Title of Invention	AN IMPROVED METHOD FOR THE SYNTHESIS OF TEMPERATURE SENSITIVE POLY (N-ISOPROPYLACRYLAMIDE) (PIPAAM) COATING ON POLYMERIC MATERIALS
Abstract	An article for tissue culture comprising a substrate cited with a temperature sensitive surface of poly (N-isopropylacrylamlde) (PNIPAAm)

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FIELD OF THE INVENTION This invention relates to an improved method for the synthesis of temperature sensitive poly (N-Isopropylacrylamlde) (PIPAAM) coating on polymeric materials. This invention further relates to an improved method for the synthesis of temperature sensitive PIPAAm coating on polymeric materials, particularly on tissue culture grade polystyrene. BACKGROUND OF THE INVENTION In vitro cell culture is usually performed in glass or commercially available cell culture grade polystyrene. Cell adhesion needs certain requirements which enable the cells to adhere and grow without any toxic effects. The commercially available Tissue Culture Grade Polystyrene (TCPS) modified for this serves the requirement. Maintenance of such culture Is done by sub-culturing by enzymatic or mechanical methods. Both of these methods affect the cell properties and morphology at least for a minimum time during such processes. For tissue engineering purposes it is advisable and preferred to obtain cells with minimum destruction. An alternative method to harvest cells without destruction Is to modify the culture surface. Many techniques have been adopted to modify the culture surface. One of the methods is to graft the polymer Poly (N- Isopropylacrylamlde) on culture surface. Poly (N-Isopropyl acrylamlde) (PIPAAm) Is a well known temperature sensitive polymer that demonstrate the property Of switching characteristics depending upon temperature. The polymer exhibits thermally reversible surface changes in aqueous system from hydrophllllc to hydrophobic. The temperature at which this phase change occurs Is called Lower Critical Solution Temperature or LCST. PIPAAm exhibits LCST at 32 °C; and the polymer is hydrophilllc below LCST and hydrophobic above LCST. Moreover the polymer exhibits narrower range of hydrophillic/hydrophobic change around 32 °C. To adjust the LCST of temperature sensitive polymer, PNIPAAm Is synthesized In the form of copolymer by Including other monomers. Hence the available types of thermo sensitive polymer are of homopolymer of PNIPAAm or copolymer of homopolymer of PNIPAAm. Both these types of polymers have been shown to be used for cell culture purpose. The polymers made so far is by UV irradiation, Electron beam bombardment or by chemical reactions. The above said methods have ;its own disadvantages. The electron beam irradiation needs sophisticated instruments like Electron beam accelerator. UV irradiation is usually used for synthesis of copolymers. Synthesis of thermo responsive polymer by chemical reaction needs thorough removal of the reactants before using it for cell culture. The system may leave unresolved reactants which can affect cell cultures. So there is a need for new easy methods for the synthesis of thermally responsive surfaces with high efficiency. OBJECTS OF THE INVENTION It is therefore an object of this invention to propose a method for the synthesis of temperature sensitive poly (N-lsopropyl-acrylamide) surface on polymeric materials which is efficient and simple. It is a further object of this invention to propose a method for the synthesis of temperature sensitive poly (N-isopropyl-acrylamide) surface on polymeric materials which does not require sophisticated instruments. These and other objects and advantages of the invention will be apparent from the ensuing description. At the outset of the description, which follows, it is to be understood that the ensuing description only illustrates a particular form of this invention. However, such a particular form is only an exemplary embodiment and the teachings of the invention is not Intended to be taken restrictively. BRIEF DESCRIPTION OF THE INVENTION According to this Invention is provided a method for the synthesis of temperature sensitive poly (N-isopropyiacrylamide) surface on polymeric materials. This invention further relates to temperature sensitive coating on polymeric substrates. In accordance with this invention, a layer of temperature sensitive homopolymer of Poly (N-Isopropylacrylamide) is grafted on to commercially available tissue culture grade polystyrene by y- ray Irradiation. The terms "low temperature' In this description refers to a temperature from 0°C to temperature below LCST, while ‘high temperature' refers to a temperature below 100 °C but above the LCST. The polymers are either homopolymers of PIPAAm or heteropolymers with PIPAAm and other cross linking agents. The synthesis of the homopolymer or copolymer on the surface is done by polymerisation and grafting in a single step by irradiating with high energy electromagnetic radiation. The irradiation done at very high dose of 0.2 to 0.3 Mgy. After grafting the monomer can be completely removed by washing with distilled water. The monomer solution is prepared by dissolving N-isopropylacrylamide (NIPMm) in alcohol a concentration of 42-50% w/w. The monomer is polymerized and grafted to any commercially available polystyrene with amount of 10-12 µl monomer pre cm2 of polystyrene. The polymerization of monomer and grafting of the polymer on the polystyrene surface is simultaneously done by irradiating with y-ray at a dose of 0.1-0.3 MGy. The unreacted monomer or the unbounded polymer is removed by washing with cold sterile deionized distilled water and the surface was immediately kept in nitrogen atmosphere. The grafted culture dishes are sterilized either by y-ray radiation or by ethylene oxide. The invention will now be explained in greater details with the help of the following non-limiting examples. MATERIALS: The monomer N-isopropylacrytamide (NIPAAm) was purchased from polyscience. (scove's Modified Oulbeccos' Medium (IMDM), Foetal Caff Serum (FCS) Trypsin and FITC conjugated phalioidin were purchased from Sigma (USA). Durapore filter membrane [0.45 J.1IT1, Hydrophllic Polyvinylidene Fluoride (PVDF)] was purchased from Millipore. All other chemicals were of reagent grade. Example: The monomer solution was prepared by dissolving NIPMm in alcohol preferably isopropyl alcohol at a concentration of 42-50% w/w. The monomer was polymerized and grafted to any commercially available polystyrene with amount of 10-12 pi monomer pre cm2 of polystyrene. This was then irradiated with y-ray at a dose of 0.1-0.3 MGy. The unreacted monomer or the unbounded polymer was removed by washing with cold sterile deionized distilled water and the surface was immediately in nitrogen atmosphere. The grafted culture dishes were sterilized either bv v-rav radiation at 2.5 MRad or by ethylene oxide. FTIR spectrophotometry analysts of modified surface of polystyrene when compared with unmodified surface, confirmed the grafting of PiPAAm on polystyrene. The presence of amide peak of PIPM, at 1650 cm-1 on grafted surface instead of the monosubstituted aromatic ring peak of polystyrene at 1600 cm-1 proves the grafting of polymer on surface. Static water contact angle on the surface of the modried surface is measured by NRI contact angle Gonliometer (Model 100.00) using Mill Q Water. Water drop of 5-10µl deposited on the surface at 37°C and 27oC is measured as soon as possible. The static contact angle may be read directly from the goniometer. A minimum of 5 different fields are measured from each sample and mean data are taken as final data and they have standard deviation of less than 5%. For SEM analysis, the grafted and ungrafted surfaces are puttered with gold (3 min, 20A) In ion sputter device (Hitachi E101). Samples are observed at high magnification using SEM (Hitachi S 2500) with till angle of 90o and 15 KV accelerating voltage. The grafting of PIPAAm on polystyrene does not show surface alteration when compared to normal ungrafted surfaces. Example- 2: L-929 (mouse subcutaneous connective tissue) fibroblast cells procured from National Centre for Cell Science (NCCS, India) were used for cell culture studies. Cells were maintained in isocovers Modified Dulbeccos Medium (1MDM) supplemented with 5-10% Foetal Calf Serum, 100 lU/ml penicillin and 100 µg/ml streptomycin, at 37oC in a humidified atmosphere with 3-5% CO2 Cells were allowed to form monolayer and then subcultured using enzymatic treatment (trypsdin-EDTA). Cells were obtained as suspension In medium and seeded on PIPM grafted and untreated dishes at a density of 1 x 105- 2 x 105 Cells per 2.826 x 103 cm2. The dishes were incubated at 37°C until it reached near confluency with media change at an interval of three days. The surface did not induce any cytotoxic response to the cells and cells on grafted dishes showed similar morphology as on ungrafted culture surface. The amount of culture medium was reduced to 100-300 \i\ by removing excess medium. A hydrophilic polyvinylidene fluoride (PVDF) membrane soaked in culture medium was placed over the confluent cell layer and was Incubated at room temperature (20±1oC) for 30 minutes. The cells detached from grafted surface and adhered on to the membrane and the membrane together with the cells was kept on a new culture dish with cell side down. 250-500µl fresh culture medium was added on membrane and incubated at 37oC for 10-15 minutes. The membrane was removed after adding sufficient medium and culture was incubated at 37oC. Cells on unmodified surfaces and PIPAAM grafted surfaces were fixed with2-4 % paraformaldehyde in Phosphate Buffered Saline (PSS) and used for staining cytoskeletal proteins. Cells were stained using FITC-conjugated phalloidin diluted in PBS, 1:100-1 :P200 for 15-25 minutes at room temperature. After rinsing the cells with PBS, it was mounted on micro slides and observed under fluorescence microscope with FITC filter. Cytoskeletal reorganization of adhered cells on PIPAAm grafted and unmodified surface under fluorescene microscope showed normal growth pattern. For SEM analysis cells were fixed in glutaraldehyde (2'-3% in 0.1 M phosphate Buffer, pH 7.0) for a minimum time of 2 hours around 25 DC. The specimens were then processed for SEM by dehydrating In graded alcohol and critical point drying (Hitachi, HCP-2). The samples were gold coated and observed under SEM as described earlier. L-929 cells at high magnification under SEM revealed intact morphology of cells on grafted surface and similar adhesion pattern in comparison with that of cells adhered on unmodified culture surface. The coated surface thus produced is used as a substrate for cell culture studies as normal by routine maintenance by enzyme treatment of cells and if necessary the same can be used for cell retrieval by temperature variation. The polymer can be used for any type of anchorage dependent cells. The process may be applied for grafting different polymeric substances other than NIPAAIW. The product can be modified to make it responsive to other type of stimuli such as ionic strength and pH. WE CLAIM: 1. An article for tissue culture comprising a substrate cited with a temperature sensitive surface of poly (N-isopropylacrylamlde) (PNIPAAm) 2. The article of claim 1 wherein the substrate is selected from a variety of polymers other than commercially available tissue culture grade polystyrene. 3. The article in claim 1 wherein the monomer Is NIPAAm dissolved in alcohol, preferably is isopropyl alcohol. 4. The article of claim 1 wherein the product is made by a process by y-ray irradiation of the monomer on substrate. 5. The article In claim 1 wherein the temperature sensitive surface is non cytotoxic to cells in vitro system and can be used for cell culture especially for tissue engineering. 6. The article of claim 1 wherein the temperature sensitive surface has advancing contact angle between 31°C to 33°C at 27°C and 34°C to 35°C at 37°C . 7. The article of claim 5 wherein the cells cultured on polymer can be detached by lowering the temperature from 37°C to 21-23°C . 8. A process for the preparation of an article for tissue culture comprising preparing a monomer solution followed by polymerisation and grafting of the monomer on a polymeric substrate by irradiating with a y-ray source and washing to remove the unbound polymer followed by sterilising the grafted surface in an inlet atmosphere to obtain the article for tissue culture. 9. The process as claimed in claim 8, wherein said monomer is N-isopropyi acrylamide (NIPAAm) 10. The process as claimed in claim 8, wherein said polymeric substrate is any polymer for example tissue culture grade polystyrene. 11. The process as claimed in claim 8, wherein the N-lsopropyl acrylamide solution is prepared In a concentration 42 to 50% w/w. 12. The process as claimed in claim 8, wherein the NiPPAm solution is prepared in an alcohol. 13. The process as claimed in claim 8, where irradiation Is alone at a dose of 0,1 to 0.3MGy. 14. The process as claimed in claim 8, wherein the grafted surface is washed with water. 15. The process as claimed in claim 8, wherein sterilisation is done using y-ray.

Documents:

816-che-2003 abstract-29-06-2009.pdf

816-che-2003 claims 27.07.2009.pdf

816-che-2003 claims-29-06-2009.pdf

816-che-2003 claims.pdf

816-che-2003 correspondence others 27.07.2009.pdf

816-che-2003 correspondence others-02-07-2009.pdf

816-che-2003 correspondence others-29-06-2009.pdf

816-che-2003 correspondence others.pdf

816-che-2003 form-1-29-06-2009.pdf

816-che-2003 others-29-06-2009.pdf

816-che-2003-claims.pdf

816-che-2003-correspondnece-others.pdf

816-che-2003-description(complete).pdf

816-che-2003-description(provisional).pdf

816-che-2003-form 1.pdf

816-che-2003-form 26.pdf

816-che-2003-form 3.pdf

816-che-2003-form 5.pdf

816-che-2003-other documents.pdf