Hypoxia responsive lipid nano delivery system as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of nano biology, and discloses a hypoxia responsive lipid nano delivery system as well as a preparation method and application thereof. The method comprises the following steps: mixing and stirring 2-(4-chloro-2-nitrophenyl) acetic acid, 2-(7-azabenzotriazole)-N, N, N ', N'-tetramethylurea hexafluorophosphate, N, N-diisopropylethylamine and pyridine to react, adding mPEG-NH2 to stir and react, then adding distearoyl phosphatidyl ethanolamine, palladium acetate, 4, 5-bis (diphenylphosphine-9, 9-dimethyl xanthene and cesium carbonate to stir and react, and finally adding a catalyst to stir and react, so as to obtain a target product. Carrying out reflux reaction at 120 DEG C overnight, dialyzing, centrifuging, collecting supernatant, and freeze-drying to obtain DNP; the preparation method comprises the following steps: dissolving distearoyl phosphatidylcholine, cholesterol, DNP, sorafenib and mitoxantrone in absolute ethyl alcohol, and stirring and dissolving in a dark place; spin-drying to form a film, adding PBS, hydrating, and carrying out ultrasonic treatment in an ice-water bath; and extruding a film to obtain the hypoxia responsive lipid nano delivery system.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanobiology, and particularly relates to a hypoxia-responsive lipid nanoparticle delivery system, a preparation method thereof, and an application thereof. Background Art

[0002] Chemotherapy is a common treatment method for cancer. Chemotherapy kills tumor cells mainly by using the cytotoxicity of synthetic drugs. However, since chemotherapy drugs are toxic to both normal cells and tumor cells, many toxic side effects will occur during chemotherapy, which is an important problem to be solved clinically in chemotherapy at present. The nano-targeted drug delivery system refers to a new drug delivery technology that uses nanoparticles as carriers and transports drugs efficiently and precisely to the diseased site through the pore channels of biological membranes or receptor-mediated transmembrane transport mechanisms. Based on the characteristics of the small size effect and surface effect of nanoparticles, this technology can penetrate the cell membrane and enter the cell interior, thereby delivering drugs to the target tissue or organ. Liposomes are a common nanomaterial that has entered clinical practice. Liposomes can more easily fuse with the cell membrane and deliver the encapsulated contents (such as drugs) into the cell, thereby improving the efficacy of drugs. However, since the structure of liposomes is close to that of the human cytoplasmic membrane, traditional liposome carriers still lack specificity and it is difficult to achieve an ideal effect in reducing toxic side effects. At the same time, due to the relatively large volume of liposomes, the targeting difficulty may increase in the complex physiological barriers of tumor tissues.

[0003] The tumor microenvironment has the characteristics of hypoxia, low pH, inflammatory response and immunosuppression, which is clinically called the tumor microenvironment. In solid tumors, due to the rapid growth of tumor tissues, high volume expansion and incomplete internal vascular systems of tumor tissues, these will lead to insufficient oxygen supply in tumor tissues. This hypoxic environment will affect the metabolism and gene expression of tumor cells, thereby changing their biological behaviors. For example, it can reduce the sensitivity of tumor cells to chemotherapy, thereby enhancing the resistance to treatment. The hypoxic environment can induce functional changes in immune cells in the tumor microenvironment, such as increasing the infiltration and activity of regulatory T cells (Tregs), inhibiting the anti-tumor immune response, and thus reducing the effect of immunotherapy; under hypoxic conditions, the metabolism and membrane transport functions of tumor cells change, resulting in reduced drug uptake in liposome carriers, thereby reducing the efficacy of chemotherapy drugs. Summary of the Invention

[0004] In order to overcome the above-mentioned disadvantages and deficiencies in the prior art, the primary object of the present invention is to provide a preparation method of a hypoxia-responsive lipid nanoparticle delivery system.

[0005] Another object of the present invention is to provide a hypoxia-responsive lipid nanoparticle delivery system prepared by the above preparation method.

[0006] Another object of the present invention is to provide an application of the above hypoxia-responsive lipid nanoparticle delivery system.

[0007] The object of the present invention is achieved by the following technical solutions:

[0008] A preparation method of a hypoxia-responsive lipid nanoparticle delivery system, comprising the following operating steps:

[0009] (1) Weigh 18 - 22 mg of 2-(4-chloro-2-nitrophenyl)acetic acid, 50 - 55 mg of 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate, and 52 - 56 mg of N,N-diisopropylethylamine into a pressure-resistant bottle containing 4 mL of pyridine, and dissolve them by ultrasonic treatment. The obtained mixture is stirred at room temperature for 0.5 - 1.5 h, then 180 - 190 mg of mPEG-NH2 with a molecular weight of 2000 is added, and the mixture is stirred at room temperature overnight. 65 - 70 mg of distearoylphosphatidylethanolamine, 0.8 - 1.2 mg of palladium acetate, 1.8 - 2.2 mg of 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene, and 80 - 85 mg of cesium carbonate are added to the obtained reaction solution, and the mixture is refluxed at 120 °C overnight. After confirming the completion of the reaction by TLC, it is dialyzed for 48 h and then centrifuged. The supernatant is collected and freeze-dried to obtain the active ingredient of the hypoxia liposome, nitrophenyl polyethylene glycol phosphate (DNP);

[0010] (2) Dissolve 3 - 5 mg of distearoylphosphatidylcholine, 0.8 - 1.2 mg of cholesterol, 10 - 14 mg of nitrophenyl polyethylene glycol phosphate (DNP) obtained in step (1), 0.8 - 1.2 mg of sorafenib (SFB), and 0.8 - 1.2 mg of mitoxantrone (MTX) in 2 mL of absolute ethanol, and stir well in the dark for 10 minutes to dissolve all the solid powders. Then, the solution is rotary evaporated to dryness to form a film, 2 mL of PBS is added, and the mixture is hydrated in a water bath at 60 °C for 15 minutes, and then ultrasonically treated in an ice-water bath for 10 minutes. The ultrasonically treated solution is extruded through a 200 nm extrusion membrane 8 - 10 times using an extruder to obtain the final product, the hypoxia-responsive lipid nanoparticle delivery system HCDNP@MS.

[0011] The dialysis in step (1) is carried out using a dialysis bag with a relative molecular weight of 1000; the centrifugation is carried out at a rotation speed of 12000 rpm for 20 min.

[0012] A hypoxia-responsive lipid nanoparticle delivery system prepared by the above-mentioned preparation method, wherein the particle size of the hypoxia-responsive lipid nanoparticle delivery system is 120 nm ± 3, the polydispersity index (PDI) is 0.16 ± 0.01, the zeta potential is -11 mV ± 1, the encapsulation efficiency is 87% ± 0.01, and the drug loading rate is 9% ± 0.01; the mitoxantrone (MTX) has a drug release rate of 70% ± 0.01 at 24 h and 86% ± 0.01 at 48 h; sorafenib (SFB) has a drug release rate of 73% ± 0.01 at 24 h and 75% ± 0.01 at 48 h.

[0013] Use of the above-mentioned hypoxia-responsive lipid nanoparticle delivery system in the preparation of anti-tumor drugs.

[0014] In this study, taking the particle size, encapsulation efficiency, drug loading rate and stability of the obtained hypoxia-responsive lipid nanoparticle delivery system as indicators, the ratios between the components of the liposome, the drug dosage and other related parameters and the preparation process were optimized; then, by monitoring the changes in the hydrated particle size, zeta potential and polydispersity index (PDI) of the lipid nanoparticle delivery system, the stability of the liposome was evaluated; then, the performance of the lipid nanoparticle delivery system was verified through the following experiments:

[0015] (1) Simulating the hypoxic environment in vitro, it was found by electron microscopy that the chemical bond conversion of the lipid nanoparticle delivery system occurred, and the material dispersed from the original complete nanostructure.

[0016] (2) By liquid-phase detection of the drug release concentration in the lipid nanoparticle delivery system at different time points, the results showed that the drug release rate increased significantly under the hypoxic environment, proving the superior hypoxia responsiveness of the delivery system.

[0017] (3) By simulating the tumor microenvironment, it was found that HCDNP@MS could penetrate deeper into the tumor sphere than HCDP@MS under hypoxia.

[0018] (4) Through in vivo experiments on mice, it was found that the HCDNP@MS group could reach a more hypoxic area far from blood vessels, proving the hypoxia responsiveness and permeability of the material.

[0019] (5) Through HE staining of mouse tumor sections, it was found that the HCDNP@MS group had the best anti-tumor effect, proving the superior anti-tumor effect of the material.

[0020] The principle of the present invention is:

[0021] ① Traditional liposomes are prone to oxidation and hydrolysis, which increases the uncontrollability of drug release. The present invention aims to create a stable carrier. Nitro groups generally have high stability. There are a large number of nitroreductases in the hypoxic microenvironment of tumors. When the enzyme reduces the nitro group in the compound, the liposome (drug-loaded nanomaterial) dissociates when it reaches the deep part of the tumor, thereby releasing the drug, increasing the drug concentration in the deep part of the tumor, effectively killing tumor cells, and achieving the therapeutic effect.

[0022] ② The tumor microenvironment is hypoxic. According to the characteristics of hypoxia, the present invention designs a hypoxia-responsive liposome, which targets and delivers chemotherapeutic drugs to the tumor site by virtue of the EPR effect, increases the effective drug concentration of the drug in the tumor, prolongs the circulation time in the body, and reduces the damage to normal tissues; at the same time, due to the hypoxia responsiveness of the liposome, free drugs can be released when it reaches the tumor microenvironment, and the smaller-sized free drugs can reach the deep tissues of the tumor, achieving a better killing effect.

[0023] The present invention has the following advantages and beneficial effects compared with the prior art:

[0024] (1) The method of the present invention enhances the stability of liposomes as drug carriers and reduces the occurrence of oxidation and hydrolysis of liposomes.

[0025] (2) The method of the present invention increases the bioavailability of the drug and improves biological safety. The hypoxia-responsive structure of the lipid nanodelivery system prepared by the present invention realizes the directional release of the drug, increases the drug concentration at the targeted position, improves the therapeutic effect, and reduces the toxic effect on normal tissues.

[0026] (3) The current drug delivery system has low bioavailability and large side effects, causing pain to patients during the treatment process. According to the hypoxic environment of tumors, the present invention obtains an improved liposome with a drug delivery system having a hypoxic structure, which can effectively reduce the side effects of chemotherapy and reduce the damage to normal organs.

[0027] (4) The hypoxia-responsive lipid nanodelivery system of the present invention can be applied to more water-soluble and lipophilic drugs: it can deliver hydrophilic anticancer drugs such as 5-fluorouracil, gemcitabine, paclitaxel and its derivatives, water-soluble ginsenoside Rg3, etc., and can also deliver hydrophobic anticancer drugs such as cyclophosphamide, thiotepa, rosuvastatin, atorvastatin, actinomycin D, daunorubicin, etc.

[0028] (5) The preparation method adopted by the present invention is economical and practical, and the technology is simple. Description of the Drawings

[0029] Figure 1Synthesis schematic diagram and 1H NMR spectrum of the active ingredient DNP prepared in the present invention.

[0030] Figure 2 Physical characteristic diagrams of the hypoxia-responsive lipid nanoparticle delivery system of the present invention, where A is the particle size distribution diagram, B is the potential diagram, and C is the transmission electron microscopy diagram.

[0031] Figure 3 For the hypoxia-responsive lipid nanoparticle delivery system of the present invention under the simulated hypoxic environment of Na2S2O4, the drug release concentrations at different time points were detected by liquid phase. Among them, A is the MTX release diagram under normoxia / hypoxia conditions, and B is the SFB release diagram under normoxia / hypoxia conditions.

[0032] Figure 4 Results diagram of the uptake ability of tumor spheres to drugs in different treatment groups detected by confocal microscopy.

[0033] Figure 5 Fluorescence imaging diagrams of the material group HCDNP@Cy7 and the free drug Cy7 group in a mouse orthotopic liver cancer model.

[0034] Figure 6 Immunofluorescence diagrams of CD31 and HIF-α markers in frozen sections of mouse tumor tissues.

[0035] Figure 7 HE staining result diagram of mouse tumor tissues. Detailed implementation manners

[0036] The content of the present invention will be further described below in conjunction with specific embodiments, but it should not be construed as a limitation to the present invention.

[0037] Example 1

[0038] (1) Weigh 20 mg of 2-(4-chloro-2-nitrophenyl)acetic acid, 53 mg of 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, and 54 mg of N,N-diisopropylethylamine into a pressure-resistant bottle containing 4 mL of pyridine, and dissolve them by ultrasound. The resulting mixture is stirred at room temperature for 1 h, and then 186 mg of mPEG-NH2 with a molecular weight of 2000 is added, and the reaction is stirred overnight at room temperature. 68 mg of distearoyl phosphatidylethanolamine, 1 mg of palladium acetate, 2 mg of 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene, and 83 mg of cesium carbonate are added to the resulting reaction solution, and the reaction is refluxed at 120 °C overnight. After confirming the completion of the reaction by TLC, dialysis is carried out for 48 h and then centrifuged, and the supernatant is collected and freeze-dried to obtain the hypoxia liposome active ingredient DNP;

[0039] The structural formula, synthesis schematic diagram and 1H NMR spectrum of the above active ingredient DNP are as Figure 1 shown, from Figure 1From the hydrogen spectrum, it can be seen that the structure of the obtained compound is consistent with the expectation. The final product contains characteristic peaks of hydrogen, proving the successful synthesis of DNP;

[0040] (2) Dissolve 4 mg of distearoyl phosphatidylcholine, 1 mg of cholesterol, 12 mg of DNP, 1 mg of sorafenib (SFB), and 1 mg of mitoxantrone (MTX) in 2 mL of absolute ethanol. Stir well for 10 minutes in the dark to dissolve all the solid powders; then use a rotary evaporator to spin-dry the solution and form a film. Add 2 mL of PBS and hydrate in a water bath at 60 °C for 15 minutes, and then place it in an ice-water bath and sonicate for 10 minutes; use an extruder to extrude the sonicated solution through a 200-nm extrusion membrane 8 - 10 times to obtain the final product, the hypoxia-responsive lipid nanodelivery system HCDNP@MS.

[0041] Comparative Example 1

[0042] (1) Synthesize the control group of phosphopolyethylene glycol (DP) without hypoxia-responsive components: Weigh 65 mg of distearoyl phosphatidylethanolamine, 25 mg of N-hydroxysuccinimide (NHS), and 35 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and dissolve them in 4 mL of pyridine. Stir at 30 °C for 1 h; then add 185 mg of mPEG-NH2 and stir at 30 °C for 24 h. Then, purify by dialysis using a 2000 KDa dialysis bag for 2 days. After freeze-drying, obtain phosphopolyethylene glycol (DP). The freezing temperature is -60 °C and the freezing time is 24 h.

[0043] (2) Replace DNP with the above-obtained DP according to the specific steps in step (2) of Example 1 to finally obtain HCDP@MS.

[0044] Example 2

[0045] The particle size, zeta potential, and transmission electron microscopy of the hypoxia-responsive lipid nanodelivery system HCDNP@MS obtained in Example 1 were detected. The particle size of the material was measured by DLS (dynamic light scattering) to be 124 nm ± 2, the PDI was 0.16 ± 0.01, and the zeta potential was -10.40 mV ± 0.01 ( Figure 2 as shown in Figures A and B). To verify that the hypoxia-responsive lipid nanodelivery system HCDNP@MS obtained in the present invention is hypoxia-sensitive, we found through transmission electron microscopy results that the liposomes dissociated completely under hypoxic conditions, proving excellent hypoxia responsiveness. Because the liposomes obtained in the present invention are hypoxia-sensitive, we simulated a hypoxic environment in vitro using Na2S2O4 and found through transmission electron microscopy that due to the electron rearrangement and carbon-oxygen bond cleavage reaction of compound 2-(4-chloro-2-nitrophenyl)acetic acid, the liposomes dissociated, and the material dispersed from the original complete nanostructure ( Figure 2of C).

[0046] Subsequently, we used Na2S2O4 to simulate the hypoxic environment and detected the drug release concentration of HCDNP@MS at different time points under hypoxic conditions through liquid phase ( Figure 3 in A-B) to prove that the material has good hypoxic-responsive drug release. In the liquid phase detection, MTX: A aqueous phase: B methanol = 60:40, SFB: A aqueous phase: B acetonitrile = 40:60.

[0047] Then, we detected the uptake ability of tumor spheres to drugs in different treatment groups (free drugs MTX + SFB, HCDP@MS obtained in Comparative Example 1, and HCDNP@MS obtained in Example 1) through confocal microscopy. The results are as Figure 4 shown. It can be found that under hypoxia, HCDNP@MS can penetrate deeper into the tumor spheres compared with HCDP@MS without nitrophenyl.

[0048] We also detected whether the material could effectively reach the tumor site in mice through in vivo imaging of animals. In this experiment, a liver orthotopic carcinoma model was constructed using luciferase-transfected mouse hepatoma cells Hepa1-6-Luc. The hypoxia-responsive lipid nanoparticle delivery system obtained in Example 1 (denoted as HCDNP@Cy7) was labeled with Cy7 by encapsulating the material. The results are as Figure 5 shown. It can be found that compared with the free drug Cy7 group, the material group HCDNP@Cy7 group can effectively accumulate in the liver orthotopic carcinoma of mice and prolong the existence time of the drug in the liver orthotopic carcinoma.

[0049] To verify the hypoxic responsiveness of the material in vivo, in this experiment, different treatment groups (PBS, free drugs MTX + SFB, i.e., M + S, HCDP@MS obtained in Comparative Example 1, and HCDNP@MS obtained in Example 1) were injected into mice through the tail vein. After 6 h, tumor tissues were taken for frozen section and immunofluorescence staining. CD31 was used to label blood vessels in the tumor tissue, and HIF-α was used to label the hypoxic region. The results are as Figure 6 shown. It can be found that the HCDNP@MS group labeled with red light can reach the hypoxic region farther away from blood vessels in the tumor tissue, proving that the material prepared by the present invention has very good hypoxic responsiveness and permeability.

[0050] Finally, we stained the tumor tissues of the mice in the above tail vein injection experiment with HE. The results are as Figure 7 shown. The HCDNP@MS group has the best tumor-killing effect, proving that the hypoxia-responsive lipid nanoparticle delivery system HCDNP@MS obtained by the present invention has superior anti-tumor effects.

[0051] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A preparation method of a hypoxia-responsive lipid nanoparticle delivery system, characterized in that It includes the following operation steps: (1) Weigh 18 - 22 mg of 2-(4-chloro-2-nitrophenyl)acetic acid, 50 - 55 mg of 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate, and 52 - 56 mg of N,N-diisopropylethylamine into a pressure-resistant bottle containing 4 mL of pyridine, and dissolve them by ultrasound. The resulting mixture is stirred at room temperature for 0.5 - 1.5 h, then 180 - 190 mg of mPEG-NH2 with a molecular weight of 2000 is added, and the reaction is stirred overnight at room temperature. 65 - 70 mg of distearoylphosphatidylethanolamine, 0.8 - 1.2 mg of palladium acetate, 1.8 - 2.2 mg of 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene, and 80 - 85 mg of cesium carbonate are added to the resulting reaction solution, and the reaction is refluxed at 120 °C overnight. After confirming the completion of the reaction by TLC, it is dialyzed for 48 h and then centrifuged, and the supernatant is collected and freeze-dried to obtain the effective component of the hypoxia liposome, phosphonitrophenyl polyethylene glycol DNP; (2) Dissolve 3 - 5 mg of distearoylphosphatidylcholine, 0.8 - 1.2 mg of cholesterol, 10 - 14 mg of the phosphonitrophenyl polyethylene glycol DNP obtained in step (1), 0.8 - 1.2 mg of sorafenib, and 0.8 - 1.2 mg of mitoxantrone in 2 mL of absolute ethanol, and stir well in the dark for 10 minutes to dissolve all the solid powders. Then, the solution is dried by rotary evaporation under vacuum to form a film, 2 mL of PBS is added, and it is hydrated in a water bath at 60 °C for 15 minutes, and then sonicated in an ice-water bath for 10 minutes. The sonicated solution is extruded through a 200 nm extrusion membrane 8 - 10 times using an extruder to obtain the final product, the hypoxia-responsive lipid nanodelivery system HCDNP@MS.

2. The preparation method according to claim 1, characterized in that: The dialysis described in step (1) is carried out using a dialysis bag with a relative molecular weight of 1000; the centrifugation is carried out at a rotation speed of 12000 rpm for 20 min.

3. A hypoxia-responsive lipid nanoparticle delivery system prepared by the preparation method according to claim 1 or 2, characterized in that: The particle size of the hypoxia-responsive lipid nanodelivery system is 120 nm ± 3, the polydispersity index of the polymer is 0.16 ± 0.01, the potential is -11 mV ± 1, the encapsulation efficiency is 87% ± 0.01, and the drug loading rate is 9% ± 0.01; among them, the drug release rate of mitoxantrone is 70% ± 0.01 at 24 h and 86% ± 0.01 at 48 h; the drug release rate of sorafenib is 73% ± 0.01 at 24 h and 75% ± 0.01 at 48 h.

4. Use of the hypoxia-responsive lipid nanodelivery system according to claim 3 in the preparation of an antitumor drug.

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