Organic molecule self-assembly body serving as material-free drug loading system and preparation method and application of organic molecule self-assembly body

Abstract

The invention belongs to the field of biological medicine, and particularly relates to an organic molecule self-assembly serving as a material-free medicine carrying system and a preparation method and application thereof. The invention provides a self-assembly body, the self-assembly body is composed of steroid series molecules, and a material-free drug loading system formed by self-assembly of the steroid series molecules is prepared by optimizing the molecular structure and critical aggregation concentration of the steroids. The material-free drug loading system can be used for loading drugs and preparing drug nano-composites. In the nano compound, the drug effect of the drug is improved, and the effects of reducing toxicity and improving efficiency can be achieved. Furthermore, if the used steroid molecules have biological activity, the pharmacological activity of the steroid molecules is not influenced after the steroid molecules are prepared into the drug nano-composite. The material-free drug loading system prepared by the invention has the advantages of no carrier and two nano preparations taking the traditional material as the carrier, and has a good prospect in the aspect of developing novel nano drug preparations.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to an organic molecular self-assembly as a material-free drug-carrying system, a preparation method and use thereof. Background Art

[0002] Whether a drug molecule can fully exert its therapeutic effect depends not only on the physiological activity brought by its single-molecule chemical structure itself, but also on its preparation form. Because the latter determines how the drug will enter the human body, and how it will be absorbed, distributed, metabolized and excreted after entering the human body, and thus determines its actual efficacy and possible side effects. With the continuous integration of nanotechnology and biomedical technology, nanoformulation has become an extremely important development direction in the field of modern drug research and development (Biomed Phys Eng Express.2023,9(5).doi:10.1088 / 2057-1976 / acedb2). Preparing drugs into nanoformulations can bring advantages that drug monomers cannot have in terms of targeting, sustained release, and improved bioavailability, thereby achieving the purpose of reducing toxicity and increasing efficacy (Molecules.2024,29(9):2073.doi:10.3390 / molecules29092073.). For example, nano-sized anti-tumor drug preparations can target tumor tissues through the enhanced permeation and retention (EPR) effect, thereby enhancing the tumor-killing effect while reducing toxicity to normal tissues (Front Pharmacol. 2024, 15: 1363346. doi: 10.3389 / fphar.2024.1363346.); preparing water-soluble local anesthetics into nano-preparations can achieve long-term sustained release, thereby meeting the needs of long-term postoperative analgesia (Drug Des Devel Ther. 2023, 17: 2639-2655. doi: 10.2147 / DDDT.S417051.).

[0003] At present, the strategies for preparing nanoformulations are mainly divided into two categories: carrier-free and carrier-based. The carrier-free strategy is to prepare the drug itself into nanoscale crystals through bottom-up crystallization or top-down physical crushing and dispersion (Recent Pat Nanotechnol. 2023, 17 (4): 307-326. doi: 10.2174 / 1872210516666220523120313.). However, this strategy is only applicable to drugs with poor water solubility, and there are many problems in the uniformity and controllability of crystal morphology and size, as well as the dispersibility and stability of the preparation, which limits its wide application. The carrier-based strategy uses various nanobiomaterials as carriers to form nanoformulations by adsorbing and loading drugs (Pharmaceutics, 2024, 16 (10): 1339. doi: 10.3390 / pharmaceutics16101339.). Although the use of carrier materials makes the preparation of nanoformulations more precise and controllable, and can obtain nanoformulations with uniform shape and size and good dispersion, the introduction of additional materials also brings new problems. For example, due to the consideration of efficacy and production cost, there are higher requirements for drug loading and encapsulation rate; most carrier materials are non-natural ingredients with large molecular weight, and their own degradability and biocompatibility bring new safety issues; additional materials and the more complex preparation production process they bring will inevitably lead to higher costs.

[0004] In addition to the above-mentioned two types of nanoformulations, carrier-free and carrier-containing (using traditional materials as carriers), recent studies have proposed a material-free drug-carrying nanosystem (Carrier-Free Nanosystems). Different from the above-mentioned classification of nanoformulations based on whether or not a carrier is included, a material-free drug-carrying nanosystem can be regarded as a category between the two. It refers to some small molecules that have physiological activity and can be used as therapeutic drugs, and can form nanostructures by self-assembly under certain conditions. Among them, the material-free drug-carrying system based on the self-assembly of drug molecules has begun to attract people's attention in recent years (Med Res Rev. Sep 2020; 40 (5): 1754-1775. doi: 10.1002 / med.21669, Emerging carrier-free nanosystems based on molecular self-assembly of pure drugs for cancer therapy). It does not rely on exogenous carrier materials, reduces the immune response caused by the carrier; has a high drug loading capacity; is simple to prepare; and can itself be used as a drug for disease treatment. The material-free drug-carrying nanosystem can also be used as a drug carrier to load other drugs, thereby obtaining a dual-active or multi-active drug preparation. The dual-active or multi-active drug preparation can not only combine the advantages of the above two nanoformulations (carrier-free and traditional material-based), but also the combination of two or more drug molecules may play a synergistic role of "reducing toxicity and increasing efficacy". Therefore, it is of great significance for the development of new material-free drug-carrying nanosystems.

[0005] Therefore, exploring new material-free drug delivery systems provides more options for the development of new nanodrug preparations and will greatly promote the development of nanodrug preparations. Summary of the invention

[0006] In view of the defects of the prior art, the present invention provides a steroid series organic molecular self-assembly, the purpose of which is to prepare a new material-free drug delivery system, providing more options for the development of new nano drug preparations.

[0007] The present invention provides an organic molecule self-assembly, which is prepared by self-assembly of steroid organic molecules; the steroid organic molecules are selected from at least one of steroid organic molecules with a pregnane skeleton structure and an estrastane skeleton structure;

[0008] When the steroid organic molecule is selected from at least one steroid organic molecule with a pregnane skeleton structure, the concentration of the steroid organic molecule is ≥1 mg / mL;

[0009] When the steroid organic molecule is selected from at least one steroid organic molecule with an estraneskeletal structure, the concentration of the steroid organic molecule is ≥0.4 mg / mL.

[0010] Preferably, when the steroid organic molecule is selected from at least one steroid organic molecule with a pregnane skeleton structure, the concentration of the steroid organic molecule is ≥2 mg / mL; when the steroid organic molecule is selected from at least one steroid organic molecule with an estraneskeletal structure, the concentration of the steroid organic molecule is ≥0.6 mg / mL.

[0011] Preferably, the steroid organic molecule with a pregnane skeleton structure is selected from at least one of hydrocortisone, betamethasone, dexamethasone, methylprednisolone, prednisolone or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable ester thereof;

[0012] And / or, the steroid organic molecule with an estranestane skeleton structure is selected from at least one of estradiol, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable ester thereof.

[0013] Preferably, the steroid organic molecule is at least one selected from hydrocortisone sodium succinate, betamethasone sodium phosphate, dexamethasone sodium phosphate, methylprednisolone sodium succinate, prednisolone sodium phosphate, and beta-estradiol 17-hemisuccinate.

[0014] Preferably, the specific conditions for the self-assembly are: stirring for 4-6 hours at a temperature of 20-25° C., a stirring speed of 600-1000 rpm, and then standing at a temperature of 2-20° C. for 12-18 hours.

[0015] The present invention provides a method for preparing any of the above-mentioned organic molecular self-assemblies, which comprises: preparing the self-assembly of steroid organic molecules; the steroid organic molecules are selected from at least one of steroid organic molecules with a pregnane skeleton structure and an estranes skeleton structure;

[0016] When the steroid organic molecule is selected from at least one steroid organic molecule with a pregnane skeleton structure, the concentration of the steroid organic molecule is ≥1 mg / mL;

[0017] When the steroid organic molecule is selected from at least one steroid organic molecule with an estraneskeletal structure, the concentration of the steroid organic molecule is ≥0.4 mg / mL.

[0018] The present invention provides use of any of the above-mentioned organic molecular self-assemblies as drug carriers and / or active ingredients in the preparation of drugs.

[0019] The present invention provides a pharmaceutical composition, comprising any one of the organic molecular self-assemblies described above loaded with a drug; the drug is selected from at least one of a pharmaceutically active compound containing a tertiary amine group and a hydrophobic aromatic ring structure, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable ester thereof;

[0020] The molar ratio of the steroid organic molecules used to form the organic molecular self-assembly to the drug is 1:2.5-40.

[0021] Preferably, the pharmaceutical composition is prepared by the following steps: the drug is mixed with the steroid organic molecules used to constitute the organic molecule self-assembly.

[0022] Preferably, the specific process of the preparation is: at a temperature of 20-25°C, the steroid organic molecule is added dropwise to the drug, mixed under stirring, the stirring speed is 600-1000 rpm, the stirring time is 4-6 hours, and then allowed to stand at a temperature of 2-20°C for 12-18 hours;

[0023] And / or, the pH of the steroid organic molecule is adjusted to ≤ 7.0 using a pH adjuster before being mixed with the drug.

[0024] The term "organic molecular self-assembly" refers to a nanostructure formed by self-assembly of organic molecules, wherein the organic molecules may be organic molecules with pharmaceutical activity or organic molecules without pharmaceutical activity.

[0025] The present invention provides a self-assembly, which is composed of steroid series molecules. By optimizing the molecular structure of these steroids and their critical aggregation concentration, a material-free drug delivery system formed by the self-assembly of steroid series molecules is prepared. The material-free drug delivery system can be loaded with drugs and used as a drug carrier to prepare drug nanocomplexes. In the nanocomplex, the efficacy of the drug is improved, and the effect of "reducing toxicity and increasing efficacy" can be achieved. Furthermore, if the steroid molecules used themselves have biological activity, then after they are prepared into the drug nanocomplex of the present invention, the pharmacological activity of the steroid molecules themselves is not affected. The material-free drug delivery system prepared by the present invention has the advantages of both carrier-free and traditional material-based nanopreparations, and has good prospects in the development of new nanopharmaceutical preparations.

[0026] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0027] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Transmission electron microscopy image of nanospheres formed by HSS self-assembly;

[0029] Figure 2 This is the change of zeta potential after HSS is loaded with different proportions of DHM;

[0030] Figure 3 Transmission electron microscopy images of nanosphere preparations formed by HSS loading different proportions of DHM;

[0031] Figure 4 The graph shows the change of zeta potential after HSS is loaded with different proportions of LB;

[0032] Figure 5 Transmission electron microscopy images of nanosphere preparations formed by HSS loading different proportions of LB;

[0033] Figure 6 Transmission electron microscopy image of nanospheres formed by BSP self-assembly;

[0034] Figure 7 The graph shows the change of zeta potential after BSP is loaded with different proportions of ATL;

[0035] Figure 8 Transmission electron microscopy images of nanosphere preparations formed by BSP loading different proportions of ATL;

[0036] Fig. 9 This is the result of the change of zeta potential after BSP is loaded with different proportions of ROP;

[0037] Fig.10 This is the result diagram of the change of zeta potential after BSP is loaded with PMZ in different proportions;

[0038] Fig.11 Transmission electron microscopy images of nanosphere preparations formed by BSP loading with different proportions of PMZ;

[0039] Fig.12 Transmission electron microscopy image of DMSP self-assembly to form nanospheres;

[0040] Fig.13 This is the result of the change of zeta potential after DMSP is loaded with different proportions of BUP;

[0041] Fig.14Transmission electron microscopy images of nanosphere preparations formed by DMSP loading different proportions of BUP;

[0042] Fig.15 This is the result of the change of zeta potential after DMSP is loaded with different proportions of OCD;

[0043] Fig.16 Transmission electron microscopy images of nanosphere preparations formed by DMSP loading different ratios of OCD;

[0044] Fig.17 Transmission electron microscopy image of MPSS self-assembly to form nanospheres;

[0045] Fig.18 Atomic force electron microscopy image of MPSS self-assembly to form nanospheres;

[0046] Fig.19 This is the change of zeta potential after MPSS is loaded with different proportions of TDZ;

[0047] Fig. 20 Transmission electron microscopy images of nanosphere preparations formed by MPSS loaded with different proportions of TDZ;

[0048] Fig.21 This is the result of the change of zeta potential after MPSS is loaded with different proportions of LB;

[0049] Fig. 22 Transmission electron microscopy images of nanosphere preparations formed by MPSS loading with different proportions of LB;

[0050] Fig.23 This is the result diagram of the change of zeta potential after MPSS is loaded with different proportions of MP;

[0051] Fig.24 This is the atomic force electron microscopy image of MPSS after loading ROP;

[0052] Fig.25 Transmission electron microscopy image of PNSP self-assembly to form nanospheres;

[0053] Fig.26 The graph shows the change of zeta potential after PNSP is loaded with different proportions of ROP;

[0054] Fig. 27 Transmission electron microscopy images of nanosphere preparations formed by PNSP loaded with different proportions of ROP;

[0055] Fig.28 Transmission electron microscopy image of BEH self-assembly to form nanospheres;

[0056] Fig.29 This is the result of the change of zeta potential after BEH is loaded with different proportions of DHM;

[0057] Fig.30 Transmission electron microscopy images of nanosphere preparations formed by BEH loading with different proportions of DHM;

[0058] Fig.31 This is a graph showing the results of determining the critical aggregation concentration of HSS;

[0059] Fig.32 This is a graph showing the results of the determination of the critical aggregation concentration of BSP;

[0060] Fig.33 This is the result of the determination of the critical aggregation concentration of DMSP;

[0061] Fig.34 This is the result of the determination of the critical aggregation concentration of MPSS;

[0062] Fig.35 This is a graph showing the results of determining the critical aggregation concentration of PNSP;

[0063] Fig.36 This is a graph showing the results of the determination of the critical aggregation concentration of BEH;

[0064] Fig.37 The results of systemic inflammatory factor levels after administration of ROP and ROP-MPSS in rats (*: p<0.05);

[0065] Fig.38 The results of LB and LB-MPSS on the survival rate of A549 cells at different drug concentrations (*: p<0.05, ***: p<0.001);

[0066] Fig.39 The results of BUP and BUP-DMSP on the survival rate of A549 cells at different drug concentrations (*: p<0.05, **: p<0.01);

[0067] Fig.40 The graph shows the survival rate of A549 cells under different drug concentrations of ROP and ROP-PNSP (*: p<0.05, **: p<0.01). DETAILED DESCRIPTION

[0068] In the following examples and experimental examples, reagents and materials not particularly described are all commercially available.

[0069] Example 1 Preparation of hydrocortisone sodium succinate nanospheres

[0070] 1. Preparation of Hydrocortisone Sodium Succinate Nanospheres

[0071] Weigh a certain amount of hydrocortisone sodium succinate (HSS, hereinafter referred to as HSS), add ultrapure water to prepare a 10 mg / mL HSS mother solution (i.e., a 1% HSS mother solution by mass), and perform water bath ultrasound for 10 minutes to fully dissolve it. Stir magnetically at 600 rpm for 4 hours at room temperature, and place at 4°C overnight to allow it to fully self-assemble.

[0072] 2. Characterization of Hydrocortisone Sodium Succinate Nanospheres

[0073] Transmission electron microscopy (TEM) was used to observe the nanostructures formed by HSS self-assembly. Figure 1 As shown, HSS forms nanospheres with a diameter of 10-50 nm in aqueous solution, which proves that the method of this example successfully prepares hydrocortisone sodium succinate nanospheres.

[0074] Example 2 Nanoformulation of hydrocortisone sodium succinate nanospheres loaded with diphenhydramine hydrochloride (DHM-HSS)

[0075] 1. Preparation of DHM-HSS Nanoformulation

[0076] The concentration of HSS was fixed at 5 mg / mL, and carrier-free nanoformulations were prepared according to the molar concentration ratios of HSS to diphenhydramine hydrochloride (DHM, molecular weight 291.82) of 1:5 and 1:10 (Table 1).

[0077] Table 1. Ratio of raw materials in DHM-HSS nanoformulation

[0078] HSS:DHM HSS concentration (mg / mL) DHM concentration (mg / mL) pH 1:5 5 15.06 6.15 1:10 5 30.12 5.92

[0079] The specific preparation process is as follows:

[0080] Weigh a certain amount of DHM, add an appropriate amount of ultrapure water, and perform water bath sonication for 15 minutes to completely dissolve it; at room temperature, the DHM solution was magnetically stirred at 600 rpm, and 0.4 mL of 1% HSS mother solution was slowly and uniformly added (20 μL / s) under stirring to prepare the preparations with the corresponding proportions shown in Table 2. After stirring for 4 hours, the mixture was left overnight to allow the system to fully complete the loading of drug molecules by self-assembled nanospheres.

[0081] 2. Characterization of DHM-HSS Nanoformulations

[0082] The zeta potential of the preparation was measured using a Malvern nanometer particle size potential analyzer. Figure 2), each sample was tested 3 times, and the results were averaged. The results showed that HSS nanospheres were negatively charged, with a zeta potential of -28.4mV. After loading DHM, the negative charge of the nanospheres in the preparation decreased, and with the increase of drug loading, the negative charge reduction also increased. In addition, it can be observed under TEM that with the increase of DHM ratio, the microstructure of the preparation is 50-100nm nanospheres ( Figure 3 ). It is proved that the method of this example successfully prepared DHM-HSS nanoformulations with different molar ratios.

[0083] Example 3 Nanoformulation of hydrocortisone sodium succinate nanospheres loaded with levobupivacaine hydrochloride (LB-HSS)

[0084] 1. Preparation of LB-HSS nanoformulation

[0085] The concentration of HSS was fixed at 2 mg / mL, and carrier-free nanoformulations were prepared according to the molar concentration ratios of HSS to levobupivacaine hydrochloride (LB) of 1:2.5, 1:10, and 1:40 (Table 2).

[0086] Table 2. Ratio of raw materials in LB-HSS nanoformulation

[0087] HSS:LB HSS concentration (mg / mL) DHM concentration (mg / mL) pH 1:2.5 2 3.35 5.89 1:10 2 13.41 5.71 1:40 2 53.65 5.39

[0088] The specific preparation process is as follows:

[0089] Weigh a certain amount of LB, add an appropriate amount of ultrapure water, and perform water bath sonication for 30 minutes to completely dissolve it; at room temperature, the LB solution was magnetically stirred at 600 rpm, and 0.4 mL of 1% HSS mother solution was slowly and uniformly added (20 μL / s) under stirring to prepare the preparations with the corresponding proportions shown in Table 3. After stirring for 4 hours, leave it overnight to allow the system to fully complete the loading of drug molecules by self-assembled nanospheres.

[0090] 2. Characterization of LB-HSS Nanoformulations

[0091] The zeta potential of the preparation was measured using a Malvern nanometer particle size potential analyzer. Figure 4 ), the results suggest that after HSS nanospheres are loaded with LB, the negative charge of HSS nanospheres is significantly reduced, and decreases with the increase of the total amount of LB. In addition, it can be observed under TEM that with the increase of LB ratio, the microstructure of the preparation is 30-150nm nanospheres ( Figure 5 ). It is proved that the method of this embodiment successfully prepared the LB-HSS nanoformulation, and the larger the proportion of LB, the larger the particle size of the LB-HSS nanoformulation.

[0092] Example 4 Preparation of Betamethasone Sodium Phosphate Nanospheres

[0093] 1. Preparation of Betamethasone Sodium Phosphate Nanospheres

[0094] Weigh a certain amount of betamethasone sodium phosphate (BSP), add ultrapure water to prepare a 10 mg / mL BSP mother solution (i.e., a 1% BSP mother solution by mass), and dissolve it completely in a water bath by ultrasonic for 20 minutes. Stir it magnetically at 600 rpm for 4 hours at room temperature, and leave it at 4°C overnight to allow it to fully self-assemble.

[0095] 2. Characterization of Betamethasone Sodium Phosphate Nanospheres

[0096] TEM was used to observe the nanostructures formed by BSP self-assembly. Figure 6 As shown, BSP forms nanospheres with a diameter of 10-50 nm in aqueous solution, which proves that the method of this example successfully prepares betamethasone sodium phosphate nanospheres.

[0097] Example 5: Nanoformulation of Betamethasone Sodium Phosphate Nanospheres Loaded with Amitriptyline Hydrochloride (ALT-BSP)

[0098] 1. Preparation of ALT-BSP Nanoformulation

[0099] A 5 mg / mL BSP solution was used to load amitriptyline hydrochloride (ATL), and carrier-free nanoformulations were prepared according to the molar concentration ratio of BSP to ATL of 1:5 and 1:10 (Table 3). The preparation method was: first, the pH value of BSP was adjusted to 6.0 with 1M hydrochloric acid, and then the preparation was carried out according to the method described in Example 2, except that the preparation was prepared into the corresponding proportions shown in Table 3.

[0100] Table 3. Ratio of raw materials in ALT-BSP nanoformulation

[0101] BSP:ATL BSP concentration (mg / mL) ATL concentration (mg / mL) pH 1:5 5 15.20 5.66 1:10 5 30.39 5.66

[0102] 2. Characterization of ALT-BSP Nanoformulation

[0103] The zeta potential of the preparation was detected using a Malvern nanoparticle size potential instrument. The BSP nanospheres were negatively charged, with a zeta potential of -24.9 mV. After loading with ATL, the negative charge of the nanospheres was significantly reduced, and they were positively charged ( Figure 7 In addition, it can be observed under TEM that with the increase of ATL ratio, the microstructure of the preparation is 40-80nm nanospheres ( Figure 8 ). This proves that the method of this example successfully prepared the ALT-BSP nanosphere preparation.

[0104] Example 6 Betamethasone sodium phosphate nanospheres loaded with ropivacaine hydrochloride (ROP-BSP) nanoformulation

[0105] 1. Preparation of ROP-BSP Nanoformulation

[0106] A 2 mg / mL BSP solution was used to load ropivacaine hydrochloride (ROP) with a molar concentration of 2.5 times and 10 times that of BSP, and a 1.333 mg / mL BSP was used to load ROP with a molar concentration of 30 times that of BSP. The specific parameter ratios are shown in Table 4. The preparation conditions are the same as those in Example 5, except that the preparations are prepared into the corresponding proportions shown in Table 4.

[0107] Table 4. Ratio of raw materials in ROP-BSP nanoformulations

[0108] BSP:ROP BSP concentration (mg / mL) ROP concentration (mg / mL) pH 1:2.5 2 3.18 6.84 1:10 2 12.74 6.26 1:30 1.333 25.47 5.99

[0109] 2. Characterization of ROP-BSP Nanoformulations

[0110] When BSP was loaded with ROP, the negative charge of the nanospheres decreased and decreased significantly with the increase of the total amount of ROP ( Fig. 9 ). It is proved that the method of this example successfully prepared the ROP-BSP nanoformulation.

[0111] Example 7 Betamethasone sodium phosphate nanospheres loaded with promethazine hydrochloride (PMZ-BSP) nanoformulation

[0112] 1. Preparation of PMZ-BSP Nanoformulation

[0113] Use 5mg / mL BSP solution to load promethazine hydrochloride (PMZ for short), and prepare carrier-free nanoformulations according to the molar concentration ratio of BSP to PMZ of 1:5 and 1:10 (Table 5). Weigh a quantitative amount of PMZ, add an appropriate amount of ultrapure water, and completely dissolve it in a water bath ultrasound for 20 minutes. At room temperature and 600rpm magnetic stirring, slowly (20μL / s) add 0.4mL of 1% BSP mother solution (pH 6.0), continue stirring for 4 hours, and obtain the nanoformulations of the corresponding proportions shown in Table 5, and place it at 4°C overnight to stabilize the system.

[0114] Table 5. Ratio of raw materials in PMZ-BSP nanoformulations

[0115] BSP:PMZ BSP concentration (mg / mL) PMZ concentration (mg / mL) pH 1:5 5 15.534 6.24 1:10 5 31.07 5.47

[0116] 2. Characterization of PMZ-BSP Nanoformulations

[0117] After loading with PMZ, the negative charge of BSP nanospheres was significantly reduced, and the PMZ-BSP nanospheres were positively charged ( Fig.10 In addition, it can be observed under TEM that with the increase of PMZ ratio, the microstructure of the preparation is 50-150nm nanospheres ( Fig.11 ). It is proved that the method of this embodiment successfully prepared PMZ-BSP nanoformulation.

[0118] Example 8 Preparation of Dexamethasone Sodium Phosphate Nanospheres

[0119] 1. Preparation of Dexamethasone Sodium Phosphate Nanospheres

[0120] A certain amount of dexamethasone sodium phosphate (DMSP) was weighed and added to ultrapure water to prepare 10 mg / mL as a mother solution, and the solution was fully dissolved by water bath ultrasound for 20 minutes. The self-assembly was completed according to the conditions of Example 1.

[0121] 2. Characterization of Dexamethasone Sodium Phosphate Nanospheres

[0122] TEM observations show that DMSP self-assembles into relatively uniform nanospheres with a diameter of 20-40 nm in aqueous solution ( Fig.12 ). It is proved that the method of this example successfully prepared dexamethasone sodium phosphate nanospheres.

[0123] Example 9 Nanoformulation of dexamethasone sodium phosphate nanospheres loaded with bupivacaine hydrochloride (BUP-DMSP)

[0124] 1. Preparation of BUP-DMSP Nanoformulation

[0125] 2 mg / mL DMSP was used to load bupivacaine hydrochloride (BUP) with a molar concentration of 2.5 and 10 times that of the DMSP, and 1.333 mg / mL DMSP was used to load BUP with a molar concentration of 30 times that of the DMSP. The specific parameter ratios are shown in Table 6. The preparation method is: first, the pH value of DMSP is adjusted to 6.0 with 1M hydrochloric acid, and then the preparation is performed according to the method described in Example 2, except that the preparation is prepared into the corresponding proportions shown in Table 6.

[0126] Table 6. Ratio of raw materials in BUP-DMSP nanoformulation

[0127]

[0128] 2. Characterization of BUP-DMSP Nanoformulations

[0129] DMSP nanospheres are negatively charged, with a zeta potential of -26.3 mV. After loading with BUP, the negative charge of DMSP nanospheres decreases, and decreases with the increase of the total amount of BUP ( Fig.13 ). It can be observed under TEM that with the increase of BUP ratio, the BUP-DMSP preparation presents a nanosphere structure with a diameter of 60-100nm ( Fig.14 ). It is proved that the method of this embodiment successfully prepared BUP-DMSP nanoformulation.

[0130] Example 10 Nanoformulation of dexamethasone sodium phosphate nanospheres loaded with oxycodone hydrochloride (OCD-DMSP)

[0131] 1. Preparation of OCD-DMSP Nanoformulation

[0132] Oxycodone hydrochloride (OCD) was loaded with 1 mg / mL DMSP, and carrier-free nanoformulations were prepared according to the molar concentration ratio of DMSP to OCD of 1:2.5 and 1:10 (Table 7). The preparation conditions were the same as those in Example 9, except that the preparations were prepared in the corresponding proportions shown in Table 7.

[0133] Table 7. Ratio of raw materials in OCD-DMSP nanoformulation

[0134]

[0135] 2. Characterization of OCD-DMSP Nanoformulations

[0136] After loading with OCD, the negative charge of the nanosphere preparation was significantly reduced, and further decreased with the increase of the total amount of OCD loaded ( Fig.15 ). It can be observed under TEM that with the increase of OCD ratio, the OCD-DMSP preparation presents a nanosphere structure with a diameter of 30-120nm ( Fig.16 ). It is proved that the method of this embodiment successfully prepared the OCD-DMSP nanosphere preparation.

[0137] Example 11 Preparation of methylprednisolone sodium succinate nanospheres

[0138] 1. Preparation of methylprednisolone sodium succinate nanospheres

[0139] A certain amount of methylprednisolone sodium succinate (MPSS) was weighed and added to ultrapure water to prepare a 10 mg / mL mother solution, and the solution was fully dissolved by water bath ultrasound for 20 minutes. The self-assembly was completed according to the conditions of Example 1.

[0140] 2. Characterization of Methylprednisolone Sodium Succinate Nanospheres

[0141] Under TEM, it can be observed that MPSS self-assembles into relatively uniform nanospheres with a diameter of 30-50 nm in aqueous solution ( Fig.17 ). It is proved that the method of this example successfully prepared methylprednisolone sodium succinate nanospheres.

[0142] Example 12 Preparation of methylprednisolone sodium succinate nanospheres

[0143] 1. Preparation of methylprednisolone sodium succinate nanospheres

[0144] Methylprednisolone sodium succinate nanospheres were prepared according to the preparation method of Example 11, except that the concentration of MPSS was 5 mg / mL.

[0145] 2. Characterization of Methylprednisolone Sodium Succinate Nanospheres

[0146] Atomic force microscopy showed that MPSS self-assembled into relatively uniform nanospheres in aqueous solution ( Fig.18 ). It is proved that the method of this example successfully prepared methylprednisolone sodium succinate nanospheres.

[0147] Example 13 Nanoformulation of methylprednisolone sodium succinate nanospheres loaded with thioridazine hydrochloride (TDZ-MPSS)

[0148] 1. Preparation of TDZ-MPSS Nanoformulation

[0149] Thioridazine hydrochloride (TDZ) was loaded with 5 mg / mL MPSS, and carrier-free nanoformulations were prepared according to the molar ratio of MPSS to TDZ of 1:5 and 1:10 (Table 8). The preparation method was the same as that in Example 2.

[0150] Table 8. Ratio of raw materials in TDZ-MPSS nanoformulation

[0151] MPSS:TDZ MPSS concentration (mg / mL) TDZ concentration (mg / mL) pH 1:5 5 20.49 5.13 1:10 5 40.99 5.18

[0152] 2. Characterization of TDZ-MPSS Nanoformulations

[0153] MPSS nanospheres are negatively charged, with a zeta potential of -41.5 mV. After loading TDZ, the negative charge disappears and the nanospheres are positively charged ( Fig.19 ). It can be observed under TEM that with the increase of TDZ ratio, TDZ-MPSS preparations present nanosphere structures with a diameter of 30-120nm ( Fig. 20 ). It proves that the method of this example successfully prepared the TDZ-MPSS nanosphere preparation.

[0154] Example 14 Nanoformulation of methylprednisolone sodium succinate nanospheres loaded with levobupivacaine hydrochloride (LB-MPSS)

[0155] 1. Preparation of LB-MPSS Nanoformulation

[0156] LB was loaded with 2 mg / mL MPSS, and carrier-free nanoformulations were prepared according to the MPSS to LB molar concentration ratio of 1:2.5, 1:10 and 1:40 (Table 9). The preparation method was the same as that of Example 3, except that the preparations were prepared in the corresponding proportions shown in Table 9.

[0157] Table 9. Ratio of raw materials in LB-MPSS nanoformulation

[0158]

[0159]

[0160] 2. Characterization of LB-MPSS Nanoformulations

[0161] After loading with LB, the negative charge of MPSS nanospheres decreased, and it decreased significantly with the increase of the total amount of loaded LB ( Fig.21 ). It can be observed under TEM that with the increase of LB ratio, the LB-MPSS preparation presents a nanosphere structure with a diameter of 30-150nm ( Fig. 22 ). It proves that the method of this example successfully prepared the LB-MPSS nanosphere preparation.

[0162] Example 15 Nanoformulation of methylprednisolone sodium succinate nanospheres loaded with morphine hydrochloride (MP-MPSS)

[0163] 1. Preparation of MP-MPSS Nanoformulations

[0164] Using 1 mg / mL MPSS to load morphine hydrochloride (MP), carrier-free nanoformulations were prepared according to the molar concentration ratio of MPSS to MP of 1:2.5 and 1:10 (Table 10). The preparation method was the same as that of Example 2, except that the preparations were prepared in the corresponding proportions shown in Table 10.

[0165] Table 10. Raw material ratios in MP-MPSS nanoformulations

[0166] MPSS:MP MPSS concentration (mg / mL) MP concentration (mg / mL) pH 1:2.5 1 1.42 4.46 1:10 1 7.57 3.73

[0167] 2. Characterization of MP-MPSS Nanoformulations

[0168] After MPSS nanospheres were loaded with MP, the negative charge they carried was significantly reduced, and gradually decreased with the increase in the total amount of loaded MP ( Fig.23). It proves that the method of this example successfully prepared MP-MPSS nanoformulation.

[0169] Example 16 Nanoformulation of methylprednisolone sodium succinate nanospheres loaded with ropivacaine hydrochloride (ROP-MPSS)

[0170] 1. Preparation of ROP-MPSS Nanoformulation

[0171] 5 mg / mL MPSS was used to load ropivacaine hydrochloride with a molar concentration 15 times that of MPSS, and the preparation method was the same as Example 2.

[0172] 2. Characterization of ROP-MPSS Nanoformulations

[0173] like Fig.24 As shown in Figure 2, it can be seen from atomic force microscopy that ROP-MPSS is in the form of nanospheres in aqueous solution, and the particle size is slightly larger than that of MPSS nanospheres prepared at the same MPSS concentration (such as Fig.18 ). It proves that the method of this example successfully prepared the ROP-MPSS nanosphere preparation.

[0174] Example 17 Preparation of prednisolone sodium phosphate nanospheres

[0175] 1. Preparation of prednisolone sodium phosphate nanospheres

[0176] A certain amount of prednisolone sodium phosphate (PNSP) was weighed and added to ultrapure water to prepare 10 mg / mL as a mother solution, and the solution was fully dissolved by water bath ultrasound for 20 minutes. The self-assembly was completed according to the conditions of Example 1.

[0177] 2. Characterization of Prednisolone Sodium Phosphate Nanospheres

[0178] TEM observations show that PNSP self-assembles into relatively uniform nanospheres with a diameter of 50-100 nm in aqueous solution ( Fig.25 ). It is proved that the method of this embodiment successfully prepared prednisolone sodium phosphate nanospheres.

[0179] Example 18 Prednisolone sodium phosphate nanospheres loaded with ropivacaine hydrochloride (ROP-PNSP) nanoformulation

[0180] 1. Preparation of ROP-PNSP Nanoformulation

[0181] 2 mg / mL of PNSP was used to load ropivacaine hydrochloride (ROP) with a molar concentration of 2.5 and 10 times, and 1.333 mg / mL of PNSP was used to load ROP with a molar concentration of 30 times. The specific parameter ratios are shown in Table 11. The preparation method is the same as in Example 3, except that the preparations are prepared into the corresponding proportions shown in Table 11.

[0182] Table 11. Ratio of raw materials in ROP-PNSP nanoformulations

[0183] PNSP:ROP PNSP concentration (mg / mL) ROP concentration (mg / mL) pH 1:2.5 2 3.39 6.78 1:10 2 13.58 6.15 1:30 1.333 27.16 4.81

[0184] 2. Characterization of ROP-PNSP Nanoformulations

[0185] PNSP nanospheres are negatively charged, with a zeta potential of -20.2 mV. After loading with ROP, the negative charge of the nanospheres decreases, and decreases significantly with the increase in the total amount of ROP ( Fig.26 ). It can be observed under TEM that with the increase of ROP ratio, ROP-PNSP preparations present nanosphere structures with a diameter of 50-150nm ( Fig. 27 ). It proves that the method of this example successfully prepared ROP-PNSP nanoformulation.

[0186] Example 19 Preparation of beta-estradiol 17-hemisuccinate nanospheres

[0187] 1. Preparation of beta-estradiol 17-hemisuccinate nanospheres

[0188] Weigh a certain amount of beta-estradiol 17-hemisuccinate (BEH), add ultrapure water, slowly add 1M sodium hydroxide until the pH is 7.0, perform water bath ultrasound for 20 minutes, and shake to fully disperse it in water to prepare a 10 mg / mL mother solution. Self-assembly is completed according to the conditions of Example 1.

[0189] 2. Characterization of beta-estradiol 17-hemisuccinate nanospheres

[0190] TEM observation showed that BEH self-assembled into relatively uniform nanospheres with a diameter of about 50 nm in aqueous solution ( Fig.28 ). It is proved that the method of this example successfully prepared beta-estradiol 17-hemisuccinate nanospheres.

[0191] Example 20 Nanoformulation of beta-estradiol 17-hemisuccinate loaded with diphenhydramine hydrochloride (DHM-BEH)

[0192] 1. Preparation of DHM-BEH Nanoformulation

[0193] 2.5 mg / mL BEH was used to load diphenhydramine hydrochloride (DHM) at 5 and 10 times the molar concentration thereof, and the specific parameter ratios are shown in Table 12. The preparation method was the same as in Example 2, except that the preparations were prepared into the corresponding proportions shown in Table 12.

[0194] Table 12. Ratio of raw materials in DHM-BEH nanoformulation

[0195] DHM:BEH BEH concentration (mg / mL) DHM concentration (mg / mL) pH 1:5 2.5 9.79 6.54 1:10 2.5 19.59 6.28

[0196] 2. Characterization of DHM-BEH Nanoformulations

[0197] BEH nanospheres are negatively charged, with a zeta potential of -30.04 mV. After loading with DHM, the negative charge of the nanospheres decreases, and decreases significantly with the increase in the total amount of DHM ( Fig.29 ). It can be observed under TEM that with the increase of DHM ratio, DHM-BEH preparations present nanosphere structures with a diameter of 100-150nm ( Fig.30 ). It proves that the method of this example successfully prepared DHM-BEH nanoformulation.

[0198] The technical solution of the present invention is further described below through experiments. The sample ROP-MPSS nanoformulation tested in the following experimental examples was prepared by the method of Example 16.

[0199] Experimental Example 1 Determination of critical aggregation concentration of self-assembling molecules HSS

[0200] 1. Experimental Methods

[0201] Take 499 μL of HSS of different concentrations (see Table 13 for specific concentrations) or ultrapure water (control), add 1 μL of pyrene mother solution (2 mM, prepared in dimethyl sulfoxide), incubate at room temperature for 2 minutes, add to a quartz cuvette, and use a fluorescence spectrophotometer to detect the emission spectrum of pyrene in HSS or water in the range of 360-440 nm under 336 nm excitation light.

[0202] Table 13. I1 / I3 ratio of pyrene in HSS solutions of different concentrations

[0203] HSS concentration (mg / mL) I1 I3 I1 / I3 0.08 19473 12677 1.54 0.1 19541 12758 1.53 0.2 35886 23370 1.54 0.4 18335 12106 1.51 0.6 21722 14384 1.51 0.8 26535 17732 1.50 1 16802 11287 1.49 2 17530 11915 1.47 4 13528 9653 1.40 6 13450 10134 1.33 8 12535 9684 1.29

[0204] 2. Experimental Results

[0205] The experimental results are shown in Table 13 and Fig.31As shown. Five characteristic peaks of free pyrene can be detected in the range of 360-440nm. When pyrene is in a hydrophobic environment, the ratio of the first peak (I1) to the third peak (I3) will decrease. When pyrene is in water with higher polarity, its I1 / I3 is 1.60; compared with the ratio in water, when the HSS concentration is higher than 0.8mg / mL, the I1 / I3 of pyrene in the HSS solution begins to drop sharply, which is significantly lower than the ratio in water. This result shows that when the HSS concentration is higher than 0.8mg / mL, a large number of hydrophobic areas begin to appear in the solution environment, indicating that HSS has the characteristics of self-assembly. From Fig.31 It can be seen that the critical aggregation concentration (CAC) of HSS is 1-2 mg / mL.

[0206] The above results show that when the concentration of HSS solution is higher than 1-2 mg / mL, HSS has the characteristics of self-assembly and can be used to self-assemble to form nanomaterials.

[0207] Experimental Example 2 Determination of the critical aggregation concentration of self-assembling molecules BSP

[0208] 1. Experimental Methods

[0209] The critical aggregation concentration of the self-assembling molecule BSP was measured according to the method of Experimental Example 1. The difference is that the concentration of BSP is as shown in Table 14.

[0210] Table 14. I1 / I3 ratio of pyrene in BSP solutions of different concentrations

[0211] BSP concentration (mg / mL) I1 I3 I1 / I3 0.06 24488 15866 1.54 0.08 15429 10000 1.54 0.1 25357 16395 1.55 0.2 12817 8397 1.53 0.4 14429 9375 1.54 0.6 20367 13219 1.54 0.8 18812 12353 1.52 1 40975 26932 1.52 2 18120 12235 1.48 4 19521 15021 1.30 6 13284 11406 1.16 8 3891 4026 0.97 10 3803 4152 0.92

[0212] 2. Experimental Results

[0213] The experimental results are shown in Table 14 and Fig.32 When the BSP concentration is higher than 0.8 mg / mL, the I1 / I3 of pyrene in it is significantly lower than the I1 / I3 value in water (1.60). Fig.32 It can be seen that the CAC for BSP self-assembly is 1-2 mg / mL.

[0214] The above results show that when the concentration of BSP solution is higher than 1-2 mg / mL, BSP has the characteristics of self-assembly and can be used to self-assemble to form nanomaterials.

[0215] Experimental Example 3 Determination of the critical aggregation concentration of self-assembling molecule DMSP

[0216] 1. Experimental Methods

[0217] The critical aggregation concentration of the self-assembling molecule DMSP was determined according to the method of Experimental Example 1. The difference is that the concentration of DMSP is as shown in Table 15.

[0218] Table 15. I1 / I3 ratio of pyrene in DMSP solutions of different concentrations

[0219]

[0220]

[0221] 2. Experimental Results

[0222] The experimental results are shown in Table 15 and Fig.33 As shown in Figure 2, with the increase of DMSP concentration, the I1 / I3 ratio decreased, especially when the concentration was greater than 0.4 mg / mL, the I1 / I3 of pyrene in it was significantly lower than the I1 / I3 value in water (1.60). Fig.33 It can be seen that the CAC for DMSP self-assembly is 1 mg / mL.

[0223] The above results show that when the concentration of DMSP solution is higher than 1 mg / mL, DMSP has the characteristics of self-assembly and can be used to self-assemble to form nanomaterials.

[0224] Experimental Example 4 Determination of critical aggregation concentration of self-assembling molecule MPSS

[0225] 1. Experimental Methods

[0226] The critical aggregation concentration of the self-assembling molecule MPSS was determined according to the method of Experimental Example 1. The difference is that the concentration of MPSS is as shown in Table 16.

[0227] Table 16. I1 / I3 ratio of pyrene in MPSS solutions of different concentrations

[0228]

[0229]

[0230] 2. Experimental Results

[0231] The experimental results are shown in Table 16 and Fig.34 When the MPSS concentration is greater than 0.5 mg / mL, the I1 / I3 ratio decreases with the increase of MPSS concentration, especially when the concentration is greater than 1 mg / mL, the I1 / I3 of pyrene in it is significantly lower than the I1 / I3 value in water (1.60). Fig.34 It can be seen that the CAC for self-assembly of MPSS is 1-2 mg / mL.

[0232] The above results show that when the concentration of MPSS solution is higher than 1-2 mg / mL, MPSS has the characteristics of self-assembly and can be used to self-assemble to form nanomaterials.

[0233] Experimental Example 5 Determination of the critical aggregation concentration of the self-assembling molecule PNSP

[0234] 1. Experimental Methods

[0235] The critical aggregation concentration of the self-assembling molecule PNSP was determined according to the method of Experimental Example 1. The difference is that the concentration of PNSP is as shown in Table 17.

[0236] Table 17. I1 / I3 ratio of pyrene in PNSP solutions of different concentrations

[0237]

[0238]

[0239] 2. Experimental Results

[0240] The experimental results are shown in Table 17 and Fig.35 As shown in Figure 2, with the increase of PNSP concentration, the I1 / I3 ratio decreased, especially when the concentration was greater than 0.8 mg / mL, the I1 / I3 of pyrene in it was significantly lower than the I1 / I3 value in water (1.60). Fig.35 It can be seen that the CAC for PNSP self-assembly is 1-2 mg / mL.

[0241] The above results show that when the concentration of PNSP solution is higher than 1-2 mg / mL, PNSP has the characteristics of self-assembly and can be used to self-assemble to form nanomaterials.

[0242] Experimental Example 6 Determination of critical aggregation concentration of self-assembling molecule BEH

[0243] 1. Experimental Methods

[0244] The critical aggregation concentration of the self-assembling molecule BEH was determined according to the method of Experimental Example 1. The difference is that the concentration of BEH is as shown in Table 18.

[0245] Table 18. I1 / I3 ratio of pyrene in BEH solutions of different concentrations

[0246]

[0247]

[0248] 2. Experimental Results

[0249] The experimental results are shown in Table 18 and Fig.36As shown in Figure 2, with the increase of BEH concentration, the ratio of I1 / I3 decreased, especially when the concentration was greater than 0.1 mg / mL, the I1 / I3 of pyrene in it was significantly lower than the I1 / I3 value in water (1.60). Fig.36 It can be seen that the CAC for BEH self-assembly is 0.4-0.6 mg / mL.

[0250] The above results show that when the concentration of BEH solution is higher than 0.4-0.6 mg / mL, BEH has the characteristics of self-assembly and can be used to self-assemble to form nanomaterials.

[0251] Experimental Example 7 Anti-inflammatory Effect of ROP-MPSS

[0252] The sample ROP-MPSS nanoformulation tested in this experimental example was prepared by the method of Example 16.

[0253] 1. Experimental Methods

[0254] 1. Experimental Grouping

[0255] This experimental example is divided into three groups, namely the control group, the ROP group, and the ROP-MPSS group. The drug of the control group is normal saline, the drug of the ROP group is ROP solution, and the drug of the ROP-MPSS group is ROP-MPSS solution. The preparation methods of the drug solutions of each group are as follows:

[0256] ROP group: ROP was dissolved in ultrapure water to form a 10 mg / mL ROP solution.

[0257] ROP-MPSS group: The pH of the ROP-MPSS nanoformulation was adjusted to 7.5-8.5 with 1 M sodium hydroxide solution, and then freeze-dried and resuspended into a ROP-MPSS solution with a ROP concentration of 10 mg / mL.

[0258] 2. Animal experiments

[0259] 0.2 mL of saline, ROP solution (10 mg / mL) and ROP-MPSS solution (ROP concentration of 10 mg / mL) were injected into the lateral thigh muscle of rats, 8 rats in each group, and the tissues at the injection site were collected 12 hours and 24 hours after administration. Tissue homogenate was prepared, the supernatant was centrifuged and stored at -80°C for detection. ELISA kits (ThermoFisher Scientific Inc., USA) were used to determine the concentrations of TNF-α, IL-1β and IL-6 according to the instructions.

[0260] 2. Experimental Results

[0261] The results are as follows Fig.37As shown. Compared with the saline control group, injection of 10 mg / mL ROP resulted in significant local inflammation, as evidenced by increased expression levels of TNF-α, IL-1β, and IL-6. In rats injected with ROP-MPSS, the expression levels of inflammatory factors were similar to those in the saline group, and the expression of TNF-α (12h: p = 0.0079, 24h: p = 0.011), IL-1β (12h: p = 0.0079, 24h: p = 0.015), and IL-6 (12h: p = 0.0119, 24h: p = 0.015) was significantly reduced compared with the ROP group.

[0262] The above results indicate that the MPSS in ROP-MPSS can not only act as a carrier to load ROP to form nanoformulations, but also exert its original anti-inflammatory effect to achieve a "detoxification" effect.

[0263] Experimental Example 8 Cytotoxicity of Nanocomplexes

[0264] 1. Experimental Methods

[0265] 1. Cells and culture conditions

[0266] This experiment used human non-small cell lung cancer cell A549, which was cultured in DMEM medium (Hyclone) supplemented with 10% fetal bovine serum (Gibco), 100 U / ml penicillin, and 100 μg / ml streptomycin at 37° C. and 5% carbon dioxide.

[0267] 2. Experimental Grouping

[0268] The experiment was divided into 7 groups, namely LB group, LB-MPSS group, BUP group, BUP-DMSP group, ROP group, ROP-PNSP group and control group. The drug in the LB group was free LB; the drug in the LB-MPSS group was the LB-MPSS nanoformulation with a molar concentration ratio of 1:40 prepared in Example 14; the drug in the BUP group was free BUP; the drug in the BUP-DMSP group was the BUP-DMSP nanoformulation with a molar concentration ratio of 1:30 prepared in Example 9; the drug in the ROP group was free ROP; the drug in the ROP-PNSP group was the ROP-PNSP nanoformulation with a molar concentration ratio of 1:40 prepared in Example 18; and the control group used fresh culture medium without drugs.

[0269] 3. Cell treatment and viability determination

[0270] (1) A549 cells were cultured at 1×10 4 The cells were seeded in 96-well plates at a density of 100 μg / mL and incubated for 16 h (to ensure that the cells adhered to the wall and entered a stable state).

[0271] (2) Replace 100 μL of fresh culture medium respectively, and add different concentrations of drugs into the culture medium according to the grouping situation. The final concentrations of the drugs in the culture medium are 0.1 mM, 0.3 mM, and 1.0 mM, respectively. Among them, the concentration of LB-MPSS nanoformulation is calculated based on the concentration of LB therein, the concentration of BUP-DMSP nanoformulation is calculated based on the concentration of BUP therein, and the concentration of ROP-PNSP nanoformulation is calculated based on the concentration of ROP therein.

[0272] (3) Incubate for 4 hours, then remove the culture medium and replace it with 100 μL fresh culture medium. Continue culturing for 24 hours, add 1×CCK8 (APExBIO) reagent, and then use a microplate reader (BioTek) to detect the absorbance (OD) at a wavelength of 450 nm. The cell viability is calculated as follows:

[0273] Cell viability (%) = (OD 样品 –OD 空白 ) / (OD 对照 –OD 空白 )×100%

[0274] The results were averaged from three experiments.

[0275] 2. Experimental Results

[0276] like Fig.38 At 0.1-1 mM drug concentration, LB and LB-MPSS both had killing effects on A549 cells. In particular, at the same LB concentration, the killing effect of LB-MPSS was significantly higher than that of free LB.

[0277] like Fig.39 At 0.1-1 mM drug concentration, BUP and BUP-DMSP both had killing effects on A549 cells. In particular, at the same BUP concentration, the killing effect of BUP-DMSP was significantly higher than that of free BUP.

[0278] like Fig.40 At 0.1-1 mM drug concentration, ROP and ROP-PNSP have killing effects on A549 cells. In particular, at the same ROP concentration, the killing effect of ROP-PNSP is significantly higher than that of free ROP.

[0279] The above experimental results show that after the material-free drug carrier of the present invention is loaded with LB, BUP and ROP respectively, the killing effect of the drug on tumor cells can be significantly enhanced.

[0280] The self-assembling molecules of the present invention are drugs themselves. Hydrocortisone sodium succinate (HSS) is an adrenal cortical hormone drug with multiple pharmacological effects such as anti-inflammatory, anti-allergic, and immunosuppressive. It can be used for toxic infections, anaphylactic shock, severe adrenocortical insufficiency, connective tissue diseases, severe bronchial asthma and other allergic diseases, prevention and treatment of acute transplant rejection, and for rheumatoid arthritis, osteoarthritis, tenosynovitis, tendon strain, etc. Betamethasone sodium phosphate (BSP) is an adrenal cortical hormone drug with multiple pharmacological effects such as anti-inflammatory, anti-allergic, and immunosuppressive; dexamethasone sodium phosphate (DM SP) is an adrenal cortical hormone drug with multiple pharmacological effects such as anti-inflammatory, anti-allergic, anti-rheumatic and immunosuppressive, and is mainly used for allergic and autoimmune inflammatory diseases; methylprednisolone sodium succinate (MPSS) is a synthetic glucocorticoid drug with multiple pharmacological effects such as anti-inflammatory, immunosuppressive and anti-allergic; prednisolone sodium phosphate (PNSP) is a synthetic intermediate-acting glucocorticoid drug with multiple pharmacological effects such as anti-inflammatory, anti-allergic and immunosuppressive; beta-estradiol 17-hemisuccinate (BEH) is an estrogen drug, mainly used to supplement estrogen deficiency or regulate estrogen levels.

[0281] The above experimental examples 1-6 respectively screened and obtained the critical aggregation concentration of the self-assembling molecules HSS, BSP, DMSP, MPSS, PNSP and BEH. When the critical aggregation concentration is exceeded, the self-assembling molecules have the characteristics of self-assembly. The above examples 1, 4, 8, 11, 12, 17, and 19 prove that under the experimental conditions of the present invention, the self-assembling molecules HSS, BSP, DMSP, MPSS, PNSP and BEH can be self-assembled to form nanostructures. The above examples 2, 3, 5-7, 9, 10, 13-16, 18, and 20 prove that the self-assembling molecules HSS, BSP, DMSP, MPSS, PNSP and BEH form nanostructures that can be used as drug carriers to load drugs. The above experimental example 7 proves that the self-assembling molecules can still exert their original pharmacological effects after self-assembly, alleviate the inflammatory response caused by the drug, and achieve the effect of "reduced toxicity". The above experimental example 8 proves that the material-free drug carrier of the present invention can significantly enhance the efficacy of the drug after further loading the drug.

[0282] Through the above embodiments and experimental examples, it can be seen that the present invention provides a self-assembly, and the self-assembly of the present invention is composed of steroid series molecules. By optimizing the molecular structure of these steroids and their critical aggregation concentration, a material-free drug delivery system formed by the self-assembly of steroid series molecules is prepared. The material-free drug delivery system can be loaded with drugs and used as a drug carrier to prepare drug nanocomplexes. In the nanocomplex, the efficacy of the drug is improved, and the effect of "reducing toxicity and increasing efficacy" can be achieved. Furthermore, if the steroid molecules used themselves have biological activity, then after they are made into the drug nanocomplex of the present invention, the pharmacological activity of the steroid molecules themselves is not affected. The material-free drug delivery system prepared by the present invention has the advantages of two nano-preparations without carriers and with traditional materials as carriers, and has good prospects in the development of new nano-drug preparations.

Claims

1. An organic molecular self-assembly, characterized in that: It is prepared by self-assembly of steroid organic molecules; the steroid organic molecules are selected from at least one of steroid organic molecules with a pregnane skeleton structure and an estranes skeleton structure; When the steroid organic molecule is selected from at least one steroid organic molecule with a pregnane skeleton structure, the concentration of the steroid organic molecule is ≥1 mg / mL; When the steroid organic molecule is selected from at least one steroid organic molecule with an estraneskeletal structure, the concentration of the steroid organic molecule is ≥0.4 mg / mL.

2. The organic molecular self-assembly according to claim 1, characterized in that: When the steroid organic molecule is selected from at least one steroid organic molecule with a pregnane skeleton structure, the concentration of the steroid organic molecule is ≥2 mg / mL; when the steroid organic molecule is selected from at least one steroid organic molecule with an estranes skeleton structure, the concentration of the steroid organic molecule is ≥0.6 mg / mL.

3. The organic molecular self-assembly according to claim 1, characterized in that: The steroid organic molecule with a pregnane skeleton structure is selected from at least one of hydrocortisone, betamethasone, dexamethasone, methylprednisolone, prednisolone or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable ester thereof; And / or, the steroid organic molecule with an estranestane skeleton structure is selected from at least one of estradiol, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable ester thereof.

4. The organic molecular self-assembly according to claim 3, characterized in that: The steroid organic molecule is selected from at least one of hydrocortisone sodium succinate, betamethasone sodium phosphate, dexamethasone sodium phosphate, methylprednisolone sodium succinate, prednisolone sodium phosphate, and beta-estradiol 17-hemisuccinate.

5. The organic molecular self-assembly according to any one of claims 1 to 4, characterized in that: The specific conditions of the self-assembly are: stirring for 4-6 hours at a temperature of 20-25° C., a stirring speed of 600-1000 rpm, and then standing at a temperature of 2-20° C. for 12-18 hours.

6. The method for preparing an organic molecular self-assembly according to any one of claims 1 to 5, characterized in that: The method comprises: being prepared by self-assembly of steroid organic molecules; the steroid organic molecules are selected from at least one of steroid organic molecules with a pregnane skeleton structure and an estranes skeleton structure; When the steroid organic molecule is selected from at least one steroid organic molecule with a pregnane skeleton structure, the concentration of the steroid organic molecule is ≥1 mg / mL; When the steroid organic molecule is selected from at least one steroid organic molecule with an estraneskeletal structure, the concentration of the steroid organic molecule is ≥0.4 mg / mL.

7. Use of the organic molecular self-assembly according to any one of claims 1 to 5 as a drug carrier and / or active ingredient in the preparation of drugs.

8. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises the organic molecular self-assembly according to any one of claims 1 to 5 loaded with a drug; the drug is selected from at least one of a pharmaceutically active compound containing a tertiary amine group and a hydrophobic aromatic ring structure, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable ester thereof; The molar ratio of the steroid organic molecules used to constitute the organic molecular self-assembly to the drug is 1:2.5-40.

9. The pharmaceutical composition according to claim 8, characterized in that The pharmaceutical composition is prepared by the following steps: the drug is mixed with the steroid organic molecules used to form the organic molecule self-assembly body.

10. The pharmaceutical composition according to claim 9, characterized in that The specific process of the preparation is: at a temperature of 20-25°C, the steroid organic molecule is added dropwise to the drug, mixed under stirring, the stirring speed is 600-1000 rpm, the stirring time is 4-6 hours, and then allowed to stand at a temperature of 2-20°C for 12-18 hours; And / or, the pH of the steroid organic molecule is adjusted to ≤ 7.0 using a pH adjuster before being mixed with the drug.

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