Coordination-strengthened Rhein/Pectin@Zn dual-network hydrogel for local antitumor therapy

Abstract

Local therapeutic platforms capable of concurrent tumor-cell elimination and antiangiogenic activity are urgently needed. This study presents a Rhein/Pectin@Zn hydrogel fabricated through supramolecular self-assembly and metal-coordination crosslinking and administered by dual-syringe in situ gelation. Hydrogels prepared under standardized ex vivo crosslinking conditions display a porous microstructure, stable gel characteristics, shear-dependent flow behavior, and accelerated cargo release under acidic conditions. Biological evaluations indicate antiproliferative and pro-apoptotic effects against tumor cells, together with reduced endothelial angiogenic signaling. Repeated intratumoral delivery in a subcutaneous B16 melanoma model reduced endpoint tumor volume and weight without overt major-organ histopathological abnormalities in the displayed fields. Integrated transcriptomic and molecular analyses implicated cell-cycle checkpoint disruption and apoptosis-associated signaling in the observed antitumor response. Collectively, this work positions Rhein/Pectin@Zn as an in situ-forming hydrogel for local cancer therapy.

Graphical abstract

1. Introduction

Local therapeutic modalities that achieve durable tumor control while minimizing systemic exposure remain a critical unmet need in solid tumor management [1,2]. Despite advances in surgery, radiotherapy, and systemic chemotherapy, incomplete eradication of primary lesions, off-target toxicity, and acquired resistance continue to limit clinical outcomes [3]. Consequently, there is growing interest in injectable drug depots that sustain high intratumoral drug concentrations and reduce peripheral exposure [4]. The tumor microenvironment (TME) presents both a barrier and an opportunity for local therapy [5]. Pathological angiogenesis sustains nutrient delivery and metastatic dissemination [[6], [7], [8]].

Injectable hydrogels have emerged as versatile platforms for localized and sustained drug delivery in oncology [9]. Their capacity for minimally invasive administration, conformal filling of irregular tissue cavities, and tunable release kinetics makes them well-suited for maintaining therapeutic payloads at the tumor site [[10], [11], [12]]. Recent studies further illustrate the breadth of biomedical hydrogel applications and design considerations across cancer therapy, polysaccharide-based biomaterials, and microenvironment-responsive tissue repair [[13], [14], [15]]. However, most existing systems function primarily as passive carriers, lacking intrinsic bioactivity or the ability to actively remodel the pathological niche. Engineering hydrogels with multifunctional capabilities to simultaneously attack tumor cells and modulate angiogenic programs remains a significant challenge.

Coordination chemistry offers a compelling strategy to address this limitation. Metal-ligand interactions can reinforce hydrogel networks beyond conventional ionic crosslinking, imparting superior mechanical stability and stimuli-responsive behavior [16]. Zinc ions, in particular, serve a dual role: as crosslinking agents that engage polysaccharide carboxyl groups in canonical “egg-box” motifs, and as bioactive moieties with inherent antiproliferative and antiangiogenic properties [[17], [18], [19], [20]]. Recent studies have highlighted the therapeutic potential of zinc in oncology beyond structural roles, as zinc homeostasis disruption can induce tumor cell apoptosis and inhibit endothelial activation [[18], [19], [20]]. However, free zinc ions suffer from rapid clearance and dose-limiting toxicity, necessitating localized delivery strategies. Rhein, a natural anthraquinone, provides an ideal building block for coordination-enhanced networks [21,22]. Its planar tricyclic structure enables self-assembly through π–π stacking under alkaline conditions, while the peri-hydroxy ketone motif offers chelation sites for divalent metal ions. Beyond its structural contributions, rhein has reported antiproliferative and antiangiogenic activities, making it both a network component and an active therapeutic agent [21]. Despite these individual advantages, integrated systems that combine supramolecular assembly, metal coordination, local antitumor activity, and antiangiogenic activity within a single platform remain scarce. Systems relying solely on ionic crosslinking often exhibit limited mechanical robustness and burst release profiles, compromising therapeutic durability. The strategic combination of rhein, pectin, and Zn²⁺ thus represents an unexplored opportunity to construct a coordination-strengthened dual-network hydrogel with intrinsic therapeutic activity.

Herein, we hypothesized that a coordination-strengthened dual-network hydrogel comprising rhein, pectin, and Zn²⁺ would function as an active therapeutic platform rather than a passive vehicle. By leveraging rhein-pectin supramolecular assembly followed by Zn²⁺-mediated crosslinking, we anticipated that the resulting depot would sustain localized codelivery of rhein and zinc ions, concurrently exerting direct cytotoxicity and antiangiogenic activity within the TME. To test this hypothesis, we systematically engineered and characterized the Rhein/Pectin@Zn hydrogel, evaluated its in vitro antitumor and antiangiogenic properties, and assessed its therapeutic efficacy and preliminary compatibility in a murine melanoma model. Transcriptomic and mechanistic analyses were further employed to elucidate the molecular underpinnings of its antitumor activity. The results identify Rhein/Pectin@Zn as a promising dual-syringe in situ-forming platform for local cancer therapy that couples controlled drug delivery with microenvironment-directed activity in the tested model (Fig. 1).

Fig. 1.

Schematic illustration of Rhein/Pectin@Zn preparation, local administration, and proposed biological responses.

2. Materials and methods

2.1. Materials

Rhein (purity > 98%) was purchased from Shanghai Acmec Biochemical Co., Ltd. Zinc acetate dihydrate (Zn(CH₃COO)₂·2H₂O, 99%) was obtained from Beijing Solarbio Science & Technology Co., Ltd. Pectin from citrus peel was acquired from Shanghai Macklin Biochemical Technology Co., Ltd. All other chemicals and solvents were of analytical grade and used as received.

2.2. Characterization of pectin: molecular weight, monosaccharide composition, and methylation analysis

The molecular weight of pectin was determined by gel permeation chromatography (GPC) on an Agilent 1260 Infinity system equipped with a refractive index detector. A 6 mg sample was dissolved in 3 mL distilled water, filtered through a 0.22 μm aqueous membrane, and injected into the system. The mobile phase was 0.1 M NaNO₃ at a flow rate of 0.8 mL/min.

Monosaccharide composition was analyzed using high-performance liquid chromatography (HPLC) on a Shimadzu LC-20AD system. Pectin was hydrolyzed with 2 M trifluoroacetic acid (TFA) at 120 °C for 6 h in a sealed vial. The hydrolysate was evaporated under nitrogen flow, redissolved in methanol, and dried again. Derivatization was performed with 0.5 M NaOH and 0.3 M 1-phenyl-3-methyl-5-pyrazolone (PMP) at 70 °C for 2 h. After neutralization with 0.3 M HCl, the sample was extracted with chloroform, and the aqueous layer was filtered (0.45 μm) and analyzed on a C18 column (4.6 × 250 mm, 5 μm) using 100 mM phosphate buffer (pH 6.4) and acetonitrile (83:17, v/v) as the mobile phase at 1.0 mL/min, with detection at 250 nm. Monosaccharides were identified by comparison with standard references.

For methylation analysis, uronic acid residues in pectin were first reduced to neutral sugars using carbodiimide activation followed by sodium borohydride (NaBH₄) reduction [23]. The reduced sample (Pectin-R) was then permethylated, hydrolyzed, reduced, and acetylated to generate partially methylated alditol acetates (PMAAs), following a standard linkage-analysis workflow [24]. PMAAs were analyzed by GC-MS on an HP-5MS column (30 m × 0.25 mm, 0.25 μm) with a temperature program from 120 °C to 280 °C at 4 °C/min.

2.3. NMR analysis

Approximately 20 mg of pectin was dissolved in 1 mL of D₂O (99.96%) and analyzed using a Bruker AVANCE III 400 MHz spectrometer. ¹H NMR spectra were acquired with 256 scans at 25 °C. Chemical shifts are reported in δ (ppm). Data were processed using MestReNova software (v14.2).

2.4. Synthesis of Rhein/Pectin@Zn hydrogel

Rhein was dissolved in HEPES buffer (0.05 M, pH 8.5) at 65 °C for 1 h to obtain solutions of 1, 2, and 4 mg/mL. Pectin (2.5 g) was added to 50 mL of the rhein solution and stirred at 65 °C for 3 h. For physicochemical characterization, the Pectin/Rhein precursor solution was introduced into an excess of 2% (w/v) zinc acetate solution to induce Zn²⁺-mediated coordination crosslinking. The resulting hydrogel specimens were collected and used for subsequent physicochemical analyses. For in vivo treatment, the Pectin/Rhein precursor and zinc acetate solution were loaded separately into a dual-syringe system and simultaneously delivered into the tumor, enabling in situ coordination crosslinking at the administration site (Section 2.7). Because these mixing environments may affect crosslinking density and network homogeneity, the physicochemical measurements should be interpreted as properties of hydrogels prepared under the standardized in vitro crosslinking conditions and do not demonstrate structural or mechanical equivalence of the in situ-formed depot. For analyses requiring dried material, the collected hydrogels were rapidly frozen at −80 °C for 2 h and lyophilized at −45 °C and 65 Pa for 48 h using a SCIENTZ-10ND freeze dryer (Ningbo Scientz, China).

2.5. Physicochemical characteristics

2.5.1. Scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS)

Lyophilized hydrogels were sectioned, mounted on conductive adhesive, and sputter-coated with gold for 90 s. Morphology and elemental composition were examined using a Hitachi SU8600 SEM equipped with an EDS detector.

2.5.2. Fourier transform infrared (FTIR) spectroscopy

FTIR spectra were recorded on a Bruker Vertex 70v spectrometer in the range of 4000–400 cm⁻¹ using the KBr pellet method.

2.5.3. X-ray diffraction (XRD)

XRD patterns were obtained on a Bruker D8 Advance diffractometer with Cu Kα radiation (λ = 1.5406 Å) over a 2θ range of 5–90° at a scanning rate of 0.5°/min.

2.5.4. Swelling behavior

The swelling ratio was measured gravimetrically. Dry hydrogel (W₀) was immersed in distilled water at 37 °C. At predetermined intervals, the sample was removed, surface water was blotted, and the weight (W_t) was recorded. The swelling ratio was calculated as:

$$S_t=(W_t‐W_0)/W_0\text{.}$$

2.5.5. Rheological testing

Rheological properties were evaluated using a HAAKE MARS III rheometer (Thermo Scientific) with a 20 mm parallel plate geometry and a gap of 1500 μm. Strain amplitude sweeps (0.1–100%) were performed at 1 Hz to determine the linear viscoelastic region (LVR). Frequency sweeps (0.1–100 Hz) and time sweeps (300 s) were conducted within the LVR. Steady-state flow behavior of the fully crosslinked hydrogels was assessed by varying the shear rate from 0.1 to 100 s⁻¹; these measurements were not used as evidence of injectability or post-extrusion recovery of a pre-formed hydrogel.

2.5.6. In vitro release study

The release profile of rhein was evaluated using dialysis bags (MWCO, 7.5 kDa). Freeze-dried hydrogel (40 mg) was immersed in 100 mL of PBS at pH 7.4 or 6.5 and maintained at 37 °C with shaking at 100 rpm. At scheduled time points, 1 mL of release medium was withdrawn and replaced with fresh PBS. Rhein concentration was quantified by UV-vis spectrophotometry at 228 nm using a pre-established calibration curve.

2.5.7. Thermogravimetric analysis (TGA)

TGA was performed on a TA Instruments Q50 analyzer. Samples were heated from room temperature to 680 °C at 20 °C/min under a nitrogen flow of 10 mL/min.

2.5.8. Zeta potential and UV measurements

Zeta potential was measured using a Malvern Zetasizer Nano ZS. UV-Vis spectra were recorded on a Shimadzu UV-2600 spectrophotometer. Samples were dispersed in PBS (pH 7.4) for analysis.

2.6. In vitro studies

2.6.1. Cell culture

Human umbilical vein endothelial cells (HUVECs), murine K7M2 osteosarcoma cells, RAW264.7 macrophages, and murine B16 melanoma cells were used. HUVECs and K7M2 cells were maintained in DMEM containing 10% FBS and 1% penicillin-streptomycin. RAW264.7 cells were maintained in DMEM containing 5% FBS and 1% antibiotics. B16 cells were maintained in RPMI-1640 containing 10% FBS and 1% antibiotics. All cells were incubated at 37 °C in 5% CO₂ and passaged at approximately 90% confluence using 0.25% trypsin-EDTA.

2.6.2. Cell proliferation, IC₅₀ determination, and viability staining

To prepare hydrogel extracts, 4.0 mg of freeze-dried hydrogel containing Rhein at concentrations of 1, 2, and 4 mg/mL was incubated in 30 mL of DMEM or RPMI-1640 medium for 24 h. The resulting extracts were used to culture B16, K7M2, and HUVEC cells (3 × 10³ cells/well) in 96-well plates. Each well received 100 μL of extract, and control groups received fresh culture medium. CCK-8 measurements were performed on days 1, 3, and 5 for HUVECs and on days 1, 2, and 3 for B16 and K7M2 cells, matching the time points displayed in Fig. 5 and S7. At each time point, the medium was replaced with 110 μL of fresh medium containing 10% CCK-8 reagent. Following 1 h of incubation, absorbance at 450 nm was measured using a microplate reader (Bio-Tek, USA). The selected hydrogel concentration was used in subsequent experiments.

Fig. 5.

CCK-8-derived relative cell-viability signals and representative calcein-AM/propidium iodide live/dead images after exposure to hydrogel extracts. (A) HUVECs on days 1, 3, and 5. (B) B16 melanoma cells on days 1, 2, and 3. Bar charts compare NC, Pectin@Zn, and Rhein/Pectin@Zn formulations prepared with 1, 2, or 4 mg/mL rhein; images show NC, Pectin@Zn, and the selected 2 mg/mL Rhein/Pectin@Zn formulation. Data are presented as mean ± SD, with individual values shown as plotted. *P < 0.05 versus NC. Scale bars, 500 μm.

For IC₅₀ determination, cells were seeded in 96-well plates and allowed to adhere for 24 h. The culture medium was then replaced with extracts of Pectin@Zn or Rhein/Pectin@Zn hydrogels at various concentrations. After 48 h of treatment, cell viability was assessed using the CCK-8 assay, and IC₅₀ values were calculated from the dose-response curves.

Cell viability after exposure to hydrogel extracts was further evaluated using a Calcein-AM/PI Double Stain Kit according to the manufacturer's instructions. B16 cells and HUVECs were seeded in 96-well plates at 3 × 10³ cells/well. After attachment, HUVECs were treated for 1, 3, or 5 days, whereas B16 cells were treated for 1, 2, or 3 days. Cells were then stained and imaged by confocal laser scanning microscopy. The representative images in Fig. 5 were obtained using the selected 2 mg/mL Rhein/Pectin@Zn formulation.

2.6.3. Quantitative real-time PCR (RT-qPCR)

RT-qPCR was used to measure transcripts associated with tumor-cell responses and endothelial angiogenic signaling in B16, K7M2, and HUVEC cells, and inflammatory and matrix-remodeling transcripts in LPS-stimulated RAW264.7 macrophages. After treatment with hydrogel extracts for 1 or 3 days, total RNA was isolated with TRIzol reagent (Invitrogen) and quantified using a NanoDrop 2000. cDNA was synthesized from 1 μg of total RNA using the PrimeScript RT Kit (TaKaRa). Reactions were performed using SYBR Green Premix Pro Taq HS qPCR Kit (AG11701). Each 20 μL reaction contained 2 μL of cDNA, 2 μL of primer mixture, 10 μL of SYBR Green mixture, 5.5 μL of RNase-free water, and 0.5 μL of ROX reference dye. Amplification was conducted for 40 cycles. Expression was normalized to GAPDH and calculated using the 2^−ΔΔCt method.

2.6.4. Cell scratch assay

B16 cells were seeded in 6-well plates and divided into three groups: negative control (RPMI 1640 with 10% FBS), Pectin@Zn, and Rhein/Pectin@Zn. After 48 h of culture at full confluence, a scratch was made in each well using a 10 μL pipette tip. The cells were washed and maintained in FBS-free RPMI 1640. Wound closure was monitored at regular intervals under a microscope.

2.6.5. Cell apoptosis assay

B16 cells were seeded in 6-well plates at 5 × 10⁵ cells/well and treated with Pectin@Zn or Rhein/Pectin@Zn extracts for 48 h. Control cells received RPMI-1640 containing 10% FBS. Cells were harvested using EDTA-free trypsin and stained with Annexin V-FITC and propidium iodide using a commercial kit (MREDA, Beijing). Apoptosis was analyzed by flow cytometry (FACSCalibur, BD, USA) and FlowJo software.

2.6.6. Cell cycle analysis

B16 cells were seeded in 6-well plates at 5 × 10⁵ cells/well and treated with Pectin@Zn or Rhein/Pectin@Zn extracts for 48 h. Control cells were maintained in complete medium. After treatment, cells were fixed in 95% ethanol at 4 °C overnight, stained with PI/RNase A solution, and analyzed by flow cytometry. Cell-cycle distributions were determined using FlowJo software.

2.7. In vivo antitumor efficacy

All animal experiments were approved by the Animal Ethics Committee of Southern University of Science and Technology (JY202305013). Male C57BL/6 mice were subcutaneously inoculated with B16 cells. When tumors reached approximately 80 mm³, mice were randomized into NC, Pectin@Zn, and Rhein/Pectin@Zn groups (n = 6 per group).

For local treatment, a dual-syringe system connected through a three-way stopcock was used to co-inject the Pectin/Rhein precursor and zinc acetate solution intratumorally, allowing Zn²⁺-mediated coordination crosslinking to occur in situ. Both precursor solutions are flowable liquids before mixing and can pass through a needle; a pre-formed hydrogel was therefore not the administered dosage form. The dual-syringe system was selected to enable in situ gelation at the target site, thereby avoiding extrusion of a pre-formed gel and reducing the risk of premature gelation within the delivery device. Mice received treatments on days 0, 2, and 4. Tumor dimensions were measured using digital calipers, and tumor volume was calculated as 0.5 × L × W². At the endpoint, tumors and major organs were collected for histological analyses.

2.8. Histology and immunohistochemistry

Tumor tissues and major organs were fixed, paraffin-embedded, sectioned, and stained with hematoxylin and eosin (H&E). Tumor apoptosis was evaluated by TUNEL staining. Immunohistochemistry was performed using antibodies against Ki-67, BAX, BCL-2, CD31, Cyclin B1, and PUMA.

2.9. Transcriptomic analysis

Transcriptomic profiling was performed on tumor samples from the indicated groups. Differentially expressed genes were visualized by heatmap and volcano plot, followed by KEGG pathway enrichment and gene set enrichment analysis (GSEA). Selected genes were subsequently validated by RT-qPCR and immunohistochemistry.

2.10. Statistical analysis

Data are presented as mean ± SD unless otherwise stated. Statistical analyses were performed using GraphPad Prism 9.4. Comparisons among multiple groups were performed by one-way ANOVA followed by an appropriate post hoc test, and two-group comparisons were performed using Student's t-test. P < 0.05 was considered statistically significant.

3. Results and discussion

3.1. Molecular characterization of pectin

HPLC analysis of acid-hydrolyzed pectin identified glucose (39.118%), galacturonic acid (20.180%), galactose (18.206%), arabinose (11.656%), and rhamnose (7.527%) as the principal monosaccharide constituents under the applied analytical conditions (Fig. 2A and Table 1). Because galacturonic acid was not the predominant measured monosaccharide and its relative content differed substantially from the supplier specification, these data do not independently support describing the analyzed sample as galacturonic-acid-enriched or HG-rich. GPC yielded an apparent molecular weight of 2.76 × 10⁵ and a polydispersity index of 4.29 (Fig. 2B), indicating a broad molecular-weight distribution.

Fig. 2.

Molecular characterization of pectin. (A) HPLC chromatograms of monosaccharide standards and acid-hydrolyzed pectin. Peaks: A, mannose; B, ribose; C, rhamnose; D, glucuronic acid; E, galacturonic acid; F, N-acetylglucosamine; G, glucose; H, N-acetylgalactosamine; I, galactose; J, xylose; K, arabinose; and L, fucose. (B) GPC profile used to determine the apparent molecular-weight distribution of pectin.

Table 1.

Monosaccharide composition of acid-hydrolyzed pectin.

Monosaccharides	Molar Mass (g/mol)	Measured concentration (μg/mL)	Relative content (%)	Content (mmol/g)
Mannose	180.16	0.291	0.647	0.00155
Ribose	150.13	0.134	0.299	0.000866
Rhamnose	164.16	5.887	7.527	0.0342
Glucuronic acid	194.14	0.709	1.208	0.00350
Galacturonic acid	194.14	11.324	20.180	0.0555
Glucose	180.16	18.855	39.118	0.0997
Galactose	180.16	8.322	18.206	0.0440
Xylose	150.13	0.059	0.151	0.0004
Arabinose	150.13	4.327	11.656	0.0275
Fucose	164.16	0.535	1.009	0.00311

Following uronic-acid reduction, the Pectin-R profile contained 40.60% 1,4-linked galactose, compared with 1.12% in untreated pectin, a change consistent with the detection of reduced 1,4-linked galacturonic-acid-derived residues (Table 2; Figs. S1 and S2). Terminal and branched rhamnose-, arabinose-, and galactose-related linkages were also detected. However, untreated pectin showed a high reported proportion of 1,4-linked glucose (84.40%), and the monosaccharide analysis measured galacturonic acid at 20.180%.

Table 2.

Linkage profiles of uronic-acid-reduced pectin (Pectin-R) and untreated pectin determined by methylation–GC–MS.

sugar	Type of linkage	Relative molar ratio (%)
		Pectin-R	Pectin
Ara
	Tf-	7.10	1.93
Rha
	Tp-	4.05	1.77
	1,2,4p-	3.42	1.94
Gal
	Tp-	8.27	2.15
	1,4p-	40.60	1.12
	1,6p-	4.01	4.99
Glu
	Tp-	3.42	0.80
	1,4p-	22.63	84.40

NMR analysis further supported this structural assignment. The ¹H and ¹³C spectra showed characteristic pectin signals, and HSQC cross-peaks were assigned to C1/H1-C5/H5 correlations of 1,4-α-D-GalpA (Fig. S3 and Table S1). Together, these results supported the expected pectic framework for subsequent Zn²⁺-mediated network formation.

3.2. Hydrogel formation and physicochemical characteristics

SEM revealed a porous microarchitecture in both Pectin@Zn and Rhein/Pectin@Zn hydrogels, and the pore morphology changed noticeably with increasing rhein content (Fig. 3A). Relative to Pectin@Zn, rhein-containing formulations showed denser and more organized internal structures, consistent with additional intermolecular interactions beyond simple ionically crosslinked pectin.

Fig. 3.

Physicochemical characterization of Pectin@Zn and Rhein/Pectin@Zn hydrogels. (A) Representative SEM images of Pectin@Zn and Rhein/Pectin@Zn formulations prepared with 1, 2, or 4 mg/mL rhein. Images were acquired at 80× magnification; scale bars, 500 μm. (B) Representative EDS spectra and reported semiquantitative elemental compositions. (C) XRD patterns of zinc acetate, rhein, pectin, Pectin@Zn, and Rhein/Pectin@Zn. (D) FTIR spectra of the indicated components and hydrogel formulations.

EDS confirmed the presence of zinc in all crosslinked formulations and showed a progressive increase in Zn content with increasing rhein loading (Fig. 3B), indicating that rhein likely contributes additional coordination sites within the network. XRD further supported hydrogel formation: characteristic crystalline peaks of the starting materials were attenuated or lost in the composite formulations, whereas Rhein/Pectin@Zn displayed a broader pattern with residual or new reflections consistent with altered molecular packing after assembly (Fig. 3C).

FTIR spectra likewise supported intermolecular association among pectin, rhein, and Zn²⁺ (Fig. 3D). Changes in the O-H and carbonyl-associated bands after crosslinking were consistent with hydrogen bonding and metal coordination, while retention of characteristic bands from the parent components indicated incorporation rather than chemical degradation. UV-Vis spectra showed the coexistence of spectral features from pectin and rhein without interference at the analytical wavelength used for rhein quantification (Fig. S4). Zeta-potential measurements remained negative after crosslinking and rhein incorporation, supporting formation of a stable colloidal or gel network with abundant anionic groups still exposed at the interface (Fig. S6). Collectively, these data support successful construction of a coordinated supramolecular hydrogel rather than a simple physical mixture.

3.3. Rheological, drug release, and thermogravimetric analysis

Rheological characterization showed that the hydrogels behaved as predominantly elastic materials. In the time-sweep test, G′ remained higher than G″ and relatively stable over the measurement period for all formulations, indicating a persistent gel state (Fig. 4A). Frequency-sweep data showed a similar trend across the linear viscoelastic region (Fig. S5). Increasing rhein content raised both moduli, suggesting that rhein reinforced the network by supplying additional noncovalent and/or coordination-mediated crosslinking points.

Fig. 4.

Rheological behavior, swelling, rhein release, and thermal response of pectin-based hydrogels prepared under standardized ex vivo crosslinking conditions. (A) Time sweeps of storage modulus (G′) and loss modulus (G″) for Pectin@Zn and Rhein/Pectin@Zn formulations containing 1, 2, or 4 mg/mL rhein. (B) Amplitude sweep of the plotted Rhein/Pectin@Zn formulation, showing the linear viscoelastic region and yielding behavior. (C) Steady-state viscosity as a function of shear rate for fully crosslinked Pectin@Zn and Rhein/Pectin@Zn; these measurements do not demonstrate injectability or post-extrusion recovery. (D) Swelling ratio over time. (E) Cumulative rhein release at pH 7.4 and 6.5. (F) Thermogravimetric profiles of the indicated pectin-based formulations.

Amplitude-sweep analysis of Rhein/Pectin@Zn identified a clear linear viscoelastic range followed by yield behavior at higher strain (Fig. 4B). In parallel, viscosity decreased sharply with increasing shear rate, indicating shear-dependent steady-state flow of the fully crosslinked hydrogels (Fig. 4C). The flow sweep analysis characterizes the steady-state rheological behavior of the fully crosslinked Rhein/Pectin@Zn hydrogels and enables comparison of the effects of Rhein concentration on the properties of the resulting hydrogel networks.

Swelling reached a plateau within the monitored time window and decreased as rhein content increased (Fig. 4D), consistent with a denser network that restricts water uptake. Release experiments further showed sustained rhein release under both neutral and mildly acidic conditions, with faster release at pH 6.5 than at pH 7.4 (Fig. 4E), indicating environmentally responsive behavior relevant to the acidic tumor milieu. TGA demonstrated improved thermal stability after hydrogel formation, and the rhein-containing formulations retained higher residual mass than pectin alone (Fig. 4F). Together, these results indicate that Rhein/Pectin@Zn combines mechanical robustness and controlled release under the standardized ex vivo preparation conditions.

In vivo injectability is conferred by the liquid state of the two precursor solutions and does not depend on the flow-sweep measurements obtained from fully crosslinked hydrogels prepared under standardized ex vivo conditions. Although both preparation procedures are based on Zn²⁺-mediated coordination crosslinking, their gelation environments are not identical. Immersion in excess zinc acetate may promote more extensive and homogeneous crosslinking than the local mixing and diffusion conditions associated with dual-syringe administration. Consequently, the crosslinking density, network uniformity, mechanical properties, and release behavior of the in situ-formed hydrogel may differ quantitatively from those of the standardized specimens characterized in vitro. The physicochemical data reported here should therefore be interpreted as reference properties obtained under the specified in vitro preparation conditions rather than as an exact representation of the hydrogel formed within the tumor. Direct comparative characterization of hydrogels produced under simulated dual-syringe conditions remains necessary and represents a limitation of the present study.

3.4. In vitro cellular response to hydrogel treatment

3.4.1. CCK-8 and LIVE/DEAD assays

We next evaluated the biological response to hydrogel extracts in endothelial and tumor cells. Pectin@Zn alone caused relatively small reductions in HUVEC and B16 cell-viability signals relative to NC. Rhein/Pectin@Zn reduced the B16 cell-viability signal in a concentration-dependent manner and also reduced HUVEC viability (Fig. 5). Because reduced HUVEC viability alone does not establish a specific antiangiogenic mechanism, endothelial angiogenic signaling was evaluated separately.

Among the tested formulations, the 4 mg/mL rhein hydrogel markedly reduced viability in both HUVECs and B16 cells, whereas the 2 mg/mL formulation retained substantial activity against B16 cells while producing an intermediate reduction in HUVEC viability. The 2 mg/mL formulation was therefore selected for subsequent studies as a compromise between antitumor activity and broad cytotoxicity. The concentration series was used as an independent formulation-optimization experiment. In the principal experiments, Pectin@Zn served as a component control for the Zn-crosslinked pectin carrier. Additional CCK-8 experiments in K7M2 osteosarcoma cells showed a similar inhibitory trend (Fig. S7). Live/dead staining was consistent with the viability assays and showed reduced B16-cell viability after Rhein/Pectin@Zn treatment (Fig. 5).

We also note that, because the pectin and Zn²⁺ concentrations are constrained by the narrow gelation window required for injectable hydrogel formation, component-omitted states that lack a stable gel architecture are not functionally equivalent comparators to the intact formulation. Within this constraint, the enhanced activity of Rhein/Pectin@Zn over the Pectin@Zn carrier control supports a cooperative contribution of rhein to the integrated system.

3.4.2. Hemocompatibility, preliminary inflammatory-response screen, cell-cycle arrest, apoptosis, and migration inhibition

The hemocompatibility of the hydrogels was evaluated by hemolysis analysis. Based on plasma-free hemoglobin levels, materials were classified as nonhemolytic (<2%), slightly hemolytic (2%–5%), or hemolytic (>5%) [25]. As shown in Fig. 6A, all hydrogel groups remained below the 2% hemolysis threshold under the tested conditions. As a preliminary in vitro screen of intrinsic inflammatory-response potential relevant to local material tolerance, LPS-stimulated RAW264.7 macrophages were treated with hydrogel extracts. Relative to LPS alone, Rhein/Pectin@Zn reduced iNOS, IL-1β, IL-6, MMP9, and MMP13 transcript levels (Fig. S8). This observation is reported together with the hemolysis and cytotoxicity data as a safety-related adjunct for an implantable local hydrogel; it does not model tumor-associated macrophages and is not interpreted as therapeutic immunomodulation in the tumor microenvironment.

Fig. 6.

Hemolysis and B16-cell response readouts after hydrogel-extract treatment. (A) Hemolysis ratios for PBS, Pectin@Zn, Rhein/Pectin@Zn formulations containing 1, 2, or 4 mg/mL rhein, and Triton X-100. (B, C) Representative Annexin V/PI flow-cytometry plots and quantified apoptotic-cell fractions for NC, Pectin@Zn, and Rhein/Pectin@Zn. (D) RT-qPCR readouts for the Bax/Bcl2 mRNA ratio and relative Tnf and Trp53 mRNA levels. (E) CCK-8 dose-response curves and fitted IC₅₀ values after 48 h; fitted values are expressed in μg/μL, and the x axes display log₁₀-transformed concentrations. (F) Cell-cycle distributions showing a higher G2/M-phase fraction after Rhein/Pectin@Zn treatment; the summary axis reports cell-cycle fraction (%). Data are presented as mean ± SD, with individual values shown as plotted. *P < 0.05 versus NC; #P < 0.05 versus Pectin@Zn.

Annexin V-FITC/PI flow cytometry further showed an increased apoptotic fraction in Rhein/Pectin@Zn-treated B16 cells (Fig. 6B and C). RT-qPCR showed an increased Bax/Bcl2 mRNA ratio and altered Tnf and Trp53 transcript levels, a pattern associated with the observed apoptotic response (Fig. 6D). In K7M2 cells, the Bax/Bcl2 mRNA ratio and Tnf and Trp53 transcript levels were also altered after treatment (Fig. S9).

B16-cell viability responses after 48 h were summarized by fitted IC₅₀ values. Rhein/Pectin@Zn showed a lower fitted IC₅₀ (18.28 μg/mL) than Pectin@Zn (360.0 μg/mL) (Fig. 6E). The x axes in panel E display log₁₀-transformed concentration values.

Cell-cycle analysis showed a higher G2/M-phase fraction after Rhein/Pectin@Zn treatment than in the control group (11.92%; Fig. 6F). This result is consistent with G2/M-phase accumulation.

Furthermore, scratch assays with B16 cells demonstrated significant inhibition of tumor cell migration by Rhein/Pectin@Zn hydrogel (Fig. S10), highlighting its multi-targeted suppression of malignant phenotypes.

3.4.3. Endothelial angiogenesis-associated readouts

We assessed endothelial angiogenic readouts in HUVECs. Rhein/Pectin@Zn reduced the expression of pro-angiogenic genes, including VEGF, EGF, HGF, and EGFL6, relative to the NC group and, in most cases, relative to Pectin@Zn alone (Fig. 7A–C). These changes were accompanied by weaker CD31 and VEGF immunofluorescence signals and lower corresponding mean fluorescence densities (Fig. 7B and C), together with a lower VEGF concentration in HUVEC culture supernatants measured by ELISA (Fig. S11). Because treatment also affected HUVEC viability, the ELISA result was not interpreted as a per-cell secretion rate. Collectively, these readouts support reduced endothelial angiogenic signaling. Primer sequences are provided in Table S2.

Fig. 7.

Endothelial angiogenesis-associated molecular and imaging readouts after Rhein/Pectin@Zn treatment in vitro. (A) RT-qPCR analysis of VEGF, HGF, and EGF transcript levels in HUVECs. (B) Representative immunofluorescence images of CD31 and VEGF. (C) RT-qPCR analysis of EGFL6 and quantification of CD31 and VEGF mean fluorescence density. Data are presented as mean ± SD. *P < 0.05 versus NC. Scale bars, 50 μm.

3.5. In vivo antitumor efficacy and biosafety

Following the experimental period, mice were euthanized humanely, and tumor tissues were excised for histopathological evaluation via H&E and TUNEL staining.

The antitumor efficacy of Rhein/Pectin@Zn was evaluated in a subcutaneous B16 melanoma model by repeated local administration. Gross inspection of excised tumors showed a clear size reduction in the Rhein/Pectin@Zn group compared with the NC group, whereas Pectin@Zn produced partial inhibition (Fig. 8A). Quantitative analysis confirmed decreases in endpoint tumor volume and tumor weight (Fig. 8B). H&E staining showed more extensive tissue damage and fewer preserved tumor structures in the Rhein/Pectin@Zn group (Fig. 8C). Ki-67 staining decreased, whereas TUNEL positivity increased, indicating reduced proliferation and increased apoptosis (Fig. 8C and D). Protein-level immunohistochemistry further showed increased BAX staining, decreased BCL-2 staining, and reduced CD31 staining in Rhein/Pectin@Zn-treated tumors (Fig. 8C and D). The BAX and BCL-2 mean-density values were obtained from matched tumor specimens and used to calculate the corresponding BAX/BCL-2 ratio. The concordant increase in the Bax/Bcl2 mRNA ratio and the BAX/BCL-2 immunohistochemical ratio supports a shift toward pro-apoptotic signaling, while the CD31 result indicates reduced tumor vascular staining.

Fig. 8.

In vivo antitumor efficacy and tumor histology of Rhein/Pectin@Zn in the subcutaneous B16 melanoma model. (A) Representative endpoint photographs of excised tumors from the NC, Pectin@Zn, and Rhein/Pectin@Zn groups. (B) Endpoint tumor volume and tumor weight. (C) Representative H&E, Ki-67, TUNEL, BAX, BCL-2, and CD31 staining of tumor sections. (D) Quantification of the mean staining density for Ki-67, TUNEL, BAX, BCL-2, and CD31. Data are presented as mean ± SD; n = 6 mice for endpoint tumor measurements, and individual values for histological quantification are shown as plotted. *P < 0.05 versus NC; #P < 0.05 versus Pectin@Zn. Scale bars: 200 μm for H&E, Ki-67, TUNEL, BAX, and BCL-2; 100 μm for CD31.

Major-organ H&E sections showed no overt treatment-associated histopathological abnormalities in the displayed heart, liver, spleen, lung, or kidney fields (Fig. S12). No grossly visible residual hydrogel was observed at dissection; dedicated degradation measurements were not performed.

3.6. The therapeutic mechanism of Rhein/Pectin@Zn hydrogel

To further define the underlying mechanism, transcriptomic analysis was performed to compare the Rhein/Pectin@Zn and control groups. Differential-expression analysis identified broad transcriptional remodeling (Fig. 9A and B), and pathway-enrichment analyses highlighted cell-cycle regulation and p53-associated apoptosis as major affected pathways (Fig. 9C and D). RT-qPCR and immunohistochemistry further showed downregulation of Cyclin B1/Ccnb1 and upregulation of PUMA/Bbc3 after Rhein/Pectin@Zn treatment (Fig. 9E–G), findings consistent with the observed G2/M arrest and apoptotic phenotype.

Fig. 9.

Transcriptomic and histological readouts associated with cell-cycle regulation and apoptosis after Rhein/Pectin@Zn treatment. (A) Heatmap of DEGs between the NC and Rhein/Pectin@Zn groups; the source sample label “ZnPerh” denotes Rhein/Pectin@Zn. (B) Volcano plot of DEGs for comparison A versus B, where A denotes Rhein/Pectin@Zn and B denotes NC. (C) KEGG pathway enrichment analysis of DEGs. (D) GSEA showing negative enrichment of the cell-cycle gene set in the Rhein/Pectin@Zn group. (E) RT-qPCR analysis of Ccnb1 and Bbc3. Data are presented as mean ± SD with individual values shown (n = 3). (F) Representative immunohistochemical staining of Cyclin B1 and PUMA in tumor sections from the indicated groups. Scale bars, 300 μm. (G) Quantification of Cyclin B1 and PUMA staining. Data are presented as mean ± SD (n = 6). *P < 0.05 versus NC; #P < 0.05 versus Pectin@Zn.

The significant downregulation of Ccnb1 (encoding Cyclin B1) and the notable upregulation of Bbc3 (encoding PUMA) after hydrogel treatment jointly explain the molecular mechanism underlying its anti-melanoma effect. Ccnb1 is a critical regulatory subunit of the M-phase promoting factor (MPF), and its decreased expression leads to the inactivation of the MPF complex, preventing the initiation of mitosis and causing cell cycle arrest at the G2/M phase [26,27]. On the other hand, Bbc3, a BH3-only protein encoded by the p53 target gene, is upregulated and antagonizes anti-apoptotic proteins, thereby activating the mitochondrial apoptosis pathway and promoting cell death in the arrested cells [28,29]. The coordinated alterations of these two key molecules correspond to the phenotypic changes of G2/M phase arrest and apoptosis induction, respectively, thus constituting the mechanistic basis for the hydrogel's anti-melanoma effect through the dual “arrest-death” mechanism.

4. Conclusions

This study developed a coordination-strengthened Rhein/Pectin@Zn hydrogel and demonstrated proof-of-concept efficacy after repeated intratumoral dual-syringe administration in a subcutaneous B16 melanoma model. Standardized ex vivo measurements characterized the bulk material, whereas the antitumor outcome was measured directly using the actual in situ delivery procedure. In vitro, the formulation reduced tumor-cell viability signals and increased apoptosis-associated readouts; the scratch-wound images were interpreted descriptively because no quantitative migration analysis was displayed. It also increased the Bax/Bcl2 mRNA ratio in treated cells and the BAX/BCL-2 immunohistochemical ratio in tumor tissue, while reducing endothelial angiogenic signaling and tumor CD31 staining. The current evidence does not establish formal component synergy, equivalence between the ex vivo and in situ gel states, comprehensive systemic safety, or prevention of postoperative recurrence. Further work requires component-resolved controls, direct gelation-comparison and recovery testing, and a resection or residual-tumor model.

CRediT authorship contribution statement

Kai Li: Writing – original draft, Validation, Methodology, Investigation, Formal analysis, Conceptualization. Qili Sun: Writing – review & editing, Methodology, Investigation, Conceptualization. Jingle Chen: Validation, Methodology, Investigation. YanSong Wang: Methodology, Investigation. Enze Zhao: Writing – review & editing, Methodology, Funding acquisition. Shuaishuai Cao: Writing – review & editing, Methodology, Funding acquisition. Chao Xie: Writing – review & editing, Validation, Supervision, Investigation, Funding acquisition. Bin Tang: Supervision, Project administration, Funding acquisition.

Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this work the authors used CHATGPT3.5 in order to check grammar and polish the English language quality. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.

Declaration of competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is/are the supplementary data to this article:

Data availability

Data will be made available on request.