The overall goal is to establish a nanoscale platform open for a universal protocol to synthesize personalized medicine on the basis of the genetic outfit of a person's disease, especially for cancer. First produce and purify poly malic acid from the microorganism frum polycephalum chemically activate the purified PMLA at its pendant. Carboxylic groups attach the small functional groups and the linker MEA by substitution of the activated groups and revert the unused activated groups to carboxylic groups by hydrolysis.
Next, add the antibodies and antisense molecules via the sulf hydro groups of the spacer, MEA and cap. The unused spacer by reaction with PDP on completion test the composition and the function of the lead. Nano drug results from an animal model like mice carrying aggressive human.
HER two positive breast cancer can determine the efficacy of the treatment and also whether the treatment is without undesired side effects. The main advantage of bioproduction over existing synthetic methods like oping polymerization is that the biopolymer retains full optical purity and is not contaminated with byproducts. This family of PolyPhen nano drugs can target any cell type based on specific targeting and regulate protein expression.
Generally, cancer treatment by PHE nano drugs is very efficient at several animal models, spearing primary brain and breast cancers. We first had the idea for this method to block the synthesis of glioblastoma specific laming in isof forms and later we proved the targeting an inhibition of the HER two and triple negative breast cancer proteins to prevent the tumor growth. The implications of this technique extend towards diagnosis of cancer by using optical and magnetic resonance imaging.
Poly nano drugs can provide insight into drug delivery for cancer treatment. It can also be applied to other pathological conditions such as infectious disease, pain relief, and neurological disorders. After growing a seed plasmodium culture from spial on auger inoculate 100 milliliters of basal culture medium and grow the culture in a 25 degrees Celsius.
Thermos stated incubator produce an amount of gravity packed microplasm modia under the exclusion of light. In order to avoid sporulation in a 10 liter bioreactor vessel, dissolve 80 grams of calcium carbonate in eight liters of basal medium, then transfer the pact microplasm modia into the mounted reaction vessel and set the bioprocess for 75 hours. At 25 degrees Celsius at a 10 liter per minute flow of filtered air and 150 RPM stirring by a segment stir.
When the culture broth has a pH of 4.8, stop the culture to measure the poly maleic acid production. Now cool the broth to 17 degrees Celsius and allow the cells to settle by gravity and remove the culture supernatant from the reactor. Adjust the pH to 7.5.
Now pump the supernatant from the bottom through a 1.5 liter DEAE cellulose column equilibrated with 20 millimolar tris hydrochloride pH 7.5 at five degrees Celsius. Then pass three columns of wash buffer containing 0.3 molar sodium chloride from the bottom to the top of the column elute the PMLA in the presence of 0.7 molar sodium chloride. Adjust the PMLA eluded fraction to 0.1 molar calcium chloride and add 80%ice cold ethanol to precipitate the PMLA calcium next size fractionate, the PMLA calcium using cidex G 25 and water.
Finally pass each fraction over an amber light column and immediately freeze the flow through poly malic acid in liquid nitrogen and lyophilize before storing at minus 20 degrees Celsius. To activate the carboxyl groups of PMLA mix one milli mole mala units of P-M-L-A-H in one milliliter of anhydrous acetone and one milli mole, each of an hydroxy CIN aide and cylo heyl carbo ide in two milliliters of dimethylformamide after stirring at 20 degrees Celsius for three hours, add 0.05. Millimoles of methyl Pega mean in one milliliter of DMF followed by 0.05 millimoles of triethylamine.
Then in a dropwise fashion add 0.4 millimoles of leucine ethyl ester and after one hour check for reaction completion by negative hyn response on thin layer chromatography. Similarly add 0.1 millimoles of two me capto, one ethylamine and 0.5 millimoles of TEA. Remove the leftover NHS ester by spontaneous hydrolysis with phosphate buffered saline.
Desalt over a PD 10 column and lyophilize to obtain a white powder store this pre conjugate dry at minus 20 degrees Celsius. Next, add 30 milligrams of antibody in 4.5 milliliters of 150 millimolar sodium chloride in 100 millimolar sodium phosphate at pH 5.5. Reduce the diss sulfide bonds with five millimolar tris, two carboxy ethyl phosphine hydrochloride, and purify over a PD 10 column.
Now dissolve the IDE PEG IDE in two milliliters of the above buffer, albeit at pH 6.3. Add the reduced antibody and stir at 20 degrees Celsius for one hour. Concentrate to 2.5 milliliters over AVEVA spin 20 with 30 kilodalton exclusion and then purify over cidex G 75.
Now verify the product over SEC HPLC. Measure the amount at 280 nanometers and also confirm the absence of impurities at other wavelengths. Next, mix 50 milligrams of the pre conjugate with 200 MLEs of the antibody conjugated PEG IDE in five milliliters of the pH 6.3 buffer.
Adjust the concentration of sulf hydro to two millimolar and incubate at 20 degrees Celsius for three hours. To obtain a 98%yield solubilize, the three prime amino antisense oligonucleotide react with one volume of SPDP, purify the A-O-N-P-D-P over cidex LH 20 in methanol, then evaporate the methanol, dissolve the product in water, and lyophilize now incubate 800 nan animals of the activated aos with the antibody PEG pre conjugate to MEA SH overnight. When the diss sulfide exchange reaction is terminated, then incorporate the fluorescent dye block, the excess sulf hydros with PDP and purify over cidex G 75.
Store the final poly malic acid based nano drug at minus 20 degrees Celsius to estimate the poly maleic acid content during production proceed as follows, to 320 microliters of sample add 160 microliters of 10%hydroxyl ammonium chloride and 160 microliters of 10%sodium hydroxide. After 10 to 15 minutes, mix with 160 microliters of 5%ferric chloride solution and visualize the formation of a dark purple color at 540 nanometers. Next, hydrolyze the nano drug overnight in two molar hydrochloric acid at 110 degrees Celsius using a sealed ampule assay for maleic acid by reversed phased chromatography.
Using samples spiked with maleic acid standards. Now cleave the nano drug with 100 millimolar dihi three etol and measure the morph antisense oligonucleotides by quantitative reversed phase HPLC against morpho a ON standards. Using the cleaved nano drug measure the antibody content using a protein assay kit to quantify the Malik PEG content.
First, react with ammonium OT thiocyanate, then extract with chloroform and measure absorbance at 510 nanometers. Test the antibody activity and co ligation by Eliza using five microliters of nano drug per well and 0.5 micrograms per well of her two as plate coated antigen. Finally, calculate the achieved percentage for antisense oligonucleotide, EG.And antibody with regard to maleic acid.
Compare with the percent intended by drug design. This bimodal strategic approach to the nano drug employs Herceptin to inhibit the HER two signaling pathway and HER two specific a ON to block transcription of the HER two receptor antibodies facilitate transcytosis via endothelial TFR of the tumor vessel. The nano conjugate platform poly maleic acid was produced by cultured micro plasmo and purified activating the PMLA carboxylic groups facilitated synthesis of the pre conjugate scaffold.
And finally, the lead nano conjugate. Eli SA assays demonstrated retention of active monoclonal antibodies throughout nano drug synthesis. With both antibodies assembled on the same polymer platform, fluorescence confocal microscopy tracked cellular uptake of the nano drug.
The superimposed images at zero and three hours were analyzed together with the labeled endosomes. Pearson's correlation coefficients for colocalization indicate nano drug release from endosomes into the cytoplasm and dissociation of a ON from the nano drug by a glutathione dependent diss sulfide cleavage in the cytoplasm. In vitro, the lead nano drug significantly inhibited growth most effectively in the HER two over expressing cell line.
In vivo experiments in mice bearing BT 4 74, HER two over expressing human breast cancer showed greater than 95%growth inhibition by the lead nano drug containing Herceptin. And HER two specific A ON ex vivo Western blotting indicated inhibition of both HER two synthesis and phosphorylation of a KT cleavage of PARP marked apoptosis. The tumor regression in response to drug treatment is demonstrated by shrinkage of the tumor and depletion of tumor cells by histological examination.
You should now have a good understanding of how to use a biocompatible nano platform to achieve efficacious personalized treatment without side effects and achieving a nano drug with a control composition and verified functional activities Once mastered, these sensor can be performed with high reproducibility After its development. This technique using poly acid acid nano platform for targeted delivery of antisense oligonucleotides paves the way for researchers in the field of personalized ano medicine to explore tumor treatment targeting specific molecular tumor markets identified by personalized genetic analysis. Pharmacologically, active agents and cells can be extremely hazardous.
Precautions such as wearing mask and glove should always be taken while performing this procedure.
