| 1 ObjectiveA series of poly(amino acid methyl ester)nanohydrogels(NPs)with different side chains were synthesized with N-acryloyl-L-amino acid methyl ester of different properties.There binding ability and interaction with proteins and biomarker polypeptides were investigate to provide preliminary basis for the development of abiotic antibodies.2 Methods2.1 Hydrophobic amino acid methyl ester(phenylalanine methyl ester,leucine methyl ester,tyrosine methyl ester)hydrochloride,negatively charged amino acid methyl ester(glutamic acid methyl ester)hydrochloride,positively charged Amino acid methyl ester(arginine methyl ester,histidine methyl ester)hydrochloride,polar amino acid methyl ester(serine methyl ester)hydrochloride were acylated by acryloyl chloride as raw materials to synthesize N-acryloyl-L-amino acid methyl esters under basic condition.2.2 N-acryloyl-L-amino acid methyl esters were copolymerized with appropriate amount of hydrophilic monomer N-isopropylacrylamide(NIPAm)and hydrophobic monomer N-t-butyl-acrylamide(TBAm)to prepare a series of NPs with different side chains and different monomer ratios,their properties such as appearance,yield,particle size,Zeta potentials were characterized and the effects of temperature,buffer solutions and pH on their particle size were investigated.2.3 The binding of NPs with proteins of different molecular weights and isoelectric points were investigated by BCA kit assay and fluorescent labeling assay to illustrate their interaction laws.2.4 The binding of NPs with small cell lung cancer biomarker pro-gastrin-releasing peptide(proGRP)and heart failure biomarker N-terminal pro B type natriuretic peptide,(NT-proBNP)were investigated by fluorescent labeling assay to illustrate their interaction laws.3 Results3.1 Seven different N-acryloyl-L-amino acid methyl ester monomers were synthesized,and their structures were confirmed by 1H-NMR.Among them,N-acryloyl-L-glutamic acid-α-methyl ester(AGlu-OMe)was a new compound.3.2 A series of NPs with different side chains and different monomer ratios were prepared by precipitation polymerization using the above monomers.NPs with different side chains are temperature-sensitive except for AGlu-OMe@NPs.The buffer solutions enhanced the temperature-sensitivity of NPs and induced their aggregation state,but different pH conditions had no obvious effect on the properties of NPs.For AArg-OMe@NPs of different monomer ratios,the temperature sensitivity was reduced by the increase of the proportion of AArg monomer,40%AArg-OMe@NPs showed no temperature sensitivity in water and buffer solutions.3.3 NPs-protein binding experiments showed that the binding rates of AArg-OMe@NPs,AHis-OMe@NPs,ASer-OMe@NPs were higher for human serum albumin,ovalbumin and α-lactalbumin with different molecular weights;the greater the molecular weight,the higher the binding rate with NPs.Among them,AArg-OMe@NPs showed the highest binding rate with above three proteins,50.69%,32.32%and 28.36%respectively.As for proteins of different isoelectric points--trypsin,elastase,chymotrypsin,and α-lactalbumin,myoglobin,lysozyme,there were no correlation of binding rates and isoelectric points.For AArg-OMe@NPs of different monomer ratios,compared to 10%AArg-OMe@NPs,20%AArg-OMe@NPs showed no significant change of binding rate due to the stereo-hindrance effect of protein and side chains of NPs.In addition,poor temperature sensitivity of 40%AArg-OMe@NPs resulted in decrease of the binding rate.3.4 NPs-biomarker polypeptide experiments showed that the binding rate of polar ASer-OMe@NPs with proGRP(40.66%)was relatively high due to the strong polarity of peptide chain,while no obvious selectivity was seen of NPs with NT-proBNP due to the untypical peptide properties.4 ConclusionNPs with different side chains showed different binding ability to proteins and biomarker polypeptides.For proteins,the interaction was related to the temperature sensitivity,the side chain properties of amino acid methyl ester monomers and the molecular weights of the proteins.For biomarker polypeptides,the interaction may be related to the proportion of different amino acids in the sequence and the properties of the peptide chain regions.These results provided a preliminary basis for the application of NPs as abiotic antibody for proteins or biomarker peptides recognition. |
