包载阿霉素的高渗透性纳米粒的制备与体外抗肿瘤研究

Preparation and in vitro anti-tumor study of highly penetrable nanoparticles loaded with doxorubicin

摘要

目的 研究高渗透性纳米递送系统协同阿霉素(doxorubicin,DOX)诱导免疫原性细胞死亡(immunogenic cell death, ICD)增强抗肿瘤疗效。方法 采用薄膜分散法制备包载 DOX 的 DOX-L 纳米粒,利用其在 pH 为中性环境下带负电荷的共聚物聚乙二醇-聚赖氨酸-2,3-二甲基马来酸酐(mPEG-PLL-DMA,PPA)包裹在 DOX-L 纳米粒外层,制得 DOX-PPA/L 纳米粒。考察粒径、Zeta 电位和包封率优化处方,且考察制剂的体外释放行为和 pH 敏感性。同时采用小鼠乳腺癌细胞 (4T1) 考察其体外抗肿瘤活性。结果 优化处方结果表明,(2,3-二油氧基丙基)三甲基氯化铵(N-[1-(2,3-dioleyloxy) propyl]-N,N,N-trimethylammonium chloride,DOTAP) 与 DOX 的质量比为 8∶1,PPA 与 DOTAP 质量比为 5∶1,粒径为 (155.39± 8.15)nm,Zeta 电位为( -13.34± 2.35)mV。体外实验结果表明,空白载体的生物安全性良好,pH 6.8 条件下 的 DOX-PPA/L 纳米粒的细胞摄取、细胞毒性和肿瘤穿透性明显高于 pH 7.4 条件下的制剂,验证了其 pH 敏感性及必要 性。另外,DOX 诱导 ICD 作用,联合纳米粒的 pH 敏感性,增强体外肿瘤免疫活性。结论 通过高渗透性纳米递送系统包 载 DOX,提高纳米粒的渗透性和肿瘤免疫治疗疗效,增强抗肿瘤作用。

Abstract

Objective To investigate the high-permeability nano-delivery system synergizes with doxorubicin (DOX) to induce immunogenic cell death (ICD) and enhance anti-tumor efficacy. Methods DOX-loaded L nanoparticles were prepared via the thin-film hydration method. The nanoparticles were further coated with the negatively charged copolymer mPEG-PLL-DMA (PPA,polyethylene glycol-polylysine-2,3-dimethylmaleic anhydride),which maintains negative charge under neutral pH conditions,to fabricate DOX-PPA/L nanoparticles. The formulation was optimized by investigating particle size,Zeta potential and encapsulation efficiency. In vitro drug release behavior and pH sensitivity of the formulation were evaluated. Additionally,the in vitro antitumor activity was investigated using murine breast cancer 4T1 cells. Results The formulation optimization results showed that the optimal mass ratio of N-[ 1-( 2,3-dioleyloxy) propyl]-N,N,N-trimethylammonium chloride (DOTAP) to doxorubicin ( DOX) was 8∶1,and the mass ratio of PPA to DOTAP was 5∶1. The prepared nanoparticles exhibited a particle size of (155.39± 8.15) nm and a Zeta potential of ( -13.34± 2.35) mV. In vitro cell experiments showed that the blank carrier had good biological safety,and the cell uptake,cytotoxicity,and tumor penetration of DOX-NPs at pH 6.8 were significantly higher than those of the formulation at pH 7.4,verifying the pH sensitivity of the formulation. In addition,DOX significantly induced immunogenic cell death (ICD) in vitro,combined with the pH sensitivity of nanoparticles,enhanced tumor immune activity,and improved anti-tumor efficacy. Conclusion Encapsulated doxorubicin ( DOX) within a high-permeability nano-delivery system can enhance the penetrability of nanoparticles,improve the efficacy of tumor immunotherapy,and further strengthen the anti-tumor effect.

关键词

免疫原性细胞死亡 / 肿瘤穿透性 / pH 敏感 / 阿霉素

Key words

immunogenic cell death / tumor penetration / pH sensitive / doxorubicin

参考文献

[1] OJHA T, PATHAK V, SHI Y, et al. Pharmacological and physical vessel modulation strategies to improve EPR-mediated drug targeting to tumors[J]. Adv Drug Deliver Rev, 2017, 119: 44-60.

[2] THEEK B, BAUES M, GREMSE F, et al. Histidine-rich glycoprotein-induced vascular normalization improves EPR-mediated drug targeting to and into tumors[J]. J Control Release, 2018, 282: 25-34.

[3] SHU M, TANG J J, CHEN L L, et al. Tumor microenvironment triple-responsive nanoparticles enable enhanced tumor penetration and synergistic chemo-photodynamic therapy[J]. Biomaterials, 2021, 268: 120574.

[4] HUANG Z Y, CHEN Y Y, ZHANG J L, et al. Laser/GSH-activatable oxaliplatin/phthalocyanine-based coordination polymer nanoparticles combining chemophotodynamic therapy to improve cancer immunotherapy[J]. Acs Appl Mater Inter, 2021, 13(33): 39934-39948.

[5] SOURI M, SOLTANI M, MORADI K, et al. Engineered strategies to enhance tumor penetration of drug-loaded nanoparticles[J]. J Control Release, 2022, 341: 227-246.

[6] XU W J, WANG J X, JIN L J, et al. A tumor acidity-driven transformable polymeric nanoassembly with deep tumor penetration and membrane-anchoring capability for targeted photodynamic therapy[J]. Biomaterials, 2021, 276: 121024.

[7] DAI L L, LI X, YAO M J, et al. Programmable prodrug micelle with size-shrinkage and charge-reversal for chemotherapy-improved IDO immunotherapy[J]. Biomaterials, 2020, 241: 119901.

[8] ZHAO L P, ZHENG R R, LIU L S, et al. Self-delivery oxidative stress amplifier for chemotherapy sensitized immunotherapy[J]. Biomaterials, 2021, 275: 120970.

[9] CHENG L L, ZHANG X G, TANG J J, et al. Gene-engineered exosomes-thermosensitive liposomes hybrid nanovesicles by the blockade of CD47 signal for combined photothermal therapy and cancer immunotherapy[J]. Biomaterials, 2021, 275: 120964.

[10] GUO J F, ZOU Y F, HUANG L. Nanodelivery of chemotherapeutic ICD inducers for tumor immunotherapy[J]. Small Methods, 2023, 7(5): e2201307.

[11] ZHANG F, CHEN F M, YANG C, et al. Coordination and redox dual-responsive mesoporous organosilica nanoparticles amplify immunogenic cell death for cancer chemoimmunotherapy[J]. Small, 2021, 17(26): e2100006.

[12] WANG L L, GUAN R L, XIE L N, et al. An ER-targeting iridium(III) complex that induces immunogenic cell death in non-small-cell lung cancer[J]. Angew Chem Int Edit, 2021, 60(9): 4657-4665.

[13] ALZEIBAK R, MISHCHENKO T, SHILYAGINA N, et al. Targeting immunogenic cancer cell death by photodynamic therapy: past, present and future[J]. J Immunother Cancer, 2021, 9(1): e001926.

[14] SHI M H, ZHANG J L, WANG Y, et al. Tumor-specific nitric oxide generator to amplify peroxynitrite based on highly penetrable nanoparticles for metastasis inhibition and enhanced cancer therapy[J]. Biomaterials, 2022, 283: 121448.

[15] JIA L, GAO Y, QIAO M X, et al. Glycyrrhetinic acid-long circulation adriamycin loaded liposomes: preparation and in vivo targeting study[J]. Journal of Shenyang Pharmaceutical University(沈阳药科大学学报), 2022, 39(5): 521-528.

[16] SUN Y, LIU H T, LIU M H, et al. Study on doxorubicin and carboplatin coloaded liposome in the treatment of ovarian cancer ascites[J]. Journal of Shenyang Pharmaceutical University(沈阳药科大学学报), 2024, 41(5): 537-543.

[17] SHI M H, ZHANG J L, WANG Y, et al. Blockage of the IDO1 pathway by charge-switchable nanoparticles amplifies immunogenic cell death for enhanced cancer immunotherapy[J]. Acta Biomater, 2022, 150: 353-366.

[18] SHI M H, WANG Y, HAN Y Y, et al. Highly penetrable nanoparticles co-loading doxorubicin and IDO1 siRNA enhance tumor immunotherapy[J]. Acta Pharmaceutica Sinica(药学学报), 2022, 57(1): 242-250.

基金资助

内蒙古自治区自然科学青年基金项目(2024QN08031)

赤峰市自然科学课题(SZR2023003)