Patients with advanced colorectal cancer (CRC) and diffuse peritoneal metastasis are not eligible for surgical intervention, and calling for more efficient and precise treatment strategies to complement the traditional systemic chemotherapy and intraperitoneal chemotherapy. Herein, an injectable two-dimensional molybdenum disulfide (2D-MoS2)-integrated adhesive N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitro-sophenoxy) butanamide-linked sodium alginate-MoS2-5-fluorouracil (AlgNB/MoS2/5-Fu) hydrogel, which can function as an near-infrared light (NIR)-triggered photothermal and drug-delivery implant for CRC treatment is introduced. Ultraviolet (UV) light-activated aldehyde groups in AlgNB bound to the surface-modified MoS2 nanosheets via a Schiff base reaction. The MoS2 nanosheets maintained superior dispersibility in the hydrogel and exhibited a highly efficient NIR-triggered photothermal effect. More importantly, the aldehyde group in AlgNB also imparted tissue adhesion to the hydrogel, the adhesive hydrogel was used to infiltrate and fix in tumor tissue. Combining applications as a 5-Fu drug delivering implant, the injectable adhesive AlgNB/MoS2/5-Fu hydrogel shows remarkable capability in the inhibition of SW480 cells and colorectal tumour regression by triggering photothermal therapy (PTT) and delivering the 5-Fu drug in both in vitro and in vivo studies. Therefore, this research provides distinct guidance strategies for the synergistic tumor therapy of localized CRC and shows enormous potential for cancer treatment in the clinical setting.
Research Article
Injectable 2D-MoS2-integrated adhesive hydrogel for NIR-triggered photothermal and drug-delivery implant in colorectal cancer therapy
https://doi.org/10.21203/rs.3.rs-4020021/v1
This work is licensed under a CC BY 4.0 License
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Adhesive hydrogel
MoS2 nanosheets
5-Fu drug
Colorectal cancer
PTT-chemotherapy synergistic therapeutic
Colorectal cancer (CRC) is a common malignant tumour of the gastrointestinal tract for which surgery is the first-line treatment [1, 2], particularly when the cancer cells have not yet metastasized. However, patients with advanced CRC and diffuse peritoneal metastasis are not eligible for surgical intervention [3]. In such cases, palliative systemic chemotherapy remains the standard treatment for maximising survival [4]. However, systemic chemotherapy fails to achieve suboptimal drug concentrations at tumor sites, and intraperitoneal chemotherapy (IPC) is limited by high clearance rates and associated complications [5]. Hence, the development of appropriate alternatives or treatment options that can be administered alongside systemic chemotherapy and IPC are urgently required.
Photothermal therapy (PTT) induced by a near-infrared (NIR) laser, which results in localized thermal damage of tumors, is a minimally- or non-invasive therapeutic method that has attracted great attention as a complementary treatment option for chemotherapy [6, 7]. The effectiveness of PPT relies heavily on the photothermal conversion efficiency of the photo-absorbing agents (PTA), which can absorb and convert the penetrated NIR laser into heat [8]. In recent years, two-dimensional (2D) nanosheets of layered transition metal dichalcogenides (TMDs), such as MoS2, MoSe2, WS2, and NbS2, which are topological insulators, have been used as PTAs with ultra-high light and heat conversion efficiencies [9, 10]. In particular, 2D MoS2 nanosheets have the advantages of good biocompatibility, high specific surface area, easy modification, and strong absorption in the NIR region; moreover, they have been thoroughly investigated and extensively utilized in PTT [11, 12]. However, PTT involving intra-tumor or intravenous injection of MoS2 nanosheets easily loses efficacy, as accumulation of MoS2 nanosheets in tumour tissue is challenging, and the penetrated nanosheets may induce potential safety threats to normal organs [6, 13]. Therefore, it is important to construct a precise intra-tumor PTA and drug-delivery system for synergistic tumor therapy with significantly mitigated side effects.
Various studies have revealed that injectable hydrogels are very suitable for use in PTAs and drug-delivering implants, highlighting advantages such as superior biocompatibility, biodegradability, minimally-invasive administration, and the ability to be controlled remotely [14-16]. More importantly, the synergistic efficiency of PPT and chemotherapy can have a greater impact than their individual anti-tumor effects, as NIR-triggered hyperthermia not only causes the significant tumor coagulation necrosis but also enhances the tumor chemotherapeutic efficiency by triggering the rapid release of encapsulated drug molecules [6]. Most delivered PTAs are dispersed in hydrogels by simple mechanical stirring or ultrasonication [17, 18]; however, its stability and dispersion in hydrogels cannot be guaranteed. Moreover, most injectable hydrogels for tumour therapy lack tissue adhesion ability and this may result in low tumour treatment efficacy owing to shedding or slipping from the tumor tissue [19, 20]. Therefore, it is highly urgent to develop a high-quality PTA and drug-carrying implant using an injectable adhesive hydrogel to enhance the efficiency of CRC treatment through synergistic PTT and chemotherapy.
In this work, an injectable two-dimensional molybdenum disulfide (2D-MoS2)-integrated adhesive hydrogel, prepared with N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitro-sophenoxy) butanamide (NB)-linked sodium alginate (AlgNB) and lipoid acid-modified-polyethylene glycol- (PEG)-sulfur acid (LA-PEG-NH2) surface-modified MoS2 nanosheets (NH2-PEG-MoS2) and infiltrated with 5-fluorouracil (5-Fu) to evaluate the effect of synergistic PTT and chemotherapy (Fig. 1). Therein, NB is a Ultraviolet (UV) light responsible molecules [21, 22], UV light-activated aldehyde groups in AlgNB bound to the surface-modified NH2-PEG-MoS2 nanosheets by Schiff base reaction, with this strategy, the MoS2 nanosheets maintained superior dispersibility in the hydrogel, and exhibits highly efficient NIR-triggered photothermal effect. More importantly, UV light-activated aldehyde groups in AlgNB is the root cause of tissue adhesion for the hydrogel. To the best of our knowledge, it is the first time, the pre-gel of injectable adhesive hydrogel was used as a “molecular glue” to infiltrate and anchor the tumor tissue, then, gelled in situ with calcium ion cross-linking and fixed in tumor tissue. 5-Fluorouracil (5-Fu) was also carried into the AlgNB/MoS2 hydrogel to evaluate the effect of synergistic PTT and chemotherapy, the hydrophilic anticancer 5-Fu drug is one of the most widely used antimetabolite drugs in the first-line therapy of colorectal carcinoma [23]. The cell bioactivity and photothermal effects of AlgNB/MoS2/5-Fu and its ability to induce SW480 cell death were investigated using in vitro measurements. Finally, an SW480 colorectal tumor-bearing nude mice model was constructed, and the anti-tumor effects of the tissue-adhesive AlgNB/MoS2/5-Fu hydrogel in vivo were further investigated using tumor growth recording, hematoxylin–eosin (H&E) staining and immunohistochemical staining.
Preparation of AlgNB hydrogel
The preparation of AlgNB was described in our previously published papers [24, 25]. As shown in Fig. 2a, Alg (1 g; Aladdin Chemical Reagent Co., Ltd., Shanghai, China; molecular weights (Mw) 20,000–50,000; M/G units 1:2) was dissolved in 2-(N-morphine) ethanesulfonic acid hydrate (MES) (0.01 mol/L, 100 mL, pH=5.3; Aladdin Chemical Reagent Co., Ltd.) buffer solution and reacted at 35℃. After Alg was completely dissolved, ultra-violet (UV) light-sensitive NB (60 mg; Haining Jurassic Biotechnology Co., Ltd., Zhejiang, China) was dissolved in 10 mL of dimethyl sulfoxide (DMSO) (Aladdin Chemical Reagent Co., Ltd.) and added to the reaction system. Then, 4-(4,6-dimethoxy-triazine-2-yl)−4-methylmoroline hydrochloride (DMTMM) (1.2 g; Aladdin Chemical Reagent Co., Ltd.) was added to the system three times at an interval of 0.5–1 h. The reaction was continued for 3 h after the final addition. The entire process was completed under stirring, and the system was treated with tin foil to avoid exposure to light. Finally, dialysis with 0.1 M NaCl solution (pH = 3.5) was performed for 2 days and then with deionised water for 2 days, and AlgNB was obtained after freeze-drying. AlgNB was sealed and preserved in the dark.
Preparation of MoS2-PEG-NH2
To improve their stability in phosphate buffered saline (PBS), single-layer MoS2 nanosheets (100nm, Shanghai Maclin Biochemical Technology Co., Ltd., Shanghai, China) were surface-modified with amine-PEG-sulfur acid (LA-PEG-NH2) (Mw=1 k; Shanghai Zhenjun Biotechnology Co., Ltd., Shanghai, China) [26]. Briefly, MoS2 (1 mg) was dispersed in deionized water (2 mL) under stirring, and LA-PEG-NH2 (10 mg) was added to the reaction system. After 20 min of ultrasonic treatment, the samples were stirred overnight. Then, the solution was transferred to an ultrafiltration tube with a blocking Mw of 100 kDa and washed several times with deionized water to remove excess PEG polymers. The prepared MoS2-PEG-NH2 was refrigerated at 4℃ until use.
Preparation of AlgNB/MoS2 hydrogel
After AlgNB was completely dissolved in PBS (10 mg/mL), UV light (365 nm, 30 mW/cm2, 1min) was applied to form a “molecular glue”, as aldehyde groups were generated on NB under UV irradiation [21]. Surface-modified MoS2 was then added to form the AlgNB/MoS2 pre-gel, and a CaCl2 solution was used for calcium ion crosslinking (to form the AlgNB/MoS2 hydrogel).
Materials characterization
The surface morphologies of the Alg, AlgNB, and AlgNB/MoS2 hydrogels were characterized by scanning electron microscopy (SEM) at an operating voltage of 3.0 kV (SU47; Hitachi, Tokyo, Japan), and energy-dispersive X-ray spectroscopy (EDS) element spectrum tests were also performed. Fourier-transform infrared spectroscopy (FTIR) (INVENIO S; Bruker, Billerica, MA, USA) was used to determine the modification of MoS2 with LA-PEG-NH2. To demonstrate the chemical composition of AlgNB hydrogel and AlgNB/MoS2 hydrogel, X-ray photoelectron spectroscopy (XPS) (Kratos AXIS Ultra DLD; Shimadzu-Kratos Co., Ltd., Kyoto, Japan; Al Kα, 1486.6 eV) was performed. In vitro tissue adhesion of the AlgNB/MoS2 pre-gel (about 200 μL) on the surface of pig skin were determined using mobile camera equipment, Alg/MoS2 pre-gel was used as a control group. To simulate the in vivo tumour tissue adhesion of AlgNB/MoS2 hydrogel, 50 μL pre-gel solution (with 5 μL CaCl2 solution [10 mg/mL], it is a safe range for gel dosage to the tumor volumes of approximately 100 mm3) was injected into the tumour, this process was recorded by mobile camera equipment, Alg/MoS2 hydrogel was used as a control group. In vitro photothermic effects of the MoS2 solution and AlgNB/MoS2 hydrogel under 808 nm NIR irradiation were recorded using an infrared thermal imaging instrument (E40; FLIR Systems, Wilsonville, OR, USA). The effects of photothermia were recorded in vivo using a handheld infrared thermal camera (HikVision, Hangzhou, China). The photothermal conversion efficiency of the AlgNB/MoS2 hydrogel was calculated as follows [27]:
where c, m, ∆T, P, S, t are the specific heat (hydrogels are about 2.5 Jg-1℃-1), mass, the change of temperature, the power density of the light, the irradiation area (1.9 cm2) and irradiation time, respectively.
5-Fu drug release in hydrogels
5-Fluorouracil (5-Fu) (2 mg/mL, the concentration was used throughout this work; 20 % drug-loading in hydrogel) was dissolved in AlgNB/MoS2 pre-gel solution, and calcium ion cross-linking was performed using CaCl2 solution. Then, the formed hydrogels were transferred to 12-well plates, 2 mL of PBS was added, and the samples were shaken in a shaker throughout the test. One-mL samples of the PBS solution were collected at different time points from the 12-well plates (Fig. S3h) and an equal volume of PBS solution was added. The cumulative release of 5-Fu with the 5-Fu/AlgNB/MoS2 hydrogel with or without 808 nm NIR laser irradiation (1.0 W/cm-2) was measured by the peak intensity at 266 nm using an UV-visible spectrophotometry (UV-vis) (UV-3150; Shimadzu-Kratos Co., Ltd.). NIR irradiation was performed at 24 h, 48 h, and 72 h. Two experimental conditions, 5-Fu/AlgNB hydrogel with NIR radiation and 5-Fu/AlgNB hydrogel without NIR irradiation, were used as the controls.
Cell lines and cell culture
The human CRC cell line, SW480, was purchased from the American Type Culture Collection (ATCC) (Manassas, VI, USA). The mutation statuses of the cell lines used in this study were obtained from the Cancer Cell Line Encyclopedia (CCLE) database. SW480 was maintained in Dulbecco’s modified essential medium (DMEM) (CellMax, Lanzhou, China) with 100 µg/mL streptomycin, 100 U/mL penicillin, and 10% foetal bovine serum (CellMax) and incubated at 37°C in a 5% humidified CO2 atmosphere.
Cytotoxicity assays in vitro
SW480 cells were cultured in a medium with pre-gel or on the surface of hydrogels to assess the biocompatibility of the AlgNB, AlgNB/MoS2, and AlgNB/MoS2/5-Fu hydrogels. For the pre-gel-treated groups (Fig. 4a), cells (20,000 cells/cm2) were cultured on the surface of 24-well dishes with 500 mL of culture medium and 100 mL of pre-gel added to the dish, and cells cultured without pre-gel were used as controls. For the groups of cells cultured on the surface of hydrogels (Fig. 4b), the hydrogels were calcium ion cross-linked on the surface of 24-well dishes; cells (20,000 cells/cm2) were then cultured on the surface of the hydrogels, and cells cultured on the surface of the PS substrate were used as controls. Next, all the groups were incubated in a humidified atmosphere with 5% CO2 at 37°C. After 1, 3, and 5 days of culture, cell proliferation was assessed using a cell counting kit-8 (CCK-8), and the absorbance value (450 nm) of each well was determined 2 h later using a microplate reader.
To evaluate the effect of PTT-chemotherapy synergistic therapy for the AlgNB/MoS2/5-Fu hydrogel in vitro, SW480 cells were cultured in medium with pre-gel or inside hydrogel, and AlgNB hydrogel and AlgNB/MoS2 hydrogel were used as controls. For the pre-gel treated groups, SW480 cells (10,000 cells/cm2) were seeded in 24-well plates. Then, 500 mL culture medium and 100 mL pre-gel were added to each plate and incubated in a humidified atmosphere with 5% CO2 at 37°C. After cells were cultured for 24 h, 808 nm NIR laser irradiation was applied for 10 min with different densities (0, 0.2, 0.5, 0.8, and 1.0 W/cm2). After incubation for 4 h, a live/dead cell staining kit (Abnova, Taipei, Taiwan) was used to measure cell viability, and the uptake of fluorescent indicators was detected using a confocal microscope. For the cells seeded inside the hydrogel, SW480 cells were uniformly suspended in a sterile AlgNB, AlgNB/MoS2, and AlgNB/MoS2/5-Fu precursor solution, and added dropwise to the 24-well plates. Calcium ion cross-linking was applied to realize the in situ gelation of the hydrogels, and cells were cultured for 24 h. Next, 808 nm NIR laser irradiation (1.0 W/cm2) was applied for 10 min, and the cell viability in the hydrogels was also determined using a live-dead cell staining kit. The groups without NIR irradiation were used as negative controls.
In vivo animal model
For the subcutaneous transplantation model, 2×106 SW480 cells were suspended in sterile PBS and subcutaneously injected into the right hind limbs of four-week-old female nude mice. Treatment was initiated 10 days after the injection. The mice were randomised into six groups (n=5 per group) and different hydrogels (AlgNB, AlgNB/MoS2, and AlgNB/MoS2/5-Fu) (50 μL precursor solution + 5 μL CaCl2 solution [10 mg/mL]) was injected in situ into the tumour, and the group treated with saline solution was set as the blank control. The mice in the AlgNB/MoS2 hydrogel and AlgNB/MoS2/5-Fu hydrogel groups were treated with 808 nm NIR irradiation (1.0 W/cm2, 10 min) after injection (on days 1, 2, and 3). The body weights of the mice were recorded every alternate day. Tumour growth was recorded every day after inoculation by measurement of two perpendicular diameters using the formula 4π/3 × (width/2)2 × (length/2). The mice were sacrificed 14 days after treatment. The tumour masses (g) derived from the various treatments were compared. Tumour tissues were harvested and fixed with phosphate-buffered formalin for further haematoxylin and eosin (H&E) and deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) assays, according to the manufacturer’s protocol.
Statistical analysis
The in vitro measurements were measured in quadruplicate (n=4) and the in vivo measurements were measured in quintuplicate (n=5) for each experimental group. The data are expressed as the mean ± standard deviation (SD). Statistical analyses were performed using Tukey’s post-hoc test and one-way analysis of variance. A student’s t-test was performed using SPSS software (IBM Corp., Armonk, NY, USA), and *p <0.05, **p <0.01 and ***p <0.001 were considered statistically significant.
Preparation and characterization of AlgNB/MoS2 hydrogel
An AlgNB/MoS2 hydrogel was fabricated using NB-linked sodium alginate (AlgNB) and LA-PEG-NH2 modified MoS2 nanosheets (NH2-PEG-MoS2). The process of grafting of NB onto Alg to synthesize the AlgNB hydrogel has been reported in our previously published work (Fig. 2a), and the results of 1H NMR spectra for Alg and AlgNB demonstrate the conjugation of NB to Alg (Fig. 2h) [24, 25]. The AlgNB hydrogel can be gelled in situ via ionic cross-linking, which allows it to be used as an injectable hydrogel. After NB was grafted onto Alg, 365 nm UV light irradiation was carried out to activate the hydroxymethyl group on NB to become an aldehyde group (-CHO) (Fig. 2b,f) [21]. The UV light-activated aldehyde group on AlgNB could be used as a “molecular glue”. Fig. 2c shows the surface modification strategy of MoS2 nanosheets with LA-PEG-NH2, where the two sulfur atoms in the LA moiety bind strongly to MoS2 [26], and NH2-PEG-MoS2 can be synthesized by simply mixing MoS2 with LA-PEG-NH2 under sonication and incubation in water. The FTIR spectra showed that the synthesized NH2-PEG-MoS2 contained the characteristic peaks of both MoS2 and LA-PEG-NH2 (Fig. 2e), indicating that PEG was grafted onto the surface of the MoS2 nanosheets. Previous studies have suggested that PEG offers MoS2 nanosheets remarkably enhanced physiological stability, and improved decentralized performance in PBS or serum [26, 28]. In our system, by simply mixing NH2-PEG-MoS2 and UV light-activated AlgNB under stirring and sonication, the amino group of PEG terminal and UV light-activated aldehyde group on AlgNB were bound together bia a Schiff base reaction (Fig. 2d). The binding state of AlgNB with MoS2 nanodots was explored using XPS spectra (Fig. 2f,g). Compared with AlgNB, for the C(1s) core levels, the percentage of -CHO for AlgNB/MoS2 reduced significantly from 11.53 mol% to 4.17 molS% and generated a -CHO (16.13 mol%) for AlgNB/MoS2. The strategy of chemically bonding MoS2 to hydrogel macromolecules could preserve the stability and restrain the agglomeration of MoS2 nanosheets in a more viscous and complex hydrogel system, which is crucial for enhancing the photothermal effect and its application in biology. In addition, the microscopic structures of the hydrogels were measured using SEM and EDS element spectrum tests. Alg, AlgNB, and AlgNB/MoS2 hydrogels all showed a visible internal porous structure, the AlgNB/MoS2 hydrogel exhibited a more fluffier state, and MoS2 nanosheets were uniformly distributed in the hydrogel (Fig. 2i and Fig. S1). These results all demonstrated the success formation of AlgNB/MoS2 hydrogel.
Owing to their excellent dispersion performance, PEG-modified MoS2 nanosheets have been proven to be a novel category of NIR photothermal transducing agents [13]. As shown in Fig. 3a, the temperature rose rapidly with 808 nm NIR irradiation (1.0 W/cm2) and reached a stable value within ten minutes. The temperature rise was positively correlated with the concentration of MoS2 solution, and the highest temperature increment was up to 45.5 °C for the MoS2 (500 μg/mL). To validate the photothermal function of AlgNB/MoS2 hydrogel, different concentration of MoS2 (300 and 500 μg/mL) were mixed with AlgNB pre-gel and gelled with calcium ion cross-linking. The fabricated AlgNB/MoS2 hydrogels were immersed in pure water and the corresponding solution temperature was recorded in real-time. The AlgNB hydrogel was set as the control. As shown in Fig. 3b, in marked contrast, insignificant heating with a temperature rise (3.8°C ) was observed for AlgNB hydrogel under NIR irradiation, but the highest temperature increment was up to 44.3 °C within 10 min for the AlgNB/MoS2 (500 μg/mL) hydrogel, and the photothermal conversion efficiency was about 19.43 %. Notably, the AlgNB/MoS2 (300 μg/mL) hydrogel also reached a temperature rise of 41.7°C, but for the Alg/MoS2 (300 μg/mL) group (without NB-grafted), the highest temperature increment was only up to 29.7°C, significantly lower than the NB-grafted group. This could be ascribed to the fact that the strategy of chemically bonding MoS2 to a hydrogel preserves its stability and maintains a highly efficient photothermal effect (Fig. S2). In the following experiments, the concentration of MoS2 was set as 300 μg/mL. Moreover, to demonstrate the photothermal stability, the AlgNB/MoS2 hydrogel was examined over four laser on/off cycles (at 1.0 W/cm2), and an insignificant reduction was observed (Fig. 3c). These results demonstrate that the obtained AlgNB/MoS2 hydrogel has outstanding photostability and photothermal properties.
As the results of XPS spectra show, the percentage of -CHO reserved for the AlgNB/MoS2 hydrogel was approximately 4.17% (Fig. 2f,g), suggesting it maintained a strong tissue adhesive ability. In order to certify whether the AlgNB/MoS2 pre-gel could be used as a “molecular glue”, the AlgNB/MoS2 pre-gel was dropped to the surface of the pig skin, and the Alg/MoS2, which had not been modified by NB, was used as a control. Surprisingly, the AlgNB/MoS2 pre-gel exhibited excellent tissue adhesion, whereas the Alg/MoS2 pre-gel showed no tissue adhesion (Fig. 3d, Movies S1 and S2). As an UV light-activated molecule, the grafted NB in the AlgNB hydrogel can generate aldehyde groups [22], some of which bind to an amino group on the NH2-PEG-MoS2, and the excess aldehyde groups can be combined with the amino group on the tissue via the Schiff base reaction (Fig. 3e). These are the most likely reasons why the AlgNB/MoS2 pre-gel could be used as a “molecular glue”. This characteristic would be beneficial for anchoring the “molecular glue” on the tumor tissue when it used as an injectable hydrogel, preventing the injected hydrogel from falling off or slipping and causing anti-cancer failure [29]. More importantly, unlike the reported adhesive injectable hydrogel [20], the strategy used in this work was to use the “molecular glue” to infiltrate and anchor the tumor tissue, and calcium ion cross-linking was carried out to make the AlgNB/MoS2 hydrogel formed and fixed in the tumor tissue (Fig. 3f, Movies S2). This process shows a good controllability, rendering it an outstanding implant candidate for oncotherapy.
Various studied have demonstrated that the surface of MoS2 nanosheets can be used for loading of drugs [26, 30], and injectable MoS2-integrated drug-delivery implants can deliver high amounts of PTA and drugs into the tumor site [6]. Therefore, the fabricated AlgNB/MoS2 hydrogel can function as a sustained-release drug implant. To investigate the 5-Fu drug release behaviors in this injectable hydrogel delivery system, the AlgNB/MoS2 hydrogel contained with 5-Fu (20% drug-loading) was immersed in PBS buffers, and the released 5-Fu molecules were collected at different time points to determine the drug-releasing kinetics with UV-vis spectra. The effect of NIR irradiation on drug release was also take into consideration, and the group of AlgNB hydrogels without PTAs was set as a control. As Fig. 3g shows, approximately 45% of the 5-Fu was released from the AlgNB hydrogel over 24 h, whereas approximately 39% of the 5-Fu was released from the AlgNB/MoS2 hydrogel (Fig. 3g,h), indicating the AlgNB/MoS2 hydrogel has a better drug sustained release effect than the AlgNB hydrogel, which was helpful for its chemotherapy application. For the groups without NIR irradiation, the percentage of 5-Fu released from the AlgNB hydrogel at different time points was higher than that from the AlgNB/MoS2 hydrogel (Fig. S3), which was likely due to the MoS2 nanosheets having stronger drug-loading ability than the AlgNB biomacromolecule. Notably, with NIR irradiation, an insignificant improvement in 5-Fu release was observed for the AlgNB hydrogel, but 5-Fu release increased remarkably with the AlgNB/MoS2 hydrogel (Fig. 3i, Fig. S3). These results indicate that the photothermal effect of the MoS2 nanosheets in the hydrogels could be useful for the controllable release of the drug, which is useful for tumour chemotherapy.
Cell bioactivity and photothermal effect of the AlgNB/MoS2/5-Fu hydrogel and its induced SW480 cell death in vitro
Cytocompatibility and tumor cytotoxicity are the two predominant factors determining the clinical translation of AlgNB/MoS2/5-Fu implants. A CCK-8 assay was used to determine the cell proliferation behavior, and as Fig. 4a shows, SW480 cells were cultured in the medium with AlgNB, AlgNB/MoS2, and AlgNB/MoS2/5-Fu pre-gels, respectively, and the cells cultured with normal medium was used as a controls. No significant differences were observed between the AlgNB and control groups, indicating the excellent biocompatibility of AlgNB. However, the addition of MoS2 nanosheets to the AlgNB/MoS2 group significantly decreased cell proliferation, which could be attributed to the exposure of the MoS2 nanosheets and can cause cytotoxicity [31]. Therefore, the strategy of loading the MoS2 nanosheets into an injectable hydrogel and in situ implantation into the tumor tissue may substantially reduce its health risks. In addition, 5-Fu-loaded AlgNB/MoS2 group (AlgNB/MoS2 group) exhibited a further decrease, even lower than the initial inoculation value at 3 days and 5 days, which could be attributed to the SW480 cell killing ability of the 5-Fu drug. For the assay of SW480 seeded on the surface of the hydrogels, similar results were obtained for cell proliferation behavior (Fig. 4b). All the results demonstrated that the biomacromolecule of AlgNB has a superior biocompatibility, and after loading of MoS2 nanosheets and 5-Fu drug to form the AlgNB/MoS2/5-Fu hydrogel, this implant showed an anticipated tumor-killing ability.
Thereafter, the in vitro synergetic effect of PTT and chemotherapy using the AlgNB/MoS2/5-Fu hydrogel was determined thereafter. As Fig. 4c shows, after SW480 cells were cultured in the medium with various pre-gels (the concentration of MoS2 was about 50 μg/mL) for 24 h, 808 nm NIR laser irradiation with different light densities (0, 0.2, 0.5, 0.8, and 1.0 W/cm2) were applied for 10 min. According to the live/dead staining test results, without NIR irradiation, approximately 30% of cells were killed in the AlgNB/MoS2/5-Fu pre-gel group, only approximately 5% of cells were killed in the AlgNB/MoS2 pre-gel group, and all the cells maintained good viability in the AlgNB pre-gel group. These results further confirmed the ability of the 5-Fu drug to kill SW480 cells. Under NIR irradiation at 1.0 W/cm2, the SW480 cells maintained a good viability in the AlgNB pre-gel group, indicating the safety of NIR irradiation within a certain light intensity. In contrast, the cell-killing percentage reached approximately 50% with NIR irradiation (1.0 W/cm2) for the AlgNB/MoS2 pre-gel group. Under the same stimulus conditions, the cell-killing percentage reached approximately 60% for the AlgNB/MoS2/5-Fu pre-gel group. Similar results could be achieved for the assay of SW480 cells seeded inside the hydrogels, and the highest cell-killing percentage could reached approximately 75% for the AlgNB/MoS2/5-Fu hydrogel under NIR irradiation (1.0 W/cm2) (Fig. 4d). These results contributed to the higher concentration of MoS2 for the AlgNB/MoS2/5-Fu hydrogel (about 300 μg/mL) compared with the pre-gel groups (about 50 μg/mL). Moreover, in our previous works, in vitro and in vivo degradation experiments have demonstrated that the AlgNB hydrogel slow degradation properties [24, 25], indicated that the hydrogel can play a long-term role in tumor treatment. These results all demonstrated that the AlgNB/MoS2/5-Fu hydrogel is an excellent candidate for the synergetic effect of PTT and chemotherapy in CRC.
In vivo colorectal cancer treatment using AlgNB/MoS2/5-Fu hydrogel
Finally, the anti-tumor effects of the tissue adhesive AlgNB/MoS2/5-Fu hydrogel in vivo were further investigated using SW480 colorectal tumor-bearing nude mice. Various pre-gels were injected into the primary tumor cavity when the tumor volumes reached approximately 100 mm3 and formed hydrogels in situ with the help of calcium ion crosslinking, the tumor injected with saline solution was used as a control (Fig. 5a). After AlgNB/MoS2/5-Fu hydrogel was intra-tumorally injected, NIR irradiation (1.0 W/cm2, 10 min/day) was followed over the next 3 days, and the temperature measured increased from 32.2℃ to 56.2℃ (Fig. 5b), and the group without 5-Fu (AlgNB/MoS2 hydrogel) was also carried with NIR irradiation, which shows the similar temperature increases (Fig. S4). As shown in Fig. 5c, the AlgNB/MoS2/5-Fu group with NIR irradiation exhibited the strongest tumor growth inhibition effect and was significantly superior to the treatment without NIR irradiation, suggesting a potentially high efficiency for PTT for oncotherapy. Moreover, under the same treatment of without or with NIR irradiation, the anti-tumor effects of the AlgNB/MoS2 hydrogel loaded with 5-Fu drug were all better than those of the groups without 5-Fu, revealing the positive anti-cancer efficacy of 5-Fu drug delivery. Furthermore, the body weight of mice from all groups remained stable for 2 weeks, indicating that the use of the AlgNB/MoS2/5-Fu hydrogel in combination with NIR irradiation did not cause clear side effects (Fig. 5d). After 14 days of treatment, the mice in all groups were sacrificed, and the tumors were harvested for further evaluation (Fig. 5e); the results are consistent with the above findings. These results demonstrate that the injectable 2D-MoS2-integrated adhesive AlgNB/MoS2/5-Fu hydrogel can function as an NIR-triggered phototherapy and drug-delivery implant for CRC treatment.
Subsequently, histological and immunohistochemical analyses of the harvested tumor on the 14th day were used to evaluate the therapeutic efficacy. Hematoxylin–eosin (H&E) staining results showed that the most evident necrosis of severe vacuolization and typical apoptotic characteristics were detected in the AlgNB/MoS2/5-Fu hydrogel with NIR irradiation, which was more damaged than the groups without NIR irradiation or 5-Fu drug loaded (Fig. 5f). TdT-mediated dUTP-biotin nick end labeling (TUNEL)-stained images further confirmed that the highest number of apoptotic cells (green color) was observed for the group AlgNB/MoS2/5-Fu hydrogel with NIR irradiation. However, the number of apoptotic cells significantly decreased in the control group and groups without NIR irradiation or drug-loaded with 5-Fu (Fig. 5g). In addition, H&E staining of the main organs (heart, lung, liver, spleen, and kidney) of the mice in all the groups exhibited no significant histopathological damage, indicating that the synergistic treatment with the AlgNB/MoS2/5-Fu hydrogel had negligible long-term toxicity in vivo (Fig. 6). These results further confirmed the superior efficacy of the AlgNB/MoS2/5-Fu hydrogel for CRC therapy, which could contributed to the synergetic effects of tumor tissue adhesion, PTT, and chemotherapy.
In this study, an injectable 2D-MoS2-integrated adhesive AlgNB/MoS2/5-Fu hydrogel which can be functioned as an NIR-triggered phototherapy and drug-delivery implant for CRC treatment was successfully synthesized. First, the XPS results confirmed that the UV light-activated aldehyde group on AlgNB binds with surface-modified NH2-PEG-MoS2 nanosheets via the Schiff base reaction and maintains a highly efficient NIR-triggered photothermal effect for MoS2 nanosheets in the hydrogel. Moreover, in vitro SW480 cell tests demonstrated that the biomacromolecule of AlgNB has a superior biocompatibility, and after loading of MoS2 nanosheets and 5-Fu drug to form the AlgNB/MoS2/5-Fu hydrogel, this implant showed an excellent tumor cell-killing ability. Finally, an SW480 colorectal tumor-bearing nude mice model was constructed, and the results of tumor growth, H&E staining and immunohistochemical staining confirmed the superior efficacy of the AlgNB/MoS2/5-Fu hydrogel for CRC therapy, which could contributed to the synergetic effects of tumor tissue adhesion, PTT, and chemotherapy. This research provides new insights to guide the designing and utilization of a synergistic tumor therapeutic platform with promising clinical translation potential for efficient localized tumor therapy.
Acknowledgements
This work is supported by the National Natural Science Foundation of China (No.82302662, 81771502 and 82273265), the Shenzhen Basic Research Project Natural Science Foundation (JCYJ20210324103210027), and the Department of Health of Zhejiang Province (No. 2018KY473).
Author contributions
XL conceived and designed the experiments; and wrote the manuscript; performed in vitro and in vivo experiments with assistance by XL, JW, HW and KH; XL, JW, HW, KH, WL and CF discussed and analyzed the data. XL, KC and ZS supervised the research and funding acquisition. All authors read and approved the final manuscript.
Data availability
No datasets were generated or analysed during the current study.
Ethics approval and consent to participate
All animal experiments and procedures were approved by Ethics Committee of Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University. All animal experiments were performed in accordance with standard animal care guidelines.
Consent for publication
All the authors have approved the manuscript and agree with submission to journal.
Competing interests
The authors declare no competing interest.
Author details
† Department of Colorectal Surgery, Key Laboratory of Biological Treatment of Zhejiang Province, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, 310016, China.
Ұ School of Materials Science and Engineering, State Key Laboratory of Silicon Materials, Cyrus Tang Center for Sensor Materials and Applications, Zhejiang University, Hangzhou 310027, China.
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No competing interests reported.