Single Domain Antibody Modified Macromolecule PEN Delivery Nucleic Acid For The Treatment Of CD66C Positive Tumors

The latest data show that cancer has become the biggest threat to the health of residents today,and its morbidity and mortality are higher than other diseases.Since the pathogenesis of cancer is very complex and extremely easy to transfer and diffuse,the exploration of cancer treatment methods has been continuing.At this stage,clinical treatment of tumors is still a relatively traditional means,including surgery,chemotherapy,radiotherapy and so on.Although these methods can significantly prolong the lifespan of patients in tumor therapy,these methods do not accurately and effectively target tumor cells.In other words,in addition to killing tumor cells during the treatment process,it also kills a large number of normal cells beneficial to the human body,and causes side effects such as damage to various organs and immune system.Attention is drawn to the fact that with the gradual understanding of the pathogenesis of tumor,the delivery system of targeted nano gene vector has become a hot topic in tumor therapy.In this paper,a single domain antibody capable of specifically recognizing carcinoembryonic antigen 6(CEACAM6 or CD66C)was extracted and modified to obtain a nanocarrier with targeted recognition of tumor cells.The vector can bind to the CEACAM6 receptor on the surface of the tumor cell and interact with it into the tumor cell,thereby achieving efficient delivery of the tumor drug and also improving the biological stability of the drug.Micro RNA(miRNA)is an endogenous single strand nucleic acid with multiple regulatory functions.At present,micro-34a(miR-34a)is considered to be a more thorough study of tumor growth inhibition factors,but it is severely downregulated in tumor cells.To explore the targeting of this nanocarrier and its delivery to miR-34 a,we designed a targeting validation experiment.The results show that the vector can only target CD66 C positive tumor cells MCF-7.The results of Western blotting showed that miR-34 a can target and efficiently enter MCF-7 cells under the support of this vector,and express theexpression of apoptosis-related proteins Bcl-2,Caspase-3 and Caspase-9.The amount changes to activate the intracellular mitochondrial pathway leading to apoptosis.In addition,we constructed a xenograft tumor lung cancer model with BALA/c-nu nude mice,and delivered the Cy3-labeled miR-34 a with the targeted nanocarrier.The results indicate that the nano delivery system has good targeting at the animal level.In order to further explore the effect of the nanocarriers on the delivery of other gene drugs,we allowed a plasmid stably expressing Pseudomonas exotoxin(PE38KDEL)to form a complex with electrostatic interaction and perform a particle size potential analysis.It was confirmed by gel electrophoresis experiments that the PE38 KDEL plasmid was able to be completely loaded by the vector at a mass ratio of 1.5:1 and was not degraded by serum.By flow cytometry,it was found that transfection of PE38 KDEL induced MCF-7 cells to undergo obvious apoptosis;and the cell cycle was arrested in G1 phase.From the results of immunoblotting,the expression of Bcl-2 in tumor cells transfected with PE38 KDEL decreased,and the expression levels of Caspase 3 and Notch-1 also changed accordingly.At the same time,the results of scratch test showed that the migration ability of cells in PE38 KDEL transfection group was significantly lower than that in other control groups.The results of Transwell experiments showed that the infiltration ability of tumor cells in the medicated group was significantly reduced.In conclusion,the delivery system obtained by the single-domain antibody modified high-molecular capable of specifically recognizing the CD66 C can be combined with the receptor of the surface of the positive tumor cell,and the delivery of the miR-34 a and the PE38 nucleic acid can be realized through the interaction between the ligand and the receptor,and further the action of the nucleic acid is exerted,and the proliferation and the migration of the tumor are inhibited.This study lays a foundation for the construction of targeted nanocarriers and the targeted therapy of tumor,and has great theoretical and clinical research value.