Dynamic covalent bond-based hydrogel for black phosphorus-loaded liver cancer drug, and preparation method therefor and application thereof
Patent Information
- Application Number
- PCT/CN2025/138991
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2025-11-14
- Filing Date
- 2025-12-01
- Publication Date
- 2026-10-22
Smart Images
Figure CN2025138991_22102026_PF_FP_ABST
Abstract
Description
A dynamic covalently bonded hydrogel loaded with black phosphorus for liver cancer, its preparation method and application Technical Field
[0001] This invention belongs to the field of biomedical materials, specifically relating to a dynamic covalently bonded hydrogel of black phosphorus loaded with liver cancer drugs, its preparation method and application. Background Technology
[0002] Liver cancer is the third leading cause of cancer death worldwide, and its incidence continues to rise, affecting more than one million people annually. A large proportion of these patients are diagnosed at an advanced stage, making surgical resection unsuitable. Currently, common treatments for liver cancer include chemotherapy, targeted therapy, immunotherapy, and combination therapy, typically administered orally or intravenously. However, the short half-life of these drugs and the first-pass effect significantly reduce their efficacy, and short-term high systemic drug concentrations can lead to severe toxic side effects. Therefore, there is an urgent need to develop new drug delivery systems to address issues such as low therapeutic efficacy, poor biodegradability, and uncontrolled drug release.
[0003] Black phosphorus is a novel two-dimensional nanomaterial composed of a wrinkled monolayer of phosphorus atoms formed by covalent bonds, with layers stacked by van der Waals forces. It can efficiently load chemotherapeutic drugs via electrostatic interactions, achieving a drug loading rate of up to 95%. It possesses photothermal / photodynamic activity, enabling photoresponsive controlled release. Under near-infrared radiation (NIR) irradiation, black phosphorus can generate photothermal effects and reactive oxygen species. Black phosphorus can be degraded into non-toxic phosphate ions in vivo, exhibiting good biocompatibility. Doxorubicin, an anthracycline antibiotic, can attack cancer cells through various methods, including DNA embedding, inhibition of topoisomerase 2, and the generation of free radicals. Therefore, black phosphorus loaded with doxorubicin can be used for the treatment of liver cancer and achieve targeted delivery.
[0004] Polyethylene glycol hydrogel (PEG hydrogel) is a three-dimensional cross-linked hydrophilic polymer network that can absorb large amounts of water without dissolving. It is soft, elastic, and highly biocompatible. A hydrogel network was synthesized using four-arm polyethylene glycol benzaldehyde (PEG-DF) and four-arm polyethylene glycol cyanopropyl (PEG-CN) as raw materials, forming dynamic covalent bonds between the benzaldehyde and cyanopropyl groups. These bonds are thermally reversible, breaking at high temperatures and reforming upon cooling. The covalent bonds respond to external stimuli such as heat, pH, or light, thereby stimulating drug release.
[0005] HCC-LM3 is a human highly metastatic stem cell cancer line derived from the Shanghai Cell Bank of the Chinese Academy of Sciences. It is often used as an in vivo and in vitro model in liver cancer research. It can express relevant in vivo indicators of liver cancer patients and has high proliferative and migration capabilities. It usually exhibits EMT-related markers (such as downregulated E-cadherin and upregulated N-cadherin and Vimentin), which are closely related to enhanced cell migration and invasion capabilities.
[0006] Traditional drug delivery systems have many limitations in the treatment of liver cancer, such as: (1) Traditional DOX is administered intravenously or orally, requiring frequent administration to maintain efficacy, resulting in high systemic drug exposure and causing serious side effects such as cardiotoxicity and bone marrow suppression. (2) Current drug delivery systems have problems such as poor biodegradability, uncontrollable drug release, and lack of responsiveness to external stimuli. Summary of the Invention
[0007] The technical problem to be solved by this invention is that traditional doxorubicin is administered intravenously or orally, requiring frequent administration to maintain efficacy, which leads to high systemic drug exposure and causes serious side effects such as cardiotoxicity and bone marrow suppression. Current drug delivery systems have problems such as poor biodegradability, uncontrollable drug release, and lack of responsiveness to external stimuli.
[0008] To address the aforementioned technical problems, this invention proposes a dynamic covalently bonded hydrogel loaded with black phosphorus for liver cancer, its preparation method, and its application; achieved through the following technical solution:
[0009] Firstly, the purpose of this invention is to provide a dynamic covalently bonded hydrogel of black phosphorus loaded with liver cancer drugs, using the following technical solution:
[0010] A dynamic covalently bonded hydrogel loaded with a black phosphorus drug for liver cancer includes: black phosphorus, doxorubicin, four-armed polyethylene glycol benzaldehyde, and four-armed polyethylene glycol cyanopropane. The four-armed polyethylene glycol benzaldehyde and four-armed polyethylene glycol cyanopropane form the matrix; black phosphorus serves as a photothermal agent and drug carrier; and doxorubicin is used as a chemotherapeutic drug.
[0011] This invention provides a hydrogel network formed by four-armed polyethylene glycol benzaldehyde and four-armed polyethylene glycol cyanopropane through dynamic covalent bonds. The hydrogel is loaded with black phosphorus and the drug doxorubicin, encapsulating doxorubicin in an injectable hydrogel for direct delivery to the tumor site via intratumoral injection. This significantly reduces the dose of drug entering the bloodstream, thereby reducing systemic toxicity. This invention also achieves near-infrared laser-triggered, on-demand controlled drug release by integrating black phosphorus and doxorubicin into a hydrogel network based on dynamic covalent bonds.
[0012] The dynamic covalently bonded hydrogel provided by this invention is formed by the condensation reaction of four-armed polyethylene glycol benzaldehyde and four-armed polyethylene glycol cyanohydrin. These bonds break upon heating and reform upon cooling, exhibiting thermal reversibility. The hydrogel network provided by this invention is composed of dynamic covalent bonds, which are temperature-sensitive. The local heat generated by black phosphorus will destroy these dynamic covalent bonds. Furthermore, black phosphorus has excellent photothermal conversion efficiency, and when irradiated by external near-infrared laser, it can convert light energy into heat energy, causing a rapid increase in local temperature, thus overcoming many limitations of traditional drug delivery systems.
[0013] In a preferred embodiment of the present invention, a dynamic covalently bonded hydrogel loaded with liver cancer drugs on black phosphorus is formed, in which four-armed polyethylene glycol benzaldehyde and four-armed polyethylene glycol cyanopropane are cross-linked to form a hydrogel network; the doxorubicin is loaded onto black phosphorus through physical adsorption and weak interactions, and the doxorubicin is uniformly dispersed on the hydrogel network through physical embedding.
[0014] Black phosphorus, as a photothermal agent, is sensitive to light and temperature. Four-armed polyethylene glycol benzaldehyde and its derivatives are the basic structural units of the hydrogel polymer network, linked by dynamic covalent bonds formed through a Knoevenagel condensation reaction. Black phosphorus and doxorubicin are physically loaded into the hydrogel network, with doxorubicin encapsulated throughout the network. Doxorubicin binds to black phosphorus through physical adsorption and weak interactions. The relationship between doxorubicin and the hydrogel network is one of physical encapsulation. Directly dissolving four-armed polyethylene glycol benzaldehyde and its derivatives in a doxorubicin solution and mixing them on a centrifuge tube cap initiates gelation, meaning that doxorubicin molecules are freely dispersed in the precursor solution containing the four-armed polyethylene glycol benzaldehyde and its polymer derivatives. The dynamic covalent hydrogel of black phosphorus loaded with liver cancer drugs exhibits a reversible response. It is formed by the condensation reaction of four-arm polyethylene glycol benzaldehyde and four-arm polyethylene glycol cyanohydrin, which break upon heating and reform upon cooling, demonstrating thermal reversibility.
[0015] Secondly, the present invention provides a method for preparing a dynamic covalently bonded hydrogel of black phosphorus loaded with hepatocellular carcinoma drugs, comprising the following steps:
[0016] Step 1: Remove the four-armed polyethylene glycol benzaldehyde and four-armed polyethylene glycol cyanopropane powders and allow them to equilibrate to room temperature;
[0017] Step 2: Prepare four-arm polyethylene glycol benzaldehyde solution and four-arm polyethylene glycol cyanopropane solution respectively. Mix the four-arm polyethylene glycol benzaldehyde solution and the four-arm polyethylene glycol cyanopropane solution in a predetermined ratio. Add auxiliary solvent dropwise during the mixing process. After mixing evenly, prepare a hydrogel precursor solution.
[0018] Step 3: Curing and cross-linking to prepare a dynamic covalently bonded hydrogel of black phosphorus loaded with hepatocellular carcinoma drug; the hydrogel precursor solution is vortexed to ensure sufficient reaction. Under neutral or near-neutral pH conditions, the four-armed polyethylene glycol benzaldehyde and four-armed polyethylene glycol cyanopropane in the hydrogel precursor solution rapidly cure and cross-link through covalent bonds to construct a hydrogel network. Black phosphorus and doxorubicin are embedded in the hydrogel network, thus obtaining a dynamic covalently bonded hydrogel of black phosphorus loaded with hepatocellular carcinoma drug. Under neutral or near-neutral pH conditions, the two spontaneously form dynamic covalent bonds through a Knoevenagel condensation reaction, rapidly constructing a hydrogel network at room temperature. The doxorubicin loaded with black phosphorus is uniformly dispersed in the hydrogel network through physical adsorption and weak interactions, forming a dynamic covalently bonded hydrogel of black phosphorus loaded with hepatocellular carcinoma drug.
[0019] This invention provides a method for preparing dynamic covalently bonded hydrogels loaded with black phosphorus for liver cancer, combining physical interactions and chemical cross-linking. The gelation process of the hydrogel in this study is relatively simple, requiring no catalyst or external energy initiation, and the Knoevenagel condensation reaction can occur at room temperature. The system gels rapidly, with vortexing taking about 15 seconds, and gelation can occur at room temperature, completing the transition from solution to gel within tens of seconds to minutes. The gelation time affects the hydrogel properties, including drug encapsulation efficiency, mechanical properties and stability, and self-healing properties.
[0020] A preferred embodiment of the present invention provides a method for preparing a dynamic covalently bonded hydrogel of black phosphorus-loaded liver cancer drugs, characterized in that, in step 2, the preparation of the four-arm polyethylene glycol benzaldehyde solution and the four-arm polyethylene glycol cyanopropane solution is selected from any of the following methods: dissolving the equilibrated four-arm polyethylene glycol benzaldehyde powder in a solution containing doxorubicin, and dissolving the four-arm polyethylene glycol cyanopropane powder in a solution containing black phosphorus; or dissolving the equilibrated four-arm polyethylene glycol benzaldehyde powder in a solution containing black phosphorus, and dissolving the four-arm polyethylene glycol cyanopropane powder in a solution containing doxorubicin. A preferred embodiment of the present invention further specifies that, in step 2, the auxiliary solvent is selected from one or more of PBS, 0.9% NaCl, HEPES buffer, or deionized water.
[0021] In a preferred embodiment of the present invention, the volume of the auxiliary solvent is 0.1-0.5 mL.
[0022] In a preferred embodiment of the present invention, the concentration of the dynamic covalently bonded hydrogel precursor solution of black phosphorus loaded with liver cancer drugs is 10%-40%; more preferably, the concentration of the hydrogel precursor solution is 20%-35%.
[0023] When the concentration of the hydrogel precursor solution is below 10%, the polymer chain spacing is too large, making it difficult to form a stable hydrogel network; while when the concentration is above 40%, the solution viscosity is too high, resulting in excessively high polymer chain density, leading to poor toughness and easy breakage of the formed hydrogel. A concentration range of 10%-40% can meet the requirements of the hydrogel in terms of stress-strain properties. Preferably, the hydrogel based on dynamic covalent bonds achieves the optimal drug release effect at a concentration of 20%-35%.
[0024] In a preferred embodiment of the present invention, the method for preparing a dynamic covalently bonded hydrogel loaded with black phosphorus for liver cancer drugs, in step 3, a four-arm polyethylene glycol benzaldehyde solution and a four-arm polyethylene glycol cyanopropane solution are mixed in a volume ratio of 3:7-7:3; the hydrogel is prepared at room temperature.
[0025] In a preferred embodiment of the present invention, the preparation method of the dynamic covalently bonded hydrogel loaded with black phosphorus for liver cancer drugs, in step 3, has a neutral or near-neutral pH value of 7.0-8.0. Under acidic or neutral conditions, the concentration of alkali is insufficient, making it difficult to effectively generate a sufficient amount of carbanions; under alkaline conditions, side reactions or hydrolysis reactions may occur. Therefore, the most suitable pH range for the dynamic covalently bonded hydrogel provided by the present invention is 7.0-8.0.
[0026] Thirdly, this invention provides the application of a black phosphorus-loaded dynamic covalently bonded hydrogel for liver cancer drugs in the preparation of a liver cancer drug delivery system, wherein the delivery system is used to deliver therapeutic drugs to the liver cancer site. The liver cancers for which the black phosphorus-loaded dynamic covalently bonded hydrogel for liver cancer drugs is applicable include, but are not limited to, alcoholic liver cancer, fatty liver cancer, and hepatitis B-related liver cancer.
[0027] The beneficial effects of this invention compared to the prior art are:
[0028] 1. By combining the emerging two-dimensional nanomaterial black phosphorus with dynamic covalent chemical hydrogels to deliver drugs, a novel composite drug delivery platform has been constructed, which overcomes many limitations of traditional drug delivery systems and provides new possibilities for drug research and development applications.
[0029] 2. The black phosphorus-loaded dynamic covalently bonded hydrogel liver cancer drug delivery system provided by this invention surpasses traditional monotherapy by combining drug therapy, photothermal therapy, and environmentally responsive autonomous drug release. Black phosphorus serves as a photothermal agent and drug carrier, while the hydrogel acts as a controllable release matrix and black phosphorus stabilizer, achieving synergistic therapy and potentially significantly improving antitumor efficacy and reducing drug resistance. Attached Figure Description
[0030] Figure 1: Formation principle and injection application of dynamic covalently bonded hydrogels loaded with black phosphorus for liver cancer; Figure 1a: Schematic diagram of the reaction principle of hydrogel network formation by four-arm PEG precursor solution; Figure 1b: Physical display of the process of hydrogel network formation by precursor solution; Figure 1c: Schematic diagram of the process of extruding drug-loaded hydrogels through syringe; Figure 1d: Macroscopic morphology of hydrogels obtained by injection.
[0031] Figure 2: Self-healing and mechanical properties of dynamic covalently bonded hydrogels loaded with black phosphorus for liver cancer; Figure 2a: Macroscopic morphology comparison of hydrogel fragments of different colors before and after self-repair; Figure 2b: Stress-strain diagram of hydrogel after self-repair under uniaxial tension; Figure 2c: Schematic diagram of the reaction principle of dynamic covalent bond breaking and recombination process at the hydrogel interface.
[0032] Figure 3: Comprehensive characterization of dynamic covalently bonded hydrogels loaded with black phosphorus for liver cancer; Figure 3a: Cell compatibility assay; Figure 3b: Relationship between precursor solution ratio and cell activity; Figure 3c: Temperature-responsive drug release curve; Figure 3d: Macroscopic morphology of the swelling process; Figure 3e: Swelling behavior curve; Figure 3f: In vitro degradation curve.
[0033] Figure 4: Biocompatibility and apoptosis-inducing effect of dynamic covalently bonded hydrogels loaded with black phosphorus for liver cancer; Figure 4a: Cell viability staining; Figure 4b: Comparison of NIH-3T3 cell viability; Figure 4c: Comparison of HCC-LM3 cell viability; Figure 4d: Comparison of cell viability in different culture media environments; Figure 4e: Flow cytometry of apoptosis; Figure 4f: Statistical graph of apoptosis rate.
[0034] Figure 5: Characterization of the antitumor efficacy and biosafety of dynamic covalently bonded hydrogels loaded with black phosphorus for liver cancer; Figure 5a: Physical image of tumor tissue excised after treatment; Figure 5b: Statistical graph of tumor inhibition rate of each group calculated based on tumor weight; Figure 5c: Quantitative statistical graph of Ki67 positive expression area in tumor tissue; Figure 5d: Curve of weight change of nude mice in each group during the experiment; Figure 5e: Statistical graph of alanine aminotransferase (ALT) level in serum of nude mice in each group after the experiment; Figure 5f: Statistical graph of aspartate aminotransferase (AST) level in serum of nude mice in each group after the experiment; Figure 5g: Ki67 immunohistochemical staining section of tumor tissue and H&E staining section of major organs. Detailed Implementation
[0035] The technical solutions of the present invention will be described in detail below with reference to the embodiments of the present invention and the accompanying drawings 1-5.
[0036] In the embodiments provided by this invention, DOX represents doxorubicin, BP represents black phosphorus, PEG hydrogel represents a hydrogel formed by a four-arm polyethylene glycol benzaldehyde and a four-arm polyethylene glycol cyanopropane copolymer, DOX-PEG hydrogel represents a PEG hydrogel loaded only with doxorubicin and without BP, BP-PEG hydrogel represents a PEG hydrogel containing only BP and not loaded with doxorubicin, and BP-DOX-PEG hydrogel represents a dynamic covalently bonded hydrogel of black phosphorus loaded with liver cancer drugs provided by this invention.
[0037] In the embodiments provided by this invention, the standard fibroblast cell line (NIH-3T3) and highly metastatic human hepatocellular carcinoma (HCC-LM3) cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences, and the tumor-bearing nude mice were purchased from Hangzhou Resource Experimental Animal Technology Co., Ltd.
[0038] Example 1: Preparation method and characterization of smart responsive hydrogels
[0039] In this embodiment, the DOX solution was purchased from Nanjing Dulai Biotechnology Co., Ltd., and the BP solution was purchased from Nanjing Xianfeng Nanomaterials Technology Co., Ltd.
[0040] As shown in Figure 1, the preparation method of black phosphorus-loaded hepatocellular carcinoma drug dynamic covalent group hydrogel includes the following steps:
[0041] (1) Take the four-armed polyethylene glycol benzaldehyde powder and the four-armed polyethylene glycol cyanopropane powder stored at -20°C out of the refrigerator, do not open the bottle cap, place them in a dry environment at room temperature for 5 minutes to allow the temperature to rise to room temperature, then open the bottle cap and weigh them.
[0042] (2) Weigh out 0.125g of tetra-arm polyethylene glycol benzaldehyde powder and 0.125g of tetra-arm polyethylene glycol cyanopropane powder after equilibration. Dissolve the tetra-arm polyethylene glycol benzaldehyde powder in a solution containing doxorubicin to obtain a tetra-arm polyethylene glycol benzaldehyde solution; dissolve the tetra-arm polyethylene glycol cyanopropane powder in a solution containing black phosphorus to obtain a tetra-arm polyethylene glycol cyanopropane solution. Then mix the two tetra-arm polyethylene glycol solutions, adding 0.1mL of deionized water dropwise during the mixing process. After mixing evenly, a hydrogel precursor solution is obtained.
[0043] (3) The hydrogel precursor solution was vortexed to allow it to react fully, at which point the pH of the precursor solution was 7.4. At room temperature, the four-armed polyethylene glycol benzaldehyde and four-armed polyethylene glycol cyanopropane in the hydrogel precursor solution rapidly solidified and crosslinked through covalent bonds to form a hydrogel network, and black phosphorus and doxorubicin were embedded in the hydrogel network, thus obtaining a dynamic covalently bonded hydrogel loaded with black phosphorus for liver cancer.
[0044] (4) Inject the hydrogel into a pattern, cut and reassemble it at room temperature and let it stand for a period of time. By stretching the hydrogel with different staining, use a digital camera to record the injectability, self-healing and stretching properties, and use thermal imaging to characterize it.
[0045] Example 2 Characterization of dynamic covalently bonded hydrogels containing black phosphorus-loaded hepatocellular carcinoma drugs
[0046] The hydrogels prepared using the hydrogel preparation method provided in Example 1 were dyed with pink and blue pigments, and the hydrogels of different colors were prepared into plum blossom shapes and squares.
[0047] As shown in Figure 2, two plum blossom-shaped hydrogels, one pink and one blue, were prepared in the experiment. They were then divided in half, reassembled at room temperature, and left to stand for 30 minutes.
[0048] Figure 2a shows that the reassembled hydrogels spontaneously healed at the interface without external stimulation, leaving no obvious cut lines, and the healed structures could be lifted with tweezers without separating.
[0049] To further evaluate the mechanical integrity of the self-healing interface, tensile tests were performed on the reconnected square hydrogels. The samples exhibited typical stress-strain characteristics under uniaxial tension.
[0050] The presence of dynamic covalent bonds formed by the reversible reaction between aldehydes and active methylene groups is evident in Figure 2c, which illustrates the re-bonding of internal dynamic covalent bonds at the interface of the black phosphorus-loaded hepatocellular carcinoma drug hydrogel. Figure 2b demonstrates that the surface-healing hydrogel exhibits sufficient mechanical strength and plastic deformation capacity under external force, confirming the reliability of the dynamic connections between hydrogel fragments.
[0051] This embodiment verifies the self-healing ability of the dynamic covalently bonded hydrogel loaded with black phosphorus for liver cancer, indicating that it can form a hydrogel network through dynamic chemical bonds.
[0052] Example 3: Antibacterial effect of dynamic covalently bonded hydrogels loaded with black phosphorus drugs for liver cancer
[0053] Figure 3 shows the comprehensive characterization of the drug-loaded hydrogel.
[0054] Figure 3a shows the cell compatibility test. Three groups of dynamically covalently bonded hydrogels loaded with different concentrations of DOX were prepared using the hydrogel preparation method provided in Example 1. Cell compatibility experiments were conducted to evaluate the safety of the hydrogels involved in this invention for cells. Figure 3a shows that the curves of the three groups of dynamically covalently bonded hydrogels with different concentrations exhibit a continuous upward trend over time, indicating that cells can proliferate normally when co-cultured with the hydrogels. This demonstrates that the dynamically covalently bonded hydrogels provided by this invention have excellent biocompatibility.
[0055] In Figure 3b, covalent hydrogels were prepared using the hydrogel preparation method provided in Example 1 by adjusting the volume ratio of the four-arm polyethylene glycol benzaldehyde and four-arm polyethylene glycol cyanopropane precursor solutions. The volume ratios of the two four-arm polyethylene glycol precursor solutions were set to 1:2, 1:4, 1:6, and 1:8, respectively. Cells were co-cultured with these hydrogels, and the change in cell OD value over time was detected. Figure 3b shows that when cells were cultured with the four groups of hydrogels with different components, the OD value continuously increased to 0.35 over time, then slowly decreased to 0.3 and remained constant. This indicates that this example experimentally verifies that the dynamic covalent hydrogel provided by the present invention has excellent biocompatibility.
[0056] Two different hydrogels were prepared using the hydrogel preparation method provided in Example 1. One group was a DOX-loaded BP hydrogel as the experimental group, and the other group was a DOX-loaded hydrogel without BP as the control group. Cells were cultured in the hydrogel culture medium of the control group and the experimental group at 0℃, 5℃, 10℃, 15℃ and 20℃, respectively. The temperature change of the BP-containing hydrogel under NIR laser irradiation was recorded using an infrared thermal imaging camera, and the DOX release rate was detected at 0℃, 5℃, 10℃, 15℃ and 20℃, respectively.
[0057] As shown in Figure 3c, the DOX release rate in both the experimental and control groups increased with ambient temperature. The experimental group showed a faster rate of increase, reaching 90% at 20℃; however, the control group showed a very slow rate of increase between 0 and 10℃, with almost no increase in the curve, and only 18% DOX release at 20℃. These experimental phenomena indicate that drug release behavior is highly coupled with temperature, and the dynamic covalent bonds are sensitive to temperature. Upon heating, these bonds break, accelerating drug diffusion. This also demonstrates that in black phosphorus hydrogels, near-infrared light irradiation triggers BP heat generation, breaking the dynamic covalent bonds and releasing the drug on demand. Because NIR can locally irradiate the tumor area, the hydrogel only heats and releases the drug in that area, without affecting other healthy tissues, achieving spatial targeted control and avoiding systemic toxicity.
[0058] Hydrogels of different concentrations were prepared using the method provided in Example 1. The hydrogel concentrations were 12.5%, 25%, and 50%, respectively. Two portions of each were taken, and the swelling kinetics and bulk degradation performance of the hydrogels were tested.
[0059] First, swelling kinetics tests were conducted, the specific process of which is as follows:
[0060] The newly prepared hydrogel was immersed in deionized water for 1.5 hours to remove unreacted monomers and salts. The washed hydrogel was then rapidly frozen and subsequently dried in a freeze dryer to constant weight. At this point, a completely dry polymer backbone with an established network structure was obtained. The dried gel was then removed from the drying oven and accurately weighed on an analytical balance, recorded as the initial dry weight W0.
[0061] Next, the swelling process was carried out. The dry gel sample with a dry weight of W0 was placed in a container containing excess solvent and placed in a constant temperature environment. The wet weight W of the hydrogel was weighed every ten minutes for one hour. t Weigh the hydrogel every 50 minutes for 2-3 hours. Specifically, carefully remove the hydrogel sample with tweezers, quickly and gently blot away any free water droplets on the sample surface with filter paper, and immediately weigh it on an analytical balance. Record the wet weight W at time point t. t After weighing, immediately return the sample to the original solvent to continue swelling. Repeat the weighing process until the mass change after several consecutive weighings is no longer significant; the wet weight at this point is recorded as W. eq Calculate the swelling ratio Q. t The calculation formula is:
[0062]
[0063] Plot the swelling kinetics curve as shown in Figure 3d, with time (t) as the x-axis and the swelling ratio (Q) as the x-axis. t Using y as the y-axis, plot each time point and its corresponding Q. t Plot points along the route and connect them to form a line.
[0064] As shown in Figure 3e, the experimental curve rises rapidly within 30 minutes with a large slope. Then, from 30 to 60 minutes, the slope gradually decreases. After 60 minutes, the slope becomes flat, which is the equilibrium swelling ratio.
[0065] The swelling ratio of the 12.5% concentration hydrogel remained at 60%, while the swelling ratios of the 25% and 50% concentration hydrogels were larger, with values maintained at 175% and 230%, respectively.
[0066] Hydrogel samples of uniform shape and size were prepared from the aforementioned 12.5%, 25%, and 50% concentrations. The samples were briefly rinsed with deionized water to remove impurities. The samples were then rapidly frozen and dried to constant weight in a freeze dryer. The dried samples were removed, weighed, and the initial dry weight was recorded as W0.
[0067] Place each decibel of dried gel sample into an individual container containing excess PBS buffer, seal the container, and place it in a constant temperature environment. Every 5 hours, remove the dried gel sample, weigh it, and record the data. The specific procedure here is the same as the hydrogel swelling ratio experiment, and will not be elaborated further. Calculate the mass retention rate using the following formula:
[0068]
[0069] The degradation curve was plotted as shown in Figure 3f, with time (t) as the x-axis and mass retention rate R as the x-axis. t (Mass Retention,%) is used as the y-axis, and each time point and its corresponding R are plotted. t Plot the (Mass Retention,%) values at points on the graph and connect them to form a line.
[0070] As shown in Figure 3f, the experimental curves exhibit a rapid downward trend, with the mass retention rate of the 12.5% concentration hydrogel being less than 10% after 24 hours. The mass retention rate curves for the 25% and 50% concentration hydrogels have smaller slopes, with mass retention rates of 23% and 55%, respectively, after 47 hours. This indicates that the bonding and degradation process of the hydrogel network structure provided by this invention is relatively slow, demonstrating the stability of the hydrogel structure.
[0071] This embodiment experimentally verifies the excellent injectability and self-healing ability of the hydrogel provided by this invention. Its mechanical properties and drug release behavior can be precisely controlled through the network structure, and it possesses the characteristic of near-infrared photothermal triggering release. Based on dynamic covalent cross-linking, the hydrogel exhibits excellent self-healing ability, capable of autonomously repairing its structure after damage to the hydrogel interface. It also achieves photoresponsiveness in BP, with temperature increases accelerating DOX release. Its mechanical and release properties can be controlled through simple formulation adjustments (polymer concentration). More importantly, by introducing a photothermal agent, BP, it achieves remotely triggered, on-demand pulsed drug release from external near-infrared light, greatly enhancing the intelligence and spatiotemporal controllability of the drug delivery system.
[0072] Example 4: Biocompatibility and Apoptosis Induction Experiment of Dynamic Covalently Bonded Hydrogels Loaded with Black Phosphorus Hepatocellular Carcinoma Drugs
[0073] 1. NIH-3T3 standard fibroblasts and HCC-LM3 human hepatocellular carcinoma cells were cultured in DMEM complete medium at 37°C and 5% CO2 using conventional methods.
[0074] 2. Experimental Groups: To verify the synergistic therapeutic effect of the dynamic covalently bonded hydrogel provided by this invention, the following six comparative groups were set up:
[0075] Comparative Example 1: Blank hydrogel, i.e., control group. NIH-3T3 cells and HCC-LM3 cells were cultured in conventional culture medium without the addition of any hydrogel or drug.
[0076] Comparative Example 2: PEG hydrogel, NIH-3T3 cells and HCC-LM3 cells were cultured in a medium containing PEG hydrogel, which was prepared according to the method in Example 1;
[0077] Comparative Example 3: DOX group, NIH-3T3 cells and HCC-LM3 cells were cultured in medium containing free DOX;
[0078] Comparative Example 4: DOX-PEG hydrogel; NIH-3T3 cells and HCC-LM3 cells were cultured in a medium containing DOX-PEG hydrogel, which was prepared according to the method in Example 1;
[0079] Comparative Example 5: BP-PEG hydrogel; NIH-3T3 cells and HCC-LM3 cells were cultured in a medium containing BP-PEG hydrogel, which was prepared according to the method in Example 1;
[0080] Example group: BP-DOX-PEG hydrogel; NIH-3T3 cells and HCC-LM3 cells were cultured in a medium containing BP-DOX-PEG hydrogel, which was prepared according to the method in Example 1.
[0081] 3. Test methods and results
[0082] (1) Observation of staining of live and dead cells
[0083] HCC-LM3 human liver cancer cells were co-cultured with hydrogels of various groups for 48 hours. During the culture period, the groups in this embodiment were irradiated with near-808 nm infrared light for 10 minutes. After culture, the culture medium was discarded, and Calcein-AM, propidium iodide (PI) staining solutions, and a mixture of Calcein-AM and propidium iodide (PI) staining solutions were added respectively. After incubation for 30 minutes, the cells were observed and photographed under a fluorescence microscope.
[0084] The test results are shown in Figure 4a.
[0085] In the fields of view of Comparative Example 1 and Comparative Example 2, dense and uniform green fluorescence was observed, with sporadic red fluorescence.
[0086] In the fields of view of Comparative Examples 3 and 4, a small amount of red fluorescence and a large amount of green fluorescence were observed to coexist.
[0087] In the field of view of Comparative Example 5, medium-to-high intensity fluorescence and medium-intensity green fluorescence coexist.
[0088] In the field of view of the example group, coexistence of medium-intensity red fluorescence and medium-to-high-intensity green fluorescence was observed.
[0089] (2) Quantitative analysis of cell viability
[0090] NIH-3T3 and HCC-LM3 cells were seeded at a density of 5 × 10³ cells per well in 96-well plates and co-cultured with their respective hydrogels. After 24 and 48 hours of culture, 10 μL of LCK-8 reagent was added to each well, and incubation was continued for 2 hours. The absorbance (OD value) at 450 nm was then measured, and the relative cell viability was calculated.
[0091] The test results are shown in Figures 4b, 4c, and 4d.
[0092] Figure 4b: The relative survival rate of NIH-3T3 cells in Comparative Example 5 BP-PEG hydrogel at 24 hours and 48 hours was not statistically different from that in the blank control group, and was always higher than 85%.
[0093] Figures 4c and 4d: After co-culturing for 48 hours, the relative survival rate of HCC-LM3 cells in the BP-DOX-PEG hydrogel of the present invention embodiment group and irradiated with NIR infrared light was significantly lower than that of all comparative groups (p<0.05).
[0094] (3) Apoptosis analysis
[0095] HCC-LM3 cells were co-cultured with BP solution at the same concentration as in Comparative Example E and Example Group for 24 hours, then treated with Annexin V-FITC / PI apoptosis detection kit, and subsequently analyzed by flow cytometry.
[0096] The test results are shown in Figures 4e and 4f.
[0097] Figure 4e shows a flow cytometry scatter plot: In the blank control group, 99.95% of cells were distributed in the Annexin V-FITC and PI double-negative regions. In the experimental group treated with BP solution, the proportion of cells distributed in the Annexin V-FITC single-positive region and the Annexin V-FITC and PI double-positive region was significantly increased. This indicates a significant increase in the proportion of cells undergoing early apoptosis and late apoptosis or necrosis.
[0098] Figure 4f: The total apoptosis rate of cells in the experimental group treated with BP solution was approximately 23%, which was significantly higher than that of the blank control group (3%).
[0099] Six groups of hydrogels were prepared using the method described in Example 4: blank hydrogel, PEG hydrogel, DOX-PEG hydrogel, BP-PEG hydrogel, DOX hydrogel, and BP-DOX-PEG hydrogel.
[0100] This embodiment used NIH-3T3 standard fibroblasts for biocompatibility testing. NIH-3T3 cells were co-cultured with hydrogel for 48 h, and cell viability was assessed using the Cell Counting Kit-8 (CCK-8). The results, shown in Figure 4b, indicate that the relative cell viability remained above 80%, demonstrating the material's good short-term biocompatibility at the tested concentration. When co-cultured with HCC-LM3 human liver cancer cells, the DOX-free BP-PEG group also maintained cell viability at approximately 80%, indicating that its direct toxicity to cancer cells is limited but present. Treatment of HCC-LM3 cells with BP and analysis using Annexin V-FITC / PI staining revealed that BP treatment significantly induced early and late apoptosis in HCC-LM3 cells compared to the control group.
[0101] Example 5: Wound Repair Effect of Dynamically Covalently Bonded Hydrogels Loaded with Black Phosphorus Drugs for Hepatocellular Carcinoma
[0102] 1. Establishment of tumor-bearing model
[0103] Male BALB / c tumor-bearing nude mice aged 4-6 weeks were selected. Under sterile conditions, HCC-LM3 human hepatocellular carcinoma cells in the logarithmic growth phase were resuspended in serum-free culture medium and subcutaneously injected into the right axillary region of the mice at a density of approximately 1 × 10^7 cells per mouse. The mice were observed daily, and tumors were used for subsequent experiments when they reached a diameter of approximately 4 mm.
[0104] 2. Experimental grouping and drug administration
[0105] Tumor-bearing nude mice were randomly divided into 4 groups of 5 mice each, and the grouping and treatment were as follows:
[0106] Control group: 50 μL of PBS buffer was injected peritumorally.
[0107] Comparative Example 1: BP-PEG group, 50 μL of BP-PEG hydrogel was injected peritumorally. The hydrogel was prepared according to the method in Example 1.
[0108] Comparative Example 2: DOX group, 50 μL of DOX hydrogel was injected peritumorally. The hydrogel was prepared according to the method in Example 1.
[0109] Example group: BP-DOX-PEG group, 50 μL of BP-DOX-PEG hydrogel was injected peritumorally. The hydrogel was prepared according to the method in Example 1. 24 hours after injection, the injection site was irradiated with 808 nm near-infrared light for 10 minutes. This operation was performed every 3 days.
[0110] 3. Test Methods and Results
[0111] (1) Evaluation of tumor growth inhibition effect
[0112] During the experiment, the long and short diameters of the tumor were measured daily using vernier calipers, and the tumor volume was calculated. After the treatment, the nude mice were euthanized, and the tumor tissue was dissected and weighed.
[0113] The test results are shown in Figures 5a and 5b.
[0114] Figure 5a is a physical image of the tumor tissue. The figure shows that the tumor volume of the embodiment group of the present invention, namely the BP-DOX-PEG group, is the smallest, followed by the DOX group in comparative example group 2, and the tumor volume of comparative example group 1 (BP-PEG group) and control group (Control group) is the largest.
[0115] Figure 5b shows the tumor inhibition rate statistics. Based on the final tumor weight, the tumor inhibition rate of the embodiment group of the present invention is as high as 81.73%, which is significantly higher than that of comparative example group 2 and comparative example group 1.
[0116] (2) Detection of tumor cell proliferation activity
[0117] The dissected tumor tissue was embedded in paraffin, sectioned, and then subjected to Ki67 immunohistochemical staining.
[0118] Test results are shown in Figures 5c and 5g.
[0119] Figure 5c shows the quantitative statistics of Ki67 expression. The percentage of Ki67 positive expression area in the embodiment group of the present invention is 8%, and in other groups...
[0120] Figure 5g shows an immunohistochemical section of Ki67. The figure shows that the Ki67 positive staining, i.e., the light purple area, is the sparsest in the group of the present invention examples, which is consistent with the quantitative statistical results.
[0121] (3) Biosafety assessment
[0122] Weight change detection
[0123] During the experiment, the weight of the nude mice was measured regularly.
[0124] The test results are shown in Figure 5d.
[0125] The body weight of the nude mice in each group did not decrease significantly throughout the experiment. Control group 2, the DOX group, experienced slight fluctuations in the middle stage but quickly recovered; the body weight of the remaining groups showed a stable trend. This indicates that none of the treatment regimens, especially those in the embodiments of this invention, caused significant systemic toxicity to the nude mice.
[0126] Liver function test
[0127] After the experiment, blood was collected from nude mice, serum was separated, and the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected using a fully automated biochemical analyzer.
[0128] The test results are shown in Figures 5e and 5f.
[0129] Figures 5e and 5f show that the ALT and AST values in comparative group 2 reached 90 IU / L and 230 IU / L, respectively, indicating that DOX chemotherapy alone caused some damage to the liver.
[0130] The ALT and AST values of the BP-DOX-PEG in the embodiment of this invention were 60 IU / L and 150 IU / L, respectively, which were significantly lower than those in the comparative example group 2 (DOX). This indicates that the present invention effectively reduces the systemic toxic side effects of DOX on the liver through the strategy of local delivery with hydrogel and photothermal triggered release.
[0131] Histological analysis of major organs
[0132] Important organs such as the heart, liver, spleen, lungs, and kidneys of nude mice were collected and stained with H&E.
[0133] The test results are shown in Figure 5g.
[0134] The 5g sample is an H&E stained section. The image shows that, compared to the control group, no obvious pathological damage structures were observed in the major organs of each treatment group. Combined with liver function indicators, this further demonstrates the good in vivo biocompatibility of the product of this invention.
[0135] This embodiment demonstrates, using a tumor-bearing nude mouse model, the synergistic anti-tumor effect of the hydrogel provided in this invention, BP-mediated photothermal conversion, and DOX chemotherapy. Quantitative analysis of Ki67 expression showed that BP-DOX-PEG treatment significantly reduced tumor proliferation activity. Systematic biosafety assessment and detection of serum biochemical markers showed elevated liver enzyme levels in the DOX group, while the livers of nude mice treated with BP-DOX-PEG hydrogel maintained normal functional parameters comparable to the control group, with no pathological lesions observed. This in vivo experiment fully demonstrates that the BP-DOX-PEG intelligent hydrogel system successfully achieves a balance between highly effective anti-tumor activity and high biosafety.
[0136] The above embodiments are only for illustrating the technical concept of this utility model and should not be construed as limiting the scope of protection of this utility model. Any modifications made to the technical solution based on the technical concept proposed by this utility model shall fall within the scope of protection of this utility model.
Claims
1. A dynamic covalently bonded hydrogel of black phosphorus loaded with hepatocellular carcinoma drug, characterized in that, include: Black phosphorus, doxorubicin, four-armed polyethylene glycol benzaldehyde, and four-armed polyethylene glycol cyanopropane; wherein the four-armed polyethylene glycol benzaldehyde and four-armed polyethylene glycol cyanopropane are the matrix; The black phosphorus is used as a photothermal agent and drug carrier; the doxorubicin is a chemotherapy drug.
2. The dynamic covalently bonded hydrogel of black phosphorus-loaded liver cancer drug according to claim 1, characterized in that, The four-armed polyethylene glycol benzaldehyde and the four-armed polyethylene glycol cyanopropane form a hydrogel network through a covalent cross-linking reaction; the doxorubicin is loaded onto black phosphorus through physical adsorption and weak interaction, and the doxorubicin loaded on black phosphorus is uniformly dispersed on the hydrogel network by physical embedding.
3. The method for preparing the dynamic covalently bonded hydrogel of black phosphorus-loaded hepatocellular carcinoma drug as described in any one of claims 1 and 2, comprising the following steps: Step 1: Remove the four-armed polyethylene glycol benzaldehyde and four-armed polyethylene glycol cyanopropane powders and allow them to equilibrate to room temperature; Step 2: Prepare four-arm polyethylene glycol benzaldehyde solution and four-arm polyethylene glycol cyanopropane solution respectively. Mix the four-arm polyethylene glycol benzaldehyde solution and the four-arm polyethylene glycol cyanopropane solution in a predetermined ratio. Add auxiliary solvent dropwise during the mixing process. After mixing evenly, prepare hydrogel precursor solution. Step 3: Curing and crosslinking to prepare a dynamic covalently bonded hydrogel of black phosphorus loaded with liver cancer drugs; the hydrogel precursor solution is vortexed to allow it to react fully. Under neutral or near-neutral pH conditions, the four-armed polyethylene glycol benzaldehyde and four-armed polyethylene glycol cyanopropane in the hydrogel precursor solution are rapidly cured and crosslinked through covalent bonds to construct a hydrogel network, and black phosphorus and doxorubicin are embedded in the hydrogel network, thereby obtaining a dynamic covalently bonded hydrogel of black phosphorus loaded with liver cancer drugs.
4. The method for preparing the dynamic covalently bonded hydrogel of black phosphorus-loaded liver cancer drug according to claim 3, characterized in that, In step 2, the preparation of the four-arm polyethylene glycol benzaldehyde solution and the four-arm polyethylene glycol cyanopropane solution is selected from any of the following methods: dissolving the equilibrated four-arm polyethylene glycol benzaldehyde powder in a solution containing doxorubicin, and dissolving the four-arm polyethylene glycol cyanopropane powder in a solution containing black phosphorus; or dissolving the equilibrated four-arm polyethylene glycol benzaldehyde powder in a solution containing black phosphorus, and dissolving the four-arm polyethylene glycol cyanopropane powder in a solution containing doxorubicin.
5. The method for preparing the dynamic covalently bonded hydrogel of black phosphorus-loaded liver cancer drug according to claim 3, characterized in that, In step 2, the auxiliary solvent is selected from one or more of PBS, 0.9% NaCl, HEPES buffer, or deionized water.
6. The method for preparing the dynamic covalently bonded hydrogel of black phosphorus-loaded liver cancer drug according to claim 5, characterized in that, The volume of the auxiliary solvent is 0.1-0.5 mL.
7. The method for preparing the dynamic covalently bonded hydrogel of black phosphorus-loaded liver cancer drug according to claim 3, characterized in that, In step 2, the concentration of the hydrogel precursor solution is 10%-40%.
8. The method for preparing the dynamic covalently bonded hydrogel of black phosphorus-loaded liver cancer drug according to claim 3, characterized in that, In step 3, the four-arm polyethylene glycol benzaldehyde solution and the four-arm polyethylene glycol cyanopropane solution are mixed in a volume ratio of 3:7 to 7:3; the hydrogel is prepared at room temperature.
9. The method for preparing the dynamic covalently bonded hydrogel of black phosphorus-loaded liver cancer drug according to claim 3, characterized in that, The neutral or near-neutral pH value described in step 3 is 7.0-8.
0.
10. The use of the dynamic covalently bonded hydrogel of black phosphorus loaded with liver cancer drug as described in any one of claims 1-9 in the preparation of a liver cancer drug delivery system.