Patent Application
20240315241
The invention concerns polymeric compositions having antimicrobial activity, in particular antiviral, with high effectiveness. Said compositions may be used to prepare personal care products, such as hand gel, as well as coatings intended for frequently touched objects, such as handles, seats, air filter, medical equipment and protective face masks.
The global COVID-19 pandemic has highlighted the need for increasingly effective methods and technologies to contain the diffusion of pathogenic microorganisms, and more specifically the diffusion of viruses. Transmission through contaminated surfaces has been recognise as an important mean of diffusion of SARS-COV-2. Among the various available antimicrobial agents, antimicrobial polymers represent a class of biocides which has become increasingly important as an alternative to known biocides. Known disinfectants are based on liquids or gases with a low molecular weight, hence their use is limited by their volatility and by the toxicity of their residues. Antimicrobial polymers can be bound to surfaces, allowing for killing of microorganisms without releasing biocides. Many of the antimicrobial polymers known in the art were focused on antibacterial properties, while less attention was given to antiviral properties.
Antimicrobial polymers have been known since 1965, when Cornell and Donaruma described polymers and copolymers that kill bacteria (Cornell, R.J.; Donaruma, L.G. J. Med. Chem. 1965, 8, 388-390). Since then, many reports have appeared in (patent and scientific) literature describing bactericidal coatings which incorporate unbound biocidal agents, such as metallic oxide particles (WO2008127200A1), chlorine dioxide (Leung, W. K.; Lau, A. P. S.; Yeung, K. L., J. Appl. Microbiol. 106 (2009) 1463-1472), iodine compounds (Ahmed, S. I.; Hasan, N.; Abid, C.Z; Mazumdar, N., J. Appl. Polymer Science 109 (2008) 775; Punyani, S; Narayanan, P.; Vasudevan, P.; Singh, H.; J. Appl. Polymer Science 103 (2007) 3334), silver nanoparticles (WO2008063680A2) or benzophenone (Hong, K. H.; Sun, G., J. Applied Polymer Science 112 (2009) 2019) mixed with a polymeric matrix.
Furthermore, coatings comprising quaternary ammonium (QA) compounds are known. QA compounds are the antibacterial agents which are most largely employed for medical and public health applications, since they have shown to be effective against both Gram negative and Gram positive bacteria (Tashiro, T., Macromol. Mater. Eng. 286 (2001) 63-87). According to the literature (Felix Siedenbiedel e Joerg C. Tiller, Polymers 2012, 4, 46-71), three types of antimicrobial polymers are known: polymeric biocides, in which the monomer is a biocide; biocidal polymers, wherein the active agent is incorporated into a macromolecule in a casual manner; polymers that release biocides, wherein the polymer matrix acts as a vehicle for the biocides, which can be transferred to the target, for example a microbial cell.
In light of the importance currently attributed to antimicrobial compositions, in particular antiviral, in the field of personal care products, such as for example hand sanitizer gels, there is a clear need for improving the effectiveness of such compositions. In particular, among the experts in the field there is a strong need for compositions having an effective antimicrobial activity, in particular antiviral.
A aim of the invention is to improve known antimicrobial compositions, in particular antiviral.
Another aim is to provide antimicrobial compositions having an effective antiviral activity.
A further aim is to provide antimicrobial compositions which have an effective antiviral activity and that can be incorporated into personal care products, such as for example hand gel.
Yet another aim is to provide antimicrobial compositions which have an effective antiviral activity and that can be used to produce objects having an antimicrobial coating, in particular antiviral.
According to the invention, it is foreseen a polymeric composition having antimicrobial activity, as defined in claim 1.
Thanks to the invention, polymeric compositions showing an effective antimicrobial activity, in particular antiviral, are provided. The polymeric compositions according to the invention comprise a silver complex and/or benzalkonium ions bound to the polystyrene sulfonate matrix. Therefore, in the polymeric compositions according to the invention, the active principle (silver complex and/or benzalkonium ions) is incorporated into a macromolecule (polystyrene sulfonate).
The polymeric compositions according to the invention are insoluble in water but are suitably soluble in volatile organic solvents, namely alcohols. In this way, it is possible preparing stable alcoholic solutions that can be readily sprayed on a solid surface. Upon evaporation of the solvent, on the surface a tightly bonded, transparent film remains, which is provided with remarkable antimicrobial properties towards Gram-positive and Gram-negative bacteria, as well as fungi (Candida albicans). Thanks to the insolubility in water of the polymeric compositions according to the invention, the antimicrobial effect can be maintained for several months (from the date of application to a surface). In light of the fact that viruses can survive on inanimate surfaces for periods which vary from hours to several days, the Applicant has investigated the antiviral activity of said polymeric compositions. As a surprisingly unexpected effect, strong experimental evidence of antiviral activity of the polymeric compositions according to the invention has been reported. In particular, the antiviral activity has been experimentally tested and verified towards two members of the Coronaviridae family, namely human Coronavirus 229E (HCoV-229E), which is the etiological agent of common cold, and human SARS-Cov-2, which is the etiological agent of COVID-19.
The polymeric compositions according to the invention can be suitably incorporated into coatings intended for frequently touched objects, such as seats, handles, door knobs or keyboards, as well as devices for professional use, such as medical equipment and protective face masks. Said polymeric compositions may be used to coat filters for air decontamination devices and for air conditioning devices, for example in cars and buildings. A treatment of surfaces with the polymeric compositions according to the invention allows to prevent unwanted and potentially dangerous microbial contamination, in particular viral, of the surfaces. Furthermore, the polymeric compositions according to the invention can be suitably included in formulations of hand sanitizer gels, the use of which has become increasingly important due to the COVID-19 global pandemic.
The invention will be better understood and implemented with reference to the attached drawings which illustrate an exemplary and non-limiting form of implementation, of which:
In the present description, as well as in the attached claims:
In one embodiment, the polymeric composition according to the invention is a polymeric salt of anionic polystyrene sulfonate (PSS) and benzalkonium cations (Bz+), wherein the benzalkonium cations are bonded to the PSS matrix.
Since in anionic PSS the acidic sulfonic groups (—SO3H) are deprotonated to sulfonate anions (—SO3−), the benzalkonium cations are electrostatically bonded to the sulfonate anions.
In another embodiment, the polymeric composition according to the invention is a polymeric salt of anionic polystyrene sulfonate (PSS) and positively charged micellar derivatives containing benzalkonium cations and a silver complex, wherein the positively charged micellar derivatives are electrostatically bonded to the sulfonate anions of the PSS matrix.
Therefore, the polymeric composition according to the invention is a polymeric salt of anionic polystyrene sulfonate (PSS) and a cationic component, wherein the cationic component is selected from the group consisting of benzalkonium cations and positively charged micellar derivatives containing benzalkonium cations and a silver complex.
The micellar derivatives can be prepared starting from a sodium salt of bi-coordinated silver complex having the formula Na+3[Ag+(MPA2−)2]3−, wherein MPA2− is the anionic bivalent form of 2-mercapto-4-methyl-5-thiazolacetic acid (MPA). MPA2− is obtained through deprotonation of carboxylic groups (—COOH) and mercapto (—SH) of MPA. The preparation of said sodium salt of the bi-coordinated silver complex is described in Example 1 below. The sodium salt of the bi-coordinated silver complex is then reacted with benzalkonium chloride, as described in Example 2 below.
With the purpose of facilitating the understanding of the invention, but without limiting its scope, the following Examples are described: preparation of a sodium salt of bi-coordinated silver complex having the formula Na+3[Ag+(MPA2−)2]3− (Example 1); preparation of micellar derivatives of (Bz+)3[Ag+(MPA2−)2]−3 (Example 2); preparation of polymeric compositions (polymeric salts M-1 and M-2) according to the invention (Example 3); evaluation of the antimicrobial activity of the sodium salt of bi-coordinated silver complex having the formula Na+3[Ag+(MPA2−)2]3− in aqueous solution (Example 4); evaluation of the antimicrobial activity of micellar derivatives of (Bz+)3[Ag+(MPA2−)2]−3 in aqueous solution (Example 5); evaluation of the antimicrobial activity of M-1 and M-2 (Example 6); evaluation of the antiviral effectiveness of Na+3[Ag+(MPA2−)2]3− in aqueous solution towards SARS-Cov2 (Example 7); evaluation of the antiviral effectiveness of M-1 towards Coronavirus 229E (Example 8); evaluation of the antiviral effectiveness of M-1 towards SARS-Cov2 (Example 9); evaluation of the antiviral effectiveness of M-2 towards SARS-Cov2 (Example 10).
With reference to the described Examples, all chemical products were bought from Sigma-Aldrich. Elemental analysis (C, H, N) was carried out with a LECO-CHN analyser. Silver was determined by plasma atomic absorption spectroscopy with an instrument Perkin Elmer Optima 3100 XL. FT-IR spectra were recorded with a spectrometer FT-IR Bruker Vertex 70 in diffuse reflectance mode and dispersing the powder of chemical products in KBr. Dimensional analysis and Zeta potential were obtained with a Z-sizer Malvern analyser.
12.36 g of a 1M NaOH solution in water were added to an amount of 1.135 g of MPA. 186.5 g of distilled water were then added and the solution was maintained under stirring for 5 min. Finally, 100 g of aqueous solution containing 0.51 g of AgNO3 were added, yielding a yellow solution which was maintained under stirring for another 30 min. The salt of bi-coordinated silver complex Na+3[Ag+(MPA2−)2]3− was precipitated by addition of an acetone excess, filtered and air dried. Ag+%=0.108%. Elemental analysis: calculated for Na3AgC12N2S4O4H10; C 26.14; N, 5.08; H, 1.83; Ag, 19.57. Found: C, 25.87; N 5.0; H 1.88; Ag 19.46.
Elemental analysis and spectroscopic data for the silver complex are coherent with the presence of two anionic ligands MPA2− coordinated to ions Ag+. The -IR spectrum of MPA shows a strong CO stretching band of the carboxylic group at 1704±2 cm−1 and a stretching band S—H at 2555±2 cm−1. The S—H stretching band disappears in the anionic silver complex, which is coherent with sulphur coordination to Ag+. Intense bands due to asymmetric and symmetric stretching modes of carboxylate appear at 1580±2 cm−1 e 1386±2 cm−1, respectively.
Benzalkonium (Bz) is a mixture of quaternary ammonium salts (cationic surfactants) and, more specifically, is a mixture of alkyl-benzyl-dimethyl-ammonium chlorides, in which the alkyl group varies from octyl (C8H17—) to octadecil (C18H37—). The average molecular weight of benzalkonium chloride salt is 370 g. Adding a small excess of benzalkonium chloride to an aqueous solution of Na+3[Ag+(MPA2−)2]3− (salt of bi-coordinated silver complex) of Example 1, the uncharged salt Bz+)3[Ag+(MPA2−)2]−3 can be suitably precipitated. The addition of benzalkonium chloride to the anionic silver complex [Ag+(MPA2−)2]3− in water gives rise to the neutralization of the negative charge with formation of a precipitate which is coherent with the stoichiometry (Bz+)3[Ag+(MPA2−)2]−3 (salt of the anionic silver complex).
Through further addition of an excess of benzalkonium chloride to the precipitated salt in water, (Bz+)3[Ag+(MPA2−)2]−3 is dissolved by formation of positively charged micellar derivatives (hereinafter referred to as “M-0”), caused by the excess of surrounding Bz+ cations, with a dimensional distribution in the range 10-500 nm.
On the laboratory scale, the above-described micellar derivatives can be prepared by addition of 100 g of a 50% benzalkonium chloride solution in water to 100 g of the Na+3[Ag+(MPA2−)2]3− solution with silver at 0.108% (prepared as described in Example 1).
Polymeric salts (neutral polymers) of positively charged micellar derivatives of (Bz+)3[Ag+(MPA2−)2]−3 (hereinafter referred to as “M-1”) of Example 2 and polymeric salts (neutral polymers) of benzalkonium ions (hereinafter referred to as “M-2”) were obtained as precipitates in distilled water by addition of poly(sodium 4-styrene sulfonate) (PSS) with an average molecular weight of 70,000 g. Practically, the polymeric salt M-1 can be obtained by adding PSS to the micellar derivatives of Example 2, said addition being performed with a quantity by weight of PSS equal to about 60% of the weight of benzalkonium chloride, while the polymeric salt M-2 can be obtained by adding an excess of benzalkonium chloride to an aqueous solution of PSS. Similarly to what described above with reference to M-1, that corresponds to an addition of a quantity by weight of PSS equal to about 60% of the weight of benzalkonium chloride.
The neutral precipitated polymers M-1, containing PSS, [Ag+(MPA2−)2]−3 and Bz+ ions electrostatically bonded to sulfonate groups, and M-2, containing PSS and Bz+ ions electrostatically bonded to sulfonate groups, were separated from the aqueous solutions by filtration and were dried at 50° C. both M-1 and M-2 are insoluble in water, but are suitably soluble in alcoholic solvents. Thus, the polymeric solids were dissolved in isopropanol or ethanol to obtain a solution of M-1, containing 1% Bz, 0.62% PSS and 0.002% Ag, and a solution of M-2, containing 1% Bz and 0.62% PSS.
The solutions of M-1 in alcohol are composed by micellar aggregates (micellar derivatives) with diameters in the order of 10 nm and zeta potential of −3.9 mV. A minority of aggregates with diameter in the order of 50 and 500 nm was also observed. Infrared spectra of the polymeric solids M-1 and M-2 are similar, with vibrational bands concordant with the overlap of the intense bands of benzalkonium and PSS. As example, the FT-IR spectrum of M-1 is shown in
The antimicrobial activity of an aqueous solution at pH 8.3 of Na+3[Ag+(MPA2−)2]3− with an ionic concentration of [Ag+]=0.1% was tested against the following blend of microorganism strains (bought from Diagnostic International Distribution S.p.A): Pseudomonas aeruginosa ATCC 15442, Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 10536, Enterococcus hirae ATCC 10541 e Candida albicans ATCC 10231. For each microbial strain, the concentration of the blend was in the range 1.5×1012−5×1012 unity forming colonies (UFC). A volume of 100 μl of the blend was seeded in Petri dishes containing a non-selective culture medium TSA (Tryptone Soya Agar) and a sample of 100 μl of said aqueous solution of Na+3[Ag+(MPA2−)2]3− was put in the centre of each Petri dish in contact with the microbial blend. The Petri dishes were then incubated at 37° C. for 24 h.
The antimicrobial activity of a diluted aqueous solution of micellar derivatives—containing benzalkonium at 1% and silver at 0.0025%—was tested against the following blend of microorganism strains (bought from Diagnostic International Distribution S.p.A): Pseudomonas aeruginosa ATCC 15442, Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 10536, Enterococcus hirae ATCC 10541 e Candida albicans ATCC 10231.
Blends of the different strains of microorganisms were prepared, having concentrations between 1.5×1012 and 5.5×1012 UFC for each strain. A sample of 100 μl of the aqueous solution of micellar derivatives was deposited at the centre of Petri dishes. 50 μl of the microbial blend were then deposited on the aqueous solution of micellar derivatives and left in contact with the latter for 5 minutes. Non-selective culture medium TSA (Tryptone Soya Agar) was then added and the dishes were then incubated at 37° C. for 24 h. After incubation, the dishes were examined in order to evaluate microbial proliferation (formation of colonies). As shown in
Therefore, the analytical result obtained confirms that the diluted aqueous solution of micellar derivatives (containing benzalkonium at 1% and silver at 0.0025%) is able to inhibit the growth of about 1011 UFC Gram-positive bacteria, Gram-negative bacteria and of the fungal species Candida albicans.
The antimicrobial activity of M-1 (alcoholic solution containing Bz 1%, Ag 0.002%, PSS 0.62% and the rest to 100 being ethanol or isopropanol) and M-2 (alcoholic solution containing Bz 1%, PSS 0.62% and the rest to 100 being ethanol or isopropanol) was tested against the following microorganisms strains (bought from Diagnostic International Distribution S.p.A.): Pseudomonas aeruginosa ATCC 15442, Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 10536, Enterococcus hirae ATCC 10541 and Candida albicans ATCC 10231.
Blends of the different strains of microorganisms were prepared, having concentrations expressed as UFC between 1.5×1012 e 5.5×1012 for each species. Samples of 100 μl of the alcoholic solutions of M-1 and M-2 were deposited at the centre of Petri dishes and dried for 2 h in an oven at 37°° C. 100 μl of the microbial blend were then added and left in contact with M-1 and M-2 for 5 minutes. Non-selective culture medium TSA (Tryptone Soya Agar) was added and the Petri dishes were then incubated at 37° C. for 24 h. After incubation, the dishes were examined in order to evaluate microbial proliferation (colonies formation). As shown in
The experiments were performed in a BSL-3 facility at the University of Ferrara (Italy). An aqueous solution containing Na+3[Ag+(MPA2−)2]3− with a concentration of Ag+ of 0.108% was sprayed onto disposable plastic Petri dishes and left to dry in a biosafety cabinet. Then, 1 ml of SARS-Cov-2 inoculum was incubated on each Petri dish for 5 minutes. After treatment, the virus for used to inoculate sensitive cells (VeroE6; ATCC® CRL-1586™). Cellular supernatants were collected at 24, 48, and 72 hours post-infection to evaluate viral replication. As a positive control a untreated viral inoculum was used. The replication of SARS-Cov-2 was estimated by qRT-PCR after extraction of RNA. The cytopathic effect in the infected cells was assessed by optical microscopy (Nikon inverted microscope).
Analytical results were expressed as threshold cycle (Ct), namely the number of cycles of qRT-PCR required for the fluorescent signal to exceed the threshold value, a number which is inversely proportional to the amount of viral RNA. In other words, Ct is the cycle in which the target becomes detectable. The average Ct is shown in the graph of
Table 1 below shows the replication of SARS-Cov2 in VeroE6 cells after treatment with aqueous solution containing Ag, a replication which is expressed as logarithmic difference between the untreated control. The pre-treatment of SARS-Cov2 with the aqueous solution containing Ag caused a significant decrease of 0.91 logarithms of the viral charge at 24 h post-infection (hpi), a decrease of 0.57 logarithms at 48 pci, while the reductions observed at a time more distant from the infection are not significant:
The graph of
Similar experiments were carried out on diluted aqueous solutions containing Na+3[Ag+(MPA2−)2]3− with a concentration of Ag+ of 0.002% and showed that the incubation of SARS-Cov2 for 5 minutes on surfaces treated with an aqueous solution containing Ag produced a reduction of 0.82 logarithms of the viral charge at 24 hours post-infection. The reduction was responsible for a small decrease of the cytopathic effect, as highlighted by morphologic observation at 72 hours post-infection.
The experiments were performed in a BSL-3 facility at the University of Ferrara (Italy). A solution in isopropanol of M-1 having the same composition reported in Example 6 (Bz 1%, Ag 0.002%, PSS 0.62%) was sprayed onto disposable plastic Petri dishes and left to dry in a biosafety cabinet. Then, 1 ml of human Coronavirus 229E (ATCC: VR-740) was incubated in each Petri dish for 5 minutes. After treatment, the virus was used to inoculate human sensitive cells (MRC-5, ATCC: CCL-171). Cellular supernatants were collected at 24 and 48 hours post-infection to evaluate viral replication. As a positive control a untreated viral inoculum was used. Viral replication was estimated by qRT-PCR after extraction of RNA.
Analytical results are expressed as threshold cycles (Ct), namely the cycle in which the target becomes detectable. Average Ct is shown in the graph of
Table 2 below shows the replication of Coronavirus 229E in MRC-5 cells after treatment with M-1, replication which is expressed logarithmic difference compared to the untreated control. The pre-treatment of Coronavirus 229E with M-1 caused a reduction of 6.1 logarithms of the viral charge at 24 hours post-infection and a reduction of 6.5 logarithms at 48 hours post-infection:
The graph of
All experiments were performed in a BSL-3 facility. A solution of M-1 in isopropanol, with the same composition reported in Example 6 (Bz 1%, Ag 0.002%, PSS 0.62%), was sprayed onto disposable plastic Petri dishes and left to dry in a biosafety cabinet. Then, 1 ml of SARS-Cov-2 (ATCC: VR-740) was incubated in each Petri dish for 5 minutes. After treatment, the virus was used to inoculate human sensitive cells (VeroE6; ATCC® CRL-1586™). Cellular supernatants were collected at 24, 48 and 72 hours post-infection to evaluate viral replication. As a positive control a untreated viral inoculum was used. The replication of SARS-Cov-2 was estimated by qRT-PCR after extraction of RNA. The cytopathic effect in the infected cells was assessed by optical microscopy (Nikon inverted microscope).
Analytical results are expressed as threshold cycles (Ct), namely the cycle in which the target becomes detectable. Average Ct is shown in the graph of
Table 3 below shows the replication of SARS-Cov-2 in VeroE6 cells after treatment with M-1, replication which is expressed as logarithmic difference compared to the untreated control. The pre-treatment of SARS-Cov-2 with M-1 cause a reduction of 2,665 logarithms of the viral charge at 24 hours post-infection (hpi), a reducation of 3,696 logarithms at 48 hours hpi and a reduction of 6,832 logarithms at 72 hpi compared to the untreated incolumum:
The graph of
All experiments were performed in a BSL-3 facility. A solution of M-2 in isopropanol, with the same composition reported in Example 6 (Bz 1%, PSS 0.62%), was sprayed onto disposable plastic Petri dishes and left to dry in a biosafety cabinet. Then, 1 ml of SARS-Cov-2 inoculum was incubated in each Petri dish for 5 minutes. After treatment, the virus was used to inoculate human sensitive cells (VeroE6; ATCC® CRL-1586™). Cellular supernatants were collected at 24, 48 and 72 hours post-infection to evaluate viral replication. As a positive control a untreated viral inoculum was used. The replication of SARS-Cov-2 was estimated by qRT-PCR after extraction of RNA. The cytopathic effect in the infected cells was assessed by optical microscopy (Nikon inverted microscope).
Analytical results are expressed as threshold cycles (Ct), namely the cycle in which the target becomes detectable. The threshold cycles are shown in the graph of
Table 4 below shows the replication of SARS-Cov-2 in VeroE6 cells after treatment with M-2, replication which is expressed as logarithmic difference compared to the untreated control. The pre-treatment of SARS-Cov-2 with M-2 caused a significant reduction of the viral charge at all time points post-infection (hours post-infection: hpi) compared to the untreated virus, with an average reduction of more than 5 logarithms, corresponding to a decrease of over 105:
The graph of
Variation and/or additions of what has been described above are possible.
Even if the polymeric composition according to the invention were prepared mainly on a laboratory scale, the skilled person will be able to appropriately adapt said polymeric compositions to produce them on industrial scale.
In particular, the skilled person will be able to suitably incorporated the polymeric compositions according to the invention into coatings intended for frequently touched objects, as well as medical equipment, face protective masks and air decontamination/air conditioning devices. Similarly, the skilled person will be able to suitably include the polymeric compositions according to the invention in formulations of personal care products, such as hand sanitizer gels.
| Number | Date | Country | Kind |
|---|---|---|---|
| 102021000003893 | Feb 2021 | IT | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/054100 | 2/18/2022 | WO |
