CN111097052A - Amphiphilic prodrug for active targeted therapy of tumors and preparation method and application of nanoparticles of amphiphilic prodrug - Google Patents

Amphiphilic prodrug for active targeted therapy of tumors and preparation method and application of nanoparticles of amphiphilic prodrug Download PDF

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CN111097052A
CN111097052A CN202010054358.8A CN202010054358A CN111097052A CN 111097052 A CN111097052 A CN 111097052A CN 202010054358 A CN202010054358 A CN 202010054358A CN 111097052 A CN111097052 A CN 111097052A
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夏雪霖
王冠春
杨晓媛
黄平
黄卫
颜德岳
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Abstract

The prodrug for active targeted therapy of tumors is an amphiphilic prodrug containing a targeted head group, which is formed by coupling a small molecule connecting group with a hydrophobic anti-tumor drug and an active targeted hydrophilic short peptide, wherein the small molecule connecting group is a small molecule connecting group containing a thioketone bond. The amphiphilic prodrug containing the targeting head group can be self-assembled in water to form nanoparticles with a shell layer of hydrophilic short peptide and a core of hydrophobic micromolecular antitumor drug. Because the hydrophilic short peptide on the surface of the nano-particles can be specifically combined with receptors on tumor cells or tissues, the nano-particles can actively target to tumor parts and be enriched, the concentration of small-molecule antitumor drugs on the tumor parts is improved, the proliferation of the tumor cells is effectively inhibited, meanwhile, the toxic and side effects of the antitumor drugs on normal cells and tissues are reduced, and the active targeted therapy of tumors is realized.

Description

Amphiphilic prodrug for active targeted therapy of tumors and preparation method and application of nanoparticles of amphiphilic prodrug
Technical Field
The invention relates to the technical field of nano-medicine, in particular to an amphiphilic prodrug for active targeted therapy of tumors.
Background
Malignant tumors (cancers) are serious diseases that seriously endanger human survival and social development, and have become a recognized public health problem all over the world. Therefore, how to effectively treat malignant tumor has become a difficult problem and a major challenge to be solved all over the world. At present, the following methods are mainly used for treating malignant tumors: surgical resection, chemotherapy, radiation therapy, immunization, and biological therapy. Among them, chemotherapy is one of the most effective therapeutic means. However, most of small molecular antitumor drugs have the disadvantages of poor water solubility, large toxic and side effects on normal tissues of organisms, short half-life in vivo and the like. In order to solve the problems, the carrier with nanometer size is adopted to deliver the hydrophobic micromolecule antitumor drug, so that the bioavailability of the micromolecule drug can be effectively improved, and the blood retention time and the tumor enrichment rate of the micromolecule drug can be improved. However, these nano-sized carriers, including water-soluble polymers, polymeric micelles, liposomes, vesicles, inorganic materials, etc., lack specific recognition of tumor tissues or cells, and the molecular structure of the adopted drug carrier is uncertain, and in addition, after the drug is delivered, the carriers may cause toxic and side effects or inflammation to normal tissues and organs during the in vivo degradation or metabolism process.
Disclosure of Invention
The invention aims to provide an amphiphilic prodrug for active targeted therapy of tumors and nanoparticles thereof, and aims to solve the problems that in the prior art, a nano antitumor drug is enriched to a tumor focus part only through the high permeability and retention (EPR) effect of a solid tumor, the targeting property of the nano antitumor drug to cancer tissues or tumor cells is weak, the selectivity is poor, and toxicity, inflammation and the like are generated to normal tissues or organs.
The second purpose of the present invention is to provide a preparation method of the above amphiphilic prodrug nanoparticles for active targeted therapy of tumors.
The third purpose of the invention is to provide the pharmaceutical application of the amphiphilic prodrug nanoparticles for active targeted therapy of tumors.
The technical scheme of the invention is as follows:
an amphiphilic prodrug for active targeted therapy of tumors is obtained by covalently bonding active targeted hydrophilic short peptides and hydrophobic antitumor drugs through micromolecular linking groups; the linking group is selected from a small molecule linking group containing a thioketal bond.
Preferably, the active targeting hydrophilic short peptide is selected from one of cyclic pentapeptide (cyclo- [ Arg-Gly-Asp-d-Phe-Cys ], RGD) targeting integrin α v β 3 receptor and follicle-stimulating hormone FSH targeting follicle-stimulating hormone receptor (FSHR), and the hydrophobic antitumor drug is selected from one or more of epothilone B and derivatives thereof, maytansine and derivatives thereof and adriamycin and derivatives thereof.
Preferably, the structure of the thioketal bond-containing small molecule linker is shown as the formula (I):
Figure BDA0002372302030000021
wherein R is1Selected from-alkyl-C (O) NH- (preferably-C1-8 alkyl-C (O) NH-, more preferably-C1-3 alkyl-C (O) NH-), -alkyl-SO2NH- (preferably-C1-8 alkyl-SO)2NH-, more preferably-C1-3 alkyl-SO2NH-), C1-8 alkoxy (preferably C1-3 alkoxy); r2Is selected from carboxyl, acyl chloride, n and m are independently selected from 2 or 3; the alkyl is selected from C1-8 alkyl, and the alkyl and the alkoxy are unsubstituted or substituted by 1, 2 and 3 substituents selected from the following group: halogen, cyano, acetyl, hydroxy, hydroxymethyl, hydroxyethyl, carboxy, C1-8 alkyl, C1-8 alkoxy, halo C1-8 alkyl, C3-8 cycloalkyl, C3-8 cycloalkoxy, halo C1-8 alkoxy, -C (O) C1-10 alkyl, -C (O) OC1-10 alkyl, -OC (O) C1-10 alkyl, -CONRa0Rb0；Ra0、Rb0Each independently hydrogen or C1-8 alkyl. R2Is connected with the antitumor drug to form an ester bond, and the maleimide group is connected with the targeting hydrophilic short peptide.
Preferably, the structure of the amphiphilic prodrug is shown as the formula (II):
Figure BDA0002372302030000031
wherein n and m are each independently selected from 2 or 3.
An amphiphilic prodrug nanoparticle for active targeted therapy of tumors is obtained by self-assembling the amphiphilic prodrug in water.
Preferably, the shell layer of the amphiphilic prodrug nanoparticle for active targeted therapy of tumors is composed of the active targeting functional short peptide, the inner core of the nanoparticle is composed of the hydrophobic antitumor drug, and the particle size of the nanoparticle is less than 200 nm.
A preparation method of amphiphilic prodrug nanoparticles for active targeted therapy of tumors comprises the following steps: (1) bonding a hydrophobic antitumor drug containing hydroxyl and/or sulfhydryl with a micromolecule connecting group containing active oxygen response, and then carrying out coupling reaction with an active targeting hydrophilic short peptide containing sulfhydryl to obtain an amphiphilic prodrug with a targeting head group; (2) dissolving the amphiphilic prodrug in the step (1) in an organic solvent, dripping the amphiphilic prodrug into water at room temperature, and removing the organic solvent to obtain the targeting amphiphilic prodrug nanoparticle aqueous solution.
Preferably, the organic solvent in step (2) is at least one selected from the group consisting of dimethyl sulfoxide, N' -dimethylformamide, methanol, ethanol and isopropanol.
The amphiphilic prodrug for active tumor targeted therapy, or the amphiphilic prodrug nano-particles for active tumor targeted therapy, which are prepared by the preparation method, are applied to the preparation of antitumor drugs.
Compared with the prior art, the invention has the following beneficial effects:
the amphiphilic prodrug with the targeting head group can be self-assembled in water to form nano particles, the shell layer is provided with active targeting short peptides, the active targeting short peptides can be specifically combined with tumor cells or tissues, the enrichment of the drug at tumor parts is enhanced, the prodrug nano particles can more effectively enter the tumor cells, and the toxic and side effects on normal cells and tissues are reduced; after the prodrug nanoparticles enter tumor cells, ester bonds connected between the small molecule connecting groups and the antitumor drugs are degraded to release the antitumor drugs, so that the toxicity of the small molecule antitumor drugs is reduced, and the purpose of targeted treatment of cancers is achieved.
The invention introduces a linker containing active oxygen response thioketal bond based on that the concentration of active oxygen in tumor cells is far higher than that in normal cells, connects hydrophilic RGD and hydrophobic epothilone to form an amphiphilic conjugate, forms nano particles with uniform size through self-assembly in water, not only has good tumor targeting capability, but also has selectivity on tumor cells because the thioketal bond can only be broken off in the tumor cells to release the epothilone, obviously reduces the toxic and side effects on normal tissues, and simultaneously has good inhibition effect on tumors.
Of course, it is not necessary for any product in which the invention is practiced to achieve all of the above-described advantages at the same time.
Drawings
FIG. 1 is example 1 of a prodrug A with a targeting head group amphiphile1HNMR spectrogram;
FIG. 2 is a TEM image of nanoparticles of example 1 with a targeted prodrug A;
FIG. 3 is a graph of the particle size distribution of nanoparticles of example 1 having a targeted prodrug A;
FIG. 4 is a schematic representation of the tumor cell growth inhibition effect of the targeted prodrug-containing nanoparticles of example 1;
FIG. 5 is a schematic diagram of in vivo imaging of the prodrug nanoparticles with targeting property in PC-3 tumor-bearing nude mice after fluorescent modification in example 1.
Detailed Description
In this context, a range of values from one value to another is a general expression avoiding any recitation of all values in the range in the specification. Thus, recitation of a range of values herein is intended to encompass any value within the range and any smaller range defined by any value within the range, as if the range and smaller range were explicitly recited in the specification.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. In practice, the invention will be understood to cover all modifications and variations of this invention provided they come within the scope of the appended claims.
Example 1
Figure BDA0002372302030000051
1.1 Synthesis of intermediate A-1
Mixing 3,3' -propane-2, 2-diylbis (sulfonamido) dipropionic acid (TK, 252.05mg), 1-ethyl- (3-dimethylaminopropyl)Carbodiimidate hydrochloride (EDCI, 287.55mg), 4-dimethylaminopyridine (DMAP, 12.2mg) and anhydrous triethylamine (TEA, 0.28mL) were charged into a reaction flask, 25mL of anhydrous dichloromethane were added thereto, and after stirring at room temperature for 1 hour, Epothilone B (Epothilone B, 507.27mg) was added thereto, followed by stirring at room temperature for 24 hours. After the reaction is finished, 20mL deionized water is added for extraction, an organic phase is collected, a mixed solution of dichloromethane and methanol in a volume ratio of (20:1) is used as an eluent, and the white powdery intermediate A-1(345.92mg, the yield is 46.6 percent) is obtained by column chromatography separation, wherein the MS M/z (ESI) is 742.3039[ M + H ]]+.1H NMR(CDCl3,400MHz)δ7.04(1H,s),6.82(1H,bs),5.34(1H,dd),5.26(1H,dd),4.21(1H,m),4.19(1H,bs),3.56(1H,dq),2.93(4H,m),2.84(1H,dd),2.75(3H,s),2.69(4H,m),2.51(1H,dd),2.35(1H,dd),2.16(1H,ddd),2.12(3H,d),1.95(1H,ddd),1.74(2H,m),1.63(6H,s),1.35(2H,m),1.32(3H,m),1.30(3H,s),1.27(3H,s),1.16(3H,d),1.13(3H,s),0.97(3H,d).
1.2 Synthesis of intermediate A-2
Intermediate A-1(74.13mg), N- (2-aminoethyl) maleimide trifluoroacetate (38.12mg), 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (HATU, 57.04mg) and N, N-diisopropylethylamine (DIPEA, 42. mu.L) were added to a reaction flask, followed by addition of 20mL of anhydrous dichloromethane, and the reaction was stirred at room temperature for 6 hours. After the reaction, 20mL of deionized water was added for extraction, the organic phase was collected and subjected to column chromatography using a mixed solution of dichloromethane and methanol at a volume ratio of (20:1) as an eluent to give intermediate A-2(74.07mg, yield 85.7%) as a white powder.1H NMR(CDCl3,400MHz)δ7.01(1H,s),6.74(1H,s),6.64(1H,bs),5.49(1H,dd),5.33(1H,dd),4.28(1H,bs),4.12(1H,m),3.75(1H,m),3.71(2H,t),3.50(2H,t),3.20(1H,dq),2.90(4H,m),2.84(1H,dd),2.72(3H,s),2.67(2H,t),2.58(1H,dd),2.49(1H,dd),2.44(2H,t),2.18(1H,d),2.12(3H,d),1.97(1H,d),1.68(2H,m),1.60(6H,s).1.47(2H,m),1.44(3H,m),1.38(3H,s),1.29(3H,s),1.16(3H,d),1.09(3H,s),0.94(3H,d).
1.3 Synthesis of prodrug A
Intermediate A-2(86.35mg) and RGD (57.82mg) were added to a reaction flask, followed by addition of 5mL of methanol and stirring at room temperatureThe reaction was carried out for 12 hours. Pouring the reaction solution into 50mL of ethyl glacial ether, filtering to obtain a crude prodrug A product, dissolving the crude prodrug A product in 5mL of methanol, dialyzing and purifying in water, and freeze-drying after dialysis to obtain a white powdery prodrug A (118.65mg, yield 82.3%),1H NMR(400MHz,DMSO-d6)δ8.45(1H,m),8.33-8.19(2H,m),8.02(2H,m),7.40(1H,s),7.36(1H,d),7.23-7.18(6H,m),6.50(1H,s),6.44(1H,s),5.46-5.32(2H,m),4.67-4.53(3H,m),4.19-4.00(4H,m),3.51-3.09(14H,m),2.81-2.65(12H,m),2.11-1.99(6H,m),1.72-1.41(20H,m),1.23-1.18(9H,m),0.97(3H,d).
the amphiphilic prodrug a prepared above is dissolved in methanol, and dropped into water at room temperature, and the methanol is removed to obtain an aqueous solution of nanoparticles of the amphiphilic prodrug, that is, the antitumor nanoparticles based on prodrug a prepared in this example.
The transmission electron micrograph of the anti-tumor nanoparticles based on the prodrug a of the embodiment is shown in fig. 2, and the average size of the particle size of the nanoparticles is about 100 nm; the dynamic light scattering data of the prodrug a-based anti-tumor nanoparticles are shown in fig. 3, and the average size of the nanoparticle size is about 100nm, which is substantially consistent with the data of the transmission electron microscope photograph. The good uniformity of the nano particles can effectively reduce the batch-to-batch difference during the preparation of the nano particles and improve the quality stability of the nano particles; secondly, good homogeneity can ensure that the drug nanoparticles have consistent pharmacokinetic properties and in vivo metabolic pathways when circulating in vivo, and is helpful for clinical evaluation.
The embodiment is based on that the concentration of active oxygen in tumor cells is far higher than that in normal cells, the design introduces a linker containing active oxygen response thioketal bonds to connect hydrophilic RGD and hydrophobic epothilone to form an amphiphilic conjugate, nanoparticles with uniform size are formed by self-assembly in water, the amphiphilic conjugate has good tumor targeting capability, and meanwhile, because the thioketal bonds can only break off and release the epothilone in the tumor cells, the amphiphilic conjugate has selectivity on the tumor cells, the toxic and side effects on normal tissues are remarkably reduced, and meanwhile, the amphiphilic conjugate has a good tumor inhibition effect on tumors. Thereby solving the key problem that the high-efficiency and high-toxicity epothilone is difficult to be clinically transformed.
In the embodiment, the antitumor drug is epothilone B, and the small molecular compound containing the thioketal bond is firstly condensed with epothilone B, amidated with maleimide trifluoroacetate and subjected to click reaction with RGD to obtain the amphiphilic prodrug containing the targeting head group. In other alternative embodiments, the anti-tumor drug may also be maytansine or its derivatives, doxorubicin or its derivatives, or other existing anti-tumor drugs, and at this time, the anti-tumor drug needs to introduce a group to achieve connection with the thioketal bond-containing small molecule compound, which is not described herein again.
Example 2
This example provides an experiment on the effect of amphiphilic prodrug nanoparticles on cancer cells for active targeted therapy of tumors.
The amphiphilic prodrug nanoparticles (Assembly of A) prepared in example 1 and the epothilone B (epothilone B) serving as the raw material drug are prepared into solutions with the concentration of 1.25, 2.5, 5, 10, 15, 20, 40 and 80nmol/L by using cell culture solutions respectively, and then are cultured with HCT116 cells (colon cancer cells) and PC-3 cells (prostate cancer cells) for 48 hours respectively, and then the MTT method is adopted for cell activity test, and the results are shown in FIG. 4. The concentration of the epothilone B at 2nmol/L shows the effect of killing cancer cells with high efficiency; meanwhile, after the concentration of the amphiphilic prodrug nanoparticles reaches 15nmol/L, the amphiphilic prodrug nanoparticles also show good capability of killing cancer cells, and the killing effect of the amphiphilic prodrug nanoparticles on the cancer cells is in a direct proportion relation with the concentration. The amphiphilic prodrug nano-particle has potential application value in treating malignant tumors.
Example 3
The amphiphilic prodrug nano-particle for active targeted tumor therapy disclosed by the invention is used for a fluorescence imaging experiment in a tumor-bearing nude mouse.
Cy5.5 is used as a fluorescent probe molecule in vivo, and is wrapped in the amphiphilic prodrug nano particle obtained in the embodiment 1, so that the targeting effect of the nano particle is evaluated. PC-3 cells in logarithmic growth phase were digested, counted, and prepared to 4.0X 106Per mL of cell suspension, inoculated subcutaneously into the right anterior axilla of nude mice, each inoculated with 200. mu.L, when the tumor grows to a volume of about 500mm3At that time, 2 mice were randomly selected. 200 μ L of Cy5.5-loaded nanoparticles were injected via tail vein into PC-3 tumor-bearing mice. After 12h of injection, mice were photographed and analyzed for whole body fluorescence intensity using the ZKKS-Mulaurora imaging system. The results are shown in FIG. 5. After the amphiphilic prodrug nanoparticles circulate in a mouse for 12 hours, other visceral organs do not exist obviously, but the amphiphilic prodrug nanoparticles still have obvious enrichment at tumor parts. The amphiphilic prodrug nano-particle has excellent tumor targeting performance and has potential application value in tumor treatment.
In light of the above teachings, those skilled in the art will readily appreciate that the materials and their equivalents, the processes and their equivalents, as listed or exemplified herein, are capable of performing the invention in any of its several forms, and that the upper and lower limits of the parameters of the materials and processes, and the ranges of values between these limits are not specifically enumerated herein.

Claims (9)

1. An amphiphilic prodrug for active targeted therapy of tumors is characterized in that the amphiphilic prodrug is obtained by covalently bonding active targeted hydrophilic short peptides and hydrophobic antitumor drugs through a small molecule connecting group, wherein the connecting group is a small molecule connecting group containing thioketone bonds.
2. The amphiphilic prodrug for active targeted therapy of tumors according to claim 1, wherein the active targeting hydrophilic short peptide is selected from one of cyclic pentapeptide RGD targeting integrin α v β 3 receptor or follicle stimulating hormone FSHR targeting follicle stimulating hormone receptor FSHR, and the hydrophobic antitumor drug is selected from one or more of epothilone B and its derivatives, maytansine and its derivatives, and doxorubicin and its derivatives.
3. The amphiphilic prodrug for active targeted tumor therapy according to claim 2, wherein the structure of the small molecule linker containing thioketal bond is shown in formula (I):
Figure FDA0002372302020000011
wherein R is1Selected from-alkyl-C (O) NH-, -alkyl-SO2NH-, C1-8 alkoxy; r2Is selected from carboxyl, acyl chloride, n and m are respectively and independently selected from 2 or 3, and the alkyl is selected from C1-8 alkyl; the alkyl and the alkoxy are unsubstituted or substituted by 1, 2 and 3 substituents selected from the following groups: halogen, cyano, acetyl, hydroxy, hydroxymethyl, hydroxyethyl, carboxy, C1-8 alkyl, C1-8 alkoxy, halo C1-8 alkyl, C3-8 cycloalkyl, C3-8 cycloalkoxy, halo C1-8 alkoxy, -C (O) C1-10 alkyl, -C (O) OC1-10 alkyl, -OC (O) C1-10 alkyl, -CONRa0Rb0；Ra0、Rb0Each independently hydrogen or C1-8 alkyl.
4. The amphiphilic prodrug for active targeted tumor therapy according to claim 1, wherein the structure of the amphiphilic prodrug is represented by formula (II):
Figure FDA0002372302020000021
wherein n and m are each independently selected from 2 or 3.
5. An amphiphilic prodrug nanoparticle for active targeted tumor therapy, wherein the amphiphilic prodrug nanoparticle is obtained by self-assembling the amphiphilic prodrug of any one of claims 1 to 4 in water.
6. The amphiphilic prodrug nanoparticle for active targeted tumor therapy according to claim 5, wherein the shell layer of the nanoparticle is composed of the active targeting hydrophilic short peptide, the inner core of the nanoparticle is composed of the hydrophobic antitumor drug, and the particle size of the nanoparticle is less than 200 nm.
7. A preparation method of amphiphilic prodrug nanoparticles for active targeted therapy of tumors is characterized by comprising the following steps:
(1) bonding a hydrophobic antitumor drug containing hydroxyl and/or sulfhydryl with a small molecule connecting group, and then carrying out coupling reaction with the active targeting hydrophilic short peptide to obtain an amphiphilic prodrug with a targeting head group;
(2) dissolving the amphiphilic prodrug in the step (1) in an organic solvent, dripping the amphiphilic prodrug into water at room temperature, and removing the organic solvent to obtain the targeting amphiphilic prodrug nanoparticle aqueous solution.
8. The preparation method of amphiphilic prodrug nanoparticles for active targeted therapy of tumors according to claim 7, wherein the organic solvent in step (2) is at least one selected from the group consisting of dimethyl sulfoxide, N' -dimethylformamide, methanol, ethanol and isopropanol.
9. Use of the amphiphilic prodrug for active tumor-targeted therapy according to any one of claims 1 to 4, or the amphiphilic prodrug nanoparticle for active tumor-targeted therapy according to claim 5 or 6, or the amphiphilic prodrug nanoparticle for active tumor-targeted therapy prepared by the preparation method according to claim 7 or 8, in the preparation of an antitumor drug.
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