WO2017092558A1

WO2017092558A1 - Polypeptide, lipoprotein-like nanoparticle and use thereof

Info

Publication number: WO2017092558A1
Application number: PCT/CN2016/105330
Authority: WO
Inventors: 费浩; 王乔; 马晓川; 贾俊丽
Original assignee: 中国科学院苏州纳米技术与纳米仿生研究所
Priority date: 2015-12-03
Filing date: 2016-11-10
Publication date: 2017-06-08
Also published as: CN106831997A; CN106831997B

Abstract

Disclosed are a polypeptide, a lipoprotein-like nanoparticle (LNP) and a use thereof. The LNP contains at least one phospholipid and at least one polypeptide. The polypeptide is the polypeptide with the structure of a helix-loop-helix, and comprises at least two amphipathic á-helixes. The two amphipathic á-helixes are connected through a connecting peptide comprising a cyclic structure.

Description

Polypeptide, lipoprotein-like nanoparticle and application thereof

Technical field

The invention particularly relates to a helix-loop-helix structure polypeptide, a lipoprotein-like nanoparticle based on a helix-loop-helix structure polypeptide and its application as a drug-loading system, and belongs to the field of nano-biotechnology.

Background technique

In the field of drug transportation, liposomes are widely used for the transport of cytotoxic drugs, genes, proteins and the like because of their good biocompatibility, low toxicity and sustained release. Nano-sized liposomes easily penetrate the vasculature and intercellular spaces of tumor tissues, thereby greatly promoting the diffusion of drugs in tumor tissues. However, the smaller the particle size of the liposome, the greater the surface tension, which tends to cause fusion of the liposome, resulting in collapse of the structure and release of the contents. In addition, liposomes are non-specifically bound to serum proteins, cells, tissues, etc., causing large side effects. In order to solve these problems, various surface modifications are emerging, and among them, polymer modification is the most common. Polymers (such as polyethylene glycol, poloxamer, dextran, polyvinyl alcohol, etc.) can be covalently cross-linked with lipids to form a hydrophilic protective layer on the surface of the liposome, reducing surface tension and non-specificity. Adsorption. In addition, based on the protection of the polymer, the surface of the liposome can modify the targeting element to further increase the specificity of the liposome. However, there are still some problems with these modifications. For example, polyethylene glycol cannot be degraded in vivo, and cytotoxicity and immune response are produced after accumulation. In addition, the function of the targeting element may be masked by adjacent polyethylene glycol molecules, resulting in loss of function. . In general, the surface modification of liposomes involves a process of covalent cross-linking and mixed preparation, which is complicated in steps and poor in controllability.

In vivo, there are a variety of natural vesicle structures composed of lipids and proteins, such as synaptic vesicles, extracellular vesicles and lipoproteins. These structures are natural transport vehicles that transport hydrophobic or hydrophilic materials in the body. Among these structures, proteins are an essential component not only for the stabilization of lipid molecules and the control of surface tension, but also to provide specific targeted recognition functions. At present, researchers extract natural membrane structures (such as intracellular bodies, bacterial outer membrane vesicles and red blood cells, etc.) as carriers for drug delivery. Such carriers have complex and fine natural structures, good biocompatibility, but relatively high cost, and it is difficult to modify according to different targets.

High-density lipoprotein is one of the smallest and tightest natural nanoparticles in the body, mainly used for the transport of cholesterol. High-density lipoproteins are mainly composed of apolipoproteins and phospholipids. Among apolipoproteins, apolipoprotein A-I (Apo A-I) is dominant, about 70%. Apo A-I consists of 243 amino acids and forms a series of highly homologous parental helix structures. This protein binds to lipids and determines the particle size and morphology of high-density lipoproteins. In order to construct high-density lipoprotein nanoparticles in vitro for drug delivery, researchers extracted from vivo or recombinantly expressed Apo A-I in vitro. protein. However, ApoA-I protein synthesis is costly and difficult to functionally modify, resulting in limited application of high density lipoprotein nanoparticles. How to realize the multi-functionalization of nanoparticles based on the stable formation of lipoprotein-like nanoparticles (high-density lipoprotein-like nanoparticles) is a problem that the industry has been eager to solve.

Summary of the invention

It is a primary object of the present invention to provide a polypeptide based on the improved lipoprotein-like nanoparticles of the polypeptide and its use to overcome the deficiencies of the prior art.

In order to achieve the foregoing object, the technical solution adopted by the present invention includes:

An embodiment of the present invention provides a polypeptide comprising at least two amphipathic α-helices, wherein the at least two amphipathic α-helices are linked by at least one linker peptide, and the linker peptide comprises a cyclic structure.

In some embodiments, the polypeptide comprises a structural unit consisting of an alpha helix polypeptide, a linker peptide, and an alpha helix polypeptide in tandem.

In some embodiments, a lipoprotein-like nanoparticle comprising at least one phospholipid and at least one of the polypeptides is provided.

In some embodiments, the amphipathic alpha helix of the polypeptide is trapped in the surface layer of the lipoprotein-like nanoparticle, while the loop structure of the linker peptide extends from the surface of the lipoprotein-like nanoparticle.

Further, the amphipathic alpha helix of the polypeptide is trapped in the lipid surface of the lipoprotein-like nanoparticles, and the cyclic structure of the linker peptide extends from the surface of the lipid.

The use of the lipoprotein-like nanoparticles, such as in the field of medicine, is also provided in some embodiments. For example in the preparation of a pharmaceutical or pharmaceutical composition.

Embodiments of the present invention also provide a medicament comprising the polypeptide or the lipoprotein-like nanoparticles.

Embodiments of the present invention also provide a composition comprising the polypeptide, the lipoprotein-like nanoparticles, or the drug.

Compared with the prior art, the advantages of the present invention are at least:

(1) using a helix-loop-helix structure polypeptide mainly composed of an α-helical polypeptide, a cyclic sequence-ligating peptide and an α-helical polypeptide, and a phospholipid to form a lipoprotein-like nanoparticle, in particular, a helical portion of the polypeptide is immersed in a lipid The surface layer of protein-like nanoparticles can promote the stable formation of lipid nanoparticles with uniform size and small particle size, and at the same time, by extending the circular sequence to the surface of lipoprotein-like nanoparticles, and facilitating the insertion of functional sequences, thereby realizing lipoprotein Multi-functionalization of sample nanoparticles;

(2) The core of the provided lipoprotein-like nanoparticles can be used for the encapsulation of hydrophobic functional substances, and further expands its use, for example, it can be used as a drug-loading system for the diagnosis and treatment of cancer, etc., the drug-loading system It has low toxicity, high stability, and easy functionalization. It provides targeted killing and cell and tissue imaging for tumor cells. Simple and effective means.

(3) The lipoprotein-like nanoparticle provided by the amphipathic α-helical polypeptide, the cyclic structural linker peptide and the amphipathic α-helical polypeptide in a spiral-loop-helical structure polypeptide, wherein the sequence of the cyclic structural linker peptide can be A plurality of targeting peptides having a cyclic structure are selected such that the lipoprotein-like nanoparticles have a function of targeting specific cells, tissues and organs.

DRAWINGS

1 is a schematic view showing the structure of a polypeptide and a lipoprotein-like nanoparticle according to an exemplary embodiment of the present invention;

2 is a comparative map showing the ability of the A polypeptide and the AL ₃ A, AL ₅ A, and AL ₇ A polypeptides to form lipoprotein-like nanoparticles in Example 1;

Figure 3 is a comparative map of the degree of helix of the A polypeptide and the AL ₃ A, AL ₅ A, and AL ₇ A polypeptides on the lipoprotein-like nanoparticles in Example 1;

4 is a particle size test chart for testing A polypeptide-based lipoprotein-like nanoparticles using a dynamic laser light scattering (DLS) system in Example 1;

5 is a transmission electron microscope image of a lipoprotein-like nanoparticle based on A polypeptide in Example 1;

6 is a particle size test chart for testing lipoprotein-like nanoparticles based on AL ₃ A polypeptide using a dynamic laser light scattering (DLS) system in Example 1;

7 is a transmission electron microscope imaging image of lipoprotein-like nanoparticles based on AL ₃ A polypeptide in Example 1;

8 is a particle size test chart for testing lipoprotein-like nanoparticles based on AL ₅ A structural polypeptide using a dynamic laser light scattering (DLS) system in Example 1;

9 is a transmission electron microscope imaging image of lipoprotein-like nanoparticles based on an AL ₅ A structural polypeptide in Example 1;

10 is a particle size test chart for testing lipoprotein-like nanoparticles based on AL ₇ A structural polypeptide using a dynamic laser light scattering (DLS) system in Example 1;

Figure 11 is a transmission electron microscope imaging image of lipoprotein-like nanoparticles based on AL ₇ A structural polypeptide in Example 1;

12 is a particle size test chart of the AL ₃ A-structured polypeptide-based lipoprotein-like nanoparticles coated with Ir(ppy) ₂ -DIP using a dynamic laser light scattering (DLS) system in Example 2;

13 is a transmission electron microscope imaging image of lipoprotein-like nanoparticles based on AL ₃ A structural polypeptide encapsulating Ir(ppy) ₂ -DIP in Example 2;

14 is a test chart showing the stability of a lipoprotein-like nanoparticle-encapsulated Ir(ppy) ₂ -DIP compound based on A and AL ₃ A structural polypeptides in Example 2;

15 is a particle size test chart for testing a fat-soluble substance Ir(ppy) ₂ -DIP lipoprotein-like nanoparticles based on A(His) ₅ A structural polypeptide using a dynamic laser light scattering (DLS) system in Example 3;

16 is a transmission electron microscope imaging image of a fat-soluble substance Ir(ppy) ₂ -DIP lipoprotein-like nanoparticles based on A(His) ₅ A structural polypeptide in Example 3;

17 is the residual fluorescence of supernatant of A(His) ₅ A-lipoprotein-like nanoparticles and AL ₅ A-lipoprotein-like nanoparticles coated with Ir(ppy) ₂ -DIP in Example 3 after incubation with Ni column for 2 hours. Comparison chart of quantity;

Figure 18 is wrapped in Example 3 Ir (ppy) ₂ -DIP of A (His) Embodiment ₅ A- lipoprotein-like nanoparticles and AL ₅ A- lipoprotein-like nanoparticles Ni-column for 2 hours, lipoprotein-like nano A comparison chart of the amount of particles combined with the Ni column;

19 is a particle size test chart for testing a fat-soluble substance Ir(ppy) ₂ -DIP lipoprotein-like nanoparticles based on A(RGD)A structural polypeptide using a dynamic laser light scattering (DLS) system in Example 4;

20 is a transmission electron microscope imaging image of the fat-soluble substance Ir(ppy) ₂ -DIP lipoprotein-like nanoparticles based on the A(RGD)A structural polypeptide in Example 4;

21 is the embodiment A (RGD) A-LNP package Ir (ppy) after _{2 -DIP} and Ir (ppy) _{2 -DIP} Comparative Example 4. FIG hemolysis;

22 is a graph comparing the killing rate of HLF cells with Ir(ppy) ₂ -DIP after A(RGD)A-LNP wrapping Ir(ppy) ₂ -DIP in Example 4;

23 is a graph comparing the killing rate of HT1080 cells with Ir(ppy) ₂ -DIP after A(RGD)A-LNP wrapping Ir(ppy) ₂ -DIP in Example 4;

FIG 24 is a A-LNP, A (SRGDS) A-LNP comparison, AL ₃ A-LNP endocytosis and cell morphology fluorescence amount in HT1080 cells wrapped in Example 5 Ir (ppy) ₂ -DIP of A (RGD) Embodiment Figure

FIG 25 is a A-LNP, A (SRGDS) A-LNP, AL different concentrations of ₃ A-LNP HT1080 cells killing effect of inclusions in Comparative Example 5. FIG Ir (ppy) ₂ -DIP of A (RGD) embodiment;

FIG 26 is wrapped in Ir (ppy) ₂ -DIP of A (RGD) Example 5 _{A-LNP, AL 3 A-} LNP at different time 20μM HT1080 cells killing effect comparison chart;

Figure 27 is a mass spectrogram of AL ₅ A in Example 1.

detailed description

Exemplary embodiments embodying the features and advantages of the present invention will be described in detail in the following description. It is to be understood that the invention is capable of various modifications in the various embodiments this invention.

Unless otherwise defined, all technical and scientific terms used in the specification are the same meaning The terms used in the description of the present invention are for the purpose of describing the specific embodiments and are not intended to limit the invention.

According to an aspect of the present invention, there is provided a polypeptide comprising at least two amphipathic α-helices, said at least two amphipathic α-helices being linked by at least one linker peptide, said linker peptide comprising Ring structure.

The polypeptide may also be considered to be a helix-loop-helical structure polypeptide, wherein the helix polypeptide is a polypeptide having a clear parent-hydrophobic surface of a parent alpha helix structure, and the amino acid configuration may be L-form or D-form, wherein the loop is The sequence is an amino acid sequence having a cyclic structure, and the cyclic sequence structurally doubles the amphiphilic α-helical polypeptide, which can further increase the degree of helix of the polypeptide.

The polypeptide may also be referred to as an AL _m A polypeptide, further collectively referred to as an ALA polypeptide, wherein A refers to an alpha helix polypeptide, L refers to a linker peptide having a cyclized sequence, and m is the number of amino acids contained in the cyclized sequence.

Wherein, the polypeptide can be synthesized by any suitable means known in the art, for example, by referring to the following methods and methods:

Document 1. Merrifield, R.B., Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide. J. Am. Chem. Soc 1963, 85.

Document 2. Barlos, K.; Gatos, D.; Kapolos, S.; Poulos, C.;

W.;Yao,WQ,Application of 2-chlorotrityl resin in solid phase synthesis of(Leu15)-gastrin I and unsulfated cholecystokinin octapeptide.Selective O-deprotection of tyrosine.Int.J.Pept.Protein Res.1991,38(6 ), 555-561.

According to an aspect of the present invention, a non-naturally occurring lipoprotein-like nanoparticle (LNP) comprising at least one phospholipid and at least one of said polypeptides (ie, said helix-loop-helix polypeptide) is provided ).

Referring to Figure 1, in some embodiments, the lipoprotein-like nanoparticles comprise:

a) at least one phospholipid;

b) at least one of said polypeptides comprising a structural unit consisting of a sequentially linked alpha helix polypeptide (referred to as "amphiphilic helical polypeptide" in the figure), a linker peptide and an alpha helix polypeptide, said linker peptide comprising Has a ring structure (referred to as "connecting ring" in the figure);

And, c) one or more hydrophobic contents, such as cholesterol oleic acid, and the like.

In some embodiments, the amphipathic alpha helix of the polypeptide is trapped in the lipid surface of the lipoprotein-like nanoparticles, and the loop structure of the linker peptide extends from the surface of the lipoprotein-like nanoparticles.

When the helix-loop-helix structure polypeptide is combined with the lipoprotein-like nanoparticles, the helix portion is trapped in the surface layer of the lipoprotein-like nanoparticle, and is used to promote the stable formation of the lipid nanoparticles having uniform size and small particle size, and the ring shape. The sequence extends out of the surface of lipoprotein-like nanoparticles to facilitate the insertion of functional sequences, thereby facilitating the multifunctionalization of lipoprotein-like nanoparticles. Further, by using a core of lipoprotein-like nanoparticles for encapsulation of hydrophobic functional substances, Expand its use in one step, especially in the medical field, for example as a drug delivery system for the diagnosis and treatment of cancer. The drug-loading system has the characteristics of low toxicity, high stability, and convenient function, and provides a simple and effective means for targeting killing of tumor cells and imaging of cells and tissues.

In some embodiments, the linker peptide is a targeting peptide or a sequence consisting of a hydrophilic amino acid. Preferably, the linker peptide is a targeting peptide sequence.

In some embodiments, the linker peptide comprises, but is not limited to, a cyclic structure formed by at least one of a disulfide bond, an amide bond, a thioester bond, and a lactone bond. Preferably, the cyclic structure of the linker peptide is formed by a disulfide bond.

In some preferred embodiments, the cyclic structure of the linker peptide contains a C(X) _n C amino acid sequence, and n is selected from any of 3 to 11. Wherein C refers to a cysteine for cyclization and X is any amino acid which can be used to form the cyclic structure.

In some preferred embodiments, the number of amino acids involved in the formation of the linked peptide is from 3 to 7.

In some specific embodiments, the amino acid sequence of the cyclic structure of the linker peptide comprises any one or a combination of two or more of CSGSC, CSGSGSC, CSGSGSGSC, CRGDC, CHHHHHC.

In some embodiments, the corresponding amino acid sequence of at least one of the amphipathic a-helices is selected from the polypeptide amino acid sequence that mimics the ApoA-I function.

In some preferred embodiments, the corresponding amino acid sequence of at least one of the amphipathic α-helices is selected from the group consisting of an AI polypeptide sequence (CGVLESFKASFLSALEEWTKK) or a reverse sequence thereof, a 18A polypeptide sequence (DWLKAFYDKVAEKLKEAF) or a reverse sequence thereof, a 4F polypeptide Sequence (DWFKAFYDKVAEKFKEAF) or its reverse sequence, GSLLSALEEWTKKLN or its reverse sequence. These peptides not only facilitate the formation of high-density lipoprotein nanoparticles, but also covalently cross-link functional sequences to impart specific functions to the nanoparticles.

In a preferred embodiment, the amino acid sequences corresponding to the two amphipathic alpha helices are GSFLSALEE WTKKLN and NLKKTWEELASLFSG, respectively.

In some embodiments, the phospholipid comprises phosphatidyl choline, phosphatidyl ethanolamine, dimyristoyl phosphatidylethanolamine (DMPE, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine Any one or a combination of two or more of phosphatidylserine, phosphatidylglycerol and phosphatidylinositol, but is not limited thereto.

For example, the phospholipid preferably uses phosphatidylcholine.

For example, the phospholipid is preferably dimyristoylphosphatidylcholine (DMPC).

More preferably, in some embodiments, the lipoprotein-like nanoparticles further comprise at least one hydrophobic content.

Wherein the at least one hydrophobic content can be encapsulated within an outer shell comprised of the at least one phospholipid and the at least one polypeptide.

In some embodiments, the hydrophobic content includes any one or a combination of two or more of cholesterol, cholesterol ester, hydrophobic drug, developer, and tracer, but is not limited thereto.

For example, the hydrophobic content is a combination of any one of cholesterol and cholesterol ester and any one selected from the group consisting of a hydrophobic drug, a developer, and a tracer.

For example, the hydrophobic content may preferably be from a cholesterol ester and/or a hydrophobic drug, for example a combination of a cholesterol ester and a hydrophobic drug.

For example, the hydrophobic content may preferably be from cholesterol oleate and/or Ir(ppy) _2- DIP, for example a combination of the two.

In some embodiments, the lipoprotein-like nanoparticles comprise an active agent, an anticancer agent, and the like.

The lipoprotein-like nanoparticles of the present invention may optionally be incorporated into a hydrophobic drug or a labeled compound, or may be used in the preparation of a formulation incorporating a drug or a labeled compound, wherein the lipoprotein-containing nanoparticles provide a reduced drug Or the benefit of labeling the side effects of the compound and/or also preventing degradation of the drug or labeled compound and/or loss of effect.

According to another aspect of the invention, there is also provided a method of making and applying the lipoprotein-like nanoparticles described herein.

Accordingly, the use of the lipoprotein-like nanoparticles, such as in the field of medicine, is also provided in some embodiments.

In some embodiments, a medicament comprising the lipoprotein-like nanoparticles is provided.

In some embodiments, a composition comprising the lipoprotein-like nanoparticles or the drug is provided.

According to still another aspect of the present invention, the lipoprotein-like nanoparticles can be used for treating or diagnosing cancer (for example, breast cancer, gastric cancer, colorectal cancer, colon cancer, pancreatic cancer, non-small cell lung cancer, small cell lung cancer, Brain cancer, liver cancer, kidney cancer, prostate cancer, bladder cancer, ovarian cancer, or hematological malignancies (eg, leukemia, lymphoma, multiple myeloma, etc.).

"α helix"

In the present specification, "alpha helix" is used to refer to a motif that is common in the secondary structure of a protein. The alpha-helix is a coiled conformation similar to a spring in which each backbone N-H group contributes a hydrogen bond to the backbone C=O group of the four residues of the earlier amino acid. Typically, the alpha-helix formed from naturally occurring amino acids will be right-handed, but the left-handed conformation is also known.

"Amphibian"

"Amphiphilic" is a term that describes a compound having hydrophilic and hydrophobic properties. Amphipathic alpha helix is usually born Secondary structure motifs commonly encountered in active peptides and proteins, and refer to alpha helices having opposite polar and non-polar faces oriented along the long axis of the helix.

"Helical-ring-helical structure"

In the present specification, "helix-loop-helical structure polypeptide" means a structural unit composed of an amphipathic α-helical polypeptide, a linked peptide having a cyclic structure, and an amphipathic α-helical polypeptide, which are sequentially connected in series.

"Amino acid"

In the present specification, both L-amino acids and D-amino acids are included, either naturally occurring amino acids or non-naturally occurring, synthetic amino acids. At the same time, these amino acids can be chemically modified at will.

"Targeting peptide"

In the present specification, the term "targeting peptide" refers to a molecule in which a specific molecule is relatively specifically bound to a specific organ or tissue after administration to a subject. Typically, the selective feature is in part that detection of at least two-fold greater selective binding of the molecule to an organ or tissue than a control organ or tissue. In some embodiments, the selective binding is at least three or four times greater than the control organ or tissue.

Amino acid sequences which have been identified as having a cyclic structure and which can serve as targeting peptides include, but are not limited to, the amino acid sequences listed in Tables 1 and 2:

Table 1. Protein targeting peptides

蛋白靶标Protein target	肽序列(名称)Peptide sequence (name)
Her2/ErB2Her2/ErB2	WTGWCLNPEESTWGFCTGSFWTGWCLNPEESTWGFCTGSF
α_vβ₃ _{v v} β ₃	CDCRGDCFC(RGD-4C)CDCRGDCFC (RGD-4C)
α₅β₁ _{5 5} β ₁	GACRGDCLGA(pIII)GACRGDCLGA (pIII)
APN/CD13APN/CD13	CNGRCCNGRC
MMP-9MMP-9	CTTHWGFTLC(CTT)CTTHWGFTLC (CTT)
TAG-72TAG-72	GGVSCMQTSPVCENNLGGVSCMQTSPVCENNL
VEGFR-3VEGFR-3	CSDSWHYWCCSDSWHYWC
Phosphatidyl SerinePhosphatidyl Serine	CLSYYPSYCCLSYYPSYC

Table 2. Tumor targeting peptides

It should be noted that the targeting peptides of the present invention are not limited to the peptide sequences found above, by adding cysteine to both ends of other known linear targeting peptides, or by other methods, It is possible to use as a linker peptide in a polypeptide of the lipoprotein-like nanoparticles of the present invention.

In some embodiments, the lipoprotein-like nanoparticles (LNP) of the invention comprise an anticancer agent. Such active agents can be chemotherapeutic agents, photodynamic therapeutics, boron neutron capture therapeutics or radionuclides for radiation therapy. In some embodiments, the anticancer agent is selected from the group consisting of an alkylating agent (alkylating agent), an anthracycline, an antibiotic, an aromatase inhibitor, a bisphosphonate, a cyclooxygenase inhibitor, and an estrogen receptor. Body regulators, folic acid antagonists, inorganic arsenates, microtubule inhibitors, modifiers, nitrosourea, nucleoside analogues, osteoclast inhibitors, molybdenum-containing compounds, retinoids, topologically different a constitutive enzyme inhibitor, a kinase inhibitor (such as, but not limited to, a tyrosine kinase inhibitor), an anti-angiogenic agent, an epidermal growth factor inhibitor, and a histone deacetylase (deacetylase) inhibitor Group.

In summary, the present invention utilizes a helix-loop-helix structure polypeptide to stabilize nanoparticles while providing a good interface and insertion site for biological functionalization, and further can be specifically added to the helix-loop-helix structure polypeptide as needed. Functional amino acid sequence, and encapsulation of hydrophobic substances in the core of the nanoparticles to achieve multi-functionalization of the nanoparticles.

The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The ALA polypeptides used in the following examples can be synthesized by referring to the methods described in the

above documents

1 and 2.

Example 1

In the helixoid-like nanoparticles based on the helix-loop-helix structure polypeptide of the present embodiment, the helix-loop-helix structure polypeptide (ALA polypeptide) is covalently bonded by the α-helical polypeptide, the circularized sequence and the α-helical polypeptide. Made in series. The amino acid sequence of the α-helical polypeptide is: GSLLSALEEWTKKLN, but is not limited to this sequence. The cyclized polypeptide sequences are SGS, SGSGS, and SGSGSGS, respectively, but are not limited to this sequence, and may be other sequences having specific functions.

The synthesis process of one of the AL ₅ A polypeptides is specifically as follows: a solid phase synthesis method (refer to Reference 1 and Document 2), using an Fmoc amino acid, a dichlorotrityl resin as a carrier, and a C-terminal to N-terminal thereon The amino acid condensation reaction is carried out according to the designed sequence, and after the final amino acid is completed in the polypeptide reaction, the polypeptide is cleaved from the resin by TFA, and purified and desalted. After the treatment, the pure product is concentrated to obtain a linear polypeptide.

Dissolving the linear polypeptide in distilled water, adjusting the pH to about 8, and slowly oxidizing by oxygen (or adding some catalysts such as potassium ferricyanide, iodine, etc.) to form the thiol group of two cysteines in the polypeptide sequence. The disulfide bond forms a crude cyclic peptide. The crude cyclic peptide is concentrated again, purified by HPLC and then lyophilized to obtain the pure product, ie, the ALA polypeptide, and the molecular weight (4048.2) is determined by mass spectrometry to be substantially consistent with the predicted molecular weight of the designed polypeptide (4048.55) (see figure). 27), to determine its sequence is correct.

AL ₃ A, AL ₇ A, and the like can also be synthesized by referring to the foregoing methods.

A method of preparing lipoprotein-like nanoparticles based on the helix-loop-helix structure polypeptide comprises the steps of:

(1) 3 μmol of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) and 0.1 μmol of Cholesteryl oleate (CO) are sufficiently dissolved in a chloroform solution;

(2) Drying the chloroform in the centrifuge tube with a steady stream of nitrogen to form a film on the bottom of the tube;

(3) adding 1 ml of PBS buffer (pH 7.4) to the centrifuge tube, and resuspending the film at the bottom of the test tube by a vortex shaker to form a milky white suspension;

(5) flushing nitrogen into the centrifuge tube, sealing, placing the centrifuge tube in the ultrasound system, and sonicating at 48 ° C for 1 h;

(6) A PBS solution containing a helix-loop-helix structure polypeptide was added to the centrifuge tube, mixed, sealed, and left at 4 ° C overnight.

(7) The next day, the lipoprotein-like nanoparticle solution was purified by ultrafiltration and the effective solution was collected for use.

The lipoprotein-like nanoparticles (AL _(3-7) A-LNP) of the helix-loop-helix structure polypeptide synthesized by the above steps can be characterized by means of FPLC or the like. Fig. 2 is a time chart of FPLC, the molar ratio of A polypeptide to phospholipid molecule is 1:5, and the molar ratio of corresponding ALA polypeptide to phospholipid molecule is 1:10. In Fig. 2, there are two peaks which are far apart from each other, and Peak1 and Peak2 are in order from the left, Peak1 is a nanoparticle formed by polypeptide assembly, and Peak2 is a free polypeptide. By comparison, it was found that the peak and peak areas of Peak1 in all ALA-LNPs were higher than that of monomer-LNP, and the peak and peak areas of Peak2 were smaller than that of monomer-LNP, indicating that the polypeptide can greatly enhance the polypeptide after bivalent cyclization. Utilization and the amount of nanoparticles formed. In addition, CD results show that the polypeptide can be effectively increased after bivalent (Figure 3). When the number of amino acids in the circular sequence is 5 or 7, the same result is exhibited as the AL ₃ A polypeptide. The results of DLS and TEM showed that the selected A polypeptide and the synthesized AL ₃ A, AL ₅ A, and AL ₇ A peptides could form lipoprotein-like nanoparticles with uniform particle size (<30 nm) and good dispersibility, as shown in Figure 4- Figure 11 shows. These results indicate that the designed AL _(3-7) A polypeptide can efficiently assemble lipid molecules, and the number of amino acids in the cyclized linkage structure can be adjusted.

Example 2 The results of Example 1 show that the synthetic AL ₃ A, AL ₅ A, AL ₇ A polypeptides can successfully stabilize lipoprotein-like nanoparticles. In this example, the drug-loading ability of lipoprotein-like nanoparticles was investigated. The AL ₃ A polypeptide was used as an example for research. In the present embodiment, the steps of the method for preparing the lipoprotein-like nanoparticles and the amount of the reactants are basically the same as those in Example 1, except that in the step (1), 3 μmol of DMPC and 0.15 μmol of cholesterol ester (Cholesteryl oleate, Referred to as CO), 0.2 μmol Ir(ppy) ₂ -DIP in chloroform solution. Fluorescent and toxic compounds Ir(ppy) ₂ -DIP are filled in the hydrophobic core of lipoprotein-like nanoparticles. Fig. 12 and Fig. 13 show that lipoprotein-like nanoparticles (AL ₃ A-LNP (Ir)) having uniform particle size and good dispersion can be formed after the AL ₃ A-LNP is coated with Ir(ppy)2-DIP. Figure 14 shows that A-LNP, which encapsulates Ir(ppy) ₂ -DIP in a storage environment, is unstable, with a release of up to 50% of Ir(ppy) ₂ -DIP at 48 hours. The AL ₃ A-LNP encapsulating Ir(ppy) ₂ -DIP can be stably stored in a storage environment or in 1640 medium, facilitating the following functional studies.

Example 3 In this example, a -HHHHH-sequence having a specific function was inserted into a circularized structure for functional study. In the present embodiment, the steps of the preparation method of the lipoprotein-like nanoparticles (A(His) ₅ A-LNP (Ir)) and the amount of the reactants are basically the same as those in the embodiment 2, except that in the step (6) The helix-loop-helix polypeptide is A(His) ₅ A. After step (6), the lipoprotein-like nanoparticles were purified using a 100 K ultrafiltration tube to remove free molecules. The results of DLS and TEM showed that A(His) ₅ A-LNP wrapped with Ir(ppy) ₂ -DIP formed nanoparticles with uniform particle size, as shown in Figures 15-16. A(His) ₅ A-LNP (Ir) was purified by ultrafiltration, and then incubated with a certain amount of Ni column at room temperature for 1 hour, centrifuged, and the supernatant was aspirated for fluorescence detection. Figure 17 shows that when the cyclic sequence is HHHHH, it can be combined with the Ni column. After centrifugation, the Ni column and A(His)5A-LNP(Ir) are precipitated to the bottom to change the fluorescence value in the supernatant. The incubated Ni column was diluted and added to a glass slide for fluorescence microscopy. According to the results of Fig. 18, the Ni column effectively binds to A(His) ₅ A-LNP (Ir) and exhibits green fluorescence. The above results show that the cyclic structure maintains a high affinity with the Ni column after the A(His) ₅ A polypeptide is combined with the lipoprotein-like nanoparticles.

Example 4 In this example, an RGD sequence having a higher affinity for integrin was inserted into a circular sequence for investigation. DLS and TEM results showed that the insertion of the RGD sequence had no significant effect on the formation of lipoprotein-like nanoparticles. The steps of the method for preparing lipoprotein-like nanoparticles and the amount of the reactants in this embodiment are basically the same as those in Example 2, except that in the step (6), the helix-loop-helix polypeptide is A(RGD)A polypeptide. The lipoprotein-like nanoparticles formed are A(RGD)A-LNP(Ir). A (RGD) A-LNP (Ir) and Ir (ppy) ₂ -DIP were incubated with 2 × 10 ⁷ red blood cells at 37 ° C for 1 h at different concentrations, and Absorbance 540 was detected. As shown in Fig. 21, the hemolysis rate of Ir(ppy)2-DIP reached 90% at 60μM, and A(RGD)A-LNP(Ir) showed no obvious hemolysis, indicating that Ir(ppy) ₂ -DIP compound encapsulated lipoprotein After the nanoparticles are used, the hemolytic property of the compound can be greatly reduced, and side effects can be reduced. Further study on the selective killing effect of cyclic RGD. By comparing the killing effects of A(RGD)A-LNP(Ir) and Ir(ppy) ₂ -DIP on HLF (α _v β ₃ ^- ) cells and HT1080 (α _v β ₃ ⁺ ) cells, Ir(ppy) can be obtained. ₂ -DIP has strong killing effect on both HLF and HT1080 cells, while A(RGD)A-LNP(Ir) is less toxic to HLF cells, and the killing effect on HT1080 cells appears to be related to Ir(ppy) ₂ - The same trend for DIP (see Figure 22 - Figure 23). These results show that the encapsulation of lipoprotein-like nanoparticles can effectively reduce the side effects of Ir(ppy) ₂ -DIP, and the introduction of RGD sequences can achieve targeted delivery of Ir(ppy) ₂ -DIP.

Example 5 In this example, the function of the cyclic structure was further verified, using an uncircularized A (SRGDS) A polypeptide as a control. The method in which the A(SRGDS)A polypeptide constitutes lipoprotein-like nanoparticle A (SRGDS) A-LNP (Ir) is substantially the same as the procedure of Example 2, except that in the step (6), the helix-loop-helix The polypeptide is an A (SRGDS) A polypeptide. First, 20 μM of the A(RGD)A-LNP(Ir), A(SRGDS)A-LNP(Ir), AL ₃ A-LNP(Ir) were incubated with HT1080 cells for 2 hours, and the cell morphology and fluorescence intensity were observed. . Referring to Figure 24, the A(RGD)A-LNP(Ir) group showed strong fluorescence intensity and rounded cells, showing obvious signs of death; A(SRGDS)A-LNP(Ir) and AL ₃ A In the LNP (Ir) group, the intracellular fluorescence was weak and the cell morphology was intact. Different concentrations of A(RGD)A-LNP(Ir), A(SRGDS)A-LNP(Ir), and AL ₃ A-LNP(Ir) were incubated with HT1080 cells for 6 h, and MTT assay was performed. Referring to Figure 25, A(RGD)A-LNP(Ir) can significantly kill HT1080 cells, and A(SRGDS)H-LNP(Ir) and AL ₃ A-LNP(Ir) have no significant killing effect on HT1080 cells. . Finally, 20 μM of A(RGD) A-LNP (Ir) and AL ₃ A-LNP (Ir) were incubated with HT1080 cells for different periods of time to observe the cell survival status. Referring to Figure 26, A(RGD)A-LNP(Ir) was incubated with the cells for 2 hours, and the cells died. At 6 hours, the cells died in a large amount, and the survival rate was about 20%, while AL ₃ A-LNP (Ir) was only 6 hours. It begins to cause cell death and the survival rate is higher than 80%. The above results illustrate that the loop structure facilitates maintaining the functionality of the targeting sequence.

It is to be understood that the present invention has been described by way of example only, and is not intended to limit the scope of the present invention. Therefore, the invention is not limited to the embodiments described above, but only by the claims.

Claims

A polypeptide, characterized in that the polypeptide comprises a structural unit consisting of an amphipathic alpha-helical polypeptide, a linker peptide and an amphipathic alpha-helical polypeptide, which are sequentially connected in series, the linker peptide comprising a cyclic structure.
The polypeptide of claim 1 wherein said linker peptide has a targeting peptide sequence.
The polypeptide according to claim 1, wherein the linker peptide has a sequence consisting of a hydrophilic amino acid.
The polypeptide according to any one of claims 1 to 3, wherein the linker peptide comprises a cyclic structure formed by at least one of a disulfide bond, an amide bond, a thioester bond, and a lactone bond.
The polypeptide according to claim 1, wherein the number of amino acids involved in the cyclic structure forming the linker peptide is 5 to 13.
The polypeptide according to claim 1, wherein the cyclic structure of the linker peptide comprises a C(X) n C amino acid sequence, n is selected from any of 3 to 11 and C is cysteine. X is any amino acid that can be used to form the cyclic structure.
The polypeptide according to claim 1, wherein the amino acid sequence of the cyclic structure of the linker peptide comprises any one or a combination of two or more of CSGSC, CSGSGSC, CSGSGSGSC, CRGDC, and CHHHHHC.
The polypeptide of claim 1 wherein the corresponding amino acid sequence of at least one of said amphipathic a-helices is selected from the polypeptide amino acid sequence that mimics ApoA-I function.
The polypeptide according to claim 1, wherein the corresponding amino acid sequence of at least one of the amphipathic α-helices is selected from the reverse sequence of an AI polypeptide sequence or an AI polypeptide sequence, a reverse of an 18A polypeptide sequence or an 18A polypeptide sequence. The reverse sequence of the sequence, the 4F polypeptide sequence or the 4F polypeptide sequence, the reverse sequence of GSLLSALEE WTKKLN or GSLLSALEE WTKKLN.
The polypeptide according to claim 1, wherein the amino acid sequences corresponding to the two amphipathic α-helices are GSLLSALEE WTKKLN and NLKKTWEELASLFSG, respectively.
a lipoprotein-like nanoparticle comprising at least one phospholipid and at least one polypeptide; wherein the polypeptide comprises a structural unit consisting of an amphipathic alpha-helical polypeptide, a linker peptide and an amphipathic alpha-helical polypeptide; The linker peptide comprises a cyclic structure.
The lipoprotein-like nanoparticle according to claim 11, wherein the linker peptide has a targeting peptide sequence.
The lipoprotein-like nanoparticle according to claim 11, wherein the linker peptide has a sequence consisting of a hydrophilic amino acid.
The lipoprotein-like nanoparticle according to claim 11, wherein the linker peptide comprises a cyclic structure formed by at least one of a disulfide bond, an amide bond, a thioester bond, and a lactone bond.
The lipoprotein-like nanoparticle according to claim 11, wherein the number of amino acids involved in the cyclic structure forming the linker peptide is 5 to 13.
The lipoprotein-like nanoparticle according to claim 11, wherein the cyclic structure of the linker peptide contains a C(X) n C amino acid sequence, n is selected from any one of 3 to 11, and C is half. Cystine, X is any amino acid that can be used to form the cyclic structure.
The lipoprotein-like nanoparticle according to claim 11, wherein the amino acid sequence of the cyclic structure of the linker peptide comprises any one or a combination of two or more of CSGSC, CSGSGSC, CSGSGSGSC, CRGDC, and CHHHHHC.
The lipoprotein-like nanoparticle according to claim 11, wherein the corresponding amino acid sequence of at least one of the amphipathic α-helices is selected from the polypeptide amino acid sequence which mimics the function of ApoA-I.
The lipoprotein-like nanoparticle according to claim 11, wherein the corresponding amino acid sequence of at least one of the amphipathic α-helices is selected from the group consisting of an AI polypeptide sequence or a reverse sequence thereof, a 18A polypeptide sequence or a reverse sequence thereof, 4F polypeptide sequence or its reverse sequence, GSLLSALEE WTKKLN or its reverse sequence.
The lipoprotein-like nanoparticle according to claim 11, wherein the amino acid sequences corresponding to the two amphipathic α-helices are GSFLSALEEWTKKLN and NLKKTWEELASLFSG, respectively.
The lipoprotein-like nanoparticle according to claim 11, wherein the amphipathic α-helix of the polypeptide is trapped in the surface layer of the lipoprotein-like nanoparticle, and the cyclic structure of the linker peptide is from the lipoprotein-like The surface of the nanoparticles protrudes.
The lipoprotein-like nanoparticle according to claim 11, wherein the phospholipid comprises any one or two of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, and phosphatidylinositol. The combination above.
The lipoprotein-like nanoparticle according to claim 22, wherein the phospholipid is phosphatidylcholine.
The lipoprotein-like nanoparticle according to claim 22, wherein the phospholipid is dimyristoylphosphatidylcholine.
The lipoprotein-like nanoparticle according to claim 11, wherein the lipoprotein-like nanoparticles further comprise at least one hydrophobic content.
The lipoprotein-like nanoparticle according to claim 25, wherein the hydrophobic content comprises any one or a combination of two or more of cholesterol, cholesterol ester, hydrophobic drug, developer, and tracer. .
The lipoprotein-like nanoparticle according to claim 26, wherein said hydrophobic content is selected From cholesterol esters and / or hydrophobic drugs.
The lipoprotein-like nanoparticle according to claim 26, wherein the hydrophobic content is selected from the group consisting of cholesterol oleate and/or Ir(ppy) 2 -DIP.
The lipoprotein-like nanoparticle according to claim 11, wherein the lipoprotein-like nanoparticle comprises an active agent, and the active agent comprises a chemotherapeutic agent, a photodynamic therapeutic agent, a boron neutron capture therapeutic agent, and the like. Any one or a combination of two or more of radioactive nuclei for radiation therapy.
The lipoprotein-like nanoparticle according to claim 11, wherein the lipoprotein-like nanoparticle comprises an anticancer agent selected from the group consisting of an alkylating agent, an anthracycline, an antibiotic, and an aromatase. Inhibitors, bisphosphonates, cyclooxygenase inhibitors, estrogen receptor modulators, folic acid antagonists, inorganic arsenates, microtubule inhibitors, modifiers, nitrosoureas, nucleoside analogues, broken A group consisting of an osteoblast inhibitor, a molybdenum-containing compound, a retinoid, a topoisomerase 1 inhibitor, a kinase inhibitor, an anti-angiogenic agent, an epidermal growth factor inhibitor, and a histone deacetylase inhibitor.
Use of a lipoprotein-like nanoparticle according to any one of claims 11 to 30 for the preparation of a medicament.
A medicament comprising the polypeptide of any one of claims 1 to 10 or the lipoprotein-like nanoparticles of any one of claims 11 to 30.
A composition, comprising the polypeptide of any one of claims 1 to 10, the lipoprotein-like nanoparticles of any one of claims 11 to 30, or The medicament of claim 32.