The human osteoblast cell line hFOB1.19 and OS cell lines MG-63, U2OS, and 143B were procured from iCell Bioscience, Inc. Cells were cultured in Dulbecco's Modified Eagle Medium/F-12 (DMEM-F12), supplemented with 10% FBS (both Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 μg/ml streptomycin, under a 5% CO₂ atmosphere at 37°C.

A total of 20 eight-week-old male BALB/c nude mice (weighted 18-20 g) were obtained from SPF (Beijing) Biotechnology Co., Ltd. and acclimatized for 1 week. The mice were housed at constant room temperature (60-65% humidity, 23±2°C) with a 12/12-h light/dark cycle and free access to standard chow and water. The research protocol received approval from the Medical Ethics Committee of the First People's Hospital of Nanning (Guangxi, China; approval no. 2021-076-01) and complied with the guidelines of the National Institutes of Health Animal Care and Use Committee (37).

A total of 700.00 4-hydroxymethyl PBAE, 946.25 DA, 76.95 4-dimethylaminopyridine (DMAP), 733.32 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 79.67 mg triethylamine were weighed and dissolved in 10 ml of dichloromethane and nitrogen was injected using a syringe to protect the system. Following 18 h reaction at room temperature, DA-B was obtained. A total of 700.00 DA-B, 761.32 NaIO₄ 190.40 mg and NH₄Cl (was dissolved in acetone (10 ml), with an equivalent volume of water. The reaction mixture was refluxed at 60°C overnight, resulting in the synthesis of terminal hydroxylated DA-B. The hydroxylated DA-B (30 mg) was reacted with DEX (115.18 mg) under 4A molecular sieve (200 mg) and nitrogen protection at 60°C for 60 h. The molecular sieve was removed via filtration and the filtrate was slowly dripped into ice-cold ethanol. A white precipitate emerged, which underwent centrifugation at 1,006 × g for 5 min at room temperature. The upper layer was discarded, and the ice-cold ethanol was added, followed by centrifugation at 1,006 × g for 3 min at room temperature. After being washed three times in ethanol, the precipitate yielded the DA-B-DEX polymer. The synthetic route of the DA-B-DEX polymer is shown in Fig. 1B.

¹H nuclear magnetic resonance spectra of the DA-B-DEX polymers were determined at room temperature using a Nuclear Magnetic Resonance Spectrometer (Bruker), with deuterium oxide (D₂O) serving as the solvent and trimethylsilane (TMS) as the internal standard, and the reaction grafting rates were calculated based on the characteristic proton peaks (38). In the spectrum of hydrogen, the ratio of the area of hydrogen peaks at specific positions on the grafting group to the area of hydrogen peaks at certain characteristic positions on the main chain is termed as the grafting rates.

A total of 100 mg of the DA-B-DEX polymer was dissolved in ultra-pure water. Dialysis bags with a molecular weight cut-off of 3,500 KD were used for dialysis in ultra-pure water for 48 h, with the water refreshed every 4 h. Then, the solution in the dialysis bag was freeze-dried in a freeze-drying machine for 24 h to yield a white solid, designated DA-B-DEX micelles.

CMC was determined utilizing a pyrene fluorescent probe, as previously described (39). A total of 0.0051 g pyrene was dissolved in 25 ml methanol, yielding a 1.01×10⁻³ mol/l pyrene solution, which was diluted with methanol to 1.616×10⁻⁶ mol/l pyrene solution. The prepared 1.616×10⁻⁶ mol/l pyrene solution (150 μl) was transferred to 10 ml bottles, and the methanol was evaporated at 50°C for 5 min. A total of 10 ml DA-B-DEX solution (1×10⁻⁴, 1×10⁻³, 1×10⁻², 1×10⁻¹, 1, 10, 100 and 1,000 mg/ml) was added to each of the 10 ml bottles containing a trace amount of pyrene. The solution underwent ultrasound treatment in a 50°C water bath for 30 min, followed by standing at room temperature for 2 h. The fluorescence spectra of each solution were prepared for analysis by fluorescence photometer. The fluorescence photometer was set with a slit of excitation and emission at 5 nm, with a scanning range of 300-360 nm and an emission wavelength of 372 nm. The intensity of the emitted light at 333 (I333) and 339 nm (I339) excitation wavelengths were measured, regression curves were plotted, and the ratio of I339/I333 was calculated. The intersection of the two regression curves was calculated.

A total of 91.5 DA-B-DEX and 8.5 mg GNE-477 (8.5 was co-dissolved and added to PBS buffer (100 ml). The resulting solution was then transferred to a dialysis bag with an interception relative molecular weight of 3,500 kDa and underwent dialysis in ultra-pure water for 48 h, with the water changed every 4 h. The suspension was filtered using 5.00 and 0.45 μm syringe filters and freeze-dried (Labconco Freezone 6), yielding a white powder named GNE-477@DBD.

To evaluate the morphological characteristics of GNE-477@DBD, samples were dropped onto a copper grid and negatively stained at room temperature for 2 min using 2% UO₂ acetate aqueous solution. Following drying, morphology was observed under a transmission electron microscope (TEM; FEI Talos F200S; Thermo Fisher Scientific, Inc.). Additionally, particle size distribution was determined by a laser particle sizer (Zetasizer Nano ZS ZEN3600, Malvern Instruments, Ltd.).

The prepared drug-loaded micelles were dissolved in the following solutions: i) Pure PBS (pH=7.4), PBS solution containing ii) 10 or iii) 50 μmol/l H₂O₂; iv) simulated body fluid (SBF, pH=7.4) and v) simulated tumor microenvironment (pH=6.5, 100 μM H₂O₂). These solutions were sealed in dialysis bags with a cut-off relative molecular weight of 3,500 kDa and dialyzed for 50 h in 3,000 ml PBS buffer at 37°C. A total of 1 ml GNE-477@DBD sample was taken and replaced with 1 ml fresh PBS solution to maintain solution volume. The released GNE-477 was quantified using high-performance liquid chromatography (HPLC; Agilent 1260, Agilent Technologies, Inc.). GNE-477 were filtered using 0.2 μm cellulose acetate filters and analyzed using an Eclipse XDB-C18 column (150.0×4.6 mm; 5 μm particle size) operated at 25°C. The mobile phase consisted of 2% (v/v) acetic acid in water (mobile phase A) and 0.5% acetic acid in water and acetonitrile (10:90 v/v) (mobile phase B), and at a flow rate of 1.0 ml/min. The injection volume was 10 μl and the wavelength of the UV detector is set at 280 nm. The quantification was performed by applying the standard calibration curve.

The uptake of GNE-477@ DBD was assessed in MG-63, U2OS and 143B cell lines following 2 and 6 h incubation. Cells were seeded in a 2-multiwell plate at a density of 2×10⁴ cells/well (2 ml/well) and cultured in DMEM for 24 h at 37°C and 5% CO₂. Culture medium was replaced with a dispersion of GNE-477@ DBD in fresh DMEM (0.25 mg/ml, 1 ml/well), and the cells were incubated for 2 or 6 h at room temperature. Upon reaching ~90% confluence, cells were treated with Nile Red (HY-D0718, MedChemExpress)-loaded GNE-477 for 1 h at room temperature, fixed and stained with DAPI for 10 min at room temperature using the method described previously (40). Finally, the slides were analyzed using a Leica TCS SP8 confocal laser scanning microscope (Leica-Microsystems GmbH). Nile Red was excited with a 560 nm laser and emission was collected at 570-620 nm (41).

hFOB1.19, MG-63, U2OS and 143B cells were cultured in a complete RPMI-1640 medium supplemented with 10% FBS, 100 IU/ml penicillin and 100 mg/ml streptomycin (all Gibco; Thermo Fisher Scientific, Inc.). The cells were seeded in 96-well plates (2×10⁴ cells/well) and incubated overnight at 37°C and 5% CO₂. At 24, 48 and 72 h, 10 μl MTT solution (Wuhan Biofavor Biotechnology Service Co., Ltd.) was added for 4 h at room temperature and the medium was removed. The formazan crystals were dissolved in 150 μl DMSO. Blank wells were used as blank groups. The optical density (OD) at 560 nm was measured by a microplate reader. Finally, the cell viability was calculated as follows: Cell viability (%)=OD of treated cells/OD of control ×100. The experiment was repeated three times.

The apoptotic rate (early + late apoptosis) and cell cycle distribution were assessed using flow cytometry. For apoptosis assessment, MG-63, U2OS and 143B cells subjected to different treatments (Control, GNE-477, DBD, and GNE-477@DBD) were suspended in binding buffer and incubated with 5 Annexin V-FITC and 10 μl propidine iodide (PI; BD Biosciences) for 15 min at room temperature away from light. The apoptotic cells were determined on an Attune™ NxT flow cytometer (Thermo Fisher Scientific, Inc.), and results were analyzed using FlowJo software (version 9.3.2; FlowJo LLC). For cell cycle distribution, cells were fixed in 75% ethanol for 2 h at 4°C and treated with RNase A for 40 min at room temperature. Staining with PI for 40 min at room temperature, followed by detection on an Attune™ NxT flow cytometer (Thermo Fisher Scientific, Inc.), and Attune NxT Software (version 3.2.1; Thermo Fisher Scientific, Inc.) to determine the percentage of cells in G0/G1, S and G2/M phases.

Protein samples were extracted from OS cells or tissue using RIPA buffer (BioTeke Corporation), and the protein content was measured using a BCA protein quantification kit (Beyotime Institute of Biotechnology). 10 μg of protein from each sample was loaded and separated by 10% SDS-PAGE (Sinopharm Chemical Reagent Co., Ltd.), followed by transfer onto PVDF membranes. The membrane was blocked with 5% skimmed milk in Tris-buffered saline at room temperature for 1 h, followed by overnight incubation at 4°C with primary antibodies (all Cell Signaling Technology, Inc.) against caspase 3 (1:1,000; cat. no. 9662), cleaved-caspase 3 (1:1,000; cat. no. 9661), Bax (1:1,000; cat. no. 2772), Bcl-2 (1:1,000; cat. no. 15071), Cyclin D1 (1:1,000; cat. no. 2922), Cyclin E (1:1,000; cat. no. 20808), AKT (1:1,000; cat. no. 9272), phosphorylated-AKT (p-AKT; 1:500; cat. no. 9271), mTOR (1:1,000; cat. no. 2972), p-mTOR (1:1,000; cat. no. 2971) and GAPDH (1:1,000; cat. no. 2118). After washing by TBST (Tris-buffered saline with 0.1% Tween 20), the membrane was incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (Abcam; 1:10,000; cat. no. ab205718) for 2 h at room temperature. Protein bands were visualized using enhanced chemiluminescence reagent (Thermo Fisher Scientific, Inc.) and semi-quantification was performed using Image J software (version 1.54i 03; National Institutes of Health).

Total RNA from OS cell lines was isolated using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.), followed by RT into cDNA using PrimeScript™ RT reagent kit (TransGen Biotech Co., Ltd.), according to the manufacturer's protocol. Next, qPCR was performed using AceQ qPCR SYBR Green Master Mix (MedChemExpress LLC). The analysis of mRNA expression levels was conducted by the 2^−ΔΔCq method (42), with GAPDH as the internal reference. The sequences of the primers were as follows: Caspase 3, forward, 5′-CGTGCTTCTAAGCCATGGTG-3′ and reverse, 5′-GTCCCACTGTCCGTCTCAAT-3′; Bax, forward, 5′-TGCCTCAGGATGCATCTACC-3′ and reverse, 5′-AAGTAGAAAAGCGCGACCAC-3′; Bcl-2, forward, 5′-AGGGCATTCAGTGACCTGAC-3′ and reverse, 5′-CGATCCGACTCACCAATACC-3′; Cyclin D1, forward, 5′-ACCCGACGAGTTACTGCAAAT-3′ and reverse, 5′-TCTGTTTGGTGTCCTCTGCC-3′; Cyclin E, forward, 5′-AGAGGAAGGCAAACGTGACC-3′ and reverse, 5′-TATTGTCCCAAGGCTGGCTC-3′ and GAPDH, forward, 5′-AGGCCGGTGCTGAGTATGTC-3′ and reverse, 5′-TGCCTGCTTCACCACCTTCT-3′. The thermocycling conditions were as follows: Initial denaturation at 96°C for 5 min, followed by 40 cycles of 95°C for 30 sec and 68°C for 20 sec.

U2OS tumor-bearing Balb/c mice were generated by subcutaneously injecting U2OS cells (2×10⁶/mouse). Then, the mice were randomly divided into four groups (n=5/group) as follows; Control, GNE-477, DBD and GNE-477@DBD. Every 2 days, the state of the mice was observed and the body weight was determined. In addition, the tumor size was measured using vernier calipers and tumor volume was calculated as follows: Tumor volume=0.5x length × width². Once the inoculated tumor volume reached 100-200 mm³, 25 mg/kg GNE-477, DBD@GNE-477 (containing 25 mg/kg GNE-477), or an equivalent volume of vehicle control (DBD) and negative control (PBS) were administered intraperitoneally. Weight loss ≥20% within a short period or the average tumor diameter exceeds 20 mm, the experiment was terminated and mice were euthanized. Mice were anesthetized after 34 days using 3% isoflurane and then euthanized by cervical dislocation. Cessation of heartbeat and breathing were considered to confirm death. The heart, liver, spleen, lung, kidney and tumor were removed, washed with saline, weighed, and fixed for 24 h at 4 °C in 4% formaldehyde for subsequent staining.

To evaluate the potential side effects of drugs on organs, H&E staining was performed. The fixed heart, liver, spleen, lung and kidney tissue was dehydrated, embedded in paraffin and cut into 4 μm-sections. Thereafter, the samples were stained with hematoxylin (Beijing Solarbio Science & Technology Co., Ltd.) for 8 min at room temperature and rinsed with running water. Samples were differentiated with 5% acetic acid for 1 min, washed with running water for 10 min, stained with eosin (Beijing Solarbio Science & Technology Co., Ltd.) for 1 min at room temperature, and rinsed three times with running water for 5 min. Finally, the images were captured under a light microscope (magnification, ×100).

All data are presented as the mean ± SD. GraphPad PRISM 8.0 software (Dotmatics) was used for statistical analysis by one- or two-way ANOVA followed by Tukey's post hoc test. Each sample was evaluated in a minimum of three experimental replicates. P<0.05 was considered to indicate a statistically significant difference.

The ¹H-nuclear magnetic resonance (NMR) spectra of DA-B, hydroxylated DA-B, DEX and DA-B-DEX conjugates are shown in Fig. 1C. In the spectrum of DA-B, the characteristic single peak of pinacol ester (1.24 ppm) and the two characteristic peaks of benzene rings were clearly discernible. The chromatogram of hydroxylated DA-B exhibited disappearance of the single peak at 1.24 ppm, confirming the removal of the protective group (pinacol ester). Hydroxylated DA-B was successfully grafted onto DEX, yielding a calculated grafting rate of ~3% (Fig. 1C), meeting the general requirements for formation of amphiphilic micelles (grafting rate <10%, ensuring the solubility of the micelles) (43).

As in Fig. 2A, the intersection of the two regression curves for the I339/I333 ratio of DA-B-DEX pyrene solution was at (−1.828,0.5965). CMC of DA-B-DEX micelles was 0.01486 mg/ml; the low CMC signified that DA-B-DEX readily formed micelles and maintained the core-shell structure under highly diluted conditions. The degradation process of DA-B-DEX micelles is shown in Fig. 2B. In the absence of H₂O₂, almost all drug-loaded micelles were spherical and intact, whereas exposure to 100 μmol/l H₂O₂ resulted in notable disruption of micelle structure, accompanied by an alteration in the particle size distribution of the micelles towards inhomogeneous (Fig. 2C). The in vitro release profile of GNE-477 revealed a significant disparity in drug release from DA-B-DEX in PBS, 10 and 50 μmol/l H₂O₂ environments. At 2 h, drug release in the PBS group was 20%, while it reached 41 and 85% at 10 and 50 μmol/l H₂O₂, respectively (Fig. 2D). TEM was conducted to assess rupture and release of GNE-477@DBD under conditions of SBF (pH=7.4) and tumor environment (pH=6.5, 100 μM H₂O₂). The micellar structure of GNE-477@DBD was more susceptible to disruption in the tumor environment (Fig. 2E), exhibiting a higher drug release rate compared with SBF (Fig. 2F). These findings indicated that the DA-B-DEX micelles ruptured and released the drug faster in the presence of H₂O₂, thus DA-B-DEX micelles were H₂O₂-responsive.

Following 2 h incubation, free GNE-477 was distributed in both cytoplasm and nucleus, while in the GNE-477@DBD group, GNE-477 was predominantly located in the cytoplasm, exhibiting a weaker fluorescence intensity compared with the GNE-477 group. Following 6 h incubation, the fluorescence intensity of GNE-477 in the GNE-477@DBD group notably increased, indicating a progressive internalization of more GNE-477@DBD into the cells, followed by sustained release of GNE-477 in the cells (Fig. 3A-C).

Normal human osteoblast hFOB1.19 and MG-63, U2OS and 143B OS cells were treated with PBS, GNE-477, DBD and GNE-477@DBD for 24, 48, and 72 h. The findings revealed that DBD and GNE-477@DBD exhibited good biocompatibility and low toxicity to normal cells (Fig. 4A) and both GNE-477 and GNE-477@DBD demonstrated inhibitory effects on OS cell viability (Fig. 4B-D). Following 24 h drug treatment, flow cytometry showed that compared with Control, both GNE-477 and GNE-477@DBD induced apoptosis in OS cell lines (Fig. 5) and elevated the proportion of cells in G1 phase, suggesting that GNE-477 and GNE-477@ DBD blocked cell division at G1 phase and prevented cell cycle progression (Fig. 6). Furthermore, protein and mRNA levels of cell apoptosis- and cell cycle-associated genes were verified by western blot and RT-qPCR in U2OS cells (Fig. 7A and B). Following 24 h drug treatment, compared with the Control group, the expression of Bcl-2 and cyclin D1 and E protein and mRNA was downregulated, while Bax and caspase 3 protein and mRNA expression levels were upregulated in the GNE-477 and GNE-477@DBD groups, indicating that GNE-477 exerted anti-tumor effects following encapsulation in H₂O₂-responsive nano micelles. Additionally, western blot analysis revealed that GNE-477 and GNE-477@DBD inhibited expression of proteins involved in the Akt/mTOR cascade response, confirmed by Akt agonist treatment (Fig. 7C).

A nude mouse model was established to evaluate in vivo anti-tumor ability of GNE-477@DBD. GNE-477 and DBD@ GEN-477 groups demonstrated tumor regression and impeded tumor growth compared with the Control group, notably, the anti-tumor efficacy of GNE-477@DBD surpassed that of free GNE-477 (Fig. 8A and B). There were no significant differences in the body weight of mice between groups, thereby indicating the safety profile of this nanoscale drug delivery system (Fig. 8C). Moreover, western blot results corroborated that protein expression of p-AKT, p-mTOR, Bcl-2 and cyclin D1 and E decreased in the GNE-477 and GNE-477@DBD groups, while protein expression of Bax and cleaved-caspase 3 increased. These results suggested that treatment with GNE-477 and GNE-477@DBD suppressed PI3K/AKT/mTOR pathway activation and facilitated apoptosis of tumor cells (Fig. 8D). Histopathological examination of major organs stained with H&E demonstrated varying degrees of damage in the GNE-477 group, whereas both the DBD and GNE-477@DBD groups exhibited no discernible organ damage, indicating that DBD nano micelles had favorable biocompatibility and mitigated organ damage caused by GNE-477 (Fig. 9).