Patent Application
20240301218
This invention relates to substrate independent coatings utilizing self-assembly of dopamine or other polymeric binders; ultra-high or high molecular weight hydrophilic polymers; and conjugated antimicrobial peptides (AMPs); and methods for making such coatings. In particular, the invention relates to coatings that may be applied onto substrates, such as medical devices and implants.
The onset of infections caused by opportunistic biofilm-forming pathogens can lead to severe infections and, in some cases, cause death, particularly in immunocompromised patients. Various approaches are currently being investigated to prevent implant/device associated infections, and the most promising approaches include both the prevention of bacterial adhesion and biofilm formation. Anti-adhesive and antimicrobial coatings have shown great promise to combat device/implant infections. These dual functional antibacterial surfaces are prepared by incorporating antimicrobial agents into non-fouling materials through covalent bonding[1-2] or layer-by-layer (LBL) deposition[3-4]. There are several configurations or structures that have been adopted in the literature, including combining biocidal sub-layers with antifouling upper layers[5-6], contact-active antimicrobial upper-layers and antifouling sub-layers[7-8] and, layering with evenly mixed antimicrobial and non-fouling components[9-14]. In principle, such surfaces should have both passive and active functions simultaneously, to improve the overall antibacterial efficacy.
With regards to antimicrobial agents, coatings containing covalently attached antimicrobial peptides (AMPs) showed excellent activity in preventing infections associated with implants in mouse models[14-16]. AMPs have broad spectrum anti-biofilm activity and minimal chance of developing resistance due to the multiple modes of action of the AMPs. However, tethered AMPs often showed decreased activity when compared to their soluble forms[17], which limits the efficient biofilm prevention by most peptides. The conjugation methods also influence the activity of tethered peptides since they can interfere with the mobility and flexibility of tethered AMPs[17-23]. Most often, the conjugation is dependent on the substrate (different biomedical plastics, metals, ceramics, hydrogels etc.), and multiple modification steps (and chemistry) are needed to achieve a stable AMP-based coating on the surface[17-23].
Mussel-inspired polydopamine-based coating provide a versatile platform to construct an antimicrobial coating on a variety of substrates. Antimicrobial peptides, including nisin[24], magainin II[25-26], synthetic antimicrobial peptide CWR11[27], cecropin B[28], SESB2V[29], and antimicrobial lipopeptide[30] have been grafted to surfaces utilizing dopamine coating. Although such AMP conjugated coatings showed antibacterial efficiency, such coatings showed poor resistance to protein fouling, adhesion of dead/live bacteria, which results in diminished anti-biofilm activity in long-term and activity in relevant animal models. Inactivation of such technologies is driven by the accumulation of proteins and dead bacteria killed by contact killing on the surface providing a pedestal for subsequent biofilm formation by live bacteria. Adhered dead bacteria and proteins that collect on a surface provide good support for eventual biofilm formation leading to infection. Therefore, coatings with dual functionality, antifouling and antimicrobial, are more desirable to combat implant/device-related infections. Reches et al. designed an AMP with three moieties (adhesive dopamine moiety, antimicrobial and antifouling motifs) and achieved dual functionality that resists biofouling[31]. Yang et al. achieved this functionality by conjugating both polylysine and copolymer with zwitterionic segments[32]. However, it is challenging to apply such coatings onto a medical device or on an implant surface due to complicated synthesis and the need for multiple chemical modifications. Accordingly, it is not an ideal platform to optimize antimicrobial and antifouling properties in a coating.
Another consideration is that previous AMP screening studies for the identification of surface-active AMPs have been built on different solid supports (for example, resin beads, pins, glass chips, tea bags, and cellulose membranes) that did not take account of non-specific adhesion of bacteria which can foul the support surface. In these methods, the AMPs were tethered so that peptide density was maximized. However, in these studies the tethered peptide easily became overwhelmed by the planktonic bacteria, which resulted in a surface fouled by the dead and/or live bacteria at the surface[16-17, 33-35].
Furthermore, these surfaces tend to get easily fouled by proteins and cells present in complex biological environment. The short-term activity readouts from different assays are thus not a true measure of their long-term activity.
The present invention is based, in part, on the surprising discovery that some polymers or polymer combinations are more useful than others in coating substrates to both prevent fouling and have antibacterial activity. In particular, coating compositions are provided, wherein the coating composition includes a polydopamine (PDA); a poly(N,N-dimethylacrylamide) (PDMA) polymer or a PDMA co-N-(3-Aminopropyl) Methacrylamide (APMA) polymer; and an antimicrobial peptide (AMP). Alternatively, there are provided coating compositions where the PDMA-co-APMA is substituted with a hydrophilic polymer co-APMA polymer as described herein. Alternatively PDA may be substituted with another polymeric binder.
Having antimicrobial peptides (AMPs) attached in a non-fouling background has the potential to manifest peptide activity in an uncompromised manner with a greater possibility for success when choosing an implant or a medical device coating. Also, a coating method that is simple, substrate-independent, and capable of generating a non-fouling background could potentially be used as a method for screening AMPs for anti-biofilm activity in an environment that more closely approximates the environment that an implant or a medical device is likely to be used, and is thus, is more likely to produce successful candidates.
The present invention is based in part on the surprising discovery that specific conjugation methods to produce antifouling coatings are able to retain the activity of AMPs. Two methods of conjugation are described herein.
In a first embodiment there is provided a coating composition, the coating composition including: (a) a polydopamine (PDA); (b) a poly(N,N-dimethylacrylamide) (PDMA) polymer or a PDMA co-N-(3-Aminopropyl) Methacrylamide (APMA) polymer; and (c) an antimicrobial peptide (AMP).
In a further embodiment there is provided a coating composition, the coating composition including: (a) a polymeric binder; (b) a poly(N,N-dimethylacrylamide) (PDMA) polymer or a PDMA co-N-(3-Aminopropyl) Methacrylamide (APMA) polymer; and (c) an antimicrobial peptide (AMP).
In a further embodiment there is provided a coating composition, the coating composition including: (a) a polymeric binder; (b) a hydrophilic polymer, and (c) an antimicrobial peptide (AMP), wherein the hydrophilic polymer is selected from one or more of the following: polyacrylamide; poly(N-hydroxyethyl acrylamide); poly(N-(tris(hydroxymethyl)methyl) acrylamide); poly(N-(2-hydroxypropyl) methacrylamide); poly(N-hydroxymethyl acrylamide); poly((3-(methacryloylamino)propyl)dimethyl(3-sulfopropyl)ammonium hydroxide) (PMPDSAH); poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC); poly(carboxybetaine methacrylate); and poly(sulfobetaine methacrylate).
In a further embodiment there is provided a coating composition, the coating composition including: (a) a polymeric binder; (b) a hydrophilic polymer co-APMA polymer; and (c) an antimicrobial peptide (AMP), wherein the hydrophilic polymer is selected from one or more of the following: polyacrylamide; poly(N-hydroxyethyl acrylamide); poly(N-(tris(hydroxymethyl)methyl) acrylamide); poly(N-(2-hydroxypropyl) methacrylamide); poly(N-hydroxymethyl acrylamide); poly((3-(methacryloylamino)propyl)dimethyl(3-sulfopropyl)ammonium hydroxide) (PMPDSAH); poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC); poly(carboxybetaine methacrylate); and poly(sulfobetaine methacrylate).
In a further embodiment there is provided a coated substrate, the coated substrate including: (a) a substrate; (b) a polydopamine; (c) a PDMA polymer or a PDMA co-polymer; and (d) an AMP.
In a further embodiment there is provided a coated substrate, the coated substrate including: (a) a substrate; (b) a polymeric binder, (c) a PDMA polymer or a PDMA co-polymer; and (d) an AMP.
In a further embodiment there is provided a coated substrate, the coated substrate including: (a) a substrate; (b) a polymeric binder; (c) a hydrophilic polymer; and (d) an AMP, wherein the hydrophilic polymer is selected from one or more of the following: polyacrylamide; poly(N-hydroxyethyl acrylamide); poly(N-(tris(hydroxymethyl)methyl) acrylamide); poly(N-(2-hydroxypropyl) methacrylamide); poly(N-hydroxymethyl acrylamide); poly((3-(methacryloylamino)propyl)dimethyl(3-sulfopropyl)ammonium hydroxide) (PMPDSAH); poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC); poly(carboxybetaine methacrylate); and poly(sulfobetaine methacrylate).
In a further embodiment there is provided a coated substrate, the coated substrate including: (a) a substrate; (b) a polymeric binder; (c) a hydrophilic polymer co-APMA polymer; and (d) an AMP, wherein the hydrophilic polymer is selected from one or more of the following: polyacrylamide; poly(N-hydroxyethyl acrylamide); poly(N-(tris(hydroxymethyl)methyl) acrylamide); poly(N-(2-hydroxypropyl) methacrylamide); poly(N-hydroxymethyl acrylamide); poly((3-(methacryloylamino)propyl)dimethyl(3-sulfopropyl)ammonium hydroxide) (PMPDSAH); poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC); poly(carboxybetaine methacrylate); and poly(sulfobetaine methacrylate).
In a further embodiment there is provided a medical device including: a structure for implantation or disposition inside a subject, the structure including at least one surface for coating; wherein the at least one surface has a coating disposed directly on the at least one surface of the medical device, the coating comprising: (a) a polydopamine; (b) a PDMA polymer or a PDMA co-polymer, and (c) an antimicrobial peptide (AMP).
In a further embodiment there is provided a coating composition, the coating composition including: (a) a polydopamine (PDA); and (b) PDMA co-polymer. The coating composition may further include an antimicrobial peptide (AMP).
In a further embodiment there is provided a coating composition, the coating composition including: (a) a polydopamine (PDA); (b) a hydrophilic polymer co-APMA polymer; and (c) an antimicrobial peptide (AMP); wherein, the hydrophilic polymer co-polymer may have the structure of Formula I:
wherein R may be selected from
and G may be H or —CH3; L may be a linking moiety; n may be an integer between woo and 20,000; and m may be an integer between 1 and 10,000. The linking moiety may be selected from: CH2I, CH2Br,
The hydrophilic polymer may be copolymerized with: an Iodoacetyl Linker (co-APMA-I); a Bromoacetyl Linker (co-APMA-Br); a Maleimide Linker (co-APMA-M); or a Pyridyl disulfide Linker (co-APMA-Pd).
In a further embodiment there is provided a substrate coating method, the method including: (a) bringing the substrate into contact with a PDA and PDMA polymer or a PDMA co-APMA polymer solution; (b) rinsing and drying; (c) bringing the substrate into contact with an AMP solution; (d) adding of a thiol containing hydrophilic compound; and (e) rinsing and drying. The substrate coating method may further include a cleaning of the substrate prior to step (a).
In a further embodiment there is provided a method of coating a substrate, wherein the substrate is immersed in a solution including the coating composition as described herein.
In a further embodiment there is provided a method of coating a substrate, wherein the substrate is sprayed with a solution or solutions including the composition as described herein.
In a further embodiment there is provided a coating composition, the coating composition including: (a) a polymeric binder; (b) a hydrophilic polymer; and (c) an antimicrobial peptide (AMP); wherein hydrophilic polymer is selected from one or more of the following: polyacrylamide; poly(N-hydroxyethyl acrylamide); poly(N-(tris(hydroxymethyl)methyl) acrylamide); poly(N-(2-hydroxypropyl) methacrylamide); poly(N-hydroxymethyl acrylamide); poly((3-(methacryloylamino)propyl)dimethyl(3-sulfopropyl)ammonium hydroxide) (PMPDSAH); poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC); poly(carboxybetaine methacrylate); and poly(sulfobetaine methacrylate).
In a further embodiment there is provided a coating composition, the coating composition comprising: (a) a polymeric binder, (b) a hydrophilic polymer co-APMA polymer; and (c) an antimicrobial peptide (AMP); wherein, the hydrophilic polymer co-polymer has the structure of Formula I:
R may be selected from
and G may be H or —CH3; L may be a linking moiety; n may be an integer between 1000 and 20,000; and m is an integer between 1 and 10,000. The polymeric binder may be selected from one or more of: polymeric dopamine (PDA); polymeric norepinephrine (PNE); polymeric epinephrine (PEPI); polymeric pyrogallol (PPG); polymeric tannic acid (PTA); polymeric hydroxyhydroquinone (PHHQ); polymeric catechin; and polymeric epigallocatechin. L may be CH2I, CH2Br,
The linking moiety may be CH2I, CH2Br,
n may be an integer between woo and 20,000. m may be an integer between 1 and 10,000. n may be an integer between 1,000 and 30,000. m may be an integer between 1 and 20,000. n may be an integer between 1,000 and 40,000. m may be an integer between 1 and 30,000. n may be an integer between 2,000 and 20,000. m may be an integer between 10 and 10,000. n may be an integer between 3,000 and 20,000. m may be an integer between 20 and 10,000. n may be an integer between 4,000 and 20,000. m may be an integer between 30 and 10,000. n may be an integer between 5,000 and 20,000. m may be an integer between 40 and 10,000. n may be an integer between 6,000 and 20,000. m may be an integer between 50 and 10,000. n may be an integer between 7,000 and 20,000. m may be an integer between 60 and 10,000. n may be an integer between 8,000 and 20,000. m may be an integer between 70 and 10,000. n may be an integer between 9,000 and 20,000. m may be an integer between 80 and 10,000. n may be an integer between 10,000 and 20,000. m may be an integer between 90 and 10,000. n may be an integer between 1,000 and 10,000. m may be an integer between 100 and 10,000. n may be an integer between 500 and 20,000. m may be an integer between 1,000 and 10,000. n may be an integer between 1 and 1000 and m may be an integer between 1 and 1000. n may be an integer between 1 and 900 and m may be an integer between 1 and 900. n may be an integer between 1 and 800 and m may be an integer between 1 and 800. n may be an integer between 1 and 700 and m may be an integer between 1 and 700. n may be an integer between 1 and 600 and m may be an integer between 1 and 600. n may be an integer between 1 and 500 and m may be an integer between 1 and 500. n may be an integer between 1 and 400 and m may be an integer between 1 and 400. n may be an integer between 1 and 300 and m may be an integer between 1 and 300. n may be an integer between 1 and 200 and m may be an integer between 1 and 200. n may be an integer between 1 and 100 and m may be an integer between 1 and 100.
In a further embodiment there is provided a substrate coating method, the method comprising: (a) bringing the substrate into contact with a hydrophilic polymer and a polymeric binder solution; (b) rinsing and drying; (c) bringing the substrate into contact with an AMP solution; (d) adding of a thiol containing hydrophilic compound; and (e) rinsing and drying. The polymeric binder may be selected from one or more of: polymeric dopamine (PDA); polymeric norepinephrine (PNE); polymeric epinephrine (PEPI); polymeric pyrogallol (PPG); polymeric tannic acid (PTA); polymeric hydroxyhydroquinone (PHHQ); polymeric catechin; and polymeric epigallocatechin. The hydrophilic polymer may be selected from one or more of the following: polyacrylamide; poly(N-hydroxyethyl acrylamide); poly(N-(tris(hydroxymethyl)methyl) acrylamide); poly(N-(2-hydroxypropyl) methacrylamide); poly(N-hydroxymethyl acrylamide); poly((3-(methacryloylamino)propyl)dimethyl(3-sulfopropyl)ammonium hydroxide) (PMPDSAH); poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC); poly(carboxybetaine methacrylate); and poly(sulfobetaine methacrylate). The substrate coating method may further including a cleaning of the substrate prior to step (a).
The PDMA polymer may be either a high-molecular-weight (hPDMA) or an ultrahigh-molecular-weight (uhPDMA). The PDMA co-APMA may further include a linker. The linker may be selected from: an Iodoacetyl Linker (PDMA-co-APMA-I); a Bromoacetyl Linker (PDMA-co-APMA-Br); a Maleimide Linker (PDMA-co-APMA-M); and Pyridyl disulfide Linker (PDMA-co-APMA-Pd). The AMP may be selected from one or more of: E6; Tet20C; Tet20LC; DJK5C; DJK5; DJK6; RI-DJK5; IDR-1018; and 3002C. The AMP may be conjugated by an amine group or a thiol group to a quinone group on the PDA. The thiol groups on the AMP may also be conjugated to one or more iodoacetamide groups on the PDMA-co-APMA-I polymer, one or more bromoacetamide groups on the PDMA-co-APMA-Br polymer, one or more maleimide groups on the PDMA-co-APMA-M polymer, or one or more 2-pyridyldithiol groups on the PDMA-co-APMA-Pd polymer. The AMP may be E6. The AMP may be Tet20C. The AMP may be Tet20LC. The AMP may be DJK5C. The AMP may be DJK5. The AMP may be DJK6. The AMP may be RI-DJK5. The AMP may be IDR-1018. The AMP may be 3002C. The poly(N,N-dimethylacrylamide) (PDMA) polymer or the PDMA co-N-(3-Aminopropyl) Methacrylamide (APMA) polymer may be uhPDMA.
The coating composition may have anti-fouling activity and antimicrobial activity. The coating composition may have anti-adhesion activity. The coating composition may be for use in coating a medical device. The medical device may be for implantation in a subject.
The substrate may be selected from: a plastic; a metal; a ceramic; a carbon based material; a metal oxide; a hydrogels; a biological tissue; a wood; a cement; a rubber, a resin; and a composite. The substrate may be selected from: poly(propylene) (PP); poly(urethane) (PU); poly(ethylene) (PE); unplasticized polyvinyl chloride (uPVC); plasticized polyvinyl chloride (pPVC); poly(imide) (PI); ethylene vinyl acetate (EVA); poly(tetrafluoroethylene) (PTFE); polydimethylsiloxane (PDMS); polyisoprene(PIP); poly(N-hydroxymethyl acrylamide) (PHMA); poly(acrylamide) (PAM); poly(N-hydroxyethyl acrylamide) (PHEA); poly{N-[tris(hydroxymethyl) methyl]acrylamide} (PTHMAM); poly(methacrylamide) (PMA); poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA); poly(vinyl pyrrolidone) (PVP); poly(ethylene oxide) (PEO); latex; titanium dioxide (TiO2), titanium or silicon dioxide (SiO2). The substrate may be PP, PU, PE, uPVC, pPVC, PI, EVA, or PTFE. The substrate may be TiO2 or SiO2. The substrate may forms part of an apparatus. The apparatus may be selected from: a urinary device; a dental fixture; an artificial joint; a vascular device; a storage device; blood storage device; a microfluidic device; a filtration membrane; a feed tube; or a diagnostic device. The vascular device may be a catheter, a lead, or a stent. The urinary device may be a urine storage device, blood storage device, catheter, or a stent. The filtration membrane may be a blood filtration membrane, a water purification membrane, or an air purification membrane. The coated substrate may reduce biofouling. The coated substrate may reduce adhesion. The PDMA polymer may be hPDMA or uhPDMA. The PDMA co-polymer may be a copolymer of N,N-Dimethylacrylamide and N-(3-Aminopropyl) Methacrylamide with: an Iodoacetyl Linker (PDMA-co-APMA-I); a Bromoacetyl Linker (PDMA-co-APMA-Br); a Maleimide Linker (PDMA-co-APMA-M); or Pyridyl disulfide Linker (PDMA-co-APMA-Pd). The AMP may be selected from one or more of: E6; Tet20C; Tet20LC; DJK5C; DJK5; DJK6; RI-DJK5; IDR-1018; and 3002C. The AMP may be conjugated by an amine group or a thiol group to a quinone group on the PDA. The AMP may also conjugated to one or more iodoacetamide groups on the PDMA-co-APMA-I polymer, one or more bromoacetamide groups on the PDMA-co-APMA-Br polymer, one or more maleimide groups on the PDMA-co-APMA-M polymer, or one or more 2-pyridyldithiol groups on the PDMA-co-APMA-Pd polymer. The PDMA co-polymer may be a copolymer of N,N-Dimethylacrylamide and N-(3-Aminopropyl) Methacrylamide with: an Iodoacetyl Linker (PDMA-co-APMA-I); a Bromoacetyl Linker (PDMA-co-APMA-Br); or a Maleimide Linker (PDMA-co-APMA-M). The PDMA co-polymer may be a copolymer of N,N-Dimethylacrylamide and N-(3-Aminopropyl) Methacrylamide with: an Iodoacetyl Linker (PDMA-co-APMA-I); or a Bromoacetyl Linker (PDMA-co-APMA-Br).
The hydrophilic polymer may be selected from one or more of the following: polyacrylamide; poly(N-hydroxyethyl acrylamide); poly(N-(tris(hydroxymethyl)methyl) acrylamide); poly(N-(2-hydroxypropyl) methacrylamide); poly(N-hydroxymethyl acrylamide); poly((3-(methacryloylamino)propyl)dimethyl(3-sulfopropyl)ammonium hydroxide) (PMPDSAH); poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC); poly(carboxybetaine methacrylate); and poly(sulfobetaine methacrylate). The hydrophilic polymer may be selected from PMPC and PMPDSAH. The hydrophilic polymer co-APMA polymer may be selected from PMPC-co-APMA-I and PMPDSAH-co-APMA-I.
The thiol containing hydrophilic molecule may be selected from: 1-thioglycerol; thioethanol; 2-mercaptoethanol; 3-mercapto-1,2-propandiol; and dimercaptosuccinic acid. The bringing the substrate into contact with the PDA and PDMA polymer or a PDMA co-APMA polymer solution, may be by immersing the substrate in the PDA and PDMA polymer or a PDMA co-APMA polymer solution. The bringing the substrate into contact with the AMP solution may be by immersing the substrate in the AMP solution. The immersing of the substrate in the AMP solution may be for between 2-12 hours. The addition of the thiol containing hydrophilic compound to the AMP solution, the substrate may remain in the AMP solution with the thiol containing hydrophilic compound for between 12-24 hours. The addition of the thiol containing hydrophilic compound to the AMP solution, the substrate may remain in the AMP solution with the thiol containing hydrophilic compound for between 20-24 hours. The addition of the thiol containing hydrophilic compound to the AMP solution, the substrate may remain in the AMP solution with the thiol containing hydrophilic compound for between 20-30 hours. The rinsing in (b) and (e) may be with water. The drying in (b) and (e) may be under a stream of argon gas or a flow of nitrogen gas. The drying in (b) and (e) may be under a stream of argon gas. The AMP may be conjugated by an amine group or a thiol group to a quinone group on the PDA.
The polymeric binder may be selected from one or more of: polymeric dopamine (PDA); polymeric norepinephrine (PNE); polymeric epinephrine (PEPI); polymeric pyrogallol (PPG); polymeric tannic acid (PTA); polymeric hydroxyhydroquinone (PHHQ); polymeric catechin; and polymeric epigallocatechin. The thiol containing hydrophilic molecule may be selected from: 1-thioglycerol; thioethanol; 2-mercaptoethanol; 3-mercapto-1,2-propandiol; and dimercaptosuccinic acid.
In a further embodiment, there is provided a composition comprising AMPs conjugated via amine or thiol groups to quinone groups of PDA in PDA/PDMA coating (BA coating).
In a further embodiment, there is provided a method comprising conjugation of AMPs directly onto PDA component in a non-fouling background generated by PDA/PDMA coating (BA coating) said method comprising a reaction between quinone groups in the underlying PDA and amine or thiol groups in the AMP.
In a further embodiment, there is provided a method comprising conjugation of AMPs via a copolymer approach (MA coating) said method comprising tethering the AMP into the coating between the reaction of iodoacetamide groups on the polymers and thiol groups on the AMP and the reaction between PDA and AMP.
In a further embodiment, there is provided an AMP modified coating structure with optimal protection against non-specific adhesion of dead/live bacteria and the bactericidal and anti-biofilm activity of conjugated AMPs.
In a further embodiment, there is provided an AMP modified coating structure (with cysteine at the C-terminus) with anti-biofilm have potent activity against bacteria including but not limited to S. saprophyticus and E. coli.
In a further embodiment, there is provided a conjugated AMP, Tet20LC, showed the best anti-biofilm activity against S. aureus.
In a further embodiment, there is provided a conjugated AMPs including E6, Tet20C, Tet20LC and DJK5C showed better broad-spectrum activity against different pathogens.
In a further embodiment, there is provided a conjugated AMP, DJK5C, in the coating showed better prevention of biofilm formation than N-terminal conjugated DJK5 for all four bacterial strains tested.
In a further embodiment, there is provided a surface conjugated with AMPs applied to the coating of polymeric surfaces including.
In a further embodiment, there is provided a combination of antimicrobial peptides conjugated to an antifouling coating.
In a further embodiment, there is provided a PDA/PDMA coating (BA coating) with AMP that has optimal protection against non-specific adhesion of dead/live bacteria and retained the bactericidal activity by an electrostatic membrane disruption mechanism.
In a further embodiment, there is provided peptide compositions comprising DJK5C and 3002C.
In a further embodiment, there is provided a peptide compositions comprising the amino acid sequence vqwrairvrvirc (SEQ ID NO:4).
In a further embodiment, there is provided a peptide compositions comprising the amino acid sequence ILVRWIRWRIQWC (SEQ ID NO:7).
The substrate may be a plastic, a rubber, a resin, a metal, a ceramic, a carbon based material, a metal oxide, a hydrogels, a biological tissue, a wood or a cement. The substrate may be poly(propylene) (PP); poly(urethane) (PU); poly(ethylene) (PE); unplasticized polyvinyl chloride (uPVC); plasticized polyvinyl chloride (pPVC); poly(imide) (PI); ethylene vinyl acetate (EVA); poly(tetrafluoroethylene) (PTFE); poly(N-hydroxymethyl acrylamide) (PHMA); poly(acrylamide) (PAM); poly(N-hydroxyethyl acrylamide) (PHEA); poly{N-[tris(hydroxymethyl) methyl]acrylamide} (PTHMAM); poly(methacrylamide) (PMA); poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA); poly(vinyl pyrrolidone) (PVP); poly(ethylene oxide) (PEO); titanium dioxide (TiO2), titanium or silicon dioxide (SiO2). The substrate may be poly(propylene) (PP); poly(urethane) (PU); poly(ethylene) (PE); unplasticized polyvinyl chloride (uPVC); plasticized polyvinyl chloride (pPVC); poly(imide) (PI); ethylene vinyl acetate (EVA); poly(tetrafluoroethylene) (PTFE); titanium dioxide (TiO2) or silicon dioxide (SiO2). The substrate may be PP, PU, PE, uPVC, pPVC, PI, EVA, PTFE, PHMA, PAM, PHEA, PTHMAM, PMA, PHPMA, PVP, or PEO. The substrate may be TiO2 or SiO2. The substrate may form part of an apparatus. The apparatus may be selected from: a urinary device; a dental fixture; an artificial joint; a vascular device; a storage device; a microfluidic device; a filtration membrane; a feed tube; or a diagnostic device or a blood storage device. The vascular device may a catheter, a lead, guide wire, sheath or a stent. The vascular device may a catheter, a lead or a stent. The urinary device maybe a urine storage device, catheter, or a stent. The filtration membrane may be a blood filtration membrane, a water purification membrane, or an air purification membrane.
The methods described herein may be for preventing: biofouling; biofilm formation; protein adsorption; protein binding; cell adhesion; cell growth; microorganism adhesion; and microorganism adhesion and growth. The methods described herein may be for preventing: biofouling; biofilm formation; protein adsorption; protein binding; cell adhesion; microorganism adhesion; and microorganism adhesion and growth. The microorganism may be bacteria. The bacteria may be Gram-positive or Gram-negative bacteria. The gram-positive bacteria may be Staphylococcus aureus (S. aureus). The gram-negative bacteria may be Escherichia coli (E. col). The microorganism may be selected from one or more of the following: E. facium, S. aureus, K. pneumonia, A. baumannii, P. aeruginosa, E. cloacae, E. coli, S. epidermidis, and S. saprophyticus.
The following detailed description will be better understood when read in conjunction with the appended figures. For the purpose of illustrating the invention, the figures demonstrate embodiments of the present invention. However, the invention is not limited to the precise arrangements, examples, and instrumentalities shown.
Any terms not directly defined herein shall be understood to have the meanings commonly associated with them as understood within the art of the invention.
The term “high molecular weight polymer” or hMWP as used herein refers to any polymer having a molecular weight between ≥100,000 daltons (i.e. greater than and equal to 100 kDa) and about ≤200 kDa and in particular refers to the hydrophilic polymers described herein, including the hydrophilic biocompatible polymer poly(N,N-dimethyl acrylamide) (PDMA). Alternatively, the HMW polymer may be selected on the basis of having a polydispersity index (PDI) of between 1 to 3.
The term “ultra-high molecular weight polymer” or uhMWP as used herein refers to any polymer having a molecular weight >200 kDa and in particular refers to the hydrophilic polymers described herein.
The present disclosure provides, in part, AMPs conjugated to polymer coating surfaces. The conjugated AMPs have broad spectrum activity against biofilms.
The polymer-AMP coatings can be deposited on diverse biomedical material surfaces.
The contact killing by the AMP, and repulsion or release of live/dead bacteria from the surface by the non-fouling component prevents biofilm formation.
The present disclosure provides, in part, a simple universal antifouling coating approach which has the ability to perform with potent surface activity and can also easily translated as a biomedical device coating
When the peptide was presented within the antifouling layer without direct attachment to the polymer chains (BA-AMP coating) potent long-term biofilm activity is achieved due to the AMPs being sterically protected.
The BA-AMP coating structure offered optimal protection of AMPs against non-specific adhesion of dead/live bacteria while retaining the bactericidal and anti-biofilm activity of conjugated AMPs. The PDMA chains in the moderate brush regime were responsible for the antifouling characteristics of the BA-AMP coating and was helpful in preventing the accumulation of bacterial debris on the surface, the current coating approach is simple, and versatile, and can be applied to diverse materials used in medical device manufacturing, making its translation potential into clinical practice high.
A “polymeric binder” as used herein may be selected from one or more of: polymeric dopamine (PDA); polymeric norepinephrine (PNE); polymeric epinephrine (PEPI); polymeric pyrogallol (PPG); polymeric tannic acid (PTA); polymeric hydroxyhydroquinone (PHHQ); polymeric catechin; and polymeric epigallocatechin. A polymeric binder may also be selected from catechol and catechol derivative polymers as well known in the art[36].
The term “peptide” as used herein includes any structure comprised of two or more amino acids, including chemical modifications and derivatives of amino acids. The amino acids forming all or a part of a peptide may be naturally occurring amino acids, stereoisomers and modifications of such amino acids, non-protein amino acids, post-translationally modified amino acids, enzymatically modified amino acids, constructs or structures designed to mimic amino acids, and the like, so that the term “peptide” includes pseudopeptides and peptidomimetics, including structures which have a non-peptidic backbone. The amino acids in a “peptide” may be in either the D or the L-configuration. Furthermore, “peptide” is meant to include dimers or multimers and peptides produced by chemical synthesis, recombinant DNA technology, biochemical, or enzymatic fragmentation of larger molecules, combinations of the foregoing or, in general, made by any other method. Peptides as used herein, optionally with non-amino acid residue groups at the N- and C-termini, such groups including acyl, acetyl, alkenyl, alkyl, N-alkyl, amine, or amide groups, among others, as described in more detail below in reference to “peptide modifications”.
The term “antimicrobial peptide” (AMP) refers to diverse group of peptide molecules, which are generally between 10 and 50 amino acids, but may extend to over a hundred amino acids. These peptides are potent, broad spectrum antibiotics which demonstrate potential as novel therapeutic agents. Some AMPs are effective against Gram-positive bacteria, Gram-negative, fungi and some viruses. AMPs are divided into subgroups on the basis of their amino acid composition and structure (i.e. anionic peptides; linear cationic α-helical peptides; cationic peptide enriched for specific amino acid; and anionic/cationic peptides forming disulfide bonds). Furthermore, AMPs are regularly used as therapeutic agents (for example, Bacitracin; Boceprevir, Dalbavancin; Daptomycin; Enfuvirtide; Oritavancin; Teicoplanin; Telaprevir, Telavancin; Vancomycin; and Guavanin 2). Non-limiting examples of AMPs as described herein are as follows: E6; Tet20C; Tet20LC; DJK5C; DJK5; DJK6; RI-DJK5; IDR-1018; and 3002C. The amino acids in the AMPs may be in either the D or the L-configuration. The AMPs may include those sequences identified in TABLE 3. However, many such peptides are known in the art[53, 54, 55] and would be suitable for use with the present compositions and coatings.
As used herein “a linking moiety” is used to attach the hydrophilic polymer co-polymer with the AMP. This may be accomplished via numerous linking groups, for example, by adding: an alkyne group (via copper assisted or copper free click reactions or a peptide with azide functionality can be used); an alkene group (thiolene click reaction, where a peptide with an —SH group can be attached); an aldehyde group (via Schiff-based reactions of aldehyde group on surface and —NH2 group on peptides); or via enzymatic ligation of peptides to the surface (e.g. transglutaminase-reaction of an AMP with glutamine with amine residues on the surface). Furthermore, the linker may be selected from one or more of the following: an iodoacetyl linker (L=CH2I); a bromoacetyl linker (L=CH2Br); a Maleimide Linker (i.e. L=
and Pyridyl disulfide Linker (L=
The term “peptide modifications” as used herein refers to any modification to a peptide improves the characteristics of the peptide to act as a bound anti-microbial peptide (AMP). For example, modifications may reduce susceptibility to proteolysis, improve binding affinities, and/or confer or modify other physicochemical or functional properties. Examples of modifications include but are not limited to phosphorylation; acetylation; N-linked glycosylation; amidation; hydroxylation; methylation; O-linked glycosylation; ubiquitylation; pyrrolidone carboxylic acid; and sulfation. Alternative peptides modifications may include: single or multiple amino acid substitutions (e.g., equivalent, conservative or non-conservative substitutions, deletions or additions) may be made in a sequence; the peptide or peptide analog is lipidated (e.g., myritoylated, palmitoylated, or other linking to a lipid moiety), glycosylated, amidated, carboxylated, phosphorylated, esterified, acylated, acetylated, cyclized, pegylated to or converted into an acid addition salt and/or optionally dimerized or polymerized, or conjugated.
One type of peptide modification is C-terminal amidation. C-terminal amidation removes the charge form the C-terminus of a peptide and may reduce the overall solubility of the peptide. Having an uncharged C-terminal amide end more closely mimics the native protein, and therefore may increase the biological activity of a peptide. Alternatives include N-terminal acetylation, which may increase peptide stability by preventing N-terminal degradation.
The term “biofilm” is used herein as it is normally understood to a person of ordinary skill in the art and often refers to any group of organisms adhering to the surface of a structure.
The term “biofouling” is used herein as it is normally understood to a person of ordinary skill in the art and often refers to the colonization of an interface by organisms, which often leads to deterioration of the interface.
The term “antifouling” is used herein as it is normally understood to a person of ordinary skill in the art and often refers to the reduction of formation of biofilms and biofouling.
The term “plastic” as used herein is meant to encompass a vast number of synthetic or semi-synthetic organic polymers that are malleable and may be molded into solid forms. Exemplary plastics are: Polyester (PES); Polyethylene terephthalate (PET); Polyethylene (PE); High-density polyethylene (HDPE); Polyvinyl chloride (PVC); Polyvinylidene chloride (PVDC); Low-density polyethylene (LDPE); Polypropylene (PP); Polystyrene (PS); High impact polystyrene (HIPS); Polyamides (PA) (Nylons); Acrylonitrile butadiene styrene (ABS); Polyethylene/Acrylonitrile Butadiene Styrene (PE/ABS a blend of PE and ABS); Polycarbonate (PC); Polycarbonate/Acrylonitrile Butadiene Styrene (PC/ABS a blend of PC and ABS); Polyurethane (PU); Polylactic acid (PIA); Polyimide; Polyetherimide (PEI); Polyetheretherketone (PEEK); phenol formaldehydes (PF); and Polymethyl methacrylate (PMMA).
The term “polydopamine (PDA)” is used herein as it is normally understood to a person of ordinary skill in the art and often refers to the pH-dependent self-polymerization of dopamine. However, “polydopamine” may be formed by any polymerisation of dopamine monomers. It should be noted that the mechanism of PDA formation is currently not understood[37-38]. Furthermore, it should be noted that the structure of the polymer product has not been elucidated yet[37].
The term “PDMA” is used herein as it is normally understood to a person of ordinary skill in the art and often refers to poly(N,N-dimethyl acrylamide).
The term “PMPDSAH” is used herein as it is normally understood to a person of ordinary skill in the art and often refers to “poly(N-(3-(methacryloylamino)propyl)-N,N-dimethyl-N-(3-sulfopropyl) ammonium hydroxide)”.
The term “PMPC” is used herein as it is normally understood to a person of ordinary skill in the art and often refers to “poly(2-methacryloyloxyethyl phosphorylcholine)”.
The term “PP” is used herein as it is normally understood to a person of ordinary skill in the art and often refers to “poly(propylene)”.
The term “PU” is used herein as it is normally understood to a person of ordinary skill in the art and often refers to “poly(urethane)”.
The term “PE” is used herein as it is normally understood to a person of ordinary skill in the art and often refers to “poly(ethylene)”.
The term “uPVC” is used herein as it is normally understood to a person of ordinary skill in the art and often refers to “unplasticized polyvinyl chloride”.
The term “pPVC” is used herein as it is normally understood to a person of ordinary skill in the art and often refers to “plasticized polyvinyl chloride”.
The term “PI” is used herein as it is normally understood to a person of ordinary skill in the art and often refers to “poly(imide)”.
The term “EVA” is used herein as it is normally understood to a person of ordinary skill in the art and often refers to “ethylene vinyl acetate”.
The term “Teflon” is used herein as it is normally understood to a person of ordinary skill in the art and often refers to “poly(tetrafluoroethylene) or PTFE”.
The term “PHMA” is used herein as it is normally understood to a person of ordinary skill in the art and often refers to “poly(N-hydroxymethyl acrylamide)”.
The term “PAM” is used herein as it is normally understood to a person of ordinary skill in the art and often refers to poly(acrylamide).
The term “PHEA” is used herein as it is normally understood to a person of ordinary skill in the art and often refers to “poly(N-hydroxyethyl acrylamide)”.
The term “PTHMAM” is used herein as it is normally understood to a person of ordinary skill in the art and often refers to “poly{N-[tris(hydroxymethyl) methyl]acrylamide}”.
The term “PMA” is used herein as it is normally understood to a person of ordinary skill in the art and often refers to “poly(methacrylamide)”.
The term “PHPMA” is used herein as it is normally understood to a person of ordinary skill in the art and often refers to “poly(N-(2-hydroxypropyl)methacrylamide)”.
The term “PVP” is used herein as it is normally understood to a person of ordinary skill in the art and often refers to “poly(vinyl pyrrolidone)”.
The term “PEO” is used herein as it is normally understood to a person of ordinary skill in the art and often refers to “poly(ethylene oxide)”.
A hydrophilic co-polymer may have the structure of Formula I
L may be selected from: CH2I; CH2Br;
The term “coating” is used herein as it is normally understood to a person of ordinary skill in the art to be a covering that is applied to the surface of an object and is to be broadly constructed to include adhesive coating, resistive coating (e.g., resistive to cellular adhesion), and protective coating. The present invention offers adhesion in “highly humid” environments (50% to 80% humidity) and “wet”, “saturated”, or “super-saturated” environments (at least 80% humidity and higher). Adhesion under dry environment is also contemplated herein.
The term “dip-coating” is used herein as it is normally understood to a person of ordinary skill in the art and often refers to the immersion of the substrate into the solution of the coating material.
The term “lubricity” is used herein as it is normally understood to a person of ordinary skill in the art and often refers to the property of “slipperiness” or “smoothness”, or “a surface with low friction”.
The coating described herein has high lubricity. These coatings are useful for medical devices where their lubrication results in reduced frictional forces when the device is introduced and moved within the body, reducing inflammation and tissue trauma as well as supporting patient comfort.
Various alternative embodiments and examples are described herein. These embodiments and examples are illustrative and should not be construed as limiting the scope of the invention.
Ultra-high molecular weight poly(N,N-dimethylacrylamide) (uhPDMA) and high molecular weight poly(N,N-dimethylacrylamide) (hPDMA) were synthesized by atom transfer radical polymerization 391. N-(3-Aminopropyl) methacrylamide hydrochloride (APMA) (98%) was purchased from Polysciences™, USA and was used as supplied. All other reagents including 1, 1, 4, 7, 10, 10-hexamethyl triethylene tetramine (HMTETA) (97%), Tris[2-(dimethylamino)ethyl]amine (Me6TREN) 97%, methyl 2-chloropropionate (97%), CuCl (99%), CuCl2 (99%), 1-thioglycerol (97%) were purchased from Sigma-Aldrich™ (Oakville, ON). A single-side-polished silicon wafer (University Wafer™, Boston, MA) deposited with titanium was prepared by e-beam evaporation of titanium. The process was progressed in a home-assembled Evaporator 2000™ system equipped with a quartz crystal microbalance to monitor the film thickness and a cryo pump to reach high-vacuum (10−7-10−6 Torr) condition. After deposition, the substrates were washed with Milli-Q water™, dried via a nitrogen gun, and stored for further usage. SurFlash™I.V. Catheter Cat. #SR*FF1451, #SR*FF2419 (14 Gauge and 24 Gauge) made of polyurethane (PU), were purchased from Terumo™. Antimicrobial peptides, E6 (RRWRIVVIRVRRC-NH2(SEQ ID NO:1)), Tet20C (KRWRIRVRVIRKC-NH2 (SEQ ID NO:2)), DJK5C (vqwrairvrvirc-NH2 (SEQ ID NO:4)) and 3002C (ILVRWIRWRIQWC-NH2 (SEQ ID NO:7)) with cysteine at the C-terminus (purity>95%) were synthesized by Canpeptide Corp.™ (Quebec, Canada). Peptide IDR-1018 (VRLIVAVRIWRR-NH2 (SEQ ID NO:6)) at >95% purity was obtained from CPC Scientific™ (Sunnyvale, CA), while all other synthetic peptides (>95% purity) were obtained from GenScript™ (Piscataway, NJ). Tet20LC (KRWRIRVRVIRK-bA-bA-C—NH2(SEQ ID NO:3)) was synthesized and purified (>90%) by the Hilpert laboratory by automated solid-phase peptide synthesis (SPPS) on a MultiPep RSI Peptide Synthesizer (INTAVIS™, Tuebingen, Germany) using the 9-fluorenyl-methoxycarbonyl-tert-butyl (Fmoc/tBu) strategy. Crude peptides were purified to homogeneity of >90% by preparative RP HPLC on a Shimadzu™ LC2020 system equipped with a Jupiter™ 10 μm Proteo C18 column (90 Å, 250×21.2 mm, Phenomenex™) using a linear gradient system containing 0.01% (v/v) TFA in H2O (solvent A) and 0.01% (v/v) TFA in acetonitrile (solvent B). Pure products were finally characterized by analytical reverse phase high performance liquid chromatography (RP-HPLC) and liquid chromatography-mass spectrometry (LC-MS). The broth microdilution method with minor modifications for cationic peptides was used for measuring the MICs of peptides.
Copper (II) chloride (CuCl2, 3 mg), copper (I) chloride (CuCl, 20 mg) and Me6TREN (120 μL) were added successively into a glass tube followed by the addition of 20 mL Milli-Q water™. The solution was degassed using three freeze-pump-thaw cycles, DMA (2 mL) and APMA (346 mg) was added into the glass tube and degassed with another freeze-pump-thaw cycle. Soluble methyl 2-chloropropionate (20 μL from a stock solution of 40 μL in 5 mL methanol) was added immediately to the reaction mixture, and the polymerization was allowed to proceed at RT (22° C.) for 24 h. The soluble polymer formed was collected and purified by dialysis (molecular weight cut off: 1000 Da) against water (pH was adjusted to 8 by using 0.1M NaOH) for 3 days with daily exchange of water. The polymer was lyophilized to obtain the final product. The absolute molecular weight of PDMA-co-APMA was determined by gel permeation chromatography (GPC) using a DAWN HELEOS II™ multi angle laser light scattering (MALLS) detector (Wyatt Technology Inc.™), an Optilab T-rEX refractive index detector and a quasi-elastic light scattering (QELS) detector (Wyatt Technology Inc.™) in 1.0 M NaNO3 (pH 7) aqueous solution. NMR spectra were recorded on a Bruker Avance™ 300 MHz NMR spectrometer using deuterated solvents (Cambridge Isotope Laboratories™, 99.8% D) with the solvent peak as a reference.
The copolymer (PDMA-co-APMA, 100 mg) was dissolved in anhydrous acetonitrile (10 mL). Iodoacetic acid N-hydroxysuccinimide ester (70 mg) was added into the solution. The reaction was allowed overnight with stirring. The solution was dialyzed against water for 3 days with daily exchange of water. The solution was finally dialyzed against 5 mM Tris buffer (at pH 8.5) and concentrated to the solid content about 12 mg/mL as measured by Thermogravimetric Analysis (TGA Q500, TA Instruments™, New Castle, DE, USA) and characterized by NMR.
Titanium substrates were initially cleaned with nitrogen gas at a flow rate of 50 SCCM (standard cubic centimeter per minute) and at a pressure of 350 mTorr. A plasma power (75 W) was used for 3 min in a M4L Plasma Processing System from PVA Tepla™ (Corona, California, USA). PDMA or PDMA-co-APMA-I solution was prepared at a concentration of 12 mg/mL in 10 mM Tris buffer, pH 8.5, respectively. Dopamine was freshly prepared at a concentration of 12 mg/mL in 10 mM Tris buffer, pH 8.5 before each experiment. The dopamine solution was then mixed with PDMA or PDMA-co-APMA-I solution with a volume ratio of 1:5 (dopamine:polymer) and used right after. The cleaned Ti substrate was dipped into a mixed solution (0.7 mL) of dopamine and polymer in the wells of 24 well-plate for 24 h. The coated substrate was then rinsed by Mill-Q water™ and dried under argon.
PU catheter with different size (14G and 24G) were initially cleaned by nitrogen plasma treatment and coated using the same protocol as Ti substrate. For the 24G catheter used for the in vivo study, a second coating (PDA/PDMA) using the same solution composition was then applied onto the catheter to increase the thickness and coverage.
Microtiter 96-well plates were initially cleaned by nitrogen plasma treatment similar to the Ti substrate. The wells in 96-well plate were then coated with dopamine/PDMA mixed solution (250 μL, 2 mg/ml and 10 mg/ml respectively in 10 mM Tris buffer, pH 8.5). A second coating was applied using 200 μL solution having the same composition. The coated 96-well plate was then rinsed by Mill-Q water™ and dried under a stream of argon gas.
AMP Conjugation onto PDA/PDMA and PDA/PDMA-Co-APMA-I Coated Surfaces
The Ti surface or catheter coated with PDA/PDMA or PDA/PDMA-co-APMA-I was fully immersed into the peptide solution (0.6 mL, 0.1 mg/mL in 10 mM phosphate buffer (pH ˜8) overnight followed by adding 1-thioglycerol (6 μL, at a final concentration of 10 μL/mL) for 24 h. For the 96 well-plate, 250 μL peptide solutions (0.1 mg/mL in 10 mM phosphate buffer, pH ˜8) were added into the wells and incubated overnight followed by addition of 1-thioglycerol (2.5 μL, final concentration of 10 μL/mL) for 24 h. The peptide immobilized substrates were thoroughly rinsed with Milli-Q water™ consecutively and dried under argon. The mass of peptides grafted on to the surface was calculated by the following equation m=ρ·h·A: where h is the increase in thickness after peptide conjugation measured by ellipsometry; ρ is the volumetric mass density of antimicrobial peptide (1.5 mg/cm3);48 A is the surface area (cm2).
Pseudomonas aeruginosa (luminescence tagged strain PAO1 Tn7:Plac-lux), and S. saprophyticus strain (ATCC 15305) were sub-cultured for testing the anti-biofilm activity of the coating. An initial concentration 5×105 CFU/mL was used for these analyses. The coated Ti substrate along with the pristine Ti substrate were placed into a 24-well plate and was sterilized by submerging in 1 mL of 70% ethanol for 5 min. Ethanol was then removed, and samples were each rinsed with 1 mL of sterile phosphate-buffered saline (PBS) for a total of 3 times. After the removal of LB media from the last rinse, 1 mL of the prepared bacterial culture (˜5×105 CFU/mL) of S. saprophyticus in TSB or P. aeruginosa in LB was added to each sample. The 24-well plate was incubated at 37° C. with shaking at 50 rpm for 6 h or 24 h. The samples were then thoroughly rinsed with PBS buffer, stained with green SYTO9 for all bacteria and red propidium iodide (red PI) for dead bacteria. The stained bacteria on the Ti surface were examined by fluorescence microscopy (Zeiss Axioskop™ 2 plus, Thornwood, NY) equipped with a fluorescence illumination system (AttoArc™ 2 HBO) and appropriate filter sets. Images were randomly acquired on different spots by using a 10× and 20× objective lens. Images were taken using the filters for fluorescein isothiocyanate and rhodamine to visualize the presence of live and dead bacteria on the surface. The images taken using two different filters were overlaid to generate the merged image by using imageJ v153a at an opacity of 50%. The total number of adhered bacteria was counted using imageJ™. The antimicrobial activity was calculated by dividing the number of dead bacteria by the total number of bacteria. The samples that were incubated for 24 h also analyzed using C2+ confocal microscope (Nikon™) with the 488 and 561 nm channels. All images were acquired using identical acquisition settings. The experiments were repeated 3 times, and representative results from an experiment are shown.
Catheters with different sizes, 14G and 24G PU, were used in this study. The coated and uncoated catheter samples were cut into 1 cm section and suspended in 70% ethanol for 5 min. The ethanol was removed, and samples were rinsed in sterile PBS for a total of 3 times. After the last rinse, each sample was introduced to culture medium containing approximately 1 mL of 5×105 CFU/mL bacteria (S. saprophyticus in TSB and P. aeruginosa in LB) per sample in Eppendorf tubes. All samples were incubated at 37° C. on a 360° rotator. At 24 h, post-incubation, samples (N=5 per condition) were rinsed with sterile PBS buffer 3 times. Rinsed samples were then transferred to 1 mL of sterile PBS and sonicated for 10 min in a water bath. Samples were vortexed at high speed for 10 s, then serially diluted and plated on LB agar for CFUs. Plates were incubated at 37° C. overnight until visible colonies form. The experiment has been repeated 3 times with similar results, and the representative results from one experiment are shown.
Screening and Identification of Surface Conjugated AMPs with Anti-Biofilm Activity on a 96-Well Plate
Bioluminescence-tagged bacterial strains used in this study included S. aureus (Xen36), P. aeruginosa (PAO1.lux), E. coli (E38.lux), and S. saprophyticus (ATCC 15305). Bacteria were cultured in LB, with shaking at 250 rpm, at 37° C. Bacterial growth was monitored using a spectrophotometer at the optical density of 600 nm (OD600). S. aureus Xen36 was purchased from PerkinElmer Inc.™ while P. aeruginosa PAO1.lux and E. coli E38.lux were generated by conjugating in plasmids that constitutively-expressed lux reporter genes[40]. The biofilm inhibitory activity of AMPs conjugated onto coated polypropylene surfaces were evaluated in a static microtitre plate assay as previously described with some modifications[41]. For all experiments, 96-well Costar™ polypropylene plates (Corning™) were used. An overnight culture of selected bacterial strains in LB was diluted to an OD600=0.01 in TSB or basal medium 2 (BM2) supplemented with 0.1% or 0.4% glucose and 0.5 mM MgSO4, (the biofilm growth medium for different bacteria is listed in TABLE 1).
E. facium
S. aureus
K. pneumonia
A. baumannii
P. aeruginosa
E. cloacae
E. coli
S. epidermidis
S.
saprophyticus
For inhibition experiments, 100 μL of the diluted overnight culture was added to each well in the plate coated with control coating and AMP coating, respectively. After overnight growth under static condition at 37° C., planktonic cells were removed, adhered biomass rinsed three times with distilled water and subsequently the remaining adhered total biomass was quantified by measuring the luminescence (Total white light) using the Synergy H1™ multimode microtitre plate reader (BioTek™). The remaining adhered S. saprophyticus biomass was quantified using the crystal violet (CV) staining. Briefly, each well was added and incubated with 125 μL of 0.1% (v/v) CV solution for 30 minutes at room temperature with moderate shaking. Then, CV was discarded, and each well was rinsed three times with distilled water. The remaining CV stain was resuspended in 150 μL of 70% ethanol for 30 minutes at room temperature with moderate shaking and then quantified by measuring OD59, using the Synergy H1™ multimode microplate plate reader (BioTek™). The percent biofilm inhibition was calculated in relation to the amount of biofilm grown in the absence of the coating (defined as 100%) and the media sterility control (defined as 0% growth). The experiment was repeated 3 times with 3 technical replicates per biological replicate.
A total of 48 male C57BL/6 mice (Harlan™) at 10 weeks of age were included in experiments. Twenty mice were included in the control group (bare catheter) and 14 mice for each treated group (AMPs conjugated catheter, E6 and Tet20LC). The implantation of the catheter followed the procedure published previously 421. Briefly, prior to animal procedures, 4 mm section from the tip of PU catheter was cut using sterile blades and re-assembled back onto the original needle. The assembled catheter was sterilized using ethylene oxide. All mice were anesthetized using 3% isoflurane. The abdominal area was shaved, and the area around the mouse bladder was secured. Sterile ultrasound gel was applied to visualize the bladder. The 24G PU catheter assembly was positioned at a 30-degree angle just above the pubic bone with the bevel directed to the anterior. The catheter assembly was carefully inserted towards the bladder and left the 4 mm catheter segment inside while the ‘pusher’ was pushed slightly inward. One day after catheter implantation, all mice were anaesthetized and S. saprophyticus (1×107 CFU/mL in 50 μL PBS) was percutaneously injected into the bladder. Mice were kept anaesthetized with 1% isoflurane for 1 h on a heating pad to allow time for bacteria to adhere onto the implanted catheter. At 7 days post-instillation of S. saprophyticus, all mice were sacrificed by CO2 asphyxiation. The presentation of urine in the bladder was examined by ultrasound. If present, urine samples were collected from the bladder. In the case of urine not presenting in the bladder, 50 μL PBS buffer was injected into the bladder to rinse the bladder wall. The number of bacteria in the urine was quantified via serial dilutions and CFU counts. Indwelling catheters were collected, rinsed in 200 μL of sterile PBS three times and finally placed in 100 μL PBS. Seventeen from 20 explanted catheter samples in the control group and eleven from 14 for the coated group were sonicated for 10 minutes to aid biofilm dispersal. Samples were then vortexed at high speed for 10 sec, and bacterial numbers were determined by serial dilutions and CFU counts. Three catheters from each group were fixed in 2.5% glutaraldehyde (200 μL) for 1 h, and then dehydrated using an ethanol/water gradient (50%, 70%, 90%, 100%) for 10 min each. The samples were dried in the ambient condition, sputtered with Au/Pd (˜10 nm) and viewed using a scanning electron microscope (SEM, Hitachi™ SU3500) to visualize biofilm formation on catheter surface. There were 5 cases of unsuccessful infection with S. saprophyticus in the control group and 4 in the coated group of E6 and 2 in the coated group with Tet20LC as the CFU reading on both catheter surface and in urine was zero.
ATR-FTIR analysis: Absorption spectra of different surface coatings (both control and AMP conjugated) on catheter surface were collected on a Bruker 670 TensorII™ with a MCT/A liquid nitrogen cooled detector, a KBr beam splitter, and a VariGATR™ Grazing Angle accessory. Spectra were recorded at 4 cm−1 resolution, and 128 scans were collected. Water contact angle measurements: A water droplet (6 uL) was placed on the surface and an image of the droplet was taken with a digital camera (Retiga 1300™, Q-imaging Co.™). The contact angle was analyzed using Northern Eclipse™ software. Over three different sites were tested for each sample. X-ray photoelectron spectroscopy(XPS): Measurements were carried out at Nanofabrication and Characterization Facility (nanoFAB™), University of Alberta. The spectra were collected using a Kratos Axis Ultra™ X-ray photoelectron spectrometer operated in energy spectrum mode at 210 W Spectra were fit using CasaXPS (VAMAS) software and were calibrated to the lowest binding energy component of the Cis emission at 284.6 eV. Ellipsometry measurements: The variable-angle spectroscopic ellipsometry (VASE) spectra were collected on an M-2000 V spectroscopic ellipsometer (J. A. Woollam, Lincoln, NE) at 50°, 60°, and 70° at wavelengths from 480 to 700 nm with an M-2000 50W quartz tungsten halogen light source. The VASE spectra were then fitted with a multilayer model utilizing WVASE32 analysis software based on the optical properties of a generalized Cauchy layer to obtain the dry thickness of the deposited layers. OCM measurements: AMP immobilization on the surfaces were quantitatively monitored in real-time by QCM-D™ (Q-sense AB™, Sweden) at room temperature. Briefly, the BA coating was deposited on SiO2 (˜50 nm) coated sensors. For the evaluation of AMP E6 binding to the BA functionalized surface, the sensors were mounted into the QCM-D chamber. After stabilization of the baseline with buffer, a 0.1 mg/mL AMP E6 solution (pH 8.0) was flowed (50 μL/min) over the sensor. Finally, phosphate buffered saline (pH 8.0, 10 mM) was perfused to remove physically absorbed peptides. The measurements of the resonant frequency were carried out continuously. The mass was fitted through the Sauerbrey equation by using Qsense Dfind 1.2.2. Atomic force microscopy analysis: Measurements were performed on a commercially available multimode system (Dimension 3100™) with an atomic head of 130×130 μm2 scan range which used a NanoScope IIIa™ controller (Digital Instruments™, Santa Barbara, CA). The surface morphology and adhesive force in PBS buffer was collected in the contact mode using commercially manufactured V-shaped silicon probe (Bruker™, NP—S10) with a spring constant ˜0.06 N/m.
The uhPDMA was prepared at a concentration of 36 mg mL−1 in sodium acetate buffer (50 mM, pH=5). Dopamine was freshly prepared at a concentration of 12 mg mL−1 in sodium acetate buffer (50 mM, pH=5) before each experiment. Sodium periodate (NaIO4) was freshly prepared at a concentration of 28 mg mL-1 in sodium acetate buffer (50 mm, pH=5). The dopamine solution was then mixed with uhPDMA solution with a volume ratio of 1:5 (dopamine:uhPDMA). Sodium periodate solution was then added to generate a final concentration of uhPDMA at 30 mg mL−1, dopamine at 2 mg mL−1, and NaIO4 at 0.9 mg mL−1. Mixed solution (200 μL) was added into the wells of 96-well polypropylene plate. The plate was placed onto a rocking platform with shaking at 50 rpm for 2 hr. The coated plate was then rinsed by Milli-Q™ water and dried for further characterization.
Antimicrobial Peptide (AMP) Conjugation onto the PDMA/PDA Coated 96-Well Polypropylene Plate
Peptide solutions (200 μL, 0.25 mg/mL in 10 mM phosphate buffer, pH ˜8) were added into the wells and incubated for 24 h. The peptide-conjugated plate were rinsed with Milli-Q™ water thoroughly and dried under argon flow.
Bacterial strains used in this study included S. epidermidis (isolated from contaminated platelet units by Canadian Blood Services research laboratory in Ottawa), and S. saprophyticus (ATCC 15305). Bacteria were cultured in LB, with shaking at 250 rpm, at 37° C. for overnight. An overnight culture of the bacterial strain was diluted to a 500 CFU/mL in Mueller Hinton Broth (MHB). For antimicrobial activity experiments, 120 μL of the diluted overnight culture was added to each well in the bare PP plate, PP plate coated with BA and BA-AMPs. After 24 hrs growth under shaking condition at room temperature, bacteria culture (100 μL) was transferred to sterile clear polystyrene plate and the optical density was read using UV-vis spectrometer at wavelength 595 nm. Planktonic bacteria were then serially diluted and plated on LB agar for CFUs.
All the data values were expressed as mean±standard deviation (SD). Statistically significant value was set as p<0.05 based on Student's two-tailed unpaired t-test.
In the first approach, we initially constructed a non-fouling surface coating using a rapid assembly of polydopamine (PDA) and ultra-high molecular weight PDMA (uhPDMA) (800 KDa, PDI 1.3), which is highly stable and can be prepared on any substrate[39]. The coating was highly hydrophilic and enriched with uhPDMA chains on the surface. The coating was further modified with AMP E6. The conjugation (
In the second approach, a copolymer PDMA-co-APMA (N-(3-aminopropyl) methacrylamide) (Mn 630 000, PDI L3) was synthesized with APMA molar content of 10% (
The coating formation and peptide conjugation were initially investigated using ATR-FTIR analysis (
The conjugation of AMP to BA and MA coatings was further analyzed using water contact angle measurements. The water contact angle of BA-AMP coating was 36.2±0.4° in comparison to the BA coating 31±0.9°. In the case MA-AMP coating, the water contact angle was 37.9±0.9° compared to 29.2±1° for MA coating (
To gather the information on surface structure for two different coatings (BA-AMP and MA-AMP), we utilized AFM force measurements in wet conditions. The representative force curves for BA and BA-AMP coatings are shown in
The representative AFM force curves for the MA and MA-AMP (E6) coatings are shown in
The data clearly show that the structure of PDMA chains and presentation of AMPs (E6) were quite different for both BA-AMP and MA-AMP coatings. This allowed us to further investigate the role of presentation of AMP and coating structure to their anti-biofilm activity.
We further evaluated the influence of AMP presentation and coating structure on the anti-biofilm activity. A live/dead assay was used to examine the level of bacterial adhesion as well as the viability of adhered bacteria in early stage biofilm formation.
We further evaluated the early stage biofilm formation at 24 h.
A similar observation was made for P. aeruginosa (
There are changes in membrane integrity and morphology upon interaction of the bacteria with the surface tethered AMPs on the coating. The bacteria on the control and BA coated substrates were observed to have a smooth surface (
We further analyzed the efficiency of the coating to prevent biofilm formation on a biomedical plastic surface relevant to catheter-associated urinary tract infections. We adapted the coating methodology on the surface of 14G PU catheters. The pristine catheter, BA, BA-AMP, MA and MA-AMP coating on PU catheters were tested by assessing the number of adhered bacteria on the surface by assessing CFUs after 24 h of incubation.
From these results of bacterial adhesion and early stage biofilm formation, it was determined that presentation of AMPs on the coating and the coating structure had a significant influence on their anti-biofilm activity. The BA-AMP coating, where the AMP is conjugated on the PDA layer and protected by the non-fouling uhPMDA chains showed significantly higher activity than the coating where AMPs were presented throughout the coating (MA-AMP). Since the synthesis method for BA-AMP coating is very simple, we anticipate that this method could be easily and rapidly adapted to diverse surfaces and diverse peptides rapidly both as a screening method as well as an infection resistant coating.
To develop a robust screening protocol in a realistic environment which could be potentially used to test implant and device modifications, we initially tested a small library of AMPs peptides (TABLE 3) with broad-spectrum activity. Besides E6, Tet20C and its variant Tet20LC, IDR-1018, 3002, and D-enantiomeric peptide DJK5 and its variant, were also included in the study. Tet20C (KRWRIRVRVIRKC (SEQ ID NO:2)) and Tet20LC (KRWRIRVRVIRK-bA-bA-C—CONH2 (SEQ ID NO:3)) are variants of AMP Opt5 (KRWRIRVRVIRK-CONH2 (SEQ ID NO: 10)), which showed high antimicrobial activity in soluble and tethered form against various pathogens.
P.
E.
S.
S.
aeruginosa
coli
aureus
saprophyticus
Although the C-terminus of the tested peptides was amidated, modifications to the peptides are not a requirement for activity, but may have benefits to the activity of the AMP and assist in linking the AMP to the hydrophilic polymer or co-polymer. Adding a cysteine at C-terminus of Opt5 resulted in Tet20C. Tet20LC has a linker consisting of two beta-alanine added before the cysteine. Tet20C showed strong anti-biofilm activity while tethered in the brush coating on surface 1491. Peptide IDR-1018 was developed based on bactenecin and possessed both immunomodulatory activity and anti-biofilm activity[50]. Peptide 3002 as a variant of IDR-1018, was discovered with computer aiding and exhibited stronger anti-biofilm activity than IDR-1018[51]. DJK5 and its variants was recently developed D-enantiomeric protease-resistant peptide with a more potent activity in inhibiting biofilm formation relative to L-amino acid IDR-1018[52]. This proven initial library of peptides (TABLE 3) was used to test our concept on the application of BA-AMP coating as a screening tool to identify optimal surface tethered peptide that prevents biofilm formation in an antifouling background.
Initial studies were performed using a silicon wafer coated with titanium and the data showed that different AMPs could be successfully conjugated onto the BA coating (
Overall, the 96-well-based anti-biofilm screening assay developed by conjugation of AMPs to a non-fouling background helped us to identify AMPs with better anti-biofilm activity. We determined that peptides, E6, Tet20C, Tet20LC and DJK5C showed better broad-spectrum activity than the other tested peptides from the library against the most common uro-pathogens.
Hemolysis analysis was performed to determine the hemolytic properties of the AMP conjugated coating in direct contact with blood. The result of hemolysis analysis showed that the BA and BA-E6 coating has very low hemolysis, 0.4% and 0.3%, respectively (
We next investigated whether our new coating chemistry and data from the screening studies could be utilized for the prevention of implant/device infection. We adapted the coating chemistry to 24G catheters using two different peptides that showed greater promise (BA-E6) and BA-Tet20LC). The generated coatings on catheters were studied for their in vitro activity first before testing in mouse infection models (
The efficiency in reducing biofilm formation was assessed using an ultrasound-guided percutaneous model of catheter-associated urinary tract infections[42]. The reduction in bacterial numbers of S. saprophyticus both on the catheter sample as well as in the urine was accessed by CFU counts. The average CFU counts for mice bearing untreated control catheters was (3±0.7)×107 CFU/mL while those implanted with catheters bearing BA-AMP (E6) and BA-AMP (Tet20LC) coatings were (2.1±0.7)×107 and (1.8±0.9)×107 CFU/mL respectively, indicating that catheters from different groups were exposed to similarly infectious conditions (
The biofilm formation on the catheter surface was also examined by scanning electron microscopy (SEM).
Conjugated peptides on the BA platform showed different inhibition capacities in association with planktonic bacteria growth and bacteria-killing activity (
Although these results showed variable effects depending on the bacteria tested and the AMP used in the assay, effectiveness was shown for all AMPs tested for some bacteria. Accordingly, depending on the particular use (i.e. duration, location and microbiology of the individual, either the BA or MA polymer coating may be selected and the most suitable AMP or AMPs may be selected.
The disclosure may be further understood by the following non-limiting examples. Although the description herein contains many specific examples, these should not be construed as limiting the scope of the disclosure but as merely providing illustrations of some of the embodiments of the disclosure. For example, thus the scope of the disclosure should be determined by the appended aspects and their equivalents, rather than by the examples given.
Many of the molecules disclosed herein contain one or more ionizable groups [groups from which a proton can be removed (e.g., —COOH) or added (e.g., amines) or which can be quaternized (e.g., amines)]. All possible ionic forms of such molecules and salts thereof are intended to be included individually in the disclosure herein. With regard to salts of the compounds herein, one of ordinary skill in the art can select from among a wide variety of available counterions those that are appropriate for preparation of salts of this disclosure for a given application. In specific applications, the selection of a given anion or cation for preparation of a salt may result in increased or decreased solubility of that salt. Every formulation or combination of components described or exemplified herein may be used to practice the disclosure, unless otherwise stated.
Whenever a range is given in the specification, for example, a temperature range, a time range, or a composition or concentration range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. It will be understood that any subranges or individual values in a range or subrange that are included in the description herein can be excluded from the aspects herein.
Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numeric ranges are inclusive of the numbers defining the range. The word “comprising” is used herein as an open ended term, substantially equivalent to the phrase “including, but not limited to”, and the word “comprises” has a corresponding meaning. As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a thing” includes more than one such thing. Citation of references herein is not an admission that such references are prior art to an embodiment of the present invention. The invention includes all embodiments and variations substantially as hereinbefore described and with reference to the examples and drawings. Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which a disclosed disclosure belongs.
Xu, Antimicrobial Peptide-Conjugated Hierarchical Antifouling Polymer Brushes for Functionalized Catheter Surfaces, Biomacromolecules 20 (2019) 4171-4179.
Rodriguez, Antibacterial Properties of hLf1-11 Peptide onto Titanium Surfaces: A Comparison Study Between Silanization and Surface Initiated Polymerization, Biomacromolecules 16 (2015) 483-496.
This application claims the benefit of U.S. Provisional Patent Application Ser. No. 63/195,836 filed on 2 Jun. 2021 entitled “HIGH EFFICIENCY ANTIFOULING COATING WITH ANTIMICROBIAL PEPTIDES”.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2022/050883 | 6/2/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63195836 | Jun 2021 | US |
