Hypoxia targeting hyaluronic acid nano delivery system as well as construction method and application thereof

Abstract

The invention relates to a hypoxia targeting hyaluronic acid nano delivery system and a construction method and application thereof, the construction method comprises the following preparation method: S1, dissolving 2-nitroimidazole and K2CO3 in dimethylformamide, carrying out high temperature alkali treatment, adding N-Boc-bromoethylamine, and carrying out high temperature reaction to obtain Boc-NIH; s2, dissolving the Boc-2-NIH in dichloromethane, adding trifluoroacetic acid for deprotection, and adjusting the pH value to be neutral, so as to obtain 2-NIH; s3, dissolving HA in deionized water, sequentially adding EDC and NHS to activate carboxyl, then adding 2-NIH, reacting at room temperature, and dialyzing to obtain a hypoxic targeting hyaluronic acid nano delivery carrier HA-NIH; s4, HA-NIH loads a drug, and the hypoxia targeted hyaluronic acid nano delivery system is formed. According to the invention, a bifunctional nano system with CD44 active targeting and hypoxia response release is successfully constructed, a dynamic regulation and control mechanism of HA molecular weight on a targeting-permeation-retention effect is provided, and a synergistic effect of tumor stem cell microenvironment specific response and drug space-time release is realized.

Description

Technical Field

[0001] This invention belongs to the field of nanomedicine delivery system technology, and relates to an oxygen-deficient targeted hyaluronic acid nanodelivery system, its construction method and application. Background Technology

[0002] Precision treatment of malignant tumors is a major challenge in the biomedical field. Colorectal cancer, a globally prevalent malignant tumor, faces a treatment bottleneck primarily due to recurrence, metastasis, and drug resistance mediated by colorectal cancer stem cells (CCSCs). CCSCs maintain their stemness characteristics through signaling pathways such as Wnt / β-catenin and exhibit inherent resistance to traditional chemotherapy drugs. Targeted elimination of CCSCs has become a core strategy for overcoming the treatment challenges of colorectal cancer.

[0003] As a core surface marker of CCSCs, the CD44 receptor's specific binding to hyaluronic acid (HA) provides an ideal pathway for targeted delivery. In existing technologies, HA-based nanocarriers mostly focus on single molecular weight design, lacking systematic research on the dynamic relationship of "molecular weight-targeting-retention", and generally have the following defects: (1) they do not integrate the tumor microenvironment response mechanism, and cannot achieve intelligent release of drugs in CCSC-rich areas; (2) they do not solve the problem of non-specific adsorption of HA in normal tissues; (3) it is difficult to balance the targeting binding strength and in vivo circulation stability. Summary of the Invention

[0004] The purpose of this invention is to overcome the problems of insufficient targeting specificity, poor tumor permeability, and difficulty in long-term retention in tumor areas in existing targeted delivery technologies, thereby providing a hypoxia-targeted hyaluronic acid nanodelivery system, its construction method, and its application.

[0005] The objective of this invention can be achieved through the following technical solutions: One of the technical solutions of the present invention is to provide a method for preparing a hypoxia-targeted hyaluronic acid nanocarrier, comprising the following steps: S1. Synthesis of Boc-2-NIH: 2-nitroimidazole and K2CO3 were dissolved in dimethylformamide (DMF), and then N-Boc-bromoethylamine (NBoc-BHA) was added. The mixture was reacted at high temperature and extracted to obtain Boc-2-NIH. S2. Synthesis of 2-NIH: The Boc-2-NIH obtained in step S1 was dissolved in dichloromethane, and trifluoroacetic acid (TFA) was added to remove protection. After adjusting the pH to neutral, 2-NIH was obtained by extraction. S3. Synthesis of HA-NIH: Hyaluronic acid (HA) was dissolved in deionized water, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were added sequentially to activate the carboxyl group. Then, 2-NIH obtained in step S2 was added, and the reaction was carried out at room temperature. Dialysis was then performed to obtain the hypoxia-targeted hyaluronic acid nanodelivery carrier HA-NIH.

[0006] In some specific embodiments, in step S1, the mass ratio of 2-nitroimidazole, K2CO3 and 6-(Boc-amino)bromohexane is 1:(1.2~1.9):2.6; The high-temperature reaction was carried out at a temperature of 80℃ for a time of (4~8) h.

[0007] In some specific embodiments, in step S2, the ratio of Boc-2-NIH to trifluoroacetic acid is 1 mmol: 1 mL, and the de-reaction time is (3~5) h.

[0008] In some specific embodiments, in step S3, the mass ratio of hyaluronic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and 2-NIH is 1:(1.9~2.0):(1.15~1.2):(2.1~2.2). The molecular weight of the hyaluronic acid is 3 kDa to 30 kDa.

[0009] In some specific embodiments, in step S3, the reaction time at room temperature is (20~24) h, and the temperature is 15℃~25℃; The dialysis process is as follows: methanol-water mixture and pure water are used sequentially, and the dialysis time is greater than or equal to 2 days. The volume ratio of methanol to water in the methanol-water mixture is 1:1.

[0010] The second technical solution of the present invention is to provide a hypoxia-targeted hyaluronic acid nanodelivery system, comprising a hypoxia-targeted hyaluronic acid nanodelivery carrier and a compound to be acted upon loaded thereon, wherein the hypoxia-targeted hyaluronic acid nanodelivery carrier is prepared by the preparation method described in one of the above technical solutions.

[0011] In some specific embodiments, the mass ratio of the hypoxia-targeted hyaluronic acid nanodelivery carrier to the compound to be acted upon is (1~1000):1.

[0012] In some specific embodiments, the preparation method of the hypoxia-targeted hyaluronic acid nanodelivery system includes the following steps: S401. Dissolve HA-NIH in an aqueous solution of dimethyl sulfoxide to obtain an HA-NIH solution; S402. Add the drug to be used dropwise to the HA-NIH solution in step S401 to obtain a mixed solution; S403. The mixed solution from step S402 is added dropwise to water and mixed, and then dialyzed to obtain an oxygen-deficient targeted hyaluronic acid nanodelivery system.

[0013] In some specific embodiments, in step S401, the volume ratio of dimethyl sulfoxide to water in the dimethyl sulfoxide aqueous solution is 4:1.

[0014] This invention also provides a curcumin-loaded hypoxia-targeting hyaluronic acid nanodelivery system, which achieves specific clearance of tumor stem cells through a synergistic effect of HA-CD44 receptor-mediated active targeting and nitroreductase (NTR)-triggered hypoxia-responsive release. This system can intelligently release drugs in the hypoxic tumor microenvironment, significantly improving drug penetration depth in the tumor core region, and downregulates stem cell markers such as LGR5 and Oct4 by inhibiting the Wnt / β-catenin signaling pathway.

[0015] The third technical solution of the present invention is to provide an application of the hypoxia-targeted hyaluronic acid nanodelivery system as described in the second technical solution above in the preparation of antitumor drugs.

[0016] In some specific embodiments, the compound to be acted upon is an antitumor compound.

[0017] In some specific embodiments, the compound to be acted upon includes curcumin.

[0018] In some specific embodiments, the tumor includes colon cancer, breast cancer, lung cancer, stomach cancer, gallbladder cancer, liver cancer, pancreatic cancer, and drug-resistant CD44-overexpressing solid tumors.

[0019] This invention successfully combines nitroimidazole units with HA molecular weight regulation technology to construct a bifunctional nanosystem that combines active CD44 targeting and hypoxia-responsive release. This delivery system has a dynamic regulation mechanism of HA molecular weight on the "targeting-penetration-retention" effect, a synergistic design of tumor stem cell microenvironment-specific response and drug spatiotemporal release, and a multi-level synergistic therapeutic strategy of nanocarrier and chemotherapeutic drugs.

[0020] Compared with the prior art, the present invention has the following advantages: (1) Achieving synergistic optimization of targeting, penetration, and drug release: By regulating hyaluronic acid carriers of different molecular weights (3-30 kDa) combined with CD44 active targeting and nitroimidazole hypoxia response units, the specific recognition of tumor stem cells (CCSCs), deep tumor penetration (particle size < 200 nm, positive charge), and nitroreductase (NTR) triggered controlled release capabilities are simultaneously enhanced, overcoming the bottlenecks of insufficient targeting, poor permeability, and uncontrollable drug release in traditional systems. This upper size limit ensures that the particles can cross the tumor vascular endothelial space, while the lower limit (usually > 10 nm) avoids excessive renal clearance and maintains the EPR effect. The positively charged delivery system can enhance tissue retention and promote penetration behavior through electrostatic interactions.

[0021] (2) Overcoming the drug resistance barrier of tumor stem cells: The present invention uses a hypoxia-targeted hyaluronic acid nanodelivery system loaded with curcumin to efficiently deliver curcumin to the CCSCs enrichment area. By continuously inhibiting the Wnt / β-catenin / NF-κB / STAT3 pathway and downregulating stem cell markers such as LGR5 / Oct4 / Nanog / Sox2, and in conjunction with the spatiotemporal drug release characteristics of the carrier itself, it significantly reverses the inherent drug resistance of CCSCs.

[0022] (3) Establish a multi-level synergistic treatment paradigm: Based on the intelligent response characteristics of the hypoxia-targeted hyaluronic acid nanodelivery system of this invention, a spatiotemporal synergistic strategy is constructed with chemotherapy drugs (such as 5-FU) (5-FU clears proliferating cancer cells, and this system targets resting CCSCs) to achieve complete tumor suppression and reduce the risk of recurrence; at the same time, it overcomes the clinical obstacles of poor water solubility and low bioavailability of curcumin, and provides a universal drug resistance reversal solution for CD44-high expression solid tumors (such as colon cancer and breast cancer). Attached Figure Description

[0023] Figure 1 The hypoxia-nitroreductase-responsive nitroimidazole-modified hyaluronic acid delivery carrier HA-NIH prepared in Example 1 was characterized. Figure a shows a schematic diagram of the synthesis steps of HA-NIH; Figure b shows the 1H NMR spectrum of Boc-2-NIH; Figure c shows the 1H NMR spectrum of 2-NIH; Figure d shows the 1H NMR spectrum of HA (7 kDa); and Figure e shows the 1H NMR spectrum of HA-NIH.

[0024] Figure 2Characterization of the different molecular weight (3 kDa, 7 kDa, 10 kDa and 30 kDa) nanodelivery systems (ICG@HN) prepared in Example 2. Figures a and b show the SEM images of ICG@HN (3 kDa), ICG@HN (7 kDa), ICG@HN (10 kDa), and ICG@HN (30 kDa), respectively; Figure e shows the particle size distribution of ICG@HN (3 kDa), ICG@HN (7 kDa), ICG@HN (10 kDa), and ICG@HN (30 kDa); Figure f shows the Zeta potentials of HA, ICG, and ICG@HN systems with different molecular weights (3 kDa, 7 kDa, 10 kDa, 30 kDa); Figure g shows the UV-Vis absorption spectra of ICG solutions with different concentrations (2, 3, 4, 6, 8, 10 μg / mL); Figure h shows the UV-Vis absorption spectra of HN, ICG, and ICG@HN systems with different molecular weights (3 kDa, 7 kDa, 10 kDa, 30 kDa).

[0025] Figure 3 The uptake efficiency of the nanodelivery system (ICG@HN) with different molecular weights (3 kDa, 7 kDa, 10 kDa and 30 kDa) prepared in Example 2 in CD44-overexpressing tumor cells (CT26).

[0026] Figure 4 Characterization of the hyaluronic acid (HA)-based tumor microenvironment-responsive nanodelivery system (Cur@HN) prepared in Example 3. Figure a shows the SEM image of Cur@HN, Figure b shows the TEM image of Cur@HN, Figure c shows the DLS image of Cur@HN, Figure d shows the particle size change of Cur@HN before and after Cur loading: HN before loading, Cur@HN after Cur loading; Figure e shows the UV-Vis absorption spectra of Cur@HN before and after Cur loading: HN before loading, Cur@HN after Cur loading; Figure f shows the Fourier transform infrared spectra of HA, HA-NIH, Cur, and Cur@HN.

[0027] Figure 5 This study investigated the cell death mechanism of the hyaluronic acid (HA)-based tumor microenvironment-responsive nanodelivery system (Cur@HN) prepared in Example 3. Figure a shows laser confocal microscopy images of cell death in the Control, HN, Cur, and Cur@HN groups under hypoxic and normoxic environments; Figure b shows apoptosis in the Control, HN, Cur, and Cur@HN groups under hypoxic and normoxic environments, as shown in Figure a.

[0028] Figure 6This study investigated the inhibitory effect of the hyaluronic acid (HA)-based tumor microenvironment-responsive nanodelivery system (Cur@HN) prepared in Example 3 on the proliferation of colon cancer stem cells. Figure a shows the Western blot results after treatment in the Control group, HN group, Cur group, and Cur@HN group; Figures b and c show the quantitative analysis of LGR5, Oct4, Nanog, and Sox2 protein expression after treatment in the Control group, HN group, Cur group, and Cur@HN group.

[0029] Figure 7 This study investigated the in vivo antitumor effect of the hyaluronic acid (HA)-based tumor microenvironment-responsive nanodelivery system (Cur@HN) prepared in Example 3 in a SW480 colon cancer-bearing BALB / c nude mouse model. Figure a shows the tumor growth fluorescence images of tumor-bearing nude mice in the Control group, HN group, Cur group, Cur@HN group, 5-Fu group, and 5-FU + Cur@HN group at 0, 5, 10, and 15 days; Figure b shows the weight changes of tumor-bearing nude mice in each group from 0 to 16 days; Figure c shows the tumor volume growth curves of tumor-bearing nude mice in each group from 0 to 16 days; Figure d shows the tumor photographs of tumor-bearing nude mice in each group from 0 to 16 days; Figure e shows the tumor weight of tumor-bearing nude mice in each group from 0 to 16 days.

[0030] Figure 8 This is a schematic diagram of the present invention. Detailed Implementation

[0031] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0033] Unless otherwise specified, all embodiments and optional embodiments of this application can be combined to form new technical solutions. Unless otherwise specified, all technical features and optional technical features of this application can be combined to form new technical solutions.

[0034] Unless otherwise specified, all steps in this application may be performed sequentially or randomly, but sequentially is preferred.

[0035] Example 1 This embodiment provides a method for preparing the hypoxia-targeted hyaluronic acid nanocarrier HA-NIH, including the following steps: (1) Synthesis of Boc-2-NIH: 2-nitroimidazole (3 mmol, 340 mg) and K2CO3 (3 mmol, 414 mg) were dissolved in DMF (5 mL), and N-Boc-bromoethylamine (NBoc-BHA) (884 mg) was added. o After reacting at C for 8 hours, the product was washed three times with pure water and extracted with ethyl acetate, and then concentrated by vacuum distillation to obtain Boc-2-NIH. (2) Synthesis of 2-NIH: Boc-2-NIH (1 mmol) was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (1 mL) was added to deprotect the reaction for 4 h. After adjusting the pH to neutral, the mixture was washed three times with pure water and extracted with ethyl acetate. The mixture was then concentrated by vacuum distillation to obtain 2-NIH. (3) Synthesis of HA-NIH: Hyaluronic acid (HA, 100 mg, 0.25 mmol) with molecular weights of 3 kDa, 7 kDa, 10 kDa and 30 kDa respectively was dissolved in deionized water (5 mL), and EDC (192 mg) and NHS (115 mg) were added sequentially to activate the carboxyl group. Then 2-NIH (212 mg) was added and reacted at room temperature of 25°C for 24 hours. The mixture was dialyzed with methanol-water mixture (methanol to water volume ratio of 1:1) and pure water for 2 days. After freeze drying, the hypoxia-targeted hyaluronic acid nano-delivery carrier HA-NIH, abbreviated as HN, was obtained.

[0036] Figure 1 (a) is a schematic diagram of the synthesis steps of HN. Figure 1 (b)-(e) are the proton NMR spectra of Boc-2-NIH, 2-NIH, HA (7 kDa), and HN (7 kDa HA), respectively. 1 1H NMR nuclear magnetic resonance spectroscopy analysis confirmed the successful synthesis of Boc-2-NIH, 2-NIH and HN.

[0037] Example 2 Based on the hypoxia-targeted hyaluronic acid nanocarrier HN constructed in Example 1, nanodelivery systems for hyaluronic acid with different molecular weights (3 kDa, 7 kDa, 10 kDa, and 30 kDa) were constructed, including the following preparation steps: (1) Dissolve 20 mg of HN completely in 2 mL of a mixed solvent of dimethyl sulfoxide and water (the volume ratio of dimethyl sulfoxide to water is 4:1) to obtain an HN solution.

[0038] (2) Dissolve 2 mg of ICG (indocyanine green) in 0.1 mL of water to prepare a 20 mg / mL ICG solution, and add it dropwise to the HN solution according to the loading amount, and stir the reaction for 3 hours.

[0039] (3) Slowly add the obtained solution to 8 mL of water and stir for 24 hours; (4) The product obtained above was then dialyzed sequentially with a mixture of methanol and water (methanol to water volume ratio 1:1) and pure water. The dialyzed solution was then subjected to 4... o By storing at C, we obtained ICG@HN nanodelivery systems in which ICG is encapsulated by hyaluronic acid of different molecular weights.

[0040] Figure 2 (a)- Figure 2 (d) SEM images of nanodelivery systems (ICG@HN) loaded with 2 µg / mL ICG and hyaluronic acid molecular weights of 3 kDa, 7 kDa, 10 kDa and 30 kDa, respectively.

[0041] Figure 2 (e) shows the particle size distribution of the ICG@HN nanoparticles with hyaluronic acid molecular weights of 3 kDa, 7 kDa, 10 kDa, and 30 kDa. The results show that the hydrodynamic diameters of the nanoparticles are 113 nm (3 kDa), 122 nm (7 kDa), 135 nm (10 kDa), and 167 nm (30 kDa), respectively, indicating that the particle size increases significantly with increasing HA molecular weight.

[0042] Figure 2 (f) shows the Zeta potentials of HA (7 kDa), HN (7 kDa HA), ICG (2 µg / mL), and ICG@HN with molecular weights of hyaluronic acid of 3 kDa, 7 kDa, 10 kDa, and 30 kDa. The results show that free HA exhibits a strong negative charge (-25.8 mV) due to carboxyl dissociation, while free ICG exhibits a positive charge (+9.9 mV) due to amino protonation. After loading ICG (2 µg / mL), the potential value of ICG@HN falls between the two, indicating that ICG binds to HA through electrostatic interactions. As the molecular weight of HA increases from 3 kDa to 30 kDa, the potential value of ICG@HN gradually increases, indicating that the loading capacity of ICG for high molecular weight HA is lower than that for low molecular weight HA, further verifying the successful loading of ICG.

[0043] Figure 2 (g) shows the UV-Vis absorption spectra of ICG@HN loaded with different concentrations of ICG (2, 3, 4, 6, 8, 10 μg / mL) and with a hyaluronic acid molecular weight of 7 kDa. Figure 2(h) shows the UV-Vis absorption spectra of HN (7 kDa HA), ICG (2 µg / mL), and ICG@HN with molecular weights of 3 kDa, 7 kDa, 10 kDa, and 30 kDa, respectively. The results show that the characteristic peak intensity of free ICG at 780 nm is linearly correlated with concentration; under the same concentration gradient, the absorption peak of ICG@HN at 780 nm exhibits a significant blue shift. This blue shift is attributed to the aggregation effect of ICG molecules, further confirming the stable encapsulation of ICG in nanoparticles.

[0044] The prepared hyaluronic acid molecules with molecular weights of 3 kDa, 7 kDa, 10 kDa, and 30 kDa were characterized as follows: Validation of the molecular weight-dependent cell uptake kinetics and mechanism of action of HA in mouse colon cancer CT26 cells: Cell uptake of the four materials mentioned above was analyzed by flow cytometry. CT26 cells were seeded in 12-well plates and incubated for 12 h to allow cell adhesion. After washing the cells three times with PBS, ICG@HN (3 kDa), ICG@HN (7 kDa), ICG@HN (10 kDa), ICG@HN (30 kDa), or ICG (2 µg / mL) were added, respectively, and a blank control group was set up. After incubation for 0, 1, 2, and 4 h, the supernatant was discarded, and the cells were washed three times with PBS to remove unuptaken materials. The cells were digested, filtered through a sieve, and transferred to flow cytometry tubes. Fluorescence intensity was detected by flow cytometry to assess cell uptake.

[0045] The results showed that ICG ( Figure 3 The fluorescence signal of a) reached its peak at 2 h and then did not increase further; ICG@HN (3 kDa) and ICG@HN (7 kDa) Figure 3 The signal value of b-3c) reached its peak at 3 hours and then stopped increasing; ICG@HN (10 kDa) and ICG@HN (30 kDa) Figure 3 The signal value of d-3e) continued to increase until 4 h, indicating that the larger the molecular weight of HA, the weaker its binding ability to CD44. Figure 3 As shown in f, after 4 h of incubation, the cell uptake of the four ICG@HN materials was significantly higher than that of the ICG group, indicating that HA encapsulation can effectively improve the cell uptake efficiency of the probe; and as the molecular weight of HA increases, the closer the fluorescence signal value is to that of the ICG group, the lower the cell uptake efficiency of the material.

[0046] Example 3 Based on the hypoxia-targeted hyaluronic acid nanocarrier HN constructed in Example 1, a curcumin (Cur) delivery system Cur@HN was constructed, comprising the following steps: 20 mg HN (7 kDa HA) was completely dissolved in 10 mL of a mixed solvent of DMSO:water (4:1, v / v). 0.1 mL of Cur solution (20 mg / mL) was added dropwise to the above solution and stirred for 3 h. Subsequently, the solution was slowly added dropwise to 8 mL of water and the reaction continued for 24 h. The reaction product was dialyzed sequentially with a methanol:water (1:1, v / v) mixture for 24 h and with pure water for 48 h (the molecular weight cutoff of the dialysis bag was 7000 kDa), and the dialyzed solution was placed in a 4-degree Celsius environment. o Save as C.

[0047] Figure 4 (a) and Figure 4 (b) SEM and TEM images of Cur@HN, respectively. The results show that Cur@HN is a regular sphere with a particle size distribution of 100 ± 5.2 nm. Figure 4 (c) is the DLS diagram of Cur@HN, with a hydration dynamic diameter of 126.3 ± 3.8 nm. Figure 4 (d) Showing the particle size change of Cur@HN before and after loading with Cur: Compared with the unloaded blank material, the hydrated particle size of Cur@HN was significantly reduced, which is attributed to the increased hydrophobicity of nanoparticles and the shrinkage caused by the enhanced hydrophobic interaction due to drug loading. Figure 4 (e) shows the UV-Vis absorption spectra of Cur@HN before and after loading Cur: the complex shows a nitro characteristic absorption peak at 325 nm (originating from the HA-NIH support), and the characteristic absorption peak of Cur is significantly blue-shifted to 420 nm; this spectral shift indicates that π-π stacking interaction occurs between the drug molecule and the polymer support. Figure 4 (f) Fourier transform infrared spectra of HA, HA-NIH, Cur, and Cur@HN: at 3430 cm⁻¹ -1 A broadened OH stretching vibration peak (ν) was observed at [location]. O-H ), 1655 cm -1 An amide bond NH bending vibration peak (δ) appears at this location. N-H Both of these results demonstrate the integrity of the chemical structure of Cur@HN. All of the above results confirm the successful synthesis of Cur@HN.

[0048] The prepared Cur@HN was characterized as follows: (1) Evaluation of the cytotoxic effect of the hypoxia-responsive nanodelivery system (Cur@HN) on the SW480 colon cancer cell line: After SW480 cells were seeded and cultured for 24 hours, they were incubated for 12 hours under normoxic or hypoxic conditions with medium containing Cur@HN (10 μM), Cur (10 μM), HN, or DMEM (control) alone. After incubation, the cells were washed three times with PBS. Live / dead cell staining: Cells were co-incubated with Calcein-AM and propidium iodide (PI) for 20 minutes, and the green / red fluorescence was observed under a confocal microscope. Apoptosis detection by flow cytometry: Cells were digested with trypsin, collected by centrifugation, resuspended in binding buffer, mixed with Annexin-FITC, and then PI dye was added. The cells were incubated in the dark for 20 minutes, and the cell state was analyzed by flow cytometry.

[0049] like Figure 5 As shown in (a), the live / dead staining experiment revealed that the Cur@HN treatment group (Hypoxia + Cur@HN) exhibited significant PI red fluorescence (dead cells) under hypoxic conditions, with fluorescence intensity significantly higher than that of the free Cur group, the normoxic Cur@HN group, and the control group. This indicates that the nanosystem can effectively release drugs and disrupt cell membrane integrity in a simulated tumor hypoxic microenvironment. Further flow cytometry analysis using Annexin V-FITC / PI dual staining revealed (… Figure 5 (b) The total apoptosis rate in the Hypoxia + Cur@HN group was significantly higher than that in the normoxic Cur@HN group, and the proportion of late-stage apoptotic cells was significantly higher than in other experimental groups, confirming that this delivery system can enhance the apoptosis-inducing effect. The mechanism may involve triggering mitochondrial membrane potential depolarization and caspase protease cascade activation. The experimental results further validated the hypoxia-responsive characteristics of Cur@HN and its ability to precisely regulate drug release in hypoxic tumor regions.

[0050] (2) Evaluation of the inhibitory effect of Cur@HN on colon cancer stem cells: After SW480 colon cancer cells were seeded and cultured for 24 hours, they were incubated for 12 hours under normoxic or hypoxic conditions in media containing Cur@HN (10 μM), Cur (10 μM), HN, or DMEM (blank control) alone. After incubation, the cells were digested and collected by centrifugation. The cell pellet was lysed on ice with RIPA lysis buffer to obtain total protein, which was separated by gel electrophoresis and then transferred to a membrane. The protein expression levels of colon cancer stem cell markers LGR5, Oct4, Nanog, and Sox2 were detected by Western blotting.

[0051] Quantitative analysis results ( Figure 6The results showed that, compared with the control group, the protein expression levels of LGR5, Oct4, Nanog, and Sox2 in the Cur@HN treatment group were significantly downregulated (by 62.3%, 27.8%, 53.2%, and 79.5%, respectively), indicating that Cur@HN effectively inhibits the self-renewal and proliferation of colon cancer stem cells through multi-target regulation. No significant inhibition of protein expression was observed in the free Cur group. Based on existing research, this phenomenon may stem from the inherent physicochemical defects of Cur: its high hydrophobicity leads to poor water solubility and low bioavailability, severely limiting cellular uptake efficiency and thus failing to effectively inhibit colon cancer stem cells. In contrast, the Cur@HN nanodelivery system achieves highly efficient targeted delivery via CD44 receptor-mediated endocytosis through the specific binding of surface hyaluronic acid to the tumor CD44 receptor, with significantly higher cellular uptake efficiency than the free drug. In conclusion, the results confirm that Cur@HN can effectively inhibit the proliferation of colon cancer stem cells.

[0052] (3) Evaluate the in vivo antitumor efficacy of Cur@HN: Construction of a subcutaneous tumor model: SW480 cells were digested, centrifuged, and resuspended in PBS solution, adjusting the cell concentration to 1 × 10⁻⁶. 6 mL -1 (Cells were stored on ice). The cell suspension (100 μL) was then injected subcutaneously into the thigh of mice. Tumor growth was continuously monitored until the tumor volume reached approximately 100 mm². 3 Subsequent in vivo treatment experiments were conducted. SW480 tumor-bearing mice were divided into 6 groups (n = 3) and injected via tail vein with 100 μL of saline, HN (9 mg / kg), Cur (3 mg / kg), Cur@HN (12 mg / kg), 5-FU (12 mg / kg), and Cur@HN + 5-FU (12 mg / kg), respectively. Administered the drugs every two days, while simultaneously measuring mouse body weight and tumor volume. Tumor bioluminescence imaging was performed every five days. After 14 days of treatment, the mice were sacrificed, and the tumors were harvested for further analysis.

[0053] Figure 7 (a) The growth of tumors in mice was dynamically monitored using a small animal in vivo imaging system. Figure 7 (b) Body weight changes showed that the weight growth rate of mice in each experimental group was basically the same as that in the control group, and the fluctuation range was controlled within ±5% of the initial body weight, which confirmed the in vivo safety of Cur@HN and showed that the material system has good biocompatibility. Figure 7(c) Tumor growth kinetics results showed that the tumor proliferation rate in the HN group was not significantly different from that in the control group, indicating that the carrier material itself had no anti-tumor activity; the tumor inhibition effect in the Cur group was limited due to the low bioavailability of curcumin; the tumor volume in the Cur@HN group was reduced by 58.3% compared with the control group through the EPR effect and CD44 receptor-mediated active targeting; although the 5-FU monotherapy group could inhibit tumors in the rapid proliferation phase, drug resistance escape occurred on the 6th day after treatment; the combination therapy group (Cur@HN + 5-FU) adopted a time-sequential dosing strategy (5-FU clears differentiated cancer cells, Cur@HN targets drug-resistant stem cells), which achieved complete cessation of tumor growth and no recurrence was observed. Figure 7 (d) and Figure 7 (e) The results showed that the tumor volume in the Cur@HN + 5-FU group was significantly smaller than that in the control group, which was basically consistent with the tumor growth kinetics curve monitored by in vivo imaging. These results indicate that Cur@HN synergistically with 5-FU has good anti-tumor efficacy.

[0054] In this embodiment, a tumor microenvironment-responsive nanodelivery system (Cur@HN) based on hyaluronic acid (HA) was administered intravenously to an SW480 tumor-bearing mouse model as a formulation targeting colon cancer stem cells. In the hypoxic tumor microenvironment, Cur@HN responded to overexpressed nitroreductase (NTR), achieving intelligent controlled release of curcumin. Its HA shell, through CD44 receptor-mediated active targeting, significantly improved the drug accumulation efficiency at the tumor site, effectively overcoming the problem of poor tumor penetration of traditional chemotherapy drugs. In vitro experiments confirmed that the released curcumin significantly inhibited the Wnt / β-catenin signaling pathway, downregulating the expression of stem cell markers such as LGR5, Oct4, Nanog, and Sox2. In the in vivo model, although Cur@HN monotherapy reduced tumor volume by more than 50%, its effectiveness was limited by tumor heterogeneity. By combining 5-fluorouracil (5-FU) with a synergistic treatment strategy, 5-FU preferentially eliminates proliferating cancer cells, while Cur@HN targets and eliminates drug-resistant stem cell populations, ultimately achieving complete inhibition of tumor growth without observing systemic toxicity. As an innovative nanodelivery strategy, Cur@HN combines microenvironment-responsive properties with the spatiotemporal synergistic effect of chemotherapeutic drugs, providing a new approach to overcoming tumor stem cell resistance and comprehensive treatment of solid tumors.

[0055] Curcumin is a multi-target natural anti-tumor compound whose mechanism of action includes inhibiting key cancer signaling pathways such as NF-κB and STAT3, downregulating tumor stem cell (CCSC) surface markers (such as CD133, CD44, and ALDH1), and inducing CCSC differentiation and apoptosis. However, curcumin's inherent poor water solubility and low bioavailability (oral bioavailability less than 1%) severely limit its clinical application.

[0056] The hyaluronic acid (HA)-based nanodelivery system developed in this invention effectively improves the solubility of curcumin through its excellent water solubility. Furthermore, by specifically binding to the CD44 receptor highly expressed on the surface of CCSCs, it achieves active targeted drug delivery and efficient accumulation in tumor regions. The rationally designed nanocarriers, through optimized particle size (< 200 nm) and surface charge (positive charge), enhance penetration into the dense tumor matrix and adhesion to the CCSC cell membrane. The nanoparticle-mediated sustained-release properties can continuously inhibit key signaling pathways in CCSCs (such as Wnt / β-catenin), helping to overcome their drug resistance.

[0057] The hyaluronic acid-based curcumin nanodelivery system targeting CCSCs developed in this embodiment significantly enhances the inhibitory effect of curcumin on the stemness of CCSCs (manifested as downregulation of stem cell markers such as LGR5, Oct4, Nanog, and Sox2), and effectively overcomes the main obstacles to its clinical application. This strategy, by improving drug solubility, enhancing tumor-targeted enrichment, and achieving controlled sustained release, provides new directions and theoretical support for the clinical application of curcumin and the development of nanomedicines targeting tumor stem cells.

[0058] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A method for preparing an oxygen-deficient targeted hyaluronic acid nanocarrier, characterized in that, Includes the following steps: S1. Synthesis of Boc-2-NIH: 2-nitroimidazole and K2CO3 were dissolved in dimethylformamide, N-Boc-bromoethylamine was added, the reaction was carried out at high temperature, and Boc-2-NIH was obtained by extraction. S2. Synthesis of 2-NIH: The Boc-2-NIH obtained in step S1 was dissolved in dichloromethane, and trifluoroacetic acid was added to remove protection. After adjusting the pH to neutral, 2-NIH was obtained by extraction. S3. Synthesis of HA-NIH: Hyaluronic acid was dissolved in deionized water, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added sequentially to activate the carboxyl group. Then, 2-NIH obtained in step S2 was added, and the reaction was carried out at room temperature. Dialysis was then performed to obtain the hypoxia-targeted hyaluronic acid nanodelivery carrier HA-NIH.

2. The method for preparing the hypoxia-targeted hyaluronic acid nanocarrier according to claim 1, characterized in that, In step S1, the mass ratio of 2-nitroimidazole, K2CO3 and N-Boc-bromoethylamine is 1:(1.2~1.9):2.6; The high-temperature reaction was carried out at a temperature of 80℃ for a time of (4~8) h.

3. The method for preparing the hypoxia-targeted hyaluronic acid nanocarrier according to claim 1, characterized in that, In step S2, the ratio of Boc-2-NIH to trifluoroacetic acid was 1 mmol: 1 mL, and the de-reaction time was (3~5) h.

4. The method for preparing the hypoxia-targeted hyaluronic acid nanocarrier according to claim 1, characterized in that, In step S3, the mass ratio of hyaluronic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and 2-NIH is 1:(1.9~2.0):(1.15~1.2):(2.1~2.2). The molecular weight of the hyaluronic acid is 3 kDa to 30 kDa.

5. The method for preparing the hypoxia-targeted hyaluronic acid nanocarrier according to claim 1, characterized in that, In step S3, the reaction time at room temperature is (20~24) h, and the temperature is 15℃~25℃; The dialysis process is as follows: methanol-water mixture and pure water are used sequentially, and the dialysis time is greater than or equal to 2 days. The volume ratio of methanol to water in the methanol-water mixture is 1:

1.

6. A hypoxia-targeted hyaluronic acid nanodelivery system, characterized in that, The invention includes a hypoxia-targeted hyaluronic acid nanocarrier and the compound loaded thereon, wherein the hypoxia-targeted hyaluronic acid nanocarrier is obtained by the preparation method described in any one of claims 1-5.

7. The hypoxia-targeted hyaluronic acid nanodelivery system according to claim 6, characterized in that, The mass ratio of the hypoxia-targeted hyaluronic acid nanocarrier to the compound to be acted upon is (1~1000):

1.

8. The application of the hypoxia-targeted hyaluronic acid nanodelivery system as described in claim 6 or 7 in the preparation of antitumor drugs.

9. The application according to claim 8, characterized in that, The compound to be acted upon includes curcumin.

10. The application according to claim 8, characterized in that, The tumors include colon cancer, breast cancer, lung cancer, stomach cancer, gallbladder cancer, liver cancer, and pancreatic cancer.

Images