Patent Application
20150197644
1. Field of the Invention
The present invention is generally related to a biomimetic agent, and more particularly to a biomimetic agent for anti-biofouling networks and application thereof.
2. Description of the Prior Art
At present, anti-biofouling surface treatment is one of the important techniques in various applications. An anti-biofouling surface indicates a biomolecule adhering resistant surface where the biomolecule is, for example, protein, blood, cells, bacteria, etc. The coating agent used in anti-biofouling surface treatment is generally considered to make a surface become hydrophilic in assisting of anti-biofouling according to prior arts, but the anti-biofouling effect in practice is not enough.
Furthermore, it have been reported that zwitterionic molecules or polymers are used as the anti-biofouling agent. For example, Wu et al. (L. Wu, J. Jasinski, S. Krishnan, J. Appl. Polymer Scienec, Vol. 124, 2154(2012)) disclosed a block copolymer comprising a betaine moiety, sulfobetaine moiety or carboxybetaine moiety as an anti-biofouling agent and anti-biofouling surface treatment by spin-coating the anti-biofouling agent. However, from
According to prior arts, although in general a surface being hydrophilic or having zwitterionic moieties shows anti-biofouling, an excellent anti-biofouling surface to be useful should be treated by a novel anti-biofouling agent comprising a specific structure.
In accordance with the present invention, a biomimetic agent for anti-biofouling coating and a method for making the same are provided. The present invention utilizes a polymer comprising a specific structure and zwitterionic or pseudo-zwitterionic moieties as an effective component of anti-biofouling to achieve anti-biofouling effect.
One objective of the present invention is to provide a biomimetic agent for anti-biofouling coating to apply to various surfaces, such as polymeric, metallic, ceramic and porous surfaces, by a simple well-known coating method, such as dipping, spin-coating and so forth.
One objective of the present invention is to provide a method for making a biomimetic agent for anti-biofouling coating to use a simple well-known coating method, such as dipping, spin-coating and so forth, to be easily applied to surfaces with a large area and porous surfaces. Therefore, mass-production can be easily implemented and no special or large-scaled equipment is required to reduce production cost.
Another objective of the present invention is to provide a method for preventing from fouling of a biomolecule on a substrate, the method comprising applying a copolymer comprises poly(vinylpyrolidone)-block-poly(sulfobetaine methacrylate), poly(vinylpyrolidone)-random-poly(sulfobetaine methacrylate) and poly(vinylpyrolidone)-alternating-poly(sulfobetaine methacrylate); and performing a thermal process at 120° C. above to have the copolymer to form a network on the substrate surface so as to prevent from fouling of a biomolecule on the substrate.
Still another objective of the present invention is to provide a antibiofouling network, the antibiofouling network has the mole mass ratio of poly(vinylpyrolidone) to poly(sulfobetaine methacrylate) in a range of 70/30˜42/58 and is derived from a copolymer comprises poly(vinylpyrolidone)-block-poly(sulfobetaine methacrylate), poly(vinylpyrolidone)-random-poly(sulfobetaine methacrylate) and poly(vinylpyrolidone)-alternating-poly(sulfobetaine methacrylate) by heating the copolymer at 120° C. above.
Accordingly, the present invention discloses a biomimetic agent for anti-biofouling coating, comprising a block, random or alternating copolymer having a general formula (1): AUnBUm wherein AU represents a repeating unit comprising an anchoring moiety, being a methylene moiety with substituents R1 and R2 having a structure of formula (2): —CR1R2—; BU represents a repeating unit comprising a zwitterionic moiety or a pseudo-zwitterionic moiety, being an ethylene moiety with a substituent R3 having a structure of formula (3): —CH2CR3H— or a propylene moiety with substituents R4 and R5 having a structure of formula (4): —CR4HCH2CR5H—; m is an integer of 5˜120; n is an integer of 5˜120; R1 represents a C3˜18 chained, branched or cyclic alkyl moiety, ester moiety (i.e. —COORx, where Rx represents a C3˜18 chained, branched or cyclic alkyl moiety, aryl moiety having substituent(s) of C1˜18 alkyl, or C5˜12 heteroaryl moiety), phenyl moiety having substituent(s) of C1˜18 alkyl, or C5˜12 heteroaryl moiety; R2 represents a hydrogen atom (H) or methyl moiety (CH3); R3 represents —COOR′ or —CONR″H; R5 represents —COOR′ or —CONR″H when R4 represents a hydrogen atom or R5 represents a cationic moiety when R4 represents a carboxylic moiety; R′ and R″ individually represent a betaine moiety, sulfobetaine moiety or carboxybetaine moiety.
Furthermore, the present invention discloses a method for making a biomimetic agent for anti-biofouling coating, comprising the following steps: providing a monomer comprising an anchoring moiety; providing a monomer comprising a zwitterionic moiety or a pseudo-zwitterionic moiety; and performing atomic transfer radical polymerization under existence of a catalyst and a polymerization initiator in nitrogen environment to have the monomer comprising an anchoring moiety and the monomer comprising a zwitterionic moiety or a pseudo-zwitterionic moiety react to form a block, random or alternating copolymer having a general formula (1): AUnBUm.
Moreover, the present invention discloses a coating composition for anti-biofouling, comprising: the above mentioned biomimetic agent and a solvent.
In addition, the present invention discloses a method for preventing from fouling of a biomolecule on a substrate, the method comprising: applying a copolymer containing a poly(vinylpyrolidone) and a zwitterionic moieties and performing a thermal process at 120° C. above to have the copolymer to form a network on the substrate so as to prevent from fouling of a biomolecule on the substrate. The biomolecules comprises fibrinogen, platelets, erythrocytes, fibroblast, and E. coli.
Finally, the present invention discloses an antibiofouling network which has the mole mass ratio of poly(vinylpyrolidone) to poly(sulfobetaine methacrylate) in a range of 70/30˜42/58 and is derived from a copolymer comprises poly(vinylpyrolidone)-block-poly(sulfobetaine methacrylate), poly(vinylpyrolidone)-random-poly(sulfobetaine methacrylate) and poly(vinylpyrolidone)-alternating-poly(sulfobetaine methacrylate) by heating the copolymer at 120° C. above.
According to the biomimetic agent for anti-biofouling coating and networks and both of the method for making the same of the present invention and application, a polymer comprising a specific structure and zwitterionic or pseudo-zwitterionic moieties is used as an effective component of anti-biofouling to achieve excellent anti-biofouling effect. Besides, the biomimetic agent can be applied to surfaces with a large area and porous surfaces easily by a simple well-known coating method, such as dipping, spin-coating and so forth. Therefore, mass-production can be easily implemented and no special or large-scaled equipment is required to reduce production cost.
a) to
a) to
FIG. 8(A)˜(D) show application of poly(VP-co-SBMA) coating on the metal stent, where (A) shows the stainless stain disk coated by copolymer gels and networks, (B) shows the results of fibrinogen adsorption test of copolymer gels/networks coated stainless steel disk, (C) shows the results of whole blood cell attachment test of copolymer gels/networks coated stainless steel disk and (D) shows the result of the stability of copolymer gels and networks coated on stainless steel disks in 30 days.
What is probed into the invention is a biomimetic agent for anti-biofouling coating and networks. Detail descriptions of the structure and elements will be provided in the following in order to make the invention thoroughly understood. Obviously, the application of the invention is not confined to specific details familiar to those who are skilled in the art. On the other hand, the common structures and elements that are known to everyone are not described in details to avoid unnecessary limits of the invention. Some preferred embodiments of the present invention will now be described in greater detail in the following. However, it should be recognized that the present invention can be practiced in a wide range of other embodiments besides those explicitly described, that is, this invention can also be applied extensively to other embodiments, and the scope of the present invention is expressly not limited except as specified in the accompanying claims.
The biomimetic agent for anti-biofouling coating according to the present invention can be coated on a surface to perform anti-biofouling surface treatment so as to achieve the effect of anti-biofouling. For example, the treated surface becomes resisting adherence of biomolecules like protein, blood, cell, bacteria, etc. The biomimetic agent according to the present invention is a compound having a structure imitating nature of a biomolecule. The biomimetic agent according to the present invention has a specific structure and zwitterionic or pseudo-zwitterionic moieties to possess the anti-biofouling characteristic. Besides, usually the coating agent usually has a problem of adhering to a treating surface. The coating agent according to the present invention uses a specific anchoring moiety not only to solve the adherence problem but also to provide excellent properties of coating, film-forming, and surface adherence.
In one embodiment of the invention, a biomimetic agent for anti-biofouling coating is disclosed. The biomimetic agent comprising a block, random or alternating copolymer having a general formula (1): AUnBUm wherein AU represents a repeating unit comprising an anchoring moiety, being a methylene moiety with substituents R1 and R2 having a structure of formula (2): —CR1R2—; BU represents a repeating unit comprising a zwitterionic moiety or a pseudo-zwitterionic moiety, being an ethylene moiety with a substituent R3 having a structure of formula (3): —CH2CR3H— or a propylene moiety with substituents R4 and R5 having a structure of formula (4): —CR4HCH2CR5H—; m is an integer of 5˜120; n is an integer of 5˜120.
Specifically, the biomimetic agent has the following structure shown by formula (A) or (B).
In the formula (A) and (B), R1, R2, R3, R4 and R5 represent the same moieties as the above.
R1 represents a C3˜18 chained, branched or cyclic alkyl moiety, ester moiety (i.e. —COORx, where Rx represents a C3˜18 chained, branched or cyclic alkyl moiety, aryl moiety having substituent(s) of C1˜18 alkyl, or C5˜12 heteroaryl moiety), phenyl moiety having substituent(s) of C1˜18 alkyl, or C5˜12 heteroaryl moiety. “C3˜18”, “C1˜18” or “C5˜12” means the number of carbon atoms in the moiety. That is, for example, C3˜18 chained, branched or cyclic alkyl moiety means alkyl having three carbon atoms˜eighteen carbon atoms, such as propyl, isopropyl, butyl, isobutyl, pentyl, hexyl, heptyl, octyl, nonyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, cyclopentyl, cyclohexyl, cycloheptyl, etc. The ester moiety (i.e. —COORx), for example, is phenyl, tolyl, benzyl, 2-pyrrolidone-1-yl, 2-pyridyl, phenoxycarbonyl (—C(═O)—OC6H5), benzyloxycarbonyl (—C(═O)—OCH2C6H5) and N,N-diphenylamino-para-phenoxycarbonyl
etc. The aryl moiety, for example, is phenyl, tolyl, benzyl, etc. The C5˜12 heteroaryl moiety, for example, is 2-pyrrolidone-1-yl, 2-pyridyl, etc. The open dashed symbol “-” indicates the linkage bond and the following “-” also has the same meaning.
R2 represents a hydrogen atom (H) or methyl moiety (CH3). R3 represents —COOR′ or —CONR″H. R5 represents —COOR′ or —CONR″H when R4 represents a hydrogen atom or R5 represents a cationic moiety when R4 represents a carboxylic moiety. R′ and R″ individually represent a betaine moiety, sulfobetaine moiety or carboxybetaine moiety. Preferably, m is an integer of 10˜80; n is an integer of 10˜80. More preferably, m is an integer of 20˜40; n is an integer of 20˜40.
The cationic moiety represented by R5, for example, is N,N-dimethylammnio-ethylene-1-amino-vinyl (—C(═CH2)NH2CH2CH2N(CH3)2H), N,N-dimethylammnio-propylene-1-amino-vinyl (—C(═CH2)NH2CH2CH2CH2N(CH3)2H), N,N-dimethylammnio-butylene-1-amino-vinyl (—C(═CH2)NH2CH2CH2CH2CH2N(CH3)2H), and N,N-dimethylammnio-pentylene-1-amino-vinyl (—C(═CH2)NH2CH2CH2CH2CH2CH2N(CH3)2H).
Specifically, the biomimetic agent has the following structure shown by formula (A-1)˜(A-21).
The above biomimetic agents with formula (A-1)˜(A-21) comprise rod-like anchoring moieties where m and n have the same meaning as the above. The biomimetic agents with formula (A-1)˜(A-21) can be a block, random or alternating copolymer.
Furthermore, the biomimetic agent has the following structure shown by formula (A-22)˜(A-33). The following biomimetic agents with formula (A-22)˜(A-33) comprise comb-like anchoring moieties where m and n have the same meaning as the above. The biomimetic agents with formula (A-22)˜(A-33) can be a block, random or alternating copolymer.
Furthermore, the biomimetic agent has the following structure shown by formula (B-1) or (B-2).
The biomimetic agents with formula (B-1)˜(B-2) can be an alternating copolymer.
Furthermore, the biomimetic agent has the following structure shown by formula (B-3).
The biomimetic agents with formula (B-3) can be a block or random copolymer.
Furthermore, the biomimetic agent has the following structure shown by formula (C-1)˜(C-6).
The biomimetic agents with formula (C-1)˜(C-6) can be a block, random or alternating copolymer comprising a cross-linkable moiety. After the biomimetic agents with formula (C-1)˜(C-6) is coated on a surface, the cross-linkable moiety can cross-link with each other by heating, Therefore, an antibiofouling network can be formed from the copolymer with formula (C-1)˜(C-6) by a thermal process. The thermal process is annealing process operated at 200° C.
In general, zwitterionic polymer, such as poly(sulfobetaine methacrylate) (PSBMA) exhibited the expected bio-inert nature, but the thermo-setting zwitterionic polymer (PSBMA or poly(carboxybetaine methacrylate) polymer networks showed the lost of fouling resistant control with the high-temperature annealing process. However, the present invention disclose that the modulated composition of thermo-setting poly(vinylpyrolidone)-co-poly(sulfobetaine methacrylate) copolymer networks having the specific mole mass ratio of poly(vinylpyrolidone) to poly(sulfobetaine methacrylate) effectively resist fibrinogen adsorption, platelet adhesion, erythrocytes attachment, bacteria attachment, cell attachment, hemolysis in 100% human plasma and 100% human whole blood. Accordingly, a method for preventing from fouling of a biomolecule on a substrate by forming a network on the substrate and an antibiofouling network are disclosed in the following embodiments of the invention.
In another embodiment of the invention, a method for preventing from fouling of a biomolecule on a substrate is provided. The method comprising applying a copolymer comprises poly(vinylpyrolidone)-block-poly(sulfobetaine methacrylate), poly(vinylpyrolidone)-random-poly(sulfobetaine methacrylate) and poly(vinylpyrolidone)-alternating-poly(sulfobetaine methacrylate) to a substrate; and performing a thermal process at 120° C. above to have the copolymer to form a network on the substrate surface so as to prevent fouling on the substrate from a biomolecule. Preferably, the copolymer further comprises (Poly((octadecyl acrylate)-alt-((acrylic acid)-(N-(3-(dimethylamino)propyl) acrylamide))), poly(vinylpyrolidone)-block-poly(carboxybetaine methacrylate), poly(vinylpyrolidone)-random-poly(carboxybetaine methacrylate), and poly(vinylpyrolidone)-alternating-poly(carboxybetaine methacrylate).
In one example of another embodiment, the substrate is selected from the group consisting of metal, glass, wafer, polymer and ceramic. Preferably, the substrate is a metal which is selected from Fe and Fe alloys.
In one example of another embodiment, the copolymer has the mole mass ratio of poly(vinylpyrolidone) to poly(sulfobetaine methacrylate) being 68/32˜39/61
In one example of another embodiment, the network has the mole mass ratio of poly(vinylpyrolidone) to poly(sulfobetaine methacrylate) being 70/30˜42/58.
In one example of another embodiment, the thermal process is annealing process. Preferably, the annealing process is operated at 200° C. or above.
In one example of another embodiment, the network forming on all or part of a stent surface.
In one example of another embodiment, the network forming on all or part of surgical instruments surface which comprises a scalpel surface and an endoscope surface.
In one example of another embodiment, the network forming on all or part of a catheter surface.
In one example of another embodiment, the network forming on all or part of a lens surface which comprises intraocular lens surface.
In one example of another embodiment, the network forming on all or part of a blood separation device surface.
In one example of another embodiment, the network forming on forming on all or part of a marine device surface.
In one example of another embodiment, the network forming on forming on all or part of dental instruments surface and dentures surface which comprises dental implants surface.
In one example of another embodiment, the network forming on all or part of an artificial joint surface.
In one example of another embodiment, the biomolecule comprising fibrinogen, platelets, erythrocytes, fibroblast, and E. coli.
In still another embodiment of the invention, an antibiofouling network is disclosed. The antibiofouling network has the mole mass ratio of poly(vinylpyrolidone) to poly(sulfobetaine methacrylate) in a range of 70/30˜42/58 and is derived from a copolymer comprises poly(vinylpyrolidone)-block-poly(sulfobetaine methacrylate), poly(vinylpyrolidone)-random-poly(sulfobetaine methacrylate) and poly(vinylpyrolidone)-alternating-poly(sulfobetaine methacrylate) by heating the copolymer at 120° C. above.
In one example of still another embodiment, the copolymer has the mole mass ratio of poly(vinylpyrolidone) to poly(sulfobetaine methacrylate) being 68/32˜39/61.
In one example of still another embodiment, the antibiofouling network being all or part of a stent.
In one example of still another embodiment, the antibiofouling network being all or part of surgical instruments which comprises a scalpel and an endoscopy.
In one example of still another embodiment, the antibiofouling network being all or part of a catheter.
In one example of still another embodiment, the antibiofouling network being all or part of a lens which comprises intraocular lens.
In one example of still another embodiment, the antibiofouling network being all or part of a blood separation device.
In one example of still another embodiment, the antifouling networks being all or part of a marine device.
In one example of still another embodiment, the antifouling networks being all or part of dental instruments and dentures which comprises dental implants.
In one example of still another embodiment, the antifouling networks being all or part of an artificial joint.
Production example 1: preparing A-33 (PODA-b-PSBMA Octadecyl acrylate (ODA) was dissolved in toluene to form a 40 wt % solution. By atomic transfer radical polymerization (ATRP), under existence of CuBr/2,2′-bipyridine (CuBr/bpy) as the catalyst and methyl 2-bromopropionate (MBrP) as the initiator, in nitrogen environment, [ODA]/[MBrP]/[CuBr]/[bpy](molar ratio) being 6/1/12˜120/1/1/2 was used to react for 24 hrs at 120° C. Then, by removing the catalyst and initiator, PODA was obtained.
By ATRP, poly(2-(dimethylamino)ethyl methacrylate) (DMAEMA) and PODA reacted with each other with a molar ratio of [DMAEMA]/[PODA]/[CuBr]/[bpy] being 5/1/1/2˜100/1/1/2 for 24 hrs at 120° C. to polymerize. Then, by removing the catalyst and initiator, the copolymer A-33P (Poly(octadecyl acrylate)-b-poly(2-(dimethylamino)ethyl methacrylate)) was obtained. 1,3-propanesultone and the copolymer A-33P were dissolved in THF to betainize the copolymer A-33P in atmospheric environment at room temperature for 24 hrs to obtain A-33.
2 ml of 10 wt % PMAO (poly(maleic anhydride alt 1-octadecene)) solution (solvent is THF) was prepare. Dimethylethylenediamine (DMEA) 3.2 g was dissolved in THF (8 ml) to form a DMEA solution. PMAO solution was then added into DMEA solution to form precipitate. By centrifugal separation and extraction, the copolymer B-3 was obtained.
Other biomimetic agents for anti-biofouling coating in the above can be made or synthesized by similar method shown in the production examples 1 and 2 or modified method together with well-known polymerization methods. According to experiments conducted by the inventors, the biomimetic agents for anti-biofouling coating according to the invention show good resistance in adherence of biomolecules like protein (plasma protein, Fibrinogen, bovine serum albumin (BSA)), blood (such red blood, leukocyte, platelet), cell (human cell, Fibroblasts, Keratinocytes), bacteria (S. epidermidis, E. coli), etc. That is, less than 20% of biomolecules will adhere on the surface treated by the biomimetic agent for anti-biofouling coating according to the invention. Under the better condition, less than 2% of biomolecules will adhere on the treated surface. The invention also discloses a coating composition for anti-biofouling, comprising: the above mentioned biomimetic agent according to the invention and a solvent. The solvent can be, for example, water, phosphate-buffered saline, ethanol, methanol, etc. The biomimetic agent for anti-biofouling coating according to the invention can be applied to a surface such as polymeric surface like polytetrafluoroethylene (PTFE), polyvinylidene difluoride (PVDF), polystyrene (PS), polypropylene (PP), polyvinyl chloride (PVC) or inorganic oxide surface like SiO2, Al2O3. In addition, a metallic surface like Ti, Fe can be treated by the biomimetic agent for anti-biofouling coating according to the invention.
The cross-linker N,N′-methylenebisacrylamide (MBAA) was dissolved in DI water at 1.6 wt % and stirred for 6 hr at 50° C. [2-(Methacryloyloxy)ethyl]-dimethyl-(3-sulfopropyl)-ammonium hydroxide (SBMA) and 1-vinyl-2-pyrrolidone (VP) of varying molar ratios were added to the MBAA solution, and the solution mixture was stirred for 10 min or until it was completely dissolved. The copolymeric reaction of the hydrogels was initiated by 0.2 wt % of Ammonium persulfate (APS) and 0.2 wt % of N,N,N′,N-tetraethylmethylenediamine (TEMED), and the reacting solution was then immediately transferred to a container to form a layer with the thickness of 0.2 mm. After polymerizing for 3 hr at 25° C., the gels were immersed in a water base for 48 hr to remove the chemical impurities. All the gels were made in disk-form with a diameter of 10.0 mm (10 mm biopsy punch, Acuderm Inc.) and stored in DI water at 4° C. till ready to use. The prepared gels were later dried in an evaporated oven to remove extra water to form a consistent shape. Finally, the polymer and copolymer networks were annealed at 200° C. to fix their shape with thermo-setting.
Characterizations of Poly(VP-co-SBMA) Copolymer Gels and Networks with Thermo-Setting Control.
Chemical characterization of prepared poly(VP-co-SBMA) copolymer samples (Vm-Sn) are analyzed by Fourier transform infrared spectroscopy (FT-IR) and X-ray photoelectron spectroscopy (XPS). The FT-IR spectrums of PVP, poly(VP-co-SBMA), and PSBMA (co)polymer gels and networks were shown in
V0-S100
aReaction molar ratio of co-monomers to initiator was fixed at 90 to 1. The total reactant mass percentage was 20 wt % in the prepared reaction solution.
bThe mole mass ratio of PVP to PSBMA in the poly(VP-co-SBMA) copolymer gels and networks was determined by XPS in the dry state from the spectral area ratio of the atomic percentages based on the N 1s of the pyrrolidone group from PVP segments and S 2p of the sulfonate group from PSBMA segments at the BE of 399 and 168 eV, respectively.
Human Plasma Protein Adsorption, Human Blood Platelets and whole blood cell Adhesion.
The adsorption of fibrinogen, a human plasma protein, was evaluated by the enzyme-linked immunosorbent assay (ELISA) to improve the sensitivity of the target proteins. Human blood platelets and whole blood cells attachments were tested using blood samples from healthy volunteers. The protocol for human protein adsorption and human blood cell attachment was followed by the procedure described in Jiang, S. Blood-Inert Surfaces via Ion-Pair Anchoring of Zwitterionic Copolymer Brushes in Human Whole Blood. Adv. Funct. Mater. 2013, 23, 1100-1110.
a) to
Red Blood Cell Hemolysis.
The red blood cell (RBC) hemolysis assay was performed to evaluate the antihemolytic activity and blood compatibility of prepared copolymer gels and networks. When red blood cells encounter incompatible environments, such as a hydrophobic surface or a non-isotonic environment, the blood cell membrane might be destroyed and thus hemoglobin proteins are released out from the disrupted membranes of blood cells. By detecting the absorbance of hemoglobin at 541 nm, it is easy to identify the amount of released hemoglobin and clarify the hemolysis level. DI water (+) and PBS (−) were set as positive and negative controls for the hemolysis of RBCs, as shown in
Cell Attachment Assay.
Human HT-1080 fibroblasts (ATCC, Manassas, Va.) modified with stable luciferase and EGFP expression by using the plasmid pAAVluciferase-EGFP were used to examine the attachment behavior of cells on the polymeric gel surfaces. The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37° C. in a humidified atmosphere with 5% CO2. A cell suspension at 2×104 cells/mL prepared, and 1 mL the cell suspension was allocated onto wither the copolymer gels or network disks, which were then incubated for three days at 37° C./5% CO2. A Nikon TS 100 microscope with a 10× objective lens and a blue excitation fluorescence (450-490 nm) filter was used to observe the cells after proliferation and the fluorescence images were used to quantify the amount of cell attachment on the surfaces of the six independent disks (n=6 in total) for each gel substrate, and the average result was reported.
Fibroblast Cell Attachment on Poly(VP-co-SBMA) Copolymer Gels and Networks.
Bacteria Adhesion Assay.
Escherichia coli (E. coli), was used to investigate bacterial adhesion behavior on the surface of poly(VP-co-SBMA) copolymer gels without thermo-setting and copolymer networks with thermo-setting. The cultures of E. coli were incubated in the medium (3.0 mg/mL beef extract and 5.0 mg/mL peptone) at 37° C. and shaken at 100 rpm for 12 hr to reach the stationary phase then plated on either the prepared copolymer gel or network at concentration of 106 cells/mL. The samples were cleaned before use by submerging the prepared copolymer gels or networks in 75 wt % ethanol for 1 hr and washed by PBS solution for three times. Each gel was then put into the well a 24 well plate, to which 1 mL of bacteria suspension was added. The samples were then incubated in an oven for 24 hr at 37° C. After incubation, the bacterial suspensions were removed from the wells the all samples were washed with PBS for three times to remove the bacteria that had accumulated but not adhered on the surfaces. Live/Dead Baclight was added to the samples to stain the adhering bacteria and the stained samples were washed with PBS three times then observed with the fluorescence microscope using 450-490 nm excitation, an Olympus BX51 CCD camera and a 10× objective lens.
Antibacterial Efficacy of Poly(VP-co-SBMA) Copolymer Gels and Networks.
According to
Obviously many modifications and variations are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims the present invention can be practiced otherwise than as specifically described herein. Although specific embodiments have been illustrated and described herein, it is obvious to those skilled in the art that many modifications of the present invention may be made without departing from what is intended to be limited solely by the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 101144643 | Nov 2012 | TW | national |
This application is a Continuation In Part of applicant's earlier application Ser. No. 13/690,202, filed Nov. 30, 2012.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13690202 | Nov 2012 | US |
| Child | 14661332 | US |
