Title of Invention	PREPARATION OF AGAR BIONANOPARTICLES
Abstract	A process of preparation of agar bionanoparticles measixring 50mn to 80nm is described. These bionanoparticles prevent calcium induced clotting of human blood plasma, inhibits the growth of onion roots and human epidermoid carcinoma HEp2 cells.

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Agar is extracted from sea weeds belonging to the group rhodophyta (red algae). Agar is a polymer of alternating D - galactose and 3,6-anhydro-L-galactose linked by altemating beta 1 -> 4 and alpha 1-^3 bonds. Agar is a polymer like starch and is made up of agarose and agaropectin like amylose and amylopectin in starch. While starch nanoparticles were prepared and used reports and lacking in agar. Starch nanoparticles below 400nm was produced by plasticizing the polymer using shear forces and with the addition of the cross linking agent and the biopolymer is dispersed in aqueous medium (US Patent 6, 677, 386). A nanoparticle / nanosphere enclosing a magnetic material not exceeding 1500 nm has been patented (US Patent 450 1726). Similarly the nanoparticles of chitosan measuring 383nm and its improved polydispersity by ultrasonication has been reported. (Tang 2003)^ Though polyglucose and aminated glucose nanoparticles were made, preparation of agar nanoparticles has not been reported. Summary of the prior art: Nanoparticles of oligoglucose (starch) and oligoglucosamines (chitosan) were prepared ranging in size from 300nm to 400nm. Though oligosaccharides were generated from mushrooms by acid hydrolysis, no nanoparticles were made. Similarly oligogalactose nanoparticles were also not made. Objectives of the invention To prepare agar bio-nanoparticles measuring less than 100 nm in size. -2- Summary of the invention: Bionanoparticles of agar measuring 50nm to 80nm were prepared by controlled acid hydrolysis of agar, selective size fractionation by ultrafiltration and particle preparation by spray drying at 100°C. 4. Description of the invention • lOOmg/ml of agar in water was taken in sahxlet apparatus and treated with 0.5N HCl for 30 to 60 minutes at 100°C. • Acid hydrolyzed agar was brought down to 25°C. • Characterized with the addition of IM NaHOa solution to give a final concentration of 0.IM NaHCOa for 30 minutes. • The suspension was filtered through 0.45 micron filter and then either filtered through 0.22 micron filters or through ultra filtration membranes with a cut off molecular weight of 3000 daltons. • Agar bionanoparticles of 50nm to 80nm were prepared by spray drying at 100°C. The bionanoparticles prepared were viewed under the atomic force microscope and the image is presented in figure 1. The size of the bionanoparticles were between 50nm to 80nm. The efficacy of agar bionanoparticles were elucidated. Example 1: Effect of agar bionanoparticles on the growth of onion roots. • Fresh onions were placed in test tubes containing sterile tap water for a period of 2-3 days • Bionanoparticles (lOmg/ml) were serially diluted in distilled water upto 10"^ dilutions. • Bioactivity of nanoparticles were tested by placing onions in test tubes containing appropriate dilutions. • The root tips were totally immersed in the bionanoparticle suspension and the initial length of the root tips were noted. • The increase in length of the root tips were recorded in each dilution after 24 hours • Onions kept in sterilized tap water served as control. Agar bionanoparticles have brought about 100% and 99% inhibition of root grovrth at lOmg/ml and Img/ml concentrations respectively and about 80% at lOO^ig/ml (Fig.2). Legend to Fig. 2 : Effect of agar bionanoparticles on the grov^h of onion roots X axis represents the concentration of bionanoparticle additions Y axis represents the % inhibition. The numbers in the X axis denotes the following: 1 : 1 Omg of bionanoparticles / ml 2 : Img of bionanoparticles / ml 3 : 100|ig of bionanoparticles / ml 4 : 10|ag of bionanoparticles / ml 5 : 1 |ag of bionanoparticles / ml ANTS (8-amino naphthalene - 1, 3, 6 trisulfonic acid) labeled agar bionanoparticles when treated to onion root tip binds to them and this could be ascertained by their ability to fluoresce (Fig.3). Fig. 3 The fluorescence emission by the onion roots treated with bionanoparticles stained with ANTS and viewed under the fluorescence microscope. Example 2: Anti coagulant assay • 300|il of human blood plasma was taken in a sterile eppendorff tube. • 3mM CaCb was added to 300|il of plasma to induce clotting. • l|ig of agar bionanoparticle with 3mM Cacb was added to 300|al of plasma. • 100|ig of agar was added along with 3mM Cac^ to 300|il of plasma. The effect of bionanoparticles on the clotting of the blood plasma is presented in Fig 4. V, Explanation for Fig.4 Tube 1: 300|il of plasma + 3mM Cach (control) Tube2: 300|il of plasma + 3mM Cach + l|ig bionanoparticles Tube3: 300|il of plasma + 3mM Cacb + lOOjig of agar Calcium chloride induced clot in the control. While the bionanoparticles inhibited the formation of clot, agar could not prevent the formation of clot. Example 3: Influence of agar bionanoparticles on HEp2 cells • HEp2 cells (1x10^ cells) were subcultured to attain confluent growth in 4 days. • lOOjil of bionanoparticles (20|ag/ml) were added and incubated at 37°C and grown for 4 days. While the cells in the control divided from 1x10"* cells to 40 x 10"^ cells , the bionanoparticle treated cells divided from 1x10'* cells to 8 x 10"^ cells indicating the ability of bionanoparticles to inhibit the cell division of HEp2 cells. Reference: Ultrasonication of chitosan and chitosan nanoparticles. Tang ES, Huang M, Lim Ly. Int.J Pharm 2003, 265, 103-14. 5. We claim: A process to prepare 50nm to SOnm agar bionanoparticles comprising the following steps of, (i) lOOmg/ml of agar in water was taken in sahxlet apparatus and treated with 0.5N HCl for 30 to 60 minutes at 100°C, (ii) acid hydrolyzed agar was brought down to 25°C, (iii) characterized with the addition of IM NaHCOa solution to give a final concentration of O.IM NaHCOa for 30 minutes, (iv) the suspension was filtered through 0.45 micron filter and then either filtered through 0.22 micron filters or through ultrafiltration membrane with a cut off molecular weight of 3000 daltons and (v) agar bionanoparticles of the size SOnm to SOnm were prepared by spray drying at 100°C

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376-che-2003-abstract.pdf

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376-che-2003-correspondnece-others.pdf

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

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