Endostar was obtained from Jiangsu Simcere Pharmaceutical Co., Ltd (China). Docetaxel injection (Taxotere®) was purchased from Sanofi-Aventis Deutschland GmbH (Germany) and Cisplatin (CDDP) was purchased from Jiangsu Hengrui Pharmaceuticals Co., Ltd (China). Docetaxel injection was configured as a mother liquor according to the instructions, and then diluted with normal saline to the corresponding working solution concentration. Others were all diluted in normal saline.

Docetaxel polymeric micelles HT001 was synthesized using protocols described by Zhang et al. (2022). Briefly, mPEG-PDLLA (480 mg) and DTX (20 mg) were co-dissolved in methanol, mixed, and heated over 47°C rotary steam for at least 1 h to remove the solvent. Normal saline was added and hydrated at 60°C to prepare HT001. The HT001 solution was obtained, filtered, sterilized, and lyophilized to obtain a lyophilized preparation of HT001.

The morphology of HT001 was observed by transmission electron microscopy (TEM, HT7700 Exalens, Hitachi, Japan). One drop of micelles was dispensed on the 300-mesh copper grid, and the excessive liquid was removed using filter paper. The DTX PMs were negatively stained with 3% (w/v) phosphotungstic acid and dried at room temperature before observation.

The human breast cancer cell line MCF-7 was purchased from the American Type Culture Collection (ATCC) and maintained in Minimum Essential Medium Non-Essential Amino Acids (MEM-NEAA) medium (HyClone, United States) containing 10% fetal bovine serum (FBS) (Gibco, United States) and 1% penicillin-streptomycin (P/S) (Gibco, United States). The human breast cancer cell line MDA-MB-231 was purchased from Shanghai Institute of Biological Sciences, Chinese Academy of Sciences, and maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, United States) containing 10% FBS and 1% P/S. The human breast cancer cell line MDA-MB-435, human non-small cell lung cancer cell lines A549 and H460, and human ovarian cancer cell lines SKOV-3 and ES-2 were purchased from Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, and maintained in RPMI 1640 medium containing 10% FBS and 1% P/S. The human embryonic lung fibroblast cell line MRC-5 was purchased from Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, and maintained in MEM-NEAA (Gibco, United States) containing 10% FBS and 1% P/S. Mouse hepatoma cell line H22 was purchased from Nanjing Cobioer Biosciences CO., LTD (Nanjing, China) and maintained in RPMI 1640 medium containing 10% FBS and 1% P/S. The cell lines mentioned above were in their passages 10–15 during the experiments.

A549, H460, MCF-7, MDA-MB-231, MDA-MB-435, SKOV-3, or MRC-5 cell suspension was seeded in a 96-well flat-bottom tissue culture plate with 5000 cells per well. After 72-h incubation with HT001 or DTX, cell viability was assessed by Cell Counting Kit (CCK)-8 assay. Specifically, the culture medium was replaced with 100 μL fresh medium containing 10 μL CCK-8 solution (Dojindo Laboratories, United States), and cells were incubated at 5% CO₂ in a humidified incubator at 37°C for 0.5–1.5 h. The absorbance at a wavelength of 450 nm was detected by a SpectraMax M5 microplate reader (Molecular Devices, United States). The IC₅₀ value was calculated based on the luminescence at different concentrations by GraphPad Prism 8.0 (GraphPad Software Inc., San Diego, CA, United States).

Female Balb/c mice, Balb/c nude mice, and CB-17 SCID mice at 6–8 weeks were all purchased from Shanghai Lingchang Biotechnology Co., Ltd (Shanghai, China). Eight-week-old Sprague-Dawley (SD) rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Beagle dogs (male/female) weighing 7.73–8.37 kg at about 6–8 months old were purchased from Mars Biological Technology Co., Ltd (Beijing, China). All experimental procedures involving animals and their care were conducted in accordance with the State Council Regulations for Laboratory Animal Management (Enacted in 1988) and were approved by the Institutional Animal Care and Use Committee of the Jiangsu Simcere Pharmaceutical Co. Ltd.

To establish A549, MCF-7, and SKOV-3 xenograft mouse models, 5^∗10⁶ A549, MCF-7, and SKOV-3 cells per mouse were injected subcutaneously into the right flank of female CB-17 SCID mice, Balb/c nude mice, and Balb/c nude mice respectively. Mice were sacrificed when tumor volume reached 300 mm³. Tumors were collected and divided into small pieces (2^∗2^∗2 mm). The resulting small tumor pieces were implanted subcutaneously into the right flank of mice. As average tumor volume reached 100–300 mm³, the mice were randomly assigned to experimental groups according to the tumor volume and treated with normal saline, HT001 (5, 10, 20, or 40 mg/kg), or DTX (5, 10, 20, or 40 mg/kg) intravenously. The tumor volume and the bodyweight of the animals were measured twice a week. The long diameter a) and the short diameter b) of the tumor were measured using a caliper, and the tumor volume was calculated using the following formula: V = 0.5^∗a^∗b².

To establish the H22 ascites mouse model, Balb/c mice were intraperitoneally injected with 1^∗10⁷ cells H22 cells. After 7 days, the ascites cells were harvested from mice and re-suspended in saline and each naïve Balb/c mouse was injected intraperitoneally with 5^∗10⁵ ascites cell suspension. Three days after H22 tumor cells inoculation, the mice were randomly assigned to experimental groups (n = 16) according to the bodyweight, and treated with normal saline, CDDP (5 mg/kg), HT001 (1.5, 5, or 20 mg/kg), Endostar (8 mg/kg), or combination intraperitoneally. The bodyweight of the animals was measured three times a week and the survival of mice was monitored daily. After 8 days of treatment, 6 mice from each group were sacrificed to collect plasma and ascites for analysis. The weights of ascites were recorded, and cells with ascites were counted. In addition, VEGF levels in plasma and ascites were measured by Quantikine™ mouse VEGF immunoassay kit (R&D Systems, United States). The remaining mice were used for monitoring survival.

To establish the ES-2 ascites mouse model, Balb/c nude mice were intraperitoneally injected with 1^∗10⁷ cells ES-2 cells. Seven days after ES-2 cells inoculation, the mice were randomly assigned to experimental groups (n = 16) according to the bodyweight and treated with normal saline, CDDP (5 mg/kg), or HT001 (5 or 20 mg/kg) intraperitoneally. The bodyweight of the animals was measured three times a week and the survival of mice was monitored daily. After 7 days of administration, 6 mice from each group were sacrificed to collect plasma and ascites for analysis. The weights of ascites were recorded, and cells with ascites were counted. In addition, VEGF levels in plasma and ascites were measured by Quantikine™ mouse VEGF immunoassay kit (R&D Systems, United States). The remaining mice were used for monitoring survival.

Eight-week-old Sprague-Dawley (SD) rats were intravenously administered with a single dose of 2.5, 5, 10 mg/kg HT001 or 5 mg/kg DTX (six rats per group, male: female = 1:1). Blood was collected from the jugular vein in heparinized tubes at 0.083, 0.25, 0.5, 1, 2, 3, 4, 6, 8, and 24 h after administration. Blood samples were centrifuged at 3,000 rotations per minute (rpm) for 10 min to obtain plasma and analyzed by LC-MS/MS (Waters Corp., Manchester, United Kingdom). Beagle dogs were intravenously administrated with a single dose of 1 mg/kg of DTX or HT001 (6 dogs per group, male: female = 1:1). Blood was collected from the forelimb vein in heparinized tubes at 0.25, 0.5, 0.55, 1, 2, 4, 6, 8, and 24 h after administration. Blood samples were centrifuged at 3000 rpm for 10 min to obtain plasma and analyzed by LC-MS/MS.

Biodistribution studies were performed in MCF-7 tumor-bearing Balb/c nude mice. After the tumor volume reached 100–300 mm³, mice were randomly assigned to two groups (24 mice per group, male to female ratio 1:1). Mice were intravenously administered with 10 mg/kg HT001 or DTX. At the various time points (0.5, 2, 6, and 24 h, n = 6, male: female = 1:1), mice were anesthetized and sacrificed. The plasma and major organs, including brain, heart, lung, liver, kidney, spleen, stomach, small intestine, fat, muscle, gonads, tumor, and marrow were harvested. Organs were weighed and homogenized with 400 μL of PBS/10 international units (IU) of heparin solution and extracted. The concentration of compounds was analyzed by LC-MS/MS.

Eight-week-old SD rats were intraperitoneally administered with normal saline, 5, 10, or 16 mg/kg HT001 once a week (ten rats per group, male: female = 1:1). The bodyweight and survival of rats were monitored during the experiments. On the day after the fifth dose, rats were sacrificed. The liver, kidney, and spleen samples from normal saline and 16 mg/kg HT001 groups were harvested and fixed in Zn fixing buffer for about 36 h at room temperature, changed to ddH₂O for 5 min, dehydrated, and embedded in paraffin using the Leica ASP300S system and EG 1150H+C system (Leica, Wetzlar, Germany). The liver, kidney, and spleen sections were stained using Hematoxylin and Eosin method. Each stained slide was observed via a light microscope (Nikon Labophot, Japan) in a blind manner to evaluate histopathological changes in liver, kidney, and spleen.

All results are expressed as the mean ± standard error of mean (SEM). The statistical analysis of data was performed using two-way ANOVA followed by Tukey’s multiple comparisons test in efficacy studies using GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, United States). Original spectrum concentration and accuracy were outputted using UNIFI (Waters, United States). The pharmacokinetic parameters were calculated using non-chamber model (NCA) method in WinNolin (V6.2) (Certara, Inc., United States). The student’s-t test was conducted to evaluate the statistical significance in pharmacokinetic studies and biodistribution assessment studies using Microsoft Office EXCEL (2007) (Microsoft, United States). p-values < 0.05 were considered statistically significant.

The structure of polyethylene glycol monomethyl ether polylactic acid block polymer (mPEG-PDLLA) was shown in Figure 1A. The obtained copolymers were characterized by nuclear magnetic resonance, and the results reveal that the molecular weight is 3647 g/mol and the polydispersity coefficient value was 1.2 (data not shown). The synthesis schematic of HT001 was shown in Figure 1B. Lyophilized HT001 preparation was analyzed by transmission electron microscope. As shown in Figure 1C, polymer structures were approximately spherical and the micelles were formed. The solubility of HT001 in normal saline was 2.0 mg/ml. The basic chemical and structural features of HT001 (also known as DTX-mPEG-PDLLA) were characterized and the results published recently (Zhang et al., 2022). The drug loading efficiency, Zeta potential and DLS of HT001 were 4.27 ± 0.36%, −3.13 mV and 25 nm, respectively (Zhang et al., 2022). The result of drug release assay showed that less than 30% and 58% of HT001 was released in 6 and 72 h, respectively (Zhang et al., 2022). In comparison, DTX had a much faster release kinetics, e.g., ∼45% of DTX was released in the 1 h, and >90% of DTX was released in 6 h (Zhang et al., 2022).

Since DTX is effective in treating human non-small cell lung cancer, breast cancer, and ovarian cancer, human non-small cell lung cancer cells A549 and H460, human breast cancer cells MCF-7, MDA-MB-231, and MDA-MB-435, and human ovarian cancer cells SKOV-3 were chosen to study the cytotoxicity of HT001 using CCK-8 assay. The cytotoxicity of DTX and HT001 on human embryonic lung fibroblast cell MRC-5 was also studied. Upon treatment of DTX or HT001, cell viability decreased dose-dependently (Figures 2A–G). The IC₅₀ values of HT001 and DTX in MCF-7, MDA-MB-231, MDA-MB-435, A549, H460, SKOV-3, and MRC-5 cells are listed in Table 1. These results reveal that comparable cytotoxic activities between HT001 and DTX are observed in majority of cell lines tested in vitro while in some cell lines (MCF-7, H460, and MDA-MB-435), HT001 exhibits weaker cytotoxicity than DTX.

The anti-tumor effects of HT001 and DTX were also compared in the A549, MCF-7, and SKOV-3 xenograft mouse models. Compared with normal saline group, tumor growth was inhibited in a dose-dependent manner when treated with HT001 or DTX in three tumor mouse models, and generally the anti-tumor effect of HT001 was comparable or superior to DTX at the same dose and frequency (Figures 3A,C,E). Tumor photographs of all animals at end of studies are shown in Supplementary Figures S1A–C. Generally, tumor bearing mice presented a decrease in bodyweight at higher doses of HT001 or DTX, and Q2W dosing frequency resulted in a lower bodyweight loss than QW (Figures 3B,D,F). HT001 presented a lower animal bodyweight loss compared with DTX at same dose and dosing frequency. Notably, SKOV-3 tumor bearing mice treated with 40 mg/kg HT001 biweekly presented better anti-tumor efficacy and lower bodyweight loss than mice treated with 40 mg/kg DTX biweekly. This result indicates that the efficacy of HT001 is comparable to DTX while the safety of HT001 is superior than DTX, at least in some tumor mouse models that have been evaluated.

Malignant ascites is caused by intraperitoneal spread of solid tumor cells and results in a poor quality of life. Here, we further investigated the anti-tumor effects of HT001 in mouse models of malignant ascites to provide evidence for clinical development of HT001 to treat cancer patients with malignant ascites. Cisplatin (CDDP) is widely used in malignant ascites therapies. Thus, in vivo efficacies upon HT001 or CDDP treatment were compared in mouse hepatocyte cancer H22 and human ovarian cancer ES-2 ascites-bearing mice, respectively. Both CDDP and HT001 could prolong the survival of H22 ascites-bearing mice but HT001 showed a significantly better efficacy than CDDP at 5 mg/kg in both lifetime extension and bodyweight (reflects ascites weight) reduction (Figures 4A,B). In addition, a dose-dependent efficacy of HT001 was been observed. In a more aggressive ES-2 ascites mouse model, 5 mg/kg HT001 and 5 mg/kg CDDP showed comparable slight prolongation of mice survival while 20 mg/kg HT001 significantly prolonged the survival of ascites-bearing mice, compared with both normal saline group and 5 mg/kg CDDP group (Figure 4C). In summary, HT001 demonstrated dose-dependent activities in survival extension of ascites-bearing mice and HT001 was apparently more efficacious than CDDP.

To analyze the effects of HT001 on ascites formation, 6 ascites-bearing mice from each group were sacrificed 8 or 7 days after treatment in H22 or ES-2 ascites mouse model shown in Figure 4. As shown in Figures 5A,B, treatment of either CDDP or HT001 significantly reduced the ascites weight and cell counts compared with normal saline group in the H22 ascites mouse model. Notably, there was no ascites observed in all the mice analyzed after 8 days treatment of 20 mg/kg HT001. Similar results were observed in ES-2 ascites mouse model (Figures 5E,F).

The VEGF levels in plasma and ascites from H22 or ES-2 ascites-bearing mice after CDDP or HT001 treatment were measured by ELISA. Both CDDP and HT001 reduced the VEGF levels in plasma and ascites from H22 ascites-bearing mice (Figures 5C,D). Only HT001 but not CDDP reduced the VEGF levels in ascites from the more aggressive ES-2 ascites-bearing mice (Figure 5G). No reduction of VEGF levels was observed in plasma from ES-2 ascites-bearing mice upon CDDP or HT001 treatment (Figure 5H).

Together, the results demonstrate that HT001 dose-dependently inhibited ascites formation and reduced VEGF levels in H22 and ES-2 ascites-bearing mice, correlating with prolonging survival in those two ascites mouse models.

Due to potential linkages between tumor angiogenesis and ascites formation, the therapeutic effect of HT001 combined with Endostar was explored in the H22 ascites-bearing mice. As shown in Figure 6A, treatment with either CDDP or HT001 prolonged the survival of the H22 ascites-bearing mice similar to the effect seen in previous experiment. Combination of Endostar and HT001 further prolong the survival of mice. Accordingly, treatment of either CDDP or HT001 reduced ascites weight and cell counts while combination of Endostar and HT001 further inhibited ascites formation in mice (Figures 6B,C). Similar effects on VEGF reduction were also observed (Figures 6D,E). Thus, HT001 presented a synergistic anti-ascites efficacy in combination with Endostar in H22 ascites mouse model.

The mean plasma concentration-time curves of docetaxel after administration with a single dose of HT001 or DTX via intravenous injection in SD rats are shown in Figure 7A. The PK parameters in SD rats are listed in Table 2. There was no significant difference in the main PK parameters between different genders in each group of rats (data not shown). In the dose range of 2.5–10 mg/kg of HT001, the values of C_max and AUC_last both increased with increasing dose, and the ratio of increased exposure was higher than that of increased dose. At a dose of 5 mg/kg, the AUC_last values of docetaxel in rats after DTX administration were significantly lower than that of HT001 treatment group.

The mean plasma concentration-time curves of docetaxel after administration with a single dose of HT001 or DTX are shown in Figure 7B. The PK parameters in beagle dogs are listed in Table 2. There was no significant difference in the main PK parameters between different genders in each group of dogs (data not shown). At a dose of 1 mg/kg, the C_max, AUC_last, AUC_inf, Vd, and Cl values of docetaxel after HT001 treatment were approximately half of that in the DTX treatment group. To be noted, allergic reactions, such as skin flushing, swelling and redness of the eyelids, and shortness of breath, were observed in each dog of DTX group during administration, although this phenomenon was not observed in HT001 group.

The in vitro drug release behavior of HT001 was described previously and HT001 had a significantly slower drug release than DTX when evaluated by a dialysis method (Zhang et al., 2022). To further investigate the drug release in vivo, biodistribution of docetaxel in major organs was assessed in MCF-7 tumor-bearing Balb/c nude mice at 0.5, 2, 6, and 24 h after intravenous injection of 10 mg/kg HT001 or DTX, and the results are illustrated in Figures 8A–D, respectively. At 0.5 and 6 h, the docetaxel biodistributions between the HT001 group and the DTX group were approximately the same. At 2 and 24 h, the docetaxel biodistributions were slightly lower in HT001 groups in some of the organs. Overall, similar trend and drug distributions in various tissues of tumor-bearing mice were observed between HT001 and DTX.

In tolerability studies in SD rats treated with multiple doses of HT001, all rats received low (5 mg/kg) and middle (10 mg/kg) doses tolerated well to the drug with no mortality. In high (16 mg/kg) dose group, the animals experienced 33% bodyweight loss and one rat died on the last day of the study. Pathological analysis revealed that in high dose group, an increased mitosis in liver hepatocytes and renal pelvis epithelium, a decreased white pulp lymphocytes population and an increased lymphocyte mitosis in the spleen were present, while these abnormal changes were rarely observed in middle dose-treated rats and not observed at all in low dose-treated animals (Supplementary Figures S2A–C). All rats received multiple doses of 5 mg/kg and 10 mg/kg tolerated well. The toxicological findings of 16 mg/kg dosing group are likely due to the very high dose level of the drug. This dosage (16 mg/kg) represents approximately 22-fold of the equivalent minimal efficacious dose, therefore, it will less likely be reached in clinical trials.