GB2592369A - Formation of antimicrobial surfaces using molecular films via quaternary salts ion pairing attachment and incorporation of metal nanoparticles - Google Patents

Abstract

The use of negatively charged functionalities on surfaces for the attachment of positively charged quaternary ammonium antibacterial compounds via ion pairing for their application as effective and tuneable antimicrobial and antibiofilm surfaces. Preferably the surface is first functionalised through chemical or electrochemical reduction of a diazonium salt with carboxyphenyl functionality which is then used to immobilise the quaternary ammonium salts. The ion pairs may serve as a backbone for the formation of antibacterial metal nanoparticles, particularly silver and/or copper nanoparticles. The modified surfaces can be used in medical devices, wound dressings and general surface hygiene materials such as tissues, towels, wipes and beddings.

Description

Formation of antimicrobial surfaces using molecular films via quatemary salts ion pairing attachment and incorporation of metal nanoparticles

1. Background of the Invention

[01] In the recent years, bacterial resistance to antibiotics has become a major challenge in the healthcare sector. In addition, it is estimated that 60% of the human infections are caused by bacteria able to form biofilms. Biofilm formation is also a major issue in various sectors including food and beverage industry, corrosion and environmental sectors etc. These biofilms are creating lots of economic and health problems due to the interfering effects they have with industrial and environmental apparatus and resistant to antibiotics and immune system actions. Biofilms can be defined as an association of microorganisms o (between one or more species), attached to surfaces. This adhesion and association confers C\I them new properties such as antibiotic resistance, which is probably the greatest problem on biofilm's related infections.

o [02] Biofilms are more aggressive in patients who have suffered burn injuries due to the absence of natural skin barrier protection. After 48h of hospitalization, the majority of these patients present poly-microbial infection being Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus the most common pathogens involved in.

[3] Biofilms start to develop when bacteria or other microorganism attach to a surface. The polymers produced by the microbial cells play a critical role in the adhesion process, sometimes creating a "polymer bridge" between the cells and the molecules adsorbed at the surface that strengthens the attached layer [Simees, M., L. C. Simees and M. J. Vieira "A review of current and emergent biofilm control strategies." LWT -Food Science and Technology 2010, 43(4), 573]. A way for avoiding biofilm formation is the surface modification in order to alter the surface properties and thus bacterial adhesion [L. Rizzello, R. Cingolani & P. P. Pompa, Nanomedicine (2013) 8(5), 807].

[4] Modification of the surfaces of such materials, for example by grafting of functional groups, has applications in a wide variety of fields. For instance, organic layers on electrode surfaces can be used to modify the electrocatalytic behaviour of the electrode and/or to improve the uniformity of subsequent metal deposition. Such modified electrode surfaces can be used in fuel cells, batteries, solar cells, metal corrosion, electrocatalysis, sensors, drug delivery and in other electrochemical applications [R. C. Alkire, D. M. Kolb J. Lipkowski P. N. Ross (Eds), Chemically Modified Electrodes, 2011, Wiley].

[5] Many other materials benefit from surface modification in order to alter their properties or to immobilise specific chemical functionalities which permit coupling to other moieties. For example, organic groups can be grafted onto metal surfaces to impart the ability to bind to other chemical or biological entities; carbon fibres can be modified by binding to epoxy resins to form carbon composite materials having improved mechanical properties; carbon nanotubes can be functionalised to modify their properties (e.g. to prevent bundling); and silicon wafer surfaces can be functionalised to modify their performance. Current methods for surface modification include the grafting of organic groups to carbon or metal surfaces via the chemical or electrochemical reduction of the corresponding diazonium derivatives [M. M. Chehimi (Ed.), Aryl Diazonium Salts, 2012, Wiley-VCH].

[6] The binding of organic contaminants to dissolved carboxylic acids via ion pairing have been used for reducing the free concentration of the contaminants such as surfactants in the environment. Fixing of these contaminants through binding surfaces via functionalities with opposite charges is a solution to prevent their release to the environment. In addition,

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C\I their properties can be enhanced by fixing to the surface in contact with the target environment. Binding of cationic surfactants to anionic polymers have been studied before [M. lshiguro, L.K. Koopal, Colloids and Surfaces A: Physicochem. Eng. Aspects 347 (2009)

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69]. Aqueous mixtures of ionic surfactants and oppositely charged polyelectrolytes have been reported in good details [K. Hayakawa, J.C.T. Kwak, in D.N. Rubingh, P.M. Holland (Eds.), Surfactant Science Series, vol. 37, Marcel Dekker, 1991, p. 189]. Binding of cationic surfactants to aromatic carboxylic acids such as natural poly-electrolytes of Humic and Fulvic acids has been proposed [L.K. Koopal, T.P. Goloub, T.A. Davis, J. Colloid Interface Sci. 275 (2004) 360]. Polyelectrolyte multilayer (PEM) technology alone covers the entire widespread spectrum of functionalization possibilities. PEMs are obtained through the alternating deposition of polyanions and polycations on a substrate, and the great advantages of PEMs are that (i) they can be applied to almost any type of substrate whatever its shape and composition; (ii) various chemical, physicochemical, and mechanical properties of the coatings can be obtained; and (iii) active compounds can be embedded and released in a controlled manner [Sean, L. et al. Langmuir, 2015, 31, 12856].

[7] Surface engineering protocols in order to tune the interactions between microorganisms and highly organized/ordered micro-and nano-patterns have been reviewed in details [Nanomedicine, 2013 8(5), 807]. It has been shown that ordered nano-patterns can control biofilm formation. Templated deposition of metal nanoparticles has shown great control on particle size, stability and shape of monodispersed nanoparticles by controlled electrochemical reduction. Electrodes modified with these N Ps have been used for catalytic application. For instance, silver nanoparticles on a polyphenyl acetate layer anchored to an electrode [J.M. Noel, D. Zigah, J. Simonet, P. Hapiot (2010), Langmuir, 26, 7638], nitrophenyl film on HOPG for the electrodeposition of platinum NPs for the oxidation of methanol and carbon monoxide [M. Bayati, J. M. Abad, C. A. Bridges, M.J. Rosseinsky and D.J. Schiffrin, (2008) J. Electroanal. Chem, 623, 19] and nitrophenyl films on glassy carbon surfaces for the electroreduction of gold nanoparticles for selective oxygen reduction reaction [F. Mirkhalaf, K. Tammeveski and D.J. Schiffrin (2009), Phys. Chem. Chem. Phys. 11, 3463]. Silver nanoparticles (Ag NPs) have been extensively used as antibacterial materials although their health and safety concerns are still under debates.

[8] The incorporation of some antibiotics onto solid supports via covalent attachment may lead to loss of their antibacterial activity. Considering this problem, polyelectrolytes were used via non-covalent electrostatic interactions. This invention relates to the fixation of charged antibacterial surfactants onto surfaces by the interaction with species containing opposite charges already attached on the surfaces. The ion pair thus formed can also host for the further formation of metal nanoparticles in order to improve antibacterial/antibiofilm

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C\I properties.

[9] In the invention filed hereby, attachment of positively charged cations including quaternary ammonium salts (Scheme 1) on solid surfaces by ion pairing with the previously

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immobilised anionic carboxyphenyl functionality after its attachment onto the surface following the reduction of P-benzoic acid diazonium salt is disclosed (Scheme 2). The ion pair thus formed on the surface can also host silver and/or copper nanoparticles for further tailoring antibacterial/antibiofilm properties of the surface. The immobilisation of quaternary ammonium groups on functionalised surfaces offers several advantages including enhanced antibacterial effects, strong surface attachment, no requirement for solvents, no mobile counter anion and thus no negative environmental, health or corrosive effects.

Scheme 1. Alkyl Benzyldimethylammonium Chloride (BAC) Detergent Scheme 2. Immobilisation of Alkyl Benzyldimethylammonium cations on carboxyphenyl functionalities on the surface by electrostatic attractions (left) and incorporation of metal nanoparticles(right).

2. Surface modification of solid supports with diazonium compounds [9] Modification of the surfaces of such materials, for example by grafting of functional groups, has applications in a wide variety of fields. For instance, organic layers on electrode surfaces can be used to modify the electrocatalytic behaviour of the electrode and/or to improve the uniformity of subsequent metal deposition. Such modified electrode surfaces can be used in fuel cells, batteries, solar cells, metal corrosion, electrocatalysis, sensors, drug delivery and in other electrochemical applications.

[10] Many other materials benefit from surface modification in order to alter their properties or to immobilise specific chemical functionalities which permit coupling to other moieties. For example, organic groups can be grafted onto metal surfaces to impart the ability to bind to other chemical or biological entities; carbon fibres can be modified by binding to epoxy resins to form carbon composite materials having improved mechanical properties; carbon nanotubes can be functionalised to modify their properties (e.g. to prevent bundling); and silicon wafer surfaces can be functionalised to modify their performance. C\I

[11] Current methods for surface modification include the grafting of organic groups to LCD carbon or metal surfaces via the chemical or electrochemical reduction of the corresponding CD diazonium derivatives. Due to their spontaneous surface reactivity in the presence of reducing agents, diazonium derivatives have also been reported for use in electroless grafting of organic residues to metal nanoparticles (Mirkhalaf et al. J. Am. Chem. Soc. 128: 7400-7401, 2006) and Indium Tin Oxide (ITO) surfaces (Mirkhalaf eta,'., Langmuir, 27: 18531858, 2011).

[12] Electrochemical reduction of diazonium derivatives is the most widely used method for coating metal and carbon-containing surfaces, especially electrode materials. In such methods, a diazonium salt of the desired organic group is electrochemically reduced to form a highly reactive organic radical with the spontaneous elimination of dinitrogen. The resulting radical can then form a covalent bond with a conducting or semi-conducting surface.

[13] For example, US 2002/0144912 discloses a method in which organic diazonium salts are electrochemically reduced in a protic solvent in acidic medium leading to binding of the organic group to the surface of the carbon-containing material. In the electrochemical process, the carbon-containing substrate forms the negative electrode and is maintained at a potential value such that it can donate an electron to the diazonium salt. Cathodic reduction of the diazonium salt is effected either by repetitive cyclic voltammetry in a potential range in which the diazonium salts are reduced, or by electrolysis at a potential which is more negative than the reduction potential of the diazonium salt. The resulting organic radical binds to a carbon of the carbon-containing electrode. A disadvantage of this particular method is that the substrate to be coated must be conducting (or semi-conducting). Such electrochemical methods are thus not suitable for modifying the surfaces of non-conducting substrates, e.g. non-conducting polymeric and inorganic materials.

[14] There are also further disadvantages associated with electrochemical methods, which involve the reduction of diazonium species. The radicals formed on electrochemical reduction of diazonium salts are highly reactive and have a tendency to react with one another to form dimers or other polymeric species before bonding to the substrate surface. This can lead to the formation of multi-layered coatings (which may, in some cases, be undesirable) and, more generally, to difficulties in controlling the extent of surface coverage. Furthermore, the conventional use of both chemical and electrochemical reducing agents leads to the formation of contaminants during the grafting process which need to be removed. These contaminants can also result in undesirable side-reactions taking place.

[15] Any diazonium salt whose organic residue, R, in radical form is stable enough to CD covalently bind to a substrate surface may be used in the method of the invention. Suitable C\I diazonium salts are those of the formula RN2*X-where R is an organic residue and X-is an organic or inorganic counter-anion (e.g. selected from halides, sulphates, phosphates,

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perchlorates, tetrafluoroborates, carboxylates and hexafluorophosphates, etc.). Ultrasound-assisted reduction of such salts may also be applied and can be shown as follows: RN2*-> RN2. -> IR. + N2 [16] Diazonium salts can easily be prepared using well known syntheses. These may, for example, be prepared in a one-step process from a wide range of anilines (diazotisation of the aniline derivative readily leads to the formation of an aryl diazonium compound). Alternatively, the diazonium salts may be prepared by reaction of the corresponding amine with sodium or potassium nitrite in a mineral acid. If required, the desired diazonium compound can be generated in situ (S. Branton and D. Belanger, J. Phys. Chem. 2005, 109, 24401).

[17] The diazonium salt concentration may be varied according to the desired extent of surface modification and the nature of the substrate surface. Suitable concentrations may readily be determined by those skilled in the art.

[18] Typically, the molar concentration of the diazonium salt will be between about 1 mM and 20 mM, preferably around 10 mM.

[19] Substrate surfaces which may be modified according to the invention include carbon-containing surfaces such as those comprising carbon materials (carbon black, glassy carbon, carbon powder, the surfaces of carbon nanotubes, diamond, synthetic diamond nanopowders, doped diamond substances and graphite), metal oxides (e.g. aluminium oxide, titanium oxide, zinc oxide, iron oxide etc) and silicon dioxide (silica) particles. Such materials may take any form, such as fibres, powders, felts, fabric, beads, composites, particulates, etc. Metal surfaces and metal nanoparticles (including those which comprise alloys) may also be modified according to the methods herein described and may include, in particular Pt, Ag, Au, Pd, Zn, Ti, Fe, Cu, Co, Ni or stainless steel. Semi-conducting and nonconducting surfaces may also be suitably modified. Semi-conducting surfaces include those comprising silicon or indium doped tin oxide (ITO) surfaces (Mirkhalaf et al., Langmuir, 2010, 27, 1853) and non-conducting textiles such as cotton fabrics. The term "substrate surface" as used herein is to be construed broadly and is considered to encompass the surfaces of particles (e.g. nanoparticles), metals, textiles, beads etc. 3. Experimental Protocols 3.1. Surface Modification of Fabrics, silica or alumina particles with p-calboxyphenyl

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C\I functionality [20] In order to functionalise fabrics and particles with covalent attachment, the reduction CD of the corresponding diazonium salts has been used. This is a known protocol to functionalise solid surfaces (M. M. Chehimi, Aryl Diazonium Salts, Wiley-VCH, 2012). The diazonium derivatives was synthesised and separated or made in situ followed by the attachment to the supports.

Procedure 1: Synthesis of 4-carboxybenzene diazonium tetrat1ouroborate salt (CBD) [21] 12 gr p-aminobenzoic acid is dispersed in 50 ml water to form a suspension. In order to improve dissolving, a few drops of 4 M NaOH is added with stirring. Then 200 ml HCI (1:1) is added with strong stirring in an ice bath. Then 9.8 gr Nallo2 is added slowly in several portions with strong stirring in the ice bath. After one hour stirring, 100 ml HBF4 is added with continued stirring in the ice bath for further one hour. The solution was left to the yellow precipitate to be settled down and then filtered on a sintered glass funnel and dried under the vacuum (yield=4.7 gr).

[22] The product is soluble in alkaline water and its reaction with alkaline beta-Naphtol solution gives a deep red precipitate confirming the presence of diazonium groups. ATRFTIR and Raman spectra of the product show diazonium group.

Figure 1. ATR FTIR spectrum of the product.

[23] Peak at 2309 cm-1 represents the presence of -N=N (diazonium) groups.

Figure 2. Raman reflectance spectra of the product (excitation laser= 785 nm).

[24] Again, peak at 2309 cm-1 represents diazonium groups and peaks at 1710 cm-1 show carboxylic acid functionality. Peaks correspond to the ring can also be assigned.

[25] The product was used for the attachment on various solid supports including: carbon powder and fibre, silica, alumina and dressing fabrics. The reduction of the diazonium salt on various supports was achieved by using either strong or weak reducing agents. C\I

LCD Figure 3. ATR FT-IR spectrum of a fabric made of viscose and wool (45%:55%) modified with 4-carboxybenzene diazonium tetraflouroborate salt (CBD) followed by reduction in aqueous solution.

[26] For a fabric modified with CBD as indicated in Fig 3, no peak between 2000-2500 cm-1 region is observed representing no remaining diazo band (-N=N) after the attachment on the fabric.

Procedure 2.

Example 1: Reduction with strong reducing agent [27] 0.5 gr CBD is dissolved in either iso-propanol or water containing a few drops of surfactant (Triton-X100) to improve solubility. Solid samples to be functionalised were immersed in this solutions and the reduction of CBD is taken place by adding droplets of sodium borohydride (0.05 gr NaBH4 in 50 ml water) in a sonicating bath. The complete reduction of diazonium leads to evolving nitrogen gas and the covalent attachment of carboxyphenyl functionalities. The modified fabrics are washed with water in the sonication bath to remove physically adsorbed materials and have a final light yellow colour. Modified powders are vacuum filtered and washed with water several times.

Example 2: Reduction with a user friendly procedure; [28] This procedure does not require strong reducing agents and is user and environmental friendly. The formation of reactive radicals from polymers and alcohols and their reductive effects for the reduction of metal ions leading to the formation of metal NPs have previously been reported (C. E. Hoppe, Langmuir, 2006, 22, 7027; J. Kou, Chem Comm., 2013, 49, 692).

[29] Herein, we report the reduction CBD by poly vinyl pyrrolidone (PVP-40) and/or glycerol together with microwave radiations instead of using hazardous strong chemical reducing agents.

[30] 0.5 gr CBD is dissolved in 50 ml water containing few drops of surfactant (Triton-X100) with stirring, then 1 gr PVP-40 (Aldrich) is added and after complete dissolving, solid samples immersed in the solution. The container is then covered with a lid and left in a microwave, high power for 5 min. Then, the samples are washed with water. The modified samples are then used for the templated attachment of N Ps and the quaternary ammonium salt.

Procedure 3: Modification with in situ diazonium production

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C\I [031] In order to avoid preparation and separation of CBD, in situ diazotisation of p-aminobenzoic acid (ABA) can also be carried out as outlined below: [32] 0.2 gr of (ABA) was dispersed in 20 ml water and HCI 1:3 (5m1 HCI(c) +15 ml water) CD is added drop wise with stirring until full dissolution of ABA. Then, 0.1 gr NaNO2 is added in several portions with strong stirring. After stirring for 30 min., dressing fabrics are dipped in this solution and reduced with either strong reducing agent (NaBH4) and sonication or weak reducing agent in microwave as outlined above. After completing the modification cycle, the samples are washed with pure water several times. The modified fabrics have yellow colours and are not affected with further washing and in ultrasonic bath.

Procedure 4: Functional/sat/on of silica surfaces [33] 0.3 gr silica (Si02) is dispersed in 50 ml water and 1 ml Triton-X100 is added with sonication, then 0.3 gr CBD is added until complete dissolution of CBD. Then 2 ml sodium borohydride reducing agent is added drop by drop with stirring within 30 min and further stirring for 2 hours. The yellow/orange product is separated by filtering or centrifuging and washed with pure water three times.

Procedure 5: Functionalisation of Alumina surfaces [34] 0.5 gr Alumina (Alumina, 0.3 micron) is dispersed in 50 ml water and 1 ml Triton-X100 is added with sonication, then 0.3 gr CBD is added until complete dissolution of CBD.

Then 2 ml sodium borohydride reducing agent is added drop by drop with stirring within 30 min and further stirring for 2 hours. The yellow/orange product is separated by filtering or centrifuging and washed with pure water three times.

Procedure 6: Functional/sat/on of Titania surfaces [35] 0.4 gr titanium dioxide (Ti02, 50 nm) nanopowder is dispersed in 50 ml water and 1 ml Triton-X100 is added with sonication, then 0.3 gr CBD is added until complete dissolution of CBD. Then 2 ml sodium borohydride reducing agent is added drop by drop with stirring within 30 min and further stirring for 2 hours. The yellow/orange product is separated by filtering or centrifuging and washed with pure water three times 3.2. Attachment of quatemary ammonium cation via ion pairing with surface anions Procedure: The support functionalised with carboxyphenyl groups are dipped in an alkaline O solution (e.g. 0.1 M KOH). The supports are then washed with water and dipped in a solution C\I containing 0.5 gr alkylbenzyl dimethyl ammonium chloride in 50 ml water and sonicated for min. The samples are then washed with water several times and let them to dry. For O powders (e.g. carbon, silica, alumina, titania), they are modified with CBD as mentioned above, then made alkaline and dispersed in a 1% solution of quaternary ammonium salt as mentioned above and sonicated for 30 min. The particles are then filtered and washed with water and dry in air.

3.3. Antibacterial and Antiblofilm determination [36] Three types of bacterial species were selected for the measurements: S. Epidennidis RP62A (ATCC 35984); P. aeruginosa (PA01) and a alb/cans.

[37] To evaluate the adhesion capacity to different surfaces of biofilm forming bacteria, the bacteria (1.5x108 CFU/ml in Phosphate Buffered Saline (PBS 1X)) was incubated in contact with the surfaces of titanium slides in a 6-well plate for 18-24 h at 37°C. After incubation, the titanium slides were gently washed to remove non-adhered or weakly-adhered bacteria in 3 mL of PBS 1X.

[38] For removing the adhered cell from the surface of the titanium slides, each slide was sonicated into a falcon tube with 3 ml of PBS 1X for 20 min. 100 pl of sonication liquid was inoculated in a Luria-Bertani agar plate (Sabouraud agar in case of C. alb/cans) and cultured overnight at 37°C for counting the adhered bacteria.

4. Antibacterial Results 4.1. Dressing Fabrics directly modified with surface ion pairs * No Silver (sample 1) [039] 0.15 gr CBD dissolved in 20 ml water and dressings dipped in this solution and reduced with 0.1 gr PVP-40 and two drops of glycerine in microwave for 5 min high power. The light yellow dressing are then washed and dipped in 20 ml 0.01M NaOH and then washed. The dressings were then dipped in solution containing 0.15 gr Benzenealkanium Chloride (BAC) in 20 ml water with sonication and then washed several time and dry in air.

Figure 4. Logarithmic reduction of bacterial population for Sample 1 compared to uncoated dressing (blank) for each bacterial species: SA (S. Aurous); PA (P. Aeruginosa); EC (E. Coli); CA (c. Alb/cans; AN (Antrobacter Agilis); CP(C. Perfringenes); BF (Bacillus Fragilis), EF (E. Faecalis); SP (S. Pneumonia): MRSA (Meth:Giffin-resistant Staphylococcus Aureus); SE (S. Epiciermidis): BS (Bacillus Subtills) O * With Sliver (sample 2) C\I [040] Similar to 1 but before drying the dressings are dipped in 5 ml 0.01 M AgNO3 in 20 ml water with sonication for 10 min. The dressings were then washed several times and O reduced with either a few drops of dilute NaBH4 0.01M or in microwave with the presence of PVP-40 and a few drops of glycerine in 20 ml water followed by washing.

Figure 5. Logarithmic reduction of bacterial population for Sample 2 compared to uncoated dressing (blank) for each bacterial species: SA (S. Aurous); PA (P. Aeruginosa); EC (E. Coli); CA (c. Alb/cans; AN (Antrobacter Agilis); CP(C. Perfringenes); BF (Bacillus Fragilis), EF (E. Faecalis); SP (S. Pneumonia): MRSA (Meth:Giffin-resistant Staphylococcus Aure.us); SE S. Epidermidis); BS (Bacillus Subtilis).

4.2. Samples based on CBD coated Alumina No silver (sample 3) [041] 1.2 gr Alumina (0.05 micron) modified with CBD and 0.2 gr Benzenealkanium Chloride (BAC) are dispersed in 50 ml water and the gauze dressings are sonicated in this solution for 10 min. Then remove and wash them several times and dry in air.

Figure 6. Logarithmic reduction of bacterial population for Sample 3 compared to uncoated dressing (blank) for each bacterial species: SA (S. Aurous); PA (P. Aeruginosa); EC (E. Coli); CA (C. Alb/cans; AN (Antrobacter Agilis); CP(C. Perfringenes); BF (Bacillus Fragilis), EF (E. Faecalis); SP (S. Pneumonia); MRSA (Meth:Giffin-resistant Staphylococcus Aureus): SE (S. Epideraidis); BS (Bacillus Subtilis).

[42] From the above results, it is obvious that sample 3 that is dressing with Alumina functionalised with CBD and BAC have maximum logarithmic reduction of bacterial growth compared with uncoated dressing for all 12 bacterial species tested. Dressings with silver nanoparticles have shown less activity compared to those without silver although they are still active against MRSA and Bacillus Fragilis (BF). This suggests that by careful selecting of modification procedure, the coating can be tuned for certain bacteria and application.

5. Anti-biofilm Properties of Selected Samples 5.1. Methods for treated dressings C\I * No silver (ST1) [43] 1.2 gr Alumina-ABA and 0.2 gr sodium carbonate and 0.2 gr BAG were grounded to

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fine powder. 0.2 gr of the powder was dispersed in water and sonicated with two drops of Triton in 20 ml water in the presence of dressing.

* With silver (ST2) [44] 0.2 gr of ST1 was dispersed in 100 ml water and two drops of Triton-X100 was added and sonicated for fine dispersion. Then, 5 ml AgNO3 in 100 ml water was added and the solution was reduced with NaBH4 with sonication. Gauze dressings were dispersed in this solution with shaking (sonication) followed by washing several times.

Dressing strips [45] Dressing strips were prepared cutting strips size 1.5cm x 0.5 cm with sterile scissors. The strips contain the nanoparticles to test in front of different microorganisms. Growth medium [46] Microorganisms were stored on milk medium at -80°C. A primary culture was grown on blood agar medium at 37°C for 24 hours. (the most common growth media for bacteria). Luria broth medium (LB) was used to grow and to test the compounds activity.

lnoculum [017] Inoculum concentration is important to test the activity of new compounds. Lower or higher concentrations can have influence in positive or false results. An inoculum of 105 CFU/ml in bacterial species is adequate for working. Mc Farland 0.5 was used to get 108 CFU/ml. 1 rriL of Mc Farianci 0.5 was inoculated in 1 niL of LB culture media betting about 105cluirnl. The tube containing the inoculurn media and the dressing strip was inoculated at 37°C for 24 hours without shaking to avoid disrupting the biofilm's formation.

Washing dressing stfips [48] To avoid contamination on the final CFU's count is important to wash dressing strips gently, as inefficient washes can show false compound inactivity. Washes must be effective but non aggressive at the same time. To do it, dressing strips were introduced in Eppendoff tubes with saline solution and were washed for 4 times (washed by inversion for ten seconds).

Son/cation [49] After correct washing, samples were sonicated for twenty minutes in a water bath to transfer microorganisms from strips to medium and to detect the antimicrobial activity. Antimicrobial activity evaluation CD [050] Antimicrobial activity was detected by different dilutions from Eppendoris tubes and C\I cultured in LB agar at 37°C for 24 hours. Finally, Cfu's were determined by counting them.

The growth of planktonic bacteria were tested following the above procedure and the

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average growth population was measured by spectrophotometric method. The following results are obtained: B. S. S. E. S. C. A. niger C. B. fragilis subtilis pyogenes epider faecalis aureus albica perfring midis MRSA ns ens 511 0.0 0.0 1.2x101 0.0 7.0x103 0.0 1.3x104 0.0 0.0 512 0.0 0.0 6.0x103 1.2x104 0.0 0.0 2.0x104 0.0 1.2x107 Table 1: Growth of planktonic bacterial in the presence of samples ST1 and ST2. [051] The following results are for antiblofilm testing for the above samples: Staphylococcus aureus sample ng mean cfu/m110E-1 positive control Cfu/positive control ST1 1,22E+02 1,040E-F11 1,17E-09 1,17E-07 512 2,78E+01 1,040E+11 2,68E-10 2,68E-08 Table 2. Antibiofilm results of Samples ST1 and ST2 for Staphylococcus aureus Escherichia coli sample n2 mean cfu/m110E-1 positive control Cfu/positive control ST1 2,93E+01 6,350E+11 4,62E-11 4,62E-09 S12 2,22E+07 6,350E+11 3,49E-05 3,49E-03 Table 3. Antibiofilm results of Samples ST1 and S12 for Escherichia coil Pseudomonas aeruginosa CDsample mean cfu/m110E-1 positive control Cfu/positive control % ng ST1 1,09E+07 1,150E+12 9,50E-06 9,50E-04 LCD ST2 6,26E+07 1,150E+12 5,44E-05 5,44E-03 Table 4. Antibiofilm results of Samples ST1 and ST2 for Pseudomonas aeruglnosa [052] In conclusion, samples ST1, and ST2 have strong bactericidal and antibiofilm properties, in addition, ST1 has the highest antibiofilm property against E. Coli and P. aeruginosa but ST2 has highest property against Staphylococcus aureus. Therefore, based on application, products for the selective activity against planktonic bacteria and antibiofilms of selective species can be prepared.

Claims (15)

1. Claims 1. The use of negatively charged functionalities on surfaces for the attachment of positively charged quaternary ammonium antibacterial compounds via ion pairing for their application as effective and tuneable antimicrobial and antibiofilm surfaces is claimed.
2. 2. The procedure in claim 1 uses strong or weak reducing agents for the reduction of p-benzoic acid diazonium ion leading to the attachment of carboxyphenyl functionalities on solid surfaces as monolayer coatings.
3. 3. The carboxylic acid functionality in claim 2 is used for the immobilisation of any type of antibacterial quaternary ammonium salts leading to strong attachment of these agents to the substrate demonstrating enhanced antibacterial properties.
4. 4. The substrate as described in claim 1 is applicable on surfaces including metal CD surfaces, cotton fabrics, carbon, silica, alumina, Titania or metal oxide particles. C\I
5. 5. The ion pairing formed on the surface as claimed in 1-4 offers extended capabilities asCDbackbones for the further formation of antibacterial metal nanoparticles.
6. 6. The composite nanofilms formed as claimed in 1-5 are used for tuning antibacterial/antibiofilm properties of desired surface.
7. 7. The composite nanofilms formed as claimed in 1-5 are used for tuning maximum effect on specifically targeted bacteria.
8. 8. The modified surfaces as claimed in 1-7 can be used for applications in medical devices, wound dressings and general surface hygiene materials such tissues, towels, wipes and beddings.
9. 9. The effective antibacterial (quaternary ammonium) as claimed in 1-8 is strongly bound to the surface preventing its release into the open wound and environment, offering long lasting effectiveness of the modified surface.
10. 10. The procedures as claimed in 1-3 minimise the usage of antibacterials offering cost effective production and low impact on environment and human health.
11. 11. The formation of the ion pairs in claim 2 and 3, removes the chloride ions as negative counter ions associated with the quaternary ammonium surfactants after washing preventing adverse effects of these anions.
12. 12. The solvents used in the procedures in claims 1-3 are subsequently removed to obtain dry products.
13. 13. The ion pairs as in procedure in claims 1, 4,5 are used as backbones for further deposition of antibacterial silver and/or copper nanoparticles on desired substrates.
14. 14. Substrates in claim 13, are formed as alternative coatings on fabric materials offering extended effects.C\I
15. 15. Substrates in claim 13, are applied as liquid suspensions in aqueous media. LtD