Preparation of doxorubicin-loaded polyphyllin H liposomes and synergistic anti-tumor activity against non-small cell lung cancer <italic style="font-style: italic">in vitro</italic>

LIU Yining; ZHOU Dawei²; GAI Shouchang³; SUI Lu⁴; SUN Xue⁴; TONG Zhenhua⁴; WANG Yuhang; ZHAO Jing⁵; YUAN Xiaofeng⁵; XIANG Yong⁵

doi:10.16016/j.2097-0927.202504089

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Preparation of doxorubicin-loaded polyphyllin H liposomes and synergistic anti-tumor activity against non-small cell lung cancer in vitro

Biomedical Engineering | 更新时间：2025-09-11

- Preparation of doxorubicin-loaded polyphyllin H liposomes and synergistic anti-tumor activity against non-small cell lung cancer in vitro
- Journal of Army Medical University Vol. 47, Issue 17, Pages: 2134-2144(2025)
- 作者机构：
  
  1.联勤保障部队第九六七医院，卫勤处，辽宁大连
  2.联勤保障部队第九六七医院，急诊医学科，辽宁大连
  3.陆军第七十九集团军医院药剂科，辽宁大连
  4.北部战区总医院药剂科，辽宁沈阳
  5.联勤保障部队第九六七医院，药剂科，辽宁大连
- 作者简介：
  
  YUAN Xiaofeng， E-mail： xiaofeng012345@163.com
  XIANG Yong， E-mail： peterbird88@163.com
- 基金信息：
- DOI：10.16016/j.2097-0927.202504089
  CLC： R734.2;R944.9;R979.14
- Received：22 April 2025，
  
  Revised：2025-07-26，
  
  Accepted：29 July 2025，
  
  Published：15 September 2025
- 稿件说明：
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刘轶宁，周大伟，盖守昌，等.载阿霉素的重楼皂苷脂质体制备及其体外协同抗非小细胞肺癌的活性研究［J］.陆军军医大学学报，2025，47（17）：2134-2144.DOI：10.16016/j.2097-0927.202504089.

LIU Y N，ZHOU D W，GAI S C，et al.Preparation of doxorubicin-loaded polyphyllin H liposomes and their synergistic anti-tumor activity against non-small cell lung cancer in vitro［J］.J Army Med Univ，2025，47（17）：2134-2144.DOI：10.16016/j.2097-0927.202504089.
刘轶宁，周大伟，盖守昌，等.载阿霉素的重楼皂苷脂质体制备及其体外协同抗非小细胞肺癌的活性研究［J］.陆军军医大学学报，2025，47（17）：2134-2144.DOI：10.16016/j.2097-0927.202504089. DOI：

LIU Y N，ZHOU D W，GAI S C，et al.Preparation of doxorubicin-loaded polyphyllin H liposomes and their synergistic anti-tumor activity against non-small cell lung cancer in vitro［J］.J Army Med Univ，2025，47（17）：2134-2144.DOI：10.16016/j.2097-0927.202504089. DOI：

摘要

目的

以重楼皂苷H（polyphyllin H，ppH）替代胆固醇作为脂质体膜材，制备具有葡萄糖转运蛋白1（glucose transporter 1，Glut1）靶向性的载阿霉素（doxorubicin，DOX）脂质体（doxorubicin/polyphyllin H-liposomes，DOX/ppH-LPs），并对其体外协同抗非小细胞肺癌（non-small cell lung cancer，NSCLC）活性进行研究。

方法

采用薄膜分散法制备DOX/ppH-LPs，并采用单因素考察法优化处方。采用透射电镜、动态光散射仪等手段，对优化后的DOX/ppH-LPs脂质体的形态、粒径、粒度分布（polydispersity index，PDI）及Zeta电位进行表征。采用HPLC法测定载药量（drug loading，DL）。同时考察了脂质体在4 ℃ PBS中的7 d储存稳定性及在37 ℃含10%胎牛血清（fetal bovine serum，FBS）DMEM中的48 h血清稳定性；并分别对其在pH 7.4和pH 5.0的PBS中的释药性进行了体外研究。以人非小细胞肺癌细胞A549为细胞模型，利用MTT比色法检测不同浓度（0.5、5.0、15.0 μg/mL）的DOX/ppH-LPs及对照组（ppH+DOX/LPs，即游离ppH与不含ppH的载DOX脂质体物理混合）对肿瘤细胞的增殖抑制作用；通过荧光显微镜评价A549细胞对含等量DOX（5 μg/mL）的DOX/ppH-LPs和DOX/LPs在15 min和2 h的摄取情况，并通过活/死细胞染色评估含等量DOX（15 μg/mL）的制剂孵育48 h诱导细胞凋亡/死亡的效果；通过Transwell实验评估其抑制细胞迁移侵袭的能力，从而综合评价DOX/ppH-LPs的靶向性及体外协同抗NSCLC的活性。

结果

DOX/ppH-LPs的最优处方为水化温度50 ℃，DOX为6 mg，ppH为2 mg，卵磷脂为24 mg。制备得到的DOX/ppH-LPs外观呈圆球形，分布均匀，其平均粒径和PDI分别为（145.13±22.14） nm和0.15±0.05，电位为（-23.92±1.73）mV，DOX和ppH的载药量分别为（10.13±0.71）%和（1.22±0.21）%。 DOX/ppH-LPs在4 ℃ PBS中储存7 d及在37 ℃含10%FBS DMEM中孵育48 h，其粒径、PDI和Zeta电位均未发生显著变化，表现出良好的物理稳定性和血清稳定性。两种脂质体在pH 7.4下释放缓慢，而在pH 5.0下释放显著加速（

0.05），表现出pH敏感释放特性。MTT结果表明：DOX/ppH-LPs对A549细胞的毒性显著强于ppH+DOX/LPs组（

0.05）。相较于DOX/LPs，DOX/ppH-LPs在A549细胞中的摄取作用显著增强（

0.05），且更多DOX定位在细胞核内，活/死细胞染色结果显示，在相同DOX浓度（15 μg/mL）下，DOX/ppH-LPs处理组诱导的细胞凋亡/死亡比例显著高于ppH+DOX/LPs组。Transwell小室实验结果显示，DOX/ppH-LPs组穿过滤膜的细胞数（迁移和侵袭）均显著少于ppH+DOX/LPs组。

结论

ppH替代胆固醇构建的Glut1靶向载阿霉素脂质体可靶向NSCLC细胞，显著增强DOX与ppH的体外协同抗NSCLC活性。

Abstract

Objective‍　‍To prepare glucose transporter 1 (Glut1) -targeted doxorubicin (DOX)-loaded liposomes (doxorubicin/polyphyllin H-liposomes

DOX/ppH-LPs) using polyphyllin H (ppH) instead of cholesterol as the liposomal membrane material

and to investigate their

in vitro

synergistic anti-tumor activity against non-small cell lung cancer (NSCLC). Methods‍　‍DOX/ppH-LPs were prepared using thin-film hydration

and the formulation was optimized by single-factor investigation. The optimized DOX/ppH-LPs were characterized for morphology

particle size

polydispersity index (PDI)

and zeta potential with transmission electron microscopy (TEM) and dynamic light scattering (DLS). Drug loading DL% was determined by high-performance liquid chromatography (HPLC). The storage stability was evaluated by observing in PBS at 4 ℃ for 7 d

and the serum stability was observed in DMEM containing 10% fetal bovine serum (FBS) at 37 ℃ for 48 h.

In vitro

drug release was studied in PBS at pH 7.4 and pH 5.0 values

respectively. Human NSCLC A549 cells were subjected as the model

MTT assay was performed to detect the proliferation inhibition by DOX/ppH-LPs at different concentrations (0.5

5.0

15.0 μg/mL) and the control group (ppH + DOX/LPs

a physical mixture of free ppH and DOX-loaded liposomes). Fluorescence microscopy was used to observe cellular uptake of DOX/ppH-LPs and DOX/LPs (containing 5 μg/mL DOX) at 15 min and 2 h. Live/dead cell staining was applied to assess apoptosis/necrosis induced by formulations (15 μg/mL DOX) after 48 h incubation. Transwell assay was conducted to evaluate inhibitory effect on cell migration and invasion

and the targeting property and

in vitro

synergistic anti-NSCLC activity of DOX/ppH-LPs were then comprehensively evaluated. Results‍　‍The optimal formulation of DOX/ppH-LPs was determined as hydration temperature at 50 ℃

6 mg DOX

2 mg ppH

and 24 mg lecithin. The prepared DOX

/ppH-LPs were in spherical shape

uniform distribution

and at an average particle size of 145.13±22.14 nm

a PDI of 0.15±0.05

a zeta potential of -23.92±1.73 mV

and a DL of 10.13±0.71 % for DOX and (1.22±0.21)% for ppH. DOX/ppH-LPs maintained stable particle size

PDI

and exhibited significantly unchanged zeta potential after storage in PBS at 4 ℃ for 7 d or incubation in DMEM containing 10% FBS at 37 ℃ for 48 h

demonstrating excellent physical and serum stability. Both liposomes showed slow release at pH 7.4 value

while drug release was significantly accelerated at pH 5.0 value (

0.05)

indicating pH-sensitive release characteristics. MTT assay revealed that DOX/ppH-LPs exerted significantly stronger cytotoxicity against A549 cells than the ppH+DOX/LPs control group (

0.05). Compared with ppH+DOX/LPs

DOX/ppH-LPs showed remarkably enhanced cellular uptake in A549 cells (

0.05)

with more DOX localized in the nucleus. Live/dead cell staining showed that at the same DOX concentration (15 μg/mL)

the proportion of apoptotic/necrotic cells induced by DOX/ppH-LPs was significantly higher than that of the DOX/LPs control group. Transwell assay demonstrated that there were significantly less cells migrating and invading through the membrane in the DOX/ppH-LPs group than the ppH+DOX/LPs group. Conclusion‍　‍Glut1-targeted doxorubicin-loaded liposomes (DOX/ppH-LPs) constructed by substituting cholesterol with ppH can target NSCLC cells

significantly enhance the in vitro synergistic anti-NSCLC activity of DOX and ppH.

关键词

Keywords

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Department of Oncology, Daping Hospital, Army Medical University(Third Military Medical University)

⁰

Preparation of doxorubicin-loaded polyphyllin H liposomes and synergistic anti-tumor activity against non-small cell lung cancer in vitro

DOI：10.16016/j.2097-0927.202504089

摘要

Abstract

关键词

Keywords

references