Tumor penetration type carrier-free nanomedicine and preparation method and application thereof

Abstract

The application discloses a tumor-penetrating carrier-free nanomedicine as well as a preparation method and application thereof, and belongs to the field of nanomedicine. The carrier-free nanomedicine can significantly improve water solubility of a water-insoluble compound, has mitochondrion targeting and tumor active penetration capabilities, and under the action of a tertiary amine nitrogen oxygen group, the carrier-free nanomedicine can be mostly transported into mitochondria, which is beneficial to inducing a mitochondrion membrane potential drop and cell apoptosis. The carrier-free nanomedicine also has a good blood long-circulation effect and tumor accumulation capability in a body, and an antitumor effect is better than that of a clinical drug, and the carrier-free nanomedicine also has good biological safety. The preparation method is simple, and can be used for preparation of an antitumor drug.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of nanomedicine, and particularly relates to a tumor-penetrating carrier-free nanomedicine and a preparation method and application thereof. BACKGROUND

[0002] At present, most of the carrier designs of nanomedicine are based on carrier delivery systems (CDDS), however, CDDS has the problems of low drug loading capacity and certain systemic toxicity of the carrier itself, such as high toxicity and severe inflammation. Based on this, researchers design carrier-free nanomedicine, which is self-assembled from carrier-free prodrugs or amphiphilic drug-drug conjugates, and has the advantages of low systemic toxicity and high drug loading capacity. At present, a variety of carrier-free nanomedicine has entered the clinical phase II test, such as NCD (Wang Y, Yang P, Zhao X, et al. Multifunctional cargo-free nanomedicine for cancer therapy [J]. International Journal of Molecular Sciences, 2018, 19(10): 2963.). However, the clinical efficacy of carrier-free nanomedicine has not been significantly improved. Carrier-free nanomedicine is facing the following problems: 1) Carrier-free nanomedicine has no carrier and relies on non-covalent interactions to drive its self-assembly, which leads to poor stability and is difficult to ensure long blood circulation; 2) Carrier-free nanomedicine composed of therapeutic drugs lacks tumor-specific targeting ability and mostly depends on the EPR effect to accumulate in the tumor site, while the EPR effect has tumor heterogeneity and is affected by the complex tumor microenvironment; 3) Carrier-free nanomedicine lacks rapid intracellular drug release, so that after the carrier-free nanomedicine is internalized by tumor cells, it cannot release drugs quickly to kill tumor cells. In order to solve these problems, it is necessary to modify the carrier-free nanomedicine so that it has excellent blood circulation effect, effective tumor targeting and accumulation, and rapid intracellular drug release, etc. to achieve efficient tumor treatment.

[0003] Mitochondria are the "energy engine" of cells, producing ATP to maintain life and organ normal operation, and also involved in the management of active oxygen free radicals (ROS) and the biosynthesis of amino acids. It has been proved that inducing mitochondrial dysfunction by drugs is an effective tumor treatment strategy, and the most commonly used method is to use triphenylphosphine (TPP) modified carriers to obtain drug mitochondrial targeting function (Fulda S, Galluzzi L, Kroemer G. Targeting mitochondria for cancer therapy [J]. Nature Reviews Drug Discovery, 2010, 9 (6): 447-464.). However, due to the hydrophobicity and positive charge of TPP, it is contrary to the "concealment" characteristics required for the blood circulation of nanodrugs, so more complex modification of the carrier is needed, which greatly reduces the possibility of clinical conversion of the drug.

[0004] Therefore, how to prepare a simple structure and tumor efficient penetration of mitochondria targeting of carrier-free nanodrug is urgent to be solved. SUMMARY

[0005] In view of this, the present application aims to provide a tumor penetration type carrier-free nanodrug and its preparation method and application. The carrier-free nanodrug is prepared by bonding a water-insoluble chemotherapeutic small molecule drug and an amphiphilic tertiary amine nitrogen oxygen group to form a small molecule prodrug. The prodrug can self-assemble to form water-soluble nanoparticles in an aqueous solution, which has mitochondria targeting and tumor active penetration ability, and lays a foundation for the preparation of antitumor drugs.

[0006] The technical scheme of the present application is as follows:

[0007] A tumor penetration type carrier-free nanodrug, the tumor penetration type carrier-free nanodrug is formed by self-assembly of a small molecule prodrug, wherein the small molecule prodrug is prepared by connecting a drug molecule and a tertiary amine nitrogen oxygen group through a linker, and the structure is shown as formula (I):

[0008]

[0009] In the formula, A is a drug molecule, Y is a linker, R1 and R2 are respectively independent alkyl, substituted alkyl, aromatic group or substituted aromatic group, wherein the drug molecule includes but is not limited to at least one of camptothecin, doxorubicin, paclitaxel, curcumin, triptolide, methotrexate, mitoxantrone, vinblastine and oxaliplatin.

[0010] The introduction of the linker in the present application can accelerate the rapid response release of the carrier-free nanodrug in tumor cells, and these linkers can respond to the tumor acid pH or the abnormally up-regulated ROS level in the tumor.

[0011] Preferably, the tertiary amine nitroxide group is at least one of N,N-dimethylaminoethyl acrylate, N,N-diethylaminoethyl acrylate, N,N-dipropylaminoethyl acrylate, pyrrole ethanol acrylate, piperidine ethanol acrylate, morpholine ethanol acrylate, N,N-dimethylethanolamine, N,N-diethylethanolamine, N,N-dipropylethanolamine, N-hydroxyethylpyrrole, N-hydroxyethylpiperidine, and N-hydroxyethylmorpholine containing an N-oxide group. The tertiary amine nitroxide group is an amphiphilic group with excellent water solubility; its water solubility can be improved by bonding with hydrophobic small molecule chemotherapeutic drugs.

[0012] The small molecule chemotherapeutic drugs mentioned are all at least one of the following: camptothecin, doxorubicin, paclitaxel, curcumin, tripterygium wilfordii, methotrexate, mitoxantrone, vincristine, and oxaliplatin, which have antitumor efficacy and poor water solubility. In one embodiment of the present invention, camptothecin is selected as an example, but it is not limited to camptothecin. The structure of the small molecule prodrug is shown in formula (II):

[0013]

[0014] In the formula, R1 and R2 are independent alkyl, substituted alkyl, aromatic, and substituted aromatic groups, respectively, and n is the number of structural units, n = 2 to 8.

[0015] X is -S-, -N-(CH3)2C(=O)O(CH3)2R1R2N + O - -, -NR3-, thioketal bond, ketal bond, -C(=O)OC(=O)O-, -O-, -SS-, -OC(CH3)2O-, -SC(CH3)2S-, -C(=O)C(=O)-, wherein R3 is an alkyl group, a substituted alkyl group, an aromatic group, or a substituted aromatic group.

[0016] Preferably, the structure of the small molecule prodrug is shown in any one of formulas (III) to (V):

[0017]

[0018]

[0019] In the formula, R1 and R2 are independent alkyl, substituted alkyl, aromatic, and substituted aromatic groups, respectively; R3 is an alkyl, substituted alkyl, aromatic, or substituted aromatic group; and n is the number of structural units, n = 2 to 8.

[0020] More preferably, in formula (V), R1 is methyl, R2 is methyl, and n = 2.

[0021] The application also provides a preparation method of the tumor-penetrating carrier-free nanomedicine, which comprises any of the following steps:

[0022] (1) the small molecule chemotherapeutic drug is subjected to carboxylic acid reaction with a thiol or amino group containing a protective group, and after the protective group is removed, a small molecule chemotherapeutic drug with a thiol or amino functional group is obtained, then a Michael addition reaction is performed on the small molecule prodrug with a tertiary amine nitrogen oxygen group containing an acrylic ester under the catalysis of an organic base to obtain a small molecule prodrug, and the small molecule prodrug is self-assembled to form the tumor-penetrating carrier-free nanomedicine;

[0023] (2) the small molecule chemotherapeutic drug is subjected to esterification reaction with a dicarboxylic acid or anhydride containing a linker group, and then esterification is performed on the small molecule prodrug with a tertiary amine nitrogen oxygen group containing a hydroxyl group to obtain a small molecule prodrug, and the small molecule prodrug is self-assembled to form the tumor-penetrating carrier-free nanomedicine.

[0024] The step of self-assembling the small molecule prodrug to form the tumor-penetrating carrier-free nanomedicine is any of the following:

[0025] (a) the small molecule prodrug is dispersed in a buffer solution, and the small molecule prodrug is self-assembled by ultrasonic method to obtain the tumor-penetrating carrier-free nanomedicine;

[0026] (b) the small molecule prodrug is dissolved in an organic solution, and is added dropwise into water to form nanoparticles, and then the organic solvent contained is removed by dialysis to obtain the tumor-penetrating carrier-free nanomedicine.

[0027] The buffer solution is a PBS buffer solution, a HEPES buffer solution or a Tris buffer solution; and the organic solvent is dimethyl sulfoxide, dimethyl formamide, ethanol or methanol.

[0028] The organic base is triethylamine, pyridine, N,N-diisopropyl ethylamine or diethylamine.

[0029] In a specific embodiment of the application, a tertiary amine nitrogen oxide camptothecin carrier-free nanomedicine CPT-S-NO is prepared, and the specific preparation method is as follows:

[0030] Step one: a tertiary amine nitrogen oxide monomer ODMA is prepared by oxidizing methyl methacrylate-2-(N,N-dimethylamino) ethyl ester with meta-chloroperoxybenzoic acid (mCPBA), and the crude product is purified by column to obtain the tertiary amine nitrogen oxide monomer ODMA;

[0031] Step two: 3-triphenyl thio propionic acid is prepared by reacting 3-mercapto propionic acid with triphenylmethyl chloride at room temperature, and then CPT-SH is synthesized by reacting the 3-triphenyl thio propionic acid with CPT under the carboxyl activation of DCC and DMAP;

[0032] Step three: Michael addition reaction of CPT-SH and ODMA under the catalysis of triethylamine base to generate the target product CPT-S-NO.

[0033] Compared with the prior art, the prepared tertiary amine nitroxide camptothecin has the following advantages:

[0034] (1) The preparation method is simple, the tertiary amine nitroxide group is bonded to camptothecin CPT by using a sulfide bond, and the water solubility of camptothecin CPT can be significantly improved, and the CPT-S-NO containing a sulfide bond can self-assemble into nanoparticles with a diameter of about 100 nm in an aqueous solution.

[0035] (2) The tertiary amine nitroxide camptothecin carrier-free nanomedicine CPT-S-NO has mitochondrial targeting and tumor active penetration capacity, and under the action of the tertiary amine nitroxide group, CPT-S-NO can be mostly transported into mitochondria, which is beneficial to induce mitochondrial membrane potential to drop and cause cell apoptosis, and CPT-S-NO also has rapid tumor active penetration capacity.

[0036] (3) The tertiary amine nitroxide camptothecin carrier-free nanomedicine CPT-S-NO has good blood long-circulation effect and tumor accumulation capacity.

[0037] (4) The in-vivo anti-tumor activity experiment proves that under the same dosage of camptothecin CPT, the tumor inhibition effect of the tertiary amine nitroxide camptothecin carrier-free nanomedicine CPT-S-NO on the MDA-MB-231 subcutaneous tumor model is better than that of the clinical drug CPT-11, and CPT-S-NO also has good biological safety.

[0038] The application further provides application of the tumor penetration type carrier-free nanomedicine in preparation of an anti-tumor drug.

[0039] The application has the following beneficial effects:

[0040] The carrier-free nanomedicine can significantly improve the water solubility of a poorly water-soluble compound, has mitochondrial targeting and tumor active penetration capacity, and under the action of the tertiary amine nitroxide group, the carrier-free nanomedicine can be mostly transported into mitochondria, which is beneficial to induce mitochondrial membrane potential to drop and cause cell apoptosis; the carrier-free nanomedicine also has good blood long-circulation effect and tumor accumulation capacity in the body, and has better tumor inhibition effect than a clinical drug and also has good biological safety. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 It is a synthesis route map of the carrier-free nanomedicine CPT-S-NO in the application.

[0042] Figure 2 Figure 1 is a 1H NMR chart of the carrier-free nanodrug CPT-S-NO in the present application;

[0043] Figure 3 Figure 2 is a morphology and particle size characterization of CPT-S-NO in the present application: a is a TEM observation of the morphology of CPT-S-NO in an aqueous solution; b is a DLS measurement of the particle size of the nanoparticles formed by CPT-S-NO;

[0044] Figure 4 Figure 3 is a release curve of CPT-S-NO in different media in the present application;

[0045] Figure 5 Figure 4 is an in vitro anti-cell proliferation curve of CPT-S-NO and the clinical camptothecin control CPT-11 on each cell line in the present application;

[0046] Figure 6 Figure 5 is a distribution characterization of CPT-S-NO in mitochondria in the present application: a is a laser confocal microscope observation of the subcellular distribution of CPT-S-NO and mitochondria green dye; b is an Image J calculation of the degree of coincidence of CPT-S-NO and mitochondria green fluorescent dye;

[0047] Figure 7 Figure 6 is a laser confocal microscope observation of the penetration of CPT-S-NO in tumor spheres MCF-7 in the present application, wherein the scale is 200 μm;

[0048] Figure 8 Figure 7 is a plasma clearance curve of CPT-S-NO and the clinical camptothecin control CPT-11 in the present application;

[0049] Figure 9 Figure 8 is a tumor volume (a) and body weight (b) change chart of CPT-S-NO, the clinical camptothecin control CPT-11 and PBS buffer four groups of preparations in the tumor inhibition experiment of MDA-MB-231 tumor-bearing mice in the present application. DETAILED DESCRIPTION

[0050] The present application provides a tumor-penetrating carrier-free nanodrug, which is formed by self-assembly of a small molecule prodrug, wherein the small molecule prodrug is prepared by connecting a small molecule chemotherapeutic drug with a tertiary amine nitroxy radical group through a linker, and the structure is shown as formula (I):

[0051]

[0052] In the formula, A is a drug molecule, Y is a linker, and R1 and R2 are independent alkyl, substituted alkyl, aromatic or substituted aromatic groups, respectively. The drug molecule includes at least one of camptothecin, doxorubicin, paclitaxel, curcumin, tripterygium wilfordii, methotrexate, mitoxantrone, vincristine, and oxaliplatin.

[0053] In the technical solution of the embodiments of the present invention, the small molecule chemotherapy drug camptothecin is selected as an example, but is not limited to camptothecin, and the structure of the small molecule prodrug is shown in formula (II):

[0054]

[0055] In the formula, R1 and R2 are independent alkyl, substituted alkyl, aromatic, and substituted aromatic groups, respectively, and n is the number of structural units, n = 2 to 8.

[0056] X is -S-, -N-(CH3)2C(=O)O(CH3)2R1R2N + O - -, -NR3-, thioketal bond, ketal bond, -C(=O)OC(=O)O-, -O-, -SS-, -OC(CH3)2O-, -SC(CH3)2S-, -C(=O)C(=O)-, wherein R3 is an alkyl group, a substituted alkyl group, an aromatic group, or a substituted aromatic group.

[0057] Preferably, the structure of the small molecule prodrug is shown in any one of formulas (III) to (V):

[0058]

[0059]

[0060] In the formula, R1 and R2 are independent alkyl, substituted alkyl, aromatic, and substituted aromatic groups, respectively; R3 is an alkyl, substituted alkyl, aromatic, or substituted aromatic group; and n is the number of structural units, n = 2 to 8.

[0061] Preferably, in formula (V), R1 is methyl, R2 is methyl, and n = 2.

[0062] To make this invention clearer and easier to understand, the preparation and application of carrier-free nanomedicines containing tertiary amine nitrogen oxidase camptothecin will be further explained below with reference to embodiments. The carrier-free nanomedicines containing one of the following—doxorubicin, paclitaxel, curcumin, triptolide, methotrexate, mitoxantrone, vincristine, or oxaliplatin—are prepared using the same method as the carrier-free nanomedicine containing tertiary amine nitrogen oxidase camptothecin. However, this invention is not limited to these embodiments. Non-essential modifications made by those skilled in the art under the guidance of the core ideas of this invention are still within the scope of protection of this invention.

[0063] Example 1

[0064] Synthesis of tertiary amine nitroxide camptothecin of formula (III) without carrier nanomedicine:

[0065] As preferred, R3 of formula (III) in this example is hydrogen atom, n = 2.

[0066] 1. Synthesis of tertiary amine oxide ODMA:

[0067] Methyl acrylate-2-(N,N-dimethylamino) ethyl (2.36 g, 15 mmol) was dissolved in 60 mL of anhydrous dichloromethane (DCM), and m-chloroperbenzoic acid (mCPBA, 5.18 g, 30 mmol) was added dropwise using a constant pressure dropping funnel under ice bath condition. After the addition was completed, the ice bath was removed, and the reaction was stirred at room temperature (25 °C) for 2 h. After the reaction was completed, the reaction solution was directly purified by column chromatography using a neutral alumina column (dichloromethane:methanol = 5:1) after removing the excess solvent by rotary evaporation, to obtain a waxy solid ODMA.

[0068] 2. Synthesis of Boc-protected β-alanine:

[0069] β-Alanine (0.80 g, 8.90 mmol) was dissolved in 6 mL of dry dichloromethane solution, and triethylamine (1.28 g, 12.70 mmol) was added while stirring under ice bath. Then, di-tert-butyl dicarbonate (1.66 g, 7.63 mmol) was added dropwise using a constant pressure dropping funnel. After the addition was completed, the ice bath was removed, and the reaction solution was returned to room temperature and stirred for 2 h. After the reaction was completed, the excess solvent was removed by rotary evaporation, and then the reaction product was re-dissolved in a small amount of DCM, washed with pure water and saturated NaCl in sequence, dried with anhydrous Na2SO4, and the excess solvent was removed by reduced pressure distillation. Boc-protected β-alanine was obtained by vacuum drying.

[0070] 3. Synthesis of tertiary amine nitroxide camptothecin of formula (III) without carrier nanomedicine:

[0071] Boc-protected β-alanine (0.27 g, 1.43 mmol) and camptothecin (CPT, 0.50 g, 1.44 mmol) were dissolved in 10 mL of N,N-dimethylformamide (DMF), and N,N-dicyclohexylcarbodiimide (DCC) (0.36 g, 1.73 mmol) and 4-dimethylaminopyridine (DMAP) (0.16 g, 1.44 mmol) were added, and the reaction was stirred at room temperature for 24 h. After the reaction was completed, the reaction solution was filtered and the filtrate was concentrated with an oil pump, precipitated into a large amount of diethyl ether, and washed repeatedly with diethyl ether, and dried to obtain Boc-protected aminopropionyl camptothecin. The product (0.52 g, 1 mmol) was dissolved in 3 mL of a mixed solution of DCM and trifluoroacetic acid (TFA) (v / v = 2:1), stirred at room temperature for 4 h, and dried after the solvent was removed by rotary evaporation to obtain aminopropionyl camptothecin.

[0072] Aminopropionyl camptothecin (0.34 g, 0.8 mmol) and ODMA (0.13 g, 0.8 mmol) were weighed and dissolved in 10 mL of tetrahydrofuran (THF), and triethylamine (4.04 mg, 0.04 mmol) was further added, and the reaction was stirred at room temperature overnight, and then precipitated and washed with n-hexane, and dried in vacuo to obtain the compound of formula (III) in which R3 is a hydrogen atom and n = 2.

[0073] Example 2

[0074] Synthesis of a camptothecin nanomedicine without a carrier of a tertiary amine N-oxide of formula (IV):

[0075] As a preferred embodiment, R1 and R2 of formula (IV) are methyl groups and n = 2 in the present embodiment.

[0076] The synthesis of the tertiary amine oxide ODMA and aminopropionyl camptothecin in the present embodiment was the same as in Example 1.

[0077] Synthesis of a camptothecin nanomedicine without a carrier of a tertiary amine N-oxide of formula (IV):

[0078] Aminopropionyl camptothecin (0.34 g, 0.8 mmol) and ODMA (0.13 g, 1.6 mmol) were weighed and dissolved in 10 mL of THF, and triethylamine (8.08 mg, 0.08 mmol) was further added, and the reaction was stirred at room temperature overnight, and the crude product was precipitated and washed with n-hexane, and the reaction solution containing the crude product was concentrated and purified by column chromatography (silica gel column, developing solvent: dichloromethane:methanol = 10:1), and dried in vacuo to obtain the compound of formula (IV) in which R1 and R2 are methyl groups and n = 2.

[0079] Example 3

[0080] Synthesis of tertiary amine nitroxide camptothecin nanodrug CPT-S-NO without carrier:

[0081] The synthesis method of the tertiary amine oxide ODMA in this embodiment is the same as that in Embodiment 1.

[0082] To further optimize, it is determined that R1 and R2 of the tertiary amine nitroxide camptothecin nanodrug of formula (I) are both methyl, and the X linker is a sulfide bond, that is, R1 and R2 of the tertiary amine nitroxide camptothecin nanodrug of formula (V) are both methyl, and n = 2, and the nanodrug without carrier is recorded as CPT-S-NO.

[0083] The synthesis route of CPT-S-NO is shown in Figure 1 The specific preparation method of the synthesis of CPT-S-NO includes: 3-mercapto propionic acid (2.40 g, 22.61 mmol) is dissolved in 10 mL of dichloromethane and placed in an ice water bath, and a solution of triphenylmethyl chloride (6.96 g, 24.97 mmol) in dichloromethane (20 mL) is added dropwise with a constant pressure dropping funnel under nitrogen protection. After the dropwise addition is completed, the ice water bath is removed, and the reaction is carried out at room temperature for 20 h. A large amount of white solid is observed to precipitate, the white solid is collected by suction filtration, and washed repeatedly with ice ether 3 times, and dried under vacuum to obtain 3-triphenylthio propionic acid.

[0084] 3-triphenylthio propionic acid (0.50 g, 1.43 mmol) and CPT (0.50 g, 1.44 mmol) are dissolved in 10 mL of DMF, and DCC (0.36 g, 1.73 mmol) and DMAP (0.16 g, 1.44 mmol) are added, and the reaction is stirred at room temperature for 24 h. After the reaction is completed, the reaction solution is filtered and the filtrate is concentrated with an oil pump, poured into a large amount of ether for precipitation, and washed repeatedly with ether, and dried to obtain 3-triphenylthio propyl camptothecin. 3-triphenylthio propyl camptothecin (0.68 g, 1 mmol) is dissolved in 5 mL of dry dichloromethane, TFA (1.54 mL, 20 mmol) is added dropwise with a dropper, stirred at room temperature for 2 h, then poured into a large amount of ether for precipitation, and the solid is collected and purified by chromatography (silica gel column, developing agent dichloromethane:methanol = 95:5) to obtain light yellow solid CPT-SH.

[0085] CPT-SH (0.35 g, 0.8 mmol) and ODMA (0.13 g, 0.8 mmol) are weighed and dissolved in 10 mL of THF, and triethylamine (4.04 mg, 0.04 mmol) is added, and the reaction is stirred at room temperature overnight, then precipitated and washed with n-hexane, and dried under vacuum to obtain CPT-S-NO. 1 The H NMR spectrum is shown in Figure 2

[0086] ​TEM observation of the morphology of CPT-S-NO in aqueous solution:

[0087] Place a 200-mesh copper grid on filter paper. Use a pipette to apply 20 μL of CPT-S-NO aqueous solution (1 mg / mL) onto the copper grid. Absorb any excess liquid with absorbent paper. Observe the CPT-S-NO speciation using TEM. Figure 3 As shown in a, CPT-S-NO can form spherical nanoparticles in aqueous solution, and its particle size was determined to be 100 nm using DLS. Figure 3 b).

[0088] Test Example 1

[0089] The samples used in the test examples were all CPT-S-NO prepared in Example 3.

[0090] (1) In vitro release experiment:

[0091] CPT-S-NO was dissolved in 2 mL of PBS buffer (pH 7.4 or containing 1 mM H₂O₂) containing 0.5% (w / v) Tween 80, resulting in a CPT equivalent concentration of 50 μg / mL. The solution was then placed in a constant-temperature shaker at 37°C and 120 rpm. At 2, 4, 6, 8, 12, 24, and 36 h, 200 μL of buffer solution was collected and replenished with an equal volume of buffer. An equal volume of methanol was added to the collected solution for dilution. The CPT release at each time point was determined by HPLC. The HPLC mobile phase was methanol:water = 6:4, the flow rate was 1 mL / min, and the detection wavelength was 360 nm.

[0092] from Figure 4 It can be seen that the oxidizing agent (H2O2) can accelerate the release of CPT-S-NO. This is because the thioether bond in CPT-S-NO is ROS responsive, which is conducive to the rapid release of CPT-S-NO in the tumor microenvironment with high ROS levels, while it remains stable in the normal physiological environment.

[0093] (2) In vitro anti-cancer cell proliferation experiment:

[0094] Using CPT-S-NO and the control CPT-11 as samples, experiments were conducted to assess their anti-cancer cell proliferation effects on human HepG2 liver cancer cells, human Bxpc-3 pancreatic adenocarcinoma cells, human HeLa cervical cancer cells, and human MDA-MB-231 breast cancer cells (all purchased from ATCC) after 48 hours. Isoequivalent CPT concentrations were 50 μg / mL, 10 μg / mL, 1 μg / mL, 0.1 μg / mL, 0.01 μg / mL, 0.001 μg / mL, and 0.0001 μg / mL. The experimental results are as follows: Figure 5 As shown, their respective ICs 50 The values ​​are shown in Table 1.

[0095] Table 1

[0096]

[0097] In these four tumor cells, the half lethal amount IC 50 The value of CPT-S-NO is greater than that of CPT-11, indicating that the in vitro cytotoxicity of CPT-S-NO is superior to that of CPT-11.

[0098] (3) Mitochondrial targeting ability:

[0099] On the logarithmic phase growth of human breast cancer MDA-MB-231 cell line, CPT-S-NO was added for incubation for 3 h, and then mitochondrial dye MitoTracker Green and nuclear dye were added in turn for staining for 20 min, and then washed twice with PBS, and the distribution of CPT-S-NO in mitochondria was observed under laser confocal, and the results are shown in Figure 6

[0100] As shown in Figure 6 CPT-S-NO can quickly enter cells and be distributed in mitochondria in large quantities Figure 6 (a). The degree of coincidence of CPT-S-NO and mitochondrial green fluorescent dye (Pearsen coefficient) was calculated by Image J, which was 0.82, indicating that the fluorescence of the two was highly correlated Figure 6 (b).

[0101] (4) Tumor sphere penetration:

[0102] According to the hanging drop method, dense MCF-7 tumor spheres were established, and MCF-7 tumor spheres or with endocytosis inhibitors (Filipin), chlorpromazine (Chlorpromazine) or with efflux inhibitors (Brefeldin A (BFA)) or without any inhibitors were incubated for 2 h, and then CPT-S-NO was added for further incubation for 2 h (CPT equivalent concentration was 10 μg / mL). After incubation, the tumor spheres were carefully transferred to a confocal imaging dish containing 1 mL PBS with a pipette, and CLSM was used for imaging. Z-stack tomography was used, and a photo was taken every 30 μm. The imaging parameters were kept consistent to compare the penetration intensity of CPT-S-NO in each layer of the tumor sphere.

[0103] As shown in Figure 7 CPT-S-NO has a certain tumor penetration ability, and this tumor penetration ability is affected by endocytosis or efflux inhibitors, indicating that it is an active penetration behavior.

[0104] (5) Pharmacokinetics:

[0105] ​Select 9 6-8 weeks old ICR mice (purchased from Shanghai Slek Experimental Animal Co., Ltd.), randomly divided into 3 groups, 3 in each group, respectively, 200 μL CPT-11 and CPT-S-NO (CPT equivalent concentration of 10 mg / kg) tail vein injection, blank control group injection of the same dose of PBS, 2 min, 30 min, 1 h, 2 h, 4 h, 6 h and 8 h from the mouse orbit blood (50 μL) to the centrifuge tube containing a small amount of heparin sodium. After the completion of the point, to each tube of blood samples added equal volume of 0.1M NaOH, mixed evenly, placed in 37℃ oven incubated overnight. After incubation, add 1 mL acetonitrile to each sample, ultrasonic oscillation so that CPT can be completely extracted into the organic phase, followed by 5000 rpm centrifugation for 5 min, 200 μL supernatant, add equal volume of hydrochloric acid solution (0.1M), ultrasonic mixing after 5000 rpm centrifugation for 5 min, take supernatant, HPLC detection of CPT concentration in blood at each time point. HPLC mobile phase is methanol: water = 6:4, flow rate of 1 mL / min, ultraviolet detection wavelength of 360 nm. The DAS software is used to calculate the pharmacokinetic parameters of each group, summarized in table 2.

[0106] Table 2

[0107] Parameter Units CPT-11 CPT-S-NO C max ]]> mg / L 0.366±0.130 27.97±6.84 CL z ]] h 20.1±5.34 0.660±0.078 AUC 0-t ]] mg / L x h 0.410±0.118 13.85±1.33

[0108] C max : maximum concentration in plasma; CL z : plasma clearance; AUC 0-t : area under the plasma drug concentration-time curve;

[0109] As Figure 8 shown, CPT-11 is quickly eliminated after being injected into the body, and CPT-S-NO shows a certain long circulation effect. According to the calculation, the area under the plasma drug concentration-time curve of CPT-S-NO is 33.78 times that of CPT-11, and the plasma clearance is only 0.033 times that of CPT-11, which shows that CPT-S-NO has better blood long circulation effect than CPT-11.

[0110] (6) In vivo tumor inhibition activity experiment:

[0111] Select 20 6-8 weeks old female BALB / c nude mice (purchased from Shanghai Slek Experimental Animal Co., Ltd.), adopt tumor block inoculation method, MDA-MB-231 tumor block is inoculated uniformly to the right abdominal side of the mouse with a volume of 8 mm 3 , and the tumor volume is grown to 50 mm 3When the experiment was finished, the mice were killed and the tumors were taken out, photographed and weighed.

[0112] The results are shown in Table 1. Figure 9 As shown in Table 1, the therapeutic effect of CPT-S-NO was better than that of CPT-11. During the administration period, the tumors in the CPT-S-NO treatment group hardly grew, and during the observation period after the administration was stopped, the tumor growth in the CPT-S-NO treatment group was slower than that in the other two groups. Moreover, CPT-S-NO had no obvious effect on the body weight of the mice during the treatment period, which indicated that CPT-S-NO could guarantee biological safety while exerting the tumor inhibition activity.

Claims

1. A tumor-penetrating, carrier-free nanomedicine, characterized by, The tumor-penetrating carrier-free nanomedicine is formed by self-assembly of a small molecule prodrug, wherein the small molecule prodrug is prepared by connecting a drug molecule with a tertiary amine nitroxide group through a linker, and the drug molecule is camptothecin, The structure of the small molecule prodrug is shown in formula (V): Formula (V); R1 is methyl, R2 is methyl, and n=2.

2. The method for preparing tumor-penetrating carrier-free nanomedicine according to claim 1, characterized in that, The method comprises the following steps: Camptothecin is subjected to carboxylic acid reaction with a sulfhydryl group containing a protective group, and after removal of the protective group, a small molecule chemotherapeutic drug with a sulfhydryl functional group is obtained, and then subjected to Michael addition reaction with a tertiary amine nitroxide group containing an acrylic ester under catalysis of an organic base to obtain a small molecule prodrug, and the small molecule prodrug is formed into the tumor-penetrating carrier-free nanomedicine by self-assembly.

3. The production method according to claim 2, characterized by, The step of forming the tumor-penetrating carrier-free nanomedicine by self-assembly of the small molecule prodrug is any one of the following: (a) dispersing the small molecule prodrug in a buffer solution, and self-assembling the small molecule prodrug by ultrasonic method to obtain the tumor-penetrating carrier-free nanomedicine; (b) dissolving the small molecule prodrug in an organic solution, adding dropwise into water to form nanoparticles, and removing the contained organic solvent by dialysis to obtain the tumor-penetrating carrier-free nanomedicine.

4. The production method according to claim 3, characterized by, The buffer solution is PBS buffer, HEPES buffer or Tris buffer; and the organic solvent is dimethyl sulfoxide, dimethyl formamide, ethanol or methanol.

5. The preparation method according to claim 2, characterized in that, The organic base is triethylamine, pyridine, N,N-diisopropyl ethylamine or diethylamine.

6. Use of the tumor-penetrating carrier-free nanomedicine according to claim 1 in preparation of an antitumor drug.

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