Injectable “cocktail” hydrogel with dual‐stimuli‐responsive drug release, photothermal ablation, and drug‐antibody synergistic effect

The combination of the first‐line standard chemotherapeutic drug doxorubicin hydrochloride (DOX) and the molecular‐targeted drug Herceptin (HCT) has emerged as a promising strategy for human epidermal growth receptor 2 (HER‐2) overexpressing breast cancer treatment. However, insufficient drug accumulation and severe cardiotoxicity are two major challenges that limit its clinical application. Herein, an in situ forming gold nanorods (AuNRs)‐sodium alginate (ALG) hybrid hydrogel encapsulating DOX and HCT was engineered for tumor synergistic therapy involving injectable, dual‐stimuli‐responsive drug release, photothermal ablation, and drug‐antibody synergistic therapy. The photothermal agent AuNRs, anticancer drug DOX, and anticancer antibody HCT were mixed in ALG solution, and after injection, the soluble ALG was quickly transformed into a hydrogel in the presence of Ca2+ in the body. Significantly, the hybrid hydrogel exhibits an extremely high photothermal conversion efficiency of 70% under 808 nm laser irradiation. The thermal effect can also provide photothermal stimulation to trigger the drug release from the gel matrix. In addition, the drug release rate and the releasing degree are also sensitive to the pH. In vitro studies demonstrated that the PEI‐AuNR/DOX/HCT/ALG hydrogel has facilitated the therapeutic efficiency of each payload and demonstrated a strong synergistic killing effect on SK‐BR‐3 cells. In vivo imaging results showed that the local drug delivery system can effectively reduce the nonspecific distribution in normal tissues and increase drug concentration at tumor sites. The proposed hydrogel system shows significant clinical implications by easily introducing a sustainable photothermal therapy and a potential universal carrier for the local delivery of multiple drugs to overcome the challenges faced in HER‐2 overexpressing cancer therapy.


| INTRODUCTION
Breast cancer is a common malignancy with high morbidity and mortality in female cancer patients. 1,2ystemic administration, as the commonly used clinical administration method, has significant toxic and side effects on normal tissues. 3Local therapy can precisely deliver anticancer drugs to tumor tissue, which is essential to improve antitumor efficacy and reduce adverse effects. 4Injectable hydrogel-based drug delivery systems may be a promising strategy to achieve spatiotemporal control of drug release and minimize the disadvantages associated with the systemic mode of drug delivery. 5,6Hydrogels are three-dimensionally cross-linked hydrophilic polymers endowed with biocompatibility and hydrophilic drug encapsulation potential due to their porous structure, high water absorption similar to human tissue, and tunable mechanical and flow properties. 7,80][11] Furthermore, physicochemical reactions related to various biochemical signals (such as temperature and pH) in the tumor microenvironment, can be engineered in the hydrogel to control the drug release and degradation rates.0][21] Photothermal therapy (PTT) has attracted much attention in the field of cancer therapy, as a noninvasive therapeutic strategy, which induces tumor cell apoptosis by converting light to localized heat based on near-infrared (NIR) photo-response nanomaterials. 22At present, a variety of photothermal conversion nanomaterials have emerged.Gold nanorods (AuNRs), as a typical PTT material, have the advantages of good biocompatibility, abundant biological activities, easy preparation, and easy surface functionalization. 18In particular, AuNRs can easily change their surface plasmon resonance absorption in the near-infrared spectral region by adjusting the aspect ratio. 23,24AuNRbased nano-delivery nanoplatforms have been widely used for multimodal cancer therapy, such as drug delivery, 25 chemodynamic therapy, 26 and gene therapy. 27However, the light penetration depth of PTT alone is limited, and we expect to achieve enhanced anticancer efficiency by combining AuNRs-based PTT with the simultaneous application of anticancer drugs.
2][33] However, to maintain the therapeutic effect of each drug on target tumors, a high dose of systemic administration is often required, leading to systemic toxicity, especially cardiotoxicity. 34,35n this context, local administration within tumor tissue becomes a safer and more effective option, which not only achieves optimal drug accumulation at the tumor site but also minimizes side effects on normal tissues. 36,37herefore, an injectable hydrogel that can carry multiple therapeutic agents and provide sustained release in targeted tumors, may be an ideal combination drug delivery system, which can overcome many shortcomings related to combination chemotherapy. 38o address these above challenges, herein we have developed a multifunctional injectable "cocktail" hydrogel composed of photothermal agent AuNRs, chemotherapeutic drug DOX, anticancer antibody HCT, and sodium alginate (ALG) for localized HER-2 overexpressing breast tumors (Scheme 1).0][41][42][43] The AuNRs were modified with polyethyleneimine (PEI-AuNRs), and its abundant amine groups could form electrostatic coupling with the carboxylic group of ALG and HCT so that the AuNRs and HCT were uniformly dispersed in the ALG aqueous solution. 44,45irst, the precursor liquid was prepared by simply mixing ALG with PEI-AuNRs, DOX, and HCT.The hybrid solution was then injected into the tumor site in a minimally invasive manner.Sodium alginate could rapidly react with multivalent cations in the tumor site to form in situ hydrogels, [46][47][48] encapsulating DOX and HCT in the colloid, effectively immobilizing drugs and avoiding premature degradation.Under 808 nm laser irradiation, the gold nanorods absorb NIR light to generate heat, which can trigger on-demand drug release from the hydrogel and synergistically damage the tumor cells.In vitro experiments demonstrated that PEI-AuNR/ DOX/HCT/ALG hydrogel could effectively inhibit the growth and proliferation of HER-2 overexpressing breast tumor cells and induced their apoptosis.Furthermore, in vivo imaging results illustrated that PEI-AuNR/DOX/ HCT/ALG hydrogel can provide high local drug concentration and limit drug diffusion to surrounding normal tissues.

| RESULTS AND DISCUSSION
2.1 | Preparation and characterization of PEI-AuNR/DOX/HCT/ALG hydrogel First, AuNRs were prepared using a seed-mediated growth procedure.As observed in transmission electron microscopy (TEM) images, the average length and diameter of the as-prepared AuNRs were about 58 and 14 nm (Figure 1A and Supporting Information: Figure S1), respectively.Figure 1B showed the absorption spectrum of AuNRs, and the strong absorption peak at 808 nm proves that AuNRs are a potential PTT reagent.X-ray diffraction (XRD) pattern of AuNRs is consistent with that of Au (JCPDS 04-0784), illustrating that AuNRs are the only crystalline product (Figure 1C).To improve the stability and biocompatibility of gold nanorods, we carried out surface modification on gold nanorods.The lipoic acid-polyethyleneimine (LA-PEI) was prepared by a simple amide coupling reaction and immobilized on the surface of AuNRs via Au-S bonds. 49The PEI grafted on the surface of AuNRs can form electrostatic coupling with ALG and HCT, resulting in the uniform dispersion of AuNRs and HCT in the sodium alginate solution.Figure 1D showed the fourier transform infrared (FT-IR) spectra of LA-PEI and PEI-AuNR, the transmission bands at 1645 and 1550 cm −1 could be attributed to the amide I and II bands generated by the reaction of LA and PEI, respectively. 50In addition, the zeta potential of the particles was changed from −2 to +45 mV (Figure 1E), further indicating that PEI was successfully bound to the surface of AuNRs.
Next, we investigated the PEI-AuNR/DOX/HCT/ALG hydrogel formation process.Previous studies have shown that hydrogels are rapidly formed when sodium alginate solutions at concentrations greater than or equal to 5 mg/mL are injected into phosphate buffered saline (PBS) containing 1.8 mmol/L Ca 2+ (physiological concentration). 39To facilitate the injection and make the prepared hydrogels have a flexible shape and relatively weak mechanical strength, an ALG solution of 5 mg/mL was used for subsequent experiments.First, we prepared the PEI-AuNR/DOX/HCT/ALG hybrid fluid by simply mixing the ALG aqueous solution and various therapeutic agents.As shown in Figure 1F, PEI-AuNRs, DOX, and HCT were uniformly stored in the ALG aqueous solution due to the strong electrostatic interaction.Then, the PEI-AuNR/DOX/HCT/ALG hybrid fluid was injected into PBS containing 1.8 mmol/L Ca 2+ , and the PEI-AuNR/DOX/ HCT/ALG hydrogel formed within 30 s (Figure 1G).It was observed from the SEM images that the prepared PEI-AuNR/DOX/HCT/ALG hydrogel exhibited a porous network microstructure and that the AuNRs were uniformly cross-linked in the hydrogel network (Figure 1H and Supporting Information: Figure S2).Further rheological analysis of the Ca 2+ -mediated PEI-AuNR/DOX/HCT/ALG hydrogel showed that the storage modulus (G′) was greater than the loss modulus (G″), which was indicative of typical viscoelastic properties (Figure 1I). 39Meanwhile, the homogeneous PEI-AuNR/DOX/HCT/ALG hybrid fluid has good injectability and can be easily pumped out through a standard 1 mL syringe (Supporting Information: Video S1).This is critical for its potential clinical application because it requires to be injected into the tumor site using a traditional puncture needle.

| Photothermal performance
PEI-AuNR/ALG hydrogel retained the photothermal features of PEI-AuNRs, which occurred in a time-and concentration-dependent manner (Figure 2A and Supporting Information: Figure S3A).After irradiation with 808 nm laser for 10 min, the temperature of PEI-AuNR/ALG hydrogel containing 6.25, 12.5, 25 and 50 µg/mL PEI-AuNRs could raise from 28.0 °C to 46.5 °C, 52.9 °C, 66.6 °C, and 77.1 °C, respectively.Corresponding infrared thermal images are shown in Figure 2B.Moreover, the laser power density could be used to regulate the photothermal efficiency (Figure 2C and Supporting Information: Figure S3B).
After irradiation with 808 nm laser at a power density of 0.5, 1.0, and 1.5 W/cm 2 for 10 min, respectively, the temperature of PEI-AuNR/ALG hydrogel (50 µg/mL PEI-AuNRs) raised from 28 °C to 58.3 °C, 77.1 °C, and 90.9 °C, respectively.The temperature elevations of both PEI-AuNRs and PEI-AuNR/ALG hydrogel displayed negligible fluctuations during four repeated cycles, exhibiting excellent photothermal stability (Figure 2D and Supporting Information: Figure S3C).By calculating the photothermal conversion efficiency (PCE), it was found that the PCE increased from 65% to 70% after incorporating PEI-AuNRs into the hydrogel (Figure 2E and Supporting Information: Figure S3D), indicating that the porous structure of the hydrogel contributes to the improvement of photothermal performance.The mesopore structure facilitates multilight scattering/reflection, resulting in enhanced harvesting of the exciting light and eventually higher light absorption. 51At the same time, the hierarchical porosity could promote mass transport, causing improved photothermal performance. 52,53

| pH/NIR dual stimulus-responsive drug release
The porous morphology of PEI-AuNR/DOX/HCT/ ALG hydrogel is beneficial to promote the release of encapsulated DOX and HCT from the hybrid hydrogel.We simulated the tumor and normal tissue microenvironments in PBS buffer at pH 6.5 and 7.4, respectively, to explore the release behavior of DOX and HCT from hydrogels.The released DOX and HCT were quantified according to the standard absorption curves (Supporting Information: Figure S4).The results showed that in an acidic environment, the drug release rate was faster and the releasing degree was higher.Significantly, the sustained release of DOX and HCT lasted for more than 72 h, favoring the long-term sustained cancer cell killing at tumor sites (Figure 2F and Supporting Information: Figure S5).Next, the effect of laser irradiation on the drug release of hydrogel was further investigated (Figure 2G).We first immersed the hybrid gel in PBS and irradiated it under an 808 nm laser for 10 min.After 10 min, the supernatant was slightly orange, and the absorption spectra showed an increase in DOX absorbance in the supernatant after NIR treatment (Figure 2H).Subsequently, we tested the DOX release rate at each time point from 1 to 10 min and found that the hybrid gel released a large amount of DOX after the application of the NIR laser, whereas little DOX was released without NIR laser irradiation (Supporting Information: Figure S6).These results indicated that the PEI-AuNR/DOX/HCT/ALG hydrogel can realize the controllable release of the drug using the NIR laser as a trigger.Therefore, the PEI-AuNR/DOX/HCT/ ALG hydrogel has dual responsiveness to pH and NIR laser irradiation, accelerating the release of drugs in the tumor microenvironment, and bursting drug release during laser irradiation.

| Cellular uptake of DOX and HCT
Confocal laser scanning microscope and flow cytometry were used to investigate the intracellular uptake and intranuclear accumulation of the drugs released from PEI-AuNR/DOX/HCT/ALG hydrogel.Supporting Information: Figure S7 was the fluorescence microscope photograph of SK-BR-3 cells incubated with the prepared hydrogel for 4 h.The red fluorescence signal was from DOX, and the green fluorescence signal was from FITC-labeled HCT, demonstrating the efficient internalization of DOX and HCT into cells by the PEI-AuNR/DOX/HCT/ALG hybrid hydrogel.In addition, the SK-BR-3 cells were cultured together with the asprepared hydrogels under different conditions (1, 2, 3, or 3 h + laser), and the fluorescence signal of intracellular DOX was quantitatively detected by flow cytometry.As shown in Figure 3A, the fluorescence intensity increased most obviously at 1 h, and the fluorescence intensity increased with the incubation time, further proving the effective accumulation of intracellular drugs.The fluorescence of intracellular DOX was also enhanced after NIR laser irradiation because under the stimulation of the photothermal effect, DOX release was enhanced and the fluidity of the tumor cell membrane was accelerated, which collectively resulted in increased cellular uptake.

| In vitro anticancer activity of PEI-AuNR/DOX/HCT/ALG hydrogel
To verify the synergistic effect of DOX and HCT, we first tested the cytotoxicity of the pure drugs.The results showed that the IC 50 of pure DOX against SK-BR-3 cells was 4.132 µg/mL (Table 1), and pure HCT had almost no toxicity.To achieve the best effect of combination treatment, we further mixed DOX and HCT with different mass ratios and tested their cytotoxicity.Supporting Information: Figure S8 and the corresponding IC 50 values (Table 1) showed that the viability of the SK-BR-3 cells declined with the increase in HCT.Nevertheless, to avoid drug resistance caused by using a too high amount of drugs, we chose DOX:HCT = 1:5 for the follow-up experiments.
The photothermal responsive cytotoxicity of the PEI-AuNR/ALG hydrogel was assessed using a CCK8 assay with the SK-BR-3 cell.At the highest evaluated PEI-AuNRs concentration of 100 µg/mL, cell viability was maintained at more than 90% (Figure 3B), indicating the good biocompatibility of PEI-AuNR/ALG hydrogel.Furthermore, with increasing concentrations of PEI-AuNRs in the hydrogel, the proliferation of SK-BR-3 cells was significantly inhibited after 10 min of 808 nm laser irradiation at 1.0 W/ cm 2 .Specifically, less than 10% of viable cells were retained when treated with a hydrogel containing 100 µg/mL PEI-AuNRs, suggesting that the PEI-AuNR/ALG hydrogel has significant photothermal efficiency.
Based on the excellent photothermal properties of PEI-AuNRs and the synergetic effect of the drugantibody combined utilization, we further investigated the multifunctional synergistic antitumor ability of the as-prepared PEI-AuNR/DOX/HCT/ALG hybrid This synergistic therapeutic effect of the PEI-AuNR/ DOX/HCT/ALG hydrogel was further qualitatively and quantitatively proved using trypan blue staining and flow cytometric apoptosis.The results were shown in Figure 3D,E, the cell viability followed the order of PEI-AuNR/DOX/HCT/ALG + laser < DOX/HCT/ALG < DOX/ ALG < PBS, further confirming the photothermal ablation and synergistic antitumor effects of PEI-AuNR/ALG hybrid hydrogel encapsulating DOX and HCT. and surrounding tissues due to the strong fixation capability of the hydrogel, and the hydrogel showed persistent retention and sustainable release of DOX at the tumor site within the same time duration.The network structure of the hydrogel enables the retention of DOX at the tumor site and inhibits drug diffusion.To gain a clearer insight into fluorescence distribution in both conditions, mice were killed after 72 h intratumoral administration, and tumors and major organs (heart, liver, spleen, lung, and kidney) were excised for fluorescence imaging (Figure 4B,C).The results showed that the accumulation of DOX in the tumor tissue of the hydrogel group was greater than that of the solution group, while the accumulation of DOX in normal organs was smaller than that of the solution group.In conclusion, our developed PEI-AuNR/DOX/HCT/ALG hydrogel could be injectable and efficiently deliver multiple drugs into the tumor with high local retention and sustainable release.This shows excellent potential in improving PTT efficacy and reducing side effects caused by rapid drug leakage.

| CONCLUSION
In this study, we proposed a multifunctional "cocktail" therapeutic platform based on the injectable PEI-AuNR/DOX/HCT/ALG hydrogel to achieve photothermal ablation and drug-antibody synergistic therapy for HER-2 overexpressing breast cancer.The hybrid hydrogel exhibited excellent biocompatibility, high PCE (70%), and photothermal stability.The in situ forming gel matrix can serve as a good reservoir to immobilize DOX and HCT, and realize the spatial and tunable release of therapeutic drugs in a NIR laser and pH-controllable manner.Such a material design strategy based on AuNRs/ALG stimuli-responsive hydrogel is very effective and versatile in the treatment of malignant tumors.On the one hand, it can be used for the encapsulation of various therapeutics, reducing the dependence on single chemotherapy and enabling precise "on/off " controlled drug release.On the other hand, after being injected into solid tumors, in situ gelations of hydrogel matrix would lead to local retention and sustainable application of the combined therapeutic effects to reduce their systemic toxicity and at the same time improve the anticancer efficacy.This PEI-AuNR/DOX/HCT/ALG hydrogel system not only provides a new line of thought for effective breast cancer treatment where HCT and DOX coadministration has severe side effects in patients but also offers considerable promise in facilitating the rational design of combination therapy in clinical studies.

F
I G U R E 2 (A) Temperature curves of PEI-AuNR/ALG hydrogel containing different concentrations of PEI-AuNRs under 808 nm laser irradiation (1.0 W/cm 2 ).(B) Corresponding infrared thermal images.(C) Temperature curves of PEI-AuNR/ALG hydrogel containing 50 µg/mL PEI-AuNRs irradiated with 808 nm laser irradiation at different power densities.(D) Photothermal conversion curves of PEI-AuNR/ALG hydrogel containing 50 µg/mL PEI-AuNRs under four cycles of 808 nm laser irradiation (1.0 W/cm 2 ).(E) Linear regression curve in the cooling stage of the second cycle in (D).(F) Cumulative release of DOX from hydrogel in PBS buffer with pH values of 7.4 and 6.5.Data represent the mean ± SD (n = 3).(G) Schematic diagram of DOX and HCT release from PEI-AuNR/DOX/HCT/ALG hydrogel under 808 nm laser irradiation.(H) Photographs of DOX released from PBS containing the PEI-AuNR/DOX/HCT/ALG hydrogel before 808 nm laser application and after 10 min of NIR laser application and the absorption spectra of DOX in the supernatant.ALG, alginate; AuNR, gold nanorod; DOX, doxorubicin; HCT, Herceptin; PEI, polyethyleneimine.

2. 6 |
In vivo fluorescence imaging of PEI-AuNR/DOX/HCT/ALG hydrogel Breast (SK-BR-3) tumor xenograft mice were established as a model to assess the drug immobilization ability of the as-prepared hydrogel.The PEI-AuNR/DOX/HCT solution and PEI-AuNR/DOX/HCT/ALG hybrid fluid were injected into the tumor site, respectively, and the fluorescence signal of DOX at tumor sites at different times was observed by in vivo fluorescence imaging (Figure 4A and Supporting Information: Figures S9 and S10).Due to the continuous circulation of body fluids, free DOX is rapidly distributed throughout the body and almost disappears at approximately 24 h after injection, because water-soluble drugs are easily eliminated by the body.In contrast, DOX in the PEI-AuNR/ DOX/HCT/ALG hydrogel slowly penetrated the bloodstream F I G U R E 4 (A) In vivo fluorescence imaging of PEI-AuNR/DOX/HCT solution and PEI-AuNR/DOX/HCT/ALG hydrogel.(B) In vitro fluorescence imaging of major organs.(C) Semiquantitative analysis of the fluorescence intensity in major organs at 72 h.ALG, alginate; AuNR, gold nanorod; DOX, doxorubicin; HCT, Herceptin; PEI, polyethyleneimine.