Self-assembled Nanowires Bispecific Aptamers For Enhanced Immune Cell-Cancer Cell Interactions

Background&ObjectiveCancer has become a global public health problem and a serious threat to human life and health.Traditional cancer treatment methods include surgery,radiotherapy and chemotherapy,but they are difficult to eliminate all cancer cells and are prone to recurrence,side effects and drug resistance.In recent years,cancer immunotherapies,represented by Adoptive T-cell therapy and immune checkpoint inhibitors,have brought hope to some patients with haematological cancers,but at the same time,the need for complex in vitro engineering,time-consuming and costly drawbacks have limited their use in the clinical practice.In addition,the response rate to immunotherapy is low due to the high heterogeneity of solid tumours or the lack of tumour-specific antigens.How to enhance the ability of immune cells to recognize and bind cancer cells is important to the success of cell-based immunotherapy.The development of aptamer-targeted immunotherapeutic strategies allows for high affinity,specific recognition of cancer cells,particularly bispecific aptamers that bind to cancer cells and immune T cells,and recruitment of immune cells to the tumour site to enhance the anti-tumour activity of the immune system.In addition,the success of immunotherapeutic strategies developed on the basis of DNA nanotechnology hybridization chain reactions has provided new ideas for immunotherapy.Overall,the establishment of a simple and effective aptamer and DNA nanotechnology-based cellular immunotherapy may be a promising approach to cancer treatment.Here,we designed aptamers targeting highly expressed PD-L1 cancer cells and LD201t1 aptamers targeting T-cell CD62L,and assembled them into nanowires bispecific aptamers using a hybridization chain reaction to recognize and adhere to PD-L1 in cancer cells,blocking immune checkpoints while recruiting and binding more immune T cells,thereby enhancing immune cell-cancer cell interactions.Methods1.Construction of nanowires bispecific aptamers self-assembly system.1.1 Design of the aptamers Apt-PD-L1 and Apt-LD201t1 and the hairpin nucleic acid sequences H1 and H2 in the hybridization chain reaction,and free energy calculations and stability assessment of their structures via Oligo Analyzer as well as the NUPACK website,respectively.1.2 The results of the reactions of the NWBi-Apts self-assembly system were analysed using PAGE electrophoresis to verify the successful assembly and triggering of the Bi-apts and NWBi-Apts self-assembly reactions.1.3 The stability of individual aptamers and NWBi-Apts after incubation with 10%FBS was analysed using PAGE electrophoresis.2.NWBi-Apts for enhanced immune-cell-cancer cell interactions.2.1 The cellular targeting ability of Apt-LD201t1 and Apt-PD-L1 was investigated by confocal microscopy and flow cytometry to verify the feasibility of aptamer binding to target cells.2.2 Design of Apt-PD-L1-FAM assemblies with fluorescent moieties NWBi-Apts were used to bind different cell lines such as human breast cancer MDA-MB-231 cells,human cervical cancer cell line Hela cells and human bronchial epithelial line 16HBE cells to verify the specificity of NWBi-Apts binding to cancer cells.2.3 To verify the effects of different reaction buffers,reaction times and aptamer concentrations on the binding of NWBi-Apts to target cells and ultimately to select the optimal reaction conditions.2.4 Under optimal reaction conditions,fluorescence confocal microscopy and flow cytometry were used to explore whether NWBi-Apts could mediate the binding of MDA-MB-231 cells to Jurkat cells to produce cell-linked complexes and to assess their binding efficacy.2.5 The biocompatibility of NWBi-Apts was assessed using the CCK-8 Cell Proliferation Colorimetric Assay Kit.2.6 Fluorescence confocal microscopy was used to investigate the efficacy of NWBiApts in mediating the binding of MDA-MB-231 cells to mouse spleen cells at different E/T(1:1,3:1).Results1.A theoretical analysis followed by nucleic acid electrophoresis has led to the conclusion that self-assembled NWBi-Apts have a stable secondary structure that remains stable in a 10%FBS biological environment for 24 h without degradation by nucleases.2.the designed aptamers can target and bind MDA-MB-231 cells and Jurkat cells;and the constructed NWBi-Apts can specifically target cancer cells that are high in PD-L1 expression;Optimal reaction conditions that mediate the formation of more cell-linked complexes between MDA-MB-231 cells and Jurkat cells;It has good biocompatibility and mediates the formation of higher order cell clusters between MDA-MB-231 cells and mouse splenocytes at an E/T of 3:1,resulting in higher binding efficiency.ConclusionIn this work,we have established an aptamer-based and hybridization chain reaction self-assembly technique to construct multivalent NWBi-Apts that are stable in the biological environment,have good biocompatibility,can recognize and adhere to PD-L1 of cancer cells and block immune checkpoints while recruiting to bind more immune T cells.This technique is simple to use and may in the future allow for the recognition of different cancer cells by varying the aptamer,providing a new idea for the development of cell-based immunotherapy.