Heparanase and hypoxia dual-response amphiphilic nano-carrier and application thereof

Abstract

The invention discloses a heparanase and hypoxia dual-response amphiphilic nano-carrier and application thereof, an imidazole compound NI and heparan sulfate HS are coupled to form an HS-NI compound, and then a TET target head is modified to the HS-NI compound to form a TET-HS-NI amphiphilic nano-carrier. Under high-level HPSE, HS can be responsively cut, and nitro of NI can be responsively reduced in a tumor hypoxia environment, so that the amphipathy of the micelle carrier HS-NI can be effectively damaged in the tumor environment, and the chemotherapeutic drug realizes double-response release under the HPSE and hypoxia conditions of an ATC tumor microenvironment.

Description

Technical Field

[0001] The present invention relates to the technical field of drug carrier production, and particularly relates to a heparanase and hypoxia dual-responsive amphiphilic nanocarrier and its application. Background Art

[0002] Heparanase (HPSE) is overexpressed in various tumors. The abnormal high expression of HPSE in tumors is related to its malignancy. Heparan sulfate (HS) is the cleavage substrate of HPSE, and HPSE is the only enzyme in mammals that can degrade heparan sulfate. Through literature research and data query, there are few reports on HPSE-responsive nanomaterials. Among them, only individual reports use heparan sulfate-calcitriol conjugates to prepare HPSE-responsive prodrugs for the treatment of breast cancer.

[0003] There are also reports that use nanostructured lipid carriers with a positively charged core and solid lipid nanocarriers coated with heparin to generate a negative surface charge. When encountering heparanase, heparin undergoes enzymatic cleavage, thus emphasizing the acetyl heparase-reactive charge conversion to overcome intestinal barriers and enhance cell uptake (Int J Pharm. 2024;651:123817. DOI:10.1016 / j.ijpharm.2024.123817.).

[0004] There is little existing literature and data on HPSE-responsive delivery systems, and the existing work still has many defects and deficiencies. On the one hand, carriers are constructed based on the idea of prodrugs. The release of prodrugs requires complete cleavage of the carrier to play an antitumor role. It is difficult to ensure the effective release of DOX only relying on HPSE response. On the second hand, based on the charge principle of HPSE, the quaternary ammonium surface of cetyltrimethylammonium in the lipid core is positively charged and effectively interacts with the negatively charged cell membrane, which may lead to an increase in cell uptake after HPSE cleaves heparin on the surface of the nanocarrier. The heparin surface decoration is negatively charged, which promotes the stability of the carrier and reduces mucus interaction during the process of reaching epithelial cells. However, its therapeutic effect has not been verified on animal models, and the results are limited and unconvincing.

[0005] Currently, the research reports on drug delivery systems for delivering DOX to treat anaplastic thyroid cancer (ATC) are extremely limited, and the existing reports have many drawbacks, such as being limited to in vitro evaluation, low drug loading, and adverse reactions caused by in vivo accumulation. There are few reports on systematic in vivo studies with ideal results. In addition, there are even fewer reports on designing delivery systems based on the background of high expression of HPSE in the tumor microenvironment. Summary of the Invention

[0006] The object of the present invention is to provide a heparanase and hypoxia dual-responsive amphiphilic nanocarrier and its application. Under high levels of HPSE, HS can be responsive to cleavage, and the nitro group of NI can undergo responsive reduction in the tumor hypoxic environment. Therefore, the amphiphilicity of the micelle carrier HS-NI can be effectively disrupted, and the chemotherapeutic drug can achieve dual-responsive release under the HPSE and hypoxic conditions in the ATC tumor microenvironment.

[0007] The technical solution adopted by the present invention to solve its technical problems is as follows: An amphiphilic nanocarrier responsive to heparanase and hypoxia. First, an imidazole compound NI is coupled with heparin sulfate HS to form an HS-NI complex, and then a TET targeting head is modified onto the HS-NI complex to form a TET-HS-NI amphiphilic nanocarrier. The present invention is an amphiphilic nanocarrier responsive to heparanase and hypoxia based on the ATC tumor microenvironment.

[0008] In order to construct a safe and effective delivery system responsive to HPSE in the ATC tumor microenvironment, the present invention prepared a delivery system TET-HS-NI / DOX with excellent efficacy.

[0009] Based on the characteristics of high expression of HPSE in anaplastic thyroid carcinoma (ATC) tissues and hypoxia in the tumor microenvironment, a tumor microenvironment-responsive nanomicelle carrier was designed by coupling the hydrophilic backbone HS with the lipophilic compound NI carrying a nitroimidazole group. Taking advantage of the characteristic of high expression of integrin αvβ3 in the ATC tumor microenvironment, TET was used as the targeting head of the drug delivery system carrier for ATC, and a HPSE-responsive degradable HS-coupled NI was constructed and then targeted modification with TET was carried out to construct the amphiphilic carrier TET (tetraiodothyroacetic acid)-HS (heparin sulfate)-NI. Under high levels of HPSE, HS can be responsive cleaved, and the nitro group of NI can undergo responsive reduction in the tumor hypoxic environment. Therefore, the amphiphilicity of the micelle carrier HS-NI can be effectively disrupted in the tumor environment, and the chemotherapeutic drug can achieve dual-responsive release under the conditions of HPSE and hypoxia in the ATC tumor microenvironment. In order to better achieve the effective localization of the drug delivery system at the ATC tumor site and avoid differences in tumor permeability and difficulties in drug diffusion, the carrier of the present invention was subjected to targeted modification. Studies have shown that Integrin αvβ3 is highly expressed in ATC, and tetraiodothyroacetic acid (3,3',5,5'-tetraiodothyro-acetic Acid, TET) can specifically bind to it. We also used this carrier to load doxorubicin, the first-line chemotherapeutic drug for ATC, and constructed TET-HS-NI / DOX to increase the effective load of DOX. We simulated the HPSE conditions in vitro and used the new zebrafish PDX model and the thyroid orthotopic tumor-bearing mouse model to multiply verify the superior efficacy and safety of this carrier for delivering DOX for ATC chemotherapy.

[0010] Generally speaking, the present invention is based on the characteristics of the abnormal abundance of HPSE in the ATC tumor microenvironment and the general hypoxia of tumors, synthesized the delivery system TET-HS-NI / DOX loaded with the first-line chemotherapeutic drug DOX for ATC, and fully verified its synergistic effect in vitro and in vivo, which can provide a reference for solving the difficult problems of ATC chemotherapy.

[0011] The molar ratio of HS to NI in the feed is 5:1 - 3.

[0012] The specific preparation method of the HS-NI complex is as follows: Mix NI, EDC, NHS and DMSO, then add an aqueous solution of HS and stir at room temperature overnight. Place the reaction solution in a dialysis bag, dialyze with deionized water, and freeze-dry to obtain HS-NI.

[0013] The molar ratio of NI:EDC:NHS:HS = 5:3:3:1 - 3.

[0014] The specific preparation method for modifying the TET targeting head onto the HS-NI complex to form the TET-HS-NI amphiphilic nanocarrier is as follows: An aqueous solution of HS-NI was added to a DMSO solution containing TET, EDC, and NHS, and the mixture was stirred overnight at room temperature. The reaction solution was placed in a dialysis bag and dialyzed against deionized water, followed by freeze-drying to obtain the amphiphilic nanocarrier TET-HS-NI.

[0015] The molar ratio of TET:EDC:NHS:HS-NI = 5:2:2:1.

[0016] An amphiphilic nanodrug complex with heparanase and hypoxia dual-response was obtained by mixing the above-mentioned TET-HS-NI amphiphilic nanocarrier with a chemotherapeutic drug, followed by dialysis and freeze-drying.

[0017] The chemotherapeutic drug is doxorubicin.

[0018] Use of the amphiphilic nanocarrier with heparanase and hypoxia dual-response in the preparation of drugs for treating anaplastic thyroid cancer.

[0019] Use of the amphiphilic nanodrug complex with heparanase and hypoxia dual-response as a drug for treating anaplastic thyroid cancer.

[0020] The beneficial effects of the present invention are as follows: (1) The present invention uses a polymer such as heparan sulfate as a hydrophilic backbone and a nitro compound as a lipophilic group, and the two main components simultaneously serve as HPSE and hypoxia-sensitive groups. The raw material utilization rate is high and it has high safety; (2) The synthesis of the carrier is simple. Based on the simple micelle principle, standardized quantitative preparation and transformation can be achieved; (3) The dual-response design can better ensure that DOX in the drug-loaded nanoparticles TET-HS-NI / DOX can be better released, increasing the curative effect. Moreover, this invention has been verified in vitro and in vivo, and it can prove that the delivery system has excellent curative effects. Description of the Drawings

[0021] Figure 1 It is the synthesis route diagram of TET-HS-NI; Figure 2 It is 1 1H NMR characterization diagrams for confirming the structures of the carriers HS-NI and TET-HS-NI; Figure 3 It is the in vitro HPSE sensitivity and in vitro responsive release investigation diagram. (A) Changes in the particle sizes of each carrier under HPSE conditions. (B) TEM observation of the changes in particle size and morphology under HPSE conditions. (C) HPLC measurement of the release of each group; Figure 4 It is the IC of DOX, HS-NI / DOX, and TET-HS-NI / DOX incubated with 8505C cells for 48 h50 (n = 4); Figure 5 It is to investigate the in - vivo anti - tumor efficacy of the nano - drug - loaded complex using the zebrafish CDX model. (A) Schematic diagram of the zebrafish CDX model. (B) Comparison of tumor fluorescence on Day 0 and Day 3 after the zebrafish model was administered with E3 - PTU, DOX, HS - NI / DOX, and TET - HS - NI / DOX. (C) Fluorescence statistics of tumors in the zebrafish CDX model (n = 12); Figure 6 It is to investigate the in - vivo anti - tumor efficacy of the nano - drug - loaded complex using the ATC orthotopic tumor - bearing mouse model; (A) Small animal imager was used to image each group of ATC orthotopic tumor - bearing mice. (B) Changes in mouse body weight. (C) Changes in tumor fluorescence. (D) Size of tumor tissues in each group. (E) Statistics of excised tumor weights. (n = 5) (t - test, *p<0.05, **p<0.01, ***p<0.001). Detailed implementation manners

[0022] The technical solutions of the present invention will be further specifically described below through specific examples.

[0023] In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the following examples are conventional methods in the art unless otherwise specified.

[0024] Description of material sources: N-Boc-6-bromohexylamine (TBAB, CAS: 1643-19-2), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, CAS: 7084-11-9), N-hydroxysuccinimide (NHS, CAS: 6066-82-6), and 2-nitroimidazole (CAS: 527-73-1) were all purchased from Shanghai Sain Chemical Technology Co., Ltd. (Shanghai, China). Heparan Sulfate (HS, CAS: 9050-30-0) was purchased from Nanjing Shenglaide Co., Ltd. (Nanjing, China). 3,3,5,5-Tetraiodothyroacetic acid (TET, CAS: 67-30-1) was purchased from Macklin Biochemical Technology (Shanghai, China), and doxorubicin (DOX, CAS: 25316-40-9) was purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd. (Shanghai, China). RPMI-1640 medium (#SH30096.01) and DMEM medium (#SH30023.01) were purchased from Cytiva. Fetal bovine serum (FBS, #FBS-E500) was purchased from NEWZERUM. Phosphate buffer solution (PBS, #G4202) was purchased from Servicebio. Penicillin-streptomycin (#CR15140) was purchased from Senrui Biotechnology. Trypsin / EDTA (#BL512A) was purchased from Biosharp. 1-Phenyl-2-thiourea (PTU, #P7629), tricaine (#A5040), and dimethyl sulfoxide (DMSO, #D2650) were purchased from Sigma. Sodium chloride (NaCl, #C111533), calcium chloride (CaCl2, #C299717), magnesium sulfate (MgSO4, #M118820), and potassium chloride (KCl, #P112133) were provided by Aladdin. Fast-DiI™ oil (Cat #V22885) was purchased from ThermoFisher Scientific.

[0025] Example 1: Preparation of Nanocarrier TET-HS-NI The specific synthesis steps are as Figure 1 shown.

[0026] (1)In a round-bottom flask, successively add a DMF solution of 2-nitroimidazole (1.0 eq) (10 mL DMF), N-Boc bromohexylamine (1.0 eq), as well as K2CO3 (1.4 eq) and TBAB (0.3 eq). A substitution reaction of coupling and debromination occurs at 80 °C for 4 h. The product is preliminarily dried by freeze-drying to remove the solvent, and a tert-butyl ester amide compound of the 2-nitroimidazole group is obtained. An appropriate amount of MeOH (5.0 mL) and concentrated HCl (0.1 eq, 37% concentration) are added to the crude product (1.0 eq) to remove the BOC group, and it is stirred at room temperature overnight. The product is purified by column chromatography to obtain the imidazole compound NI (tert-butyl (7-(2-nitro-1H-imidazol-1-yl)heptyl)carbamate), 1 and its structure is characterized by 1H NMR.

[0027] (2)Mix NI (5.0 eq), EDC (3.0 eq), NHS (3.0 eq) and an appropriate amount of DMSO solution (0.5 mL), then add an aqueous solution of HS (1.0 eq) (HS is dissolved in 2 mL of water) and stir at room temperature overnight. The reaction solution is placed in a dialysis bag (MWCO 3.5 kDa) and dialyzed with deionized water for 48 h, and then freeze-dried to obtain HS-NI, 1 and its structure is characterized by 1H NMR.

[0028] (3)An aqueous solution of HS-NI (1.0 eq) (1.5 mL of water) is added to a DMSO solution (1.5 mL of DMSO) of TET (5.0 eq), EDC (2.0 eq), and NHS (2.0 eq) and stirred at room temperature overnight. The reaction solution is placed in a dialysis bag (MWCO 3.5 kDa) and dialyzed with deionized water for 48 h, and then freeze-dried to obtain TET-HS-NI, 1 and the structure of the product is characterized by 1H NMR.

[0029] 1 1H NMR confirmed the successful synthesis of HS-NI and TET-HS-NI ( Figure 2 ). The 1 1H NMR spectrum of NI: 7.11 ppm, HS: about 4.5 ppm. The above results indicate the successful coupling of NI and HS. The 1 1H NMR spectrum of TET: 9.0 - 10.5 ppm. TET reacts with HS-NI to prepare TET-HS-NI. The characteristic peaks of TET at 9.0 - 10.5 ppm and HS-NI at 3.5 - 5.0 ppm are attributed to the H of TET-HS-NI. This indicates that TET is successfully coupled to HS-NI.

[0030] Example 2: Optimization of nano - carriers and drug - loaded nanoparticles Prepare TET - HS - NI according to the method of Example 1. 1 Confirm the successful preparation of TET - HS - NI by \(^1H\) NMR method. Since the particle size of the carrier and the drug - loaded nanoparticles is related to their effective penetration into the tumor site, according to the method of Example 1, we improved the particle size of HS - NI by changing the molar ratio of HS and NI feed, reducing the particle size to less than 100 nm (Table 1). By trying multiple feed ratios, we found that when the feed ratio of HS and NI is 5:3, the effect is the best.

[0031] Table 1. Particle size of HS - NI at different feed ratios of HS and NI Serial number Molar ratio of HS and NI feed (HS:NI) Particle size (nm) 1 5：1 220 2 5：2 150 3 5：3 80 Select HS - NI with a particle size of 80 nm, and the TET - modified TET - HS - NI (97 nm) also meets the particle size requirements.

[0032] Example 3: Preparation steps of HS - NI / DOX drug - loaded nanoparticles: Dissolve HS - NI (10 mg) in 5 mL of pure water to prepare a 2 mg / mL solution and stir it on a magnetic stirrer (room temperature, 1000 rpm). Dissolve 1.5 mg of DOX (15% by weight of HS - NI) in 2 mL of DMSO, and slowly add the DMSO solution of DOX to the stirred aqueous solution of HS - NI, and continue stirring for 2 h. Transfer the well - stirred solution to a dialysis bag and dialyze for 12 h. Finally, freeze it into a solid at - 80 °C, and then remove the solvent in a freeze - dryer to obtain HS - NI / DOX drug - loaded nanoparticles.

[0033] Preparation steps of TET - HS - NI / DOX drug - loaded nanoparticles: Dissolve TET - HS - NI (10 mg) in 5 mL of pure water to prepare a 2 mg / mL solution and stir it on a magnetic stirrer (room temperature, 1000 rpm). Dissolve 1.5 mg of DOX (15% by weight of HS - NI) in 2 mL of DMSO, and slowly add the DMSO solution of DOX to the stirred aqueous solution of TET - HS - NI, and continue stirring for 2 h. Transfer the well - stirred solution to a dialysis bag and dialyze for 12 h. Finally, freeze it into a solid at - 80 °C, and then remove the solvent in a freeze - dryer to obtain TET - HS - NI / DOX drug - loaded nanoparticles.

[0034] Example 4: The particle sizes of the carriers HS-NI and TET-HS-NI were measured using a particle size analyzer and an electron microscope, and the average particle sizes of both were below 100 nm ( Figure 3 A). In the medium containing simulated HPSE, the particle sizes of HS-NI and TET-HS-NI increased sharply, aggregating from uniform spherical shapes into irregular shapes, demonstrating the responsive destruction of the carrier under simulated HPSE conditions( Figure 3 B).

[0035] Cumulative drug release experiment: Pipette 5 mL of DOX solution and HS-NI / DOX and TET-HS-NI / DOX nanoparticle solutions, and make up the volume to 1.0 mL, then place them in a dialysis bag (MWCO 3.5 kDa). Use 20.0 mL of pH 7.4 PBS containing or not containing HPSE solution (0.1 mM) as the release medium, and oscillate at a constant temperature (37 °C, 60 rpm). Take samples at preset time points and replace them with fresh release medium. Determine the DOX concentration by HPLC, and calculate the cumulative release amount and cumulative release percentage of DOX according to the standard curve. The results show that the release rate of free DOX is the highest, and the 72-hour cumulative release rates of HS-NI / DOX and TET-HS-NI / DOX in the medium containing HPSE are both higher than those in the environment without HPSE, indicating that this delivery system can release drugs in a responsive manner ( Figure 3 C).

[0036] In vitro anti-tumor efficacy study of the nano-drug complex: The killing ability of HS-NI / DOX is stronger than that of DOX, and the targeted modification of TET enhances the killing ability of TET-HS-NI / DOX. Compared with HS-NI / DOX, the efficacy of TET-HS-NI / DOX is better than that of HS-NI / DOX, and both TET-HS-NI / DOX and HS-NI / DOX have enhanced killing effects on tumor cells, and are far better than DOX ( Figure 4 A-4C).

[0037] In vivo anti-tumor efficacy study of the nano-drug delivery system using zebrafish CDX and ATC orthotopic tumor-bearing mouse models: We injected tumor cells 8505C into the perivitelline space of zebrafish eggs and successfully established a zebrafish CDX model ( Figure 5A-5B). The zebrafish CDX models were respectively given normal culture medium E3-PTU (E3 / PTU: containing 0.286 g NaCl, 0.048 g CaCl2, 0.081 g MgSO4, 0.0126 g KCl and 0.2 mM PTU per liter) and solutions of equal doses and equal concentrations of DOX, HS-NI / DOX, TET-HS-NI / DOX, etc. (all converted to contain 1.0 mg / mL of DOX). The changes in the fluorescence area of tumors in zebrafish were measured at 72 h and 0 h after injection. The anti-tumor proliferation abilities from weak to strong were Control, DOX, HS-NI / DOX and TET-HS-NI / DOX respectively ( Figure 5 C).

[0038] Subsequently, we successfully established an ATC orthotopic tumor-bearing mouse model ( Figure 6 A). Solutions of equal doses of DOX, HS-NI / DOX, TET-HS-NI / DOX, etc. (all converted to contain 3.0 mg / kg of DOX) were separately prepared, and the body weights of the mice and the fluorescence changes at the tumor sites were monitored. The results were consistent with those of the zebrafish CDX model experiment. The anti-tumor proliferation effects from weak to strong were the control group (normal saline), DOX, HS-NI / DOX and TET-HS-NI / DOX respectively ( Figure 6 B-6E).

[0039] The above-described embodiments are only a preferred solution of the present invention, and do not impose any formal limitations on the present invention. There are other variations and modifications without exceeding the technical solutions described in the claims.

Claims

1. An amphiphilic nanocarrier that responds to both heparanase and hypoxia, characterized in that: The imidazole compound NI is first coupled with heparan sulfate HS to form a HS-NI complex, and then the TET target head is modified onto the HS-NI complex to form a TET-HS-NI amphiphilic nanocarrier.

2. The heparanase and hypoxia dual-responsive amphiphilic nanocarrier according to claim 1, characterized in that: The molar ratio of HS to NI is 5:1-3.

3. The heparanase and hypoxia dual-responsive amphiphilic nanocarrier according to claim 1, characterized in that: The specific preparation method of HS-NI complex is: NI, EDC, NHS and DMSO were mixed, and then an aqueous solution of HS was added and stirred at room temperature overnight. The reaction solution was placed in a dialysis bag, dialyzed against deionized water, and freeze-dried to obtain HS-NI.

4. The heparanase and hypoxia dual-responsive amphiphilic nanocarrier according to claim 3, characterized in that: The molar ratio of NI:EDC:NHS:HS = 5:3:3:1-3.

5. The heparanase and hypoxia dual-responsive amphiphilic nanocarrier according to claim 1, characterized in that: The specific preparation method of modifying the TET target head onto the HS-NI complex to form the TET-HS-NI amphiphilic nanocarrier is as follows: An aqueous solution of HS-NI was added to a DMSO solution containing TET, EDC and NHS, and the mixture was stirred at room temperature overnight. The reaction solution was placed in a dialysis bag, dialyzed with deionized water, and freeze-dried to obtain TET-HS-NI amphiphilic nanocarriers.

6. The heparanase and hypoxia dual-responsive amphiphilic nanocarrier according to claim 5, characterized in that: The molar ratio of TET:EDC:NHS:HS-NI=5:2:2:

1.

7. An amphiphilic nano drug-carrying complex with dual response to heparanase and hypoxia, characterized in that: The amphiphilic TET-HS-NI nanocarrier according to claim 1 is mixed with a chemotherapeutic drug, dialyzed and freeze-dried to obtain the nanocarrier.

8. The heparanase and hypoxia dual-responsive amphiphilic nano-drug complex according to claim 7, characterized in that: The chemotherapy drug is doxorubicin.

9. Use of the heparanase and hypoxia dual-responsive amphiphilic nanocarrier according to claim 1 in the preparation of a drug against anaplastic thyroid cancer.

10. Use of the heparanase and hypoxia dual-responsive amphiphilic nano-drug carrier complex as claimed in claim 8 as a drug against anaplastic thyroid cancer.

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