Glioma is a highly malignant primary brain tumor, with glioblastoma (GBM) exhibiting the highest incidence and a recurrence rate near 100%, imposing severe economic and clinical burdens. Its clinical treatment is severely limited by poor blood-brain barrier (BBB) permeability and high multidrug resistance. To address these challenges, peptide-drug conjugates (PDCs) provide a promising strategy by pairing small-molecule drugs with targeting or cell-penetrating peptides to achieve precise delivery. In this study, a novel Nestin-targeting peptide (NTP) was designed by combining a GSCT peptide (AQYLNPS) that specifically binds to Nestin—a marker highly expressed in glioma stem cells—with a matrix metalloproteinase (MMP)-2/9 sensitive cleavage linker (PVGLIG) and a lysine residue providing a covalent conjugation site via its side-chain ε-amino group. Temozolomide (TMZ), a frontline chemotherapeutic payload prone to systemic toxicity, was conjugated to NTP and its alanine-scanning mutants (NTP-9 to NTP-14) to systematically elucidate the key functional hotspots and structure-activity relationships of the sequence. The synthesized PDC-5 series compounds were thoroughly evaluated across U87, U251, and LN229 glioma cell lines for cellular uptake, in vitro cytotoxicity, and BBB permeability. Western blot analysis revealed that the cellular uptake efficiency of the fluorescently labeled PDCs strongly correlated with the gradient of Nestin protein expression levels across the cell lines (U251 > LN229 > U87), confirming excellent targeting specificity. In vitro cytotoxicity assays via CCK-8 demonstrated distinct activity profiles among the mutants; notably, substituting asparagine (Asn) with alanine in the sequence showed potent inhibition of U87 glioma cells with an IC50 value of approximately 24 μM. Furthermore, MTT assays confirmed that the PDC-5 series exerted no obvious toxic effects against bEnd.3 brain microvascular endothelial cells. Transwell permeability assays showed that the prototype NTP-TMZ possessed the highest BBB penetration capability. Alanine substitutions at the N-terminal side of the Asn residue caused minor changes, whereas substituting Asn itself or its C-terminal adjacent residues significantly decreased permeability. Crucially, replacing Asn with alanine reduced the apparent permeability coefficient to less than half of the prototype, identifying Asn as a critical hotspot for maintaining BBB crossing functionality. Although in vitro antitumor efficacy faced limitations due to potential tertiary structure shielding or absence of environmental MMP enzymes for linker cleavage, these findings establish a valuable foundation for future structural optimizations, including orthotopic glioma model evaluations, offering a novel strategy for targeted glioma therapy.

Key words: Glioma, peptide-drug conjugate, temozolomide, targeting ability, in vitro activity, blood-brain barrier