Peptide nanoparticle composition

Abstract

The invention discloses a polypeptide nanoparticle composition, which relates to the gene delivery technology field in the field of gene therapy. The peptide for delivering nucleic acid has the following general structure: (Arg)n-Xa-Yb-Xc-Cys (general formula I), wherein: n+a+b+c+1=6-40, n, a, b or c are independent of each other, and n≥1, b≥1, a, c are any of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26. The invention provides a novel non-natural polypeptide having the function of encapsulating nucleic acid and selectively promoting nucleic acid to pass through cell membrane, as well as a macromolecular lipid polypeptide nanoparticle containing the polypeptide, a method for using the same for in vivo and in vitro cell gene transfection, and a method for applying the same to vaccine preparations.

Description

Technical Field

[0001] The present invention relates to the field of gene therapy, in particular to a polypeptide nanoparticle composition which can encapsulate nucleic acid and selectively promote the nucleic acid to pass through cell membrane. Background Art

[0002] Gene transfection is a technology that transfers or delivers nucleic acids with biological functions into cells and allows the nucleic acids to maintain their biological functions in cells. Gene vectors refer to a tool that introduces exogenous therapeutic genes into biological cells. At present, the gene vectors with industrial transformation potential in the world are mainly viral vectors and non-viral vectors. Viral vectors are a gene delivery tool that uses the ability of viruses to transmit their genomes into other cells for infection. Currently, lentivirus, adenovirus, retroviral vectors and adeno-associated virus vectors have good application prospects. However, due to their inherent physical and chemical properties and biological activity, viral vectors have serious disadvantages, such as high production cost, limited loading capacity, poor targeting, insertion integration and teratogenic, carcinogenic and mutagenic, which are not conducive to the development of universal and general therapies. Non-viral vectors include: liposome nanoparticles, complex nanoparticles, cationic polymer nanoparticles, direct injection, calcium phosphate co-precipitation, microinjection, electroporation, microparticle bombardment, etc. Among them, liposome nanoparticles are the main non-viral vectors currently used for RNA transfection. At present, gene drugs (the world's first RNAi drug Patisiran) have been launched using liposome nanoparticle technology, and many drugs under development using this technology have entered clinical phase II. Compared with other gene delivery methods, liposome nanoparticles have the advantages of low production cost, clear chemical structure, easy quality control, targeted drug delivery through targeted modification, theoretically unlimited encapsulation capacity, no risk of integration-induced mutations, biodegradability in the body, and convenient application for gene delivery in vivo and in vitro. However, most positively charged liposome lipids are highly toxic, liposome transfection efficiency is low, and few liposome nanoparticles have been successfully used in clinical practice.

[0003] The cell membrane is composed of a phospholipid bilayer, which is hydrophilic on the outside and hydrophobic on the inside. It is a barrier that prevents extracellular substances from freely entering the cell and can prevent a variety of bioactive molecules (including peptides, proteins, DNA, RNA, etc.) from entering the cell. Although this protection mechanism ensures the relative stability of the intracellular environment and enables various physiological and biochemical reactions to run in an orderly manner, it prevents many bioactive molecules with therapeutic activity from entering the cell in the field of drug therapy. Penetrating the cell membrane into the cell is a prerequisite for many biomacromolecules with targets in the cell to play a role. Therefore, the development of a delivery system that can encapsulate these biomacromolecules into the cell is of great significance in the pharmaceutical field. Cell penetrating peptides (CPPs) are a class of polypeptides that can directly penetrate the cell membrane and enter the cell in a non-receptor-dependent and non-classical endocytosis manner without causing cell membrane damage. They are generally no more than 30 amino acids in length and are rich in basic amino acids. The amino acid sequence is usually positively charged. Several CPPs have been discovered, such as human immunodeficiency virus type 1 transcription activator TAT (human immunodeficiency virus-1 transcription activator, HIV-1TAT) (Vives et al., J.Biol.Chem.1997;272,16010), the homeodomain (ANTP) in Drosophila melanogaster antennapedia (Derossi et al., J.Biol.Chem.1994,269,10444) and herpes simplex virus type I VP22 transcription factor (SHV1VP22) (Phelan et al., Nat.Biotech.,1998,16,440). The common properties of CPPs are: net positive charge or neutral charge, hydrophilic and hydrophobic (amphiphilic); high transmembrane delivery efficiency; low cytotoxicity; no cell type restrictions; can be introduced into cells by chemical binding or gene fusion, including small molecule compounds, dyes, peptides, peptide nucleic acids (PNA), proteins, plasmid DNA, siRNA, mRNA, smaller liposomes, phage particles and superparamagnetic particles, etc., and current research has not yet reached the limit of its carrying capacity, making it a potential multifunctional targeted drug carrier. The specific transmembrane mechanism of different CPPs is different. Studies have found that specific amino acid sequences can bind to mRNA and interfere with and reduce cell membrane stability, thereby carrying bioactive substances through the cell membrane, such as the arginine-alanine-leucine-alanine residue (RALA) sequence (Pardi et al., Curr Opin in Immunol. 2020, 65: 14-20).Compared with other delivery methods, CPPs are still less studied, and there are few reports on their use in mRNA delivery or vaccine development. Nevertheless, CPPs have demonstrated high medicinal value and broad commercial prospects. Summary of the invention

[0004] mRNA vaccine is a new type of vaccine developed in recent years. It is the fifth generation of new vaccines after inactivated vaccines, attenuated vaccines, subunit vaccines, and DNA vaccines. Its safety is more advantageous than DNA vaccines. As a new response plan, mRNA vaccine directly introduces mRNA encoding antigen protein into cells, synthesizes protein through the cell's expression system, and thus induces a specific immune response. Although most cells can spontaneously take up mRNA, the efficiency is very low and saturates at low doses. In addition, due to the presence of a large number of RNA enzymes (RNase) in nature, RNA is very unstable both in vivo and in vitro and is easily degraded. Therefore, a suitable preparation is needed to protect mRNA from extracellular RNase-mediated degradation and promote its entry into cells. In the development of viral mRNA vaccines and tumor mRNA vaccines, it is crucial to deliver mRNA of a specific sequence to dendritic cells (DC) to express it safely, efficiently, and in sufficient quantities for the vaccine to produce efficacy, which is the difficulty of mRNA vaccine technology.

[0005] An object of the present invention is to provide a suitable and effective vector for delivering biologically active molecules into cells. Another object of the present invention is to provide a suitable and effective vector for delivering RNA molecules. Another object of the present invention is to provide a suitable and effective vector for delivering mRNA molecules.

[0006] In one aspect, novel non-naturally occurring polypeptides are provided that have the function of encapsulating nucleic acids and selectively promoting nucleic acids to pass through cell membranes.

[0007] Another aspect provides a polypeptide nanoparticle composition, which includes a non-naturally occurring polypeptide and a PEGylated polymer material.

[0008] A non-naturally occurring polypeptide having the following general structure:

[0009] (Arg)n-Xa-Yb-Xc-Cys (Formula I)

[0010] wherein: n+a+b+c+1=6-40, that is, n+a+b+c+1 can be equal to the following values: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40, n, a, b or c are independent of each other, and n≥1, b≥1, a, c can be any value of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23.

[0011] (Arg)n-Xa is a hydrophilic end, rich in basic amino acids or polycationic polypeptide sequences, in some embodiments n≥1, preferably n≥3;

[0012] Yb is a hydrophobic part, Y represents a natural or non-natural hydrophobic amino acid sequence, in some embodiments, Yb is selected from one or more of Trp (W), Phe (F), Pro (P), Tyr (Y), which may be discontinuous or continuous, and b is the number of hydrophobic amino acids;

[0013] X is any one or more natural or non-natural, hydrophilic or hydrophobic, polar or non-polar, ionized or non-ionized amino acids other than Trp (W), Phe (F), Pro (P), and Tyr (Y), and they may be discontinuous or continuous. For example, in some embodiments, Xa is C, in some embodiments, Xa is K, and in some embodiments, Xa is KRRRR; in some embodiments, Xc is CGCGKR, and in some embodiments, Xc is KR.

[0014] The carbon terminus of Cys can be -COOH or -CONH2, but its -SH side chain is free;

[0015] In some preferred but non-limiting embodiments, the polypeptide of Formula I is selected from the following peptide sequences: those peptide sequences described in Table 1 or variants thereof that retain at least a portion of their ability to enhance transfection efficiency. In some embodiments, the peptide sequence of the polypeptide of Formula I is between 6 and about 40 amino acids, and the polypeptide of Formula I is characterized in that it improves the delivery efficiency of biologically active molecules into cells by at least 50% or more.

[0016] In some embodiments, the polypeptide sequence of Formula I is between 6 and about 40 amino acids, and the polypeptide of Formula I is characterized in that it improves the delivery of a biologically active molecule into a cell by at least 75% or greater.

[0017] In some embodiments, the polypeptide of Formula I is at least 75% identical to any of the peptides described in Table 1 or a variant thereof that retains at least a portion of its ability to enhance transfection efficiency, wherein the polypeptide of Formula I is characterized in that it improves the delivery of biologically active molecules into cells by at least 90% or greater. In some embodiments, the polypeptide of Formula I comprises SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ NO.13, SEQ ID NO.14, SEQ ID NO.15 or SEQ ID NO.16.

[0018] In some embodiments, the polypeptide of Formula I is selected from any one of the non-naturally occurring polypeptides listed in Table 1.

[0019] In another aspect, the present invention provides a polypeptide nanoparticle composition comprising a non-naturally occurring polypeptide represented by general formula (I) and one or more PEGylated polymer materials.

[0020] In some embodiments, the molecular weight of the PEGylated polymer material is 1000 Da or above, and the PEGylated polymer material is an amphiphilic polymer compound, which can improve the pharmacokinetic properties of nanoparticles in vivo, stabilize nanoparticles, and facilitate targeted modification.

[0021] In some embodiments, the PEGylated polymer material is a PEG derivative, poloxamer and / or poloxamine or a poloxamine derivative conjugated to a lipid.

[0022] In some embodiments, the poloxamine derivative can be selected from any terminally functionalized poloxamine derivative described in patent CN111285845B.

[0023] In some embodiments, the poloxamine derivative has the following structural formula:

[0024] (T304-T) where x is 4.3, y is 3.8,

[0025] (T304-D) where x is 4.3, y is 3.8,

[0026] (T304-RT) where x is 4.3, y is 3.8,

[0027] (T304-RC) where x is 4.3, y is 3.8,

[0028] (T701-R) where x is 14, y is 2.1,

[0029] (T901-C) where x is 18.2, y is 2.7,

[0030] (T803-RT) where x is 15, y is 11,

[0031] (T904-CR) where x is 17, y is 15,

[0032] (T904-RC) In the formula: x is 17, y is 15,

[0033] (T904-RT) where x is 17, y is 15,

[0034] (T90R4-R) where x is 17, y is 19,

[0035] (T90R4-RT) where x is 17, y is 19,

[0036] (T704-M) where x is 14, y is 13,

[0037] (T704-RT) where x is 14, y is 13,

[0038] (T704-RC) In the formula: x is 14 and y is 13.

[0039] The polypeptide nanoparticle composition can deliver bioactive molecules to the interior of the cell, and the bioactive molecules include nucleic acid molecules, such as DNA molecules or RNA molecules. Suitable DNA molecules can include DNA molecules with expressible nucleic acid sequences, such as expression vectors or cDNA molecules that include an open reading frame encoding protein. Suitable bioactive molecules that can be used as in the practice of the present invention include RNA molecules, such as mRNA molecules or RNAi molecules.

[0040] Other embodiments of the present invention are directed to methods for preparing polypeptide nanoparticle compositions and methods for delivering biologically active molecules to the interior of cells using the polypeptide nanoparticle compositions. The method for preparing polypeptide nanoparticle compositions may include contacting the biologically active molecule with at least one PEGylated polymer material and the non-naturally occurring polypeptide of Formula I of the present invention, optionally in the presence of one or more lipids, under conditions that promote the formation of a polypeptide nanoparticle composition capable of delivering the biologically active molecule to the interior of a cell.

[0041] Other embodiments of the present invention are directed to methods for transfecting cells, comprising forming a polypeptide nanoparticle composition comprising at least one biologically active molecule, at least one PEGylated polymeric material, and a non-naturally occurring polypeptide of Formula I of the present invention, optionally with one or more lipids, and contacting the polypeptide nanoparticle composition with cells under conditions that promote transfection of the cells. Other embodiments of the present invention are directed to biologically active molecule preparations, which optionally comprise biologically active molecules to be delivered to animals or human subjects, PEGylated polymeric materials, and non-naturally occurring polypeptides of Formula I of the present invention in the presence of one or more polymeric materials and / or one or more lipids, to form a pharmaceutically active complex suitable for delivering biologically active molecules to animals or human subjects in need thereof for the treatment of physiological conditions or disorders.

[0042] Other embodiments of the present invention disclose a transfection complex comprising a polypeptide nanoparticle composition and a nucleic acid.

[0043] Other embodiments of the present invention disclose a nucleic acid vaccine composition comprising a polypeptide nanoparticle composition, a nucleic acid

[0044] The nucleic acid vaccine composition is used to prevent or treat the following:

[0045] (i) infectious diseases;

[0046] (ii) allergic reactions or allergic diseases;

[0047] (iii) autoimmune disease; or

[0048] (iv) cancer or neoplastic disease.

[0049] Beneficial Effects

[0050] Combining the design concept of membrane-penetrating peptides, a polypeptide nanoparticle composition was designed using polymer materials. The combination of polymer materials and new membrane-penetrating peptides promotes the entry of mRNA-loaded nanoparticles into cells, thereby achieving effective delivery of mRNA by nanoparticles. DETAILED DESCRIPTION

[0051] In order to enable those skilled in the art to better understand the technical solution of the present invention, some non-limiting embodiments are further disclosed below to further illustrate the present invention in detail.

[0052] The present invention provides reagents and compositions suitable for cell transfection. Specifically, the present invention provides compositions and reagents that enhance the transfection efficiency of all cells (including those cell types that are considered to be generally difficult to transfect). The compositions and reagents of the present invention can generally increase their transfection efficiency by up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, up to 95%, up to 100% or more when used according to the methods described herein and with the general knowledge and expertise within the knowledge of ordinary technicians in the art. The present invention achieves this goal by providing a polypeptide nanoparticle composition as described in more detail below.

[0053] In some embodiments, the polypeptide nanoparticle composition of the present invention comprises at least one non-naturally occurring polypeptide having the following general structure:

[0054] (Arg)n-Xa-Yb-Xc-Cys (Formula I)

[0055] Wherein: n+a+b+c=5-39, n, a, b or c are independent of each other, and n≥1, b≥1, a, c can be any value of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23.

[0056] (Arg)n-Xa is a hydrophilic end, rich in basic amino acids or polycationic polypeptide sequences, in some embodiments n≥1, in some embodiments, n≥3;

[0057] In some embodiments, X is any one or more natural or non-natural, hydrophilic or hydrophobic, polar or non-polar, ionized or non-ionized amino acids other than Trp (W), Phe (F), Pro (P) or Tyr (Y). In some embodiments, X is selected from one or more of Arg (R), Lys (K), Cys (C), Gly (G), Val (V), Leu (L), Gln (Q) or His (H), which may be discontinuous or continuous. For example, in some embodiments, Xa is Cys (C), in other embodiments, Xa is Lys (K), in some embodiments, Xa is KRRRR; in some embodiments, Xc is CGCGKR, in some embodiments, Xc is KR.

[0058] In some embodiments, Yb is a hydrophobic part, and Y represents a natural or non-natural hydrophobic amino acid sequence. In some embodiments, Yb is selected from one or more of Trp (W), Phe (F), Pro (P) or Tyr (Y), and they may be discontinuous or continuous; for example, Xa or Xc may be interspersed between Trp (W), Phe (F), Pro (P) or Tyr (Y), or Trp (W), Phe (F), Pro (P) or Tyr (Y) may be repeatedly arranged continuously in the polypeptide.

[0059] In some embodiments, the content of Arg (R) in the non-natural polypeptide is at least 25%, or at least 30%, or at least 35%, and the content of Cys (C) in the non-natural polypeptide is at least 5.0%, or at least 7.0%, or at least 10.0%. In some embodiments, the carbon terminus of Cys in (Arg) n-Xa-Yb-Xc-Cys (general formula I) can be -COOH or -CONH2, but its -SH side chain is free; any amino acid therein, and any known chemically modified derivatives of these amino acids can also be used, provided that these derivatives are non-toxic to cells or organisms when provided with the amino acids listed above (the amino acid derivatives are distributed by different companies; see, for example, Sigma Aldrich (see http: / / www.sigmaaldrich.com) ).

[0060] (Arg)n-Xa-Yb-Xc-Cys (Formula I) begins with Arg and ends with Cys. Xa-Yb-Xc does not define any specific order of amino acids, but is intended to reflect the types of amino acids and their frequencies of occurrence in the polypeptide. (Arg)n When n is greater than 2, multiple Args may be continuous or discontinuous, such as Args separated by one or more Xa. In some embodiments, the values ​​a and c in Xa or Xc are not 0 at the same time.

[0061] In some non-limiting embodiments, the polypeptide of Formula I can be between about 6 and about 40 amino acids, between about 6 and about 35 amino acids, between about 6 and about 30 amino acids, between about 6 and about 20 amino acids, or between about 6 and about 15 amino acids, between about 8 and about 50 amino acids, between about 10 and about 30 amino acids, between about 10 and about 28 amino acids, between about 10 and about 28 amino acids, between about 10 and about 25 amino acids, between about 10 and about 20 amino acids, or between about 10 and about 15 amino acids, and wherein the polypeptide of Formula I is characterized in that the non-natural The presence of the non-naturally occurring peptide as a component of a transfection complex enhances the transfection efficiency of the transfection complex by up to 10%, up to 15%, 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, up to 95%, up to 100%, up to 150%, up to 200%, up to 250%, up to 300%, up to 350%, up to 400%, up to 500%, or more than 500% relative to the transfection efficiency of a transfection complex lacking the non-naturally occurring peptide.

[0062] In some non-limiting embodiments, the polypeptide of Formula I is a peptide comprising an amino acid sequence selected from the group consisting of:

[0063] RKRRRRYWCPKCRGGRC(SEQ ID NO:1);

[0064] RKRRRRYWCPLGRC(SEQ ID NO:2);

[0065] RKRYRRRFHKRGCGRC(SEQ ID NO:3);

[0066] RRRRRCPWKRGCGRC(SEQ ID NO:4);

[0067] RKRRRRCYPKRGCGRC(SEQ ID NO:5);

[0068] RRRRWFCGKCCGRC(SEQ ID NO:6);

[0069] RRRRRWCRGCGRC (SEQ ID NO: 7);

[0070] RRRRWFRCLGLPC (SEQ ID NO: 8);

[0071] RRRRWFRCLGTLC (SEQ ID NO: 9);

[0072] RRRRKFRGSSGRC(SEQ ID NO:10);

[0073] RKRRKWRGCGRC (SEQ ID NO: 11);

[0074] RRFKRC (SEQ ID NO: 12);

[0075] RRRRRRRRRRWFRCHKCKCVRRCKLKRC(SEQ ID NO:13);

[0076] RRRRHFCPQKKKRC(SEQ ID NO:14);

[0077] RRRRHFCKRLKRC(SEQ ID NO:15);

[0078] RRRRWFRCKLKRC (SEQ ID NO: 16).

[0079] In some embodiments, the polypeptide of Formula I is SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ NO.13, SEQ ID NO.14, SEQ ID NO.15 or SEQ ID Any of NO.16 has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence similarity and retains at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, at least 155%, greater than 100%, at most 115%, at most 120%, at most 130%, at most 140%, at most 150%, at most 160%, at most 170%, at most 180% variants of its function of enhancing delivery of biologically active molecules to the interior of cells.

[0080] Table 1 sets forth various peptide sequences that can be used to practice the present invention, but it will be understood by those of ordinary skill in the art that the list of peptide sequences in Table 1 is provided by way of example only and is not intended to limit the scope of the present invention to only those sequences explicitly written out. Rather, it will be readily apparent to such persons that, based on the teachings set forth above regarding the polypeptides described by Formula I, there may be a large number of peptides that are potentially suitable for practicing the present invention set forth herein. Furthermore, it is well within the purview of skilled artisans to determine whether a given peptide sequence falls within the scope of the present invention without undue experimentation using standard techniques in the art. Furthermore, it will be understood that various variants of the peptide sequences appearing in Table 1 also fall within the scope of the present invention, provided that such variants satisfy the structural and functional characteristics set forth above. Variants of the peptide sequences appearing in Table 1 or variants of any other candidate peptides that are not explicitly described in Table 1 but that satisfy the structural and functional characteristics set forth above may include deletions, insertions, substitutions with naturally occurring or non-proteinogenic amino acids.

[0081] Table 1: Polypeptides represented by general formula I

[0082]

[0083]

[0084] In another aspect, the present invention provides a polypeptide nanoparticle composition comprising a non-naturally occurring polypeptide represented by general formula (I) and one or more PEGylated polymer materials.

[0085] In some embodiments, the molecular weight of the PEGylated polymer material is 1000Da or more, and the PEGylated polymer material is an amphiphilic polymer compound, which is beneficial to improving the pharmacokinetic properties of nanoparticles in vivo, stabilizing nanoparticles and facilitating targeted modification. The PEGylated polymer material is preferably a PEG derivative, poloxamer and / or poloxamine or poloxamine derivative conjugated to a lipid.

[0086] In some embodiments, the PEG derivative conjugated to lipids can be mPEG-C-DMG, mPEG-C-CLS, DSPE-PEG-Mal, DSPE-PEG-NH2, DOPE-PEG-NH2, mPEG-DSPE, mPEG-DPPE, mPEG-DMPE. In some embodiments, the PEG derivative conjugated to lipids can be mPEG 2000 -C-DMG, mPEG 10000 -C-CLS and DSPE-PEG 2000 -Mal, DSPE-PEG 5000 -NH2、DOPE-PEG 2000 -NH2、mPEG5000 -DSPE, mPEG 2000 -DPPE, mPEG 2000 -DMPE, its structural formula is shown below:

[0087]

[0088] In some embodiments, the poloxamine is a polymer of 1,2-ethylenediaminetetraethanol with ethylene oxide and methyl ethylene oxide or a polymer of 1,2-ethylenediaminetetraisopropanol with methyl ethylene oxide and ethylene oxide. In some embodiments, the poloxamine is selected from 304 (abbreviated as T304), 701 (abbreviated as T701), 704 (abbreviated as T704), 707 (abbreviated as T707), 803 (abbreviated as T803), 901 (abbreviated as T901), 904 (abbreviated as T904), 908 (abbreviated as T908), 1107 (abbreviated as T1107), 1301 (abbreviated as T1301), 1304 (abbreviated as T1304), 1307 (abbreviated as T1307), 90R4 (abbreviated as T90R4) or 150R1 (abbreviated as T150R1) or more; the poloxamine structural formula is as shown in formula (II) or formula (III):

[0089]

[0090] In some embodiments, the poloxamer is a poly(propylene glycol)-poly(ethylene glycol)-poly(propylene glycol) copolymer or a poly(ethylene glycol)-poly(propylene glycol)-poly(ethylene glycol) copolymer, and the structural formula of the poloxamer is shown in Formula (IV) or Formula (VI):

[0091]

[0092] In some embodiments, the poloxamer is selected from 17R4 (abbreviated as 17R4), P-31R1 (abbreviated as 31R1), L-121 (abbreviated as L121), L-31 (abbreviated as L31), L-64 (abbreviated as L64), L-81 (abbreviated as L81), P-10R5 (abbreviated as 10R5), L-35 (abbreviated as L35), L-61 (abbreviated as L61), P-123 (abbreviated as P123), F127 (abbreviated as F127) or One or more of P-85 (abbreviated as P85).

[0093] On the one hand, the polymer material polypeptide nanoparticle composition of the present invention may further comprise a natural or synthetic lipid, and the lipid may be selected from the following: one or more of PC, DMPC, DPPC, DSPC, DMPE, DOTAP, DOPE, DOTMA, DOPC, DPPE or CLS (cholesterol). In some embodiments, the lipid is PC; wherein the structural formula of PC, DMPC, DPPC, DSPC, DMPE, DOTAP, DOPE, DOTMA, DOPC, DPPE or CLS is shown below:

[0094]

[0095]

[0096] The compositions of various lipid formulations according to several non-limiting embodiments of the present invention are provided in Table 2. Providing these exemplary embodiments is in no way intended to limit the scope of the present invention to only those formulations disclosed. On the contrary, it is intended only to provide a variety of possible lipid aggregate formulations that can be used to practice the present invention. Nevertheless, it will be readily apparent to those skilled in the art that the formulations can be varied or altered, and other components (such as, for example, other cationic or neutral lipids, peptide targeting moieties, etc.) can be added, or one of the high molecular lipids set forth in Table 2 can be optionally removed, and the resulting formulations will be within the spirit and scope of the present invention as described herein.

[0097] Table 2: Polymer materials and lipid codes involved in the embodiments of the present invention

[0098] Material Material Code Material Type Material Material Code Material Type T304 TA Polymer Materials 17R4 PA Polymer Materials T701 TB Polymer Materials L64 PB Polymer Materials T704 TC Polymer Materials P123 PD Polymer Materials T707 TD Polymer Materials F127 PE Polymer Materials T803 TE Polymer Materials P85 PF Polymer Materials T901 TF Polymer Materials 31R1 PG Polymer Materials T904 TG Polymer Materials L121 PH Polymer Materials T908 TH Polymer Materials L31 PI Polymer Materials T1107 TI Polymer Materials L81 PJ Polymer Materials T1301 TJ Polymer Materials 10R5 PK Polymer Materials T1304 TK Polymer Materials L35 PL Polymer Materials T1307 TL Polymer Materials L61 PM Polymer Materials T90R4 TM Polymer Materials PC LA Lipids T150R1 TN Polymer Materials DMPC LB Lipids T304-T T304-T Polymer Materials DPPC LC Lipids T304-D T304-D Polymer Materials DSPC LD Lipids T304-RT T304-RT Polymer Materials DMPE LE Lipids T304-RC T304-RC Polymer Materials DOTAP LF Lipids T701-R T701-R Polymer Materials DOPE LG Lipids T901-C T901-C Polymer Materials DOTMA LH Lipids T803-RT T803-RT Polymer Materials DOPC LK Lipids T904-CR T904-CR Polymer Materials DPPE LL Lipids T904-RC T904-RC Polymer Materials CLS LM Lipids T904-RT T904-RT Polymer Materials mPEG2000-C-DMG EA Polymer Materials T90R4-R T90R4-R Polymer Materials mPEG10000-C-CLS EB Polymer Materials T90R4-RT T90R4-RT Polymer Materials DSPE-PEG2000-Mal EC Polymer Materials T704-M T704-M Polymer Materials DSPE-PEG5000-NH2 ED Polymer Materials T704-RT T704-RT Polymer Materials DOPE-PEG2000-NH2 EE Polymer Materials T704-RC T704-RC Polymer Materials mPEG5000-DSPE EF Polymer Materials mPEG2000-DMPE EH Polymer Materials mPEG2000-DPPE EG Polymer Materials

[0099] The polypeptide nanoparticle composition prepared by the present invention has an average diameter of 10 to 500 nm, in some embodiments, an average diameter of 10 to 500 nm, 20 to 400 nm, 30 to 300 nm, 40 to 200 nm. In some embodiments, the nanoparticles (e.g., polypeptide complex nanoparticles) have an average diameter of 50 to 150 nm, 50 to 200 nm, 80 to 100 nm, or 80 to 200 nm.

[0100] The molar ratio of the polypeptide, PEGylated polymer material and lipid in the polypeptide nanoparticle composition is 1:(8-300):(0-15). In some embodiments, the molar ratio of the polypeptide, PEGylated polymer material and lipid is 1:(120-190):(1-12). In some embodiments, the molar ratio of the polypeptide, PEGylated polymer material and lipid is 1:124:(1-6).

[0101] The polypeptide nanoparticle composition as described above is used to deliver biologically active macromolecules into cells, and the efficiency of transfection of the biologically active macromolecule by the polypeptide nanoparticle composition is enhanced by up to 10%, up to 15%, 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, up to 95%, up to 100%, up to 150%, up to 200%, up to 250%, up to 300%, up to 350%, up to 400%, up to 500% or more than 500% relative to the transfection efficiency of the transfection complex lacking the non-naturally occurring polypeptide represented by general formula (I).

[0102] In another aspect, the present invention provides a transfection complex, which may include a non-naturally occurring peptide as described above, at least one bioactive molecule, and at least one PEGylated polymer material. In some embodiments, the transfection complex includes a non-naturally occurring peptide, at least one bioactive molecule, at least one PEGylated polymer material, and at least one lipid. The transfection complex of the present invention is stable in an aqueous solution and can be contacted with a cell tissue of a human or animal immediately after formation, and the transfection complex is stable and can be stored for a period of time before contacting the cell or tissue, such as being stored for at least 30 minutes, at least 45 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 10 hours, at least 15 hours, at least 20 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 5 days, at least 7 days, at least 14 days, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year. It will be appreciated that the storage period may be between any of these time periods, for example between 31 minutes and 1 hour or between 1 hour and 24 hours.

[0103] In some embodiments, the bioactive molecule can be any substance to be delivered to the interior of a cell in a culture in a laboratory or in an animal or human tissue, depending on the application. In some embodiments, the bioactive molecule can be a nucleic acid, or can be a drug or other organic small molecule. In some embodiments, the preferred biomacromolecules for forming the transfection complex are nucleic acids, such as, for example, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). In some embodiments, the preferred bioactive molecule can be a DNA molecule. DNA can be linear DNA or circular DNA, such as DNA in the form of a circular plasmid, a free gene, or an expression vector. In other embodiments, the preferred bioactive molecule can be an RNA molecule. The RNA molecule can be any type of RNA molecule (but not limited to this), including (but not limited to) mRNA, siRNA, miRNA, antisense RNA, ribonuclease, or any other type or type of RNA molecule familiar to a person skilled in the art (but not limited to this), which will need to be delivered to the interior of the cell, in some embodiments, the preferred bioactive molecule can be mRNA.

[0104] At least one RNA (molecule) of the RNA can have any length (preferably depending on the type of RNA to be used as a transfection complex according to the present invention). Without being limited thereto, the at least one RNA (molecule) can have a length of 5-20,000 nucleotides, more preferably a length of 5-10,000 or 300-10,000 nucleotides, even more preferably a length of 5-5,000 nucleotides, and most preferably a length of 20-5,000, 50-5,000, 100-5,000 or 300-10,000 nucleotides, depending on the type of RNA to be transfected.

[0105] At least one RNA (molecule) of the transfection complex of the present invention can be any RNA, preferably but not limited to, short RNA oligonucleotides (preferably 5-80 or, more preferably 20-80 nucleotides in length), coding RNA, immunostimulatory RNA, siRNA, antisense RNA, or riboswitches, ribozymes or aptamers. In addition, at least one RNA (molecule) of the transfection complex of the present invention can be single-stranded or double-stranded RNA (which can also be considered as RNA (molecule), which is attributed to the non-covalent binding of two single-stranded RNA (molecules)) or partially double-stranded RNA (which is typically formed by longer and shorter single-stranded RNA molecules or by 2 single-stranded RNA molecules of substantially equal length, one of which is partially complementary to the other single-stranded RNA molecule and the two thereby form a double-stranded RNA molecule in the region). Preferably, at least one RNA (molecule) of the transfection complex of the present invention can be single-stranded RNA. At least one RNA (molecule) of the transfection complex of the present invention can also be circular or linear RNA, preferably linear RNA. More preferably, at least one RNA (molecule) of the transfection complex of the present invention can be (linear) single-stranded RNA. At least one RNA (molecule) of the transfection complex of the present invention may be a ribosomal RNA (rRNA), a transfer RNA (tRNA), a messenger RNA (mRNA), or a viral RNA (vRNA), preferably an mRNA. The present invention allows all of these RNAs to be transfected into cells. In this context, an mRNA is typically an RNA that consists of several structural components, such as an optional 5'-UTR region, a ribosome binding site located upstream and a subsequent coding region, an optional 3'-UTR region, which may be followed by a poly-A tail (and / or a poly-C tail). The mRNA may function as a single, double or even multicistronic RNA, i.e., an RNA that carries the coding sequence of one, two or more proteins. Such coding sequences in a two or even multicistronic mRNA may be separated by at least one IRES sequence, for example as defined above.

[0106] Short RNA Oligonucleotides

[0107] In a first embodiment, at least one RNA (molecule) of the transfection complex of the present invention may be a short RNA oligonucleotide. The short RNA oligonucleotide in the context of the present invention may include any RNA as defined above. Preferably, the short RNA oligonucleotide may be a single-stranded or double-stranded RNA oligonucleotide, more preferably a single-stranded RNA oligonucleotide. Even more preferably, the short RNA oligonucleotide may be a linear single-stranded RNA oligonucleotide.

[0108] Preferably, short RNA oligonucleotides as used herein comprise a length as generally defined above for RNA molecules, more preferably a length of 5-100, 5-50, or 5-30 nucleotides, or, alternatively, 20-100, 20-80, or, even more preferably, a length of 20-60 nucleotides. Short RNA oligonucleotides can be used for a variety of purposes, for example for (non-specific) immunostimulation, or to reduce / inhibit transcription / translation of a gene.

[0109] Coding RNA

[0110] In a second embodiment, at least one RNA (molecule) of the transfection complex of the present invention may be a coding RNA. The coding RNA of the transfection complex of the present invention may be any RNA as defined above. Preferably, the coding RNA may be a single-stranded or double-stranded RNA, more preferably a single-stranded RNA, and / or a circular or linear RNA, more preferably a linear RNA. Even more preferably, the coding RNA may be a (linear) single-stranded RNA. Most preferably, the coding RNA may be a ((linear) single-stranded) messenger RNA (mRNA).

[0111] The coding RNA may also encode a protein or peptide, which may be selected from, but not limited to, for example, therapeutically active proteins or peptides, tumor antigens, antibodies, immunostimulatory proteins or peptides, etc., or any other protein or peptide suitable for specific (therapeutic) applications, where it is necessary to deliver at least one RNA (molecule) encoding the protein into a cell, tissue or organism and to subsequently express the protein in the cell, tissue or organism.

[0112] The polypeptide nanoparticle composition provided by the present invention has an encapsulation rate of RNA, especially mRNA, greater than 80%, or greater than 85%, or greater than 90%, or greater than 95%, such as between 84% and 99%. The polypeptide nanoparticle composition of the present invention has a high transfection efficiency for nucleic acid, especially mRNA, so that the delivery efficiency of nucleic acid into cells is improved by greater than 50%, greater than 100% or about 200%. Sequence Listing <110> Shenzhen Houcun Nano-Pharmaceutical Co., Ltd. <120> Peptide nanoparticle composition <130> PCT001 <141> 2021-06-25 <160> 16 <170> SIPOSequenceListing 1.0 <210> 1 <211> 17 <212> PRT <213> Artificial Sequence <400> 1 Arg Lys Arg Arg Arg Arg Tyr Trp Cys Pro Lys Cys Arg Gly Gly Arg 1 5 10 15 Cys <210> 2 <211> 14 <212> PRT <213> Artificial Sequence <400> 2 Arg Lys Arg Arg Arg Arg Tyr Trp Cys Pro Leu Gly Arg Cys 1 5 10 <210> 3 <211> 16 <212> PRT <213> Artificial Sequence <400> 3 Arg Lys Arg Tyr Arg Arg Arg Phe His Lys Arg Gly Cys Gly Arg Cys 1 5 10 15 <210> 4 <211> 15 <212> PRT <213> Artificial Sequence <400> 4 Arg Arg Arg Arg Arg Cys Pro Trp Lys Arg Gly Cys Gly Arg Cys 1 5 10 15 <210> 5 <211> 16 <212> PRT <213> Artificial Sequence <400> 5 Arg Lys Arg Arg Arg Arg Cys Tyr Pro Lys Arg Gly Cys Gly Arg Cys 1 5 10 15 <210> 6 <211> 14 <212> PRT <213> Artificial Sequence <400> 6 Arg Arg Arg Arg Trp Phe Cys Gly Lys Cys Cys Gly Arg Cys 1 5 10 <210> 7 <211> 13 <212> PRT <213> Artificial Sequence <400> 7 Arg Arg Arg Arg Arg Trp Cys Arg Gly Cys Gly Arg Cys 1 5 10 <210> 8 <211> 13 <212> PRT <213> Artificial Sequence <400> 8 Arg Arg Arg Arg Trp Phe Arg Cys Leu Gly Leu Pro Cys 1 5 10 <210> 9 <211> 13 <212> PRT <213> Artificial Sequence <400> 9 Arg Arg Arg Arg Trp Phe Arg Cys Leu Gly Thr Leu Cys 1 5 10 <210> 10 <211> 13 <212> PRT <213> Artificial Sequence <400> 10 Arg Arg Arg Arg Lys Phe Arg Gly Ser Ser Gly Arg Cys 1 5 10 <210> 11 <211> 12 <212> PRT <213> Artificial Sequence <400> 11 Arg Lys Arg Arg Lys Trp Arg Gly Cys Gly Arg Cys 1 5 10 <210> 12 <211> 6 <212> PRT <213> Artificial Sequence <400> 12 Arg Arg Phe Lys Arg Cys 1 5 <210> 13 <211> 28 <212> PRT <213> Artificial Sequence <400> 13 Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Trp Phe Arg Cys His Lys 1 5 10 15 Cys Lys Cys Val Arg Arg Cys Lys Leu Lys Arg Cys 20 25 <210> 14 <211> 14 <212> PRT <213> Artificial Sequence <400> 14 Arg Arg Arg Arg His Phe Cys Pro Gln Lys Lys Lys Arg Cys 1 5 10 <210> 15 <211> 13 <212> PRT <213> Artificial Sequence <400> 15 Arg Arg Arg Arg His Phe Cys Lys Arg Leu Lys Arg Cys 1 5 10 <210> 16 <211> 13 <212> PRT <213> Artificial Sequence <400> 16 Arg Arg Arg Arg Trp Phe Arg Cys Lys Leu Lys Arg Cys 1 5 10 BRIEF DESCRIPTION OF THE DRAWINGS

[0113] Figure 1 . The nanoparticle morphology of the nanoparticles was tested by transmission electron microscopy after the FLuc-mRNA of the present invention was mixed with complexes of different prescriptions.

[0114] Figure 2 .Shows the transfection fluorescence expression intensity of the FLuc-mRNA of the present invention after mixing with complexes of different prescriptions in DC2.4 cells in vitro.

[0115] Figure 3 .Shows the transfection fluorescence expression intensity of the FLuc-mRNA of the present invention after mixing with complexes of different prescriptions in DC2.4 cells in vitro.

[0116] Figure 4 The figure shows the transfection fluorescence expression intensity of the FLuc-mRNA of the present invention after mixing with complexes of different prescriptions in DC2.4 cells in vitro.

[0117] Figure 5 .Shows the cell viability of DC2.4 cells transfected in vitro after the FLuc-mRNA of the present invention was mixed with complexes of different formulations.

[0118] Figure 6 .Shows the transfection percentage of different cells in vitro after the EGFP-mRNA of the present invention is mixed with the prescription complex.

[0119] Figure 7 .Shows the transfection fluorescence expression intensity in different cells in vitro after the EGFP-mRNA of the embodiment of the present invention is mixed with the prescription complex.

[0120] Figure 8 .Shows the immune effect of the S-mRNA complex polypeptide nanoparticles in mice according to the embodiment of the present invention.

[0121] Definition of terms

[0122] The terms used throughout this specification generally have their general meaning in the art in the context of the present invention and in the specific circumstances in which each term is used. Certain terms are discussed below or elsewhere in the specification to provide practitioners with other guidance on describing various embodiments of the present invention and how to form and use the embodiments. It should be understood that the same concept can be expressed in more than one way. Therefore, alternative language and synonyms can be used for any one or more of the terms discussed herein, regardless of whether a term is described or discussed herein, and no specific meaning is given. Synonyms for certain terms can be provided. Narrating one or more synonyms does not exclude the use of other synonyms. The use of examples (including examples of any term discussed herein) anywhere in this specification is only illustrative, and in no way limits the scope and meaning of the present invention or any exemplified term.

[0123] The terms "hydrophilic" and "hydrophilicity" are used interchangeably herein and generally refer to the physical property of molecules with polar groups that have a strong affinity for water, can attract water molecules, or dissolve in water, and can form temporary bonds with water through hydrogen bonds.

[0124] The term "hydrophobic" is also called "hydrophobicity". Hydrophobic molecules tend to be non-polar and are therefore more soluble in neutral and non-polar solutions (such as organic solvents). Hydrophobic molecules usually clump together in water, and water forms a large contact angle on the surface of a hydrophobic solution and forms a droplet shape. The hydrophobic amino acid described herein refers to an amino acid with hydrophobicity, which can be a hydrophobic natural amino acid or a hydrophobic non-natural amino acid.

[0125] The term "natural amino acid" as used herein refers to any of the 22 "standard" amino acids naturally incorporated into peptides. Of these 22, 20 are directly encoded by the general genetic code. The remaining two (selenocysteine ​​and pyrrolysine) are incorporated into proteins through unique synthetic mechanisms. Typically, the amino acid residues of the peptides of the present invention exist as L-isomers. In some embodiments, one or more amino acid residues of the peptides of the present invention exist as D-isomers.

[0126] The term "non-natural amino acid" refers to an amino acid synthesized by artificial methods; the term "modified amino acid residue" as used herein refers to a non-standard amino acid, such as an amino acid that is post-translationally modified. Examples of post-translational modifications include phosphorylation, glycosylation, acylation (e.g., acetylation, myristoylation, palmitoylation), alkylation, carboxylation, hydroxylation, glycation, biotinylation, ubiquitination, chemical property changes (e.g., β-elimination deimidation, deamidation) and structural changes (e.g., disulfide bond formation), among others.

[0127] The term "polar or nonpolar" refers to the uneven distribution of charge in a covalent bond or a covalent molecule. If the charge is unevenly distributed, the bond or molecule is called polar; if it is evenly distributed, it is called nonpolar.

[0128] The term "ionization or non-ionization" Ionization, also known as ionization, refers to the process in which atoms and molecules form ions under the action of (physical) energy. It means that atoms or molecules gain or lose electrons to form ions, usually in combination with other chemical changes.

[0129] The term "PEGylated" PEG is polyethylene glycol, and PEGylation refers to coupling activated PEG to liposomes by chemical methods.

[0130] The term "PEGylated polymer material" refers to a PEGylated lipid with a molecular weight of more than 1000 Da.

[0131] The term "introducing" when used in the context of introducing a biologically active molecule into a cell culture refers to providing the macromolecule or compound into the culture medium, with the understanding that the goal of introducing the macromolecule is to enable the macromolecule to transfer from the extracellular compartment to the cytoplasmic compartment of the cultured cells.

[0132] The term "introducing" a biologically active molecule into at least one cell refers to providing a macromolecule or compound to a cell so that the macromolecule or compound becomes internalized in the cell. For example, the macromolecule or compound can be introduced into the cell using transfection, transformation, injection and / or liposome introduction, and can also be introduced into the cell using other methods known to those of ordinary skill in the art. Preferably, the macromolecule or compound is introduced into the cell by liposome introduction. The macromolecule is preferably a protein, peptide, polypeptide or nucleic acid. The macromolecule can be a protein. Alternatively, the macromolecule can be a peptide. Alternatively, the macromolecule can be a polypeptide. The macromolecule can also be a nucleic acid.

[0133] The term "drug" when used herein in the context of delivering a drug to the interior of a cell (e.g., by means of transfection) generally refers to any substance to be delivered to the interior of a cell (in laboratory culture or in animal or human tissue). Depending on the application, the drug may be a macromolecule, such as a nucleic acid, protein, or peptide, or may be a drug or other small organic molecule.

[0134] As used herein, the term "biologically active molecule" encompasses biomolecules. In one embodiment, the term biologically active molecule refers to nucleic acid. In a preferred embodiment, the term macromolecule refers to deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). In some embodiments, the term macromolecule refers to DNA. DNA can be linear DNA or circular DNA, such as DNA in the form of a circular plasmid, a free gene or an expression vector. In certain preferred but non-limiting embodiments, the term macromolecule refers to a complementary DNA (cDNA) with an expressible nucleic acid sequence, the expressible nucleic acid sequence including at least one open reading frame operably connected to one or more nucleic acid sequences required for transcription of mRNA by an expressible nucleic acid sequence. Macromolecules may be charged or uncharged. DNA molecules are an example of charged macromolecules. In some cases, as used herein, the term "macromolecule" can be used interchangeably with the terms "expressible nucleic acid" and "expression vector". In other embodiments, the term "macromolecule" refers to an RNA molecule. The RNA molecule can be any type of RNA molecule, including but not limited to mRNA, siRNA, miRNA, antisense RNA, ribonuclease, or any other type or class of RNA molecule familiar to those skilled in the art, which needs to be delivered to the interior of the cell.

[0135] The term "transfection" is used herein to mean the delivery of a nucleic acid, protein or other macromolecule to a target cell so that the nucleic acid, protein or other macromolecule is expressed or has a biological function in the cell.

[0136] As used herein, the term "expressible nucleic acid" includes both DNA and RNA regardless of molecular weight, and the term "expression" means any manifestation of the functional presence of nucleic acid within a cell, including (but not limited to) both transient expression and stable expression. Functional aspects include inhibition of expression by oligonucleotide or protein delivery.

[0137] The term "expression of nucleic acid" and equivalents thereof refers to nucleic acid replication in a cell, transcription of DNA into messenger RNA, translation of RNA into protein, post-translational modification of protein and / or protein transport in a cell, or variations or combinations thereof.

[0138] As used herein, the term "cell" refers to eukaryotic and prokaryotic cells of all types. In a preferred embodiment, the cell refers to a eukaryotic cell, especially a cell grown in culture, or a cell found in an animal or human tissue. In a preferred embodiment, the cell refers to a mammalian cell. In certain exemplary but non-limiting embodiments, the term "cell" is intended to refer to any cell and cell line routinely used in research and clinical settings, and may include, but is not limited to, immortalized cell lines, transformed cell lines, or primary cells.

[0139] "Cell culture" or "culturing" means maintaining cells in an artificial in vitro environment.

[0140] "Recombinant protein" refers to a protein encoded by a nucleic acid introduced into a host cell. The host cell expresses the nucleic acid. The term "expressing a nucleic acid" is synonymous with "expressing a protein from RNA encoded by a nucleic acid." As used herein, "protein" generally refers to any naturally occurring or synthetic amino acid polymer, such as a peptide, polypeptide, protein, lipoprotein, glycoprotein, etc.

[0141] As used herein, the term "polypeptide" generally refers to a naturally occurring, recombinant or synthetic amino acid polymer that is covalently coupled to one another by continuous peptide bonds, regardless of length or post-translational modification (e.g., fragmentation, phosphorylation, glycosylation, acetylation, methylation, isomerization, reduction, etc.). Although polypeptides are commonly referred to as "proteins" in the art, the terms "polypeptide" and "protein" are often used interchangeably. In general, the first amino acid residue or the first group of amino acid residues in a polypeptide is referred to as being at the "amino terminus" or "N-terminus" of the polypeptide. Similarly, the last amino acid residue or the last group of amino acid residues in a polypeptide is referred to as being at the "carboxyl terminus" or "C-terminus".

[0142] The term "polypeptide" as used herein includes a general term for short peptides (usually less than 100 amino acids), proteins (which contain one or more polypeptide chains). The peptides of the present invention generally have more than 4 amino acids; preferred peptides have more than 6 amino acids, more than 8 amino acids.

[0143] When used herein in the context of the polypeptides described herein, the term "variant" generally refers to a polypeptide that is structurally similar to a reference polypeptide, but characterized by differences in amino acid sequence between the polypeptide and the reference polypeptide (e.g., having at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, at least 85%, or at least 95% sequence identity) and / or the presence or absence of one or more biochemical modifications (e.g., post-translational modifications, substitutions, adduct additions, etc.). Although a subset of the general activities of certain variants may be similar, the structural differences that occur between variants may result in at least a portion of their activities not overlapping. "Variant" may refer to a polypeptide molecule that is altered at one or more positions in a polypeptide sequence relative to the polypeptide molecule (including addition, deletion, substitution of one or more amino acids in the sequence, and covalent modification of the molecule). Therefore, in some cases, the terms "variant" and "isoform" are used interchangeably. Illustrative examples of such variants would include, by way of example only, polypeptides in which hydrogen groups have been replaced by alkyl, acyl, thiol, amide or other such functional groups at one or more amino acid residues. Variants may have "conservative" changes, in which the substituted amino acids may have similar structural and / or chemical properties (e.g., a non-polar amino acid residue is replaced by a different non-polar amino acid residue). Variants may also have "non-conservative" changes (e.g., a polar amino acid residue is replaced by a non-polar or charged amino acid residue). Variants may also include similar minor variations in the amino acid sequence, including but not limited to deletions, truncations, insertions or combinations thereof. Guidance for determining which amino acid residues can be substituted, inserted or deleted without eliminating or otherwise substantially affecting biological activity is widely available in the art. Other guidance can be queried using computer programs well known in the art, such as DNASTAR software. In general and in the context of the present invention, variants will retain at least a subset of the biological functions commonly associated with known membrane-penetrating peptides, such as the ability to facilitate translocation of drug molecules (e.g., nucleic acid molecules) across cell membranes into their cytosolic compartments.

[0144] As used herein, the term "amino acid" generally refers to naturally occurring or synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those amino acids encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds having the same basic chemical structure (i.e., an α carbon bound to a hydrogen, carboxyl, amino group, and R group) as naturally occurring amino acids, such as homoserine, norleucine, methionine sulfoxide, methionine, and methylsulfonium. Such analogs have modified R groups (e.g., norleucine or norvaline) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to compounds having a structure that is different from the general chemical structure of an amino acid but functions in a manner similar to naturally occurring amino acids. The term "amino acid" may refer to an amino acid or a derivative thereof (e.g., an amino acid analog), as well as its D and L forms. Examples of such amino acids include glycine, L-alanine, L-asparagine, L-cysteine, L-aspartic acid, L-glutamic acid, L-phenylalanine, L-histidine, L-isoleucine, L-lysine, L-leucine, L-glutamine, L-arginine, L-methionine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine, N-acetylcysteine.

[0145] "Kit" refers to a transfection mRNA, DNA, RNAi or other drug (e.g., protein or anionic molecule) delivery or protein expression or gene knockdown kit, which includes one or more reagents of the present invention or a mixture thereof. The kit may include one or more non-naturally occurring peptides described herein, optionally together with one or more polymer materials or lipids. In some embodiments, peptide and lipid reagents can be provided in the form of a single formulation. In other embodiments, lipids and peptides can be provided separately, together with instructions for instructing the user to combine the reagents when used. Such kits may include a carrying device that is separated and holds one or more container devices (such as vials, test tubes, etc.) in a tightly constrained manner. Each of such container devices contains the components or component mixtures required for transfection. Such kits may optionally include one or more components selected from any drug molecule, such as nucleic acids (preferably one or more expression vectors, DNA molecules, RNA molecules or RNAi molecules), cells, one or more compounds of the present invention, lipid aggregate-forming compounds, transfection enhancers, biologically active substances, etc.

[0146] The culture medium, method, kit and composition of the present invention are suitable for monolayer or suspension culture, transfection and cultivation of cells, and are suitable for protein expression in monolayer or suspension cultured cells. Preferably, the culture medium, method, kit and composition of the present invention are used for suspension culture, transfection and cultivation of cells, and for protein product expression in suspension cultured cells.

[0147] As used herein, the term "RNA interference" or "RNAi" generally refers to a method of sequence-specific post-transcriptional gene silencing. RNAi is a method of degrading a specific mRNA into short RNAs. In order to mediate RNAi, a double-stranded RNA (dsRNA) that is substantially identical in sequence to the target mRNA is introduced into a cell. The target mRNA is then degraded in the cell, resulting in a decrease in the level of the mRNA and the protein it encodes.

[0148] Drug refers to any therapeutic or prophylactic agent, other than food, used in the prevention, diagnosis, mitigation, treatment or cure of disease in humans or animals.

[0149] Embodiment 1:

[0150] Methods of making peptides:

[0151] The non-naturally occurring peptides of the present invention are produced by any previously known peptide synthesis method known to those of ordinary skill in the art, including but not limited to recombinant methods or peptide synthesis chemistry, such as solid phase peptide synthesis. Solid phase synthesis methods (Marrifield, Journal of the American Chemical Society (J. Am. Chem. Soc.), 85, 2149-2154, 1963) can be noted as just one example of such peptide synthesis methods. Currently, peptides can be produced simply and in a relatively short period of time using automated universal peptide synthesizers based on those principles. In addition, peptides can be produced using well-known recombinant protein production techniques, which are widely known to skilled professionals.

[0152] The simple synthesis method and specific process of the polypeptide of the present invention are described as follows (taking sequence P13 as an example):

[0153] (1) Resin treatment

[0154] ① Resin swelling: Cys(Trt)-2-Cl-Resin (molar substitution coefficient is 0.298 mmol / g) is selected as the starting resin and added into a 300 ml reaction column, and then DCM is added to soak and drain to complete the swelling of the resin.

[0155] ② Deprotection: Add 20% piperidine in DMF solution, stir under N2 for 30 minutes, filter out the solvent; then wash the resin with DMF 6 times, drain and complete the deprotection of the resin.

[0156] (2) Amino acid coupling reaction

[0157] Reaction monitoring detection method: The reaction progress was monitored by the ninhydrin method.

[0158] The raw materials and reagents used are shown in Table 3:

[0159]

[0160] The specific operation process is as follows:

[0161] Weigh the corresponding amount of TBTU and protected amino acid into a beaker, add DMF to dissolve; then add the reaction solution to the resin, add DIEA, pass N2 to blow for about 90 minutes, and detect by ninhydrin reaction. After the reaction is completed, remove the solvent and wash the resin with DMF 3 times. Add 20% piperidine DMF solution to the resin, pass N2 to continue blowing for 30 minutes, then remove the solvent and wash the resin with DMF 6 times to complete the coupling of this amino acid.

[0162] The above reaction procedure was repeated until the condensation reaction of all protected amino acids was completed. After coupling to the last amino acid, the peptide was shrunk, washed with DMF / DCM / methanol three times in sequence, and weighed after being drained.

[0163] Condensation method: TBTU+DIEA, condensation agent: TBTU: 0.72g DIEA: 0.78ml

[0164] Table 4: Abbreviations and abbreviations of amino acids mentioned in this article and the names and molecular formulas of the corresponding amino acids

[0165]

[0166]

[0167] (3) TFA cleavage (dissociate the peptide from the resin and remove the amino acid side chain protecting groups)

[0168] Add the resin to the prepared lysis solution (86% TFA / 5% EDT / 5% thioanisole / 3% phenol / 2% pure water) and stir for 150 minutes. Then remove the resin and lysis solution, add ether to fully precipitate the peptide. Filter the peptide with a Buchner funnel and wash it with ether for 6 times to obtain a crude peptide, which is then sent to the purification group for subsequent purification. Other peptides can be synthesized in this way.

[0169] (4) Detection: Mass spectrometry was used to determine the molecular weight of the polypeptide, and HPLC was used to detect the purity of the polypeptide. The results are shown in Table 5:

[0170]

[0171]

[0172]

[0173]

[0174]

[0175] The mass spectrometry results showed that the molecular weights (MW) of P1-P16 were 2237.69, 1906.29, 2135.53, 1946.34, 2051.48, 1684.03, 1721.05, 1719.09, 1723.08, 1621.88, 1560.90, 865.06, 3915.83, 1899.30, 1815.23, and 1808.19, respectively; the HPLC results showed that P1- The peak times (min) of P16 were 9.416, 9.718, 8.999, 10.978, 9.139, 9.722, 10.707, 11.122, 11.220, 10.381, 9.567, 9.721, 11.104, 12.672, 13.316 and 10.243 min, respectively. There was no peak dragging phenomenon, indicating that the polypeptide had a high purity. The data showed that the purity was above 98.00%.

[0176] Example 2: Preparation method of the polypeptide nanoparticle composition of the present invention and composition of each prescription

[0177] PEGylated polymer materials Poloxamine or poloxamine derivatives Polymer materials Poloxamer Polymer materials Lipid materials

[0178] The polypeptide nanoparticle composition of the present invention is prepared by the following five methods:

[0179] Thin film evaporation method:The lipid material and the polymer material are taken out of a -20°C refrigerator and balanced to room temperature, weighed at a specific molar ratio at room temperature, dissolved in a round-bottom flask with chloroform at room temperature, and configured into a chloroform solution. The chloroform is removed by rotary evaporation with a rotary evaporator to form a thin film on the inner wall of the round-bottom flask, and then a nuclease-free isotonic buffer (such as 0.01 mol / L phosphate, 0.9% sodium chloride aqueous solution) containing 1ug / μl or 5ug / μl polypeptide is added, and glass beads are added and stirred for 1min, or 2min, or 5min, or 10min, or 20min, or 30min, or 60min; stand at 25°C for 1h, or 2h, or 3h, or 4h, or 5h to make the film swell; stir at 25°C for 1h, or 2h, or 3h, or 4h, or 5h, or 6h; and ultrasonicate at 25°C for 1min, or 5min, or 15min, or 20min, or 30min, or 60min. The polypeptide nanoparticle composition is filtered into a sterile bottle with a 0.22 um aqueous filter membrane to obtain the polypeptide nanoparticle composition, which is stored in a refrigerator at 4°C for use. A lyophilizing agent, sucrose, trehalose, mannitol, or a mixture thereof is added to make the final concentration of the lyophilized agent 5% (5 mg / 100 ml), and the polypeptide nanoparticle composition aqueous solution is lyophilized to prepare a lyophilized agent, which is stored in a refrigerator at 4°C for use.

[0180] Dissolution dialysis method: Take the lipid material and polymer material out of the -20℃ refrigerator and balance them to room temperature. Weigh them at a specific molar ratio at room temperature, dissolve them with DMSO at room temperature, and prepare a DMSO solution. Inject them into nuclease-free ultrapure water with a microsyringe while ultrasonicating at 50℃-60℃, continue ultrasonicating for 30 minutes, transfer them into a dialysis bag with a molecular weight cutoff (MWCO, Spectrumlabs) of 1000, 2000, 3500, 8000, or 10000, and dialyze them with nuclease-free ultrapure water for 24 hours. Replace the dialysate every 6 hours. Filter them into a sterile bottle with a 0.22um aqueous filter membrane to obtain stock solution A. Under the condition of vigorous shearing by a high-speed shearing machine, a nuclease-free isotonic buffer B (such as 0.01mol / L phosphate, 0.9% sodium chloride aqueous solution) containing 1ug / μl or 5ug / μl polypeptide is added dropwise to the reserve solution A, and the mixture is stirred at 25°C for 1h, 2h, 3h, 4h, 5h, or 6h with a stirrer; the mixture is filtered into a sterile bottle with a 0.22um aqueous filter membrane to obtain a polypeptide nanoparticle composition, which is stored in a refrigerator at 4°C for use. A lyophilizer sucrose, trehalose, mannitol, or a mixture thereof is added to make the final concentration of the lyophilizer 5% (5mg / 100ml), and the polypeptide nanoparticle composition aqueous solution is lyophilized to prepare a lyophilizer, which is stored in a refrigerator at 4°C for use.

[0181] Ultrasonic dispersion method:The lipid material is taken out of a -20°C refrigerator and equilibrated to room temperature, weighed at a specific molar amount at room temperature, dissolved with ether, ethanol, methanol or chloroform at room temperature, and configured into an organic solution. At 40°C, 45°C, 50°C, 55°C, or 60°C, the solution is injected into a nuclease-free isotonic buffer (such as 0.01 mol / L phosphate, 0.9% sodium chloride aqueous solution) with a microsyringe while ultrasonicating, and the organic solvent is removed by ultrasonication for 0.5 min, 1 min, 2 min, 5 min, 10 min, 20 min, 30 min, or 60 min, and the stock solution A is obtained by filtering with a 0.22 um aqueous filter membrane into a sterile bottle. The polymer material is taken out from the -20°C refrigerator and balanced to room temperature, weighed at a specific molar amount at room temperature, and dissolved in a nuclease-free isotonic buffer (such as 0.01mol / L phosphate, 0.9% sodium chloride aqueous solution) containing 0.001ug / μl or 0.01ug / μl or 0.1ug / μl or 1ug / μl or 5ug / μl polypeptide, and allowed to stand overnight to obtain a stock solution B. The stock solution A and the stock solution B are mixed at room temperature at a specific molar ratio, and a stirrer is added to vigorously stir at 25°C for 5min, 10min, 15min, 20min, 30min, or 60min; filtered into a sterile bottle with a 0.22um aqueous filter membrane to obtain the complex polypeptide nanoparticles, which are stored in a 4°C refrigerator for use. Add lyophilizing agent sucrose, or trehalose, or mannitol, or a mixture thereof to make the final concentration of the lyophilizing agent 5% (5 mg / 100 ml), freeze-dry the aqueous solution of the complex polypeptide nanoparticles to prepare a lyophilized agent, and store it in a refrigerator at 4°C for use.

[0182] Stirring volatilization method:The polymer material is taken out of the -20°C refrigerator and equilibrated to room temperature, weighed at a specific molar amount at room temperature, and dissolved in a nuclease-free isotonic buffer (such as 0.01 mol / L phosphate, 0.9% sodium chloride aqueous solution) containing 0.001 ug / μl or 0.01 ug / μl or 0.1 ug / μl or 1 ug / μl or 5 ug / μl polypeptide, and allowed to stand overnight to obtain a stock solution A. The lipid material is taken out of the -20°C refrigerator and balanced to room temperature, weighed at a specific molar amount at room temperature, dissolved with ethanol / ether / chloroform at room temperature, and configured into an organic solution. The organic solution is slowly injected into the reserve solution A with a microsyringe while stirring at 55°C-60°C, and a stirrer is added and vigorously stirred at 25°C for 6h, or 12h, or 18h, or 24h, or 30h, or 36h until the ethanol / ether / chloroform is completely evaporated; filtered into a sterile bottle with a 0.22um aqueous filter membrane to obtain a polypeptide nanoparticle composition, which is stored in a 4°C refrigerator for use. A lyophilizer sucrose, or trehalose, or mannitol, or a mixture thereof is added to make the final concentration of the lyophilizer 5% (5mg / 100ml), and the polypeptide nanoparticle composition aqueous solution is lyophilized to prepare a lyophilizer, and stored in a 4°C refrigerator for use.

[0183] Reverse evaporation method: The lipid material and the polymer material are taken out of a -20°C refrigerator and balanced to room temperature, weighed at a specific molar ratio at room temperature, dissolved with chloroform or ether at room temperature, and prepared into a chloroform or ether solution; added into a nuclease-free isotonic buffer (such as 0.01 mol / L phosphate, 0.9% sodium chloride aqueous solution) containing 0.001 ug / μl or 0.01 ug / μl or 0.1 ug / μl or 1 ug / μl or 5 ug / μl polypeptide (the volume ratio of aqueous solution to organic solution is 1:2 to 1:8), and ultrasonicated for 0.5 min or 1min or 5min or 10min, until a stable W / O type emulsion is formed, and the chloroform or ether is removed by vacuum evaporation with a rotary evaporator to form a colloid, and then a nuclease-free isotonic buffer containing 0.001ug / μl or 0.01ug / μl or 0.1ug / μl or 1ug / μl or 5ug / μl polypeptide is added dropwise, and vacuum rotary evaporation is continued to obtain an aqueous suspension, which is filtered into a sterile bottle with a 0.22um aqueous filter membrane to obtain an aqueous solution of a polypeptide nanoparticle composition, which is stored in a refrigerator at 4°C for use. Add lyophilizing agent sucrose, or trehalose, or mannitol, or a mixture thereof, so that the final concentration of the lyophilized agent is 5% (5mg / 100ml), and freeze-dry the polypeptide nanoparticle composition aqueous solution to prepare a lyophilized agent, and store it in a refrigerator at 4°C for use.

[0184] Table 6 shows that the materials of different prescriptions were weighed according to the molar ratio and the polypeptide nanoparticle composition prescription was prepared according to the method for preparing polymer material polypeptide nanoparticles of the present invention.

[0185] Table 6 Prescription of polypeptide nanoparticle composition

[0186] Prescription Number Peptides Molar Ratio Polymer Materials Molar Ratio Lipids Molar Ratio Nanofabrication methods Rp01 P1 1 TG 124 - 6 Ultrasonic dispersion method Rp02 P2 1 TA 124 LA 6 Ultrasonic dispersion method Rp03 P3 1 TB 124 - 6 Ultrasonic dispersion method Rp04 P4 1 - - LD 6 Ultrasonic dispersion method Rp05 P5 1 TD 124 LD 1 Ultrasonic dispersion method Rp06 P6 1 TE 124 LA 6 Ultrasonic dispersion method Rp07 P7 1 TF 124 LB 2 Ultrasonic dispersion method Rp08 P8 1 TG 124 LC 6 Ultrasonic dispersion method Rp09 P9 1 TH 124 - 6 Ultrasonic dispersion method Rp10 P10 1 TI 124 LE 6 Ultrasonic dispersion method Rp11 P11 1 TJ 124 LF 1 Ultrasonic dispersion method Rp12 P12 1 TK 124 LG 6 Ultrasonic dispersion method Rp13 P13 1 TL 128 LH 6 Ultrasonic dispersion method Rp14 P14 1 TM 124 LK 1 Ultrasonic dispersion method Rp15 P15 1 TN 124 LL 3 Ultrasonic dispersion method Rp16 P16 1 TM 124 - - Ultrasonic dispersion method Rp17 P1 1 EA:TM 1∶246 LD∶LM 5∶12 Reverse evaporation method Rp18 P1∶P3∶P4∶P14 1∶1∶1∶1 EA:TM 1∶180 - - Thin film dispersion method Rp19 P4 1 EA:TM 1∶40 LL 5 Ultrasonic dispersion method Rp20 P1 1 - - - - Ultrasonic dispersion method Rp21 P3 1 TM 288 LA 5 Ultrasonic dispersion method Rp22 P15 1 TG 288 LD 5 Ultrasonic dispersion method Rp23 P13 1 TG 150 LF:LG 1∶2 Dissolution dialysis Rp24 P16 1 EA:PA 1∶36 LG 6 Stirring volatilization method Rp25 P3 1 - - LD∶LM 5∶12 Ultrasonic dispersion method Rp26 P13 1 ED:TG 1∶36 LA:LM 5∶12 Ultrasonic dispersion method Rp27 P10 1 TA 188 LA 5 Ultrasonic dispersion method Rp28 P5 1 EA:TM 1∶7 LA 6 Ultrasonic dispersion method Rp29 P1 1 T904-RC 200 - - Ultrasonic dispersion method Rp30 P4 1 T90R4-RT 10 - - Ultrasonic dispersion method

[0187] Example 3: Characterization of the polypeptide nanoparticle composition formulation of the present invention

[0188] Particle size and potential: After the polypeptide nanoparticle compositions of each formulation were mixed with FLuc-mRNA at N / P=40 for 10 minutes, the particle size (Intensity Mean), surface potential (Zeta Potential) and polydispersity (PDI) of the polypeptide nanoparticle compositions containing FLuc-mRNA were tested by Malvern Zetasizer Nano ZSE at 25°C. The results are shown in Table 7, which show that the particle size of the nanoparticles of each formulation of the present invention ranges from 20 to 206 nm, and most of them have good dispersibility.

[0189] Nanoparticle morphology: The representative polypeptide nanoparticle composition formulations Rp29 and Rp30 aqueous solutions of the present invention were tested for their nanoparticle morphology by transmission electron microscopy (TEM, Hitachi H600-4). The sample to be tested was prepared by immersing a copper grid without any dyeing into a freshly prepared polypeptide nanoparticle composition aqueous solution and drying it at room temperature. The test was performed, as shown in FIG. Figure 1 As shown, the results show that the prescriptions Rp29 and Rp30 of the present invention have good dispersibility, present regular or irregular spherical structures, and the particle sizes are 80nm and 40nm respectively.

[0190] Encapsulation efficiency:The encapsulation efficiency of each formulation for FLuc-mRNA was determined using the Quant-iT RiboGreen RNA detection kit (ThermoFische). The specific method is referred to the kit instructions. The brief processing method of the present invention is as follows: each formulation was mixed with 200 ng of FLuc-mRNA aqueous solution at N / P = 40 for 10 minutes to prepare a polypeptide nanoparticle composition containing FLuc-mRNA. Each formulation was centrifuged at 4°C and 16000 rpm for 2 h using a low-temperature high-speed centrifuge, and the supernatant was collected and its volume was quantified with a pipette, recorded as V1; the concentration of FLuc-mRNA in the supernatant was measured with a Quant-iT RiboGreen RNA detection kit, recorded as C1; the precipitate after centrifugation was dissolved in 25 μl of chromatographically pure DMSO, and 50 μl of heparin sodium solution (6.66 mg / ml dissolved in 0.9% saline injection) was added and mixed evenly, and after standing at room temperature for 2 h, the total volume V2 was recorded and the concentration of FLuc-mRNA was measured with a Quant-iT RiboGreen RNA detection kit, recorded as C2; the encapsulation rate of each formulation was calculated as follows:

[0191] Packing rate = 100% - (V1C1) / (V1C1 + V2C2) × 100%. The results are shown in Table 7.

[0192] Prescription Number Particle size PDI Potential Encapsulation efficiency Rp15 181.13±11.48 0.19±0.06 -2.60±4.25 86.16％ Rp17 153.37±64.30 0.21±0.01 -0.63±0.81 93.48％ Rp19 205.53±8.91 0.19±0.02 -1.21±0.55 98.65％ Rp20 182.40±39.91 0.27±0.02 -3.44±1.74 99.01％ Rp22 34.90±35.61 0.19±0.01 3.13±3.66 88.73％ Rp29 148.83±3.31 0.19±0.03 -0.05±0.65 84.86％ Rp30 34.37±0.21 0.18±0.00 -0.38±0.26 90.72％

[0193] Table 7 shows the particle size (Intensity Mean), surface potential (Zeta Potential), nanoparticle polydispersity (PDI) and encapsulation efficiency of the mRNA-loaded formulations Rp15, Rp17, Rp19, Rp20, Rp22, Rp29 and Rp30.

[0194] Table 7 shows that the particle size of the prescriptions is between 34-206 nm, the particle size dispersion is good, and the nanoparticles are neutrally charged, between ±4.00 mV; the prescriptions have good encapsulation of mRNA, and the encapsulation rate is between 84.86% and 99.01%.

[0195] Example 4: Transfection of the polypeptide nanoparticle compositions of the present invention mixed with FLuc-mRNA in DC2.4 cells and investigation of cytotoxicity

[0196] 4.1 Cell transfection:

[0197] The cells in logarithmic growth phase were suspended at 4×10 4The density of each well was divided into 96-well plates and placed in a 37°C, 5% CO2 incubator for static culture. After 24 hours, the FLuc-mRNA with a concentration of 1ug / μl was diluted to 0.1ug / μl with ultrapure water without nuclease, and 200ng FLuc-mRNA per well was mixed with different prescriptions in equal volumes at a nitrogen-phosphorus ratio (N / P) of 40 to obtain 88μl of a mixed solution of polypeptide nanoparticle composition containing FLuc-mRNA. After standing for 10 minutes, 20μl per well was added to each well of a 96-well plate containing 180μl complete culture medium, and each sample was repeated 4 wells. 4 hours after administration, the culture medium in the 96-well plate was replaced with complete culture medium. After culturing for 24 hours, the complete medium was aspirated and rinsed with PBS. 100 μl of D-Luciferin working solution (working concentration of 250 ug / ml) was added to each 96-well plate and cultured in a 37°C incubator for 5 minutes. The fluorescence expression intensity of FLuc-mRNA was tested by imaging with an Omega-FLuostar microplate reader. The results are shown in Figure 2-Figure 4 shown.

[0198] Figure 2 The data showed that the transfection effect of the combination of Rp03, Rp04, Rp05, Rp18 and Rp29 was much higher than that of the control group, about 3-5 times that of the control group. Among them, Rp29 had the best effect, almost 5 times that of the control group, and the other groups all reached more than 3 times.

[0199] Figure 3 The data showed that the transfection effect of the combination of Rp06, Rp07, and Rp30 was about 1.5 to 2.5 times that of the control group, while the transfection effect of Rp08 and Rp09 was poor.

[0200] Figure 4 The data showed that the transfection effects of the combinations of Rp 10, Rp 11, and Rp 27 were not much different from those of the control group, and Rp 15 had the best transfection effect, which was more than twice that of the control group.

[0201] 4.2 Cytotoxicity assay:

[0202] The cells in logarithmic growth phase were suspended at 4×10 4The density of each well was divided into 96-well plates and placed in a 37°C, 5% CO2 incubator for static culture. After 24 hours, the FLuc-mRNA with a concentration of 1ug / μl was diluted to 0.1ug / μl with ultrapure water without nuclease, and 200ng FLuc-mRNA per well was mixed with different prescriptions in equal volumes at a nitrogen-phosphorus ratio (N / P) of 40 to obtain 88μl of a mixed solution of polypeptide nanoparticle composition containing FLuc-mRNA. After standing for 10 minutes, 20μl per well was added to each well of a 96-well plate containing 180μl complete culture medium, and each sample was repeated 4 wells. 4 hours after administration, the culture medium in the 96-well plate was replaced with complete culture medium. Continue to culture for 48 hours, aspirate the complete medium, and rinse three times with PBS. Use the cell wells without prescription as negative controls, and the CCK-8 medium wells without cells as blank controls. Add 90μl serum-free medium and 10μl CCK-8 solution to each well, and continue to incubate in the incubator for 2 hours. Use Omega-FLuostar microplate reader to measure the absorbance at 450nm. Cell viability calculation formula:

[0203] Cell viability*% = [A(drug added)-A(blank)] / [A(no drug added)-A(blank)]×100%

[0204] A (drug addition): absorbance of DC2.4 cells, prescription solution and CCK-8 solution added to each well

[0205] A (blank): absorbance of each well with only CCK-8 solution added

[0206] A (no drug added): absorbance of DC2.4 cells and CCK-8 solution added to each well

[0207] *Cell viability: cell proliferation activity or cytotoxic activity. Figure 5 shown.

[0208] Figure 5 The data showed that compared with the control group, the cell viability of the combinations Rp04, Rp05, Rp18, Rp19, Rp28, Rp29, and Rp30 was not significantly different, and the cell viability was greater than 100%, indicating that the drug-treated groups had little or no toxicity.

[0209] Example 5: Transfection of the polypeptide nanoparticle composition of the present invention in different cells

[0210] 5.1 Cells in logarithmic growth phase (C2C12, HL7702, MC3T3-E1, MG-63, NIH3T3) were divided into 5 groups (3 replicate wells in each group), 4x10 4After 24 hours of attachment, a mixture of Rp15, Rp29, Rp30, Ctrl-1 and 200 ng EGFP-mRNA was added to each group at N / P=40, and an equal volume of PBS was used as a blank control. After 4 hours of transfection, the cells were transferred into complete medium and cultured for 24 hours.

[0211] 5.2 mRNA transfection efficiency detection: aspirate the complete culture medium, add PBS to wash, digest all cells with trypsin, and centrifuge at 300x g for 5 minutes. After centrifugation, aspirate the supernatant, resuspend the cells with 500μl PBS containing 10% FBS, and then place on ice and store the cells at 4℃, waiting for flow cytometry detection. Filter with a 200-mesh filter before flow cytometry detection, and detect the percentage of successfully transfected EGFP-mRNA and EGFP fluorescence intensity in the FITC channel. The results are as follows Figure 6-Figure 7 shown.

[0212] Figure 6 The data show that the percentage of EGFP-mRNA transfected into different types of cells by prescription Rp15 is less than or equal to that of the control group. The percentage of EGFP-mRNA transfected into C2C12 and NIH3T3 cells by Rp29 is less than that of the control group, and the percentage in HL7702, MC3T3-E1, and MG-63 cells is higher than that of the control group. The percentage of EGFP-mRNA transfected into HL7702 and MG-63 cells by Rp30 is less than that of the control group, and the percentage in C2C12, MC3T3-E1, and NIH3T33 cells is higher than that of the control group. This suggests that prescriptions Rp15, Rp29, and Rp30 have certain selectivity

[0213] Figure 7 The data showed that compared with the control group, the fluorescence intensity of EGFP-mRNA transfected into various cells by prescriptions Rp15, Rp29 and Rp30 increased, indicating that the transfection of the control group was universal and non-selective, while prescriptions Rp15, Rp29 and Rp30 had certain selectivity and higher expression efficiency in cells that were successfully selectively transfected.

[0214] Example 6: Immune activation effect of the novel coronavirus S-mRNA polypeptide nanoparticle composition (novel coronavirus vaccine) in mice

[0215] S-mRNA was provided by Shanghai Zhaowei Technology Development Co., Ltd. The sequence of S-mRNA seq (cap1 structure, N1-me-pseudo U modified) is:

[0216]

[0217] The specific information of S-mRNA stock solution is

[0218] Product Name: COVID-19Spike Protein, Fμll Length-mRNA

[0219] Product description: Length:4088nucleotides

[0220] Modifications: Fμlly substituted with N1-Me-pseudo UTP

[0221] Concentration: 1.0mg / ml

[0222] Storage Conditions: 1mM Sodium Citrate pH6.4

[0223] Storage Recommendation: At or bclow-40C

[0224] Experimental process:

[0225] 6.1 First immunization of mice: 5-6 week old female C57BL / 6 mice were divided into 6 groups (5 mice in each group) and injected intramuscularly with 75 μl PBS (blank control), a combination of 5 μg S-mRNA and 5 μg S-mRNA coated with PEI (positive control), and 75 μl of Rp01, Rp14, Rp17, Rp29, and Rp30 nanoparticles coated with 5 μg S-EGFP (experimental group × 3).

[0226] 6.2 First serum collection: 28 days after the injection of the polypeptide nanoparticle composition of the present invention, serum was collected from the outer canthus. After the serum coagulated at 4°C for 1 hour, it was centrifuged at 5000 x g for 5 minutes at 4°C, and the supernatant was collected. It was then centrifuged at 10000 x g for 5 minutes at 4°C, and the supernatant was added to eight consecutive PCR tubes for aliquoting and stored at -20°C for later use.

[0227] 6.3 Second immunization of mice: After blood was collected from the lateral canthus of the mice, the first immunization process was repeated.

[0228] 6.4 Second serum collection: 14 days after the second immunization, serum was collected from the outer canthus. After the serum solidified at 4°C for 1 hour, it was centrifuged at 5000x g for 5 minutes at 4°C, and the supernatant was taken. It was then centrifuged at 10000x g for 5 minutes at 4°C, and the supernatant was added to eight consecutive PCR tubes for aliquoting and frozen at -20°C for later use. 6.5 ELISA detection of serum IgG content: S protein was diluted in PBS, and 100μl of diluent (containing 1μg S protein) was used to coat the ELISA enzyme-labeled plate at 4°C overnight. Discard the liquid in the plate, add 200μl PBST to each well to wash the plate 3 times, and then add 200μl PBS blocking solution containing 5% BSA to each well and block it at room temperature on a shaker for 2 hours. Discard the blocking solution, wash the plate 3 times with 200μl PBST per well, and then add 100μl of serum diluted 200 times with PBS, and incubate at room temperature on a shaker for 2 hours. Discard the serum, wash the plate 3 times with 200μl PBST per well, add 100μl antibody (1:1000 dilution) to each well, and incubate on a shaker at room temperature for 2h. Discard the antibody, wash the plate 3 times with 200μl PBST per well, add 50μl TMB colorimetric solution to each well to react in the dark, wait until the positive control well turns dark blue or reacts for 10 minutes, then add 50μl 2M sulfuric acid to each well to terminate the reaction, and detect the OD value at 450nm with an enzyme reader.

[0229] The results are as follows Figure 8 As shown, the OD value is an indicator for judging the level of IgG antibodies in serum. It can be seen from the results that the OD value corresponding to the prescription Rp14 was higher than that of the control group after two immunizations, indicating that this group of nanoparticles has a stronger immune activation function.

[0230] Example 7 Gene transfection kit

[0231] The gene transfection kit is a patented formula and a multi-purpose transfection reagent that can provide efficient transfection in various adherent and suspension cell lines. It is suitable for all common cell lines and many difficult-to-transfect cell lines, and can be used in culture media with or without serum. The kit of the present invention is used to transfect mammalian cells in a 96-well cell culture plate. The specific steps are:

[0232] 1. One day before transfection, add 4x10 4 The cells were inoculated in a 96-well cell culture plate so that the cell growth density reached more than 80% during transfection.

[0233] 2. For each transfection sample, prepare the following complexes

[0234] a. Dilute mRNA to 10 μl with sterile nuclease-free water and mix gently;

[0235] b. Gently mix the transfection reagent before use, and then dilute the appropriate amount to 10 μl with sterile nuclease-free water;

[0236] c. Gently mix the diluted mRNA and diluted transfection reagent (total volume = 20 μl) and incubate at room temperature for 20 minutes.

[0237] 3. Add 20 μl of the complex to the medium containing cells and culture medium, and add Opti-MEM medium to 200 μl.

[0238] 4. Incubate the cells in a CO2 incubator, change the medium after 4 hours, and keep in the incubator for 18-48 hours before gene expression assay.

Claims

1. A polypeptide, characterized in that The amino acid sequence of the polypeptide is as follows: RRRRRCPWKRGCGRC (SEQ ID NO:4).

2. A polypeptide nanoparticle composition, comprising the polypeptide according to claim 1 and a PEGylated polymer material, wherein the PEGylated polymer material is a poloxamine derivative, wherein the poloxamine derivative is T90R4-RT, and wherein the structure of T90R4-RT is as follows: (T90R4-RT) In the formula: x is 17 and y is 19.

3. A transfection complex comprising the polypeptide nanoparticle composition according to claim 2 and a nucleic acid.

4. A nucleic acid vaccine composition, comprising the polypeptide nanoparticle composition according to claim 3 and a nucleic acid.

5. The transfection complex according to claim 3 or the nucleic acid vaccine composition according to claim 4, wherein the nucleic acid is RNA.

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