Polylactide-tethered SN38 prodrugs in polymeric nanoparticles as reliable nanomedicines for the treatment of cervical cancer

Xiaomin Yu 1

Mingzhu Zhong 2

Qian Kang 3

Kaiwen Wu 1

Xiaoyan Xiang 3

Lifang Yan 3

Rongying Ou 2

Hangxiang Wang 4✉ Emailwanghx@zju.edu.cn

Liqing Zhu 3✉ Emailzhuliqing@bjmu.edu.cn

1 Department of Clinical Laboratory the First Affiliated Hospital of Wenzhou Medical University 325000 Wenzhou Zhejiang China

2 Department of Obstetrics and Gynecology the First Affiliated Hospital of Wenzhou Medical University 325000 Wenzhou Zhejiang China

3 Department of Clinical Laboratory Peking University Cancer Hospital & Institute 100142 Beijing China

4 The First Affiliated Hospital, NHC Key Laboratory of Combined Multi-Organ Transplantation, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases Zhejiang University School of Medicine Hangzhou Zhejiang Province PR China

Xiaomin Yu ^1*, Mingzhu Zhong^2*,Qian Kang^3*, Kaiwen Wu¹, Xiaoyan Xiang³, Lifang Yan³, Rongying Ou², Hangxiang Wang^4#, Liqing Zhu^3#

¹Department of Clinical Laboratory, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, 325000, China.

²Department of Obstetrics and Gynecology, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, 325000, China.

³Department of Clinical Laboratory, Peking University Cancer Hospital & Institute, Beijing, 100142, China.

⁴The First Affiliated Hospital, NHC Key Laboratory of Combined Multi-Organ Transplantation, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, PR China.

^# Correspondence:

Liqing Zhu: Department of Clinical Laboratory, Peking University Cancer Hospital & Institute, Beijing 100142, China.

E-mail: zhuliqing@bjmu.edu.cn

Hangxiang Wang: The First Affiliated Hospital, NHC Key Laboratory of Combined Multi-Organ Transplantation, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, PR China.

E-mail: wanghx@zju.edu.cn

Xiaomin Yu, Mingzhu Zhong and Qian Kang contributed equally to this work.

Abstract

Background

Cervical cancer remains a major global health burden, and current chemotherapeutic options are limited by poor efficacy and severe toxicity. Irinotecan (CPT-11) exerts its antitumor activity via its active metabolite, SN38, a topoisomerase I inhibitor that disrupts DNA synthesis. However, the clinical utility of CPT-11 is compromised by its poor aqueous solubility, low in vivo conversion efficiency to SN38, and significant systemic toxicity. Although nanotechnology has been used to improve the delivery of SN38, the existing nanocartors still have the defects of insufficient tumor targeting and low cellular uptake, highlighting the need to optimize the drug delivery system. This study aims to develop 2SN38-PLA@PEG1_0k-PLA_20k nanodrugs and evaluate their in vitro and in vivo antitumor efficacy against cervical cancer, in order to provide new insights into optimizing drug delivery systems and improving clinical outcomes in cervical cancer therapy.

Methods

We synthesized and characterized 2SN38-PLA prodrugs, then prepared three poly(ethylene glycol)-polylactic acid (PEG-PLA) copolymers with varying chain lengths (PEG_2k-PLA_2k, PEG_5k-PLA_8k, and PEG10k-PLA_20k) via nanoprecipitation to screen the optimal formulation. We then determined the physicochemical properties of these nanodrugs. In vitro evaluations were carried out to assess cellular uptake, cytotoxicity, apoptosis, and cell cycle distribution, while in vivo therapeutic efficacy and biosafety were evaluated using a TC-1 cell-derived subcutaneous xenograft model established in C57BL/6 mice.

Results

2SN38-PLA@PEG_10k-PLA_20k nanoparticles exhibited stable physicochemical stability. In vitro, the nanodrugs were efficiently internalized by cervical cancer cells, showing lower IC₅₀ values against cervical cancer cell lines and reduced toxicity compared to CPT11. Mechanistically, the nanodrugs induced DNA damage, which in turn triggered cell cycle arrest and apoptotic cell death. In vivo experiments, the nanodrugs significantly inhibited xenograft tumor growth, prolonged the mouse survival, and showed no obvious systemic toxicity, confirming the favorable biosafety.

Conclusion

2SN38-PLA@PEG_10k-PLA_20k nanoparticles emerge as a highly efficient and biocompatible nanodrug with robust antitumor activity against cervical cancer, validated both in vitro and in vivo. This study provides a solid preclinical foundation for the translational application of the nanodrug, thereby facilitating the optimization of cervical cancer therapeutic outcomes.

Key words:

2SN38-dsPLA prodrug

Nanomedicine

Drug Delivery

Cervical Cancer

Therapeutic potential

Introduction

7-ethyl-10-hydroxycamptothecine (SN38) is an active metabolite of the first-line chemotherapeutic medication irinotecan (CPT-11). It has 100–1000 times the anti-tumor efficacy of CPT-11, and inhibits several types of cancer, including rectal cancer, small cell lung cancer, breast cancer, and esophageal cancer[1, 2]. The cytotoxicity of SN38 is primarily mediated through the inhibition of DNA topoisomerase I (Top1)[3], which stabilizes the transient Top1-DNA cleavage complex through formation of a covalent 3'-phosphotyrosyl bond at single-strand DNA breaks[4]. This stabilization prevents the resealing of nicked DNA strands, leading to persistent single-strand breaks (SSBs) that are highly prone to progressing to lethal double-strand breaks (DSBs)[5, 6]. SN38 exerts its effects not only during transcription but also throughout the DNA replication phase. The formation of the DNA-Top1-SN38 ternary complex can induce replication fork stagnation and subsequent DNA replication termination, thus triggering cell cycle arrest or apoptosis[7, 8].

As the most potent derivative of irinotecan, SN38 has an extremely low conversion rate with only approximately 2–8%, which is mainly due to the complexity of its biotransformation pathways[9–11], thereby severely limitis its therapeutic efficacy. Moreover, the clinical application of SN38 is also restricted by extremely low aqueous solubility, poor plasma stability, and systemic toxicity. The extremely poor aqueous solubility of SN38 (less than 5 µg/mL) also severely affects the development of direct injection formulations[12]. The pharmacologically active lactone form of SN38 is highly susceptible to hydrolysis under physiological conditions (pH > 6), resulting in the conversion to the inactive carboxylate form, thus exacerbating its plasma instability and systemic bioavailability[13]. Besides, due to the lack of tumor selectivity, SN38 has cytotoxic effects on normal cells, so the treatment of CPT-11 is often accompanied by toxic reactions, especially neutropenia and diarrhea. The clinical administration of SN38 may also cause gastrointestinal toxicity, hepatotoxicity, and some rare cases, dysphasia or retinal degeneration[14–16]. These toxicities pose substantial challenges to the broader clinical use of SN38.

Cervical cancer is the fourth most prevalent malignancy in women globally, with more than 600,000 new diagnoses and approximately 340,000 deaths recorded each year. Nearly 85% of these cases are concentrated in low- and middle-income countries with limited medical resources[17, 18]. Sustained infection with high-risk human papillomavirus (HPV) is recognized as the major etiological agent, with about 70% of cases related to oncogenic subtypes, such as HPV 16 and HPV 18[19]. Although the introduction of HPV vaccines has significantly improved early prevention, the global vaccination coverage rate is still less than 15%, and nearly 40% of patients relapse or metastasize after initial treatment[20]. Early-stage disease can usually be cured by radical hysterectomy, whereas the 5-year survival rate of patients with advanced disease (FIGO stage III–IV) is less than 30%. Furthermore, over 60% of these patients develop secondary resistance to platinum chemotherapy drugs, including cisplatin[21]. The progress of targeted therapies has remained limited. Notably, HPV-positive cervical cancer cells exhibit DNA damage repair defects driven by E6/E7 oncoproteins, which may enhance sensitivity to topoisomerase I inhibitors by upregulating Top1 expression[22]. This provides a promising therapeutic target for SN38-based precision treatments. Therefore, the design of novel nano delivery systems with high-efficiency tumor targeting, microenvironmental response drug release, and controlled toxicity has become a critical strategy to overcome current treatment limitations.

The conjugation of nanomaterials with hydrophobic anticancer drugs has demonstrated improvement in drug solubility, enhancement of therapeutic efficacy, and increased tumor-targeting selectivity[23]. Recent advances in nanotechnology, such as nanoparticles, liposomes, polymeric micelles, and polymer–drug conjugations, show that they have considerable potential in the construction of drug delivery systems[24]. With the continuous progress of drug carriers based on nanoparticles, several formulations have entered clinical trials or have already been applied in the diagnosis and treatment of disease[25]. For instance, nanodrugs increase the cytotoxicity of breast tumor cells and inhibit drug resistance by co-delivering chemotherapy drugs and natural products, providing new opportunities for the therapy of breast cancer[26]. Moreover, nanodrugs have emerged as increasingly significant delivery platforms because of their ability to control drug release, selectively target colorectal cancer cells, and respond to microenvironmental signals[27].

In this study, we developed a self-assembled dual SN38 prodrug nanodelivery system designed to enhance antitumor efficacy while mitigating the inherent adverse effects of CPT-11 treatment. We synthesized a hydrophobic dual SN38 prodrug (2SN38-dsPLA) by conjugating two SN38 molecules to a double-sided polylactic acid (dsPLA) backbone. Subsequently, this 2SN38 prodrug was assembled with amphiphilic poly(ethylene glycol)-polylactic acid (PEG-PLA) copolymers through a one-step nanoprecipitation method, which was selected for its extensive clinical translation potential and favorable safety[28, 29]. Crucially, we optimized the nanoparticle architecture by systematically screening PEG-PLA chain lengths to form core-shell nanoparticles (2SN38-PLA NPs) with enhanced biocompatibility and drug stability. Furthermore, we assessed its efficacy in the treatment of cervical cancer and highlighted its potential as a novel strategy for SN38-based chemotherapy. Upon uptake by cervical cancer cells, these nanoparticles are engineered to facilitate endo-lysosomal escape and release active SN38 through ester bond hydrolysis, thereby directly inducing DNA damage and apoptosis. In preclinical mouse models, systemically administered 2SN38-PLA nanoparticles demonstrated significant tumor growth inhibition and prolonged survival compared to CPT-11, while also exhibiting an excellent biosafety that significantly reduced the systemic toxicity associated with conventional chemotherapy (Fig. 1).

Fig. 1

Schematic illustration of the synthesis and antitumor mechanism of the 2SN38-PLA nanosystem.

Fig. 1

Schematic illustration of the synthesis and antitumor mechanism of the 2SN38-PLA nanosystem. Created by BioRender (https://www.biorender.com/) (A) A hydrophobic bis-SN38 prodrug (2SN38-dsPLA) was synthesized using a double-sided polylactic acid (dsPLA) backbone and subsequently co-assembled with PEG-PLA copolymers via nanoprecipitation to form stable core-shell nanoparticles. Polymer chain lengths were screened (e.g., a = 2k/b = 2k, 5k/b = 8k, 10k/b = 20k) to optimize the nanocarrier architecture, yielding stable core-shell nanoparticles. (B) Following intravenous injection, the PEGylated nanoparticles accumulate in tumor tissues. Upon (i) cellular uptake, the nanoparticles mediate (ii) endosomal escape and liberate active SN38 via ester bond hydrolysis. The released SN38 translocates to the nucleus, resulting in the (iii) inhibition of DNA topoisomerase I and the (iv) activation of caspase-dependent apoptotic pathways (involving mitochondrial dysfunction). Collectively, these molecular events induce (1) cell cycle arrest and (2) promote apoptosis. In the tumor-bearing mouse model, this strategy leads to (a) significant inhibition of tumor growth, (b) a favorable safety profile with minimal systemic toxicity, and (c) extended survival duration.

Materials and Methods

Materials

Polymers, including double-sided hydroxyl-terminated polylactic acid (dsPLAn-OH) and amphiphilic poly(ethylene glycol)-block-polylactic acid copolymers (PEG-PLA) with varying chain lengths (PEG_2k-PLA_2k, PEG_5k-PLA_8k, and PEG_10k-PLA_20k), were purchased from Shenzhen Institute of Polymer Synthesis (Guangdong, China). SN38 and irinotecan hydrochloride trihydrate (CPT-11) were purchased from Shanghai Yucheng Biotechnology (Shanghai, China). All other compounds and solvents were obtained from Sigma-Aldrich or Biological Industries.

Human cervical cancer cell lines (HeLa, C33A) and murine cell TC-1 were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and verified through short tandem repeat (STR) profiling. Cells were cultured in DMEM or RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 µg/mL streptomycin at 37 ^◦C in a humidified atmosphere containing 5% CO₂.

Animal experiments

C57BL/6 mice (female, 20–25 g) were purchased from Shanghai Slack Laboratory Animal Co., Ltd. and co-housed in the SPF-grade Animal Center of the First Affiliated Hospital of Wenzhou Medical University under controlled conditions (25°C, 50% relative humidity).

All animal experiments received approval from the Institutional Animal Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University and were conducted in accordance with relevant regulations.

Synthesis and characterization of 2SN38-dsPLA Prodrug

The hydroxyl groups of dsPLA-OH (1500 mg, 0.121 mmol) were activated using succinic anhydride (96.8 mg, 0.968 mmol) in dichloromethane (DCM), with pyridine as a catalyst, resulting in carboxyl-terminated dsPLA (dsPLA-COOH). Subsequently, SN38 was conjugated to dsPLA-COOH at a molar ratio of 2.2:1 in anhydrous DCM, utilizing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and 4-dimethylaminopyridine (DMAP) as coupling agents. The reaction mixture was stirred at 45°C overnight. The crude product was purified via silica gel column chromatography with DCM/methanol as the eluent. ¹H nuclear magnetic resonance (NMR) spectroscopy was employed to confirm the chemical structure of 2SN38-dsPLA prodrug. The molecular weight and molecular weight distribution of 2SN38-dsPLA were determined by gel permeation chromatography (GPC) using a Waters Styragel® column system (HR4-HR3-HR1). The analysis used tetrahydrofuran (THF) as the mobile phase (flow rate of 1.0 mL/min at 35°C) coupled with a refractive index (RI) detector.

Preparation and characterization of 2SN38-PLA nanoparticles

Nanoparticles were prepared via nanoprecipitation through co-assembling the 2SN38-dsPLA prodrug with mPEG-PLA copolymers of varying chain lengths (2k-2k, 5k-8k, and 10k-20k) at a fixed mass ratio of 1:19. The resulting formulations were adjusted to a 1 mg/mL concentration and hereafter denoted as 2SN38-PLA@PEG_2k-PLA_2k NPs, 2SN38-PLA@PEG_5k-PLA_8k NPs, and 2SN38-PLA@PEG_10k-PLA_20k NPs, respectively.

For quantitative analysis, the prodrug concentration was determined using fluorescence spectrophotometry. Then serial dilutions of the three 2SN38-PLA NP formulations and free SN38 in DMSO were measured to generate a linear calibration curve (R² > 0.99). The hydrodynamic size, polydispersity index (PDI), and zeta potential of the nanoparticles with an equivalent SN38 concentration of 0.1 mg/mL were characterized by dynamic light scattering (DLS, Malvern Nano-ZS90). To evaluate colloidal stability, changes in size and PDI were monitored over a period of 7 days at 4°C in ddH₂O, PBS, and PBS supplemented with 10% FBS. The nanoparticle morphology was analyzed using transmission electron microscopy (TEM) on a 200 kV microscope after vitrification in liquid nitrogen.

In vitro cytotoxicity assay

The in vitro cytotoxic effects of the three 2SN38-PLA NPs, CPT-11, and free SN38 were evaluated in TC-1, C33A, and HeLa cell lines using the Cell Counting Kit-8 (CCK-8) assay. Cells were plated into 96-well plates at a density of 3 × 10³ cells per well and allowed to adhere for 24 hours. Subsequently, the cells were exposed to various concentrations of 2SN38-PLA NPs, CPT-11, or free SN38 for 72 h. Then the CCK-8 reagent (10% v/v) was added to measure cell viability, and the plates were incubated for 1–2 h. The optical density (OD) values were recorded at 450 nm utilizing a microplate reader.

Cellular uptake analysis

TC-1 and HeLa cells (1 × 10⁵ cells/dish) were incubated in confocal dishes overnight. The 2SN38-PLA@PEG10k-PLA20k NPs were labeled with the lipophilic probe DiI. Cells were treated with DiI-labeled NPs (100 nM DiI equivalent ) for 1, 2, 4, or 6 h. Subsequently, cells were rinsed three times with PBS before being stained for 15 minutes with Lyso-Tracker Green (1:20,000) and 10 minutes with Hoechst 33342 to highlight lysosomes and nuclei, respectively. Intracellular distribution and colocalization of the NPs were imaged using time-lapse confocal laser scanning microscopy (CLSM).

Cell apoptosis and cell cycle analysis

TC-1 and HeLa cells were planted in 6-well plates at a density of 2 × 10⁵ cells per well. To induce apoptosis, cells were stimulated with 2SN38-PLA@PEG10k-PLA20k NPs, CPT-11, or free SN38 at an equivalent SN38 concentration of 10 × IC₅₀ for 48 h. Cells were collected and stained using the Annexin V-FITC/PI Apoptosis Detection Kit (Vazyme) according to the manufacturer's protocol. For cell cycle analysis, the same cell lines were treated similarly, but with drug concentrations equivalent to 5 × IC₅₀. After 48 h, cells were harvested, fixed in 70% cold ethanol overnight at − 20°C, and stained with PI/RNase Staining Buffer (BD Biosciences). Flow cytometry (FCM) was used to examine both the cell apoptosis rate and the distribution of cell cycles.

Cell proliferation assay

Cell proliferation was evaluated with the 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay (BeyoClick™ EdU Cell Proliferation Kit, Beyotime). TC-1 and HeLa cells (1 × 10⁵ cells/well) were plated in 12-well plates and cultured for 24 h. These cells were treated with 2SN38-PLA@PEG10k-PLA20k NPs, CPT-11, or free SN38 (equivalent SN38 concentration: 10 × IC₅₀). After 48 hours of treatment, the cells were incubated with EdU (10 µM) for 2 h, fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature, and permeabilized using 0.3% Triton X-100 for 10 min. Incorporated EdU was identified via the Click-iT reaction using Alexa Fluor 488, and nuclei were counterstained with Hoechst 33342. The cell proliferation rates were determined by calculating the proportion of EdU-positive cells relative to the total number of cells stained with Hoechst 33342. ImageJ software was used to analyze images, and GraphPad Prism was used for statistical analysis.

In vivo antitumor efficacy and safety assessment

The cervical cancer subcutaneous xenograft model was established by injecting 100 µL of TC-1 cell suspension (5 × 10⁷ cells/mL) into the right axilla of female C57BL/6 mice. When the tumor volume grew to ~ 150 mm³, mice were randomly assigned to four groups (n = 6) and received intravenous injections of saline, 2SN38-dsPLA@PEG10k-PLA20k NPs, CPT-11 or CPT-11@LIP at an SN38-equivalent dose of 10 mg/kg on days 0, 2, 4, and 6. Body weight and tumor volume were assessed every two days. The tumor volume was determined by measuring the length (L) and width (W) with calipers and calculated using the formula V = (L × W²) / 2. On day 7, mice were euthanized, and tumors were removed, weighed, and photographed.

For histological analysis, excised tumors were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 4-µm sections. These sections were processed for H&E (hematoxylin and eosin) staining, TUNEL assay, and immunohistochemistry staining for Ki67 (Servicebio) following standard protocols. To assess the in vivo safety of the 2SN38-PLA nanoparticles, whole blood was collected for complete blood count (CBC) analysis (Mindray BC-7500). Serum was isolated to quantify levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and blood urea nitrogen (BUN), using commercial assay kits.

Statistical Analysis

Quantitative data are presented as the mean ± standard deviation (SD). Statistical analysis was conducted using Graphpad software, employing independent t-tests and one-way ANOVA. Statistical significance was denoted by p values < 0.05 (*), < 0.01 (**), < 0.001 (***), or < 0.0001 (****).

Results

Synthesis and characterization of 2SN38-dsPLA prodrug

In order to solve the inherent limitations of free SN38, including poor aqueous solubility, chemical instability, and short half-life, we designed and synthesized a novel polymer prodrug 2SN38-dsPLA. The prodrug covalently conjugated two SN38 molecules to the end of the dsPLA main chain through ester linkage (Fig. 2A). The ¹H NMR spectroscopy analysis was employed to validate the chemical structure of 2SN38-dsPLA prodrug, showing that the characteristic resonance signals of the aromatic protons of the SN38 moiety at δ 8.21, 8.01, and 7.66 ppm were distinct from the multiple signals of the PLA methine protons at δ 5.27–5.09 ppm (Fig. 2B). The integral ratio of these peaks was consistent with the theoretical stoichiometry, confirming the successful combination of drug molecules. In addition, the molecular weight distribution of 2SN38-dsPLA prodrug was characterized by GPC. The elution curve showed a symmetrical single-paek profile, indicating high purity and no obvious byproducts (Fig. 2C). The quantitative analysis revealed that the average molecular weight (Mn) was 27,673 Da and the polydispersity index (PDI) was 1.28 (Fig. 2D), indicating that the synthesized prodrug had a uniform chain length and was suitable for stable nano-assembly.

Fig. 2

Synthesis and structural characterization of the 2SN38-dsPLA prodrug.

Fig. 2

Synthesis and structural characterization of the 2SN38-dsPLA prodrug. (A) Synthetic route of 2SN38-dsPLA_n (n = 232) via esterification reaction involving dsPLA_n-OH, succinic anhydride, and free SN38. (B) ¹H NMR spectrum of dsPLA-COOH. (C) ¹H NMR spectrum of 2SN38-dsPLA, confirming the successful conjugation of SN38 onto the polymer chain. (D) Gel permeation chromatography (GPC) analysis for 2SN38-dsPLA prodrug, showing the retention time, number-average molecular weight (Mn), weight-average molecular weight (Mw), and polydispersity index (PDI).

Synthesis and optimization of 2SN38-PLA nanoparticles

To optimize the nanocarrier structure for reducing the systemic toxicity of SN38 and enhancing the stability, the 2SN38-dsPLA prodrug was assembled with three different chain lengths of PEG-PLA diblock copolymers (2k-2k, 5k-8k, and 10k-20k) via nanoprecipitation. Three distinct formulations were designated as 2SN38-PLA@PEG_2k-PLA_2k, 2SN38-PLA@PEG_5k-PLA_8k, and 2SN38-PLA@PEG_10k-PLA_20k, respectively (Fig. 1A). For the sake of simplicity, these will be referred to as the 2k-2k, 5k-8k, and 10k-20k formulations below. Subsequent physicochemical characterization showed that increasing the length of the polymer chain could significantly improve the uniformity and stability of the particles.

Visually, the aqueous suspension of the PEG_10k-PLA_20k NPs exhibited the highest transparency (Fig. 3A). The observation results were quantified by DLS analysis, revealing that particle size diminished with the increase of chain length. The PEG_10k-PLA_20k NPs yielded the most compact nanoparticles with a minimum hydrodynamic diameter (D_H) of 105.27 ± 0.49 nm and a polydispersity index (PDI) as low as 0.125 (Figs. 3C). Conversely, the formulations based on shorter copolymers (5k-8k and 2k-2k) resulted in significantly larger aggregates, 140.27 ± 1.29 nm and 206.83 ± 2.23 nm, respectively. All three formulations exhibited a net negative surface charge, consistent with the surface charge of the cell membrane (Fig. 3B). Transmission electron microscopy (TEM) images confirmed that all formulations were spherical and uniformly dispersed, with PEG_10k-PLA_20k nanoparticles exhibiting the smallest and most uniform particle size (Fig. 3E). Colloidal stability was the critical selection criterion. The stability of 2SN38-PLA nanoparticles was further evaluated by DLS analysis after storage in different media. The results demonstrated that only PEG_10k-PLA_20k NPs remained adequate stability in PBS with 10% FBS (v/v), and there was no significant change in particle size and PDI within a week. In contrast, the other groups showed indications of instability or precipitation (Fig. 3D). This enhanced stability may be attributed to the dense hydration shell formed by longer PEG segments and the robust hydrophobic nucleus created by high molecular weight PLA blocks.

Fig. 3

Characterization and in vitro cytotoxicity of 2SN38-loaded polymeric nanoparticles. (A) Macroscopic appearance of 2SN38-PLA@PEG_2k-PLA_2k NPs, 2SN38-PLA@PEG_5k-PLA_8k NPs, and 2SN38-PLA@PEG_10k-PLA_20k NPs (B) Zeta potential values of the indicated nanoparticles. (C) Hydrodynamic diameter (D_H) distribution and polydispersity index (PDI) of the three nanoparticle formulations measured by dynamic light scattering (DLS). (D) Colloidal stability of nanoparticles incubated in ddH2O, PBS, and 10% FBS over 6 days. Solid circles indicate particle size (left axis) and open circles indicate PDI (right axis). (E) Representative transmission electron microscopy (TEM) images exhibiting the spherical morphology of the nanoparticles. Scale bars: 200 nm (left) and 40 nm (right). (F) In vitro cytotoxicity of CPT11, free SN38, and different 2SN38-loaded nanoparticles against TC-1, HeLa, and C33A cell lines. Cell viability was determined using the CCK-8 assay after incubation with various concentrations of drugs for 72 h. Data are presented as mean ± SD (n = 3).

Additionally, the in vitro cytotoxicity potential of these formulations for TC-1, HeLa, and C33A cervical cancer cell lines was evaluated using the CCK-8 assay (Fig. 3F). Compared with free SN38, all nanoparticle formulations showed favorable safety, and the cytotoxicity was significantly enhanced relative to the clinical drug CPT-11. Notably, the PEG_10k-PLA_20k NPs displayed the lowest IC₅₀ values among all evaluated cell lines (Table 1), indicating that their optimized polymer chain length and improved colloidal stability promote cellular internalization and enhanced therapeutic efficacy. Therefore, the PEG_10k-PLA_20k nanoparticles were selected for subsequent study and referred to as 2SN38 nanoparticles.

Fig. 3

Characterization and in vitro cytotoxicity of 2SN38-loaded polymeric nanoparticles.

Table 1

Toxicity detection of 2SN38-PLA nanodrugs on cervical cancer cell lines
Cell lines	TC-1	HeLa	C33a
CPT11	17110 ± 2434	7758 ± 1335	320.3 ± 56.2
Free SN38	161.5 ± 27.64	22.75 ± 3.918	2.4 ± 0.4
2SN38-dsPLA_n NPs@PEG_2k-PLA_2k	4461 ± 641.5	801.4 ± 115.0	175.1 ± 20.2
2SN38-dsPLA_n NPs@PEG_5k-PLA_8k,	1041 ± 159.9	558.5 ± 113.6	112.8 ± 25.9
2SN38-dsPLA_n NPs@PEG_10k-PLA_20k	470.1 ± 68.19	404.6 ± 72.79	11.2 ± 2.2

Results are expressed in nM as IC₅₀ ± SD.

Apoptosis induction, anti-proliferation, and cell cycle arrest of 2SN38 NPs

We conducted a dual staining assay using Annexin V and propidium iodide (PI) to determine whether the cell death caused by nanoparticles was due to apoptosis. FITC-labeled Annexin V attaches to phosphatidylserine translocated in apoptotic and necrotic cells, while PI serves as a nucleic acid dye for late apoptotic and necrotic cells, thus distinguishing between apoptosis and necrosis[30]. In TC-1 cells, the total apoptotic rate in the 2SN38 NPs group reached 48.27%, significantly surpassing that of the CPT-11 group (14.09%) and the untreated control (7.01%) (Fig. 4A). A comparable result was also detected in HeLa cells, where 2SN38 NPs elicited an apoptosis rate of 48.25%, compared to 17.88% for CPT-11 (Fig. 4B). These results indicated that 2SN38 NPs exerted superior proapoptotic efficacy relative to CPT-11 in cervical cancer cells. Then we investigated the intrinsic apoptotic pathway to elucidate the mechanisms underlying the cytotoxicity of 2SN38 NPs. Compared with the CPT-11 group, cell death induced by the 2SN38-NPs depends on the classical intrinsic apoptotic pathway, which was evidenced by the escalated levels of cleaved-Caspase 3 (C-Caspase 3) and cleaved-Caspase 9 (C-Caspase 9) (Fig. 4C). As the initiator caspase of the intrinsic pathway, Caspase-9 is activated upon apoptosome assembly, and subsequently triggers the proteolytic activation of the executioner Caspase-3. The latter ultimately executes apoptosis by cleaving critical intracellular substrates[31, 32]. Thus, 2SN38 NPs may induce tumor cell apoptosis primarily through the intrinsic mitochondrial apoptotic cascade.

Fig. 4

Cell apoptosis, proliferation, and cell cycle arrest induced by 2SN38 NPs. Apoptosis analysis of TC-1 (A) and HeLa (B) cells treated with Untreated, CPT11, Free SN38, or 2SN38 NPs determined by Annexin V-FITC/PI staining flow cytometry. (C) Western blot analysis of apoptosis-related protein expression levels after different treatments in TC-1 and HeLa cells. β-actin was used as a loading control. (D) Cell proliferation of TC-1 and HeLa cells after exposure to drugs, as determined using the EdU assay (n = 3). Scale bar = 100 µm. Cell cycle distribution analysis of TC-1 (E) and HeLa (F) cells determined by flow cytometry (n = 3). Data are presented as the means ± SD. **p < 0.01, *** p < 0.001, **** p < 0.0001.

Correspondingly, we also conducted the anti-proliferation capability of 2SN38 NPs using an EdU incorporation assay. After 48 hours of incubation, TC-1 and HeLa cells were examined by fluorescence microscopy. Compared to CPT-11, treatment with 2SN38-NPs resulted in a significant decrease in EdU-positive proliferating cells in both TC-1 and HeLa cells (Fig. 4D), indicating that the 2SN38-NPs exerted a stronger antiproliferative capability in cervical cancer cells. As the bioactive metabolite of CPT-11, SN38 functions as a potent DNA topoisomerase I inhibitor that induces DNA damage during replication, typically resulting in G2/M phase cell cycle arrest[33]. To probe the nanoparticles’ impact on cell cycle progression. TC-1 and HeLa cells were stained with PI and RNase. Subsequent cell cycle analysis revealed a substantial increase in the proportion of cells in the S and G2/M phases after treatment with 2SN38 nanoparticles, accompanied by a notable decrease in the proportion of cells in the G1 phase (Fig. 4E, 4F). This result was consistent with previous cytotoxicity and anti-proliferation data. It is worth noting that 2SN38-NPs successfully induced cell cycle arrest, which can inhibit tumor cell proliferation and prolong the duration of DNA damage, thereby enhancing the anti-tumor effect of 2SN38-NPs.

Fig. 4

Cell apoptosis, proliferation, and cell cycle arrest induced by 2SN38 NPs.

Intracellular uptake endo/lysosomal escape of 2SN38 NPs

The internalization of 2SN38 NPs by tumor cells is a prerequisite for their therapeutic action. As the therapeutic target of SN38 is intranuclear, successful endosomal/lysosomal escape is crucial to the therapeutic effect of nanocarriers. We employed time-lapse Confocal Laser Scanning Microscopy (CLSM) to track the intracellular distribution and further elucidate the uptake pathway of the DiI-labeled 2SN38 NPs. TC-1 and HeLa cells were incubated with the DiI-labeled NPs, while LysoTracker Green was applied to stain endo/lysosomal compartments. Time-lapse fluorescence images in TC-1 (Fig. 5A) and HeLa (Fig. 5B) cells showed that red fluorescence signals exhibited clear colocalization with the green LysoTracker signals, indicating that most 2SN38 NPs were trafficked to endo/lysosomes during the initial 4 hours. Subsequently, the red fluorescence signals began to spread out and no longer overlapped with endo/lysosomes after 6 hours, implying that the 2SN38 NPs had effectively escaped into the cytosol.

Fig. 5

Cellular uptake of 2SN38-PLA NPs in TC-1(A) and HeLa(B) cells.

Fig. 5

Cellular uptake of 2SN38-PLA NPs in TC-1(A) and HeLa(B) cells. The cells were treated with DiI-labeled 2SN38-PLA NPs for 1, 2, 4, or 6 h and then subjected to visualization by time-lapse confocal laser scanning microscopy (CLSM). Cell nuclei were stained with Hoechst (blue), and endo/lysosomes were stained with LysoTracker (green), respectively. Scale bar = 5 µm.

In vivo antitumor efficacy and safety assessment of 2SN38-NPs against cervical cancer

HPV vaccination enables the prevention of cervical cancer, yet it still constitutes a significant global health concern. HPV-positive cells present a therapeutic opportunity due to DNA repair defects and the consequential upregulation of Topoisomerase 1 (Top1)[22]. Thus, we established a TC-1 subcutaneous cervical cancer model in C57BL/6 mice to further investigate the in vivo antitumor efficacy of 2SN38-NPs.

Upon the tumor volume reaching approximately 150 mm³, the mice were randomly sorted into four groups (n = 6) and given injections of saline, CPT-11, CPT-11@LIP, or 2SN38 NPs at an SN38-equivalent dose of 10 mg/kg on days 0, 2, 4, and 6 (Fig. 6A). After intravenous administration, 2SN38 NPs displayed superior antitumor efficacy compared with all control groups. The group treated with 2SN38 NPs presented the obvious decreased tumor volume and the slowest tumor growth (Fig. 6B). The survival rate of the 2SN38 NPs treatment group also increased significantly (Fig. 6C). Additionally, the weight changes were recorded across all treatment groups, and no significant differences were observed among the groups. (Fig. 6D). Meanwhile, we analyzed individual tumor growth curves of each group. The results showed that the 2SN38 NPs group maintained a minimal tumor volume increase relative to the control groups, which was consistent with the photograph results of the tumor volume (Fig. 6E, 6F). These results demonstrated that the 2SN38 NPs exhibited significantly higher tumor inhibition activity and the least toxicity than CPT-11 and CPT-11@LIP.

Fig. 6

In vivo antitumor activity and safety evaluation of 2SN38 NPs in preclinical C57BL/6 mouse subcutaneous models bearing subcutaneous TC-1 tumors. (A) Schematic illustration of the experimental timeline for the subcutaneous tumor model establishment and treatment regimen. Mice were intravenously injected with Saline (negative control), CPT11, CPT11@LIP, or 2SN38 NPs at an equivalent SN38 dose of 10 mg/kg on days 0, 3, and 6 (n = 6). (B) Tumor growth curves in the TC-1 subcutaneous tumor-bearing mouse model after different drug treatments (n = 6). (C) Kaplan-Meier survival curves of mice in different treatment groups (n = 6). (D) Body weight and body weight variation percentage in mice from different treatment groups (n = 6). (E) Individual tumor growth curves for mice in each group (n = 6).Representative images of mice and tumors in each group at day 0 and day 7. (F) Photograph of excised tumors in each group at the endpoint of the study. (G) H&E, Ki67, and TUNEL staining of tumor sections from mice treated with different formulations. Scale bars: 100/ 50 µm. (H) White blood cell (WBC), lymphocyte (LYM), and red blood cell (RBC) counts in mice from different treatment groups (n = 6). (I) Analysis of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and blood urea nitrogen (BUN) in mice from different treatment groups (n = 6). Data are presented as mean ± SD. ns, no significant difference, *p < 0.05; **P < 0.01; ****p < 0.0001.

To further verify the therapeutic efficacy of the formulation, we also used H&E, Ki-67, and TUNEL staining to conduct histopathological analysis of tumor tissues (Fig. 6F). HE staining revealed that the density of tumor cells in the 2SN38-NPs treatment group was substantially reduced in comparison to the other groups, indicating effective suppression of tumor cell proliferation. Consistent with these findings, Ki-67 immunostaining displayed a notable decrease in proliferating cells in the 2SN38-NPs group, corroborating its antiproliferative properties. Furthermore, TUNEL staining revealed a significant rise in apoptotic cells in the 2SN38-NPs group, demonstrating that this treatment effectively induces apoptosis in cervical cancer cells. Collectively, these results confirm that 2SN38-NPs exert antitumor activity against cervical cancer through inhibiting cell proliferation and promoting cell apoptosis.

Neutropenia and diarrhea are the main side effects of CPT-11[34]. Furthermore, with the increasing application of CPT-11 in the treatment of solid tumors, the hepatotoxicity induced by CPT-11 has emerged as a prominent adverse reaction, restricting dose increases[35]. To assess the in vivo toxicity of 2SN38 NPs, hematological parameters and serum biochemical markers were measured in mice after treatment. As shown in Fig. 6H, the results of hematological analysis were performed to evaluate myelosuppression. The CPT-11 group showed a significant decrease in white blood cell (WBC), lymphocyte (LYM), and neutrophil (NEU) counts, which are characteristic indicators of severe bone marrow suppression. In contrast, the peripheral blood cell counts were maintained at normal levels in the 2SN38 NPs group, with no significant differences in red blood cell (RBC) counts across all groups. Additionally, the CPT-11 group exhibited significantly increased levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and blood urea nitrogen (BUN), indicating severe damage to liver and kidney functions. Conversely, the biochemical markers in the 2SN38 NPs group remained within normal range, similar to those of the saline group (Fig. 6I). The results indicated that the 2SN38-PLA nanodelivery system effectively reduces the adverse effects associated with CPT-11, particularly systemic toxicity and myelosuppression, by decreasing hepatic drug exposure and inhibiting the release of systemic inflammatory factors. Importantly, the formulation maintained potent antitumor activity in vivo, while also exhibiting excellent in vivo safety.

Fig. 6

In vivo antitumor activity and safety evaluation of 2SN38 NPs in preclinical C57BL/6 mouse subcutaneous models bearing subcutaneous TC-1 tumors.

Discussion

CPT-11, a clinically established chemotherapeutic drug, has remained a frontline anticancer drug globally since its approval in 1994. It is mainly used as a first-line therapy for colorectal and pancreatic cancers[36]. CPT-11 exerts its therapeutic effect by inhibiting DNA topoisomerase I (Top I) activity, thereby preventing the formation of supercoiled DNA and inducing tumor cell apoptosis[37]. Although CPT-11 is commonly administered in clinical treatment, its dosage is carefully controlled and restricted because of its common adverse reactions, such as diarrhea and neutropenia[38]. As a result, its treatment effect is difficult to meet expectations. It has always been crucial to find an effective substitute for CPT-11 to improve clinical application. As the bioactive metabolite of CPT-11, SN38 exhibits 100–1000 times greater therapeutic efficacy than CPT-11, but its low aqueous solubility and inherent instability have severely restricted its practical application. In recent years, nanodrug delivery systems based on SN38 have greatly enhanced the drug characteristics and therapeutic qualities of SN38, overcoming its limitation of low solubility and poor stability, and significantly improving the antitumor efficacy, while reducing systemic toxicity[39].

Therefore, we developed a 2SN38 nanodelivery system by encapsulating a dimeric prodrug, 2SN38-PLA, within PEG-PLA micelles. We engineered an unsaturated fatty acid branched architecture to improve the encapsulation efficiency of SN38 by providing a more hydrophobic core for drug isolation, thereby confirming the current prodrug-carrier optimization strategy[40]. After screening, we identified that 2SN38-PLA@PEG_10k-PLA_20k NPs were the best formulation for balancing particle size (~ 105 nm), polydispersity, and colloidal stability. Consistent with the findings that high-density PEGylation (e.g., 150% surface coverage) is crucial for evading the mononuclear phagocyte system (MPS) and prolonging circulation[40]. Besides, our nanoparticle formulation maintained its hydrodynamic diameter in a serum-containing medium for over one week. This excellent stability may protect healthy tissues from harmful exposure, while ensuring adequate medication delivery to the tumor site by preventing the non-specific release of SN38 during systemic circulation. The monodispersed spherical morphology observed by TEM indicates the successful self-assembly of the prodrug-polymer system.

The 2SN38 NPs exhibited superior antitumor efficacy relative to CPT-11, while also maintaining greater safety than free SN38. Notably, 2SN38 NPs achieved equivalent cytotoxicity to CPT-11 at concentrations ~ 30-fold lower in cervical cancer cell lines, directly mitigating drug-related systemic toxicity. The enhanced potency is attributed to the efficient cellular internalization of the nanoparticles through endocytosis, followed by lysosomal escape and the release of active SN38 triggered by intracellular esterases. In comparison to CPT-11, the 2SN38 NPs exhibited markedly enhanced antiproliferative effects and proapoptotic activity in cervical cancer cells, indicating their potential to address the intrinsic resistance of CPT-11. Mechanistically, the released SN38 functioned as a robust Topoisomerase I inhibitor, inducing extensive DNA damage, distinct cell cycle arrest, and stimulating the intrinsic apoptotic pathway. In cervical cancer mouse models, the 2SN38 NPs showed enhanced antitumor efficacy, as reflected by reduced tumor size and prolonged survival. Moreover, they also support a favorable safety profile, with no significant body weight loss or organ toxicity, supporting their potential as a targeted delivery strategy for cervical cancer chemotherapy.

Conclusion

The novel 2SN38-PLA@PEG10k-PLA20k nanoparticles developed in this study demonstrated significantly improved therapeutic efficacy and safety compared to CPT-11 and represent a viable alternative treatment option. Furthermore, its superior performance in cervical cancer treatment suggests that this novel SN38 nano-delivery system provides a compelling preclinical rationale for the development of this nanotherapeutic as a potent clinical alternative.

Availability of data and material

Data will be made available on request.

Abbreviations

ALT

Alanine aminotransferase

AST

Aspartate aminotransferase

BUN

Blood urea nitrogen

C-Caspase 3

Cleaved-Cysteine Aspartic Acid Protease-3

C-Caspase 9

Cleaved-Cysteine Aspartic Acid Protease-9

CCK-8

Cell counting kit-8

CLSM

Confocal laser scanning microscopy

CPT-11

Irinotecan

DCM

Dichloromethane

DMAP

4-Dimethylaminopyridine

DMSO

Dimethylsulfoxide

DLS

Dynamic light scattering

D_H

Hydrodynamic diameter

EDCI

1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

EdU

5-Ethynyl-2'-deoxyuridine

FBS

Fetal bovine serum

FCM

Flow cytometry

GPC

Gel Permeation Chromatography

H&E

Hematoxylin and eosin

HPV

Human papillomavirus

LYM

Lymphocyte

NEU

Neutrophil

NMR

Nuclear magnetic resonance

PBS

Phosphate-buffered saline

PDI

Polydispersity index

PEG

Polyethylene glycol

PLA

Polylactic Acid

SN38

7-Ethyl-10-hydroxycamptothecin

TEM

Transmission electron microscopy

THF

Tetrahydrofuran

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Acknowledgements

Not applicable.

Funding

This work was supported by grants from the National Natural Science Foundation of China (NO. 82272784) and the Beijing Natural Science Foundation (NO. 7252006).

Contributions

Xiaomin Yu: Writing – original draft, methodology, investigation, formal analysis, data curation, visualization, conceptualization, funding acquisition. Mingzhu Zhong, Qian Kang: Methodology, investigation, formal analysis, data curation, visualization. Kaiwen Wu: Investigation, validation, formal analysis. Xiaoyan Xiang, Lifang Yan: Writing – review & editing, validation, formal analysis. Rongying Ou, Hangxiang Wang: Writing – review & editing, supervision, conceptualization. Liqing Zhu: Writing review & editing, validation, supervision, funding acquisition, conceptualization.

Ethics approval and consent to participate

All animals received care in accordance with guidelines set out in the Guidelines for the Care and Use of Laboratory Animals.

This study was approved by the First Affiliated Hospital of Wenzhou Medical University Laboratory Animal Ethics Committee.

Consent for publication

The authors declare that they have consent for publication.

Competing interests

The authors declare that they have no competing interests.