目的：骨肉瘤的治疗因化疗药物的非特异性靶向性和膜渗透能力有限而面临重大挑战。抗癌肽作为一种极具潜力的新兴治疗手段，展现出广阔的应用前景。然而，绝大多数抗癌肽存在易被蛋白酶降解等缺陷，且目前针对恶性程度高、侵袭性强的骨肉瘤仍缺乏高靶向性的抗癌肽。本研究旨在筛选具有高选择性杀伤骨肉瘤能力的多肽，并通过多肽改造克服其缺陷，增强其治疗效能。

方法：（1）基于CCK-8生物相容性实验，评估抗癌肽池、自组装多肽池及融合多肽池中各多肽对骨肉瘤细胞的选择性杀伤能力，逐步筛选出高靶向性杀伤骨肉瘤多肽。（2）通过细胞形态学评估、亚细胞定位分析、流式细胞术、细胞膜脂质组学分析、多肽与脂质体亲和实验及钙黄绿素渗漏实验，揭示抗癌肽对骨肉瘤细胞的靶向杀伤机制。（3）结合圆二色光谱和AlphaFold3预测多肽结构，利用CHARMM-GUI构建骨肉瘤和正常细胞膜模型，并通过分子动力学模拟分析多肽与细胞膜的结合能与多肽跨膜过程的自由能垒、渗透系数及跨膜时间。（4）构建自组装抗癌多肽载药凝胶，分析其倒置及挤出状态、内部网络形貌、圆二色光谱、流变行为、凝胶降解及药物释放速率。（5）通过细胞实验检测多肽对药物摄取的影响，并评估凝胶药物处理后的CCK-8细胞存活率、qRT-PCR基因表达及流式细胞周期分析。（6）通过动物实验检测活体荧光药物释放、骨肉瘤抑制效果、体重变化、H&E染色、免疫组织化学染色、TUNEL荧光染色、血常规及血生化指标。

结果：（1）通过细胞毒性逐层筛选框架，鉴定出具有骨肉瘤选择性杀伤效果的阳离子治疗肽RT2，并从自组装多肽库中筛选出生物相容性良好的自组装多肽E16和R16。将RT2分别与E16/R16融合，构建出多种新型融合多肽，最终筛选出对骨肉瘤细胞具有最佳选择性杀伤效果的融合多肽E16-RT2（ER），其选择性杀伤指数为RT2的175%。（2）多肽表征结果表明，RT2和ER均能选择性扰动骨肉瘤的阴离子磷脂膜，增强细胞膜通透性，诱导细胞裂解死亡。（3）分子模拟结果显示，骨肉瘤细胞膜的高负电性可显著增强对ER的亲和力。另外，与RT2相比，ER对正常细胞膜的扰动更弱，且在骨肉瘤细胞膜上可形成更大的扰动孔隙。（4）基于E16序列的自组装特性，通过生理离子环境触发形成自组装物理交联水凝胶，并负载靶向细胞核的阿霉素（DOX），构建出新型可注射骨肉瘤治疗材料E16+ER+DOX。该材料内部具有网络结构，可实现药物的梯度缓释。（5）细胞实验表明，ER介导的肿瘤细胞膜扰动显著促进DOX进入细胞，发挥ER与DOX的协同杀伤骨肉瘤细胞效应。ER显著增强DOX对促凋亡基因（Caspase-3、Caspase-9和p53）的上调和抗凋亡基因Bcl2的下调作用。此外，ER还放大了DOX诱导的细胞周期阻滞作用。（6）动物实验表明，E16+ER+DOX材料在局部注射后能够实现ER和DOX的长效缓释，显著抑制骨肉瘤生长。肿瘤组织切片的H&E、免疫组织化学和TUNEL荧光染色结果显示，E16+ER+DOX组肿瘤组织中坏死和凋亡最为广泛，进一步验证了ER与DOX的协同抗肿瘤作用。器官病理学评估和血液分析结果证实，该凝胶治疗材料无器官和循环系统毒性。

结论：本研究为骨肉瘤治疗提供了一种基于多肽的新型治疗策略。通过梯度缓释药物和细胞膜-细胞核共靶向机制，展现出显著的抗骨肉瘤疗效，且具有良好的生物安全性，为未来肿瘤精准治疗领域的发展提供了重要的理论支撑和实践方向。

Objective: Osteosarcoma treatment is significantly hindered by the non-specific targeting and limited membrane permeability of conventional chemotherapeutic agents. Anticancer peptides (ACPs) have emerged as a promising therapeutic alternative due to their unique mechanisms of action. However, most ACPs are prone to proteolytic degradation and lack high selectivity for highly malignant and aggressive osteosarcoma. This study aimed to identify peptide with high selective cytotoxicity against osteosarcoma and enhance its therapeutic efficacy through peptide engineering.

Methods: (1) CCK-8 biocompatibility assays were employed to evaluate the selective cytotoxicity of peptides from anticancer, self-assembling, and fusion peptide pools against osteosarcoma and normal cells, progressively identifying highly tumor-targeted peptides. (2) Cell morphology assessment, subcellular localization analysis, flow cytometry, lipidomics, peptide-liposome affinity assays, and calcein leakage experiments were utilized to elucidate the targeted killing mechanism of ACPs. (3) Peptide structures were predicted using circular dichroism spectroscopy and AlphaFold3, and osteosarcoma and normal cell membrane models were constructed with CHARMM-GUI. Subsequent molecular dynamics simulations quantified peptide-membrane binding energies, transmembrane free energy barriers, permeability coefficients, and transmembrane times. (4) Self-assembling anticancer peptide-loaded hydrogel was developed, and its inversion/extrusion states, internal network morphology, circular dichroism spectra, rheological behavior, degradation rates, and drug release profiles were characterized. (5) Peptide-mediated drug uptake was assessed, and post-gel treatment outcomes were evaluated using CCK8 assays, qRT-PCR, flow cytometry cell cycle analysis. (6) Animal experiments were conducted to monitor in vivo fluorescent drug release, osteosarcoma suppression, body weight changes, H&E staining, immunohistochemistry staining, TUNEL staining, and blood analysis.

Results: (1) A hierarchical cytotoxicity screening framework identified the cationic therapeutic peptide RT2, which selectively targets osteosarcoma cells. From a self-assembling peptide pool, the biocompatible peptides E16 and R16 were selected. Fusion of RT2 with E16/R16 generated novel hybrid peptides, among which E16-RT2 (ER) exhibited the highest selective cytotoxicity against osteosarcoma, with a selective cytotoxicity index 175% greater than RT2. (2) Peptide characterization revealed that both RT2 and ER selectively disrupt anionic phospholipid membranes in osteosarcoma, enhancing membrane permeability and inducing lytic cell death. (3) Molecular simulations revealed that the highly negative charge of osteosarcoma membranes significantly enhances their affinity for ER. Moreover, compared to RT2, ER induces weaker perturbation of normal cell membranes, while forming larger transmembrane pores in osteosarcoma membranes. (4) Physiological ionic conditions trigger the E16 peptide sequence to self-assemble into physically crosslinked hydrogel. Encapsulation of nuclear-localized doxorubicin (DOX) generates an advanced injectable therapeutic material (E16+ER+DOX), whose nanofibrillar network structure enables spatiotemporal regulation of drug release kinetics for osteosarcoma treatment. (5) In vitro studies demonstrated that ER-mediated perturbation of tumor cell membranes significantly enhanced DOX cellular uptake, resulting in synergistic antitumor effects against osteosarcoma cells. ER markedly potentiated DOX-induced upregulation of pro-apoptotic genes (Caspase-3, Caspase-9, and p53) while downregulating the anti-apoptotic gene Bcl2. Furthermore, ER amplified DOX-triggered cell cycle arrest. (6) In vivo studies demonstrated that locally administered E16+ER+DOX enabled sustained release of both ER and DOX, resulting in significant suppression of osteosarcoma progression. Histopathological analysis of tumor sections revealed extensive necrosis and apoptosis in the E16+ER+DOX group, as evidenced by H&E staining, immunohistochemistry, and TUNEL assays, confirming the synergistic antitumor effect. Comprehensive toxicological evaluation through organ histopathology and hematological analysis confirmed the absence of systemic or organ-specific toxicity.

Conclusion: This study presents a novel peptide-based therapeutic strategy for osteosarcoma, combining gradient drug release with membrane-nucleus co-targeting mechanisms. The approach demonstrates significant anti-osteosarcoma efficacy and excellent biocompatibility, offering a robust foundation for the development of future tumor-targeted therapies and providing critical theoretical and practical insights for precision medicine in oncology.