Oral Poly(N-oxide) zwitterionic nanoplatform for Gambogenic Acid Enhances Mucosal penetration for potentiated anti-angiogenic therapy.

1
Introduction
Hepatocellular carcinoma (HCC), the most prevalent form of primary liver cancer, remains a major global health burden and is characterized by aggressive biological behavior and high mortality (Bray et al., 2024; Siegel et al., 2024). As a vascular-rich malignancy, HCC is highly dependent on aberrant angiogenesis, making anti-angiogenic therapy a central element of first-line treatment for advanced HCC (Gordan et al., 2024; Gordan et al., 2020; Jiang et al., 2025a). Despite their clinical convenience and favorable safety profile, orally administered anti-angiogenic drugs achieve limited therapeutic benefit, with a five-year survival rate of only 18% (Gordan et al., 2020). This unsatisfactory efficacy is primarily attributable to poor intestinal absorption and insufficient tumoral accumulation (Baek et al., 2023; Di Benedetto et al., 2025; Liu et al., 2023). Furthermore, because tumor vasculature is the predominant route for drug delivery, vascular pruning induced by anti-angiogenic therapy may paradoxically restrict subsequent drug influx into tumors, thereby diminishing therapeutic reinforcement (de Palma et al., 2017; Teleanu et al., 2020; Wang et al., 2021b). Strategies capable of simultaneously enhancing gastrointestinal absorption and sustaining tumor accumulation are therefore urgently needed to improve clinical outcomes.
Oral nanomedicines face the particularly stringent requirement of overcoming both the mucus barrier and the intestinal epithelial barrier to achieve meaningful systemic exposure (Ensign et al., 2012; Kubiatowicz et al., 2025; Spleis et al., 2023). While PEGylation has long been used to increase the hydrophilicity and mucus diffusivity of nanocarriers (Huckaby and Lai, 2018; Yamazoe et al., 2021), PEG coatings typically reduce interactions with epithelial cell membranes, resulting in minimal enterocyte uptake and low oral bioavailability (Nishioka et al., 2025; Veider et al., 2023). To address these limitations, Shen et al. pioneered the development of poly(N-oxide) zwitterionic polymers (Chen et al., 2021; Sun et al., 2025; Wei et al., 2025), a new class of PEG-alternative materials with dual functionalities advantageous for oral delivery (Fan et al., 2022; Zhang et al., 2024). These polymers are nearly charge-neutral and highly protein-resistant, exhibiting excellent non-fouling properties that enable efficient mucus penetration. More importantly, they possess a unique ability to weakly and reversibly bind cell membranes through hydrogen bonding with phospholipid headgroups, thereby promoting adsorptive-mediated transcytosis without disrupting enterocyte integrity. This rare combination, simultaneously minimizing mucus adhesion and enhancing enterocyte uptake, has been experimentally validated, accompanied with prolonged circulation, and improved tumor accumulation (Fan et al., 2022; Zhang et al., 2024; Zhang et al., 2023a; Zheng et al., 2025). Based on these properties, we reasoned that poly(N-oxide) zwitterionic nanocarriers could serve as an ideal platform for the oral delivery of poorly absorbed anti-angiogenic agents.
Gambogenic acid (GNA), a cytotoxic compound derived from gamboge, exhibits both anti-angiogenic and vascular-disruptive activities and uniquely induces endothelial gap widening, thereby enhancing vascular permeability (Asano et al., 1996; Chen et al., 2020; Jun-Zeng Huang et al., 2021; Tang et al., 2018; Wang et al., 2022; Yan et al., 2012; Zha et al., 2020). This dual vascular modulation offers a compelling opportunity: if systemically delivered in a controlled manner, GNA could simultaneously inhibit tumor angiogenesis and facilitate self-reinforcing intratumoral drug accumulation (Deng et al., 2024b; Du et al., 2023). Previous nanoparticle-based GNA formulations have shown improved efficacy and reduced systemic toxicity; however, their injectable nature raises concerns regarding vascular irritation of GNA at the administration site (Deng et al., 2024b; Du et al., 2023; Zha et al., 2020), and oral formulations have thus far been limited by poor gastrointestinal absorption and unsatisfactory systemic exposure (Banna et al., 2010; Deng et al., 2024a; Sun et al., 2023; Wang et al., 2021a; Zhang et al., 2023b). Therefore, a rationally engineered oral delivery system capable of overcoming mucus and epithelial barriers while preserving GNA's vascular-modulating functions could substantially potentiate its therapeutic performance.
In this work, we constructed an oral poly(N-oxide) zwitterionic nanoplatform of poly[2-(oxide diethylamino)ethyl methacrylate]–polycaprolactone (PODEA-PCL, short as POC) for GNA encapsulation, yielding GNA-loaded nanoparticles termed POC-GNA. POC-GNA exhibited robust stability in the gastrointestinal tract, efficient mucus penetration, and improved enterocyte uptake, enabling markedly enhanced oral absorbability. Following systemic entry, POC-GNA modulated tumor vasculature by inducing both vascular pruning and permeability enhancement, thereby overcoming the vascular density–dependent drug delivery limitation and promoting their intratumoral accumulation. This vascular modulation further potentiated GNA's anti-angiogenesis, vascular disruption and immunoregulation for improved tumor inhibition (Scheme 1). Collectively, this work integrates poly(N-oxide) zwitterionic polymers with GNA yields an oral synergistic therapeutic platform capable of significantly improving anti-angiogenic therapy in HCC.

2
Materials and experimental methods
2.1
Materials
2-(Diethylamino)ethyl methacrylate (DEA), meta-chloroperoxybenzoic acid (mCPBA), dodecanol, ε-caprolactone (ε-CL), triethylamine, 2-bromoisobutyryl bromide and N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDETA) were purchased from Macklin. Toluene and ε-CL were dried with CaH2 for 2 h, then distilled under ordinary and reduced pressure, respectively. Polyethylene glycol-polycaprolactone (PEG-PCL) was prepared in our lab according to previous literature. Other reagents were achieved from Sinopharm and used as received.

2.2
Synthesis of ODEA
The nitrogen‑oxygen zwitterionic monomer was synthesized via oxidation of tertiary amino (Scheme 2). DEA (3.14 g, 20 mmol) and mCPBA (5.18 g, 30 mmol) were dissolved in 30 mL and 50 mL of dichloromethane, respectively. DEA solution was added dropwise to mCPBA solution under ice-bath conditions in 30 min. The reaction was carried out at room temperature for 5 h. After the reaction, the solvent was removed by rotary evaporation, and the residue was purified by passing through a neutral alumina column, yielding a pale yellow, clear liquid. This liquid was identified as 2-(oxide diethylamino)ethyl methacrylate (ODEA) by 1H NMR analysis (Bruker AVANCE NEO 600 M, Germany).

2.3
Synthesis of PCL—Br
The polycaprolactone with hydroxyl group (PCL-OH) was firstly prepared by ring-opening polymerization of caprolactone initiated by dodecanol. Specifically, ε-CL (9.12 g, 80 mmol) and dodecanol (373 mg, 2 mmol) were mixed, and dehydrated by reduced pressure co-distillation of anhydrous toluene for three times. Subsequently, 20 mL anhydrous toluene containing stannous octoate (81 mg, 0.2 mmol) was added to the reaction system, which was allowed to reacted at 105 °C for 7 h. After reaction, the resultant solution was precipitated in petroleum ether for three times and dried in vacuum. The product of PCL32-OH (7.02 g, yield 73.89%) was obtained as white solid and characterized with 1H NMR spectrum.
Polycaprolactone with α-bromide functional group (PCL—Br) was synthesized via esterification and used as macromolecular initiator (Scheme 3). 2-Bromoisobutyryl bromide (1.38 g, 6 mmol) in 10 mL dichloromethane was dropwise added to the mixture solution of PCL-OH (2.40 g, 0.6 mmol), triethylamine (0.61 g, 6 mmol) and 20 mL dichloromethane under ice-bath condition. The esterification was carried out at 0 °C for 1 h and at room temperature for a further 48 h. The reaction solution was successively filtered, distilled to remove the solvent, redissolved in tetrahydrofuran (THF), passing through Alumina column, concentrated by rotary evaporation and precipitated for three times in petroleum ether. The product of PCL32-Br (1.94 g, yield 82.03%) was achieved as white solid and characterized with 1H NMR spectrum. Its apparent number-average molar mass (Mn) and polydispersity index (PDI) were determined in N,N-dimethylformamide (DMF) at 25 °C on gel permeation chromatography (GPC) instrument (Waters 1515) equipped with three Waters Styragel columns (HR2, HR4, and HR6) and a Wyatt WREX-02 refractive index (RI) detector using a conventional universal calibration with linear polystyrene standards.

2.4
Synthesis of PODEA-PCL block copolymer
Poly[2-(oxide diethylamino)ethyl methacrylate]-polycaprolactone (PODEA-PCL) was prepared via atom transfer radical polymerization (ATRP) of 2-(oxide diethylamino)ethyl methacrylate (ODEA) using PCL32-Br as macromolecular initiator. PCL32-Br (199 mg, 0.05 mmol), ODEA (401 mg, 2 mmol) and PMDETA (8.65 mg, 0.05 mmol) were dissolved in 2 mL anhydrous DMF and charged in a 10 mL sealing tube. After three cycles of freeze–vacuum–thaw, CuBr (7.2 mg, 0.05 mmol) was added to the sealing tube and vacuumed for 15 min. The sealing tube was sealed and the reaction was carried out at 40 °C for 72 h. The resultant mixture was successively diluted with THF, oxidized in air for 5 min, passing through alumina column to remove cupric salt, concentrated by rotary evaporation, dialyzed in water and freezing dried. PODEA8-PCL32 (126 mg, yield 48.46%) was obtained as light-yellow powder and characterized with 1H NMR spectrum and GPC instrument.

2.5
Self-assembly and characterizations of oral GNA nanoparticles
The GNA was loaded in PODEA-PCL nanoparticle via co-assembly with PODEA-PCL by ultrasonic emulsification. PODEA-PCL (5 mg) and GNA (1 mg) were dissolved in 200 μL ethyl acetate, added to 1 mL water, ultrasonically emulsified at 80 W for 2 min, and finally removed ethyl acetate by rotary evaporation to achieve PODEA-PCL-GNA (short as POC-GNA) nanoparticle. In similar method, PEG-PCL-GNA (short as PEC-GNA) nanoparticle was prepared and used as nanoparticle control group. The morphologies of these GNA nanoparticles used immediately or after stored at room temperature for 24 h were observed by transmission electron microscope (TEM, Hitachi HT7700, Japan), while their sizes, zeta-potentials and stability were determined by dynamic light scattering (DLS, detected by Malvern Zetasizer, UK). The drug loading (DL) and encapsulation efficiency (EE) were measured by ultrahigh performance liquid chromatography (UPLC, Agilent 1290 Infinity II, USA) with the eluent composed of methanol: aqueous solution containing 0.1% formic acid = 9:1 and calculated via the following equations:where M0, M1 and M2 represented the quality of total GNA, loaded GNA and polymeric nanocarrier, respectively.

2.6
Drug release profiles of GNA nanoparticles
The drug release behaviors of GNA nanoparticles were executed via centrifugal precipitation method. Specifically, POC-GNA or PEC-GNA nanoparticle containing 1 mg GNA was dispersed in 10 mL different types of medium with 0.5% Tween 80, where the release medium included PBS (pH 7.4) used to simulate the normal physiological condition, simulated gastric juice (Yuanye Biotechnology, pH 1.2) and simulated intestinal juice (Yuanye Biotechnology, pH 6.8). The GNA dispersions were shaken at 37 °C with the rate of 220 rpm. At pre-set time points (5 min, 10 min, 20 min, 30 min, 45 min, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 24 h and 48 h), the GNA nanoparticle dispersions were centrifugated at 5000 rpm for 3 min to remove the sediment of liberated GNA and 0.5 mL supernatant was collected for the measurement of GNA concentration in nanoparticles via UPLC. The cumulative release of GNA (Q %) was calculated via the following equation:where m, V0, Vn and cn represented initial total quality of GNA, initial volume of GNA dispersion, the volume and concentration of GNA dispersion at the nth collection, respectively.

2.7
Cell lines, animals and ethics statement
Human Umbilical Vein Endothelial Cells (HUVECs) were purchased from the China Center for Type Culture Collection. Hepatoma cell lines of Hepa1–6 (mouse) and Hepa1–6-luc (mouse, luciferase expression) were obtained from Cell Bank of Chinese Academy of Sciences in Shanghai. The cells were cultured in DMEM supplemented with 10% fetal calf serum (FBS) and 1% penicillin–streptomycin, incubated at 37 °C in a humidified atmosphere with 5% CO₂. Healthy Sprague Dawley (SD) rats (Male, ∼8 weeks old, 200 ± 20 g), C57BL/6 mice (Male, ∼7 weeks old, 18–20 g) and New Zealand white rabbits (Male, ∼9 weeks-old, 2.5 ± 0.2 kg) were sourced from Anhui Medical University (2017–001, Hefei, Anhui, China), Hangzhou Ziyuan Experimental Animal Technology Company (2019–004, Hangzhou, Zhejiang, China) and Nanjing Pukou District Laifu Breeding Farm (SCXK (Su) 2019–0005, Nanjing, Jiangsu, China), respectively. The animals were housed in controlled conditions (20–25 °C, 60–70% humidity) with a 12-h light/12-h dark cycle, provided with sufficient food and water, and allowed to acclimate for at least one week prior to experiments. All procedures were approved by the Animal Ethics Committee of Anhui University of Chinese Medicine (AHUCM-rats-2,021,016, AHUCM-mice-2,021,031 and AHUCM-rabbit-20,230,050).

2.8
Cellular uptake assays
Hepa1–6 cells (3 × 105 cells in 2 mL culture medium per well) were seeded in 6-well plate and incubated overnight for cell attachment. Subsequently, the culture medium was replaced by 2 mL PBS buffer containing free GNA, PEC-GNA nanoparticle or POC-GNA nanoparticle at the GNA concentration of 4 μg/mL. Hepa1–6 cells were incubated for an additional 8 h, washed with PBS buffer and then 250 μL of cell lysis buffer containing protease inhibitor was added to each well. The plates were kept on ice for 40 min, and cells were gently collected into centrifuge tubes. Cell disruption was performed with ultrasonic treatment at a cycle of 5 s: 3 s: 5 s, followed by centrifugation for 10 min. A 100 μL aliquot of the supernatant was taken for the measurements of the GNA and protein concentrations by the following method. The drug uptake of the cells was calculated as the ratio of GNA concentration to protein concentration.
For the measurement of GNA concentrations, 100 μL of supernatant was transferred to a 1.5 mL centrifuge tube, and 10 μL of an 8 μg/mL GA solution was added as internal standard, followed by vortexing for 1 min. Next, 1 mL of ethyl acetate was added to each tube, vortexed for 3 min, and centrifuged at 3500 rpm for 15 min. 0.8 mL of the supernatant was transferred to a new 1.5 mL centrifuge tube and dried under nitrogen stream. The dried residue was then resuspended in 100 μL of HPLC-grade methanol, vortexed for 1 min, and centrifuged at 10000 rpm for 15 min. Finally, 70 μL of the supernatant was transferred to an insert for UPLC concentration analysis. Establishment of the GNA Standard Curve: 100 μL of blank cell lysate was mixed with GNA standard solutions to prepare a series of concentrations at 50, 20, 10, 5, 2, and 1 μg/mL. A 100 μL aliquot of each standard solution was then combined with 10 μL of an 8 μg/mL GA solution, followed by processing and detection in the same method.
For the measurement of protein concentrations, 200 μL of BCA working reagent was added to each well along with 20 μL of each protein concentration sample in a 96-well plate. The plate was then incubated at 37 °C in an oven for approximately 30 min. Following incubation, absorbance was measured at 562 nm using a microplate reader. Establishment of the GNA Standard Curve: Standard protein concentrations of 0, 0.02, 0.04, 0.08, 0.16, 0.32, and 0.64 mg/mL were prepared. In a 96-well plate, 200 μL of BCA working reagent was added to each well along with 20 μL of each protein concentration sample, followed by incubation and detection in the same method. A standard curve was generated by plotting the absorbance values against the protein concentrations.
To investigate the cellular uptake mechanisms of OPDEA-PCL nanoparticles, Hep1–6 cells or Caco-2 cells were seeded in 24-well plates at a density of 5 × 104 cells per well and cultured for 24 h to allow attachment. The cells were then pretreated for 2 h with different endocytosis inhibitors, including chlorpromazine (30 μM), methyl-β-cyclodextrin (MβCD, 5 mg/mL), amiloride (10 μg/mL), or cytochalasin D (5 μM), while untreated cells served as the control group. To evaluate the energy dependence of nanoparticle uptake, parallel experiments were performed at 4 °C under otherwise identical conditions. Following pretreatment, OPDEA-PCL nanoparticles loaded with Nile red were added to each well and incubated for 1 h at 37 °C. After incubation, cells were washed three times with cold phosphate-buffered saline (PBS) to remove non-internalized nanoparticles, detached using trypsin, and collected by centrifugation at 1000 rpm for 3 min. The cell pellets were resuspended in 0.3 mL PBS for analysis. All experiments were independently repeated three times. Cellular fluorescence intensity was quantified by flow cytometry (Beckman Coulter CytoFLEX S, USA), and data were analyzed using FlowJo software. Results are presented as mean ± standard deviation (SD).

2.9
Cell viability assays
An MTT colorimetric assay was used to assess the inhibitory effects of free GNA, PEC-GNA and POC-GNA nanoparticles on the viability and growth of Hepa1–6 cells. Hepa1–6 cells (1 × 104 cells in 100 μL culture medium per well) were seeded in 96-well plates overnight, and then treated with the DMEM medium containing free GNA, PEC-GNA and POC-GNA nanoparticles at the GNA concentrations of 0.5, 1, 2, 4, 8, 16, and 32 μg/mL for 48 h. The drug-containing medium was replaced with serum-free blank medium. Next, 10 μL of MTT solution was added to each well, and the cells were incubated in the dark for an additional 4 h. Then, 150 μL of DMSO was added to dissolve the formazan crystals, and shake for 10 min to dissolve thoroughly. The absorbance was measured using a microplate reader (Molecular Devices SpectraMax iD3, USA). Cell viability was calculated based on the absorbance readings. Additionally, the same method was employed to evaluate the toxicity of the zwitterionic nanocarrier to Hepa1–6 cells at concentrations of 25, 50, 100, 250, 500, and 1000 μg/mL.

2.10
Vascular tube formation/disruption assays
HUVECs were seeded at a density of 5000 cells per well in 100 μL medium onto a Matrigel-coated 96-well plate. Cells were then treated with phosphate-buffered saline (PBS), free GNA, PEC-GNA, POC-GNA, or Sorafenib at the GNA/Sorafenib concentration of 4 μg/mL, respectively. Following incubation for 4 h, tube formation by HUVECs was assessed and recorded using bright-field microscopy (YUESCOPE YP710, China).
To evaluate vessel disruption, HUVECs were first cultured on a Matrigel-coated 96-well plate under the same initial seeding and culture conditions as described above. After an initial incubation period of 4 h to allow the formation of clearly visible tubular structures, cells were subsequently treated with different formulations and further incubated for 5 h, after which the disruption of the preformed tubular networks was observed and documented via bright-field imaging.

2.11
Intestinal mucosal penetration studies
Caco-2 cells were seeded in the upper chambers of Transwell inserts and cultured until a confluent monolayer formed. Subsequently, sterile coverslips were placed into the lower chambers, onto which Hepa1–6 cells were seeded. Once cells in both upper and lower chambers reached the appropriate density, the culture medium from the upper chambers was discarded. Simulated intestinal mucus (Mucin solution 200 μg/mL, 100 μL per well) was then added to the upper chambers, followed by incubation at 37 °C for 30 min prior to nanoparticle treatment. Afterwards, free Nile Red (NR), NR-loaded PODEA-PCL and PEG-PCL nanoparticles, at a final NR concentration of 400 μg/mL (200 μL per well), were administered and incubated for 4 h in the dark. Following incubation, the medium in the lower chambers was discarded, and the coverslips were gently washed with ice-cold phosphate-buffered saline (PBS). Cells were subsequently fixed in 4% paraformaldehyde solution for 15 min on a shaker. After fixation, coverslips underwent three washes with PBS and then carefully detached from the wells for fluorescence microscopy. A small volume (approximately 5 μL) of DAPI staining solution was placed onto a microscope slide. The cell-containing side of the coverslip was inverted onto the DAPI solution droplet, ensuring direct contact. Excess staining solution was carefully removed, and after slight drying, the edges were sealed using nail polish. The fluorescent images of cell samples were recorded by inverted fluorescence microscope (Nikon Ecipse Ts2, Japan).

2.12
Pharmacokinetic study
Thirty SD rats (male, ∼200 g) were fasted for 12 h with free access to water prior to administration. The rats were randomly divided into five groups (n = 6): and respectively administrated with free GNA orally (GNA, p.o.), free GNA intravenously (GNA, i.v.), PEC-GNA orally (PEC-GNA, p.o.), PEC-GNA intravenously (PEC-GNA, i.v.) group and POC-GNA orally (POC-GNA, p.o.). The dosage of each administered drug was 2 mg/kg according to GNA concentration. Following administration, blood samples (∼0.5 mL) were collected from the orbital venous plexus at specified time intervals into 1.5 mL anticoagulant tubes. Blood samples were kept on ice and centrifuged at 3500 rpm at 4 °C for 10 min. The resulting plasma, a clear pale-yellow liquid, was carefully transferred into a new EP tube. Each plasma sample (100 μL) was mixed with 10 μL of an 8 μg/mL GA internal standard solution and 1 mL of ethyl acetate, then vortexed for 5 min. After centrifuging, 0.8 mL of the supernatant was collected and dried under nitrogen. The residue was reconstituted with 100 μL of methanol, vortexed for 30 s, and centrifuged at 12000 rpm for 10 min. GNA concentrations of the supernatants were measured using UPLC under chromatographic conditions described in “Section 2.8”. Drug concentration-time profiles were plotted, with sampling time points (T) on the x-axis and drug concentration on the y-axis. The pharmacokinetic parameters, including the area under the concentration-time curve (AUC), peak concentration (Cmax), and elimination half-life (t1/2), were calculated using a non-compartmental analysis (NCA) model with WinNonlin Phoenix software (Certara, USA). The relative bioavailability (Frel) of the oral formulations was calculated comparing to the intravenously administered free GNA using the following equation:where AUCn and AUCGNA, i.v. indicate the AUC of the specific treated group and the GNA (i.v.) group, respectively.

2.13
Biodistribution studies
One hundred C57BL/6 mice (male, ∼20 g) were randomly divided into five groups (n = 20) and administered at the GNA dosage of 4 mg/kg. The groups included oral GNA group, injected GNA group, oral PEC-GNA group, injected PEC-GNA group and oral POC-GNA group. After administration, four mice in each group were euthanized at each pre-set time point (1 h, 2 h, 4 h, 8 h and 24 h) and their major organs were harvested for the measurement of GNA concentration. The obtained organs (200 mg) were weighed and homogenized with 600 μL normal saline. 100 μL the homogenate was transferred to centrifuge tube, in which 10 μL of internal standard (GA, 20 μg/mL) was added and mixed by vortex. The GNA in the mixture solution was extracted with 1 mL ethyl acetate, dried under nitrogen stream at 37 °C, redissolved in 100 μL of methanol, and vortexed thoroughly. Finally, the GNA concentrations were determined by UPLC via the peak areas of GA and GNA.
The accumulation of different GNA formulations in tumor was evaluated using orthotopic liver tumor-bearing mice model. For establishing orthotopic hepatic tumor model, the mice were anesthetized via intraperitoneal injection of 0.6% pentobarbital sodium. The abdominal area was disinfected with povidone‑iodine, and a transverse incision was made below the xiphoid process to expose the left lateral lobe of the liver by gently pushing the thoracic cavity. Cell suspension (50 μL, containing 2 × 106 Hepa1–6-Luc cells) were injected into the liver and the puncture site was sealed by thermal cautery. The peritoneum and skin were then sutured with separate sutures for the peritoneum and skin. The incision and surrounding skin were cleaned with povidone‑iodine, and 0.2 mL potassium penicillin (20,000 U) was administered intramuscularly for prevention of infection. To monitor the orthotopic tumor growth, each mouse was anesthetized with isoflurane and executed intraperitoneal injection with 150 mg/kg D-Luciferin potassium solution (0.1 mL), then the bioluminescence of orthotopic hepatic tumors were measured by in vivo imaging system (PerkinElmer IVIS spectrum) in 5 min. The mouse with total bioluminescence of ∼2 × 108 p/s/sr was used for further investigation. Eighteen orthotopic hepatic tumor-bearing mice were randomly divided into six groups, including control group, GNA (p.o.) group, GNA (i.v.) group, PEC-GNA (p.o.), PEC-GNA (i.v.) group and POC-GNA (p.o.) group, and then administered at the GNA dosage of 4 mg/kg except control group. All the mice were euthanized at 8 h post-administration and their orthotopic tumors were harvested for the measurement of GNA concentration. Moreover, the tumors in control group and POC-GNA (p.o.) group were also used for immunohistochemical evaluation of microvascular density (MVD) and their integrity.

2.14
Pharmacodynamic studies
The in vivo tumor inhibitions of free GNA, PEC-GNA and POC-GNA were evaluated by subcutaneous and orthotopic Hepa1–6 tumor-bearing mice models. For subcutaneous tumor-bearing model, Hepa1–6 cells (2 × 106 cells) in 100 μL PBS buffer were subcutaneously injected into the right axillary region of each C57BL/6 mouse (male, ∼20 g). Once the tumor volume reached approximately 100 mm3, the mice were randomly divided into six groups: saline control group, oral GNA group, injected GNA group, oral PEC-GNA group, injected PEC-GNA group, oral and POC-GNA group. The saline control, injected GNA, injected PEC-GNA, and injected POC-GNA groups received treatment via tail vein injections using insulin syringes every other day. The oral GNA, oral PEC-GNA, and oral POC-GNA groups were treated every other day via gavage using a mouse gavage needle. The dosage for both administration methods was 4 mg/kg, and the treatment duration was 14 days. The body weights and tumor volumes of C57BL/6 mice were monitored every other day. The tumor volume was calculated via the equation: V = AB2/2, where V, A and B represented the volume, longest diameter and shortest diameter of the grafted tumor. All the mice were simultaneously euthanized when the average volume of grafted tumors in control groups reached ∼1000 m3. The tumors were harvested, weighed and stocked for further investigations of histological imaging, immunohistochemical analysis and enzyme-linked immunosorbent assays (Elisa). The major organs, including heart, liver, spleen, lung and kidney, were collected for histological imaging.
Eighteen orthotopic hepatic tumor-bearing mice were randomly divided into six groups, where the groups and the administrations of GNA formulations were carried out in the same method as those of subcutaneous tumor model. The bioluminescence of the orthotopic tumor was monitored according to the abovementioned method at 0, 4th, 8th and 14th day after the first administration. The orthotopic tumor-bearing mice model were euthanized at 15th day after the first administration. The liver and orthotopic tumor were harvested, imaged and used for immunohistochemical analysis.

2.15
Histological analysis
The tumor tissues and major organs (heart, liver, spleen, lung and kidney) derived from the tumor-bearing mice models were fixed in 4% paraformaldehyde, embedded in paraffin, and stained using the hematoxylin and eosin (H&E) method following standard protocols. Images of the samples were captured at 200× magnification with an Olympus CX41 light microscope (Olympus Corporation, Tokyo, Japan).

2.16
Immunohistochemical evaluation
The apoptosis (TUNEL), growth factor Ki67, vascular endothelial growth factor (VEGF), hypoxia-inducible factor-1α (HIF-1α), microvascular density (MVD), CD8+ t-cells and CD4+ t-cells of the tumors were assessed by immunofluorescence and immunohistochemical analysis. The excised tumor tissues were promptly fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned into 5 μm slices. The paraffin sections were then sequentially treated with 100%, 95%, and 80% ethanol, followed by PBS. Afterward, the sections were incubated with the appropriate monoclonal antibodies for a specified duration. Positive staining was visualized using 3,3-diaminobenzidine (DAB substrate kit) as the chromogen. Finally, the sections were counterstained with hematoxylin, dehydrated, and examined under a ScanScope CS2 (Leica CS2, Germany) microscope.

2.17
Elisa evaluation
The levels of key cytokines related to immune activation in tumor, such as interleukin 2 (IL-2), interferon γ (INF-γ), and tumor necrosis factor α (TNF-α), were quantified by enzyme-linked immunosorbent assay (ELISA). Specifically, 1.0 g of tumor tissue was homogenized with an appropriate volume of PBS, centrifuged at 3000 rpm for 20 min, and the supernatant was collected. This supernatant was then analyzed using cytokine assay kits specific for IL-2, IFN-γ and TNF-α, following the manufacturer's instructions precisely to ensure accurate cytokine quantification. After completing the ELISA procedure, the results were analyzed using ImageJ/Fiji software.

2.18
Vascular irritation assessment
To evaluate the potential inflammatory response of the GNA formulations on vascular tissues, a vascular irritation assay was conducted using New Zealand white rabbits. The dosage for rabbits was converted from the effective murine dosage (4 mg/kg) based on the body surface area (BSA) normalization method using the Meeh-Rubner formula (), resulting in an equivalent dosage of approximately 1.3 mg/kg for a rabbit. New Zealand white rabbits were randomly divided into six groups, including control group, GNA (p.o.) group, GNA (i.v.) group, PEC-GNA (p.o.), PEC-GNA (i.v.) group and POC-GNA (p.o.) group. The administrations were performed every other day for a total of four doses, where the intravenous injections were executed at the rabbits' auricular vein. During the treatment period, the injection sites were visually inspected for signs of erythema or edema. The rabbits were euthanized at 24 h after the final administration, and the auricular veins containing the injection sites were excised. The tissue samples were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) to examine histopathological changes such as inflammatory cell infiltration and thrombus formation.

2.19
Hemolysis assay and blood biochemistry
The hemocompatibility of the POC-GNA nanoparticles was assessed using a hemolysis assay. Fresh blood was collected from healthy SD rats into heparinized tubes and centrifuged at 3000 rpm for 10 min to isolate red blood cells (RBCs). The RBCs were washed three times with PBS and resuspended to obtain a 2% (v/v) RBC suspension. Subsequently, 0.3 mL of the RBC suspension was mixed with 1.0 mL of POC-GNA solutions at various concentrations (ranging from 2.5 to 100 μg/mL). PBS and deionized water were used as the negative (0% hemolysis) and positive (100% hemolysis) controls, respectively. The mixtures were incubated at 37 °C for 2 h and then centrifuged at 3000 rpm for 10 min. The supernatant was collected, and the absorbance (OD) was measured at 540 nm using a microplate reader. The hemolysis percentage was calculated using the following equation:
Furthermore, the blood chemistry was carried out to evaluate the potential toxicity towards the liver and kidney. Eighteen healthy C57BL/6 mice were randomly allocated into three groups (n = 6): control, free GNA (i.v.), and POC-GNA (p.o.). The mice received treatments every other day at the GNA dosage of 4 mg/kg for a total of three doses. Eight hours following the final administration, all animals were anesthetized via intraperitoneal injection of pentobarbital sodium (3% 50 mg/kg), and blood samples were collected via the retro-orbital plexus into non-heparinized tubes. The samples were allowed to clot at room temperature for 30 min and subsequently centrifuged at 3500 rpm for 10 min at 4 °C to isolate the serum. The serum samples were stored at −80 °C. The levels of liver function indicators (alanine aminotransferase, ALT; aspartate aminotransferase, AST) and kidney function markers (Urea, UREA; Creatinine‌, CREA) were quantified using an automated biochemical analyzer (CM-400, Getein Biotech. Inc., China).

2.20
Statistical analysis
Statistical analysis was performed by GraphPad Prism 9.5 software. One-way analysis of variance (ANOVA), Two-way ANOVA and two-tailed Student's t-test were used to determine statistical significance. Significant difference was indicated as follows: #P for non-significant differences, *P < 0.05, **P < 0.01, and ***P < 0.001.

3
Results and discussion
3.1
Synthesis of poly(N-oxide) zwitterionic block copolymer PODEA-PCL
Poly(N-oxide) zwitterionic monomer of 2-(N-oxide, N,N-diethylamino)ethyl methacrylate (ODEA) was prepared via oxidation of 2-(N,N-diethylamino)ethyl methacrylate(Chen et al., 2021), while the macroinitiator of PCL-Br was synthesized via ring-opening polymerization of ε-CL initiated by 1-dodecanol and following esterification with 2-bromoisobutyryl bromide (Scheme. 2&3). Both the zwitterionic monomer and macroinitiator were confirmed by 1H NMR (Fig. 1a&b). Subsequently, the zwitterionic diblock copolymer PODEA-PCL was prepared via atom transfer radical polymerization of ODEA initiated by PCL—Br. The polymerization of PCL block and PODEA block were determined to be 32 and 8 by 1H NMR spectra, respectively (Fig. 1c). Gel permeation chromatography curves (Fig. 1d) demonstrated the increased apparent molecular weight after the polymerization of ODEA, and low polydispersity index (PDI) of the synthetic PCL-Br (PDI: 1.21) and PODEA-PCL (PDI: 1.18).

3.2
Self-assembly and physicochemical investigations of poly(N-oxide) zwitterionic nanoparticles of gambogenic acid
The POC-GNA nanoparticles were prepared by co-assembly of PODEA-PCL and GNA via ultrasonic method. The POC-GNA nanoparticles were nearly spherical with the size of 86.99 nm (PDI: 0.134) and slightly negative zeta-potential of −9 mV (Fig. 2a, b&d), whose size, polydispersity index (PDI) and micromorphology were relatively stable after the storage in PBS at 25 °C for 24 h (Fig. 2e&f). The drug loading and encapsulation efficiency were determined to be 12.16 ± 0.19% and 73.07 ± 1.13%, respectively. POC-GNA nanoparticles prevented premature release and only liberated 26.74% of GNA within 24 h in simulated gastrointestinal conditions (Fig. 2g), which could probably be attributed to their high gastrointestinal stability (Fig. 2h&i). Moreover, GNA-loaded polyethylene glycol-polycaprolactone (PEG-PCL, short as PEC) nanoparticles, named as PEC-GNA, were introduced as a positive control of GNA nanoparticle, which displayed similar physicochemical properties with POC-GNA nanoparticles, including size (82.46 nm, PDI 0.141), zeta-potential (−4 mV), stability at PBS for 24 h and slightly faster drug release behaviors (cumulative GNA release of 37.72% with 24 h) in simulated gastrointestinal conditions (Fig. 2a&c-g).

3.3
GNA formulations inhibit tumor growth through mechanisms including anti-angiogenesis, vascular disruption and cytotoxicity
Vascular tube formation and disruption assays revealed that all tested GNA formulations, including free GNA, PEC-GNA nanoparticles, and POC-GNA nanoparticles, significantly suppressed angiogenesis and disrupted the pre-formed vasculature. Their vascular regulatory activity was comparable to that of the clinical first-line anti-angiogenic agent Sorafenib (Fig. 3a-c). At 8 h post oral administration of POC-GNA, a significant reduction of 51.3% in microvascular density (MVD) was observed in orthotopic liver tumors, indicating rapid vascular disruption (Fig. 3d&e). More importantly, the vascular extravasation barriers that prevent the extravasation of nanoparticles from the blood vessels into tumor tissues(Jiang et al., 2025b; Wang et al., 2024), became inconsecutive in POC-GNA group (Fig. 3d). The increased leakage of vascular extravasation barrier is beneficial for POC-GNA nanoparticles itself to further accumulate and take effect in tumor tissues (Du et al., 2023).
The cytotoxicity of blank poly(N-oxide) zwitterionic nanoparticles and its GNA nanoparticles was studied by MTT assays. PODEA-PCL zwitterionic nanoparticles showed minimal cytotoxicity with cell viability of nearly 80% at the concentration reached up to 1000 μg/mL (Fig. 3f). POC-GNA nanoparticles exhibited higher cellular uptake and cytotoxicity to Hepa1–6 cells than PEC-GNA nanoparticles (Fig. 3g&h), demonstrating that poly(N-oxide) zwitterionic nanoparticles should be a more efficient intracellular delivery system for GNA than PEGylated nanoparticles.

3.4
Oral poly(N-oxide) zwitterionic GNA nanoparticles enhanced mucus penetration and enterocyte uptake to improve bioavailability and tumor accumulation
To elucidate the ability of poly(N-oxide) zwitterionic nanoparticles to overcome intestinal mucus and epithelial barriers, a transwell-based in vitro transport model was established. In this model, the free Nile red (NR), NR-labelled PEC or POC nanoparticles were required to sequentially traverse a simulated intestinal mucus layer and a confluent Caco-2 cell monolayer before reaching and staining Hepa1–6 cells (Fig. 4a). Fluorescent imaging revealed that POC nanoparticles displayed much higher uptake by Hepa1–6 cells than that of free NR or PEC nanocarrier (Fig. 4b), indicating markedly improved mucus penetration and enterocyte transport capacity of the poly(N-oxide) zwitterionic nanocarrier. The cellular uptake mechanisms of NR-labelled POC nanoparticles were further investigated. Uptake was strongly suppressed at 4 °C, with reductions of 92.2% in Caco-2 cells and 84.9% in Hepa1–6 cells, confirming an energy-dependent process. Moreover, uptake inhibitors provide additional insights: chlorpromazine (a clathrin pathway inhibitor) hardly affected uptake for both cells, methyl-β-cyclodextrin (MβCD, a caveolae pathway inhibitor), decreased uptake by 19.2% for Caco-2 cells and 8.4% for Hepa1–6 cells, amiloride (macropinocytosis inhibitor) by 21.8% and 22.0%, and cytochalasin D (inhibitor of both macropinocytosis and phagocytosis) by 68.8% and 72.5%, suggesting the multiple pathways uptake mechanisms (Fig. 4c-f). The cellular uptake of POC nanoparticles was mainly medicated by caveolae-mediated endocytosis, macropinocytosis and phagocytosis for Caco-2 cells, while macropinocytosis and phagocytosis for Hepa1–6 cells.
Consistent with the enhanced mucus penetration and enterocyte uptake observed in vitro, oral administration (p.o.) of POC-GNA resulted in significantly higher plasma GNA concentrations than oral free GNA, oral PEC-GNA, and intravenously administered free GNA between 2 and 10 h in pharmacokinetic studies. POC-GNA (p.o.) group demonstrated higher plasma GNA concentration than oral free GNA group, oral PEC-GNA group and intravenously injected (i.v.) free GNA group during 2 h to 10 h in pharmacokinetic study. The elimination half-life (t1/2) of oral POC-GNA (490 min) was longer than those of oral PEC-GNA (340 min), injected free GNA (139 min) and injected PEC-GNA (344 min), nearly equal to oral free GNA (495 min). Despite with similar t1/2, the Cmax and AUC of oral POC-GNA were respectively 1.45 and 1.73 folds as those of oral free GNA (Fig. 4g, Table 1). These results demonstrate that encapsulation within a poly(N-oxide) zwitterionic nanocarrier markedly prolongs systemic exposure and enhances the oral bioavailability of GNA by facilitating efficient intestinal absorption.
The biodistribution profiles of orally or intravenously administered GNA formulations were assessed by measuring GNA concentrations in major organs and orthotopic liver tumors using ultra performance liquid chromatography (UPLC). Among all evaluated organs, the liver exhibited the highest accumulation of GNA. Specifically, minimal GNA accumulation was observed within the initial 2 h post-administration, followed by a significant increase starting at 4 h, with peak accumulation reached at 8 h. Notably, PEC-GNA (i.v.) and POC-GNA (p.o.) groups still exhibited high hepatic GNA concentrations at 24 h post-administration (Fig. 4h). Remarkably, the hepatic accumulation of GNA was substantially greater in the POC-GNA (p.o.) group compared with other treatment groups. Similarly, orthotopic liver tumor results also indicated significantly higher GNA concentrations in both liver tissues and tumors for the POC-GNA (p.o.) group (Fig. 4i). Collectively, these findings demonstrate that oral poly(N-oxide) zwitterionic GNA nanoparticles markedly improved intestinal absorption, blood circulation, and preferential accumulation in hepatic tumors, outperforming PEGylated nanoparticles and even achieving higher intratumoral GNA levels than intravenously administered PEGylated formulations.

3.5
Oral poly(N-oxide) zwitterionic GNA nanoparticles exhibited potentiated in vivo antitumor efficacy in both subcutaneous and orthotopic HCC models
In vivo antitumor efficacy of oral POC-GNA nanoparticles was assessed in the Hepa1–6 tumor-bearing mice model, and compared to free GNA and PEC-GNA nanoparticles via both oral administration and intravenous injection (Fig. 5a). The inhibition rates on subcutaneous xenograft Hepa1–6 tumors followed the order: 21% for GNA (p.o.), 33% for GNA (i.v.), 47% for PEC-GNA (p.o.), 69% for PEC-GNA (i.v.), and 83% for POC-GNA (p.o.) (Fig. 5b-d). Overall, the formulation type exerted a more pronounced influence on therapeutic efficacy than the administration route. Specifically, free GNA, PEC-GNA and POC-GNA exhibited progressively enhanced tumor suppression, while intravenous administration conferred higher efficacy than oral delivery for both free GNA and PEC-GNA. Notably, oral POC-GNA achieved the greatest tumor inhibition among all tested groups.
To further validate therapeutic performance under clinically relevant conditions, antitumor efficacy was assessed in an orthotopic HCC model established by intrahepatic implantation of bioluminescent Hepa1–6-luc cells. Compared with subcutaneous models, orthotopic tumors better recapitulate the hepatic microenvironment, vascular architecture, immune landscape, and pharmacokinetic behavior of nanomedicines. The bioluminescent intensity of hepa1–6-luc tumor-bearing mice models revealed progressive tumor growth in control and GNA (p.o.) groups, while tumor signals remained relatively stable in PEC-GNA (p.o.) group and GNA (i.v.) group. In contrast, the in vivo bioluminescent intensity markedly decreased following treatment with POC-GNA (p.o.) and PEC-GNA (i.v.), and was nearly undetectable by day 14 (Fig. 5f&g). Ex vivo imaging and tumor weight analysis further confirmed pronounced tumor suppression, with inhibition rates exceeding 60% for all GNA formulations (Fig. 5h&i), following the order: 66.91% for GNA (p.o.), 88.65% for GNA (i.v.), 90.86% for PEC-GNA (p.o.), 98.62% for PEC-GNA (i.v.), and nearly 100% for POC-GNA (p.o.).
Biodistribution studies provided mechanistic insight into the more pronounced therapeutic responses observed in orthotopic tumors, demonstrating preferential hepatic accumulation of GNA relative to other organs (Fig. 4g&h). This hepatotropic distribution strongly correlated with enhanced suppression of orthotopic HCC tumors, indicating that efficient liver accumulation contributes to the efficacy of all GNA formulations. Notably, POC-GNA (p.o.) demonstrated the most pronounced inhibition of orthotopic HCC tumor due to excellent intestinal absorption, preferential accumulation in both liver and tumor lesions. Collectively, these results demonstrate that oral poly(N-oxide) zwitterionic GNA nanoparticles outperforms PEGylated counterparts administered either orally or intravenously in both subcutaneous and orthotopic HCC models. Notably, previous studies evaluating oral GNA required substantially higher doses (e.g., 7.5–30 mg/kg daily for 14 times, or 24 mg/kg every other day for 12 times) to achieve therapeutic effects (Sun et al., 2023; Wang et al., 2021a). In contrast, the robust antitumor efficacy observed here at 4 mg/kg for 7 times highlights the improved oral bioavailability and translational potential of oral POC-GNA nanoparticles. Additionally, no significant differences in body weight were observed among groups, indicating a favorable safety profile for all GNA formulations throughout the treatment period (Fig. 5e&j).

3.6
Oral poly(N-oxide) zwitterionic GNA nanoparticles remodeled angiogenic, hypoxic and immunosuppressive tumor microenvironment
The effects of oral and injectable GNA formulations on the tumor microenvironment, including angiogenesis, hypoxia, and immune activation, were systematically evaluated using histological analysis, immunofluorescence and immunohistochemistry staining, and enzyme-linked immunosorbent assays (ELISA). Overall, the regulatory efficacy of different GNA formulations followed a consistent trend: GNA (p.o.) < GNA (i.v.) < PEC-GNA (p.o.) < PEC-GNA (i.v.) < POC-GNA (p.o.), which closely correlated with their in vivo antitumor performance.
Histological examination, TUNEL staining, and immunohistochemical analysis of subcutaneous tumor sections demonstrated that POC-GNA (p.o.) induced the highest level of tumor apoptosis and the most pronounced suppression of the proliferation marker Ki67 and vascular epithelial growth factor (VEGF), indicating potent anti-proliferative and anti-angiogenic activity (Fig. 6a-d). Given the central role of tumor vasculature in regulating hypoxia and immune suppression, we further assessed hypoxia-associated signaling. All GNA formulations reduced the expression of hypoxia-inducible factor-α (HIF-α), with POC-GNA (p.o.) producing the most significant alleviation of tumor hypoxia (Fig. 6e). The mitigation of hypoxia was accompanied by enhanced tumor necrosis and reprogramming of the immune microenvironment. The three GNA nanoparticles groups moderately increased tumor necrosis factor-α (TNF-α) levels, whereas both free GNA groups exerted minimal effects (Fig. 6f). Notably, POC-GNA (p.o.) markedly upregulated the immune-activating cytokines IL-2 and IFN-γ by approximately 4–5 fold, exceeding the effects observed with PEC-GNA administered either orally or intravenously and far surpassing those of free GNA (Fig. 6g&h). Consistent with cytokine activation, immunohistochemical analysis revealed increased infiltration of T lymphocytes in tumor tissues following GNA treatment, characterized by substantial recruitment of CD4+ T cells and CD8+ cytotoxic T cells. Among all formulations, POC-GNA (p.o.) elicited the highest CD8+ T-cell infiltration within the tumor microenvironment (Fig. 6a, i&j). Collectively, these results demonstrate that oral poly(N-oxide) zwitterionic GNA nanoparticles effectively remodel the tumor microenvironment by simultaneously inhibiting angiogenesis, alleviating hypoxia, promoting tumor apoptosis, and activating antitumor immunity, and its overall microenvironmental modulation exceeded that of intravenously administered PEGylated GNA nanoparticles, highlighting the therapeutic advantage of the poly(N-oxide) zwitterionic oral nanoplatform. Nevertheless, differences in intestinal mucus composition, immune responses, and pharmacokinetics between rodent models and humans should be considered when interpreting translational relevance.

3.7
Oral poly(N-oxide) zwitterionic GNA nanoparticles circumvented the vascular irritation and showed negligible off-target toxicity
Despite its potent antineoplastic activity, the clinical translation of GNA is substantially limited by adverse side effects, particularly severe vascular irritation. This toxicity is most pronounced following intravenous administration, where transiently high local drug concentrations and mechanical vascular injury at injection sites exacerbate inflammatory responses (Du et al., 2023; Tang et al., 2018). Histological examination of rabbit auricular veins revealed that intravenously administered free GNA induced severe vascular irritation characterized by pronounced oedema and hematoma formation, while PEC-GNA caused moderate irritation. In contrast, oral administration of GNA and PEC-GNA significantly attenuated vascular injury, and notably, oral POC-GNA produced almost no detectable vascular irritation (Fig. 7a). Importantly, this improved safety profile was achieved without compromising antitumor efficacy, as oral poly(N-oxide) zwitterionic GNA nanoparticles retained robust pro-apoptotic and anti-angiogenic activities.
The biosafety of POC-GNA nanoparticles was further evaluated by hemolysis, blood chemistry and histopathological analysis of major organs, including the heart, liver, spleen, lungs, and kidneys. POC-GNA nanoparticles showed relatively low hemolysis ratio (< 5%) when the GNA concentration reached up to 100 μg/mL (Fig. 7b&c). Notably, intravenous injection of free GNA induced acute hepatotoxicity, as evidenced by markedly increased serum ALT and AST levels, whereas oral administration of POC-GNA nanoparticles was associated with substantially lower elevations on these hepatic enzymes (Fig. 7d&e). Both GNA (i.v.) and POC-GNA (p.o.) produced minimal renal impairment, with no significant changes observed in kidney function indicators of UREA and CREA (Fig. 7f&g). Additionally, no apparent pathological abnormalities were observed in any treatment group compared with the control (Fig. 7h). Collectively, these results demonstrate that oral poly(N-oxide) zwitterionic GNA nanoparticles offers a markedly improved safety profile while preserving potent antitumor efficacy, underscoring their translational potential for anti-angiogenic therapy.

4
Conclusion
In summary, this study establishes an oral poly(N-oxide) zwitterionic nanoplatform as an effective strategy to enhance the anti-vascular therapeutic performance of gambogenic acid (GNA) against hepatocellular carcinoma. The POC-GNA nanoparticles simultaneously improved intestinal mucus penetration and enterocyte uptake, leading to enhanced oral absorption, and prolonged systemic circulation. Notably, this system is specifically designed to address the intrinsic paradox of anti-angiogenic therapy, in which vascular inhibition restricts subsequent drug accumulation and limits therapeutic durability. Mechanistically, POC-GNA induced potent anti-angiogenic and vascular-disruptive effects, increased tumor vascular permeability, alleviated hypoxia, and activated antitumor immune responses, thereby establishing a self-reinforcing intratumoral accumulation and therapeutic amplification. Importantly, oral administration of POC-GNA effectively avoided the vascular irritation associated with intravenous injection of GNA formulations for a favorable safety profile, while maintaining robust antitumor activity, outperforming oral and intravenously administered PEGylated GNA nanoparticles. By integrating oral barrier traversal with tumor vascular modulation, this work provides a problem-driven strategy to overcome vascular density–dependent drug accumulation and extend the therapeutic ceiling of oral anti-angiogenic treatment in HCC.

CRediT authorship contribution statement
Shuo Tang: Writing – original draft, Validation, Investigation, Data curation. Yu Tao: Investigation, Data curation. Laiting Gong: Investigation. Yali Wang: Investigation. Yu Cao: Writing – review & editing. Mengru Liang: Methodology. Xiangyong He: Methodology, Investigation. Yue Zhang: Investigation, Data curation. Jia-Feng Zou: Investigation. Yongfu Zhu: Writing – review & editing, Funding acquisition. Yong-Ling Wang: Writing – review & editing, Resources, Funding acquisition. Shengqi Chen: Writing – review & editing, Project administration, Funding acquisition, Conceptualization.

Declaration of competing interest
We submit the manuscript titled with “Oral Poly(N-oxide) Zwitterionic Nanoplatform for Gambogenic Acid Enhances Mucosal penetration for Potentiated Anti-Angiogenic Therapy” by Shuo Tang#, Yu Tao#, Laiting Gong, Yali Wang, Yu Cao, Mengru Liang, Xiangyong He, Yue Zhang, Jia-Feng Zou, Yongfu Zhu*, Yong-Ling Wang* and Shengqi Chen* to International Journal of Pharmaceutics: X as an article for the considerations of reviewing and publication. All authors contribute this manuscript and approve its submission. No conflict of interest exists in this manuscript. I would like to declare on behalf of my co-authors that this work is original and the whole manuscript has not been published previously or under consideration for publication elsewhere.