensor 1402 as its temperature rises through a predetermined measurement temperature (for example 70 degrees C.) and communicates the calculated beam energy flux to the dosimetry controller 1432 which then calculates a calibration of the beam energy flux as measured by the thermal sensor 1402 and the corresponding beam current measured by the beam current measurement device 1424. The dosimetry controller 1432 then parks the thermal sensor 1402 at parked position 1414, allowing it to cool and commands application of positive V.sub.D to deflection plate 1302 until the entire current I.sub.D due to the ionized portion of the GCIB 1128 is transferred to the deflection plate 1304. The current sensor 1422 measures the corresponding I.sub.D and communicates it to the dosimetry controller 1432. The dosimetry controller also moves the thermal sensor 1402 from parked position 1414 to intercept the Neutral Beam 1314 by commands relayed through thermal sensor controller 420. Thermal sensor controller 420 measures the beam energy flux of the Neutral Beam 1314 using the previously determined calibration factor and the rate of temperature rise of the thermal sensor 1402 as its temperature rises through the predetermined measurement temperature and communicates the Neutral Beam energy flux to the dosimetry controller 1432. The dosimetry controller 1432 calculates a neutral beam fraction, which is the ratio of the thermal measurement of the Neutral Beam 1314 energy flux to the thermal measurement of the full GCIB 1128 energy flux at sensor 1402. Under typical operation, a neutral beam fraction of from about 5% to about 95% is achieved. Before beginning processing, the dosimetry controller 1432 also measures the current, I.sub.D, and determines a current ratio between the initial values of I.sub.B and I.sub.D. During processing, the instantaneous I.sub.D measurement multiplied by the initial I.sub.B/I.sub.D ratio may be used as a proxy for continuous measurement of the I.sub.B and employed for dosimetry during control of processing by the dosimetry controller 1432. Thus the dosimetry controller 1432 can compensate any beam fluctuation during workpiece processing, just as if an actual beam current measurement for the full GCIB 1128 were available. The dosimetry controller uses the neutral beam fraction to compute a desired processing time for a particular beam process. During the process, the processing time can be adjusted based on the calibrated measurement of I.sub.D for correction of any beam fluctuation during the process.

[0083] In an exemplary embodiment of the invention, nickel titanium alloy, also known as nitinol, a material favored for certain types of vascular stents was treated by GCIB and Neutral Beam processing to inhibit or delay the attachment and/or activation of platelets on surfaces thereof and to inhibit subsequent formation of blood clots.

[0084] Electro-polished and cleaned nitinol coupons (10 mm diameter, 1 mm thick) were divided into the following groups (n=3 for each condition): 1) Unprocessed (except for cleaning) control; 2) cleaned and argon Neutral-Beam-processed; 3) cleaned and argon GCIB-processed; 4) cleaned and Neutral-Beam-processed using each of several source gas mixtures (each of CH.sub.4, O.sub.2, N.sub.2) each used at 10%, 5%, 2%, 1% mixture concentration with the balance argon; 5) GCIB processed using each of several source gas mixtures (each of CH.sub.4, O.sub.2, N.sub.2) each used at 10%, 5%, 2%, 1% mixture concentration with the balance argon.

[0085] For each GCIB treatment, a surface of the coupon was irradiated using a GCIB (gas or mixture indicated above) irradiation dose of 5×10.sup.14 gas-cluster ions/cm.sup.2, the beam was accelerated using V.sub.Acc of 30 keV. For each Neutral Beam treatment, a surface of the coupon was irradiated using a Neutral Beam (gas or mixture indicated above) irradiation dose of 2.5×10.sup.17 neutral atoms/cm.sup.2, the parent GCIB was accelerated using V.sub.Acc of 30 keV. The Neutral Beam was an essentially fully dissociated beam. The Neutral Beam dose of 2.5×10.sup.17 neutral atoms/cm.sup.2 was determined to be approximately the thermal equivalent of the 5×10.sup.14 gas-cluster ions/cm.sup.2.

[0086] Each of the nitinol coupons (controls and all processed conditions) were placed in individual wells of non-tissue culture plates treated 24 well plates (BD Falcon 351147). 500 μl of citrated human whole blood was placed in each well and the plates (with blood and coupons) were incubated for one hour at 37° C., 5% CO.sub.2 in humidified air. Blood, all taken from the same batch, was used in each well. Following incubation, the blood was removed from the wells and all coupons were gently rinsed 3 times with 500 μl 1× phosphate buffered saline (PBS). Washed coupons were then fixed in 2% gluteraldehyde in PBS buffer with a pH of 7.4 for 1 hour. Each coupon was then rinsed three times in 500 μl PBS for 5 minutes. Nitinol coupons were then fixed in a secondary fixative using 1% osmium tetra-oxide (OsO.sub.4) in H.sub.2O. They were then rinsed 3 times with distilled water for 5 minutes each. Following the washes, coupons were serially dehydrated in 30%, 50%, 70%, 90% ethanol concentration, 5 minutes each, followed by 2 times of 5 minutes in 100% ethanol. Coupons were then gold sputter-coated and imaged by scanning electron microscope (SEM).

[0087] In each instance the GCIB- or Neutral-Beam-processed (using argon alone or one of the gas mixtures) coupons showed reduced platelet attachment and/or activation and reduced clotting as compared to the control coupons. For this set of tests, the best results for both GCIB and Neural Beam treatment were obtained using a CH.sub.4/argon mixture at a concentration of 2.5% and 5% (both concentration results approximately the same) as the source gas employed for beam generation.

[0088] FIG. 5 is a typical SEM image 100 of a surface of a nitinol coupon from the control group. Individual erythrocytes (102, 104 indicated as examples), and leukocytes (106, 108 indicated as examples) are scattered throughout the field. Individual platelets (110 indicated as an example) and large areas of activated platelets (112 indicated as an example) interconnected by fibrin networks are widely observed. Substantial platelet agglutinations (114, 116 indicated as examples) indicate progression towards clotting. Clusters (118, 120) of erythrocytes, leukocytes, and activated platelets show instances of clotting progression.

[0089] FIG. 6 is a typical SEM image 150 of a surface of a nitinol coupon from the Neutral-Beam-processed group using a source gas mixture of 5% CH.sub.4 in argon. Individual erythrocytes (152 indicated as examples) are scattered throughout the field. Individual platelets (154 indicated as examples), occasional partially activated platelets (156 indicated as an example), and small areas of activated platelets (158 indicated as an example) interconnected by fibrin networks are occasionally observed. Occasional clusters (160) of erythrocytes, leukocytes, and activated platelets show instances of clotting progression. In general the progression of platelet attachment, activation, and clot formation is noticeably less advanced than the control case.

[0090] FIG. 7 is a typical SEM image 170 of a surface of a nitinol coupon from the GCIB-processed group using a source gas mixture of 5% CH.sub.4 in argon. Individual erythrocytes (172, 174 indicated as examples) are scattered throughout the field. Occasional individual platelets and partially activated platelets (176 indicated as an example) are observed. Small areas of activated platelets and preliminary clusters of clot formations are seldom observed (no examples in this field). The progression of platelet attachment, activation, and clot formation is substantially less advanced than either the control case or the Neutral-Beam-processed case.

[0091] In another test, nitinol was treated by GCIB and Neutral Beam processing to determine the effects of the beam processing on subsequent attachment and/or proliferation of endothelial cells on the surface.

[0092] Electro-polished and cleaned nitinol coupons (10 mm diameter, 1 mm thick) were divided into the following groups (n=3 for each condition): 1) Unprocessed (except for cleaning) control; 2) cleaned and argon Neutral-Beam-processed; 3) cleaned and argon GCIB-processed; 4) cleaned and Neutral-Beam-processed using each of several source gas mixtures (each of CH.sub.4, O.sub.2, N.sub.2) each used at 5% and 1% mixture concentration with the balance argon; 5) GCIB processed using each of several source gas mixtures (each of CH.sub.4, O.sub.2, N.sub.2) each used at 5% and 1% mixture concentration with the balance argon.

[0093] For each GCIB treatment, a surface of the coupon was irradiated using a GCIB (gas or mixture indicated above) irradiation dose of 5×10.sup.14 gas-cluster ions/cm.sup.2, the beam was accelerated using V.sub.Acc of 30 keV. For each Neutral Beam treatment, a surface of the coupon was irradiated using a Neutral Beam (gas or mixture indicated above) irradiation dose of 2.5×10.sup.17 neutral atoms/cm.sup.2, the parent GCIB was accelerated using V.sub.Acc of 30 keV. The Neutral Beam was an essentially fully dissociated beam. The Neutral Beam dose of 2.5×10.sup.17 neutral atoms/cm.sup.2 was determined to be approximately the thermal equivalent of the 5×10.sup.14 gas-cluster ions/cm.sup.2.

[0094] Each of the nitinol coupons (controls and all processed conditions) were placed in individual wells of non-tissue culture plates treated 24 well plates (BD Falcon 351147). Each nitinol coupon was seeded with 2000 human umbilical vein endothelial cells (HUVEC; Lonza Group Ltd, Muenchensteinerstrasse 38, CH-4002, Basel, Switzerland; Lonza #C2519A) in 1 ml of endothelial cell growth media (Lonza EGM-2), and the plates (with media and coupons) were incubated at 37° C., 5% CO.sub.2 in humidified air. Media in the wells was changed every 3 days. At day 7 and day 10, plates corresponding to those time points were removed, media was removed, cells were fixed for 30 minutes in 500 μl 10% buffered formalin at room temperature. Formalin was removed and 500 μl crystal violet stain (Sigma #HT90132; diluted 1:100 in 1× phosphate buffered saline) was added to each well and placed on a shaker with gentle agitation for 30 minutes. Crystal violet stain was removed and excess stain was washed off in tap water until clear. Nitinol coupons were then air dried overnight, 500 μl elution buffer (2% NaOH; 10% Trichloroacetic acid; 50% Methanol; in H.sub.2O) was placed in each well to allow dye elution from coupons. 100 μl samples of each well in duplicates (two samples from each well, thus 6 samples per condition [2×n]) were placed in a 96 well plate and absorbance at 570 nm for each well was read on a plate reader. Absorbance was compared to a standard curve and cell numbers were determined. T-tests were used to determine significance compared to controls. Endothelial cells attached and proliferated on the surface of nitinol coupons treated by either argon or mixtures of Argon with CH.sub.4, O.sub.2, or N.sub.2 using either GCIB or Neutral Beam. However, the best results were obtained using GCIB, and Table 1 shows the results for the GCIB-processed coupon.

TABLE-US-00001 TABLE 1 Day 7 Day 7 Day 10 Day 10 Cell Std. Day 7 Cell Std. Day 10 GCIB Process Count Deviation p value Count Deviation p value Control 18083 4867 15458 6096 Argon GCIB 24958 3333 0.037 28708 8247 0.014 1% CH.sub.4 in Ar GCIB 24625 1794 0.0041 21417 2078 0.015 5% CH.sub.4 in Ar GCIB 19000 4990 0.80 20958 1706 0.043 1% O.sub.2 in Ar GCIB 17125 3364 0.21 17167 8323 0.59 5% O.sub.2 in Ar GCIB 13667 3459 0.11 32792 2813 0.00073 1% N.sub.2 in Ar GCIB 10500 7112 0.18 35000 5282 0.0025 5% N.sub.2 in Ar GCIB 13625 8130 0.377692 34583 3289 0.001872
Generally, GCIB allowed better HUVEC attachment and proliferation as compared with Neutral Beam. As Table 1 shows, at day 7, only Argon GCIB and CH.sub.4 1% GCIB were significantly better than the control, all others were not significantly different from controls. By day 10, only O.sub.2 1% GCIB did not produce significant increase in HUVEC attachment and proliferation compared to the controls, all others were significantly better.

[0095] The best results for platelet and clotting inhibition were observed for GCIB treatment using CH.sub.4 mixtures in argon while the best results for endothelial cell attachment and proliferation were observed for GCIB treatment using N.sub.2 or O.sub.2 mixtures in argon. However, it is seen that nitinol coupons receiving identical GCIB processing using 5% CH.sub.4, 5% O.sub.2, or 5% N.sub.2 mixtures in argon all show significant platelet delay and/or inhibition as well as significantly enhanced endothelial cell attachment and/or proliferation. Other combinations also produce both desirable outcomes using either GCIB or Neutral Beam treatments.

[0096] Although the invention has been described, for exemplary purposes, as using a GCIB or a Neutral Beam derived from a GCIB for processing a surface of a nitinol object, it is understood by the inventors that benefits obtained by application of such surface processing are not limited to that specific metallic material and that the methods and apparatus described herein may be used for successful processing of other metals and other materials including, without limitation, ceramics, polymers, glasses, oxides, metal alloys, plastics, polymers and copolymers, solid resins, quartz, sapphire, glassy solids, titanium, titania, alloys of titanium, cobalt-chrome alloys, cobalt-chrome-molybdenum alloys, tantalum, and tantalum alloys. Although the invention has been described, for example, with reference beams derived from mixtures of argon and methane gases, it is understood by the inventors that useful treatments also result from employing noble gases, gases such as N.sub.2, O.sub.2, CO.sub.2, and other gases and from employing gas mixtures in various mixture concentrations, and it is intended that all such applications are included within the scope of the invention.

Example (Polyropylene)

[0097] 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 3×15 min washes in deionized H.sub.2O. 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.sup.[4]. 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 2.5×10.sup.17 Ar atoms per cm.sup.2.

[0098] 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

[0099] 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, 16 μl 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 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 of and at are the dispersive and polar components, respectively, of the wetting liquid's surface tension, where of and of are the dispersive and polar components, respectively, of the substrate's surface energy, and where 8 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.

[00001]$\begin{array}{cc}\mathrm{Owen}\text{s/W}\mathrm{endt}\mathrm{theory}& \\ \frac{{\sigma }_L\left(\cos \theta +1\right)}{2{\left({\sigma }_L^D\right)}^{1/2}}={\left({\sigma }_S^P\right)}^{1/2}\frac{{\sigma }_L^{P^{1/2}}}{{\sigma }_L^{D^{1/2}}}+{\left({\sigma }_S^D\right)}^{1/2}.& \mathrm{Equation}I\end{array}$

Atomic Force Microscopy (AFM)

[0100] 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/m were used (PointProbe® Plus, Nanosensors). 1 μm.sup.2 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 (CFUs)

[0101] 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.

[0102] 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 of 1×10.sup.9 CFU/ml. Bacterial concentrations were measured using a SpectraMax M3 series plate reader, whereby an absorbance output reading of 0.52 at the wavelength A.sub.a=562 nm corresponded to a bacterial density of 1×10.sup.9 CFU/ml. The microbial suspensions were diluted further in TSB to a concentration of 1×10.sup.6 CFU/ml, which were used to treat the coupons inside separate 24-well plates. Surfaces were sterilized, decontaminated and cleaned using 70% ethanol.

[0103] After the samples were inoculated with 1 ml of 1×10.sup.6 CFU/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 (3× 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 10−10.sup.6×. 10 μl 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 ImageJ.

Live/Dead and Crystal Violet Assays

[0104] 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-HCl, 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, Calif.) 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 was found that all the bacteria 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

[0105] 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 Adsorbed Proteins to Bacteria Attachment Inhibition

[0106] 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/ml) 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

[0107] 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.

Results and Discussion

Contact Angle Measurements & Surface Energy Calculations

[0108] Following from the contact angle analyses section, the angles that 16 μl 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 more wettable compared to the untreated control, as designed.

TABLE-US-00002 TABLE 1 Contact angle values (°) for Si coupons using the three-solvent system: deionized water (DI-H2O), ethylene glycol ((CH2OH)2), and glycerol (C3H8O3). Sample DI H.sub.2O (CH2OH)2 C3H8O3 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 Data = mean ± standard error of the mean. N = 3. 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 σ.sub.H2O = 72.8 mN/m (σ.sub.H2O.sup.D = 26.4 mN/m, σ.sub.H2O.sup.P = 46.4 mN/m), σ.sub.(CH2OH)2 = 47.7 mN/m (σ.sub.(CH2OH)2.sup.D = 26.4 mN/m, σ.sub.(CH2OH)2.sup.P = 21.3 mN/m), and σ.sub.C3H8O3 = 63.4 mN/m (σ.sub.C3H8O3.sup.D = 37.0 mN/m, σ.sub.C3H8O3.sup.P = 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 σ.sub.S.sup.D and σ.sub.S.sup.P. These values are shown in Table 2. Results showed that the ANAB-treated samples has significantly 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-US-00003 TABLE 2 Surface energy values (mN/m) for the Si coupons, as determined by application of the Owens/Wendt equation, broken into polar (σ.sub.sp) and dispersive (σ.sub.sd) components. Sample σ.sub.s σ.sub.S.sup.P (mN/m) σ.sub.S.sup.D (mN/m) Control 21.73 7.72 14.01 ANAB-treated 35.58 31.56 4.02 sample

Atomic Force Microscopy Analysis

[0109] Results of the present study demonstrated significantly increased nanoscale surface roughness, as measured by atomic force microscopy for the ABAN-treated compared to control samples (FIG. 1). Specifically, the Ra values increased 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 increased from 56.02 nm 2.78 nm to 45.04 nm±5.25 nm on ANAB-treated compared to coupons, respectively.

[0110] The FIGS. 8a and 8b AFM imaging shows 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 nm on the control to 3.80 nm±0.14 nm on the ANAB-treated samples, p<0.025; Rz decreased from 56.02 nm±2.78 nm on the control to 45.04 nm±5.25 nm on the treated coupons, p=0.135

Bacterial Assays

[0111] 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 FIGS. 2, 3, and 4 for colony forming units, live/dead assays, and crystal violet staining. Specifically, for the colony forming results, counts (in 10.sup.5 cells/cm.sup.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.

[0112] 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 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.

[0113] FIG. 9 shows colony counting data after 24 h treatment by Staphylococcus epidermidis.

[0114] FIGS. 10 and 10a show 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].

[0115] FIG. 11 shows results after crystal violet staining 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

[0116] Results of the present study also confirmed the significantly less S. aureus colonization on the ANAB-treated compared to control samples after 24 h of culture (FIG. 10a).

Mechanisms of Bacteria Colonization

[0117] Results of protein adsorption studies showed significantly greater levels of both mucin and casein adsorption of ANAB-treated samples compared to controls (0.9 μg/ml compared to 0.1 μg/ml for mucin and 0.4 μg/ml compared to 0.1 μg/ml for casein respectively). There were no differences observed for another key protein which decreases bacteria colonization, lubricin.

[0118] Lastly, results supported significantly less bacteria colonization after 24 hours onto ANAB-treated samples coated with mucin and casein compared to controls (FIGS. 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

[0119] FIG. 12 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 ANAB-treated samples were significantly less (p<0.01) than controls.

[0120] FIG. 13 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 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.

[0121] 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

[0122] 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.

[0123] The present invention is applicable to objects such as medical devices and natural or synthetic body parts implanted temporarily or indefinite duration (e.g., hernia mesh, catheter, stent, port, knee or hip replacement), in humans or other mammals or other life forms and also to usage in other ways including laboratory and industrial processes and apparatus for control of platelet formation and/or inhibition of bacterial colonization from the usage environments thereof such as membrane surfaces or of processing equipment the treated surface may be of material described above.