CN115154419A - Nano micelle precursor, nano micelle, preparation method and application thereof - Google Patents

Nano micelle precursor, nano micelle, preparation method and application thereof Download PDF

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CN115154419A
CN115154419A CN202110360627.8A CN202110360627A CN115154419A CN 115154419 A CN115154419 A CN 115154419A CN 202110360627 A CN202110360627 A CN 202110360627A CN 115154419 A CN115154419 A CN 115154419A
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ligand
protein degradation
degradation agent
precursor
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CN115154419B (en
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于海军
裴明亮
祝奇文
高晶
李俊豪
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Shanghai Institute of Materia Medica of CAS
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Abstract

The invention belongs to the field of medicines, and particularly relates to a nano micelle precursor, a nano micelle, a preparation method and application thereof. The nano-micelle of the present invention is formed by self-assembly of nano-micelle precursors. The nano micelle precursor comprises a protein degradation agent, a connecting group and a polymer skeleton; one end of the polymer skeleton is connected with the protein degradation agent through a covalent bond through a connecting group, and the other end is connected with a ligand or a terminal group, wherein the ligand is selected from ligand groups of targeted tumor cells or tumor matrixes. The nano micelle can selectively target tumor cells, improve the uptake capacity of the tumor cells to the protein degradation agent, maintain the protein degradation activity of the tumor cells, improve the tissue distribution condition and the pharmacokinetic property of protein degradation agent molecules in organisms, widen the treatment window and improve the tumor inhibition effect of the protein degradation agent molecules.

Description

Nano micelle precursor, nano micelle, preparation method and application thereof
Technical Field
The invention belongs to the field of medicines, and particularly relates to a nano micelle precursor, a nano micelle and a preparation method and application thereof.
Background
The proteolysis targeting chimera (PROTAC) technology is taken as a new generation of protein degradation technology, the targeted protein can be subjected to ubiquitination marking by simultaneously combining molecules with the targeted protein and E3 ubiquitin ligase, the target protein is degraded by virtue of an intracellular ubiquitin-proteasome system, the removal of the target protein which cannot be used as a medicine is favorably realized, and the proteolysis targeting chimera (PROTAC) technology is a hotspot in the field of current medicine research and development. Compared with the traditional small molecule inhibitor, PROTAC and other protein degradation agent molecules have catalytic activity and relatively low toxicity, and the degradation effect of the PROTAC and other protein degradation agent molecules does not depend on the affinity action of molecules and target proteins independently, so that good selectivity can be realized at a cellular level, and the method is favorable for improving the drug resistance of the small molecule inhibitor in organisms, particularly the drug resistance in tumor treatment. However, such protein degradation agent molecules generally have large molecular weight, relatively poor membrane permeability and relatively low bioavailability, and toxic and side effects caused by off-target effects hinder the development of protein degradation technologies in clinical applications.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The primary object of the present invention is to provide a nanomicelle precursor.
The second purpose of the invention is to propose a preparation method of the nano-micelle precursor.
The third invention of the present invention is to provide a nano-micelle.
The fourth invention of the present invention is to propose a method for preparing the nano-micelle.
The fifth invention of the present invention is to propose the use of the nanomicelle.
In order to realize the purpose of the invention, the technical scheme is as follows:
the invention provides a nano micelle precursor in a first aspect, which comprises a protein degradation agent, a connecting group and a polymer skeleton; the polymer skeleton is selected from polyethylene glycol or polyethylene glycol derivatives, and comprises at least one of the structural units shown in formula i1, formula i2 and formula i 3:
Figure BDA0003005412010000021
wherein m is selected from the group consisting of integers from 10 to 145, preferably from 100 to 140, more preferably 113; q is selected from integers from 0 to 100, preferably 20 to 50, more preferably 38; p is an integer of 0 to 100, preferably 20 to 50, more preferably 38; and m, q and p are not zero or not 0 at the same time;
one end of the polymer skeleton is connected with the protein degradation agent through a covalent bond through a connecting group; the other end of the polymer skeleton is connected with a ligand or a terminal group; the ligand is selected from a ligand targeting tumor cells or tumor stroma; the terminal group is preferably methoxy.
The second aspect of the present invention provides a method for preparing a nano-micelle precursor, which at least comprises the following steps:
reacting the precursor of the linking group with the protein degradation agent molecule to form covalent linkage between the two and obtain a functional group-derivatized protein degradation agent molecule;
covalently linking a functional group-derivatized protein degradation agent molecule to a polymer backbone having a terminal group or an unblocked polymer backbone to obtain a polymer-protein degradation agent conjugate having a terminal group or an unblocked polymer-protein degradation agent conjugate;
the product of the non-end-capped polymer-protein degradation agent conjugate and the ligand through the bioorthogonal reaction and the polymer-protein degradation agent conjugate with a terminal group are the precursor of the nano micelle.
The third aspect of the present invention provides a nano micelle, which is self-assembled from the nano micelle precursor.
The fourth aspect of the invention provides a preparation method of the nano micelle, which at least comprises the following steps: and (3) dissolving the nano micelle precursor in an organic solvent to obtain a solution, dispersing the solution in water, and performing ultrafiltration or dialysis to obtain the nano micelle.
The fifth aspect of the invention provides an application of the nano-micelle in preparing a medicament for treating malignant tumors, wherein the malignant tumors are preferably selected from breast cancer, cervical cancer, liver cancer, gastric cancer, pancreatic cancer, ovarian cancer, colon cancer or prostate cancer.
The technical scheme of the invention at least has the following technical effects:
the nano micelle obtained by self-assembling the nano micelle precursor can selectively target tumor cells, improve the uptake capacity of the tumor cells to protein degradation agent molecules, improve the tissue distribution condition and the pharmacokinetic property of the protein degradation agent in organisms, widen the treatment window and improve the tumor inhibition effect of the protein degradation agent, and lay a foundation for the clinical application of the protein degradation agent.
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FIG. 1 is a graph showing a dynamic light scattering particle size distribution of CPEG-PLGA-ssV-JQ1 nano-micelles prepared in preparation example 6;
FIG. 2 is a cumulative release curve of JQ1-p-VHL under the in vitro simulated normal cell physiological microenvironment and tumor cell physiological microenvironment for CPEG-PLGA-ssV-JQ1 nanomicelle prepared in preparation example 6, wherein squares represent 0.5% Tween 80+10 μ M GSH (normal cell physiological microenvironment), and dots represent 0.5% Tween 80+10mM GSH (tumor cell physiological microenvironment);
FIG. 3 shows the uptake of the CPEG-PLGA-ssV-JQ1 fluorescent nanomicelles and PEG-PLGA-ssV-JQ1 fluorescent nanomicelles prepared in preparation example 7 into MDA-MB-231 breast cancer cells. Wherein, the black background column represents PEG-PLGA-ssV-JQ1 fluorescent nano micelle, and the white background column represents CPEG-PLGA-ssV-JQ1 fluorescent nano micelle;
FIG. 4 shows the results of experiments on the inhibition of cell proliferation potency of the JQ1-p-VHL prepared in preparation example 1 (left panel) and CPEG-PLGA-ssV-JQ1 nanomicelle prepared in preparation example 6 (right panel) in MDA-MB-231 breast cancer cells;
FIG. 5 shows the degradation of BRD4 protein in MDA-MB-231 breast cancer cells by the JQ1-p-VHL molecule prepared in preparation example 1 (left panel) and the CPEG-PLGA-ssV-JQ1 nanomicelle prepared in preparation example 6 (right panel).
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. Furthermore, in the following detailed description, numerous specific details are set forth in order to provide a better understanding of the present invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In some embodiments, materials, elements, methods, means, and the like that are well known to those skilled in the art are not described in detail in order to not unnecessarily obscure the present invention.
Throughout the specification and claims, unless explicitly stated otherwise, the term "comprise" or variations such as "comprises" or "comprising", etc., will be understood to imply the inclusion of a stated element or component but not the exclusion of any other element or component.
Aiming at improving the application of protein degradation technology in tumor treatment, the embodiment of the invention provides a protein degradation agent nano micelle and a precursor thereof so as to realize the targeted delivery of a medicament. By forming the drug-loaded nano-particles, the uptake capacity of cells to drugs can be improved by means of the endocytosis of tumor cells to nano-micelles, and the influence of the membrane penetrating capacity of drug molecules on the uptake of cells can be reduced; improve the pharmacokinetic property of the protein degradation agent and finally improve the tumor treatment effect of the protein degradation agent. Meanwhile, under the protection effect of the nano particles, the half-life period of the medicament can be effectively prolonged, and the tissue distribution and the pharmacokinetic property of the medicament can be improved. The nano micelle of the embodiment of the invention is formed by self-assembling nano micelle precursors. The nanomicelle precursor provided by the embodiment of the invention is characterized in that a protein degradation agent group, a linking group and a polymer skeleton are coupled by the linking group in a covalent connection manner to obtain the polymer skeleton, and the polymer skeleton is used for adjusting the ratio of a lipophilic segment to a hydrophilic segment of a conjugate so as to promote the formation of the nano prodrug. One end of the polymer skeleton is connected with the protein degradation agent through a covalent bond through a connecting group, and can trigger response in a specific physiological microenvironment (comprising pH value difference and glutathione level (GSH)) to release the protein degradation agent; the other end of the polymer skeleton is connected with a ligand or a terminal group; the ligand group may be selected from ligands targeting tumor cells or tumor stroma; the terminal group is an inert group without reactivity. The protein degradation agent is connected to the polymer skeleton through a covalent bond, and compared with the micelle obtained by adopting the conventional encapsulation technology, the micelle is favorable for realizing targeted delivery and slow release of the protein degradation agent and reducing the influence of concentration on the action effect of the medicament.
In certain embodiments, the nanomicelle precursors of embodiments of the invention have the structural formula shown in formula I and formula II:
Figure BDA0003005412010000051
wherein Ligand represents a Ligand, F-PEG represents a polymer skeleton for functionalization, linker represents a connecting group, PROTAC represents a protein degradation agent group, R represents a terminal group, and the groups are connected through covalent bonds.
Specifically, the ligand is selected from at least one of fusion protein with tumor cell or tumor matrix targeting, a nano antibody, a targeting peptide molecule and a small molecule targeting ligand.
Preferably, the terminal group is methoxy.
In certain embodiments, the fusion protein is selected from at least one of an antibody fusion protein (e.g., an Fc fusion protein), a diphtheria toxin fusion protein (e.g., DT389-hIL13-13E 13K), an immunoglobulin fusion protein (e.g., lg fusion protein);
preferably, the nanobody is selected from, but not limited to: at least one of an anti-CD 44 nanobody, an anti-Carbonic Anhydrase IX (CAIX) nanobody, an anti-Epidermal Growth Factor Receptor (EGFR) nanobody;
wherein, the targeting peptide molecule is at least one of peptides with amino acid sequences shown as SEQ ID NO. 1-5, and the amino acid sequences are shown as table 1:
TABLE 1
Figure BDA0003005412010000052
Figure BDA0003005412010000061
In certain embodiments, the small molecule targeting ligand and its derivatives are selected from at least one of the following structural formulae:
Figure BDA0003005412010000062
in certain embodiments, the ligand comprises a linking group A for coupling to the polymer backbone,
alternatively, the linking group a is used to link with a derivatised functional group on the polymer backbone, selected from the group formed after reaction of a thiol or alkynyl functional group. For example, the linking group A formed by the mercapto group is an-S-, the linking group A formed by the alkynyl functional group is
Figure BDA0003005412010000063
The free sulfydryl or disulfide bond existing in the ligand such as fusion protein, nano antibody and targeting peptide molecule is reduced to generate sulfydryl, and alkynyl functional group existing at the tail end of the micromolecule ligand can react with the derivative functional group on the polymer skeleton.
In certain embodiments, the polymer backbone used to modulate the ratio of lipophilic to hydrophilic segments of the conjugate to promote nanomicelle formation is selected from polyethylene glycol or polyethylene glycol derivatives comprising at least one structural unit according to formula i1, formula i2, and formula i 3:
Figure BDA0003005412010000064
wherein m is selected from integers from 10 to 145, preferably from 100 to 140, more preferably 13;
q is selected from integers from 0 to 100, preferably 20 to 50, more preferably 38;
p is an integer of 0 to 100, preferably 20 to 50, more preferably 38;
and m, q and p are not zero or 0 at the same time.
In certain embodiments the polymer backbone may have structural units as shown in formula ii1 or formula ii2,
Figure BDA0003005412010000071
wherein m is the number of ethylene glycol units in the polymer, and can be an integer of 10 to 145, preferably an integer of 100 to 140, and more preferably 113;
q is the number of lactic acid units in the polymer, and may be an integer of 0 to 100, preferably an integer of 20 to 50, more preferably 38;
p is the number of glycolic acid units in the polymer, and may be an integer of 0 to 100, preferably an integer of 20 to 50, more preferably 38;
and m, q and p are not zero at the same time or are not zero at all.
R a Denotes the residue of a derivatized functional group on the polymer after reaction with a ligand to form a covalent bond, R b Represents a terminal group.
In certain embodiments, R a Selected from: -NH-,
Figure BDA0003005412010000072
R b selected from methoxy;
wherein R is a And the functional groups on the ligand form a one-to-one corresponding relationship, and the specific relationship is as follows:
when the polymer derivative functional group is NH 2 When it is desired, to form a covalent bond with it, it is the carboxyl function of the ligand, R of the polymer a forming-NH-CO-with a linking group A on the ligand, thereby linking the polymer and the ligand through covalent bond;
when the polymer-derivatizing functional group is
Figure BDA0003005412010000081
When the covalent bond is formed, the thiol functional group on the ligand or the thiol generated by the reduction of disulfide bond in the molecule, R on the polymer a Form with the linking group A on the ligand
Figure BDA0003005412010000082
Thereby covalently linking the polymer to the ligand;
when the polymer derivative functional group is-N 3 Covalently bonded thereto is a derivatized alkynyl group on the ligand, R on the polymer a Form with the linking group A on the ligand
Figure BDA0003005412010000083
Thereby covalently linking the polymer to the ligand.
In certain embodiments, when p is 0 and q is other than 0, the polymer backbone is polyethylene glycol-polylactic acid;
when q is 0 and p is not 0, the polymer skeleton is polyethylene glycol-polyglycolic acid;
when q and p are not 0, the polymer skeleton is polyethylene glycol-polylactic acid-polyglycolic acid.
Preferably, m is preferably 113, q is preferably 38, p is preferably 0 or 38.
In certain embodiments, the precursor of the polymer backbone has the structure shown in formula iii1 or formula iii 2:
Figure BDA0003005412010000084
wherein, m, q, p, R b The meaning is the same as that of the formulae ii1, ii 2; r a-p Represents a derivatized functional group for covalent binding to a ligand, wherein R a-p Selected from: NH (NH) 2 -、
Figure BDA0003005412010000085
-N 3 。
In certain embodiments, the linking group is selected from the group consisting of groups shown as formula iv1, formula iv2, and formula iv 3;
Figure BDA0003005412010000091
wherein, M 1 、M 2 、R 3 Independently selected from alkylene groups having 1 to 4 carbon atoms, preferably R 1 、R 2 Independently selected from carbonyl, -O-C (O) -, -NH-C (O) -;
preferably, R 3 is-C (CH) 3 ) 2 -，
Further preferably, M 1 And M 2 The same is true.
In certain embodiments, the group of formula iv2 can be obtained by reacting a dithiodiacid starting material, such as 4, 4-dithiodibutyrate. The group of formula iv1 can be obtained by reacting a thiodiacid, such as thiodiacetic acid, as a starting material. The group of formula iv3 can be derived from thioketal diacid, for example 2,2' - [ propane-2, 2-diylbis (thio) ] diacetic acid.
In certain embodiments, the structural formula of the linking group may be selected from the group represented by the following formula, without limitation:
Figure BDA0003005412010000092
Figure BDA0003005412010000101
in certain embodiments, the precursor of the linking group is selected from the group consisting of compounds of formula iv1-p, formula iv2-p, and formula iv 3-p;
Figure BDA0003005412010000102
wherein, M 1 、M 2 、R 3 The meanings indicated are the same as in iv1 to iv 3;
L 1 、L 2 each independently selected from-COOH, -NCO and-NH 2 、–OH；
Further, the compound represented by iv1-p is preferably thiodiacetic acid, the compound represented by iv2-p is preferably 4, 4-dithiodibutanoic acid, and the compound represented by iv3-p is preferably 2,2' - [ propane-2, 2-diylbis (thio) ] diacetic acid.
In certain embodiments, the protein degrading agent is selected from protein degrading agent molecules that bind to a Von Hippel-Lindau receptor-containing E3 ubiquitin ligase and are covalently linked to the linker group through a proline hydroxyl site present in the molecule.
In certain embodiments, the protein degrading agent is selected from, but not limited to, the following molecules:
Figure BDA0003005412010000111
Figure BDA0003005412010000121
the embodiment of the invention also relates to a preparation method of the nano micelle precursor, which comprises the following two modes:
in a first way,
S1, reacting a protein degradation agent molecule with a precursor of a connecting group to form covalent connection between the protein degradation agent molecule and the precursor of the connecting group, so as to obtain a functional group derived protein degradation agent molecule;
s2, covalently connecting the obtained protein degrading agent molecules with the derived functional groups with the uncapped polymer skeleton to obtain the uncapped polymer-protein degrading agent conjugate;
and S3, forming covalent connection by performing bio-orthogonal reaction on the non-terminated polymer-protein degradation agent conjugate and a ligand to obtain the ligand modified polymer-protein degradation agent conjugate.
Wherein the polymer skeleton without end capping is a polymer skeleton with a derivatizing functional group.
In certain embodiments, the ligand is modified prior to reaction with the derivatized functional group on the polymer backbone, preferably, the modification comprises reduction of a disulfide bond to produce a thiol group.
The second method,
S1, reacting a protein degradation agent molecule with a precursor of a connecting group to form covalent connection between the protein degradation agent molecule and the precursor of the connecting group, so as to obtain a functional group derived protein degradation agent molecule;
and S2, covalently connecting the protein degradation agent molecule with the obtained functional group derivatization with a polymer with a terminal group.
The embodiment of the invention also provides a nano micelle which is formed by self-assembling the nano micelle precursor.
In certain embodiments, the average hydrated particle size of the nanomicelles is from 10 to 150nm, more preferably from 50 to 60nm.
In some embodiments, the polymer of the nanomicelle of the embodiments of the invention has a dispersity index of 0.01-0.3.
In some embodiments, the nanomicelle is loaded with a fluorescent probe for labeling.
The embodiment of the invention also provides a preparation method of the nano micelle, which comprises the following steps:
dissolving the nano-micelle in an organic solvent to obtain a solution, dispersing the polymer-protein degradation agent conjugate solution in water under an ultrasonic condition, and obtaining the nano-micelle through ultrafiltration or dialysis.
According to the present invention, the ultrasonic conditions are conditions that are conventional in the art for the preparation of nanomicelles.
In certain embodiments, the water is deionized water and the organic solvent is selected from the group consisting of haloalkane organic solvents, alcohol organic solvents, ether organic solvents, ketone organic solvents, nitrile organic solvents, and at least one of N, N-dimethylformamide and dimethylsulfoxide; the halogenated alkane organic solvent is selected from dichloromethane and trichloromethane, the alcohol organic solvent is selected from methanol and ethanol, the nitrile organic solvent is selected from acetonitrile, ether organic solvents, diethyl ether and tetrahydrofuran, and the ketone organic solvent is selected from acetone. The organic solvent is preferably dimethyl sulfoxide.
The cut-off molecular weight of the dialysis bag in the dialysis process can be 800, 1000, 3500 and 14000, and one of the cut-off molecular weight can be reasonably selected. The solvent used for dialysis can be at least one selected from deionized water, methanol, tetrahydrofuran, and N, N-dimethylformamide.
The ultrafiltration process can be carried out by adopting an ultrafiltration tube centrifugal ultrafiltration mode or an ultrafiltration mode of an ultrafiltration machine, the cut-off molecular weight of the ultrafiltration tube or the ultrafiltration membrane used in the process can be 1500D, 3kD, 5kD and 10kD, and one of the cut-off molecular weight and the cut-off molecular weight can be reasonably selected.
On the other hand, the invention also provides application of the nano-micelle in a medicament for treating malignant tumors, wherein the malignant tumors include but are not limited to breast cancer, cervical cancer, liver cancer, gastric cancer, pancreatic cancer, ovarian cancer, colon cancer or prostate cancer.
Detailed Description
The reagents and instrumentation used in the following examples are as follows:
the CRGDK small peptides used in the examples were purchased from gangrenm biotechnology limited; mal-PEG 5k -PLGA 5k -OH (PEG, mn =5000Da, PLGA, mn =5000Da, lactic acid: glycolic acid = 50), mPEG 5k -PLGA 5k -OH (mPEG, mn =5000da, plga, mn =5000da, lactic acid: glycolic acid = 50) purchased from dendri, china handle biotechnology limited; mPEG 5k -NH 2 From Xibao Biotech GmbH, (S) - (+) -2- (4- (4-chlorophenyl) -2,3, 9-trimethyl-6H-thieno [3,2-F ]][1,2,4]Triazolo [4,3-A][1,4]Diazepin-6-yl) ethylThe acid ((+) -JQ 1) was purchased from Sigma Aldrich trade company, inc. (2S, 4R) -1- [ (2S) -2-amino-3, 3-dimethylbutyryl)]-4-hydroxy-N- [ (1S) -1- [4- (4-methyl-1, 3-thiazol-5-yl) phenyl ] methyl]Ethyl radical]Pyrrolidine-2-carboxamide hydrochloride was purchased from Jiangsu Aikang biomedical research & development Co., ltd, tert-butyl 2- (2- (2- (2-aminoethoxy) ethoxy) acetate was purchased from Acete Biotech Co., ltd, suzhou, 4-dithiodibutanoic acid, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI), 4-Dimethylaminopyridine (DMAP), diisopropylethylamine (DIEA), dichloromethane (DCM), tetrahydrofuran (THF), N, N-Dimethylformamide (DMF), dimethyl sulfoxide, 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea Hexafluorophosphate (HATU) was purchased from Tokyo Bailingwei Tech Co., ltd; the remaining reagents and solvents were purchased from the national pharmaceutical group (Shanghai) Chemicals, inc., unless otherwise specified.
MDA-MB-231 breast cancer cells were purchased from Shanghai cell bank of Chinese academy of sciences, and RPIM1640 medium and fetal bovine serum were purchased from Gibco.
The hydrodynamic particle size of the protein degradant nanomicelles was measured by a malvern laser particle sizer (ZEN 3690, malven, USA). Electronic balances (Quintix 224-1, sartorius Germany); rotary evaporator (B-40, buchi, switzerland); a constant temperature magnetic stirrer (DF-101S, shanghai Provisioning instruments, ltd.); nuclear magnetic resonance spectrometer (Mercury Plus 400, varian, USA); isolation and purification was performed by Waters preparative liquid chromatograph (Waters e2695 chromatography pump, xbridge C18 μm 19X 250mm column, waters 2998 UV detector). Flow experimental data were measured by a BD FACS CALIBUR flow cytometer. Unless otherwise indicated, the equipment and methods employed are conventional in the art.
In the present invention, the equipment and the test method are those which are conventional in the art, unless otherwise specified.
Preparation example 1: synthesis of protein degradation agent molecule (JQ 1-p-VHL)
Figure BDA0003005412010000161
JQ1 (1.37 g) was dissolved in 12mL of 50% trifluoroacetic acid/dichloromethane and reacted at room temperature for 4h. After completion of the reaction, the solvent was distilled off under reduced pressure, and the resulting pale yellow solid powder was separated by column chromatography to obtain compound 1 (1.15g, 95.83%). 1 H NMR(400MHz,CDCl 3 )δ10.19(s,1H),7.45(d,J＝8.4Hz,2H),7.35(d,J＝8.6Hz,2H),4.64(t,J＝7.0Hz,1H),3.73(dd,J＝17.0,7.0Hz,1H),3.61(dd,J＝17.0,6.9Hz,1H),2.73(s,3H),2.43(s,3H),1.71(s,3H).
Compound 1 (596mg, 1.48mmol) was dissolved in 5.0mL of Dichloromethane (DCM), and 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (HATU, 678mg, 1.78mmol), N, N-diisopropylethylamine (DIEA, 479mg, 3.72mmol) and compound 2 (411mg, 1.56mmol) were added thereto and reacted at room temperature for 24h. After completion of the reaction, the reaction mixture was diluted with dichloromethane, washed with water and a saturated ammonium chloride solution, dried over anhydrous sodium sulfate, and subjected to column separation to obtain Compound 3 (756.5mg, 79.11%). 1 H NMR(500MHz,CDCl 3 )δ7.42(d,J＝8.5Hz,2H),7.33(d,J＝8.8Hz,2H),7.08(s,1H),4.66(t,J＝7.0Hz,1H),4.04(s,2H),3.77–3.71(m,4H),3.71–3.65(m,4H),3.65–3.58(m,2H),3.57–3.48(m,3H),3.39(dd,J＝14.6,7.2Hz,1H),2.67(s,3H),2.41(s,3H),1.68(s,3H),1.47(s,9H).
660mg of Compound 3 (1.02 mmol) was dissolved in 10mL of a 50% trifluoroacetic acid/dichloromethane solution and reacted at room temperature for 4 hours. After the reaction is finished, the solvent is removed by spinning, and the compound 4 (532) is obtained by column separation
mg,88.27％)。 1 H NMR(500MHz,CDCl 3 )δ7.77(t,J＝4.9Hz,1H),7.43(d,J＝
8.5Hz,2H),7.35(d,J＝8.8Hz,2H),4.75(t,J＝7.1Hz,1H),4.26–4.14(m,2H),3.86–3.82(m,1H),3.78(dd,J＝8.6,3.7Hz,2H),3.76–3.75(m,2H),3.74–3.71(m,1H),3.70–3.68(m,2H),3.65(dd,J＝6.8,3.8Hz,1H),3.62(dd,J＝7.8,4.5Hz,2H),3.60–3.55(m,1H),3.52–3.47(m,2H),2.70(s,3H),2.47–2.38(m,3H),1.76–1.65(m,3H).
Compound 4 (1.0g, 1.7mmol) and compound 5 (828mg, 1.86mmol) were dissolved in 15mL of dichloromethane, and 2- (7-azabenzotriazole was added thereto) N, N, N ', N' -tetramethyluronium hexafluorophosphate (965mg, 2.54mmol), N, N-diisopropylethylamine (1.47mL, 8.47mmol), reacted at room temperature for 12h. After completion of the reaction, the reaction mixture was diluted with methylene chloride, washed with water and a saturated ammonium chloride solution, dried over anhydrous sodium sulfate, and subjected to column separation to obtain JQ1-p-VHL (1.12g, 64.85%). 1 H NMR(500MHz,CDCl 3 )δ8.67(s,1H),7.87(s,1H),7.55(d,J＝7.8Hz,1H),7.41(d,J＝8.4Hz,2H),7.36(dd,J＝8.8,2.5Hz,4H),7.32(dd,J＝9.0,4.3Hz,3H),5.12–5.05(m,1H),4.82(t,J＝7.7Hz,1H),4.73(d,J＝9.3Hz,1H),4.68(dd,J＝8.6,5.5Hz,1H),4.46(s,1H),4.31(d,J＝15.8Hz,1H),4.13(d,J＝15.9Hz,1H),4.08(d,J＝11.1Hz,1H),3.75–3.60(m,12H),3.59–3.47(m,2H),3.42–3.33(m,2H),2.64(s,3H),2.52(s,3H),2.46–2.41(m,1H),2.40(s,3H),2.10(t,J＝10.1Hz,1H),1.67(s,3H),1.47(d,J＝6.9Hz,3H),1.07(s,9H).
Preparation example 2: synthesis of JQ1-p-VHL-ssCOOH and Mal-PEG-PLGA-ssJQ1-p-VHL
Figure BDA0003005412010000181
4, 4-Dithiosuccinic acid (140mg, 0.59mmol), N, N-dimethylaminopyridine (24mg, 0.197mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (113mg, 0.59mmol) and diisopropylethylamine (205. Mu.L, 1.18 mmol) were dissolved in 2.0mL of anhydrous dichloromethane and reacted at room temperature for 1.5 hours, then JQ1-p-VHL (200mg, 0.197mmol) prepared in preparation example 1 was added and reacted at room temperature for 48 hours. After the reaction is finished, washing with water and 1M hydrochloric acid aqueous solution in sequence, combining organic phases and anhydrous Na 2 SO 4 Drying and column chromatography gave compound JQ1-p-VHL-ssCOOH (115mg, 47.32%). 1 H NMR(500MHz,CDCl 3 )δ8.69(s,1H),7.69(d,J＝7.6Hz,1H),7.43(d,J＝8.3Hz,2H),7.35(dd,J＝12.0,7.9Hz,7H),5.39(s,1H),5.14–5.06(m,1H),4.82(t,J＝7.7Hz,1H),4.70(t,J＝6.9Hz,1H),4.63(d,J＝9.1Hz,1H),4.16(dd,J＝15.8,6.9Hz,3H),3.87(dd,J＝11.5,4.2Hz,1H),3.79–3.68(m,8H),3.64(s,2H),3.55(ddd,J＝13.8,12.1,5.8Hz,3H),3.45(dd,J＝14.8,7.3Hz,1H),2.75(dt,J＝10.5,7.1Hz,4H),2.67(s,3H),2.65–2.58(m,1H),2.53(s,3H),2.52–2.45(m,3H),2.43(s,3H),2.30–2.24(m,1H),2.02(dd,J＝18.9,11.8Hz,5H),1.70(s,3H),1.46(d,J＝4.9Hz,4H),1.30(d,J＝14.6Hz,3H),1.09(s,9H),0.91–0.86(m,4H).
The compound JQ1-p-VHL-ssCOOH (24.5mg, 0.02mmol), N, N-dimethylaminopyridine (2.5mg, 0.02mmol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (3.8mg, 0.02mmol) were dissolved in 5.0mL of anhydrous dichloromethane, and diisopropylethylamine (3.3. Mu.L) was added and reacted at room temperature for 1.5 hours. Mal-PEG-PLGA-OH (67 mg) was then added and the reaction continued for 48h. After the reaction, impurities are removed by a dialysis method, and the polymer Mal-PEG-PLGA-ssJQ1-p-VHL (PEG-PLGA-ssV-JQ 1 for short) 45mg is obtained by freeze drying.
Preparation example 3: synthesis of mPEG-PLGA-ssJQ1-p-VHL
After dissolving the compound JQ1-p-VHL-ssCOOH (24mg, 0.02mmol) prepared in preparation example 2, N, N-dimethylaminopyridine (2.5mg, 0.02mmol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (7.7 mg, 0.04mmol) in anhydrous dichloromethane, diisopropylethylamine (10. Mu.L) was added and reacted at room temperature for 1.5 hours. Then, a solution of mPEG-PLGA-OH (67 mg) was added dropwise thereto, and the reaction was continued for 48 hours. After the reaction, 50mg of polymer mPEG-PLGA-ssJQ1-p-VHL (mPEG-PLGA-ssV-JQ 1 for short) is obtained by removing impurities by dialysis and freeze drying.
Preparation example 4: synthesis of mPEG-ssJQ1-p-VHL
After dissolving the compound JQ1-p-VHL-ssCOOH (24mg, 0.02mmol) prepared in preparation example 2, N, N-dimethylaminopyridine (2.5mg, 0.02mmol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (3.8mg, 0.04mmol) in anhydrous dichloromethane, diisopropylethylamine (10. Mu.L) was added and the reaction was carried out at room temperature for 1.5 hours. Followed by the addition of mPEG 5k -NH 2 (50 mg) solution and the reaction was continued for 48h. After the reaction, impurities are removed by a dialysis method, and the polymer mPEG-ssJQ1-p-VHL (mPEG-ssV-JQ 1 for short) is obtained by freeze drying, wherein the concentration of the polymer mPEG-ssJQ1-p-VHL is 35mg.
Preparation example 5: preparation of CRGDK modified PEG-PLGA-protein degradation agent
Figure BDA0003005412010000191
CRGDK (12.6 mg, 0.03mmol) and Mal-PEG-PLGA-ssJQ1-p-VHL (100 mg) prepared in preparation example 2 were dissolved in 5.0mL of N, N-Dimethylformamide (DMF) and reacted at 40 ℃ for 8h under a nitrogen atmosphere. Removing impurities by dialysis, and lyophilizing to obtain compound CRGDK-PEG-PLGA-ssJQ1-p-VHL (CPEG-PLGA-ssV-JQ 1 for short) 98.5mg.
Preparation example 6: preparation of nanomicelle
CPEG-PLGA-ssV-JQ1 (20 mg) prepared in preparation example 5 was dissolved in 1mL of dimethylsulfoxide solvent, and 50. Mu.L of the above solution (20 mg/mL) was slowly dropped into 1mL of deionized water in an ultrasonic (power 70W, 2min) apparatus. And dialyzing the obtained solution in deionized water (the cut-off molecular weight of a dialysis bag is 3500), thus obtaining the CPEG-PLGA-ssV-JQ1 nano micelle. And measuring the hydrodynamic radius and the particle size distribution of the nano micelle by using dynamic light scattering.
As shown in FIG. 1, the average hydrated particle size of the constructed CPEG-PLGA-ssV-JQ1 nano micelle is about 55.5nm, and the PDI is 0.096.
Preparation example 7: preparation of fluorescent probe ICG marked nano micelle
CPEG-PLGA-ssV-JQ1 prepared in preparation example 5 or PEG-PLGA-ssV-JQ1 (20 mg) prepared in preparation example 2 and ICG (0.1 mg) were mixed and dissolved in 1mL of dimethylsulfoxide solvent, and 50. Mu.L of the above solution was slowly dropped into 1mL of deionized water solution under ultrasonic conditions (power 70W, 2min). And dialyzing the obtained solution in deionized water (the cut-off molecular weight of a dialysis bag is 3500), thus obtaining the CPEG-PLGA-ssV-JQ1/PEG-PLGA-ssV-JQ1 nano micelle marked by the fluorescent probe ICG, which is used for detecting the cell uptake condition of the nano micelle.
Test example 1: release of JQ1-p-VHL in CPEG-PLGA-ssV-JQ1 Nanocollages
CPEG-PLGA-ssV-JQ1 nano-micelles prepared in preparation example 6 were dispersed in dialysis bags (14 kD), and 2 different buffer solutions were prepared to simulate the normal cell physiological environment (0.5% Tween 80+ 10. Mu.M GSH, control group) and the tumor cell physiological microenvironment (0.5% Tween 80+10mM GSH), respectively. The buffers were collected at 0, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours at 37 ℃ with shaking, and the cumulative release of JQ1-p-VHL was calculated by high performance liquid chromatography.
The result is shown in figure 2, the CPEG-PLGA-ssV-JQ1 nano micelle can realize high-efficiency sustained release of protein degradation agent molecules in the presence of 10mM GSH, which indicates that the micelle can effectively release JQ1-p-VHL molecules in the tumor microenvironment, and the cumulative release amount of the drug can reach 80% in 36 h.
Test example 2: determination of uptake capacity of tumor cells to CPEG-PLGA-ssV-JQ1 nano-micelles and PEG-PLGA-ssV-JQ1 nano-micelles
MDA-MB-231 cells were plated at 5X 10 4 Was seeded in 24-well plates and used after 24h when the cells were in logarithmic growth phase. The CPEG-PLGA-ssV-JQ1 (product of preparation example 5) fluorescent nano-micelle containing ligand CRGDK prepared in preparation example 7 and the PEG-PLGA-ssV-JQ1 (product of preparation example 2) fluorescent nano-micelle containing no ligand CRGDK are respectively incubated with cells for 2h, 4h, 8h, 12h and 24h, after the incubation is finished, the cells are washed 3 times by PBS, and then the fluorescence intensity is detected by using a flow cytometer.
The test result is shown in fig. 3, compared with the PEG-PLGA-ssV-JQ1 nano micelle without ligand CRGDK, the uptake of the MDA-MB-231 breast cancer cells to the CPEG-PLGA-ssV-JQ1 nano micelle is obviously increased along with the time extension, which indicates that the targeting ability of the nano micelle to the tumor can be effectively improved through the modification of the ligand CRGDK, and the improvement of the tumor cell targeting of the protein degradation agent molecule is facilitated.
Test example 3: determination of anti-tumor cell proliferation capacity of CPEG-PLGA-ssV-JQ1 nano micelle
MDA-MB-231 cells were plated at 2X 10 3 The density of (2) was seeded in a 96-well plate, 24 hours later, the CPEG-PLGA-ssV-JQ1 nanomicelle prepared in preparation example 6 and the molecule JQ1-p-VHL were incubated with the cells at a constant concentration (0.0001. Mu.M, 0.001. Mu.M, 0.01. Mu.M, 0.1. Mu.M, 0.5. Mu.M and 1.0. Mu.M) for 72 hours respectively, the medium was removed, PBS was washed 3 times, a medium containing 10 parts of CCK8 was added, and after an appropriate time of incubation, the measurement was carried out at 450nm using a microplate readerAbsorbance and calculating the cytotoxicity of the nanomicelle.
The test result is shown in fig. 4, the CPEG-PLGA-ssV-JQ1 nano micelle shows stronger cytotoxicity to MDA-MB-231 breast cancer cells than JQ1-p-VHL small molecules, and shows that JQ1-p-VHL molecules can be effectively released in the tumor cells by improving the uptake capacity of the tumor cells to the JQ1-p-VHL molecules, and the growth and proliferation inhibition of the protein degradation agent molecules JQ1-p-VHL to the tumor cells can be facilitated.
Test example 4: determination of BRD4 protein degradation capacity of CPEG-PLGA-ssV-JQ1 nano micelle
MDA-MB-231 cells were plated at 5X 10 5 After 24h of culture in a 6-well plate, the nanomicelle CPEG-PLGA-ssV-JQ1 and the molecule JQ1-p-VHL prepared in preparation example 6 were incubated with the cells at a concentration for a certain time (4 h and 1693 h. And (3) treating the cells with a lysis solution to obtain a corresponding protein sample, and analyzing the change of the expression quantity of the related protein in a gel electrophoresis mode.
As shown in fig. 5, the nanomicelle (right side) has BRD4 protein degradation ability as the protein degradation agent molecule (left side), and protein degradation is also time-and concentration-dependent.
Although the present application has been described with reference to preferred embodiments, it is not intended to limit the scope of the claims, and many possible variations and modifications may be made by one skilled in the art without departing from the spirit of the application.
Sequence listing
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Claims (10)

1. A nanomicelle precursor comprising a protein degradation agent, a linker, and a polymer backbone; wherein, the first and the second end of the pipe are connected with each other,
the polymer skeleton is selected from polyethylene glycol or polyethylene glycol derivatives, and comprises at least one of structural units shown as a formula i1, a formula i2 and a formula i 3:
Figure FDA0003005412000000011
wherein m is selected from integers from 10 to 145, preferably from 100 to 140, more preferably 113;
q is selected from integers from 0 to 100, preferably 20 to 50, more preferably 38;
p is an integer of 0 to 100, preferably 20 to 50, more preferably 38; and m, q and p are not zero at the same time;
one end of the polymer backbone is covalently linked to the protein degrading agent through the linking group;
the other end of the polymer skeleton is connected with a ligand or a terminal group;
the ligand is selected from a ligand targeting tumor cells or tumor stroma;
the terminal group is preferably methoxy.
2. The nanomicelle precursor of claim 1, wherein the ligand is selected from at least one of a fusion protein, a nanobody, a targeting peptide molecule, and a small molecule targeting ligand;
preferably, the fusion protein is selected from at least one of an antibody fusion protein, a diphtheria toxin fusion protein, an immunoglobulin fusion protein; the antibody fusion protein is preferably an Fc fusion protein, the diphtheria toxin fusion protein is preferably DT389-hIL13-13E13K, and the immunoglobulin fusion protein is preferably Lg fusion protein;
preferably, the nanobody is selected from at least one of anti-CD 44 nanobody, anti-carbonic anhydrase IX nanobody, anti-epidermal growth factor receptor nanobody;
preferably, the targeting peptide molecule is selected from at least one of peptides with amino acid sequences shown as SEQ ID NO. 1-5;
preferably, the small molecule targeting ligand is selected from at least one of the following structural formulas:
Figure FDA0003005412000000021
3. the nanomicelle precursor of claim 1, wherein the ligand comprises a linker group A for coupling to the polymer backbone,
optionally, the linking group a is used to link with a derivatized functional group residue on the polymer backbone, selected from a group formed after reaction of a thiol, azide, or alkyne functional group.
4. The nanomicelle precursor of claim 1, wherein the polymer backbone has structural units according to formula ii1 or formula ii2,
Figure FDA0003005412000000022
wherein m, q and p are not zero or not 0 at the same time a Denotes a derivatized functional group residue, R b Represents a terminal group;
the derivative functional group is used for forming a covalent bond with the ligand, and the derivative functional group residue is a residue of the derivative functional group after the derivative functional group forms the covalent bond with the ligand;
preferably, R a Selected from the group consisting of: -NH-,
Figure FDA0003005412000000023
R b selected from methoxy;
m is preferably 113, q is preferably 38, p is preferably 0 or 38.
5. The nanomicelle precursor of claim 1, wherein the linker group is selected from the group consisting of groups represented by formula iv1, formula iv2, and formula iv 3;
Figure FDA0003005412000000031
wherein M is 1 、M 2 、R 3 Each independently selected from alkylene groups R having 1 to 4 carbon atoms 1 、R 2 Each independently selected from carbonyl, -O-C (O) -, -NH-C (O) -;
preferably, R 3 is-C (CH) 3 ) 2 -，
Further preferably, M 1 And M 2 The same;
more preferably, the linking group is selected from the group represented by the following formula:
Figure FDA0003005412000000032
Figure FDA0003005412000000041
further preferably, R 1 、R 2 Independently selected from-COOH, -NCO, -NH before reaction 2 or-OH.
6. The nanomicelle precursor of claim 1, wherein the protein degradation agent is selected from the group consisting of protein degradation agent molecules capable of binding to the E3 ubiquitin ligase of the Von Hippel-Lindau receptor and is covalently linked to the linker through the hydroxyl group of proline in the protein degradation agent molecule;
preferably, the protein degrading agent is selected from the following molecules:
Figure FDA0003005412000000042
Figure FDA0003005412000000051
7. the method for preparing nanomicelle precursor according to any one of claims 1 to 6, comprising at least the steps of:
reacting the precursor of the linking group with a protein degradation agent molecule to form covalent linkage therebetween and obtain a functional group-derivatized protein degradation agent molecule;
covalently linking the functional group-derivatized protein degradation agent molecule to a polymer backbone having terminal groups or an uncapped polymer backbone to obtain a polymer-protein degradation agent conjugate having terminal groups or an uncapped polymer-protein degradation agent conjugate;
the product obtained by the biological orthogonal reaction or condensation reaction of the polymer-protein degradation agent conjugate without the end capping and the ligand and the polymer-protein degradation agent conjugate with the end group are the precursor of the nano micelle;
preferably, the uncapped polymer backbone is a polymer backbone with derivatized functional groups;
further preferably, the ligand is modified and then reacted with a derivatised functional group on the polymer backbone;
even more preferably, the modification comprises subjecting the disulfide bond to a reduction reaction to produce a thiol group.
8. A nanomicelle which is characterized by being self-assembled from the nanomicelle precursor according to any one of claims 1 to 6;
preferably, the average hydrated particle size of the nano-micelle is 10 to 150nm, more preferably 50 to 60nm;
further preferably, the dispersion index of the nano-micelle is 0.01 to 0.30;
further preferably, the nanomicelle is also loaded with a fluorescent probe.
9. The method for preparing nanomicelle according to claim 8, comprising at least the steps of:
dispersing the solution obtained by dissolving the nano micelle precursor in an organic solvent into water, and obtaining the nano micelle through ultrafiltration or dialysis;
preferably, the solution is dispersed in water under ultrasonic conditions;
further preferably, a fluorescent probe is added to the organic solvent.
10. Use of the nanomicelle of claim 8 for the preparation of a medicament for the treatment of a malignant tumor, preferably selected from the group consisting of breast cancer, cervical cancer, liver cancer, gastric cancer, pancreatic cancer, ovarian cancer, colon cancer, or prostate cancer.
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