The Preparation Of Pseudo-biospecific Immunoadsorbent Using PAMAM As Spacer-arm By Click Chemistry And Study Of Its Performance

In this work, using amino acids as ligands and polyamidoamine (PAMAM) dendrimersas spacer-arms, a novel immunoadsorbent possessing pseudo-biospecific affinity forimmunoglobulin (IgG), which is cheap, safe and reliable and can have an adsorptionperformance comparable to protein A immunoadsorbent, was designed and prepared via clickchemistry. The IgG adsorption performance of the immunoadsorbents with different spacer-arms from human plasma were systematically investigated and the effects of the length,structures and rigidity/flexibility of the spacer-arms on their adsorption performance werealso studied, thus, exploring a novel route to preparing pseudo-biospecific non-proteinousimmunoadsorbents by a simple, efficient and controllable method.As biomimetic or pseudo-biospecific ligands, small molecular amino acids with low cost,high chemical and physical stability, good safety and modifiability are promising alternativesto protein A ligand for preparing immunoadsorbents, which can help to overcome the defectsof proteinaceous immunoadsorbents in the cost and potential safety hazards. Click chemistry—the Huisgen1,3-dipolar cycloaddition reaction used to achieve the coupling between theligand and the activated support can greatly enhance the reaction selectivity, therebyincreasing the density and stability of the ligands immobilized onto the support. Besides, clickreaction can be conducted under mild conditions and it is inert to most chemicalfunctionalities and stable to wide ranges of solvent, temperature and pH. Consequently, itgenerates little or no by-products and the integrity and activity of the functional group of theligand immobilized onto the support via the click reaction can be maintained as far as possible,so keeping the affinity of the ligand. On the other hand, choosing the PAMAM dendrimerswith a great number of reactive groups on the peripheral ends as spacer-arms will make itpossible to bond much more ligands by use of these functional groups than the linear spacer-arms with one reactive end groups, thus leading to an increase in the ligand density and animprovement in the adsorption performance of the designed and prepared immunoadsorbent.Based on the above-mentioned research background and ideas, the main research workincludes the following aspects:(1) A group of immunoadsorbents, Sep-AA, were prepared by a conventional method usingsepharose (Sep) as a support and different amimo acids (AA) as pseudo-biospecificligands, respectively. The ligand density of the prepared immunoadsorbents wasdetermined, and the adsorption performance of them and the protein A immunoadsorbent Sep-PA prepared by the same method was compared and analyzed through an adsorptionexperiment of IgG from human plasma.(2) According to the principle of Huisgen1,3-dipolar cycloaddition reaction, sepharose as asupport and amino acids as ligands were transformed into the―clickable‖reactive supportor azidated sepharose (Sep-N₃) and the―clickable‖alkyne-containing ligands via suitablechemical modifications, respectively; and then each of the―clickable‖ligands wascoupled with the―clickable‖reactive support to prepare pseudo-biospecific non-proteinous immunoadsorbents Sep-triazole-AA. The ligand density and IgG adsorptionperformance of Sep-triazole-AA were characterized and compared with those of theimmunoadsorbents Sep-AA prepared by the conventional method. The influence of clickchemistry on the structure and the properties of the immunoadsorbents was also discussed.(3) A series of PAMAM dendrimers with terminal alkyne from generation0.5(G0.5) togeneration4.0(G4.0) was synthesized by the divergent strategy using2-propynylamineas the core; and the structure of the products was characterized and analyzed, therebylaying the foundation for using the PAMAM dendrimers as the spacer-arms ofimmunoadsorbents.(4) The―clickable‖ligands combined with alkyne-containing dendritic spacer-arms wereprepared by the reaction between the functional groups on the peripheral ends of thePAMAM dendrimers and an amino acid ligand, and coupled with the―clickable‖reactivesupport Sep-N₃to prepare the immunoadsorbents Sep-PAMAM-AA bearing dendriticspacer-arms. Their structure, ligand density and adsorption performance werecharacterized and compared with those of the corresponding immunoadsorbents Sep-triazole-AA whose spacer-arm is a linear molecular chain. The relationship between thestructure of the PAMAM dendritic spacer-arms and the IgG adsorption performance of theimmunoadsorbents Sep-PAMAM-AA was also studied.The main conclusions drawn from this study are as follows:(1) In the conventional preparation of immunoadsorbents Sep-AA, the content of activegroups in the sepharose support will decrease gradually as a series of reactions, includingthe epoxidization of Sep and the amination, hydroformylation and coupling reaction ofmodified Sep, are conducted, thus resulting in low ligand density of the products Sep-His,Sep-Phe and Sep-Trp, whose ligands are L-histidine (His), L-phenylalanine (Phe) and L-tryptophan (Trp), respectively. The IgG adsorption capacity of Sep-His, Sep-Phe and Sep-Trp was measured to be2.88,2.62and2.53mg/g, respectively, which are much lower than that of the protein A immunoadsorbent Sep-PA, or22.97mg/g. All of the preparedimmunoadsorbents Sep-AA, Sep-His, Sep-Phe and Sep-Trp, can exhibit high adsorptionselectivity for IgG from human plasma and almost have no non-specific adsorption. Itproves the feasibility of using amino acids as the pseudo-biospecific ligand ofimmunoadsorbents.(2) The immunoadsorbents Sep-triazole-His, Sep-triazole-Phe and Sep-triazole-Trp weresuccessfully prepared by click chemistry using three kinds of amino acids His, Phe andTrp as the ligand, respectively. They are obviously superior in the ligand density and theadsorption capacity to the corresponding immunoadsorbents Sep-AA prepared by aconventional method. Among the three immunoadsorbents, Sep-triazole-His shows thehighest IgG adsorption capacity, which is as high as16.49mg/g, but still lower than thatof Sep-PA. The further studies prove that Sep-triazole-His can exhibit high adsorptionselectivity for IgG comparable to Sep-PA and that the1,2,3-triazole ring in its spacer-arm, which is produced via the click reaction between Sep-N₃and L-histidine withterminal alkyne, can facilitate the binding of IgG without non-specific adsorption.(3) Generation1.0⁴.0(G1.0⁴.0) PAMAM dendrimers with terminal alkyne and multipleamino groups on the peripheral ends were successfully designed and synthesized; and thenthe immunoadsorbents with different dendritic spacer-arms and different amino acids asligands, Sep-PAMAM-AA, were prepared via click chemistry. The structure analyses ofthe products show that among the four spacer-arms, G1.0⁴.0PAMAM dendrimers, G3.0PAMAM can covalently combine with the most number of ligands and achieve thehighest bonding efficiency; and that the click reaction between the dendritic PAMAMmodified amino acids, PAMAM-AA, and the activated support Sep-N₃possesses veryhigh selectivity and the reaction efficiency is almost close to100%. The preparedimmunoadsorbents Sep-PAMAM-AA all possess high adsorption capacity of IgG fromhuman plasma, and most of them can exhibit an IgG adsorption capacity comparable tothat of Sep-PA. By comparison, the immunoadsorbent Sep-G3-His, whose spacer-arm isG3.0PAMAM, possesses an excellent adsorption selectivity for IgG and the adsorptioncapacity up to28.43mg/g, so it is an ideal immunoadsorbent.In a word, a simple, efficient and controllable method has been explored in this study todesign and prepare safe, effective and inexpensive immunoadsorbents. This method can notonly overcome the inherent defects of conventional methods, but also improve theimmobilization efficiency and adsorption performance of the immunoadsorbents. Theobtained research results lay a solid theoretical and experimental foundation for the design and preparation of novel immunoadsorbents and their applications in the fields of IgGseparation and blood purification, which will be expected to generate great social andeconomic benefits.