阅读说明：本技术 一种淀粉样蛋白β短肽介导的脑靶向递送系统 (Brain targeting delivery system mediated by amyloid beta short peptide ) 是由 占昌友 张醉 于 2018-06-14 设计创作，主要内容包括：本发明属药学领域,涉及一种可特异性吸附血浆中载脂蛋白,且能介导药物跨血脑屏障的多肽修饰的复合物和靶向递送系统及其在制备诊断与治疗外周肿瘤、脑肿瘤及其它脑部疾病的制剂中的应用。所述的淀粉样蛋白β(amyloidβ,Aβ)的多肽片段经共价键修饰荧光探针、药物分子和脂质体递药系统,经试验显示,所修饰的递送系统在与血浆蛋白作用形成蛋白冠后,增加其被血管内皮细胞的摄取；所修饰的脂质体递送系统更有效的将药物递送至病灶部位,显著提高药物治疗效果。所述的Aβ多肽经血浆蛋白吸附后,可介导药物跨血脑屏障、靶向肿瘤新生血管和肿瘤细胞,其修饰的药物和递送系统在治疗外周肿瘤、脑部肿瘤及脑内其它疾病时获得更好的治疗效果。(The invention belongs to the field of pharmacy, and relates to a polypeptide modified compound and a targeted delivery system which can specifically adsorb apolipoprotein in blood plasma and mediate a medicament to cross a blood brain barrier, and application of the polypeptide modified compound and the targeted delivery system in preparation of preparations for diagnosing and treating peripheral tumors, brain tumors and other brain diseases. The polypeptide fragment of amyloid beta (amyloid beta, Abeta) modifies a fluorescent probe, a drug molecule and a liposome delivery system through covalent bonds, and experiments show that the modified delivery system increases the uptake of the polypeptide fragment by vascular endothelial cells after the polypeptide fragment reacts with plasma protein to form a protein crown; the modified liposome delivery system can more effectively deliver the drug to the focus part, and obviously improve the therapeutic effect of the drug. After the A beta polypeptide is adsorbed by plasma protein, the A beta polypeptide can mediate a medicine to cross a blood brain barrier and target tumor neovessels and tumor cells, and the modified medicine and a delivery system of the A beta polypeptide can obtain better treatment effect when treating peripheral tumors, brain tumors and other diseases in the brain.)

1. An application of amyloid beta short peptide in preparing a preparation for mediating drug molecules, fluorescent probes or delivery systems to target peripheral tumors, brain tumors or other disease focuses of the brain, wherein the amyloid beta polypeptide is the amyloid beta polypeptide which specifically adsorbs apolipoprotein in blood plasma.

2. Use according to claim 1, wherein amyloid β polypeptide is covalently linked to imaging substance X to produce a β -X for the tracking of peripheral tumors, brain tumors and other disease foci in the brain that highly express LRP-1.

3. Use according to claim 2, wherein in said a β -X, X is a fluorescent molecule fluoroescein or a near infrared dye molecule Cy5, Cy5.5, Cy7, IR820, ICG, DiR, DiD, DiI.

4. The use of claim 1, wherein said amyloid β fragment polypeptide is covalently linked to an anti-tumor drug Y to produce Α β -Y for use in targeted intervention in peripheral tumors, brain tumors and other disease lesions in the brain that highly express LRP-1.

5. The use of claim 4, wherein in said A β -Y, Y is a paclitaxel, docetaxel, cabazitaxel, doxorubicin, epirubicin, camptothecin, hydroxycamptothecin, 9-nitrocamptothecin, or vincristine small molecule antineoplastic agent.

6. The use of claim 4, wherein in said A β -Y, Y is a p53 activating peptide or polypeptide toxin polypeptide as an anti-tumor agent.

7. The use of claim 1, wherein said amyloid β polypeptide is a β -peg-Z prepared by covalently linking peg-Z to a β -peg-Z for the preparation of a nano-delivery system.

8. Use according to claim 7, wherein in said A β -PEG-Z, Z is a phospholipid, polylactic acid (PLA), PLGA or Polycaprolactone (PCL).

9. Use according to claim 8, wherein said A β -polyethylene glycol-phospholipid is used for the preparation of a liposome delivery system, a micellar delivery system or a nanociscal delivery system.

10. Use according to claim 8, wherein said A β -polyethylene glycol-polylactic acid, CTB-polyethylene glycol-lactic glycolic acid copolymer, CTB-polyethylene glycol-polycaprolactone, are used for the preparation of micellar delivery systems and polymeric nanoparticle delivery systems.

11. Use according to claim 9 or 10, wherein said liposome, micelle, nanocolumnar or polymeric nanoparticle delivery system is used for entrapment of diagnostic molecules for tracking peripheral tumors, brain tumors or other disease lesions of the brain which highly express LRP-1.

12. Use according to claim 11, wherein the diagnostic molecule carried by the delivery system is 5-carboxyfluorescein 5-FAM or the near infrared dyes Cy5, Cy5.5, Cy7, IR820, ICG, DiR, DiD, DiI.

13. The use according to claim 9 or 10, wherein the nanoparticle delivery system, liposome delivery system, micelle delivery system, polymer nanoparticle delivery system or nanocolumnar delivery system is used for encapsulating anti-tumor drugs for targeted intervention of peripheral tumors, brain tumors or other disease foci of the brain with high LRP-1 expression.

14. The use of claim 13, wherein said drug delivery system comprises a drug selected from the group consisting of paclitaxel, docetaxel, cabazitaxel, doxorubicin, epirubicin, camptothecin, hydroxycamptothecin, 9-nitrocamptothecin, vincristine, p 53-activating peptide, and polypeptide toxins.

Technical Field

The invention belongs to the field of pharmacy, relates to a brain targeting delivery system mediated by amyloid beta short peptide, and particularly relates to a polypeptide modified compound and a targeting delivery system which can specifically adsorb apolipoprotein in blood plasma and can mediate a medicament to cross a blood brain barrier, and application of the polypeptide modified compound and the targeting delivery system in preparation of preparations for diagnosing and treating peripheral tumors, brain tumors and other brain diseases.

Background

Disclosure of Invention

The invention aims to provide a brain targeting delivery system mediated by amyloid beta short peptide aiming at the defects in the prior art, in particular to a polypeptide modified compound and a targeting delivery system which can specifically adsorb apolipoprotein in plasma and mediate a medicament to cross a blood brain barrier, and application thereof in preparing preparations for diagnosing and treating peripheral tumors, brain tumors and other brain diseases. The polypeptide-modified targeted delivery system overcomes the defects of the traditional targeted nano-drug by regulating and controlling the components and the biological activity of the protein corona formed by the nano-drug in the plasma, constructs an A beta-drug compound capable of crossing a blood brain barrier and simultaneously targeting vascular endothelial cells and tumor cells and an A beta-modified nano-drug delivery system, and realizes the targeted diagnosis of brain tumors and the drug delivery of the treatment of other brain diseases.

In the invention, the biomimetic intracerebral A beta protein takes apolipoproteins (such as Apoliprotein E, Apoliprotein A1 and Apoliprotein J) as molecular chaperones and a clearance mechanism for receptor-mediated transport to the periphery, takes A beta (Amyloid beta) protein as a template, designs a polypeptide capable of specifically binding to an apolipoprotein lipid binding domain, and by modifying the polypeptide on the surface of a liposome, the polypeptide specifically adsorbs the apolipoprotein in plasma and maintains the biological activity thereof in the in vivo blood circulation process, and further utilizes the binding of the apolipoprotein with various receptors on the blood brain barrier (including Low-dense lipid-related protein 1(LRP-1), Scavenger receptor class B member 1(SRB1) and Low-dense lipid-related protein 2 (LRP-2)), so as to mediate the liposome to be transported into the brain; the polypeptide-modified liposome can overcome the defects of the traditional targeting nano-drug by regulating and controlling the components and the biological activity of the protein corona formed by the nano-drug in plasma, construct an A beta-drug compound which can cross a blood brain barrier and simultaneously target vascular endothelial cells and tumor cells and an A beta-modified nano drug delivery system, and realize the targeted diagnosis of brain tumors and the drug delivery for treating other brain diseases.

In the invention, the A beta polypeptide is modified on materials such as polyethylene glycol-distearoyl phosphatidyl ethanolamine (PEG-DSPE), polyethylene glycol-polylactic acid (PEG-PLA), polyethylene glycol-lactic glycolic acid copolymer (PEG-PLGA), polyethylene glycol-polycaprolactone (PEG-PCL) and the like by covalent bonds to construct delivery systems such as liposome, micelle, nano disc, nanoparticle and the like modified by the A beta polypeptide.

The A beta polypeptide modified nano delivery system can entrap paclitaxel, docetaxel, cabazitaxel, adriamycin, epirubicin, camptothecin, hydroxycamptothecin, 9-nitrocamptothecin, vincristine, p53 activation peptide, melittin, scorpion venom peptide and other antitumor drugs; and fluorescent substances such as FAM, near-infrared dyes Cy5, Cy5.5, Cy7, IR820, ICG, DiR, DiD, DiI, etc. may be entrapped.

In the invention, the A beta polypeptide modified drug or probe comprises a pH sensitive hydrazone bond formed by reacting maleimide hexylhydrazine derivatives, relates to drugs such as adriamycin, epirubicin, p53 activated peptide and polypeptide toxin, or disulfide bond formed by reacting 3- (2-pyridinedithiol) propionic acid derivatives, relates to drugs such as paclitaxel, docetaxel, cabazitaxel, camptothecin, hydroxycamptothecin, 9-nitrocamptothecin, vincristine, p53 activated peptide and polypeptide toxin, or connects a fluorescent probe by stable chemical bonds, and relates to Fluorescein, Cy5, Cy5.5, Cy7, ICG and IR 820.

Furthermore, the invention provides an application of the amyloid beta short peptide in preparation of a preparation for mediating drug molecules, fluorescent probes or delivery systems to target peripheral tumors, brain tumors or other disease focuses of the brain, wherein the amyloid beta polypeptide is the amyloid beta polypeptide specifically adsorbing apolipoprotein in blood plasma.

The amyloid beta polypeptide is connected with an imaging substance X by a covalent bond to prepare Abeta-X which is used for tracing peripheral tumors, brain tumors and other disease focuses in the brain with high LRP-1 expression; in the A beta-X, X is fluorescent molecule Fluorescein or near infrared dye molecules Cy5, Cy5.5, Cy7, IR820, ICG, DiR, DiD and DiI;

the amyloid beta fragment polypeptide is connected with an anti-tumor drug Y through a covalent bond to prepare Abeta-Y which is used for the target intervention of peripheral tumors, brain tumors and other disease focuses in the brain with high LRP-1 expression; in the A beta-Y, Y is paclitaxel, docetaxel, cabazitaxel, adriamycin, epirubicin, camptothecin, hydroxycamptothecin, 9-nitrocamptothecin or vincristine micromolecule antitumor drug; in the A beta-Y, Y is p53 activation peptide or polypeptide toxin polypeptide anti-tumor drug;

the amyloid beta polypeptide is connected with polyethylene glycol-Z by a covalent bond to prepare Abeta-polyethylene glycol-Z which is used for preparing a nano delivery system; in the A beta-polyethylene glycol-Z, Z is phospholipid, polylactic acid (PLA), lactic-co-glycolic acid (PLGA) or Polycaprolactone (PCL);

the A beta-polyethylene glycol-phospholipid is used for preparing a liposome delivery system, a micelle delivery system or a nano disc delivery system;

the Abeta-polyethylene glycol-polylactic acid, the CTB-polyethylene glycol-lactic glycolic acid copolymer and the CTB-polyethylene glycol-polycaprolactone are used for preparing a micelle delivery system and a polymer nanoparticle delivery system;

the liposome delivery system, the micelle delivery system, the nano disc delivery system or the polymer nanoparticle delivery system are used for encapsulating diagnostic molecules and tracing peripheral tumors, brain tumors or other disease focuses of the brain with high LRP-1 expression; the delivery system comprises diagnostic molecules such as 5-carboxyfluorescein 5-FAM or near infrared dyes Cy5, Cy5.5, Cy7, IR820, ICG, DiR, DiD and DiI;

the nanoparticle delivery system, the liposome delivery system, the micelle delivery system, the polymer nanoparticle delivery system or the nano disc delivery system are used for encapsulating anti-tumor drugs and carrying out targeted intervention on peripheral tumors, brain tumors or other disease focuses of the brain with high LRP-1 expression; the encapsulated drug is paclitaxel, docetaxel, cabazitaxel, adriamycin, epirubicin, camptothecin, hydroxycamptothecin, 9-nitrocamptothecin, vincristine, p53 activation peptide or polypeptide toxin.

More specifically, the invention is realized by the following technical proposal,

1. preparation of Abeta polypeptide modified liposome delivery System

Reacting the A beta polypeptide containing cysteine sulfydryl with the maleamide-PEG-DSPE in a phosphate buffer solution with the pH of 7.4, dialyzing by using a semipermeable membrane with the aperture of 8000-10000Da, freeze-drying a product to prepare the A beta-PEG-DSPE for later use,

weighing a certain amount of natural phospholipid, cholesterol, methoxy-PEG-DSPE (meo-PEG-DSPE), Abeta-PEG-DSPE, model drug or probe molecule, dissolving in solvent, forming film, hydrating, passing liposome extruder through film, removing free probe molecule or drug by column chromatography to obtain Abeta polypeptide modified liposome, and characterizing particle size and surface potential by dynamic light scattering particle size analyzer.

Experiments on the effect of A.beta.polypeptide modified delivery systems on their binding activity after incubation with plasma to form protein corona,

after incubation of the Α β polypeptide-modified delivery system with fresh plasma for a period of time, its binding activity to the LRP-1 receptor was detected using western blot and radiolabelling methods, with Α β polypeptide-modified delivery system and unmodified Α β polypeptide delivery system not incubated with serum as negative controls.

Experiments on the effect of Abeta polypeptide modification on the uptake of a delivery system by the vascular endothelial cell system,

comparison of the uptake of a β polypeptide by endothelial cells (e.g., bnd 3 cells) by the delivery system before and after incubation with serum compares the amount of a β polypeptide taken by endothelial cells with unmodified delivery system.

Evaluation of the ability of the A.beta.polypeptide-modified delivery system to cross the BBB in normal mice,

normal mice (e.g., Kunming, C57BL/6, etc.) were injected tail vein with fluorescein-loaded delivery systems, and the amount of A.beta.polypeptide modified and unmodified delivery systems in the mouse brain was compared at different time points.

Evaluation of the pharmacokinetics of A.beta.polypeptide-modified delivery systems in normal rats

Normal SD rats were injected tail vein with fluorescein DiI-labeled polypeptide modified and unmodified delivery systems, blood was collected at different time points, plasma was isolated, and the DiI content in the plasma was quantitatively determined by fluorescence to evaluate the pharmacokinetics of the polypeptide modified delivery system in rats.

Evaluation of immunogenicity of A.beta.polypeptide-modified delivery systems in Normal mice

Preparing a polypeptide modified and unmodified delivery system containing Lipid A, injecting the polypeptide modified and unmodified delivery system into Balb/c mice intraperitoneally, injecting the polypeptide once every seven days within four weeks, taking blood from eye sockets on the seventh day after each injection, collecting blood plasma and freezing the blood plasma for later use. Immunogenicity was assessed by ELISA to detect the amount of IgG and IgM produced in mice at different time points against PEG and Α β.

Evaluation of anti-tumor Effect in vivo by Abeta polypeptide-modified delivery System

The in-vivo anti-tumor effect of the A beta polypeptide modified delivery system is evaluated by injecting an A beta polypeptide modified delivery system, an unmodified polypeptide delivery system, free drugs and normal saline into a nude mouse tail vein of a Homocoma model in situ U87 by taking the median survival time, tumor tissue cell apoptosis and new blood vessel density of a nude mouse as indexes.

Experiments show that the A beta polypeptide-mediated drug or drug delivery system targets LRP-1 high-expression cells and tissues, has the capacity of crossing a biological membrane barrier, particularly the capacity of crossing a Blood Brain Barrier (BBB), and can be used for targeted diagnosis and treatment of peripheral tumors, brain tumors and other diseases.

Experiments show that the Abeta polypeptide is modified on the surface of a drug delivery system and is combined with apolipoprotein in plasma to increase the uptake of vascular endothelial cells after forming a protein crown; the Abeta polypeptide can simultaneously mediate drugs to cross blood brain barriers and target tumor neovessels and tumor cells, and the modified drugs and the drug delivery system can obtain better treatment effect when treating peripheral tumors, brain tumors and other diseases in the brain.

Drawings

FIG. 1 in vitro binding assay of Abeta polypeptide modified liposomes to plasma proteins

Modifying the A beta polypeptide on the surface of the liposome by a chemical coupling mode to obtain A beta-LP, mixing the A beta-LP with mouse plasma, incubating for 1h at 37 ℃, and centrifuging at high speed to obtain a liposome precipitate containing a protein crown. Plasma protein components adsorbed by liposomes were analyzed by Biorad 4% -20% gradient SDS-PAGE and rapid silver staining (FIG. 1A). As a result, it was found that the bands of the A.beta. -LP-adsorbed plasma protein were significantly increased at 45kDa (Fraction 1), 38kDa (Fraction 2) and 25kDa (Fraction 3) as compared with methoxyliposomes. And (3) analyzing the two PAGE gels corresponding to the same positions in three positions by LC-MS/MS and western blot results, and identifying that the proteins in the three positions are respectively ApoJ, ApoE and ApoA1 derived from mice.

FIG. 2 identification of the adsorption of fluorescently labeled meo-LP and Abeta-LP to plasma functional apolipoproteins in vivo

In order to verify that the target small peptide modified liposome can still adsorb related target protein after systemic administration in an animal body, the same amount of the DiI fluorescence labeled liposome is injected into a mouse body through tail vein, blood is taken after 1h, and the liposome containing the protein corona in the plasma is separated. As shown in figure 2A, the content of three apolipoproteins (ApoJ, ApoE, ApoA1) adsorbed in mice by the same amount of liposomes was significantly different as detected by the western blot method. This result demonstrates that compared to normal liposomes, a β polypeptide modified liposomes can rapidly and specifically adsorb apolipoprotein from plasma proteins in mice.

FIG. 3, meo-LP and A β -LP evaluation of binding Activity to LRP-1 receptor after adsorption of plasma proteins

It is known that a β is transported from the brain into the blood circulation by binding to LRP-1, and that a β -modified liposomes are themselves capable of specifically adsorbing the recombinant protein LRP-1 in solution (as shown in fig. 3A). It was demonstrated that a β -LS retains the ability to bind to LRP-1 after preincubation with ApoE. And the ELISA experiment in the attached figure 3E proves that the A beta-LP after the plasma incubation loses the capacity of competing for the A beta antibody, which shows that after the A beta shown by the liposome is combined with plasma proteins such as ApoE and the like, the functional domain of the A beta is blocked, so that the binding function of the A beta is lost. Therefore, we believe that after plasma incubation, A β -LP loses its binding to BBB surface receptors, thus eliminating the possibility that A β -LP will enter the blood brain barrier via the classical pathway RAGE. In contrast, apolipoproteins such as ApoE, which specifically bind to the surface of a β -LP, have binding activity to the BBB receptor LRP-1, and are an effective way to mediate a β -LP into the brain across the BBB.

FIG. 4 uptake of NanoDeliver systems by vascular endothelial cells before and after plasma incubation

After incubation of the nano-delivery system with plasma to form a protein corona, its uptake by endothelial cells is increased.

FIG. 5 evaluation of pharmacokinetic parameters and immunogenicity of Abeta polypeptide modified liposomes in mice

The A beta polypeptide is modified on the surface of the liposome, so that the pharmacokinetic parameters of the A beta polypeptide in vivo are not influenced, and the immunogenicity generated by the liposome is not influenced (IgG and IgM in blood are used as evaluation indexes, and are shown in figures 5 b-c).

FIG. 6 evaluation of the brain-entry efficiency of A.beta.polypeptide-modified liposomes in mice

Through in vivo detection of fluorescein in mouse brain, the amount of the A beta-LP group crossing BBB is found to be significantly higher than that of meo-LP group, which indicates that the A beta can mediate the delivery system to cross blood brain barrier.

FIG. 7 in vivo efficacy of doxorubicin-loaded liposomes against brain glioma

The median survival time of mice in the saline group, DOX group, meo-LP/DOX group, and Abeta-LP/DOX group were: the A beta polypeptide modified nano delivery system can remarkably prolong the median survival period of a brain orthotopic tumor model mouse by 27 days, 31 days, 33 days and 50 days.

FIG. 8 Effect of Doxorubicin-loaded liposomes on neovascularisation in tumor tissue

The density of blood vessels in the tumor tissues of the adriamycin group encapsulated by the liposome modified by the A beta polypeptide is obviously lower than that of the unmodified group, which indicates that the A beta polypeptide can mediate a delivery system to target tumor neovascularization.

FIG. 9 Effect of Doxorubicin-loaded liposomes on glioma cell apoptosis

The apoptosis number of the A beta polypeptide modified liposome delivery system group in the tumor tissue is obviously higher than that of an unmodified group, which indicates that the A beta polypeptide can mediate a delivery system to target tumor cells.

FIG. 10 evaluation of safety

The A beta polypeptide does not show cytotoxicity on a nerve cell line PC12 cultured in vitro, and meanwhile, tissue sections of mouse organs of the liposome group modified by the A beta polypeptide have no obvious abnormality, which indicates that the A beta polypeptide is modified on the surface of the liposome and does not cause toxic or side effect.

Detailed Description

The following examples will help to understand the present invention, but the present invention is not limited to the scope of the following description