CN106831997B

CN106831997B - Polypeptide, lipoprotein-like nanoparticles and application thereof

Info

Publication number: CN106831997B
Application number: CN201510881747.7A
Authority: CN
Inventors: 费浩; 王乔; 马晓川; 贾俊丽
Original assignee: Suzhou Institute of Nano Tech and Nano Bionics of CAS
Current assignee: Suzhou Institute of Nano Tech and Nano Bionics of CAS
Priority date: 2015-12-03
Filing date: 2015-12-03
Publication date: 2020-04-24
Anticipated expiration: 2035-12-03
Also published as: CN106831997A; WO2017092558A1

Abstract

The invention discloses a polypeptide, a lipoprotein-Like Nanoparticle (LNP) and application thereof, wherein the LNP comprises at least one phospholipid and at least one polypeptide, the polypeptide is a spiral-ring-spiral structure polypeptide which comprises at least two amphiphilic α spirals, and the two amphiphilic α spirals are connected through at least one connecting peptide containing a ring structure.

Description

Polypeptide, lipoprotein-like nanoparticles and application thereof

Technical Field

The invention particularly relates to a helix-loop-helix structure polypeptide, lipoprotein-like nanoparticles based on the helix-loop-helix structure polypeptide and application of the lipoprotein-like nanoparticles as a drug delivery system, and belongs to the technical field of nano biology.

Background

In the field of drug delivery, liposomes are widely used for delivery of cytotoxic drugs, genes, proteins and other substances due to their advantages of good biocompatibility, low toxicity, sustained release and the like. The nano-scale liposome easily penetrates through the vascular system and intercellular spaces of tumor tissues, thereby greatly promoting the transportation and diffusion of the medicament in the tumor tissues. However, the smaller the particle size of the liposome, the greater the surface tension, which easily causes fusion of the liposome, resulting in collapse of the structure and release of the contents. In addition, liposomes bind non-specifically to serum proteins, cells, tissues, etc., causing major side effects. To address these problems, various surface modifications emerge in numerous ways, with polymer modifications being 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, thereby reducing surface tension and nonspecific adsorption. In addition, the surface of the liposome can be modified with a targeting element on the basis of polymer protection, so that the specificity of the liposome is further improved. However, these modifications still have some problems, such as the inability of polyethylene glycol to degrade in vivo, cellular toxicity and immune response after accumulation, and in addition, the function of the targeting element may be masked by adjacent polyethylene glycol molecules, resulting in loss of function. Generally, the surface modification of the liposome involves the processes of covalent crosslinking and mixed preparation, and has complex steps and poor controllability.

In the living body, there are various natural vesicle structures composed of lipids and proteins, such as synaptic vesicles, extracellular membrane vesicles and lipoproteins. These structures are natural transport vehicles that transport hydrophobic or hydrophilic substances in vivo. Among these structures, proteins are indispensable components not only for stabilization of lipid molecules and control of surface tension, but also can provide specific target recognition functions. Researchers are currently extracting natural membrane structures (e.g., endosomes, bacterial outer membrane vesicles, erythrocytes, etc.) as carriers for drug delivery. Such carriers have a complicated and fine natural structure, are good in biocompatibility, but are relatively expensive and difficult to modify according to different targets.

High density lipoproteins are a smallest and most compact natural nanoparticle found in the body, primarily for cholesterol transport. High density lipoproteins are composed mainly of apolipoproteins and phospholipids. Among apolipoproteins, apolipoprotein A-I (ApoA-I) predominates, at about 70%. Apo A-I consists of 243 amino acids, forming a series of highly homologous amphipathic helical structures. The protein binds to lipids, which determine the particle size and morphology of high density lipoproteins. In order to construct high-density lipoprotein nanoparticles in vitro for drug delivery, researchers either extract Apo A-I protein from living organisms or express Apo A-I protein recombinantly in vitro. However, ApoA-I protein is expensive to synthesize and difficult to functionally modify, resulting in limited application of high density lipoprotein nanoparticles. How to realize the multifunction of the nanoparticle on the basis of ensuring the stable formation of the lipoprotein-like nanoparticle (high-density lipoprotein-like nanoparticle) is a problem that the industry is eagerly to solve.

Disclosure of Invention

The invention mainly aims to provide a polypeptide, an improved lipoprotein-like nanoparticle based on the polypeptide and application thereof, thereby overcoming the defects in the prior art.

In order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:

in some embodiments, a polypeptide is provided comprising at least two amphipathic α helices joined by at least one linking peptide comprising a cyclic structure.

In some embodiments, the polypeptide comprises a building block consisting of an α helix polypeptide, a linker peptide, and a α helix polypeptide connected in series.

In some embodiments there is provided a lipoprotein-like nanoparticle comprising at least one phospholipid and at least one polypeptide as described.

In some embodiments, the amphipathic α helix of the polypeptide is embedded in the surface layer of the lipoprotein-like nanoparticle, and the loop structure of the linker peptide protrudes from the surface of the lipoprotein-like nanoparticle.

Further, the amphipathic α helix of the polypeptide is embedded in the lipid surface layer of the lipoprotein-like nanoparticle, and the cyclic structure of the linker peptide extends from the lipid surface.

Also provided in some embodiments are uses of the lipoprotein-like nanoparticles, for example in the medical field. For example in the manufacture of a medicament or pharmaceutical composition.

In some embodiments, there is provided a medicament comprising said lipoprotein-like nanoparticle.

In some embodiments, a composition comprising said lipoprotein-like nanoparticle or said drug is provided.

Compared with the prior art, the invention has the advantages that:

(1) the lipoprotein-like nano particle is constructed by adopting a helix-loop-helix structure polypeptide mainly comprising α helix polypeptide, cyclic sequence connecting peptide and α helix polypeptide, phospholipid and the like, particularly, the helix part of the polypeptide is sunk into the surface layer of the lipoprotein-like nano particle, so that the stable formation of the lipid nano particle with uniform size and smaller particle size can be promoted, and meanwhile, the cyclic sequence extends out of the surface of the lipoprotein-like nano particle, the insertion of a functional sequence is facilitated, and the multi-functionalization of the lipoprotein-like nano particle is realized;

(2) the core of the provided lipoprotein-like nano particle can be used for wrapping hydrophobic functional substances, and further can further expand the application of the lipoprotein-like nano particle, for example, the lipoprotein-like nano particle can be used as a drug delivery system to be applied to diagnosis and treatment of cancer and the like, the drug delivery system has the characteristics of low toxicity, high stability, convenience in functionalization and the like, and a simple and effective means is provided for targeted killing of tumor cells and cell and tissue imaging.

(3) In the provided spiral-ring-spiral structure polypeptide consisting of the amphiphilic α spiral polypeptide, the cyclic structure connecting peptide and the amphiphilic α spiral polypeptide which are connected in series, the sequence of the cyclic structure connecting peptide can be selected from various targeting peptides with cyclic structures, so that the lipoprotein-like nanoparticle has the function of targeting specific cells, tissues and organs.

Drawings

FIG. 1 is a schematic diagram of the structures of a polypeptide and a lipoprotein-like nanoparticle in an exemplary embodiment of the invention;

FIG. 2 shows the polypeptide A and AL of example 1₃A、AL₅A、AL₇A comparative profile of the ability of the polypeptide to form lipoprotein-like nanoparticles;

FIG. 3 shows the polypeptide A and AL of example 1₃A、AL₅A、AL₇A comparative profile of the degree of helicity of the polypeptide on the lipoprotein-like nanoparticle;

FIG. 4 is a graph showing a particle size test pattern of lipoprotein-like nanoparticles based on A polypeptide tested using a dynamic laser light scattering (DLS) system in example 1;

FIG. 5 is a transmission electron microscopy imaging of lipoprotein-like nanoparticles based on polypeptide A of example 1;

FIG. 6 is an AL-based test using a dynamic laser light scattering (DLS) system as in example 1₃A is the particle size test chart of lipoprotein-like nanometer particle of the polypeptide;

FIG. 7 shows the results of example 1 based on AL₃A transmission electron microscopy imaging of lipoprotein-like nanoparticles of polypeptide a;

FIG. 8 is an AL-based test using a dynamic laser light Scattering (DLS) System as in example 1₅A particle size test chart of lipoprotein-like nanoparticles of A-structure polypeptide;

FIG. 9 shows the results of example 1 based on AL₅A transmission electron microscope imaging image of lipoprotein-like nanoparticles of a polypeptide of structure a;

FIG. 10 is an AL-based test using a dynamic laser light Scattering (DLS) System as in example 1₇A particle size test chart of lipoprotein-like nanoparticles of A-structure polypeptide;

FIG. 11 shows AL-based data in example 1₇A transmission electron microscope imaging image of lipoprotein-like nanoparticles of a polypeptide of structure a;

FIG. 12 shows the use of dynamic laser light in example 2Scattering (DLS) System test Package Ir (ppy)₂AL-based on DIP₃A particle size test chart of lipoprotein-like nanoparticles of A-structure polypeptide;

FIG. 13 is the example 2 parcel Ir (ppy)₂AL-based on DIP₃A transmission electron microscope imaging image of lipoprotein-like nanoparticles of a polypeptide of structure a;

FIG. 14 shows the results of example 2 based on A and AL₃Lipoprotein-like nanoparticle encapsulation of A-structural polypeptides Ir (ppy)₂-a test chart for the stability of DIP compounds;

FIG. 15 is a graph of the results of example 3 based on A (His) using dynamic laser light scattering (DLS) system₅Encapsulation of A Structure Polypeptides fat-soluble substance Ir (ppy)₂-a particle size test plot of DIP lipoprotein-like nanoparticles;

FIG. 16 shows a graph based on A (His) in example 3₅Encapsulation of A Structure Polypeptides fat-soluble substance Ir (ppy)₂-transmission electron microscopy imaging of DIP lipoprotein-like nanoparticles;

FIG. 17 is the envelope Ir (ppy) in example 3₂-DIP A (His)₅A-lipoprotein-like nanoparticles and AL₅Comparative graph of residual fluorescence of supernatant after 2 hours incubation of A-lipoprotein-like nanoparticles with Ni column;

FIG. 18 is the package Ir (ppy) in example 3₂-DIP A (His)₅A-lipoprotein-like nanoparticles and AL₅A-a graph comparing the amount of lipoprotein-like nanoparticles bound to a Ni column after 2 hours incubation of the lipoprotein-like nanoparticles with the Ni column;

FIG. 19 shows the measurement of A (RGD) A structure polypeptide-based encapsulated lipid-soluble substance Ir (ppy) using dynamic laser light scattering (DLS) system in example 4₂-a particle size test plot of DIP lipoprotein-like nanoparticles;

FIG. 20 shows the encapsulation of the lipid-soluble substance Ir (ppy) based on the A (RGD) A structural polypeptide in example 4₂-transmission electron microscopy imaging of DIP lipoprotein-like nanoparticles;

FIG. 21 shows the A (RGD) A-LNP wrapper Ir (ppy) in example 4₂Post DIP with Ir (ppy)₂-comparative graph of the hemolysis rate of DIP;

FIG. 22 shows an embodimentExample 4A (RGD) A-LNP Package Ir (ppy)₂Post DIP with Ir (ppy)₂-comparative graph of DIP versus HLF cell killing rate;

FIG. 23 shows A (RGD) A-LNP wrapper Ir (ppy) in example 4₂Post DIP with Ir (ppy)₂-comparative graph of killing rate of HT1080 cells by DIP;

FIG. 24 is the parcel Ir (ppy) of example 5₂DIP A (RGD) A-LNP, A (SRGDS) A-LNP, AL₃A graph comparing the amount of cellular fluorescence and cell morphology of a-LNP in HT1080 cells;

FIG. 25 is the example 5 parcel Ir (ppy)₂DIP A (RGD) A-LNP, A (SRGDS) A-LNP, AL₃A graph comparing the killing effect of different concentrations of a-LNP on HT1080 cells;

FIG. 26 is the parcel Ir (ppy) of example 5₂A (RGD) A of DIP-LNP, AL₃A graph comparing the killing of HT1080 cells by a-LNP at different times under 20 μ M;

FIG. 27 shows AL in example 1₅Mass spectrometry pattern of a.

Detailed Description

Exemplary embodiments that embody features and advantages of the invention are described in detail below. It is to be understood that the invention is capable of other and different embodiments and its several details are capable of modification without departing from the scope of the invention, and that the description and drawings are to be regarded as illustrative in nature and not as restrictive.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

According to one aspect of the present invention, there is provided a polypeptide comprising at least two amphipathic α -helices connected by at least one linker peptide comprising a cyclic structure.

The polypeptide can also be regarded as a helix-loop-helix structure polypeptide, wherein the helix polypeptide is a polypeptide with a definite hydrophilic and hydrophobic amphiphilic α helix structure, the configuration of the amino acid can be L type or D type, the loop sequence is an amino acid sequence with a loop structure, and the loop sequence structurally bivalences the amphiphilic α helix polypeptide, so that the helix degree of the polypeptide can be further improved.

The polypeptide may also be referred to as AL_mPolypeptide A, further collectively called ALA polypeptide, wherein A refers to α helix polypeptide, L refers to linker peptide with cyclized sequence, and m is the number of amino acids contained in the cyclized sequence.

Wherein the polypeptide can be synthesized by any suitable method known in the art, for example, by reference to the following references:

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.; kaponos, s.; poulos, c.;

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

according to one aspect of the present invention there is provided a non-naturally occurring lipoprotein-Like Nanoparticle (LNP) comprising at least one phospholipid and at least one said polypeptide (i.e. said helix-loop-helix polypeptide).

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

a) at least one phospholipid;

b) at least one of said polypeptides comprising structural units consisting of an α helix polypeptide (referred to as an "amphipathic helix polypeptide" in the figure), a linker peptide comprising a loop structure (referred to simply as a "linker loop" in the figure), and a α helix polypeptide connected in series;

and, c) one or more hydrophobic content, such as cholesteryl oleic acid and the like.

In some embodiments, the amphipathic α helix of the polypeptide is entrapped on the lipid surface layer of the lipoprotein-like nanoparticle, and the cyclic structure of the linker peptide protrudes from the surface of the lipoprotein-like nanoparticle.

When the spiral-ring-spiral structure polypeptide is combined with the lipoprotein-like nanoparticles, the spiral part is sunk into the surface layer of the lipoprotein-like nanoparticles and is used for promoting the stable formation of lipid nanoparticles with uniform size and smaller particle size, and the annular sequence extends out of the surface of the lipoprotein-like nanoparticles, so that the insertion of a functional sequence is facilitated, and the multi-functionalization of the lipoprotein-like nanoparticles is facilitated. Furthermore, the core of the lipoprotein-like nano particle is adopted to encapsulate the hydrophobic functional substance, so that the application of the lipoprotein-like nano particle can be further expanded, particularly the application in the field of medicine, for example, the lipoprotein-like nano particle can be used as a medicine carrying system for diagnosing and treating cancers. The drug-loading system has the characteristics of low toxicity, high stability, convenience for functionalization and the like, and provides a simple, convenient and effective means for targeted killing of tumor cells and cell and tissue imaging.

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

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

In some preferred embodiments, the cyclic structure of the linker peptide comprises C (X)_nC amino acid sequence, n is any integer selected from 3-11. Wherein C denotes cysteine for cyclization and X is any amino acid that can be used to form the cyclic structure.

In some preferred embodiments, the number of amino acids involved in forming the cyclic structure of the linker peptide is 3 to 7.

In some embodiments, the amino acid sequence of the cyclic structure of the connecting peptide comprises any one or a combination of two or more of CSGSC, CSGSGSGSC, csgsgsgsgsgsc, CRGDC, chhhhhhhc.

In some embodiments, the corresponding amino acid sequence of at least one of the amphipathic α -helices is selected from the group consisting of polypeptide amino acid sequences that mimic 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 A-I polypeptide sequence (CGVLESFKASFLSALEEWTKK) or its reverse, 18A polypeptide sequence (DWLKAFYDKVAEKLKEAF) or its reverse, 4F polypeptide sequence (DWFKAFYDKVAEKFKEAF) or its reverse, GSFLSALEEWTKKLN or its reverse.

In a preferred embodiment, the amino acid sequences corresponding to the two amphipathic α helix are GSFLSALEEWTKKLN and NLKKTWEELASLFSG, respectively.

In some embodiments, the phospholipid includes any one or a combination of two or more of phosphatidylcholine (phosphatidylcholine), phosphatidylethanolamine (phosphatidylethanolamine), dimyristoyl phosphatidylethanolamine (DMPE,1, 2-dimyristoyl-sn-glycerol-3-phosphoethanolamine), phosphatidylserine, phosphatidylglycerol, and phosphatidylinositol, but is not limited thereto.

For example, phosphatidylcholine is preferably used as the phospholipid.

For example, Dimyristoylphosphatidylcholine (DMPC) is preferably used as the phospholipid.

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

Wherein the at least one hydrophobic content may 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, imaging agent, 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 a hydrophobic drug, an imaging agent, and a tracer.

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

For example, the hydrophobic content may preferably be selected from cholesterol oleate and/or Ir (ppy)₂DIP, for example, a combination of the two.

In some embodiments, the lipoprotein-like nanoparticles comprise active agents, anti-cancer agents, and the like.

The lipoprotein-like nanoparticles of the present invention may optionally incorporate a hydrophobic drug or labeling compound, or may be used in the preparation of a formulation incorporating a drug or labeling compound, wherein the lipoprotein-like nanoparticles provide the benefit of reducing side effects of the drug or labeling compound and/or also prevent degradation and/or loss of effect of the drug or labeling compound.

According to another aspect of the invention, methods of making and using the lipoprotein-like nanoparticles described herein are also provided.

Accordingly, there is also provided in some embodiments the use of the lipoprotein-like nanoparticles, for example in the medical field.

According to yet another aspect of the present invention, the lipoprotein-like nanoparticles can be used for the treatment or diagnosis of cancer (e.g., breast cancer, gastric cancer, colorectal cancer, colon cancer, pancreatic cancer, non-small cell lung cancer, brain cancer, liver cancer, kidney cancer, prostate cancer, bladder cancer, ovarian cancer, or hematological malignancies (e.g., leukemia, lymphoma, multiple myeloma, etc.).

"α helix"

The "α helix" is used in this specification to refer to the motif common in the secondary structure of proteins α -helices are coiled-coil conformations, similar to springs, in which each backbone N-H group contributes a hydrogen bond to the earlier four residue backbone C ═ O groups.

"amphiphilic"

Amphipathic α helix is generally a secondary structural motif commonly encountered in biologically active peptides and proteins, and refers to a α helix with opposite polar and non-polar faces oriented along the long axis of the helix.

Helix-loop-helix structure "

In the present specification, "helix-loop-helix polypeptide" refers to a structural unit composed of an amphiphilic α helix polypeptide, a linker peptide having a loop structure, and an amphiphilic α helix polypeptide, which are sequentially connected in series.

"amino acids"

In the present specification, both L-amino acids and D-amino acids are included, and they may be naturally occurring amino acids or non-naturally occurring, artificially synthesized amino acids. And these amino acids may be optionally chemically modified.

"targeting peptides"

In the present specification, the term "targeting peptide" refers to a molecule that binds relatively specifically to a molecule present in a particular organ or tissue after administration to a subject. Typically, selectivity is characterized in part by detecting selective binding of the molecule to an organ or tissue that is at least two times larger than a control organ or tissue. In some embodiments, the selective binding is at least three or four times greater than a control organ or tissue.

Cyclic structures containing amino acid sequences that may be used as targeting peptides have been identified including, but not limited to, the amino acid sequences listed in tables 1 and 2:

TABLE 1 protein targeting peptides

Protein target	Peptide sequence (name)
		Her2/ErB2	WTGWCLNPEESTWGFCTGSF
α_vβ₃	CDCRGDCFC(RGD-4C)
		α₅β₁	GACRGDCLGA(pIII)
APN/CD13	CNGRC
		MMP-9	CTTHWGFTLC(CTT)
TAG-72	GGVSCMQTSPVCENNL
		VEGFR-3	CSDSWHYWC
Phosphatidyl Serine	CLSYYPSYC

TABLE 2 tumor targeting peptides

It should be noted that the targeting peptide of the present invention is not limited to the above-mentioned peptide sequence, and it is possible to use the peptide as a linker peptide in the polypeptide of the lipoprotein-like nanoparticle of the present invention by adding cysteine to both ends of other known linear targeting peptides or by looping them in other ways.

In some embodiments, a lipoprotein-Like Nanoparticle (LNP) of the invention comprises an anti-cancer agent. Such active agents may be chemotherapeutic agents, photodynamic therapeutic agents, boron neutron capture therapeutic agents, or radionuclides for radiotherapy. In some embodiments, the anti-cancer agent is selected from the group consisting of alkylating agents (alkylating agents), anthracyclines, antibiotics, aromatase inhibitors, bisphosphonates, cyclooxygenase inhibitors, estrogen receptor modulators, folic acid antagonists, inorganic arsenates, microtubule inhibitors, modifiers, nitrosoureas (nitrosurea), nucleoside analogs, osteoclast inhibitors, molybdenum containing compounds, retinoids, topoisomerase 1 inhibitors, kinase inhibitors (such as, but not limited to, tyrosine kinase inhibitors), anti-angiogenic agents, epidermal growth factor inhibitors, and histone deacetylase (deacetylase) inhibitors.

In summary, the invention can provide good interfaces and insertion sites for biological functionalization while stabilizing the nanoparticles by using the helix-loop-helix structure polypeptide, and further can add an amino acid sequence with specific functions into the helix-loop-helix structure polypeptide according to requirements, and wrap a hydrophobic substance in the core of the nanoparticles, thereby realizing the multi-functionalization of the nanoparticles.

The present invention will be described in further detail with reference to the accompanying drawings and embodiments. The ALA polypeptides used in the following examples can be synthesized in the manner described in the above documents 1 and 2.

Example 1

In the lipoprotein-like nanoparticle based on the helix-loop-helix polypeptide of the embodiment, the helix-loop-helix polypeptide (ALA polypeptide) is formed by connecting α helix polypeptide, cyclized sequence and α helix polypeptide in series in a covalent bond mode, wherein the amino acid sequence of α helix polypeptide is GSFLSALEEWTKKLN, but is not limited to this sequence, the cyclized polypeptide sequences are SGS, SGSGSGSGS and SGSGSGSGSGS respectively, but is not limited to this sequence, and other sequences with specific functions can also be adopted.

One of them₅The synthesis process of the polypeptide A is as follows: the solid phase synthesis method (see documents 1 and 2) is adopted, Fmoc amino acid and dichlorotrityl resin are used as carriers, amino acid condensation reaction is carried out on the Fmoc amino acid and dichlorotrityl resin according to a designed sequence from C terminal to N terminal, after the polypeptide reaction is finished, TFA is used for cleaving the polypeptide from the resin, purification and desalination treatment are carried out, and after the treatment is finished, pure products are concentrated to obtain the linear polypeptide.

Dissolving the linear polypeptide in distilled water, adjusting pH value to about 8, and slowly oxidizing with oxygen (or adding some catalysts such as potassium ferricyanide, iodine, etc.) to make the sulfhydryl of two cysteines in the polypeptide sequence form disulfide bond, i.e. form crude cyclopeptide. The crude cyclic peptide is concentrated again, purified by HPLC and lyophilized to obtain a pure product, namely the ALA polypeptide, and the molecular weight (4048.2) of the ALA polypeptide is determined to be basically consistent with the predicted molecular weight (4048.55) of the designed polypeptide by adopting a mass spectrometry technology (refer to figure 27), so that the sequence of the ALA polypeptide can be determined to be correct.

And AL₃A、AL₇A and the like can also be synthesized by the above-mentioned method.

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

(1) dissolving 3 μmol of DMPC (1, 2-dimyristoyl-sn-glyco-3-phoschooline) and 0.1 μmol of cholesterol ester (Cholesteryl ester, abbreviated as CO) in chloroform solution;

(2) blowing the chloroform in the centrifugal tube by using stable nitrogen flow to dry the mixture to form a layer of film at the bottom of the tube;

(3) adding 1ml of PBS buffer solution (pH value is 7.4) into a centrifuge tube, and fully suspending the thin film at the bottom of the test tube by using a vortex oscillation instrument to form milky suspension;

(5) filling nitrogen into the centrifugal tube, sealing, placing the centrifugal tube in an ultrasonic instrument, and carrying out ultrasonic treatment at 48 ℃ for 1 h;

(6) adding PBS solution containing the spiral-ring-spiral structure polypeptide into the centrifuge tube, mixing uniformly, sealing, and standing overnight at 4 ℃.

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

Lipoprotein-like nanoparticles (AL) of helix-loop-helix structure polypeptide synthesized by the above steps_(3-7)a-LNP) can be characterized by FPLC or the like. FIG. 2 is a time plot of FPLC with a 1:5 molar ratio of polypeptide A to phospholipid molecules, corresponding to a 1:10 molar ratio of ALA polypeptide to phospholipid molecules. In fig. 2, there appear two peaks with a relatively large difference in position, in order from left, Peak1 and Peak2, Peak1 is a nanoparticle formed by polypeptide assembly, and Peak2 is a free polypeptide. The comparison shows that the Peak value and the Peak area of Peak1 in all ALA-LNP are higher than those of monomer-LNP, and the Peak value and the Peak area of Peak2 are smaller than those of monomer-LNP, which shows that the utilization rate of the polypeptide and the quantity of formed nanoparticles can be greatly improved after the polypeptide is bivalent by a cyclization sequence. In addition, CD results show that the degree of helicity of the polypeptide can be effectively increased after the polypeptide is bivalent (FIG. 3). With AL when the number of amino acids in the cyclic sequence is 5 or 7₃The a polypeptide exhibited the same results. The results of DLS and TEM showed that the selected A polypeptide and the synthesized AL₃A、AL₅A、AL₇Polypeptide A can form particles with uniform size<30nm) and lipoprotein-like nanoparticles with good dispersibility, as shown in FIGS. 4-11. These results indicate designed AL_(3-7)The polypeptide A can assemble lipid molecules with high efficiency, and the number of amino acids in a cyclized linker can be adjusted.

Example 2

The results according to example 1 show that the AL synthesized₃A、AL₅A、AL₇A is muchPeptides can successfully stabilize lipoprotein-like nanoparticles. The drug loading capacity of the lipoprotein-like nanoparticles was studied in this example. With AL₃Polypeptide A was used as an example for the study. The procedure and the amount of reactants used in the preparation of the lipoprotein-like nanoparticles in this example were substantially the same as those in example 1, except that 3. mu. mol of DMPC, 0.15. mu. mol of Cholesteryl ester (CO), and 0.2. mu. mol of Ir (ppy) were used in step (1)₂-a chloroform solution of DIP. Filling a fluorescent and toxic compound Ir (ppy) in a hydrophobic core of a lipoprotein-like nanoparticle₂-DIP. FIGS. 12 and 13 show AL₃The A-LNP can form lipoprotein-like nanoparticles (AL) with uniform particle size and good dispersity after wrapping Ir (ppy)2-DIP₃A-LNP (Ir)). FIG. 14 illustrates the packaging of Ir (ppy) in a storage environment₂A-LNP instability of DIP, 48 hours Ir (ppy)₂-DIP release amounts up to 50%. Package Ir (ppy)₂AL of-DIP₃A-LNP can stably exist in a storage environment or in a 1640 culture medium, and is convenient for the following functional research.

Example 3

In this example, functional studies were carried out by inserting a-HHHHHHH-sequence having a specific function into the cyclized structure. Lipoprotein-like nanoparticles (A (His) in this example₅The steps of the A-LNP (Ir) preparation process and the amounts of reactants used are essentially the same as in example 2, except that in step (6), the helix-loop-helix polypeptide is A (His)₅A. After step (6), the lipoprotein-like nanoparticles were purified using a 100K ultrafiltration tube to remove free molecules. Detection by DLS and TEM with results showing Package Ir (ppy)₂-DIP A (His)₅a-LNP formed nanoparticles of uniform size, see fig. 15-16. A (His)₅Performing polypeptide quantification after ultrafiltration and purification of A-LNP (Ir), then incubating with a certain amount of Ni column for 1 hour at room temperature, centrifuging, and sucking supernatant for fluorescence detection. FIG. 17 shows that the cyclic sequence is HHHHH, which can be bound to Ni column, and after centrifugation, Ni column and A (His)5A-LNP (Ir) are precipitated at the bottom to change the fluorescence value in the supernatant. Diluting the incubated Ni column, and then dripping the diluted Ni column on a glass slide for fluorescence microscopyTest, based on the results in FIG. 18, it was shown that the Ni column was effective with A (His)₅A-LNP (Ir) binds and exhibits green fluorescence. The above results show that A (His)₅After the polypeptide A is combined with the lipoprotein-like nano particles, the ring structure keeps high affinity with the Ni column.

Example 4

In this example, the RGD sequence having a higher affinity with integrin was inserted into the cyclic sequence for study. DLS and TEM results show that the insertion of the RGD sequence has no significant effect on the formation of lipoprotein-like nanoparticles. The steps of the method for preparing lipoprotein-like nanoparticles and the amounts of reactants in this example are substantially the same as those in example 2, except that in step (6), the helix-loop-helix polypeptide is a (rgd) a polypeptide, and the lipoprotein-like nanoparticles formed are a (rgd) a-lnp (ir). A (RGD) A-LNP (Ir) and Ir (ppy)₂-DIP at different concentrations with 2X 10⁷The individual erythrocytes were incubated at 37 ℃ for 1h and examined for Absorbance 540. FIG. 21 shows that the hemolysis rate of Ir (ppy)2-DIP reaches 90% at 60 μ M, and A (RGD) A-LNP (Ir) has no obvious hemolysis, which indicates that Ir (ppy)₂After the-DIP compound is wrapped in the lipoprotein-like nano particles, the hemolytic property of the compound can be greatly reduced, and the side effect is reduced. Further study on the selective killing effect of cyclic RGD. By comparing A (RGD) A-LNP (Ir) with Ir (ppy)₂-DIP pair HLF (α)_vβ₃ ^-) Cells and HT1080(α)_vβ₃ ⁺) The killing effect of the cells was found to be Ir (ppy)₂DIP has stronger killing effect on HLF and HT1080 cells, while A (RGD) A-LNP (Ir) has smaller toxicity on HLF cells and the killing effect on HT1080 cells is shown to be similar to that of Ir (ppy)₂The same trend for DIP (see FIGS. 22-23). These results show that the encapsulation of lipoprotein-like nanoparticles can effectively reduce Ir (ppy)₂The side effects of DIP, while the introduction of the RGD sequence makes it possible to achieve Ir (ppy)₂Targeted delivery of DIP.

Example 5

In this example, the function of the cyclic structure was further verified, using the non-cyclized A (SRGDS) A polypeptide as a control. A (SRGDS)The method of constructing lipoprotein-like nanoparticles a (srgds) a-lnp (ir) with a polypeptide is essentially the same as the procedure of example 2, except that in step (6), the helix-loop-helix polypeptide is the a (srgds) a polypeptide. First 20 μ M of said A (RGD) A-LNP (Ir), A (SRGDS) A-LNP (Ir), AL₃And (3) incubating A-LNP (Ir) and HT1080 cells for 2h, and observing cell morphology and fluorescence intensity. Referring to FIG. 24, cells in group A (RGD) A-LNP (Ir) have stronger fluorescence intensity, and cells are shrunken and rounded, showing obvious signs of death; a (SRGDS) A-LNP (Ir) and AL₃The A-LNP (Ir) group has weak intracellular fluorescence brightness and intact cell morphology. A (RGD) A-LNP (Ir), A (SRGDS) A-LNP (Ir), AL in different concentrations₃A-LNP (Ir) was incubated with HT1080 cells for 6h for MTT assay. As shown in FIG. 25, A (RGD) A-LNP (Ir) can significantly kill HT1080 cells, A (SRGDS) H-LNP (Ir), AL₃A-LNP (Ir) has no great killing effect on HT1080 cells. Finally, 20 μ M of A (RGD) A-LNP (Ir), AL₃A-LNP (Ir) and HT1080 cells are incubated for different times, and the survival state of the cells is observed. Referring to FIG. 26, A (RGD) A-LNP (Ir) cells incubated with cells began to die 2h, and cells died massively at 6h with a survival rate of about 20%, while AL₃A-LNP (Ir) only starts to cause cell death at 6h, and the survival rate is higher than 80%. The above results indicate that the cyclic structure is advantageous for maintaining the functionality of the targeting sequence.

It should be understood that the described embodiments of the present invention are for illustrative purposes only and are not intended to limit the scope of the present invention, and that various other substitutions, alterations, and modifications may be made by those skilled in the art within the scope of the present invention, and thus, the present invention is not limited to the above-described embodiments but only by the claims.

Claims

1. The polypeptide is characterized by being formed by sequentially connecting an amphiphilic α helical polypeptide, a connecting peptide and a reverse sequence of the amphiphilic α helical polypeptide in series, wherein the connecting peptide is of a cyclic structure;

wherein the sequence of the amphiphilic α helix polypeptide is GSFLSALEEWTKKLN, and the sequence of the linker peptide is SGS, SGSGSGS, sgsgsgsgsgs, RGD or HHHHH.

2. A lipoprotein-like nanoparticle comprises at least one phospholipid and at least one polypeptide, and is characterized in that the polypeptide is formed by sequentially connecting an amphiphilic α helical polypeptide, a connecting peptide and a reverse sequence of the amphiphilic α helical polypeptide in series, wherein the connecting peptide is of a cyclic structure;

3. The lipoprotein-like nanoparticle of claim 2 in which the amphiphilic α -helix of the polypeptide is embedded in the surface layer of the lipoprotein-like nanoparticle and the cyclic structure of the linker peptide protrudes from the surface of the lipoprotein-like nanoparticle.

4. Lipoprotein-like nanoparticles according to claim 2, characterized in that: the phospholipid comprises one or more of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol and phosphatidylinositol.

5. Lipoprotein-like nanoparticles according to claim 4, characterized in that: the phospholipid is phosphatidylcholine.

6. Lipoprotein-like nanoparticles according to claim 4, characterized in that: the phospholipid adopts dimyristoyl phosphatidylcholine.

7. Lipoprotein-like nanoparticles according to claim 2, characterized in that: the lipoprotein-like nanoparticles also include at least one hydrophobic content.

8. Lipoprotein-like nanoparticles according to claim 7, characterized in that: the hydrophobic content comprises any one or the combination of more than two of cholesterol, cholesterol ester, hydrophobic drug, developer and tracer.

9. Lipoprotein-like nanoparticles according to claim 8, characterized in that: the hydrophobic content is selected from cholesterol esters and/or hydrophobic drugs.

10. Lipoprotein-like nanoparticles according to claim 8, characterized in that: the hydrophobic content is selected from cholesterol oleate and/or Ir (ppy)₂-DIP。

11. Use of the lipoprotein-like nanoparticle of any one of claims 2-10 for encapsulating a hydrophobic drug.