Biometallic peptide-drug conjugates in photo-crosslinkable hydrogels enable combined photothermal-chemotherapy against breast cancer.

Introduction
Conventional chemotherapy (CT) for cancer treatment is often hindered by systemic toxicity, limited tumor specificity, and the development of multidrug resistance [1, 2]. Various delivery systems, such as emulsions, lipid micelles, and nanospheres, have been employed to improve the solubility of hydrophobic drugs by encapsulating them for in vivo administration. However, these carriers still suffer from insufficient targeting capability and unintended off-target effects, which significantly limit their therapeutic efficacy [3, 4]. To address these challenges, peptide-drug conjugates (PDCs) have emerged as a promising strategy for achieving targeted and precise drug delivery in cancer therapy [5–8]. In addition, engineered PDCs via chemical modification strategies have been demonstrated comprehensively, which have revealed great potential for preclinical and clinical studies [9]. PDCs are composed of therapeutic agents that are covalently or non-covalently linked to peptides, enabling tumor-specific accumulation through ligand-receptor recognition or stimuli-responsive mechanisms triggered by changes in pH [10], temperature, light, or enzymatic activity [11, 12].
Unlike conventional PDCs, which are typically constructed by covalently linking individual peptide molecules with small-molecule drugs in a one-to-one manner, the present study employs self-assembled peptide nanofibers (PNFs) as a supramolecular scaffold for drug conjugation. This hybrid PNF–drug architecture offers several distinct advantages [13, 14]. Assembled from motif-designed peptide sequences, PNFs exhibit high biocompatibility, tunable mechanical properties, and versatile functionalization potential [15, 16]. These features render PNFs ideal scaffolds for efficient drug encapsulation and controlled, sustainable release. Moreover, their fibrous architecture closely mimics the extracellular matrix (ECM), promoting enhanced cell adhesion and facilitating deeper penetration into tumor tissues [17–19]. Unlike traditional carriers, PNFs possess abundant surface functional groups (e.g., amino and carboxyl groups) that enable efficient drug loading through electrostatic interactions or covalent bonding, resulting in higher loading capacities and stimuli-responsive release profiles [20–22]. For instance, β-sheet-forming peptides can be engineered to respond to tumor microenvironmental cues, thereby enabling selective drug release and minimizing off-target effects [23]. Self-assembled RADA16-I PNFs, for example, could load the anticancer drug SN-38 through π–π stacking interactions, achieving drug loading efficiencies of up to 38.2% [24]. Despite such advancements, conventional nanocarriers still suffer from inadequate drug loading capacities and limited penetration in solid tumor tissues. Therefore, the development of appropriate carriers for the effective delivery of active therapeutic agents remains essential.
Injectable hydrogels, owing to their tunable mechanical properties, can localize at the target site and act as reservoirs for sustained and controlled drug release [25, 26]. PNF-based hydrogels, in particular, can dynamically alter their properties in response to external stimuli, facilitating targeted drug accumulation in specific regions [27, 28]. Through rational peptide design, injectable hydrogels can be endowed with biological and environmental responsiveness, thereby broadening their applicability across a wide range of biomedical applications [29].
Photothermal therapy (PTT), alternatively, offers a non-invasive and controllable approach for tumor ablation by converting absorbed near-infrared (NIR) light into localized heat through photothermal agents (PAs) [30, 31]. Advanced photothermal platforms have demonstrated that localized hyperthermia can not only directly ablate tumor cells but also enhance drug penetration, cellular uptake, and therapeutic efficacy when combined with chemotherapy [32]. Among them, gold nanoparticles (GNPs) have been extensively utilized in PTT owing to their pronounced surface plasmon resonance (SPR), outstanding photostability, and facile surface modification [33]. In addition, their unique optical and electronic properties render GNPs highly suitable for multifunctional biomedical applications, including cell imaging, targeted drug delivery, and tumor PTT [34, 35].
In this study, we present a novel biometallic PNF-GNPs-doxorubicin (DOX) conjugate (PGDC)-based hydrogel system for breast cancer therapy (Scheme 1). We hypothesize that the designed PGDCs enhance therapeutic efficacy through the combination of physical (PTT) and chemical (CT) modalities. Meanwhile, the injectable hydrogel system offers flexible handling and enables the controlled release of anticancer drugs for sustained and localized breast cancer treatment.

To test this hypothesis, hyaluronic acid (HA) is first modified with methacrylic anhydride (MA) to introduce UV sensitivity, followed by the synthesis of oxidized photo-responsive hyaluronic acid (O-HAMA). This modification enabled rapid gelation of the precursor solution within 90 s under exposure to 365 nm UV light. UV light-induced cross-linking of HA ensured reproducible in-situ formation of bioactive hydrogels. The rapid gelation rendered the system suitable for minimally invasive injection, forming a stable three-dimensional (3D) hydrogel scaffold at the tumor site. Subsequently, PNFs are employed as biomimetic architecture to immobilize GNPs and DOX through both covalent and non-covalent interactions, forming PGDCs. The PNFs are then linked to O-HAMA through dynamic Schiff base bonds, enabling pH-responsive drug release in the acidic tumor microenvironment. Moreover, the incorporated GNPs demonstrate strong photothermal stability and high light-to-heat conversion efficiency upon 808 nm NIR laser irradiation. The heat generated during PTT enhanced vascular permeability and facilitated deeper DOX penetration, ultimately achieving a combined PTT/CT therapeutic effect. Unlike conventional PDCs that primarily focus on peptide-drug linkage for targeted drug delivery, our platform uniquely combines biomimetic nanofiber scaffolding, biometalization, and injectable hydrogel fabrication, resulting in exceptional tumor inhibition and minimal off-target toxicity.

Experimental section

Materials and reagents
The KIIIIKNNCCY peptide (purity = 95%) was purchased from Synpeptide Co., Ltd (Nanjing, China). HA (Mw: 400–800 kDa), MA (94%), Irgacure 2959 (98%), NaIO4 (96%), trisodium citrate dihydrate (98%), HAuCl4 (99%), doxorubicin hydrochloride (98%), and polyethylene glycol (PEG, 98%) were obtained from the Macklin Biochemical Co., Ltd. (Shanghai, China). α-MEM (Cat. No. L570KJ) was purchased from the BasalMedia Technologies, China., while and fetal bovine serum (FBS) was acquired from the LONSERA, Suzhou Shuangru Biology Science Technology Co., Ltd. Penicillin-streptomycin (PS) (Cat. No. 60162ES76) was purchased from the Yeasen Biotechnology (China). The FITC-Annexin V/PI Apoptosis Kit (Cat. No. SK2070) was obtained from Beijing Coolaber Science＆Technology Co., Ltd. The Calcein-AM/PI double-staining kit and hematoxylin and eosin (H&E) (Cat. No. G1121) stain kit were purchased from the Solarbio, China. Confocal dish (Cat. No. BDD012035) was purchased from the Jet Biofil,China. Anti-Ki67 Rabbit anitbody (Cat. No. R013743) was purchased from Shanghai Epizyme.

Synthesis of PNFs
The peptide with the sequence of KIIIIKNNCCY was selected for the synthesis of PNFs. A peptide solution (1 mg/mL) was prepared using ultrapure water and incubated in a 47 ℃ water-bath for 1–3 days to form PNFs. Similarly, a 2 mg/mL peptide solution was prepared and incubated under the same conditions for 1–2 days to obtain PNFs with different assembly characteristics. The self-assembly process was monitored at various time intervals to elucidate the formation mechanism of the PNFs.

Synthesis of GNPs
The synthesis of GNPs was carried out with slight modifications to a previously reported method [36]. In brief, 97.1 mg of trisodium citrate dihydrate was dissolved in 150 mL of deionized water and heated under continuous stirring. After the solution reached boiling for 15 min, 1 mL of HAuCl4·3H2O (25 mM) was added, and the reaction was maintained under stirring. Once the solution developed a characteristic burgundy color, both stirring and heating were stopped, yielding the GNP colloidal solution. The resulting solution was then concentrated to an appropriate volume using a PEG solution (here PEG serves as an agent to adsorb water but not react with GNPs). The concentration of the obtained GNP solution was determined by inductively coupled plasma (ICP) analysis.

Synthesis of PGDCs
An appropriate amount of DOX was added into a 1 mg/mL PNF solution to create a final DOX concentration of 100 µg/mL under continuous stirring. Subsequently, an equal volume of the PNFs-DOX solution was mixed with the GNP solution (600 µg/mL) to obtain the PGDC solution. The resulting PGDC solution, including 0.5 mg/mL PNFs, 25 µg/mL DOX, and 300 µg/mL GNPs, was stored at 4 °C in the dark to preserve its stability.

Synthesis of O-HAMA and photo-crosslinked hydrogels
The preparation of O-HAMA was adapted from previously reported methods [37]. Briefly, 80 mL of a 2 wt% HA solution was prepared and maintained at 0 °C. 4.8 mL of MA (94%) was then added to create a final MA molar concentration of 0.36 M, followed by the slow addition of 2.4 mL of NaOH (5.0 M) after thorough mixing. The reaction mixture was allowed to react for 24 h at 0 °C. Upon completion, the mixture was dialyzed against deionized water for 4 days and subsequently freeze-dried to obtain HAMA. The final product was stored at 4 °C for further use.
The introduction of aldehyde groups in O-HAMA was qualitatively verified using aldehyde-specific chemical reactions. Briefly, non-oxidized HAMA and O-HAMA samples were separately dispersed in aqueous solution. For general carbonyl detection, samples were reacted with 2,4-dinitrophenylhydrazine (DNPH) reagent, and the formation of yellow–orange precipitates was visually inspected.
To specifically detect aldehyde groups, Tollens’ reagent was freshly prepared and added to the samples. The reaction mixtures were gently incubated at room temperature, and the formation of a characteristic silver mirror on the inner wall of the reaction tube was recorded. Non-oxidized HAMA was used as a negative control.
Then, 1 g of HAMA was dissolved in water to prepare a 2 wt% solution. After that, 0.5 g of NaIO4 (96%) was added, and the mixture was stirred at room temperature in the dark. After 6 h, 1 mL of ethylene glycol (98%) was introduced to quench the reaction. The resulting O-HAMA product was further purified by dialysis against deionized water for 3 days. Finally, the purified O-HAMA solution was freeze-dried under vacuum and stored at 4 °C for subsequent use.
The O-HAMA/PGDC hydrogel precursor solution was prepared by dissolving 40 mg of O-HAMA in 1 mL of the PGDC solution that synthesized in Sect. Synthesis of PGDCs. Then, 1.5 mg of Irgacure 2959 (98%) was added and stirred until fully dissolved. The mixture was then exposed to 365 nm UV irradiation to induce gelation.

Swelling test
Three hydrogels of identical size were prepared and weighed to obtain the initial weight (W₀). The samples were then immersed in PBS at pH 5.0, 6.5, and 7.4 (same volume for each). At predetermined time points, the hydrogels were removed, and surface water was gently blotted using filter paper. The weight of each hydrogel (Wi) was recorded, and the swelling ratio was calculated using the formula:

Rheological properties
Cylindrical hydrogels with a diameter of 25 mm and a height of 2 mm were prepared for rheological characterization. A circular plate with a diameter of 25 mm was used for the measurements. To minimize sample slippage, 400 grit sandpaper was affixed to the plate surface. The strain response of the hydrogels was first assessed in oscillatory mode, and the amplitude sweep tests (ranging from 1 to 250%) were conducted at a constant frequency of 1.0 Hz to determine the linear viscoelastic region. To evaluate the strain stability, alternating strain experiments were performed by cycling the strain between 5% and 250% at 2-minute intervals, repeated four times, with the angular frequency fixed at ω = 10 rad/s.

Photothermal characteristics of hydrogels
The photothermal properties were evaluated by irradiating 1 mL samples of H2O, GNPs, and PGDCs using an 808 nm laser at a power density of 2 W cm−2. The corresponding temperature-time curves were recorded to assess their photothermal conversion efficiency. Subsequently, the O-HAMA/PGDC hydrogels were tested to investigate the concentration-dependent effect of GNPs. Hydrogels containing 200, 250, and 300 µg/mL of GNPs were prepared (1 mL each) and placed into cuvettes. Each sample was irradiated with 808 nm NIR light (power of 2 W cm−2) for 10 min. Real-time infrared thermal images and temperature-time profiles were simultaneously recorded to monitor the photothermal response.
Power-dependent photothermal tests were performed on O-HAMA/PGDC hydrogels containing 300 µg/mL of GNPs. The hydrogels were irradiated with an 808 nm laser at power densities of 1.5, 2.0, and 2.5 W cm⁻2 for 10 min. During irradiation, real-time infrared thermal images were captured, and the corresponding temperature-time curves were recorded to evaluate the photothermal performance under varying laser intensities.
The photothermal stability of the O-HAMA/PGDC hydrogels was evaluated by irradiating the hydrogel (with a 300 µg/mL concentration of GNPs) for 10 min using an 808 nm laser at a power of 2 W cm−2. Following each irradiation, the laser was turned off, and the hydrogel was allowed to cool at room temperature for 10 min. This heating-cooling cycle was repeated seven times, and the temperature-time curves were recorded to monitor the maximum temperature reached during each cycle, thereby evaluating the hydrogel’s photothermal stability.

Drug release of hydrogels
Three sets of photo-crosslinked hydrogels with identical specifications were prepared and immersed in equal volumes of PBS at pH 5.0, 6.5, and 7.4 to evaluate their drug release behavior. At predetermined time intervals, 100 µL of the release medium was collected, diluted to 5 mL, and the DOX concentration was determined spectrophotometrically. The cumulative drug release profiles were subsequently obtained by plotting the percentage of drug released versus time.

Biocompatibility test
To evaluate the biocompatibility of the materials, L929 fibroblast cells were used as the model cell line. Briefly, L929 cells were seeded into 96-well plates at a density of 5,000 cells/well and incubated overnight at 37 ℃ in a humidified atmosphere containing 5% CO2 to allow cell adhesion. The cells were then divided into three groups: a control group (treated with PBS), an O-HAMA/PG group, and an O-HAMA/PGDC group. After co-culturing for 1 and 3 days, cell viability was assessed using Calcein-AM/PI double staining. Calcein-AM was hydrolyzed by intracellular esterases in viable cells to produce green fluorescence, whereas PI penetrated compromised cell membranes and bound to nucleic acids in dead cells, emitting red fluorescence. Following staining, the supernatant was discarded, and the cells were washed three times with PBS to remove residual dyes. Fluorescence images were captured using inverted fluorescence microscope, and the live/dead cell was quantitatively analyzed to assess the biocompatibility of each hydrogel formulation. To assess apoptosis, L929 cells were treated under the specified experimental conditions and subsequently analyzed by flow cytometry. Apoptotic cells were stained using the Annexin V–FITC Apoptosis Detection Kit (Vazyme, China) according to the manufacturer’s protocol and as previously described. Briefly, after treatment, the cells were collected, washed twice with cold PBS, and resuspended in 1× binding buffer. Annexin V–FITC and PI were added to the cell suspension and incubated for 15 min at room temperature in the dark. The samples were then analyzed using a flow cytometer (BD FACSCanto II, USA). The percentage of live, early apoptotic, late apoptotic, and necrotic cells was determined using FlowJo software.

In vitro PTT of tumor cells
To evaluate the in vitro antitumor efficacy of the materials, 4T1 breast cells were selected as the tumor model. Briefly, 4T1 cells were seeded into 96-well plates at a density of 5000 cells/well and incubated overnight at 37 ℃ in a humidified atmosphere containing 5% CO2 to allow cell adhesion. The cells were then divided into three treatment groups: control (treated with PBS), O-HAMA/PG, and O-HAMA/PGDC. After exposure to NIR light irradiation at a power density of 2 W cm−2 for 5 min, the cells were stained using Calcein-AM/PI double staining to distinguish viable and dead cells. Following staining, the supernatant was discarded, and the cells were washed three times with PBS to remove unbound dyes. Finally, fluorescence microscopy was used to image the cells in each group, and the ratios of live to dead cells were quantitatively analyzed to assess the antitumor performance of the materials. The apoptosis analysis was also performed by flow cytometry as described in Sect. Biocompatibility test.

Cellular uptake
4T1 cells were seeded into confocal dishes and incubated overnight at 37 °C in a humidified atmosphere containing 5% CO2 to ensure proper cell adhesion. Subsequently, DOX and O-HAMA/PGDC solutions were diluted with culture medium to achieve a final DOX concentration of 10 µg/mL. The diluted solutions were co-incubated with the cells for 4 h. After incubation, the cells were gently washed three time with pre-cooled PBS to remove uninternalized drug, and then fixed with 4% paraformaldehyde. The cell nuclei were stained with Hoechst 33,258, and the cytoskeleton was labeled with phalloidin. Finally, the stained samples were observed using an inverted confocal fluorescence microscope to analyze the intracellular uptake and subcellular distribution of DOX. The mean fluorescence intensity of intracellular DOX was quantified from confocal microscopy images using ImageJ software. Statistical significance was determined using an unpaired two-tailed Student’s t-test.

PTT of tumors in vivo and biosafety analysis
Thirty healthy female BALB/c nude mice (its weight was about 20 ± 2 g with 4 weeks old and the sex of the animals did not influence the outcomes of this study) were selected to establish a subcutaneous 4T1 tumor xenograft model. Each mouse was subcutaneously injected with 100 µL of 4T1 cell suspension at a concentration of 5 × 106 cells/mL. After 7 days, when the tumor volume reached approximately 100 mm3, the mice were randomly divided into five groups (n = 6): control group (treated with PBS), laser group, laser + O-HAMA/PG group (PTT group), O-HAMA/PGDC group (CT group), and laser + O-HAMA/PGDC group (PTT/CT group). On day 7, 50 µL of the corresponding hydrogel (DOX concentration = 25 µg/mL) was injected around the tumor site (PBS for the control group). The applied dose of drug for these mice groups was 0.0625 mg/kg. The tumor areas of mice in the laser, PTT, and PTT/CT groups were then locally irradiated with an 808 nm NIR laser (2 W cm− 2) for 10 min. Tumor volumes were measured every two days using the formula V = (L × W2)/2, where L and W represent the longest and shortest tumor diameters, respectively. On 21-day post-inoculation, all mice were euthanized, and tumor tissues and blood from each group were collected, photographed, weighed, and subjected to Ki-67 immunohistochemical staining to assess tumor cell proliferation. Major organs were also collected for hematoxylin and eosin (H&E) staining to evaluate the in vivo biocompatibility and systemic toxicity of the materials.

Animal experiments and ethics statement
Animal studies were performed according to the National Research Council’s Guide for the Care and Use of Laboratory Animals. The experimental protocols were performed in accordance with the ARRIVE guidelines. The experimental protocol was reviewed and approved by the Animal Ethics Committee of Qingdao University (Approval No. 20241022BALB/cA-nu3020241130130).

Characterization techniques, software, and statistical analysis
Atomic force microscopy (AFM) measurements were performed in tapping mode under ambient conditions using an FM-Nanoview 6800 AFM system (FSM-Precision Instruments, China). AFM images were obtained with Tap300Al-G silicon probes (300 kHz, 40 N·m−1). Image acquisition and analysis were carried out using Gwyddion software (version 2.57). The structural morphology of the samples (PNFs, PNFs-DOX, and PGDCs) was examined via transmission electron microscope (TEM, Tecnai G2 F20, FEI Co.). The zeta potential and dynamic light scattering (DLS) of the material were determined by a nanoparticle size-zeta potential meter (Nano ZSE, Malern). The microstructure of the hydrogels was observed using scanning electron microscope (SEM, Hitachi Regulus 8100, Japan). X-ray photoelectron spectroscopy (XPS) analysis was conducted with a PHI 5000 VersaProbe III spectrometer (ULVAC-PHI, Japan). The gold content in the solution was quantified using inductively coupled plasma mass spectrometry (ICP-MS, Avio 200, USA). FT-IR spectroscopy of the samples was performed with a Nicolet iS10 spectrometer (USA). UV-vis spectrophotometry was carried out using a UV-2600 spectrophotometer (China). An infrared thermography camera (Fotric 223 s, PR China) was employed to capture the in-situ temperature of the material, and a photothermal warming curve was generated using photothermal analysis software (AnalyzIR).

Results and discussion

Preparation and characterizations of PNFs
To construct PNFs with specific functional properties, the peptide sequence used in this study was designed to incorporated two motifs: KIIIIK and NNCCY (Fig. 1a). The KIIIIK segment serves as an amphiphilic domain that facilitates peptide self-assembly into PNFs [38, 39], while the NNCCY motif enhances the hydrophilicity of the formed PNFs and introduces multiple sulfhydryl (-SH) groups, providing active binding sites for the subsequent conjugation of GNPs with PNFs [40]. The interplay between these two motifs ensures that self-assembly and biometallization occur in a sequential and structurally compatible manner: first, KIIIIK-driven nanofiber formation establishes a robust template; then, the thiol-rich NNCCY domain enables selective nucleation and anchoring of GNPs without disrupting the underlying fibrillar architecture. This synergistic molecular design leads to well-dispersion of GNPs, enhanced photothermal conversion efficiency, and improved structural integrity of the PGDCs.

To investigate the self-assembly behavior of the designed peptide, a 1 mg/mL aqueous peptide solution was incubated at 47 ℃ for 1–3 days. Samples were collected daily and characterized by AFM to monitor morphological evolution over time (Fig. 1b-d). After 1 day of incubation (Fig. 1b), a limited number of short nanofibers were observed, along with a large proportion of unassembled peptide monomers. As shown in Fig. 1c, the length of nanofibers increased after 2 days, indicating progressive peptide self-assembly. After 3 days, the peptides had self-assembled into morphologically stable PNFs with extended fiber lengths (Fig. 1d). The corresponding statistical analysis (Fig. 1j and k) indicates that the length of the formed nanofibers increased from 0.29 ± 0.1 μm to 0.78 ± 0.29 and 1.02 ± 0.52 μm after incubating 1, 2, and 3 days, respectively, while the height of the nanofibers decreased from 10.83 ± 3.09 nm to 6.21 ± 1.07 and 5.81 ± 0.87 nm, correspondingly. Based on these results, it can be found that the peptide monomers tended to form short aggregates at the beginning, and with the self-assembly process, the conformation of peptide aggregates transited and formed longer nanofibers with lower height. However, a noticeable number of peptide aggregates were still present, suggesting incomplete conversion of peptide monomers into ordered nanofibers.
An aqueous peptide solution at a concentration of 2 mg/mL was incubated at 47 ℃ and monitored using AFM. After 1 day of incubation, numerous short fibers were observed, indicating initial peptide self-assembly (Figs. 1e). Figure 1f presents a view of the peptide self-assembly into PNFs after 2 days of incubation in water, along with a typical AFM height image. The image showed that peptides cultured under these conditions almost entirely self-assemble into PNFs with a height of approximately 6 nm. Comparison with the 1 mg/mL solution demonstrated that increasing peptide concentration enhanced the efficiency of self-assembly into PNFs. Additionally, the length of PNFs increased with incubation time. The peptide solution (2 mg/mL) incubated for 2 days was further characterized by TEM, which revealed PNFs with lengths ranging from a few hundred nanometers to a few micrometers, consistent with the AFM observations (Fig. 1g).
Then, we considered the temperature effect on the peptide self-assembly and the formation of nanofibers. The peptide aqueous solution (1 mg/mL) was incubated at 25 and 37 °C for three days and the prepared samples were characterized with AFM, and the results are shown in Fig. 1h and i. It can be found that at both conditions, only very short nanofibers were formed. In previous studies, we have proved that high temperature (such as 47 °C) was crucial for promoting peptide self-assembly and formation of long nanofibers [41]. Also, we have noticed that the formed PNFs and nanohybrids were stable at this temperature, and formed PNF-based nanomaterials have been successfully applied for cancer therapy, wound healing, and antibacterial applications.
Based on these results, the peptide self-assembly mechanism in water was inferred (Fig. 1l). We propose that, at 47 °C in aqueous solution, the designed peptide monomers initially self-assemble into shorter PNFs, which subsequently elongate over time to form long, stable fibers.

Synthesis and characterizations of biometallic PGDCs
Chemically reduced GNPs were initially characterized using TEM. The resulting image indicates that GNPs synthesized via sodium citrate reduction possessed a uniform spherical morphology with stable structural features. Particle size analysis reveals an average diameter of approximately 10 nm (Fig. 2a). DLS measurements showed hydrodynamic diameters of GNPs range from 20 to 80 nm, with a polydispersity index (PDI) of 0.194, indicating good monodispersity and colloidal stability in aqueous media (Fig. 2b). Together, these results confirm that the synthesized GNPs exhibit a narrow size distribution and high stability.

The theoretical isoelectric point of the motif-designed peptide was 9.02, resulting in a highly positive surface charge (zeta potential = 36.85 ± 0.49 mV) under aqueous conditions. In contrast, DOX exhibited a negative surface charge (zeta potential=−3.18 ± 0.30 mV) in the same environment, as confirmed by the zeta potential analysis (Fig. 2c). Therefore, this electrostatic complementarity facilitated the formation of sTable 1D PDCs via ionic interactions, referred to as PNFs-DOX conjugates (zeta potential = 36.27 ± 1.16 mV). The PNFs-DOX in this study possess abundant surface -SH groups from cystine (C) and a positive charge, which enabled both covalent via forming Au-S bonds and non-covalent attachment (electrostatic interaction) of GNPs (zeta potential=−17.17 ± 0.92 mV), thereby forming stable PGDCs. TEM image confirmed the successful immobilization of GNPs onto the PNFs-DOX structure (Fig. 2d). However, after the conjugation of GNPs with PNFs-DOX, it can be found that the diameter of GNPs did not change but showed clear aggregation along the PNFs (Table 1), due to the formation of strong Au-S bonds. As shown in Fig. 2e, DLS analysis showed that hydrodynamic diameter of the PGDCs centers around 1500 nm, with a PDI of 0.205. Although particle uniformity is slightly reduced compared to free GNPs, the PGDCs maintain satisfactory dispersion and colloidal stability [42]. An ideal drug delivery system requires not only an appropriate hydrodynamic diameter to achieve desired release rate and in vivo distribution, but also a sufficiently low PDI value to ensure consistent behavior across all particles, thereby delivering safe, effective, and reliable therapeutic outcomes. In this study, the PDI of 0.205 (relatively low) and the hydrodynamic diameter (1500 nm) of PGDCs are helpful to release the DOX slowly and stably.

To further confirm the successful formation of PGDCs, XPS was conducted on the PGDC materials. The full XPS spectrum reveals characteristic peaks corresponding to C1s, N1s, O1s, Au4f, and S2p, validating the presence of relevant chemical components (Fig. 2f). The Au4f spectrum shows distinct peaks at 86.2 (Au4f5/2) and 82.4 eV (Au4f3/2), confirming the elemental state of gold in the PGDCs (Fig. 2g). As shown in Fig. 2h, the characteristic XPS peaks of S2p1/2 and S2p3/2 are 167.8 and 162.8 eV, respectively. UV-vis spectroscopy was employed to compare the optical behavior of GNPs alone and in the PGDCs (Fig. 2i). The results indicate that the UV-vis characteristic absorption peak of GNPs shifted from 529 to 561 nm upon binding with PNFs, while the increased absorbance at 808 nm provides a solid foundation for the subsequent PTT mediated by the PGDCs [43, 44].

Stability, injectability, and rheological behavior of O-HAMA/PGDC hydrogels
HA was first modified with MA to introduce photo-crosslinkable vinyl groups, and subsequently oxidized with sodium periodate (NaIO4) to yield O-HAMA. FT-IR analysis revealed the presence of a carbonyl-related absorption band around ~1720 cm⁻¹ in HAMA and O-HAMA, corresponding to ester/carbonyl stretching vibrations associated with methacrylation and oxidation. After periodate oxidation, a slight increase in the intensity of this band was observed, suggesting the introduction of additional carbonyl functionalities in O-HAMA (Fig. 3a). To avoid ambiguity arising from spectral overlap, aldehyde formation was further verified by aldehyde-specific chemical reactions, emphasizing functional accessibility rather than exhaustive structural elucidation (inset of Fig. 3a). As a result, O-HAMA serves as a dual-functional macromer containing aldehyde (–CHO) groups for dynamic Schiff-base crosslinking and methacrylate groups for robust photo-induced polymerization. This combination of chemical functionalities enables versatile hydrogel formation strategies with improved mechanical stability and tunable network properties.
Upon mixing O-HAMA with the PGDC solution, the aldehyde groups on O-HAMA reacted with the amino groups of the peptide molecules to form dynamic Schiff base bonds [45], enabling pH-responsive release of PGDC in the acidic tumor microenvironment (Fig. 3b). The mixed solution underwent rapid gelation within 90 s upon exposure to 365 nm UV light. During this process, the vinyl group (-CH=CH2) underwent free radical reaction under UV light irradiation, resulting in chemical cross-linking and promoting hydrogel formation [18]. The resulting O-HAMA/PGDC hydrogel exhibits sufficient mechanical stability, retaining its shape even when tilted at 45° for 2 min (Fig. 3d). When immersed in PBS, the hydrogel remained stable for 7 days (Fig. 3e). Additionally, the O-HAMA/PGDC hydrogel could be extruded from the microtiter syringe in the form of a micro-hydrogel in PBS solution (Fig. 3f). SEM image of the freeze-dried samples presents a rich and homogeneous porous structure (Fig. 3c), providing an ideal environment for efficient encapsulation and sustained retention of PGDCs.

The viscoelastic properties of O-HAMA/PGDC hydrogels were evaluated using strain sweep and cyclic strain tests on a rheometer. The modulus variations of the hydrogel at the strain pressure ranging from 1% to 250% was measured at a fixed frequency using the strain scanning mode, providing insights into its mechanical properties. As shown in Fig. 3h, the storage modulus (G’) of the hydrogel is greater than the loss modulus (G’’) at strain pressures below 186%, indicating that the hydrogel maintained stable structural integrity and elastic behavior within this range. At strains exceeding 186%, G′ dropped below G″, suggesting a breakdown or rearrangement of the hydrogel network structure [46]. To assess the reversibility of the hydrogel, alternating low (1%) and high (250%) strains were applied in dynamic strain scanning to evaluate the viscoelastic recovery (Fig. 3i). The results show that at smaller strains, where G’ > G’’, the internal hydrogel structure remained intact, exhibiting good elasticity. However, at high strains, where G’ < G’’, the internal structure of the hydrogel was destroyed. After four consecutive strain cycles, both G’ and G’’ returned to their initial values, indicating that the mechanical properties of the hydrogel are recoverable and supporting its suitability for injectable applications.
In addition, the swelling behavior of the O-HAMA/PGDC hydrogels was further evaluated. Hydrogels with identical size were incubated in PBS at different pH values at 37 °C for 48 h to monitor their swelling behavior. Swelling equilibrium was achieved after approximately 36 h. The hydrogels exhibited swelling ratios ranging from 50% to 60% in PBS across the pH values of 5.0, 6.5, and 7.4 (Fig. 3g). These results demonstrate favorable swelling characteristics, highlighting the O-HAMA/PGDC hydrogel system’s environmental responsiveness, biocompatibility, and potential to enhance therapeutic efficacy.
It should be noted that the physicochemical properties of photo-crosslinked HA-based hydrogels are closely related to the degree of crosslinking, which can be modulated by UV exposure conditions and photoinitiator parameters. Previous studies have shown that increased crosslinking density generally leads to reduced swelling, enhanced mechanical strength, and slower drug diffusion. In this work, a fixed curing condition was adopted to ensure rapid gelation and experimental consistency [45].

Photothermal properties of O-HAMA/PGDC hydrogels
Photothermal experiments were performed to assess the influence of PNFs on the photothermal performance of GNPs. A 1 mL solution of GNPs and PGDCs (both 300 µg/mL) was irradiated with an 808 nm laser (2 W cm−2) for 10 min. Although the maximum absorption of PGDCs occurs at 561 nm, 808 nm irradiation was selected due to its superior tissue penetration and widespread use in in vivo PTT. As shown in Fig. 4a, the PGDC solution exhibited a higher temperature increase compared to the GNPs solution alone. This enhancement can be attributed to the synergistic interactions between amino acid residues and the SPR of GNPs, which boosts PGDC absorption at 808 nm and improves light-to-heat conversion efficiency. Furthermore, the -SH groups on the PNF side chains act as effective biological templates that promote uniform binding with GNPs. This prevents GNP aggregation, amplifies the photothermal effect, and improves the biocompatibility of the PGDCs effectively.

The photothermal performance of the O-HAMA/PGDC hydrogels was further evaluated in terms of both concentration and power dependence. O-HAMA/PGDC hydrogels containing 200, 250, and 300 µg/mL of GNPs were irradiated with an 808 nm laser at 2 W cm−2 for 10 min. Real-time photothermal images show a gradual and uniform temperature increase over time (Fig. 4b). As shown in the corresponding temperature-time curves (Fig. 4d), the final temperature of the hydrogel rose proportionally with GNP concentration, with the hydrogel containing 300 µg/mL of GNPs reaching approximately 50℃ after 10 min of laser irradiation. Based on this screening, 300 µg/mL GNP concentration was used in the subsequent cell experiments because it could reproducibly raise the temperature to ~50 °C in our experimental conditions, providing sufficient photothermal heating for biological evaluation without relying on excessively ablative conditions. To further assess power dependence, the O-HAMA/PGDC hydrogel containing 300 µg/mL GNPs was exposed to laser powers of 1.5, 2.0, and 2.5 W cm−2. The resulting real-time photothermal images and temperature-time profiles were recorded (Fig. 4c&e) demonstrate a clear positive correlation between irradiation power and temperature elevation. Moreover, the O-HAMA/PGDC hydrogel presents remarkable photothermal stability over seven multiple heating-cooling cycles under 808 nm laser irradiation (Fig. 4f). This excellent stability is attributed to the formation of a protective matrix surrounding the GNPs by the PNFs and O-HAMA, which effectively prevents nanoparticle aggregation and structural degradation during the photothermal process, thereby maintaining consistent light-to-heat conversion efficiency across multiple cycles.

In vitro drug release and cellular drug uptake
The drug release behavior of the O-HAMA/PGDC hydrogels was then evaluated by immersing the samples in PBS at varying pH values for 120 h, followed by quantification of the released DOX using UV-vis spectroscopy.
As shown in Fig. 5a, under physiological environment (pH = 7.4), the amide bond between the peptide and O-HAMA remained stable, leading to a slow and sustained drug release. In contrast, in the mildly acidic tumor microenvironment (pH = 6.5), the cumulative drug release reached approximately 70% within 5 days. This accelerated release was attributed to the partial hydrolysis of the amide bond and the detachment of PGDCs from the O-HAMA network under acidic conditions. Furthermore, the protonation of acidic groups such as carboxyl groups loosened the hydrogel network structure, accelerating drug release. At pH 6.5, more drug molecules were released from the hydrogel due to acid-induced hydrolysis. At pH 5.0, the enhanced hydrolysis of amide bonds further facilitated greater PGDCs dissociation from the hydrogel matrix, thereby accelerating drug release, as illustrated in Fig. 5b.

Next, we explored the cellular uptake of DOX in 4T1 cells using both free DOX and the O-HAMA/PGDC hydrogel formulation. The uptake of DOX by 4T1 cells was visualized by fluorescence microscopy, taking advantage of autofluorescence properties of DOX [47]. As shown in Fig. 5c, blue fluorescence represents the cell nucleus of 4T1 cells, red represents DOX autofluorescence, and green represents the cytoskeleton stained with phalloidin. It is clear that cells treated with O-HAMA/PGDCs exhibited markedly stronger intracellular DOX fluorescence compared with free DOX, indicating enhanced cellular uptake and intracellular accumulation. Quantitative analysis of the confocal fluorescence images revealed a significant increase in intracellular DOX fluorescence intensity in the O-HAMA/PGDC group compared with the free DOX group (p = 0.0063), indicating enhanced cellular uptake efficiency of DOX when delivered via the hydrogel formulation (Fig. 5d). While we did not delineate the specific endocytic pathway, these results provide direct evidence of efficient drug internalization in the O-HAMA/PGDC system. Compared with free DOX, cells treated with the O-HAMA/PGDC hydrogel exhibited markedly stronger red fluorescent signals, particularly within the nuclei after 4 h of incubation, confirming efficient DOX internalization and nuclear localization. This enhanced drug uptake is attributed to the negatively charged surface of cancer cell membranes and the release of positively charged PGDCs from the O-HAMA/PGDC hydrogel in the acidic tumor environment. Electrostatic interactions between PGDCs and the cancer cell membrane, enhance cellular adhesion and promote endocytosis. Furthermore, the hydrogel’s sustained-release behavior ensures prolonged DOX availability within the tumor microenvironment, thereby maximizing anticancer efficacy while minimizing systemic toxicity and reducing the likelihood of drug resistance.

Biocompatibility and in vitro photothermal properties of hydrogels
Good biocompatibility is essential for the biomedical application of materials within living systems. To evaluate the cytocompatibility of the engineered O-HAMA/PGDC hydrogels, L929 fibroblast cells were co-cultured with different hydrogel formulations for 3 days. Calcein-AM/PI staining was subsequently performed to visualize cell viability, morphology, and potential cytotoxicity in vitro. The distribution and morphology of stained cells provide a reliable indication of whether the O-HAMA/PGDC hydrogel components interfere with cell proliferation, attachment, or survival, thereby reflecting the intrinsic biocompatibility of the material.

As shown in Fig. 6a and b, after 1 day of co-culture with L929 cells, the cell viability in both the control and O-HAMA/PG (hydrogel without DOX) groups exceeded 90% (n = 3), indicating that the O-HAMA/PG hydrogel possesses good biocompatibility. In contrast, the survival rate in the DOX-containing O-HAMA/PGDC hydrogel group decreased to 77% (n = 3), which can be attributed to the intrinsic cytotoxicity of DOX. After 3 days of co-culture, the viability in the O-HAMA/PG and O-HAMA/PGDC groups showed a slight further decrease but remained at an acceptable level, indicating no obvious progressive toxicity from the hydrogel matrix itself (Fig. 6c and d). To more accurately determine the proportion of live and dead cells, flow cytometry was performed (Fig. 6e). The Control and O-HAMA/PG groups predominantly consisted of live cells (Q4: 97.1% and 97.3%), with minimal early apoptosis (Q3: 0.84% and 0.95%), late apoptosis (Q2: 1.06% and 0.93%), and necrosis (Q1: 0.97% and 0.87%). In comparison, the O-HAMA/PGDC group exhibited a modest decrease in live cells (Q4: 95.3%) accompanied by slightly increased late apoptosis (Q2: 1.83%) and necrosis (Q1: 1.78%), consistent with DOX-induced cytotoxicity. Collectively, these results confirm that the O-HAMA hydrogel is biocompatible and that the observed reduction in viability in the DOX-loaded group mainly arises from the drug component rather than the hydrogel matrix.
To further validate the tumor-killing efficacy of the O-HAMA/PGDC hydrogel, various hydrogels were tested using 4T1 cells. As shown in Fig. 7a, after 10 min of irradiation with the 808 nm NIR laser, the three treatment groups showed a significant cancer cell-killing effect compared to the control and laser-only groups. In particular, the O-HAMA/PG + laser group showed a large number of dead cells (indicated by red coloration) in, demonstrating the good PTT performance of the material. Meanwhile, in the O-HAMA/PGDC group, the high cell death rate of 97.1% (Fig. 7b) was mainly attributed to the chemotherapeutic effect of DOX. The strong red fluorescence observed throughout the field further confirmed the combined enhancement of the antitumor activity. To further quantify the proportion of live and dead cells, flow cytometry analysis was performed (Fig. 7c). The Control and Laser-only groups showed negligible apoptosis, remaining mostly viable (Q4: 96.1% and 95.9%). Treatment with O-HAMA/PGDC or O-HAMA/PG + Laser increased apoptotic populations, with late apoptosis (Q2) being the dominant component (9.68% and 13.8%, respectively). Notably, O-HAMA/PGDC + Laser induced the highest apoptotic response (Q2: 29.8% with a modest increase in early apoptosis, Q3: 2.09%), while necrosis remained low across groups (Q1 ≤ 0.68%). These data confirmed the strong cytotoxic effect of combined treatment. These results were consistent with the fluorescence imaging data, further validating the enhanced antitumor efficacy of the O-HAMA/PGDC hydrogel under NIR irradiation.

These findings confirmed the potent in vitro antitumor performance of the O-HAMA/PGDC hydrogel and highlight its potential for combined PTT/CT treatment. The significant photothermal effect observed in the O-HAMA/PG+laser group indicates that the incorporated PA efficiently converted NIR light into localized heat, triggering apoptosis in tumor cells. In the O-HAMA/PGDC group, the combined interaction between DOX and the photothermal effect likely improved cellular uptake and increased chemosensitivity, resulting in remarkably improved therapeutic efficacy. Notably, the O-HAMA/PGDC hydrogel exhibited distinct biological responses toward normal and tumor cells, indicating a favorable therapeutic window. While L929 fibroblasts maintained high viability under the tested conditions, 4T1 tumor cells showed pronounced sensitivity, especially under photothermal activation. This difference can be attributed to the higher proliferative activity and drug susceptibility of tumor cells, as well as the tumor-associated acidic pH microenvironment, which collectively promote drug release and cellular uptake. In contrast, under non-irradiated and neutral conditions, both normal and tumor cells experienced limited drug exposure, contributing to good cytocompatibility. These results highlight the tumor-selective therapeutic potential of the O-HAMA/PGDC hydrogel system under localized treatment conditions. The apoptosis-dominant cell death observed in this study suggests that the antitumor effect is not merely attributable to nonspecific thermal injury but is closely associated with intracellular apoptotic signaling. PTT has been reported to trigger mitochondrial stress and mitochondrial membrane potential disruption, facilitating intrinsic apoptosis activation [48]. Meanwhile, hyperthermia can enhance chemotherapeutic efficacy by increasing membrane permeability and intracellular drug diffusion/uptake, thereby promoting DOX internalization and retention [49]. Once accumulated in the nucleus, DOX primarily induces cytotoxicity through DNA intercalation and topoisomerase II inhibition, leading to DNA damage and apoptosis amplification [50]. It is clear that, mitochondrial stress, enhanced DOX nuclear delivery, and DNA damage signaling likely converge to yield the observed apoptosis-dominant cell death and combined chemo–photothermal efficacy [51].

In vivo cancer therapy
The in vivo antitumor effect of the O-HAMA/PGDC hydrogel was further evaluated using a 4T1 tumor subcutaneous transplantation model in BALB/c nude mice. The nude mice were randomly divided into five groups (n = 6), including PBS control group, laser group, laser + O-HAMA/PG group (PTT group), O-HAMA/PGDC group (CT group), and laser + O-HAMA/PGDC group (PTT/CT group). When the tumor volume reached approximately 60 mm3, 50 µL of physiological saline or hydrogel (DOX concentration = 25 µg/mL) was injected directly in the tumor site, and 808 nm laser irradiation (2 W cm− 2, 10 min) was applied according to the groups. During the irradiation, real-time photothermal images and temperature-time curves were recorded to monitor local temperature changes. Although identical laser power densities were applied in vitro and in vivo, the effective photothermal response in vivo was modulated by tissue scattering and heat dissipation, and local temperature was carefully monitored to ensure safety and reproducibility. It should be noted that the present study does not aim to achieve systemic active targeting via intravenous administration. Instead, effective tumor cell accessibility is realized through a localized delivery strategy combined with material-specific interactions. The O-HAMA hydrogel provides tumor-preferential cell interaction through CD44-related affinity, while the nanoscale PGDC architecture and photothermal activation facilitate cellular internalization and intracellular drug release [52]. This localized and microenvironment-responsive approach enables efficient tumor therapy while avoiding overreliance on systemic targeting mechanisms.

The in vivo results demonstrated that the hydrogel exhibited excellent photothermal conversion performance in the 4T1 tumor model in BALB/c nude mice. Following intratumor injection and 808 nm NIR light irradiation, the local tumor temperature increased rapidly, reaching a maximum of 62 °C, which was significantly higher than that of the laser-only irradiation alone group (Fig. 8a and b). This temperature range was sufficient to induce irreversible thermal damage to the tumor cells while simultaneously providing thermal stimulation for localized release of DOX [53, 54]. The sustained heating properties of the hydrogel indicated good photothermal stability. The significant increase in localized temperature exemplifies its potential in spatiotemporally controllable therapy. This drug release behavior contributed to the effective enrichment of DOX at the tumor site while reducing the risk of systemic toxicity. Although photothermal ablation at temperatures above 60 °C can completely eradicate tumors, such conditions may cause severe thermal injury to adjacent normal tissues. In this study, the integration of DOX within the O-HAMA/PGDC hydrogel enabled potent tumor inhibition under heating, indicating that PDT enhanced the therapeutic efficacy while lowering the photothermal threshold. This combination therefore achieves combined PTT/CT synergy, balancing therapeutic efficiency and biosafety. It should be noted that photothermal treatment conditions in this study were intentionally set at sub-ablative levels and can not totally reflect the true situation of tumor therapy. Therefore, further measurement of the tumor volume is necessary.
In addition, the hydrogel’s ability to maintain stable and reproducible heating under laser irradiation ensures consistent therapeutic outcomes. As shown in Fig. 8c and e, compared with the control group, the PTT group, the CT group, and the PTT/CT group all significantly inhibited tumor growth, with the PTT/CT group showing the most pronounced effect. We understand that tumor volume measurement alone may be insufficient to fully reflect tumor status after incomplete tumor ablation, due to the complexity of biological tissues. Therefore, we analyze the tumor growth data using normalized relative tumor volume (V/V0) to evaluate the tumor growth situation (Fig. 8c). By day 14, the relative tumor volume in the PTT/CT group was significantly lower (0.20 ± 0.11, n = 3) compared to the control (5.52 ± 0.35) and single treatment groups (PTT: 0.58 ± 0.12; CT: 0.82 ± 0.18). Statistical analysis using one-way ANOVA followed by Tukey’s post hoc test confirmed the significance of these differences (p < 0.001). These relative tumor volume (V/V0) results agree well with the true tumors collected from mice, as presented in the camera photo (Fig. 8e). Notably, the enhanced therapeutic efficacy in the PTT/CT group is likely attributed to heat-induced apoptosis combined with increased DOX uptake, facilitated by hyperthermia-induced membrane permeability and enhanced cellular internalization.
To further evaluate the anti-tumor efficacy of the O-HAMA/PGDC hydrogel, Ki-67 immune histochemical staining was performed to analyze the proliferative activity of tumor cells [55]. As shown in Fig. 8d, compared with the control group, the Ki-67-positive cell rate in the laser + O-HAMA group was significantly reduced, suggesting that this treatment group could effectively inhibit tumor cell proliferation. These results were consistent with the observed trends in tumor volume and weight, further verifying the excellent antitumor activity of the PTT/CT group. To assess in vivo biocompatibility, major main organs (heart, liver, spleen, lung, and kidney) of the nude mice were analyzed via H&E staining. As shown in Fig. 8d, tissue morphology in all treatment groups appeared normal, with no detectable inflammatory cell infiltration or pathological changes detected. In addition, serum biochemical analysis was performed using the available frozen plasma samples. As shown in Fig. 8f and g, alanine aminotransferase (ALT) and blood urea nitrogen (BUN) levels in all treated groups remained comparable to those of the control group, indicating no obvious hepatic or renal dysfunction under the tested conditions. These findings indicate that the O-HAMA/PGDC hydrogel exhibits good in vivo biocompatibility and does not cause significant damage to vital organs.

Performance and sustainability analysis
To better illustrate the effectiveness and potential of the O-HAMA/PGDC hydrogel in the combined treatment of cancer with PTT/CT, we analyzed the sustainability of this hydrogel material using the Sustainability Footprint (SF) method developed by Mezzenga and co-workers [56]. To assess the sustainability of the O-HAMA/PGDC hydrogel prepared in this study, we employed the Overall Sustainability Footprint (OSF) method to analyze its performance. This method involves evaluating different sustainability criteria based on three levels: low (i = 1), medium (i = 2), and high (i = 3). The OSF values were then calculated by comparing the O-HAMA/PGDC hydrogel to previously reported hydrogels, such as GOD@SiO2-Arg hydrogel [57], APPF hydrogel [58], and PDA NPs/pNIPAM hydrogel [59]. The OSF values for each hydrogel were calculated using the following equation:

As shown in Fig. 9, the analysis of the four hydrogels was conducted based on eight key properties: Swelling rate, Multifunction, Degradability, Biosafety, Stability, Economy, Effectiveness, and Mechanical properties. The results revealed that the O-HAMA/PGDC hydrogel outperformed the other three materials in all categories. The OSF values of O-HAMA/PGDC, GOD@SiO2-Arg, APPF, and PDA NPs/pNIPAM hydrogels were calculated to be 88.3%, 62.5%, 75.0%, and 66.6%, respectively. The analysis clearly shows that the O-HAMA/PGDC hydrogel offers significant advantages over the other materials, particularly in terms of tumor killing ability and potential. Furthermore, its superior photothermal conversion and drug delivery capabilities highlight its value as a functional nanomedicine platform for cancer therapy.
This comparison suggests that the O-HAMA/PGDC hydrogel provides an integrated and highly efficient solution for combined tumor PTT and CT, making it a promising nanoplatform for advancing cancer treatment. Furthermore, the high OSF value demonstrates their superior overall sustainability and effectiveness, providing a critical benchmark for the design of future hydrogel-based therapeutics.

Conclusion
In summary, this study presents an innovative biometallic PGDC delivery platform based on photo-crosslinked hydrogels, which strategically combines the advantages of CT and PTT. This approach addresses critical challenges in cancer treatment, such as low drug delivery efficiency and high systemic toxicity in conventional therapies, while overcoming the limitations of monotherapy-based treatments. The development process began with the construction of PGDCs using electrostatic interactions between peptides and chemotherapeutic agents. Following this, GNPs were integrated into the system via Au-S bonds and electrostatic forces, resulting in a multifunctional PGDCs that precisely integrates various therapeutic functionalities. The hydrogel system took advantage of the photo-crosslinkable properties of O-HAMA and the biomimetic characteristics of PNFs, resulting in a pH- and photo-responsive bioactive hydrogel. This system not only demonstrated outstanding photothermal conversion efficiency and thermal stability but also enabled controlled drug release upon exposure to specific stimuli. In vitro and in vivo experiments confirmed that, under NIR irradiation, the hydrogel system achieved a tumor cell ablation rate exceeding 97% for 4T1 cells, significantly enhanced DOX uptake, and demonstrated good biocompatibility with low toxicity. Overall, this study introduces a highly integrated, responsive, and therapeutically effective multimodal PTT/CT cancer treatment strategy, offering a promising platform for combinatorial tumor therapy. The results not only advance the development of next-generation cancer therapeutics but also offer new insights into multifunctional drug delivery systems with dual therapeutic efficacy. Future studies will focus on the rational design of peptide sequences to develop multifunctional PGDCs with enhanced self-assembly, tumor recognition, cell penetration, and stimuli-responsive capabilities.