Immunoassay And Application Of Nano-drug Carrier PSI_OAm Loaded With Anticancer Drugs Or Pesticides

At present,many anticancer drugs and traditional pesticides were hydrophobic,which leaded to its low utilization and poor targeting,they had limited the development of cancer treatment and agriculture.Nano drug carriers could effectively solve these problems because to their high biocompatibility,high drug loading rate,and controlling drug release.As a new nano drug delivery system,nano drug carriers played a decisive role in cancer treatment and pesticide delivery.Therefore,based on the advantages of the nano drug carrier PSI_OAm,it was very important to establish a highly sensitive analysis scheme and multi-functional application for anticancer drugs or pesticides-loaded.In this paper,polymer nano drug carrier oleylamine-functionalized polysuccinimide(PSI_OAm)was used as a drug loading system.We first prepared polymer nano drug carrier PSI_OAm and its holoantigen,and obtained high titer anti-PSI_OAm antibody of our laboratory.Based on this,the drug carrier PSI_OAm not only could load anticancer drugs,but also pesticides.Based on the high specificity and selectivity of antigen-antibodies,fluorescence immunoassay and multifunctional application with performance research on nano drug carrier-loaded anticancer drugs or pesticides was established.This provided a broader application value for the quantitative detection and performance research of nano drug carrier-loaded drugs.The specific work was as follows:1.Preparation and characterization of drug carrier PSI_OAm and its antigen and antibodyFirstly,the nano drug carrier carrier PSI_OAm hapten and whole antigen were successfully prepared,and high titer anti-PSI_OAmAm antibodies were obtained.The PSI_OAmAm hapten and whole antigen were characterized,and the PSI_OAm antiserum titer was determined.Good titer antibodies meet the subsequent immunoassay determination,which provided an important basis for the nano drug carrier PSI_OAmAm loaded with the anticancer drug camptothecin and the pesticide avermectin.2.A fantastic antibody which can react with drug-loaded nano carriers antigen and its application forsensitivedetection of camptothecine-loaded nano drug carriers by fluorescence immunoassayOne of nature of Ag/Ab reactions is the ability of a population of antibody molecules to react with only one antigen.Here,we found a fantastic antibody which not only can combine with free-drug nano carriers,but also react with drug-loaded nano carriers antigens,thus achieving a breakthrough from�0 to 1�.A pH-responsive nano drug carrier PSI_OAm was prepared as an immunogen to obtain high titer anti-PSI_OAm antibodies.More interesting,when anticancer drug camptothecine?CPT?was loaded into PSI_OAm(termed CPT@PSI_OAm),not only can anti-PSI_OAm antibodies combine highly specificity with PSI_OAm,but also capture CPT@PSI_OAm in immune reaction.The anti-PSI_OAmAm antibodies became a bridge from free drug to drug-loaded systems,we established a direct competition fluorescence immunoassay for CPT@PSI_OAm,fluorescein isothiocyanate?FITC?was applied to label antibodies as a fluorescent marker.Under the optimal conditions,the fluorescent immunoassay exhibited a wide linear range of0.12?1.6�10³ng/mL?R²=0.991?for CPT@PSI_OAm with a low detection limit of0.04ng/m L.The recoveries ranged from 91%to 104%demonstrated that insertion of the fantastic antibodies provided a feasible approach to detect drug-loaded nano carriers.3.Fluorescence resonance energy transfer?FRET?fluorescent probe for highly sensitive and quantitative detection of camptothecin-loaded nano drug carrierIn order to further improve the sensitivity of the fluorescent immunoassay method,in this work,a piperazine-RhB FRET fluorescence immunoassay scheme was established for high sensitivity quantitative detection of CPT@PSI_OAm.A non-toxic rigid piperazine fluorescent dye was used as a donor for FRET,and Rhodamine B?RhB?was used as a fluorescent acceptor.Two substances could produce resonance energy transfer,which increase the excitation range of RhB.Optimizing the conditions of piperazine-RhB FRET fluorescent probe which effectively improved the fluorescence intensity of the probe.Finally,a high sensitivity detection of CPT@PSI_OAm by piperazine-RhB FRET fluorescence immunoassay was established,and the conditions of the immunoassay wereoptimized.The optimal antigen concentration was 25?g/m L,the antibody concentration was 30?g/m L,and the optimal reaction time was 2h.The blocking agent concentrationwas 1.0%,and the PBS buffer solution was 0.005mol/L,pH 7.0.Under the above optimal conditions,a competitive standard curve was established with a linear equation F=23066-3689.7logc?R²=0.993?,the linear range was 0.027-698.1ng/m L,and the detection limit(IC₁₀)was 5pg/m L.The detection sensitivity(IC₅₀)was 4.39pg/m L and the recovery was 96.3-107%.The results showed that the piperazine-RhB FRET fluorescence immunoassay achieved highly sensitive detection of CPT@PSI_OAm,and had a lower detection limit?5.0pg/mL?compared with FITC immunoassay.This method effectively overcame the disadvantages of FITC photodegradability,low fluorescence intensity,and narrow emission wavelength range.4.Preparation of avermectin-loaded nano drug carrier PSI_OAm through flash nanoprecipitation:controllable size,uniform size,and leaves adsorptionConventional pesticides caused serious pollution to the environment and food due to low utilization and weak affinity for plant leaves.However,because of the complexity of the nanomaterial preparation process,the preparation of nanomaterials still faced many problems in terms of morphology,size uniformity,and controllability of particles.Especially for nano-pesticides with specific functions,its morphology,particle size and size uniformity played a very important role in its adsorption on the leaves surface and the transport of pesticides.In this article,in order to solve the above problem,we selected green and biodegradable polysuccinimide,functionalized with oleylamine(PSI_OAm),which as a pesticide delivery system,uniform avermectin?AVM?-loaded PSI_OAm(AVM@PSI_OAm)nanoparticles were prepared through flash nanoprecipitation.The results showed that the particle size of AVM@PSI_OAm nanoparticles was 70-150 nm under optimal conditions.The obtained nanoparticles had the following advantages,such as rapid preparation,uniform size,controlling pesticide release,great stability and high drug encapsulation efficiency?66.4%?.AVM@PSI_OAm exhibited excellent adsorption performance compared to commercially available avermectin emulsion on corn leaves,and highly depended on functional groups of nanoparticles.As a scalable nano pesticide formulation,AVM@PSI_OAm showed attractive application prospects in future agriculture.