Construction Of Chiral Y₁ Ligand Nanoassemblies For Glioma Imaging And Therapy

Glioma accounts for about 80%of malignant brain tumours,with a 5-year survival rate of less than 5%,and is characterised by high morbidity,high mortality and low cure rate.Since most gliomas are infiltrative in nature and poorly demarcated from surrounding normal tissues,the difficulty in determining tumour boundaries and identifying micro-infiltrating foci during surgery is a major challenge in the clinical management of gliomas.Drug therapy for glioma remains challenging because the blood-brain barrier restricts the entry of drugs into brain tissue.As gliomas are difficult to cross the barrier and highly heterogeneous,there is an urgent need to find more new targets and develop highly specific probes to achieve precise diagnosis and treatment of gliomas.In this thesis,we take the neuropeptide Y₁ receptor,a new target of glioma,as the starting point,and design and prepare chiral Y₁ ligand peptide nano-self-assemblies to improve their in vivo circulation stability,tumour targeting and tissue penetration depth,and carry out imaging and therapeutic evaluation of glioma from nano-self-assemblies to cellular and animal levels.The research aims to achieve a breakthrough in the application of chiral Y₁ ligand-controlled nano-self-assemblies in the precise diagnosis and treatment of glioma.The main research elements are summarised as follows.First,to address the problem that peptide ligands are susceptible to degradation by multiple proteases in vivo,resulting in poor circulating stability and low concentration of peptides reaching the glioma site,a chiral amino acid mutation strategy was designed for glioma overexpression of neuropeptide Y（NPY）Y₁ receptor based on the blood-brain barrier and glioma co-expression biomarkers to improve The chiral Y₁ ligand ^D/L[Asn28,Pro30,Trp32]-^DNPY（25-36）(referred to as ^D/LAPT)was designed to improve ligase stability;based on molecular docking and molecular dynamics calculations,it was found that the number of hydrogen bonds interacting with the Y₁receptor was 2.5-fold higher for ^DAPT compared to the L-type amino acid ligand（^LAPT）;The chiral Y₁ ligand linear peptide nanoassemblies were constructed using the chiral Y₁ ligand(^D/LAPT),thereby demonstrating that the chiral Y₁ ligand linear peptide nanoassemblies constructed from ^DAPT crossed the blood-brain barrier and specifically targeted gliomas significantly better than ^LAPT in an in vitro blood-brain barrier model and a mouse model of U87-MG glioma in situ.The chiral Y₁ ligand linear peptide nano-assemblies constructed from ^DAPT were able to achieve photothermal therapy and photoacoustic imaging of gliomas.This study demonstrates the importance of the stability and affinity of the chiral Y₁ ligand linear peptide nanoassemblies in the Y₁receptor-mediated transcytosis process across the blood-brain barrier.Secondly,peptide nano-assemblies constructed in vitro are prone to structural collapse in the in vivo blood circulation,and therefore the concentration of peptides reaching the glioma site is low,making it more difficult to achieve targeted imaging of specific tumour subcellular organelles.The formation of nanostructures in glioma subcellular organelles through in situ self-assembly can achieve deep tumour penetration and effective enrichment of peptide ligands,further improving the efficiency of diagnostic and therapeutic drugs in tumour imaging and treatment,but how to achieve controlled in situ self-assembly in glioma subcellular organelles is also a key problem to be solved.To address these key issues,this thesis designed chiral Y₁ligand branched peptide nano-self-assemblies coupled to the near-infrared two-region fluorescent molecule IR1048(^D/LNPY（15）-ENTK-IR1048)based on theα-helical structure of the NPY（25-36）fragment,and evaluated by in vitro cellular and in vivo animal levels,^DNPY（15）-ENTK-IR1048 was found to be effective in The nanofibrous structure formed by controlled in situ self-assembly of ^DNPY（15）-ENTK-IR1048induced by mitochondrial overexpression of intestinal kinase（ENTK）in glioma cells allowed the fluorescence emission wavelength to be red-shifted from 1048 nm to 1300nm,enhancing the tissue penetration depth and effective imaging time（retained for at least 7 days）for optical imaging of glioma.This study enables in situ self-assembly of chiral Y1 ligand branched peptide nanofibers for enhanced and highly specific imaging of glioma subcellular organelles.Finally,the introduction of enzyme-recognised peptide fragments would inevitably be hydrolysed by hydrolases,forming nanostructures in a non-essential time and space,as well as affecting the biological activity and secondary structure of the chiral peptides.Based on the[Asn⁶,Pro³⁴]-NPY（AP-NPY）Y₁ ligand peptide as anα-helical peptide,the construction of the Y₁ ligand as a leucine zip(^D/LLAP-NPY（36）)can induce the stacking of peptide chains to form an ordered assembly structure.The NAD+-dependent deacetylase family protein SIRT5 expressed in the mitochondria of glioma cells was used as a target,and ^D/LLAP-NPY（36）was chemically modified by succinate(SUC^D/LLAP-NPY（36）),and the succinate group was removed after encountering SIRT5,thus achieving in situ mitochondrial self-assembly while avoiding the introduction of self-assembly precursors into the enzyme recognition fragments to accelerate hydrolysis in vivo.Simulations revealed that ^DLAP-NPY（36）formed two more hydrogen bonds than the ^LLAP-NPY（36）dimer,resulting in a better self-assembly capacity.The self-assembly concentration of ^DLAP-NPY（36）was 14.5 times lower than that required for ^LLAP-NPY（36）,as measured by the self-assembly threshold concentration,and the formation of in situ nanofibres affected and generated mitochondrial ROS correspondingly weakening mitochondrial activity,thus enhancing the photothermal therapeutic effect of IR1048 on gliomas.