A nano-targeted drug-loaded micelle and a preparation method and application thereof

Abstract

The application discloses a kind of nano targeting drug-loaded micelles and preparation method and application thereof, the nano targeting drug-loaded micelles is core-shell structure, the inner core is by adipohydrazide group connection on the hyaluronic acid straight chain dihydrogen porphyrin e6 group;The shell is hyaluronic acid;The outer surface of the shell also has endoplasmic reticulum targeting signal peptide by adipohydrazide group connection on the hyaluronic acid straight chain.The nano targeting drug-loaded micelles prepared by the application has active targeting, immunosuppressive microenvironment improvement, immunotherapy and photodynamic diagnosis and treatment integration.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical preparations, in particular to a nano-targeted drug-loaded micelle and a preparation method and application thereof. BACKGROUND

[0002] With the development of nanobiotechnology, multifunctional intelligent polymer nanoparticles have been paid more and more attention, especially in the field of cancer cell treatment. Multifunctional intelligent nanocarriers as a new type of drug carrier have been widely used in the field of targeted drug delivery and have good development prospects. Intelligent nanocarriers can selectively deliver drugs to specific targets and respond to chemical signals or temperature or pH triggers to release the loaded drugs. Such nanocarriers can be divided into active targeting carriers and passive targeting carriers.

[0003] Active targeting carriers have attracted more and more attention in the medical field due to their strong targeting ability and small side effects. For example, hyaluronic acid is a high molecular weight polymer composed of D-glucuronic acid and N-acetylglucosamine. It has unique physical and chemical properties and physiological functions and has been widely used in medicine and biological materials. Cell surface receptors are diverse, and selecting appropriate receptors and their ligands is the key to achieving active drug targeting. CD44 is a widely studied cell surface receptor and plays an important role in the occurrence and development of tumors.

[0004] Although nanopolymer drug carriers have been proven to have great development prospects in delivering antitumor drugs, there are still many shortcomings. For example, most of the carriers used at present have single function and only play a role in encapsulation. The drug loading stability is poor, and most of them rely on passive transport to enrich in tumor sites. These shortcomings greatly limit the clinical application of polymer nanocarriers. SUMMARY

[0005] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application provides a nano-targeted drug-loaded micelle. The hyaluronic acid has active targeting ability, which can better enrich around the tumor and enter the tumor cells. At the same time, the targeting signal peptide can induce the nano-photosensitizer to target the endoplasmic reticulum, generate ROS through photodynamic therapy, produce endoplasmic reticulum stress, kill tumors while inducing tumor immunogenic cell death (ICD) effect, activate tumor immunity, improve the immunosuppressive microenvironment, and realize the photodynamic / immune synergistic therapy of the particles. The near-infrared optical properties of Ce6 can realize real-time tracking of nanoparticles in vivo and realize the integration of diagnosis and treatment.

[0006] The second aspect of the present application further provides a preparation method of the nano-targeted drug-loaded micelle.

[0007] The third aspect of the present application also provides an antitumor drug.

[0008] The nano-targeting drug-loaded micelles provided by the first aspect of the present application have a core-shell structure, the inner core is a dihydrophenyl e6 group connected to a hyaluronic acid straight chain through a adipic dihydrazide group, the outer shell is hyaluronic acid, and the outer surface of the outer shell has an endoplasmic reticulum targeting signal peptide connected to the hyaluronic acid straight chain through an adipic dihydrazide group.

[0009] The nano-targeting drug-loaded micelles provided by the embodiments of the present application have at least the following beneficial effects:

[0010] The present application connects the dihydrophenyl e6 group and the endoplasmic reticulum targeting signal peptide to the skeleton of hyaluronic acid through an adipic dihydrazide group, forms a core-shell structure, the hydrophobic dihydrophenyl e6 group is the inner core, hyaluronic acid is the shell, and the endoplasmic reticulum targeting signal peptide is connected to the outer surface of the shell. This makes it have active targeting, improved immunosuppressive microenvironment, immunotherapy, and integrated photodynamic diagnosis and treatment. Because the endoplasmic reticulum targeting signal peptide is on the outer surface of the nano-targeting drug-loaded micelles, it can effectively induce the aggregation of the nano-photosensitizer dihydrophenyl e6 in the endoplasmic reticulum, and produce endoplasmic reticulum stress through the cooperation of near-infrared laser irradiation and dihydrophenyl e6, kill tumors, induce tumor immunogenic death (ICD) effect, activate tumor immunity, and improve the immunosuppressive microenvironment to realize the synergistic antitumor function of immunity and photodynamics.

[0011] Immunogenic cell death (ICD) is an important type of regulation that stimulates anti-cancer immune response and enhances the effect of immunotherapy. By causing tumor ICD effect, activating the immune cells in situ of the tumor has become the most potential cancer treatment strategy.

[0012] The endoplasmic reticulum (ER) stress caused by the overexpression of reactive oxygen species (ROS) can effectively induce immunogenic cell death (ICD), thereby improving the tumor immunosuppressive microenvironment.

[0013] According to some embodiments of the present application, the mass ratio of the dihydrophenyl e6 group, the endoplasmic reticulum targeting signal peptide and the hyaluronic acid is (0.2-3):(0.1-3):1.

[0014] According to some embodiments of the present application, the average particle size of the nano-targeting drug-loaded micelles is 50-200 nm. The nano-particles of this particle size are more conducive to entering cells to realize their biological properties.

[0015] According to some embodiments of the present application, the endoplasmic reticulum targeting signal peptide is Fmoc-RACR (arginine-alanine-cysteine-arginine) polypeptide.

[0016] According to some embodiments of the present application, the weight average molecular weight of the hyaluronic acid is 10K-100K. The hyaluronic acid has good biocompatibility, is biodegradable and can be absorbed or excreted out of the body through normal physiological pathways; the hyaluronic acid has the function of actively targeting tumor cells, and is rich in a large amount of hyaluronidase in the tumor microenvironment, thereby controlling the slow release of anticancer drugs at the tumor site by degrading the hyaluronic acid chain.

[0017] According to the second aspect of the present application, a preparation method of a nano-targeting drug-loaded micelle is provided, comprising the following steps:

[0018] S1, using adipohydrazide (ADH) to chemically modify hyaluronic acid in an acidic aqueous solution to obtain a HA-ADH derivative;

[0019] S2, reacting the HA-ADH derivative, dihydrophoeophorbide e6 activated by 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and endoplasmic reticulum targeting signal peptide and solvent, after the reaction is completed, the reaction solution is dialyzed to obtain the nano-targeting drug-loaded micelle.

[0020] According to some embodiments of the present application, the solvent is selected from at least one of formamide, DMF or DMSO.

[0021] According to some embodiments of the present application, the method of using adipohydrazide to chemically modify hyaluronic acid in an acidic aqueous solution is as follows:

[0022] After adding the adipohydrazide to the hyaluronic acid solution, the pH value of the aqueous solution is adjusted to 4.5-4.8, a condensing agent is added, and the pH value is maintained at 4.5-4.8, so that the adipohydrazide and the hyaluronic acid are subjected to condensation reaction.

[0023] According to some embodiments of the present application, the condensing agent is 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC), 1,3-dicyclohexyl carbodiimide (DCC).

[0024] According to some embodiments of the present application, in step S2, the reaction temperature is room temperature.

[0025] According to some embodiments of the present application, in step S2, the reaction time should at least ensure that the reactants are fully reacted, such as stirring for 24 hours.

[0026] The third aspect of the present application provides an anti-tumor drug, comprising a hydrophobic anticancer drug and a carrier loaded with the hydrophobic anticancer drug, wherein the carrier is the nano-targeting drug-loaded micelle described above, and the hydrophobic anticancer drug is loaded in the hydrophobic inner core of the nano drug-loaded micelle.

[0027] The hyaluronic acid modified hydrophobic chlorin e6 group and the endoplasmic reticulum targeting signal peptide form a nano-targeting micelle, which can well encapsulate the hydrophobic anticancer drug to form a stable, particle size controllable, and combined therapy functional nano drug-loaded micelle, and can increase the water solubility of the hydrophobic anticancer drug, reduce the toxicity, and specifically target and enhance the half-life of the drug-loaded micelle in the body circulation.

[0028] According to some embodiments of the present application, the mass ratio of the hydrophobic anticancer drug to the carrier is (0.1-0.5):1.

[0029] According to some embodiments of the present application, the hydrophobic anticancer drug is at least one of doxorubicin, paclitaxel or camptothecin.

[0030] According to some embodiments of the present application, the above hydrophobic anticancer drug only needs to dissolve the above nano-targeting drug-loaded micelle and the hydrophobic anticancer drug in an organic solvent to realize automatic loading of the hydrophobic anticancer drug, and the preparation method has simple process, controllable conditions, high yield, and controllable drug particle size.

[0031] According to some embodiments of the present application, after the above nano-targeting drug-loaded micelle and the hydrophobic anticancer drug are dissolved in an organic solvent, the concentration of the nano drug-loaded micelle is preferably 10-500 g / L, and the concentration of the hydrophobic anticancer drug is in the range of 1-100 g / L.

[0032] Other features and advantages of the present application will be set forth in the description that follows, and in part will become apparent to those skilled in the art upon examination of the following or can be learned by practice of the application. BRIEF DESCRIPTION OF DRAWINGS

[0033] The above and / or additional aspects and advantages of the present application will become apparent and be readily understood from the following description, taken in conjunction with the following drawings, in which:

[0034] Figure 1 is a structural schematic diagram of the nano-targeting drug-loaded micelle of Example 1 of the present application;

[0035] Figure 2 is a dynamic light scattering particle size distribution diagram of the nano-targeting drug-loaded micelle of Example 1 of the present application;

[0036] Figure 3 is a transmission electron microscope diagram of the nano-targeting drug-loaded micelle of Example 1 of the present application;

[0037] Figure 4is the distribution diagram of the nanometer targeted drug-loaded micelles of embodiment 1 of the present application in cells;

[0038] Figure 5 is a killing effect diagram of the nanometer targeted drug-loaded micelles of embodiment 1 of the present application on tumor cells. DETAILED DESCRIPTION

[0039] The following are specific embodiments of the present application, and the technical solutions of the present application are further described in combination with the embodiments, but the present application is not limited to these embodiments.

[0040] The reagents, methods and equipment used in the present application are all conventional reagents, methods and equipment in the technical field unless otherwise specified.

[0041] Embodiment 1

[0042] Embodiment 1 provides a kind of nanometer targeted drug-loaded micelles (HA-Ce6-RACR), and its preparation method is as follows:

[0043] S1, 20g hyaluronic acid (HA, MW10K) is dissolved in 5L deionized water, 30g adipic dihydrazide (ADH) is added to its aqueous solution, and the pH value is adjusted to 4.75 with 0.1M hydrochloric acid solution under rapid stirring. 19g EDC is added to the above reaction solution, and the pH value is adjusted to 4.75- after using 0.1M hydrochloric acid solution, and then reacted at room temperature for 1h to obtain white solid HA-ADH. 10g HA-ADH is dissolved in 2.4L formamide for standby;

[0044] S2, 24g chlorin e6 (Ce6) and 29g signal peptide Fmoc-RACR are dissolved in formamide solution after activation by EDC and NHS, wherein the molar ratio of EDC, NHS and chlorin e6 is 1.2:1.2:1;And the mixture is added dropwise to the formamide solution of the above HA-ADH, and after the dropwise addition is completed, it is reacted at room temperature for 24h. After the reaction is completed, the liquid is reduced pressure suction filtration, and the insoluble substances are removed. The filtrate is placed in a cellulose dialysis bag with a molecular weight cut-off of 7000Da, and dialyzed in distilled water for 24h to obtain nanometer targeted drug-loaded micelles with an average particle size of 180nm.

[0045] The structure diagram of the nanometer targeted drug-loaded micelles is shown in Figure 1 , the nanometer targeted drug-loaded micelles are core-shell structure, the inner core is chlorin e6 group connected to the hyaluronic acid straight chain through adipic dihydrazide group; the outer shell is hyaluronic acid; and the outer surface of the outer shell has endoplasmic reticulum targeting signal peptide Fmoc-RACR connected to the hyaluronic acid straight chain through adipic dihydrazide group.

[0046] Embodiment 2

[0047] Example 2 provides a nano-targeting drug-loaded micelles, the preparation method is as follows:

[0048] S1, 20g hyaluronic acid (HA, MW10K) is dissolved in 5L deionized water, 40g adipic dihydrazide (ADH) is added to the aqueous solution, and the pH value is adjusted to 4.75 with 0.1M hydrochloric acid solution under rapid stirring. 25g EDC is added to the above reaction solution, and the pH value is adjusted to 4.75 with 0.1M hydrochloric acid solution, and then reacted at room temperature for 1h to obtain white solid HA-ADH. 10g HA-ADH is dissolved in 2.4L formamide for use.

[0049] S2, 18g chlorin e6 (Ce6) and 21g signal peptide Fmoc-RACR are dissolved in formamide solution after activation by EDC and NHS, wherein the molar ratio of EDC, NHS and chlorin e6 is 1.2:1.2:1; and the mixture is added dropwise to the above HA-ADH formamide solution, and after the dropwise addition is completed, it is reacted at room temperature for 24h. After the reaction is completed, the liquid is reduced pressure filtration to remove large particle insoluble substances. The filtrate is placed in a cellulose dialysis bag and dialyzed in distilled water for 24h to obtain nano-targeting drug-loaded micelles.

[0050] Example 3

[0051] Example 3 provides a nano-targeting drug-loaded micelles, the preparation method is as follows:

[0052] S1, 20g hyaluronic acid (HA, MW10K) is dissolved in 5L deionized water, 40g adipic dihydrazide (ADH) is added to the aqueous solution, and the pH value is adjusted to 4.75 with 0.1M hydrochloric acid solution under rapid stirring. 25g EDC is added to the above reaction solution, and the pH value is adjusted to 4.75 with 0.1M hydrochloric acid solution, and then reacted at room temperature for 1h to obtain white solid HA-ADH. 10g HA-ADH is dissolved in 2.4L formamide for use.

[0053] S2, 18g chlorin e6 (Ce6) and 21g signal peptide Fmoc-RACR are dissolved in formamide solution after activation by EDC and NHS, wherein the molar ratio of EDC, NHS and chlorin e6 is 1.2:1.2:1; and the mixture is added dropwise to the above HA-ADH formamide solution, and after the dropwise addition is completed, it is reacted at room temperature for 24h. After the reaction is completed, the liquid is reduced pressure filtration to remove large particle insoluble substances. The filtrate is placed in a cellulose dialysis bag and dialyzed in distilled water for 24h to obtain nano-targeting drug-loaded micelles.

[0054] Example 4

[0055] Example 4 provides a nano-targeting drug-loaded micelles, the preparation method is as follows:

[0056] S1, 20 g of hyaluronic acid (HA, MW 100 K) was dissolved in 5 L of deionized water, 3 g of adipohydrazide (ADH) was added to the aqueous solution, and the pH was adjusted to 4.75 with a 0.1 M hydrochloric acid solution under rapid stirring. 1.9 g of EDC was added to the above reaction solution, and the pH was adjusted to 4.75 with a 0.1 M hydrochloric acid solution, and then the reaction was carried out at room temperature for 1 h to obtain white solid HA-ADH. 10 g of HA-ADH was dissolved in 2.4 L of formamide for use.

[0057] S2, 2.4 g of chlorin e6 (Ce6) and 1.4 g of signal peptide Fmoc-RACR were dissolved in a formamide solution after activation with EDC and NHS, wherein the molar ratio of EDC, NHS, and chlorin e6 was 1.2:1.2:1; and the mixture was added dropwise to the formamide solution of HA-ADH described above, and after the dropwise addition was completed, the reaction was carried out at room temperature for 24 h. After the reaction was completed, the liquid was filtered under reduced pressure to remove insoluble large particles. The filtrate was placed in a cellulose dialysis bag with a molecular weight cut-off of 7000 Da, and dialyzed in distilled water for 24 h to obtain nanometer targeted drug-loaded micelles.

[0058] Example 5

[0059] Example 5 provides an anti-tumor drug, and the preparation method is as follows:

[0060] The nanometer targeted drug-loaded micelles prepared in Example 1 and the hydrophobic drug paclitaxel were dissolved in formamide, and the reaction was carried out at 20°C for 24 h to obtain an anti-tumor drug. The mass ratio of the nanometer targeted drug-loaded micelles and the hydrophobic drug was 1:0.2.

[0061] Example 6

[0062] Example 6 provides an anti-tumor drug, and the preparation method is as follows:

[0063] The nanometer targeted drug-loaded micelles prepared in Example 1 and the hydrophobic drug doxorubicin were dissolved in formamide, and the reaction was carried out at 20°C for 24 h to obtain an anti-tumor drug. The mass ratio of the nanometer targeted drug-loaded micelles and the hydrophobic drug was 1:0.15.

[0064] Example 7

[0065] Example 7 provides an anti-tumor drug, and the preparation method is as follows:

[0066] The nanometer targeted drug-loaded micelles prepared in Example 1 and the hydrophobic drug paclitaxel were dissolved in formamide, and the reaction was carried out at 20°C for 24 h to obtain an anti-tumor drug. The mass ratio of the nanometer targeted drug-loaded micelles and the hydrophobic drug was 1:0.2.

[0067] Comparative Example 1

[0068] Comparative Example 1 also provides a nano-targeting drug-loaded micelles (HA-Ce6) which is prepared by substantially the same method as Example 1, except that it does not contain the endoplasmic reticulum targeting signal peptide Fmoc-RACR.

[0069] Performance test

[0070] 1. The nano-targeting drug-loaded micelles prepared in Example 1 were dissolved in an appropriate amount of PBS solution with a pH of 7.4 to prepare a 1 mg / mL solution. The above solution was diluted and measured using a dynamic light scattering particle size tester. The above solution was also repeatedly immersed in the sample with a copper mesh containing a cellulose acetate film. After standing for 2 minutes, the nanoparticles were deposited on the copper mesh, and the excess water was removed with filter paper, and 1% phosphotungstic acid was added dropwise for negative staining. After standing for 3 minutes, the excess dye solution was removed and the morphology of the nanoparticles was observed using a transmission electron microscope (TEM). As shown in the dynamic light scattering particle size tester, the average particle size of the nano-targeting drug-loaded micelles was 180 nm, and the distribution was uniform; the morphology of the nanoparticles was observed by transmission electron microscopy (TEM) (Figure 2), and the size of the HA-Ce6-RACR nanoparticles was 150-250 nm. Figure 2 Figure 3

[0071] 2. 4T1 cells were inoculated into cell culture dishes, and after the cells adhered overnight, the old culture medium was removed, and fresh culture medium containing 5 μM Ce6 concentration of polymer micelles prepared in Comparative Example 1 and nano-targeting drug-loaded micelles prepared in Example 1 was added, respectively. After 2 h of incubation, the culture medium was removed, washed twice with PBS, stained with Hoechst 33342, washed twice with PBS, and 200 μl of PBS solution was added. The distribution of Ce6 molecules in the cells was observed using a laser confocal microscope. As shown in Figure 3, compared with Comparative Example 1 without endoplasmic reticulum targeting, the nano-targeting drug-loaded micelles prepared in Example 1 had better co-localization effect with the endoplasmic reticulum. Figure 4

[0072] 3. The killing effect of the nano-targeting drug-loaded micelles prepared in the present application on A549 cells was detected by CCK8 method. Blank control group, Ce6 group, Comparative Example 1 group, and Example 1 group were set. A549 cells (200 μL, 2×105 / well) were inoculated into a 96-well plate and cultured in a CO2 incubator; after 12 h of plate culture, 2.5 μg / mL of Ce6 was added to each group, and incubated for 4 h. 10 μL of Cell Counting Kit-8 (CCK8) reagent was added to each well, and incubated for another 4 h. The absorbance at 450 nm was measured by a multifunctional enzyme label instrument.

[0073] The results are shown in Figure 4. Figure 5 ​​​As shown, the HA-Ce6-RACR nanoparticles prepared in Example 1 have stronger killing ability to tumor cells compared to free Ce6 and HA-Ce6 particles without endoplasmic reticulum targeting ligand. The test results of the drug-loaded materials prepared in other examples are basically consistent with Example 1, and to avoid redundancy, they are not shown one by one.

[0074] The above is described in detail in combination with the embodiments of the present application, but the present application is not limited to the above-described embodiments, and various changes can be made within the knowledge range possessed by those skilled in the art without departing from the purpose of the present application.

Claims

1. A nano-targeted drug-loaded micelle, characterized in that, The nano-targeting drug-loaded micelles have a core-shell structure, the core is a dihydrophenyl e6 group connected to a hyaluronic acid straight chain through a adipohydrazide group; the shell is hyaluronic acid; the outer surface of the shell also has an endoplasmic reticulum targeting signal peptide connected to the hyaluronic acid straight chain through an adipohydrazide group; the mass ratio of the dihydrophenyl e6 group, the endoplasmic reticulum targeting signal peptide and the hyaluronic acid is (0.2-3):(0.1-3):1; The endoplasmic reticulum targeting signal peptide is Fmoc-RACR polypeptide; The nano-targeting drug-loaded micelles are prepared by the following steps: S1, using adipohydrazide to chemically modify hyaluronic acid in an acidic aqueous solution to obtain a HA-ADH derivative; S2, mixing HA-ADH derivative, dihydrophenyl e6 activated by EDC and NHS, endoplasmic reticulum targeting signal peptide and solvent to react, after the reaction is completed, the solution after reaction is dialyzed to obtain the nano-targeting drug-loaded micelles.

2. The nano-targeted drug-loaded micelles according to claim 1, characterized in that, The average particle size of the nano-targeting drug-loaded micelles is 50-200 nm. 3.The nano-targeting drug-loaded micelles of claim 1, wherein, The method for chemically modifying hyaluronic acid with adipohydrazide in an acidic aqueous solution is as follows: After adding the adipohydrazide to the hyaluronic acid solution, the pH value of the solution is adjusted to 4.5-4.8, a condensing agent is added, and the pH value is kept at 4.5-4.8 to make the adipohydrazide and hyaluronic acid condense. 4.The nano-targeting drug-loaded micelles of claim 1, wherein, The solvent is at least one selected from formamide, DMF or DMSO.

5. An antitumor agent, characterized by comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof. A hydrophobic anticancer drug and a carrier loaded with the hydrophobic anticancer drug, the carrier is the nano-targeting drug-loaded micelles of any one of claims 1-4, and the hydrophobic anticancer drug is loaded in the hydrophobic core of the nano drug-loaded micelles.

6. The antitumor drug according to claim 5, wherein The mass ratio of the hydrophobic anticancer drug to the carrier is (0.1-0.5):1.

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