CN115521359A

CN115521359A - Polypeptide nanoparticle composition

Info

Publication number: CN115521359A
Application number: CN202110713076.9A
Authority: CN
Inventors: 张龙贵
Original assignee: Shenzhen Houcun Nano Pharmaceutical Co ltd
Current assignee: Shenzhen Houcun Nano Pharmaceutical Co ltd
Priority date: 2021-06-25
Filing date: 2021-06-25
Publication date: 2022-12-27

Abstract

A polypeptide nanoparticle composition relates to the technical field of gene delivery in the field of gene therapy, and a peptide for delivering nucleic acid has the following general structure: (Arg) n-Xa-Yb-Xc-Cys (formula I) wherein: n + a + b + c +1=6-40, n, a, b or c are independent of each other, and n.gtoreq.1, b.gtoreq.1, a, c is any of values 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 present invention provides novel non-naturally occurring polypeptides having the functions of encapsulating nucleic acids and selectively promoting the passage of nucleic acids across cell membranes, as well as macromolecular lipid-like polypeptide nanoparticles comprising the polypeptides, as well as methods for their use in gene transfection of cells in vivo and in vitro, and methods for their use in vaccine formulations.

Description

Polypeptide nanoparticle composition

Technical Field

The invention relates to the field of gene therapy, in particular to a polypeptide nanoparticle composition which entraps nucleic acid and selectively promotes the nucleic acid to pass through cell membranes.

Background

Gene transfection is a technique by which a nucleic acid having a biological function is transferred or transported into a cell and the nucleic acid is maintained in the cell for its biological function. A gene vector refers to a means for introducing an exogenous therapeutic gene into a cell of an organism. At present, the gene vectors with industrial transformation potential internationally are mainly viral vectors and non-viral vectors. The viral vector is a gene delivery tool for transmitting the genome of a virus into other cells for infection, and has better application prospects such as lentivirus, adenovirus, retrovirus vector, adeno-associated virus vector and the like. However, due to its inherent physicochemical properties and biological activities, viral vectors have serious disadvantages, such as high production cost, limited loading capacity, poor targeting, insertion integration, teratogenic and mutagenic properties, and are not conducive to the development of universal and general therapies. And non-viral vectors include: liposome nanoparticles, composite nanoparticles, cationic polymer nanoparticles, direct injection method, calcium phosphate coprecipitation method, microinjection method, electroporation method, microparticle bombardment method, etc. The liposome nanoparticles are the main non-viral vectors currently applied to RNA transfection, the existing gene drugs (first RNAi drug Patisiran in the world) are on the market by using the liposome nanoparticle technology, and a plurality of drugs in research using the technology enter the clinical stage II. Compared with other gene delivery methods, the liposome nanoparticle has the advantages of low production cost, definite chemical structure, convenience in quality control, capability of realizing targeted drug delivery through targeted modification, no limit on the theoretical loading capacity, no risk of integration induced mutation, capability of being biodegraded by organisms, convenience in application to gene delivery inside and outside the body and the like, but most of positively charged liposome lipid has high toxicity and low liposome transfection efficiency, and less liposome nanoparticles are successfully applied to clinic.

The cell membrane consists of phospholipid bilayers, the outer part of which is hydrophilic and the inner part of which is hydrophobic, is a barrier for preventing substances outside the cell from freely entering the cell and can prevent various bioactive molecules (including polypeptides, proteins, DNA, RNA and the like) from entering the cell. Although the protection mechanism ensures the relative stability of the intracellular environment and enables various physiological and biochemical reactions to be orderly operated, the protection mechanism prevents a plurality of bioactive molecules with therapeutic activity from entering cells in the field of drug therapy. The penetration of cell membranes into cells is a prerequisite for the action of many biological macromolecules with target sites in cells, and therefore, the development of delivery systems capable of entrapping these biological macromolecules into cells is of great importance in the pharmaceutical field. Cell Penetrating Peptides (CPPs) are a class of polypeptides that are capable of passing directly across a cell membrane into a cell in a receptor-independent, non-classical endocytic manner without causing damage to the cell membrane, and are generally no more than 30 amino acids in length and rich in basic amino acids, the amino acid sequence being generally positively charged. Several CPPs have been found, such as the homeodomain (ANTP) in the human immunodeficiency virus type 1 transcriptional activator TAT (human immunodeficiency virus-1transcription activator, HIV-1 TAT) (Vives et al, J.biol.chem.1997;272, 16010), drosophila melanogaster Zygen peptide (anticannapedia) (Derossi et al, J.biol.chem.1994,269, 10444) and the herpes simplex virus type I VP22 transcription factor (SHV 1VP 22) (Phelan et al, nat.Biotech.,1998,16, 440), among others. The common properties of CPPs are: a net positive charge or charge neutrality, while being hydrophilic and hydrophobic (amphiphilic); the transmembrane delivery efficiency is higher; low cytotoxicity; no limitation of cell type; bioactive substances of different sizes and properties can be introduced into cells by chemical bonding or gene fusion, and the like, including small molecule compounds, dyes, polypeptides, polypeptide Nucleic Acids (PNAs), proteins, plasmid DNA, siRNA, mRNA, smaller liposomes, phage particles, superparamagnetic particles, and the like, and current research has not yet reached its carrying capacity limit, making it potentially a multifunctional targeted drug carrier. The specific transmembrane mechanisms of different CPPs differ, and it has been found that specific amino acid sequences bind to mRNA and interfere with and decrease cell membrane stability, thereby carrying bioactive substances across the cell membrane, such as arginine-alanine-leucine-alanine Residue (RALA) sequences (Pardi et al, curr Opin in immunol.2020, 65-20. Compared with other delivery methods, the research on CPPs is still relatively rare, and there are few reports on the research in the field of mRNA delivery or vaccine development. Nonetheless, CPPs have demonstrated high pharmaceutical value and broad commercial prospects.

Disclosure of Invention

The mRNA vaccine is a novel vaccine developed in recent years, is a 5 th generation novel vaccine after inactivated vaccine, attenuated vaccine, subunit vaccine and DNA vaccine, and has more advantages than the DNA vaccine in safety. mRNA vaccines are a new approach to induce specific immune responses by introducing mRNA encoding antigenic proteins directly into cells and synthesizing the proteins using the expression system of the cells. Although most cells can spontaneously take up mRNA, the efficiency is low and at low doses they saturate. In addition, because of the existence of a large amount of RNase in nature, RNA is unstable in vitro and in vivo and is easily degraded. Thus, there is a need for suitable agents to protect mRNA from extracellular RNase mediated degradation and to facilitate its entry into cells. In the development process of viral mRNA vaccines and tumor mRNA vaccines, it is difficult to deliver mRNA with a specific sequence to Dendritic Cells (DCs) so that the mRNA can be expressed safely, efficiently and sufficiently, which is important for the efficacy of the vaccines.

It is an object of the present invention to provide suitable and efficient vectors for the delivery of biologically active molecules into cells, a further object of the present invention to provide suitable and efficient vectors for the delivery of RNA molecules, and a further object of the present invention to provide suitable and efficient vectors for the delivery of mRNA molecules.

In one aspect, novel non-naturally occurring polypeptides are provided that have the function of encapsulating nucleic acids and selectively facilitating passage of nucleic acids across cell membranes.

In another aspect, a polypeptide nanoparticle composition is provided that includes a non-naturally occurring polypeptide, a pegylated polymeric material.

A non-naturally occurring polypeptide having the general structure:

(Arg) n-Xa-Yb-Xc-Cys (general formula I)

Wherein: n + a + b + c +1=6-40, i.e. n + a + b + c +1 may be equal to the following value: 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.gtoreq.1, b.gtoreq.1, a, c can be 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 or 23.

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

yb is a hydrophobic moiety, Y represents a natural or non-natural hydrophobic amino acid sequence, and in some embodiments Yb is selected from one or more of Trp (W), phe (F), pro (P), tyr (Y), which may be interrupted or continuous therebetween, and b is the number of hydrophobic amino acids;

x is any one or more amino acids, other than Trp (W), phe (F), pro (P), tyr (Y), that is natural or unnatural, hydrophilic or hydrophobic, polar or nonpolar, ionized or non-ionized, and may be interrupted or continuous therebetween, e.g., 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, in some embodiments, xc is KR.

The carbon end of Cys can be-COOH or-CONH ₂ But the-SH side chain is free;

in some preferred but non-limiting embodiments, the polypeptide of formula i is selected from the group consisting of the peptide sequences: those peptide sequences set forth 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 the biologically active molecule into a cell by at least 50% or more.

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

In some embodiments, the polypeptide of formula i is at least 75% identical to any of the peptides set forth 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 delivery of a biologically active molecule into a cell 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 ID NO.13, SEQ ID NO.14, SEQ ID NO.15 or SEQ ID NO.16.

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

In another aspect, the present invention provides a polypeptide nanoparticle composition comprising a non-naturally occurring polypeptide of formula (I) and one or more pegylated polymeric materials.

In some embodiments, the molecular weight of the pegylated polymeric material is 1000Da or more, and the pegylated polymeric material is an amphiphilic polymeric compound, which can improve pharmacokinetic properties in the nanoparticle, can stabilize the nanoparticle, and facilitates targeted modification.

In some embodiments, the pegylated polymeric material is a PEG derivative conjugated to a lipid, a poloxamer and/or a poloxamine derivative.

In some embodiments, the poloxamine derivative may be selected from any of the materials described in patent CN111285845B for the end-functionalized poloxamine derivatives.

In some embodiments, the poloxamine derivative has the structural formula shown below:

(T304-T) wherein: x is 4.3And y is 3.8, in the formula,

(T304-D) wherein: x is 4.3, y is 3.8,

(T304-RT) wherein: x is 4.3, y is 3.8,

(T304-RC) wherein: x is 4.3, y is 3.8,

(T701-R) wherein: x is 14, y is 2.1,

(T901-C) wherein: x is 18.2, y is 2.7,

(T803-RT) wherein: x is 15, y is 11,

(T904-CR) wherein: x is 17 and y is 15,

(T904-RC) wherein: x is 17 and y is 15,

(T904-RT) wherein: x is 17 and y is 15,

(T90R 4-R) wherein: x is 17 and y is 19,

(T90R 4-RT) wherein: x is 17 and y is 19,

(T704-M) wherein: x is 14, y is 13,

(T704-RT) wherein: x is 14, y is 13,

(T704-RC) wherein: x is 14 and y is 13.

The polypeptide nanoparticle compositions can deliver biologically active molecules, including nucleic acid molecules, such as DNA molecules or RNA molecules, to the interior of a cell. Suitable DNA molecules may include DNA molecules having expressible nucleic acid sequences, such as expression vectors or cDNA molecules comprising an open reading frame encoding a protein. Suitable biologically active molecules that may serve in the practice of the present invention include RNA molecules, such as mRNA molecules or RNAi molecules.

Other embodiments of the invention are directed to methods for preparing polypeptide nanoparticle compositions and to methods for delivering biologically active molecules to the interior of cells using the polypeptide nanoparticle compositions. Methods for preparing polypeptide nanoparticle compositions can comprise contacting a biologically active molecule with at least one pegylated polymeric material and a non-naturally occurring polypeptide of formula i of the 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.

Other embodiments of the present invention are directed to methods for transfecting a cell 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 a cell under conditions that promote transfection of the cell. Other embodiments of the present invention are directed to bioactive molecule formulations comprising a bioactive molecule to be delivered into an animal or human subject, a pegylated polymeric material, and a non-naturally occurring polypeptide of formula i of the present invention, optionally in the presence of one or more polymeric materials and/or one or more lipids, to form a pharmaceutically active complex suitable for delivering the bioactive molecule into an animal or human subject in need thereof for the treatment of a physiological condition or disorder.

Other embodiments of the invention disclose transfection complexes comprising polypeptide nanoparticle compositions, nucleic acids.

Other embodiments of the invention disclose a nucleic acid vaccine composition comprising a polypeptide nanoparticle composition, a nucleic acid

The nucleic acid vaccine composition for use in the prevention or treatment of:

(i) Infectious diseases;

(ii) Allergies or allergic diseases;

(iii) Autoimmune diseases; or

(iv) Cancer or neoplastic disease.

Advantageous effects

By combining the design idea of the cell-penetrating peptide, the polypeptide nanoparticle composition is designed by adopting a high molecular material, and the high molecular material is combined with the novel cell-penetrating peptide to promote mRNA-loaded nanoparticles to enter cells, so that the nanoparticles can effectively deliver mRNA.

Detailed Description

In order to make the technical solutions of the present invention better understood by those skilled in the art, the following further discloses some non-limiting examples to further explain the present invention in detail.

The present invention provides improved reagents and compositions suitable for cell transfection. In particular, the invention provides compositions and agents that enhance the transfection efficiency of all cells, including those cell types that are considered to be generally difficult to transfect. The compositions and agents of the invention, when used according to the methods described herein and with the general knowledge and expertise of those of ordinary skill in the art, 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 over 100%. The present invention accomplishes this goal by providing compositions with polypeptide nanoparticles as described in more detail below.

In some embodiments, the polypeptide nanoparticle compositions of the invention comprise at least one non-naturally occurring polypeptide having the general structure:

(Arg) n-Xa-Yb-Xc-Cys (general formula I)

Wherein: n + a + b + c =5-39, n, a, b or c are independent of each other, and n.gtoreq.1, b.gtoreq.1, a, c may 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.

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

in some embodiments, X is any one or more amino acids other than Trp (W), phe (F), pro (P), or Tyr (Y), either natural or non-natural, hydrophilic or hydrophobic, polar or non-polar, ionized or non-ionized, in some embodiments, X is selected from one or more of Arg (R), lys (K), cys (C), gly (G), val (V), leu (L), gin (Q), or His (H), which may be intermittent or continuous therebetween, e.g., 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.

In some embodiments, yb is a hydrophobic moiety and Y represents a natural or non-natural hydrophobic amino acid sequence, and in some embodiments Yb is selected from one or more of Trp (W), phe (F), pro (P), or Tyr (Y), which may be interrupted or continuous therebetween; for example, xa or Xc can be interspersed between Trp (W), phe (F), pro (P), or Tyr (Y), or a repeating sequential arrangement of Trp (W), phe (F), pro (P), or Tyr (Y) in a polypeptide.

In some embodiments, arg (R) is present in the non-naturally occurring polypeptide in an amount of at least 25%, or at least 30%, or at least 35%, and Cys (C) is present in the non-naturally occurring polypeptide in an amount of at least 5.0%, or at least 7.0%, or at least 10.0%. In some embodiments, (Arg) n-Xa-Yb-Xc-Cys (formula I) may have a Cys carbon end that is-COOH or-CONH ₂ But the-SH side chain is free; any of the amino acids therein, any of the known chemically modified derivatives of these amino acids may also be used, provided that these derivatives are non-toxic to cells or organisms when provided together with the amino acids as listed above (the amino acid derivatives are distributed by different companies; see, e.g., sigma Aldrich (Sigma Aldrich) (seehttp://www.sigmaaldrich.com))。

(Arg) n-Xa-Yb-Xc-Cys (formula I) beginning with Arg and ending with Cys, xa-Yb-Xc does not determine any particular order of amino acids, but is intended to reflect the type of amino acid and its frequency of occurrence in the polypeptide. (Arg) n when n is greater than 2, the Arg groups may be continuous or discontinuous, for example, the Arg groups may be interrupted by one or more Xa. In some embodiments, the values a and c in Xa or Xc are not both 0.

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

In some non-limiting embodiments, the polypeptide of formula i is a peptide comprising an amino acid sequence selected from the group consisting of:

RKRRRRYWCPKCRGGRC(SEQ ID NO:1)；

RKRRRRYWCPLGRC(SEQ ID NO:2)；

RKRYRRRFHKRGCGRC(SEQ ID NO:3)；

RRRRRCPWKRGCGRC(SEQ ID NO:4)；

RKRRRRCYPKRGCGRC(SEQ ID NO:5)；

RRRRWFCGKCCGRC(SEQ ID NO:6)；

RRRRRWCRGCGRC(SEQ ID NO:7)；

RRRRWFRCLGLPC(SEQ ID NO:8)；

RRRRWFRCLGTLC(SEQ ID NO:9)；

RRRRKFRGSSGRC(SEQ ID NO:10)；

RKRRKWRGCGRC(SEQ ID NO:11)；

RRFKRC(SEQ ID NO:12)；

RRRRRRRRRRWFRCHKCKCVRRCKLKRC(SEQ ID NO:13)；

RRRRHFCPQKKKRC(SEQ ID NO:14)；

RRRRHFCKRLKRC(SEQ ID NO:15)；

RRRRWFRCKLKRC(SEQ ID NO:16)。

in some embodiments, the polypeptide of formula i is a variant that 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 to any one of 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 ID No.13, SEQ ID No.14, SEQ ID No.15 or SEQ ID No.16 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% or at most 170% of its function of enhancing delivery of the bioactive molecule to the interior of a cell.

Table 1 sets forth various peptide sequences useful in practicing 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. Rather, it will be readily apparent to such persons that, based on the teachings set forth above with respect to the polypeptides described by general formula i, there may be a large number of peptides that are potentially useful in practicing the invention set forth herein. Moreover, determining whether a given peptide sequence falls within the scope of the present invention using standard techniques in the art without undue experimentation is well within the knowledge of the skilled artisan. Furthermore, it is to be understood that various variants of the peptide sequences presented in table 1 are also within the scope of the present invention, as long as such variants meet the structural and functional characteristics set forth above. Variants of the peptide sequences presented in table 1 or any other candidate peptide not specifically recited in table 1 but satisfying the structural and functional requirements set forth above may include deletions, insertions, use of naturally occurring or non-protein amino acid substitutions.

Table 1: a polypeptide of formula I

In some embodiments, the molecular weight of the pegylated polymeric material is 1000Da or more, and the pegylated polymeric material is an amphiphilic polymeric compound, which facilitates improving pharmacokinetic properties in the nanoparticle, stabilizing the nanoparticle, and facilitating targeted modification. The PEGylated high molecular material is preferably a PEG derivative, poloxamer and/or poloxamine or a poloxamine derivative conjugated with lipid.

In some embodiments, the lipid-conjugated PEG derivative may be mPEG-C-DMG, mPEG-C-CLS, DSPE-PEG-Mal, DSPE-PEG-NH2, DOPE-PEG-NH2, mPEG-DSPE, mPEG-DPPE, mPEG-DMPE, and in some embodiments, the lipid-conjugated PEG derivative may be mPEG ₂₀₀₀ -C-DMG、mPEG ₁₀₀₀₀ -C-CLS and DSPE-PEG ₂₀₀₀ -Mal、DSPE-PEG ₅₀₀₀ -NH ₂ 、DOPE-PEG ₂₀₀₀ -NH ₂ 、mPEG ₅₀₀₀ -DSPE、mPEG ₂₀₀₀ -DPPE、mPEG ₂₀₀₀ -DMPE having the formula:

in some embodiments, the poloxamines are polymers of 1, 2-ethylenediaminetetraacetic acid with ethylene oxide and methyl ethylene oxide or 1, 2-ethylenediaminetetraacetic acid with methyl ethylene oxide and ethylene oxide, and in some embodiments, the poloxamines are 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 T90R 4) or

150R1 (abbreviated as T150R 1) or more; the structural formula of the paloxamide is shown as a formula (II) or a formula (III):

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, wherein the poloxamer has a structural formula as shown in formula (IV) or formula (VI):

in some embodiments, the poloxamer is selected from the group consisting of

17R4 (abbreviated to 17R 4),

P-31R1 (abbreviated to 31R 1),

L-121 (abbreviated as L121),

L-31 (abbreviated as L31),

L-64 (abbreviated as L64),

L-81 (abbreviated as L81),

P-10R5 (abbreviated as 10R 5),

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).

In one aspect, 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 PC, DMPC, DPPC, DSPC, DMPE, DOTAP, DOPE, DOTMA, DOPC, DPPE or CLS has the structural formula shown as the following formula:

the compositions of various lipid formulations according to several non-limiting embodiments of the present invention are provided in table 2. These exemplary examples are provided in no way intended to limit the scope of the invention to only those formulations disclosed. Rather, it is merely intended to provide a variety of possible lipid aggregate formulations that may be used in the practice of the present invention. Nonetheless, it will be readily apparent to those skilled in the art that variations or modifications can be made to the formulation, and that other components (such as, for example, other cationic or neutral lipids, peptide targeting moieties, etc.) can be added, or one of the polymeric lipids set forth in table 2 can optionally be removed, and the resulting formulation will be within the spirit and scope of the invention as described herein.

Table 2: the embodiments of the present invention relate to a polymer material and a lipid code

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

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

The polypeptide nanoparticle composition comprises the polypeptide, the PEG high molecular material and the lipid in a molar ratio of 1: 8-300: 0-15, in some embodiments, the polypeptide, the PEG high molecular material and the lipid in a molar ratio of 1: 120-190: 1-12, and in some embodiments, the polypeptide, the PEG high molecular material and the lipid in a molar ratio of 1: 124: 1-6.

The polypeptide nanoparticle compositions described above are used to deliver a biologically active macromolecule into a cell with an increased efficiency of transfecting the biologically active macromolecule relative to the transfection efficiency of a transfection complex lacking a non-naturally occurring polypeptide of formula (I) of at most 10%, at most 15%, 20%, at most 25%, at most 30%, at most 35%, at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 95%, at most 100%, at most 150%, at most 200%, at most 250%, at most 300%, at most 350%, at most 400%, at most 500%, or at most 500%.

In yet another aspect, the invention provides a transfection complex that may include a non-naturally occurring peptide as described above, at least one bioactive molecule, at least one pegylated polymeric material. In some embodiments, the transfection complex comprises a non-naturally occurring peptide, at least one bioactive molecule, at least one pegylated polymeric material, and at least one lipid. The transfection complexes of the invention are stable in aqueous solution and can be contacted with cell tissue of a human or animal immediately after formation, the transfection complexes being stable and can be stored for a period of time prior to contact with the cells or tissue, such as can be stored for a period of time of 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.

In some embodiments, the biologically active molecule may be any substance to be delivered to the interior of a cell in culture in a laboratory or in animal or human tissue, and depending on the application, in some embodiments, the biologically active molecule may be a nucleic acid, or may be a drug or other small organic molecule. In some embodiments, preferred biological macromolecules for forming transfection complexes are nucleic acids, such as, for example, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). In some embodiments, the preferred bioactive molecule may be a DNA molecule. The DNA may be linear DNA or circular DNA, such as DNA in the form of circular plasmids, episomes or expression vectors. In other embodiments, the preferred biologically active molecule may be an RNA molecule. The RNA molecule can be any type of RNA molecule (but is not limited to) including, but not limited to, mRNA, siRNA, miRNA, antisense RNA, ribonuclease, or any other type or species of RNA molecule familiar to those of skill in the art (but not limited to) that will require delivery into the interior of a cell, and in some embodiments, the preferred biologically active molecule can be mRNA.

At least one RNA (molecule) of the RNA may be of any length (preferably depending on the type of RNA to be applied as a transfection complex according to the invention). Without being limited thereto, the at least one RNA (molecule) may have a length of 5-20000 nucleotides, more preferably a length of 5-10000 or 300-10000 nucleotides, even more preferably a length of 5-5000 nucleotides, and most preferably a length of 20-5000, 50-5000, 100-5000 or 300-10000 nucleotides, depending on the type of RNA to be transfected.

The at least one RNA (molecule) of the transfection complex of the present invention may be any RNA, preferably but not limited to, a short RNA oligonucleotide (preferably 5-80 or, more preferably 20-80 nucleotides in length), coding RNA, immunostimulatory RNA, siRNA, antisense RNA, or riboswitches, ribozymes or aptamers. Furthermore, at least one RNA (molecule) of the transfection complex of the invention may be a single-stranded or double-stranded RNA (which may also be considered as an RNA (molecule) due to the non-covalent binding of two single-stranded RNAs (molecules)) or a partially double-stranded RNA (which is typically formed by a longer and a shorter single-stranded RNA molecule or by 2 single-stranded RNA molecules of substantially equal length, wherein one single-stranded RNA molecule is partially complementary to the other single-stranded RNA molecule and both thus form a double-stranded RNA molecule in this region). Preferably, at least one RNA (molecule) of the transfection complex of the present invention may be a single-stranded RNA. The at least one RNA (molecule) of the transfection complex of the invention may also be a circular or linear RNA, preferably a linear RNA. More preferably, at least one RNA (molecule) of the transfection complex of the present invention may be a (linear) single-stranded RNA. At least one RNA (molecule) of the transfection complex of the invention may be ribosomal RNA (rRNA), transfer RNA (tRNA), messenger RNA (mRNA), or viral RNA (vRNA), preferably mRNA. The present invention allows for the transfection of all of these RNAs into cells. In this context, an mRNA is typically an RNA which consists of several structural components, such as an optional 5'-UTR region, a ribosome binding site located upstream and a coding region followed by an optional 3' -UTR region, which may be followed by a poly-a tail (and/or a poly-C-tail). mRNA may be present as single, double or even polycistronic RNA, i.e. RNA carrying the coding sequence of 1,2 or more proteins. Such coding sequences in a di-or even polycistronic mRNA may be separated by at least one IRES sequence, e.g., as defined above.

Short RNA oligonucleotides

In a first embodiment, at least one RNA (molecule) of the transfection complex of the invention may be a short RNA oligonucleotide. Short RNA oligonucleotides in the context of the present invention may comprise 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.

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

Coding RNA

In a second embodiment, at least one RNA (molecule) of the transfection complex of the invention may encode an RNA. The coding RNA of the transfection complex of the invention may be any RNA as defined above. Preferably, the coding RNA may be single-stranded or double-stranded RNA, more preferably single-stranded RNA, and/or circular or linear RNA, more preferably 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).

The coding RNA may also encode a protein or peptide, which may be selected from, for example and without limitation, a therapeutically active protein or peptide, a tumor antigen, an antibody, an immunostimulatory protein or peptide, etc., or any other protein or peptide suitable for specific (therapeutic) applications, wherein it is desired to transport 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.

The polypeptide nanoparticle composition provided by the invention has an encapsulation rate of more than 80%, or more than 85%, or more than 90%, or more than 95%, such as between 84% and 99% for RNA, especially mRNA. The polypeptide nanoparticle composition has high efficiency of nucleic acid transfection, particularly mRNA transfection, and improves the delivery of nucleic acid into cells by more than 50%, or more than 100% or about 200%.

Drawings

FIG. 1 shows the morphology of nanoparticles of FLuc-mRNA of the present invention mixed with different formulations of compounds and tested by transmission electron microscopy.

FIG. 2 shows the intensity of transfection fluorescence expression in DC2.4 cells in vitro after mixing FLuc-mRNA of the present example with different prescribed complexes.

FIG. 3 shows the intensity of transfection fluorescence expression in DC2.4 cells in vitro after mixing FLuc-mRNA of the present example with different prescribed complexes.

FIG. 4 shows the intensity of transfection fluorescence expression in DC2.4 cells in vitro after mixing FLuc-mRNA of the present example with different prescribed complexes.

FIG. 5 shows the cell viability of FLUc-mRNA of the present example after transfection in DC2.4 cells in vitro, mixed with different prescribed complexes.

FIG. 6 shows the percent transfection efficiency of EGFP-mRNA in different cells in vitro after mixing with the prescribed complex according to the example.

FIG. 7 shows the intensity of transfection fluorescence expression in different cells in vitro after mixing EGFP-mRNA of the present example with the prescribed complex.

FIG. 8 shows the immune effect of the S-mRNA complex polypeptide nanoparticles loaded in the mouse.

Definition of terms

The terms used throughout this specification generally have their ordinary meanings in the art within the context of the present invention and in the specific circumstances where each term is used. Certain terms are discussed below or elsewhere in the specification to provide additional guidance to the practitioner in describing various embodiments of the invention and how to make and use such embodiments. It should be appreciated that the same concept can be expressed in more than one way. Thus, alternative language and synonyms may be used for any one or more of the terms discussed herein, regardless of whether a term is detailed or discussed herein, nor is any particular meaning assigned. Synonyms for certain terms may be provided. Reciting one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification, including examples of any terms discussed herein, is illustrative only and in no way limits the scope and meaning of the invention or any exemplified terms.

The terms "hydrophilic" and "hydrophilicity" are used interchangeably herein and generally refer to a molecule with polar groups that has a large affinity for water, can attract water molecules, or dissolve in water, and is capable of forming transient bonds with water through hydrogen bonding.

The term "hydrophobic" also refers to "hydrophobic molecules that are biased to be non-polar and therefore more soluble in neutral and non-polar solutions (e.g., organic solvents). Hydrophobic molecules generally gather together in water, and water forms a large contact angle and is in a drop shape when being on the surface of a hydrophobic solution.

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

The term "unnatural amino acid" is an amino acid that is synthesized by artificial means; the term "modified amino acid residue" as used herein refers to non-standard amino acids, such as post-translationally modified amino acids. Examples of post-translational modifications include, inter alia, phosphorylation, glycosylation, acylation (e.g., acetylation, myristoylation, palmitoylation), alkylation, carboxylation, hydroxylation, saccharification, biotinylation, ubiquitination, changes in chemical properties (e.g., β -elimination deimination, deamidation), and changes in structure (e.g., formation of disulfide bonds)

The term "polar or non-polar" polarity refers to the non-uniformity of charge distribution in a covalent bond or a covalent molecule. If the charge distribution is not uniform, the bond or molecule is said to be polar; if uniform, it is referred to as non-polar.

The term "Ionization" or "non-Ionization" Ionization (Ionization), or Ionization and Ionization, refers to a process in which atoms and molecules form ions under the action of (physical) energy. Refers to the taking up or losing of a negative or positive charge of an atom or molecule to form an ion, usually in combination with other chemical changes

The term "pegylated" PEG is polyethylene glycol, pegylation refers to chemically coupling activated PEG to liposomes.

The term "pegylated polymeric material" refers to a pegylated lipid having a molecular weight of greater than 1000 Da.

The term "introducing" when used in the context of introducing a biologically active molecule into a cell culture is directed to providing a macromolecule or compound in the culture medium and it is understood that the goal of introducing the macromolecule is to enable the macromolecule to be transferred from the extracellular compartment to the cytoplasmic compartment of the cultured cell.

The term "introducing" a biologically active molecule into at least one cell refers to providing a macromolecule or compound to a cell such 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 liposomal 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 may be a protein. Alternatively, the macromolecule may be a peptide. Alternatively, the macromolecule may be a polypeptide. The macromolecule may also be a nucleic acid.

The term "drug" when used herein in the context of delivering a drug into the interior of a cell (such as by 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.

As used herein, the term "bioactive molecule" encompasses a biomolecule. In one embodiment, the term bioactive molecule refers to a 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. The DNA may be linear DNA or circular DNA, such as DNA in the form of circular plasmids, episomes or expression vectors. In certain preferred but non-limiting embodiments, the term macromolecule refers to a complementary DNA (cDNA) having an expressible nucleic acid sequence comprising at least one open reading frame operably linked to one or more nucleic acid sequences required for transcription of an mRNA by the expressible nucleic acid sequence. The macromolecule may be charged or uncharged. A DNA molecule is an example of a charged macromolecule. In some cases, as used herein, the term "macromolecule" may 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 may be any type of RNA molecule, including but not limited to mRNA, siRNA, miRNA, antisense RNA, ribonuclease, or any other type or species of RNA molecule familiar to those of skill in the art (but not limited to) that would require delivery into the interior of a cell.

The term "transfection" is used herein to mean the delivery of a nucleic acid, protein, or other macromolecule to a target cell such that the nucleic acid, protein, or other macromolecule is expressed or biologically functional in the cell.

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

The term "expression of a nucleic acid" and its equivalents refers to the replication of a nucleic acid in a cell, the transcription of DNA into messenger RNA, the translation of RNA into protein, post-translational modification of protein, and/or the trafficking of protein in a cell, or a variant or combination thereof.

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

By "cell culture" or "culturing" is meant maintaining cells in an artificial in vitro environment.

"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 an RNA encoded by a nucleic acid". As used herein, "protein" generally refers to any naturally occurring or synthetic polymer of amino acids, such as peptides, polypeptides, proteins, lipoproteins, glycoproteins, and the like.

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

The term "polypeptide" as used herein includes the generic term for short peptides (typically less than 100 amino acids), proteins (which contain one or more polypeptide chains). The peptides of the invention typically have more than 4 amino acids; preferred peptides have more than 6 amino acids, more than 8 amino acids.

The term "variant" when used herein in the context of the polypeptides described herein generally refers to the following polypeptides: it is similar in structure to a reference polypeptide, but is characterized by amino acid sequence differences (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, and the like) between the polypeptide and the reference polypeptide. While some variants may be similar in subsets of their general activities, structural differences that arise between variants may result in at least some of their activities not overlapping. A "variant" may refer to a polypeptide molecule that is altered (including the addition, deletion, substitution, and covalent modification of a molecule of one or more amino acids in the sequence) relative to the polypeptide molecule at one or more positions in the polypeptide sequence. Thus, in some cases, the terms "variant" and "isoform" are used interchangeably. Illustrative examples of such variants will include, by way of example only, polypeptides in which the replacement of a hydrogen group by an alkyl, acyl, thiol, amide or other such functional group has occurred at one or more amino acid residues. Variants may have "conservative" changes, wherein the substituted amino acid may have similar structural and/or chemical properties (e.g., a non-polar amino acid residue is replaced with a different non-polar amino acid residue). Variants may also have "non-conservative" changes (e.g., a polar amino acid residue is replaced with a non-polar or charged amino acid residue). Variants may also include similar minor variations in amino acid sequence, including (but not limited to) deletions, truncations, insertions, or combinations thereof. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing or otherwise substantially affecting biological activity is widely available in the art. Other guidance may be provided using computer programs well known in the art, such as DNASTAR software queries. In general, and in the context of the present invention, a variant will retain at least a subset of the biological functions normally associated with known membrane-penetrating peptides, such as the ability to facilitate translocation of a drug molecule (e.g., a nucleic acid molecule) across the cell membrane into its cytosolic compartment.

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 the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, γ -carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refers to compounds having the same basic chemical structure as a naturally occurring amino acid (i.e., the alpha carbon bound to a hydrogen, a carboxyl group, an amino group, and an R group), such as homoserine, norleucine, methionine sulfoxide, methionine, and methyl sulfonium. Such analogs have modified R groups (e.g., norleucine or norvaline) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. The term "amino acid" may refer to amino acids or derivatives thereof (e.g., amino acid analogs) as well as D and L forms thereof. 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-acetyl cysteine.

"kit" refers to a transfection mRNA, DNA, RNAi or other drug (e.g., protein or anionic molecule) delivery or protein expression or gene knock-down (knock down) kit comprising one or more reagents of the invention or mixtures thereof. The kit may include one or more non-naturally occurring peptides described herein, optionally together with one or more polymeric materials or lipids. In some embodiments, the peptide and lipid agents may be provided in a single formulation. In other embodiments, the lipid and peptide may be provided separately, along with instructions directing the user to combine the agents at the time of use. Such kits may comprise a carrier device partitioned to hold one or more container devices (e.g., vials, tubes, etc.) in a tightly constrained manner. Each of such container devices contains the component or mixture of components required for performing the transfection. Such kits may optionally include one or more components selected from any drug molecule, such as a nucleic acid (preferably one or more expression vectors, DNA molecules, RNA molecules, or RNAi molecules), a cell, one or more compounds of the invention, a lipid aggregate forming compound, a transfection enhancer, a biologically active substance, and the like.

The media, methods, kits and compositions of the invention are suitable for monolayer or suspension culture, transfection and incubation of cells, and for expression of proteins in monolayer or suspension cultured cells. Preferably, the media, methods, kits and compositions of the invention are used for suspension culture, transfection and incubation of cells, and for expression of protein products in suspension cultured cells.

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 specific mrnas into short RNAs. To mediate RNAi, double-stranded RNA (dsRNA) that is substantially sequence identical to the target mRNA is introduced into the cell. The target mRNA is subsequently degraded in the cell, resulting in a reduction in the levels of the mRNA and the protein it encodes.

A medicament refers to any therapeutic or prophylactic agent other than food used in the prevention, diagnosis, alleviation, treatment or cure of a disease in a human or animal.

The first embodiment is as follows:

method for producing the peptide:

the non-naturally occurring peptides of the 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, J.Am. Chem. Soc.) -85, 2149-2154, 1963) may be labeled 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 the skilled artisan.

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

(1) Resin treatment

(1) Swelling resin: cys (Trt) -2-Cl-Resin (molar substitution coefficient of 0.298 mmol/g) is selected as the starting Resin, added into a 300ml reaction column, added with DCM for soaking, and pumped to dry, thereby completing the swelling of the Resin.

(2) Deprotection: adding 20% piperidine in DMF, introducing N ₂ Stirring for 30 minutes, and filtering to dry the solvent; the resin was washed 6 times with DMF and the deprotection of the resin was completed by suction drying.

(2) Amino acid coupling reaction

The reaction monitoring and detecting method comprises the following steps: the reaction progress was monitored by the indetrione method.

The raw materials and reagents used are shown in table 3:

the specific operation process is as follows:

weighing corresponding amount of TBTU and protected amino acid in a beaker, and adding DMF for dissolving; then adding the reaction solution to the resinAdding DIEA, and introducing N ₂ The mixture was blown for about 90 minutes and detected by ninhydrin reaction. After the reaction was complete, the solvent was removed and the resin was washed 3 times with DMF. Then 20% piperidine in DMF was added to the resin and N was added ₂ Blowing is continued for 30 minutes, then the solvent is removed, and the resin is washed 6 times by DMF, namely the current coupling of the amino acid is completed.

The above reaction procedure is repeated until the condensation reaction of all protected amino acids is completed. After the coupling to the last amino acid, the polypeptide was contracted, sequentially washed 3 times with DMF/DCM/methanol, drained and weighed.

The condensation method comprises the following steps: TBTU + DIEA, condensing agent: TBTU 0.72g DIEA

Table 4: abbreviations, and names and formulae of corresponding amino acids as referred to herein and in the context of the text

(3) TFA cleavage (cleavage of the polypeptide from the resin and removal of the amino acid side chain protecting group)

Adding the resin to the previously prepared lysis solution (86%/5%/EDT/5% thioanisole/3% phenol/2% pure water), and stirring for 150min. Then the resin and the lysate are extracted, and the polypeptide is fully precipitated by adding ether. The polypeptides were filtered using a buchner funnel and washed thoroughly 6 times with ether to give crude peptides, which were then taken to the purification group for subsequent purification. And the like to synthesize other polypeptides.

(4) And (3) detection: the molecular weight of the polypeptide is determined by mass spectrometry and the purity of the polypeptide is determined by HPLC. The results are shown in Table 5:

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, 1808.19; HPLC results show that the peak-off times (min) of P1-P16 are respectively: 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 10.243min, no peak dragging phenomenon exists, which indicates that the polypeptide has higher purity, and the data show that the purity is more than 98.00%.

Example two: the preparation method of the polypeptide nanoparticle composition and the composition of each prescription

PEGylated high polymer material poloxamer high polymer material lipid material

The polypeptide nanoparticle composition is prepared by the following five methods:

thin film evaporation method:taking out the lipid material and the high molecular material from a refrigerator at the temperature of-20 ℃ to balance to room temperature, weighing according to a specific molar ratio at room temperature, dissolving the lipid material and the high molecular material in a round-bottom flask at room temperature by using chloroform to prepare a chloroform solution. Removing chloroform by rotary evaporation with rotary evaporator to form a thin film on the inner wall of the round bottom flask, and adding nuclease-free isotonic buffer containing 1ug/μ l or 5ug/μ l polypeptideAdding rinsing solution (such as 0.01mol/L phosphate, 0.9% sodium chloride water solution), adding glass beads, and stirring for 1min, or 2min, or 5min, or 10min, or 20min, or 30min, or 60min; standing at 25 ℃ for 1h, or 2h, or 3h, or 4h, or 5h to make the film swell; stirring at 25 deg.C for 1h, or 2h, or 3h, or 4h, or 5h, or 6h; performing ultrasonic treatment at 25 deg.C for 1min, or 5min, or 15min, or 20min, or 30min, or 60min. Filtering with 0.22um water-based filter membrane to sterile bottle to obtain polypeptide nanoparticle composition, and storing in 4 deg.C refrigerator. Adding lyophilized agent sucrose, trehalose, mannitol, or their mixture to make the final concentration of lyophilized agent 5% (5 mg/100 ml), lyophilizing the polypeptide nanoparticle composition water solution to obtain lyophilized agent, and storing in 4 deg.C refrigerator.

Dissolution dialysis method:taking out the lipid material and the high polymer material from a refrigerator at the temperature of-20 ℃ to balance to room temperature, weighing according to a specific molar ratio at room temperature, dissolving by using DMSO at room temperature to prepare a DMSO solution. Injecting ultra-pure water with enucleation enzyme into micro-injector under ultrasound at 50-60 deg.C, continuing ultrasound for 30min, transferring into dialysis bag with molecular cut-off (MWCO, spectrumlabs) of 1000, 2000, 3500, 8000 or 10000, dialyzing with ultra-pure water with nuclease for 24 hr, and replacing dialysate every 6 hr. Filtering with 0.22um water-based filter membrane to obtain stock solution A. Adding nuclease-free isotonic buffer solution B (such as 0.01mol/L phosphate, 0.9% sodium chloride aqueous solution) containing 1 ug/ul or 5 ug/ul polypeptide dropwise into the stock solution A under severe shearing with high speed shearing machine, and stirring with a stirrer at 25 deg.C for 1 hr, 2 hr, 3 hr, 4 hr, 5 hr, or 6 hr; filtering with 0.22um water-based filter membrane to sterile bottle to obtain polypeptide nanoparticle composition, and storing in refrigerator at 4 deg.C. Adding lyophilized agent sucrose, trehalose, mannitol, or their mixture to make the final concentration of lyophilized agent 5% (5 mg/100 ml), lyophilizing the polypeptide nanoparticle composition aqueous solution to obtain lyophilized agent, and storing in 4 deg.C refrigerator.

An ultrasonic dispersion method:taking out lipid material from refrigerator at-20 deg.C, balancing to room temperature, weighing at room temperature according to specific molar weight, and mixing with the above materialsDissolving in diethyl ether, ethanol, methanol or chloroform at room temperature to obtain organic solution. Injecting isoosmotic buffer solution (such as 0.01mol/L phosphate and 0.9% sodium chloride aqueous solution) of enucleating enzyme into micro-syringe under ultrasonic treatment at 40 deg.C, 45 deg.C, 50 deg.C, 55 deg.C, or 60 deg.C, continuously ultrasonic treating for 0.5min, or 1min, or 2min, or 5min, or 10min, or 20min, or 30min, or 60min to remove organic solvent, and filtering with 0.22um water-based filter membrane to obtain stock solution A. Taking out the high molecular material from a refrigerator at the temperature of minus 20 ℃ to balance to room temperature, weighing at the room temperature according to a specific molar weight, adding a nuclease-free isotonic buffer solution (such as 0.01 ug/L phosphate and 0.9% sodium chloride aqueous solution) containing 0.001 ug/mul or 0.01 ug/mul or 0.1 ug/mul or 1 ug/mul or 5 ug/mul polypeptide to dissolve, and standing overnight to obtain a stock solution B. Mixing the stock solution A and the stock solution B at room temperature according to a specific molar ratio, adding a stirrer, and vigorously stirring at 25 deg.C for 5min, or 10min, or 15min, or 20min, or 30min, or 60min; filtering with 0.22um water-based filter membrane to sterile bottle to obtain compound polypeptide nanoparticles, and storing in 4 deg.C refrigerator. Adding lyophilized agent sucrose, trehalose, mannitol, or their mixture to make the final concentration of lyophilized agent 5% (5 mg/100 ml), lyophilizing the composite polypeptide nanoparticle water solution to obtain lyophilized agent, and storing in 4 deg.C refrigerator.

Stirring and volatilizing:taking out the high molecular material from a refrigerator at the temperature of minus 20 ℃ to balance to room temperature, weighing at the room temperature according to a specific molar weight, adding a nuclease-free isotonic buffer solution (such as 0.01 ug/L phosphate and 0.9% sodium chloride aqueous solution) containing 0.001 ug/mul or 0.01 ug/mul or 0.1 ug/mul or 1 ug/mul or 5 ug/mul polypeptide to dissolve, and standing overnight to obtain a stock solution A. Taking out the lipid material from a refrigerator at the temperature of-20 ℃ to balance to room temperature, weighing the lipid material according to a specific molar weight at the room temperature, dissolving the lipid material by using ethanol/diethyl ether/chloroform at the room temperature to prepare an organic solution, slowly injecting the organic solution into the stock solution A by using a micro-injector while stirring at the temperature of 55-60 ℃, adding a stirrer, and violently stirring at the temperature of 25 ℃ for 6 hours, or 12 hours, or 18 hours, or 24 hours, or 30 hours, or 36 hours until the ethanol/diethyl ether/chloroform is volatilized completely; filtering with 0.22um water-based filter membrane to sterile bottle to obtain polypeptide nanoparticle groupThe compound was stored in a refrigerator at 4 ℃ until use. Adding lyophilized agent sucrose, trehalose, mannitol, or their mixture to make the final concentration of lyophilized agent 5% (5 mg/100 ml), lyophilizing the polypeptide nanoparticle composition water solution to obtain lyophilized agent, and storing in 4 deg.C refrigerator.

A reverse evaporation method:taking out lipid material and polymer material from refrigerator at-20 deg.C, balancing to room temperature, weighing at room temperature according to specific molar ratio, dissolving with chloroform or diethyl ether at room temperature, and making into chloroform or diethyl ether solution; adding a nuclease-free isotonic buffer solution (such as 0.01mol/L phosphate and 0.9% sodium chloride aqueous solution) containing 0.001 ug/ul or 0.01 ug/ul or 0.1 ug/ul or 1 ug/ul or 5 ug/ul polypeptide (the volume ratio of the aqueous solution to the organic solution is 1. Adding lyophilized agent sucrose, trehalose, mannitol, or their mixture to make the final concentration of lyophilized agent 5% (5 mg/100 ml), lyophilizing the polypeptide nanoparticle composition aqueous solution to obtain lyophilized agent, and storing in 4 deg.C refrigerator.

Table 6 shows that the polypeptide nanoparticle composition is prepared by weighing the materials according to the molar ratios of the different prescriptions and using the preparation method of the polymer material polypeptide nanoparticles of the present invention.

TABLE 6 polypeptide nanoparticle composition recipe

Prescription number

Polypeptides

Molar ratio of

Polymer material

Molar ratio of

Lipid

Molar ratio of

Nano preparation method

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 method

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

Example three: characterization of the polypeptide nanoparticle composition of the invention

Particle size and potential:after each prescription of polypeptide nanoparticle composition was mixed with FLuc-mRNA for 10min with N/P =40, the size of the dynamic light scattering nanoparticles (Intensity Mean), surface Potential (Zeta Potential) and Polydispersity (PDI) of the FLuc-mRNA containing polypeptide nanoparticle composition were tested at 25 ℃ using Malvern Zetasizer Nano ZSE. The results are shown in table 7, and the results show that the particle size of the nanoparticles of each prescription of the invention ranges from 20 nm to 206nm, and most of the nanoparticles have better dispersibility.

The morphology of the nanoparticles:the representative polypeptide nanoparticle composition formula Rp of the invention29. The morphology of the nanoparticles of the Rp30 aqueous solution is tested by a transmission electron microscope (TEM, hitachi H600-4), a sample to be tested is prepared by immersing a copper grid without any dyeing into a freshly prepared aqueous solution of the polypeptide nanoparticle composition and drying at room temperature, and the test is carried out, as shown in figure 1, the results show that the formulas Rp29 and Rp30 have good dispersibility, are in regular or irregular spherical structures, and have particle sizes of 80nm and 40nm respectively.

The encapsulation efficiency is as follows:the method uses a Quant-iT RiboGreen RNA detection kit (ThermoFische company) to determine the entrapment rate of each prescription to the FLuc-mRNA, and the specific method refers to the kit specification, and the brief processing method of the invention comprises the following steps: and mixing each prescription with 200ng of FLuc-mRNA aqueous solution according to N/P =40 for 10min to prepare the FLuc-mRNA-containing polypeptide nanoparticle composition. Centrifuging each prescription for 2h at the temperature of 4 ℃ and the speed of 16000rpm by using a low-temperature high-speed centrifuge, collecting supernatant, and quantifying the volume by using a pipettor, wherein the volume is marked as V1; measuring the concentration of the Fluc-mRNA in the supernatant by using a Quant-iT RiboGreen RNA detection kit, and recording the concentration as C1; dissolving the centrifuged precipitate in 25. Mu.l of chromatographic pure DMSO, continuously adding 50. Mu.l of heparin sodium solution (6.66 mg/ml dissolved in 0.9% physiological saline injection), uniformly mixing, standing at room temperature for 2h, recording the total volume V2, and determining the concentration of FLUc-mRNA (recorded as C2) by using a Quant-iT RiboGreen RNA detection kit; the package carrying rate calculation formula of each prescription is as follows:

the entrapment rate =100% - (V1C 1)/(V1C 1+ V2C 2) × 100%, and the results are shown in table 7.

Prescription number	Particle size	PDI	Potential of	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％

Table 7 shows the particle size (Intensity Mean), surface Potential (Zeta Potential), nanoparticle Polydispersity (PDI) and encapsulation efficiency of each of the mRNA-carrying formulations Rp15, rp17, rp19, rp20, rp22, rp29 and Rp 30.

Table 7 shows that the particle size of the prescription is between 34 and 206nm, the particle size dispersibility is good, and the nano-particles are biased to be neutral in electricity and are between +/-4.00 mV; the prescription has better encapsulation to mRNA, and the encapsulation efficiency is between 84.86% and 99.01%.

Example four: the polypeptide nanoparticle composition disclosed by the invention is transfected in DC2.4 cells after being mixed with FLuc-mRNA in each prescription and examined in cytotoxicity

4.1 cell transfection:

the logarithmic growth phase cell suspension is divided into 4X10 ⁴ The density of each well was divided into 96-well plates, charged at 37 ℃ and 5% CO ₂ And (5) standing and culturing in an incubator. Diluting the Fluc-mRNA with the concentration of 1 ug/mul to 0.1 ug/mul by using nuclease ultrapure water after 24h, respectively mixing the Fluc-mRNA with different prescriptions in equal volumes according to the nitrogen-phosphorus ratio (N/P) of 40 and 200ng in each hole to respectively obtain 88 mul of polypeptide nanoparticle composition mixed liquor containing the Fluc-mRNA, standing for 10min, respectively adding the polypeptide nanoparticle composition mixed liquor into a 96-well plate containing 180 mul of complete culture medium in each hole according to the volume of 20 mul in each hole, and repeating 4 holes in each sample. After 4h of administration, the aspirated 96-well plate was replaced with complete medium. The culture was continued for 24h, the complete medium was aspirated and rinsed once with PBS and 100. Mu.l of D-Luciferin working solution (working concentration: working concentration)250 ug/ml) was added to each 96-well plate, incubation was continued in an incubator at 37 ℃ for 5min, and the fluorescent expression intensity of FLuc-mRNA was measured by imaging with an Omega-FLuostar plate reader. The results are shown in FIGS. 2 to 4.

The data in FIG. 2 shows: the transfection effect of the combination Rp03, rp04, rp05, rp18 and Rp29 is far higher than that of the control group and is about 3-5 times of that of the control group, wherein the Rp29 effect is preferably almost 5 times of that of the control group, and the other groups reach more than 3 times.

The data in FIG. 3 shows: the transfection effect of the combination Rp06, rp07 and Rp30 is about 1.5 to 2.5 times of that of the control group, and the transfection effects of Rp08 and Rp09 are poor.

The data in fig. 4 shows: the transfection effect of the combination Rp 10, rp 11 and Rp 27 is not much different from that of the control group, and the transfection effect of Rp15 is best and is more than 2 times of that of the control group.

4.2 cytotoxicity assay:

cell suspension in logarithmic growth phase at 4X10 ⁴ The density of each well was divided into 96-well plates, charged at 37 ℃ and 5% CO ₂ And (5) standing and culturing in an incubator. Diluting the Fluc-mRNA with the concentration of 1 ug/mul to 0.1 ug/mul by using nuclease ultrapure water after 24h, respectively mixing the Fluc-mRNA with different prescriptions in equal volumes according to the nitrogen-phosphorus ratio (N/P) of 40 and 200ng in each hole to respectively obtain 88 mul of polypeptide nanoparticle composition mixed liquor containing the Fluc-mRNA, standing for 10min, respectively adding the polypeptide nanoparticle composition mixed liquor into a 96-well plate containing 180 mul of complete culture medium in each hole according to the volume of 20 mul in each hole, and repeating 4 holes in each sample. After 4h of dosing, the aspirated 96-well plate was replaced with complete medium. The culture was continued for 48h, the complete medium was aspirated and rinsed three times with PBS, wells without the prescription cell were used as negative controls and wells with CCK-8 medium without cells were used as blank controls, and 90. Mu.l serum-free medium and 10. Mu.l CCK-8 solution were added to each well and the incubation was continued in the incubator for 2h. Absorbance at 450nm was measured using an Omega-Fluostar microplate reader. Cell viability calculation formula:

cell viability = [ a (dosed) -a (blank) ]/[ a (not dosed) -a (blank) ] × 100%

A (dosing): absorbance of 2.4 DC cells, prescription solution and CCK-8 solution in each well

A (blank): absorbance of CCK-8 solution added to each well

A (no drug addition): absorbance of the solution containing DC2.4 cells and CCK-8 in each well

* Cell viability: cell proliferation activity or cytotoxic activity. The results are shown in FIG. 5.

The data in FIG. 5 shows: compared with the control group, the cell viability of the combinations Rp04, rp05, rp18, rp19, rp28, rp29 and Rp30 is not obviously different, and the cell viability is more than 100%, which indicates that the administration group has little or no toxicity.

Example five: transfection of the polypeptide nanoparticle composition of the invention in different cells

5.1 cells in logarithmic growth phase (C2C 12, HL7702, MC3T3-E1, MG-63, NIH3T 3) were divided into 5 groups (3 multiple wells per group) with 4X10 per well ⁴ And (4) cells. After 24h of attachment, a mixture of the prescriptions Rp15, rp29, rp30, ctrl-1 and 200ng EGFP-mRNA was added to each group at N/P =40, and an equal volume of PBS was used as a blank control, and after 4h of transfection, the complete medium was removed and incubated for 24h.

5.2mRNA transfection efficiency test: the complete medium was aspirated, washed with PBS and then all cells were trypsinized and centrifuged at 300 Xg for 5min. After centrifugation, the supernatant was aspirated, the cells were resuspended in 500. Mu.l of 10% FBS-containing PBS, and then placed on ice, the cells were stored at 4 ℃ and the flow assay was awaited. Filtration through a 200 mesh screen and FITC channel detection of the percentage of successfully transfected EGFP-mRNA and EGFP fluorescence intensity was performed prior to flow-on-machine detection. The results are shown in FIGS. 6 to 7.

The data in FIG. 6 shows: the percentage of the prescription Rp15 transfected EGFP-mRNA into cells of different species was less than or equal to that of the control group. The percentage of Rp29 transfected EGFP-mRNA into C2C12, NIH3T3 cells was less than the control group, and the percentage in HL7702, MC3T3-E1, MG-63 cells was higher than the control group. The percentage of the Rp30 transfected EGFP-mRNA into HL7702 and MG-63 cells is smaller than that of the control group, and the percentage of the EGFP-mRNA in C2C12, MC3T3-E1 and NIH3T33 cells is higher than that of the control group. Prompting that the prescriptions Rp15, rp29 and Rp30 have certain selectivity

The data in FIG. 7 shows: the fluorescence intensity of the prescriptions Rp15, rp29 and Rp30 for transfecting EGFP-mRNA into various cells was increased compared with the control group, suggesting that the transfection of the control group was general and non-selective, the prescriptions Rp15, rp29 and Rp30 were selective, and the expression efficiency in the cells in which the selective transfection was successful was higher.

Example six: the immune activation effect of the new crown S-mRNA polypeptide nanoparticle-loaded composition (new crown vaccine) in mice

The S-mRNA is provided by Shanghai Mv science and technology development Limited, and the sequence of the S-mRNA seq (cap 1 structure, N1-me-pseudo U modified) is as follows:

GAGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGUUCGUGUUCCUGGUGCUGCUGCCUCUGGUGUCCAGCCAGUGUGUGAACCUGACCACCAGAACACAGCUGCCUCCAGCCUACACCAACAGCUUUACCAGAGGCGUGUACUACCCCGACAAGGUGUUCAGAUCCAGCGUGCUGCACUCUACCCAGGACCUGUUCCUGCCUUUCUUCAGCAACGUGACCUGGUUCCACGCCAUCCACGUGUCCGGCACCAAUGGCACCAAGAGAUUCGACAACCCCGUGCUGCCCUUCAACGACGGGGUGUACUUUGCCAGCACCGAGAAGUCCAACAUCAUCAGAGGCUGGAUCUUCGGCACCACACUGGACAGCAAGACCCAGAGCCUGCUGAUCGUGAACAACGCCACCAACGUGGUCAUCAAAGUGUGCGAGUUCCAGUUCUGCAACGACCCCUUCCUGGGCGUCUACUACCACAAGAACAACAAGAGCUGGAUGGAAAGCGAGUUCCGGGUGUACAGCAGCGCCAACAACUGCACCUUCGAGUACGUGUCCCAGCCUUUCCUGAUGGACCUGGAAGGCAAGCAGGGCAACUUCAAGAACCUGCGCGAGUUCGUGUUUAAGAACAUCGACGGCUACUUCAAGAUCUACAGCAAGCACACCCCUAUCAACCUCGUGCGGGAUCUGCCUCAGGGCUUCUCUGCUCUGGAACCCCUGGUGGAUCUGCCCAUCGGCAUCAACAUCACCCGGUUUCAGACACUGCUGGCCCUGCACAGAAGCUACCUGACACCUGGCGAUAGCAGCAGCGGAUGGACAGCUGGUGCCGCCGCUUACUAUGUGGGCUACCUGCAGCCUAGAACCUUCCUGCUGAAGUACAACGAGAACGGCACCAUCACCGACGCCGUGGAUUGUGCUCUGGACCCUCUGAGCGAGACAAAGUGCACCCUGAAGUCCUUCACCGUGGAAAAGGGCAUCUACCAGACCAGCAACUUCCGGGUGCAGCCCACCGAAUCCAUCGUGCGGUUCCCCAAUAUCACCAAUCUGUGCCCCUUCGGCGAGGUGUUCAAUGCCACCAGAUUCGCCUCUGUGUACGCCUGGAACCGGAAGCGGAUCAGCAAUUGCGUGGCCGACUACUCCGUGCUGUACAACUCCGCCAGCUUCAGCACCUUCAAGUGCUACGGCGUGUCCCCUACCAAGCUGAACGACCUGUGCUUCACAAACGUGUACGCCGACAGCUUCGUGAUCCGGGGAGAUGAAGUGCGGCAGAUUGCCCCUGGACAGACAGGCAAGAUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGUGUGAUUGCCUGGAACAGCAACAACCUGGACUCCAAAGUCGGCGGCAACUACAAUUACCUGUACCGGCUGUUCCGGAAGUCCAAUCUGAAGCCCUUCGAGCGGGACAUCUCCACCGAGAUCUAUCAGGCCGGCAGCACCCCUUGUAACGGCGUGGAAGGCUUCAACUGCUACUUCCCACUGCAGUCCUACGGCUUUCAGCCCACAAAUGGCGUGGGCUAUCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAACUGCUGCAUGCCCCUGCCACAGUGUGCGGCCCUAAGAAAAGCACCAAUCUCGUGAAGAACAAAUGCGUGAACUUCAACUUCAACGGCCUGACCGGCACCGGCGUGCUGACAGAGAGCAACAAGAAGUUCCUGCCAUUCCAGCAGUUUGGCCGGGAUAUCGCCGAUACCACAGACGCCGUUAGAGAUCCCCAGACACUGGAAAUCCUGGACAUCACCCCUUGCAGCUUCGGCGGAGUGUCUGUGAUCACCCCUGGCACCAACACCAGCAAUCAGGUGGCAGUGCUGUACCAGGACGUGAACUGUACCGAAGUGCCCGUGGCCAUUCACGCCGAUCAGCUGACACCUACAUGGCGGGUGUACUCCACCGGCAGCAAUGUGUUUCAGACCAGAGCCGGCUGUCUGAUCGGAGCCGAGCACGUGAACAAUAGCUACGAGUGCGACAUCCCCAUCGGCGCUGGAAUCUGCGCCAGCUACCAGACACAGACAAACAGCCCUCGGAGAGCCAGAAGCGUGGCCAGCCAGAGCAUCAUUGCCUACACAAUGUCUCUGGGCGCCGAGAACAGCGUGGCCUACUCCAACAACUCUAUCGCUAUCCCCACCAACUUCACCAUCAGCGUGACCACAGAGAUCCUGCCUGUGUCCAUGACCAAGACCAGCGUGGACUGCACCAUGUACAUCUGCGGCGAUUCCACCGAGUGCUCCAACCUGCUGCUGCAGUACGGCAGCUUCUGCACCCAGCUGAAUAGAGCCCUGACAGGGAUCGCCGUGGAACAGGACAAGAACACCCAAGAGGUGUUCGCCCAAGUGAAGCAGAUCUACAAGACCCCUCCUAUCAAGGACUUCGGCGGCUUCAAUUUCAGCCAGAUUCUGCCCGAUCCUAGCAAGCCCAGCAAGCGGAGCUUCAUCGAGGACCUGCUGUUCAACAAAGUGACACUGGCCGACGCCGGCUUCAUCAAGCAGUAUGGCGAUUGUCUGGGCGACAUUGCCGCCAGGGAUCUGAUUUGCGCCCAGAAGUUUAACGGACUGACAGUGCUGCCUCCUCUGCUGACCGAUGAGAUGAUCGCCCAGUACACAUCUGCCCUGCUGGCCGGCACAAUCACAAGCGGCUGGACAUUUGGAGCAGGCGCCGCUCUGCAGAUCCCCUUUGCUAUGCAGAUGGCCUACCGGUUCAACGGCAUCGGAGUGACCCAGAAUGUGCUGUACGAGAACCAGAAGCUGAUCGCCAACCAGUUCAACAGCGCCAUCGGCAAGAUCCAGGACAGCCUGAGCAGCACAGCAAGCGCCCUGGGAAAGCUGCAGGACGUGGUCAACCAGAAUGCCCAGGCACUGAACACCCUGGUCAAGCAGCUGUCCUCCAACUUCGGCGCCAUCAGCUCUGUGCUGAACGAUAUCCUGAGCAGACUGGACCCUCCUGAGGCCGAGGUGCAGAUCGACAGACUGAUCACAGGCAGACUGCAGAGCCUCCAGACAUACGUGACCCAGCAGCUGAUCAGAGCCGCCGAGAUUAGAGCCUCUGCCAAUCUGGCCGCCACCAAGAUGUCUGAGUGUGUGCUGGGCCAGAGCAAGAGAGUGGACUUUUGCGGCAAGGGCUACCACCUGAUGAGCUUCCCUCAGUCUGCCCCUCACGGCGUGGUGUUUCUGCACGUGACAUAUGUGCCCGCUCAAGAGAAGAAUUUCACCACCGCUCCAGCCAUCUGCCACGACGGCAAAGCCCACUUUCCUAGAGAAGGCGUGUUCGUGUCCAACGGCACCCAUUGGUUCGUGACACAGCGGAACUUCUACGAGCCCCAGAUCAUCACCACCGACAACACCUUCGUGUCUGGCAACUGCGACGUCGUGAUCGGCAUUGUGAACAAUACCGUGUACGACCCUCUGCAGCCCGAGCUGGACAGCUUCAAAGAGGAACUGGACAAGUACUUUAAGAACCACACAAGCCCCGACGUGGACCUGGGCGAUAUCAGCGGAAUCAAUGCCAGCGUCGUGAACAUCCAGAAAGAGAUCGACCGGCUGAACGAGGUGGCCAAGAAUCUGAACGAGAGCCUGAUCGACCUGCAAGAACUGGGGAAGUACGAGCAGUACAUCAAGUGGCCCUGGUACAUCUGGCUGGGCUUUAUCGCCGGACUGAUUGCCAUCGUGAUGGUCACAAUCAUGCUGUGUUGCAUGACCAGCUGCUGUAGCUGCCUGAAGGGCUGUUGUAGCUGUGGCAGCUGCUGCAAGUUCGACGAGGACGAUUCUGAGCCCGUGCUGAAGGGCGUGAAACUGCACUACACAUGAGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA。

the specific information of the S-mRNA stock solution is

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

Product description：Length:4088nucleotides

Modifications：Fμlly substituted with N1-Me-pseudo UTP

Concentration：1.0mg/ml

Storage Conditions：1mM Sodium Citrate pH6.4

Storage Recommendation：At or bclow-40C

The experimental process comprises the following steps:

6.1 first immunization of mice: female C57BL/6 mice at 5-6 weeks were divided into 6 groups (5 mice per group) and intramuscularly injected with 75. Mu.l PBS (blank control), 5. Mu.g S-mRNA and a combination of 5. Mu.g S-mRNA coated with PEI (positive control), 75. Mu.l of nanoparticles Rp01, rp14, rp17, rp29, rp30 coated with 5. Mu.g S-EGFP (experimental group X3), respectively.

6.2 first serum collection: after 28 days of injection of the polypeptide nanoparticle composition of the invention, serum was taken from the outer canthus. After the serum is solidified for 1h at 4 ℃, centrifuging for 5 minutes at 4 ℃ at 5000x g of rotation speed, taking the supernatant, centrifuging for 5 minutes at 4 ℃ at 10000x g of rotation speed, taking the supernatant, adding the supernatant into eight rows of PCR tubes, subpackaging and preserving for later use at-20 ℃.

6.3 Secondary immunization of mice: the first immunization procedure was repeated after the mice were bled from the outer canthus.

6.4 second serum collection: sera were taken from the outer canthus 14 days after the second immunization. After the serum is solidified for 1h at 4 ℃, centrifuging for 5 minutes at 4 ℃ at 5000x g of rotation speed, taking the supernatant, centrifuging for 5 minutes at 4 ℃ at 10000x g of rotation speed, taking the supernatant, adding the supernatant into eight rows of PCR tubes, subpackaging and preserving for later use at-20 ℃.6.5 detection of serum IgG content by ELISA: the S protein was diluted in PBS and the ELISA plate was coated with 100. Mu.l of the dilution (containing 1. Mu.g of S protein) per well and overnight at 4 ℃. Discard the plate liquid, add 200. Mu.l PBST per well to wash the plate 3 times, add 200. Mu.l BSA 5% in PBS blocking solution per well and block for 2h with a warm shaker. The blocking solution was discarded, 200. Mu.l of PBST per well was washed 3 times, 100. Mu.l of serum diluted 200-fold with PBS was added, and the mixture was incubated for 2 hours at room temperature in a shaker. Serum was discarded, and after washing the plate 3 times with 200. Mu.l PBST per well, 100. Mu.l antibody (1 diluted 1000) was added per well and incubated for 2h at room temperature in a shaker. Discarding the antibody, washing the plate with 200. Mu.l PBST per well for 3 times, adding 50. Mu.l TMB color development solution per well for light-proof reaction, adding 50. Mu.l 2M sulfuric acid per well for stopping reaction after the positive control well turns deep blue or reacts for 10 minutes, and detecting OD value at 450nm by an enzyme-labeling instrument.

The result is shown in fig. 8, the OD value is an index for determining the level of IgG antibody in serum, and it can be seen from the result that the OD value corresponding to the prescription Rp14 is higher than that of the control group after two immunizations, which indicates that the nanoparticles in this group have a stronger immune activation function.

Example 7 Gene transfection kit

The gene transfection reagent kit is a patent formula, is a multipurpose transfection reagent, and can provide high-efficiency transfection in various adherent and suspension cell lines. Suitable for all common cell lines and many difficult to transfect cell lines, and can be used in medium with or without serum. The use method of the kit provided by the invention comprises the following steps: mammalian cells were transfected in 96-well cell culture plates. The method comprises the following specific steps:

1. one day before transfection, 200. Mu.l of medium per well contained 4X10 ⁴ The individual cells were seeded in 96-well cell culture plates to achieve a cell growth density of 80% or greater upon transfection.

2. For each transfection sample, the following complexes were prepared

a. Diluting mRNA to 10 μ l with sterile nuclease-free water, and gently mixing;

b. the transfection reagents were gently mixed prior to use and then diluted with sterile nuclease-free water in an appropriate amount to 10 μ l;

c. diluted mRNA and diluted transfection reagent were mixed gently (total volume =20 μ Ι) and incubated at room temperature for 20 min.

3. Mu.l of the complex was added to the medium containing cells and opti-MEM to 200. Mu.l.

4. Cells were incubated in a carbon dioxide incubator, media changed after 4 hours, and stored in the incubator for 18-48 hours prior to gene expression detection.

Sequence listing

<110> Shenzhen Shenzheng Nanyao pharmaceutical Co Ltd

<120> polypeptide nanoparticle composition

<130> PCT001

<141> 2021-06-25

<160> 16

<170> SIPOSequenceListing 1.0

<210> 1

<211> 17

<212> PRT

<213> Artificial Sequence (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (Artificial Sequence)

<400> 12

Arg Arg Phe Lys Arg Cys

1 5

<210> 13

<211> 28

<212> PRT

<213> Artificial Sequence (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 (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 (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 (Artificial Sequence)

<400> 16

Arg Arg Arg Arg Trp Phe Arg Cys Lys Leu Lys Arg Cys

1 5 10

Claims

1. A polypeptide having the 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.gtoreq.1, b.gtoreq.1, a, c is any of the values 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;

yb is selected from one or more of Trp (W), phe (F), pro (P) and Tyr (Y), and can be interrupted or continuous;

x is any one or more of natural or non-natural, hydrophilic or hydrophobic, polar or non-polar, ionized or non-ionized amino acids except Trp (W), phe (F), pro (P), tyr (Y), and can be interrupted or continuous.

2. The polypeptide of claim 1, wherein X is selected from the group consisting of Arg (R), lys (K), cys (C), gly (G), val (V), leu (L), gln (Q), and His (H), and is optionally interrupted or continuous.

3. Polypeptide according to claim 2, characterized in that Xa is Cys, lys or LysArgArgArgArg and Xc is LysArg or CysGlyCysGlyLysArg.

4. The polypeptide of claim 1, wherein Arg is present in the polypeptide of formula (I) in an amount of at least 25%, or at least 30%, or at least 35%, and Cys is present in the polypeptide of formula (I) in an amount of at least 5.0%, or at least 7.0%, or at least 10.0%

5. The polypeptide of claim 1, wherein the peptide molecule has an amino acid sequence selected from the group consisting of:

RKRRRRYWCPKCRGGRC(SEQ ID NO:1)；

RKRRRRYWCPLGRC(SEQ ID NO:2)；

RKRYRRRFHKRGCGRC(SEQ ID NO:3)；

RRRRRCPWKRGCGRC(SEQ ID NO:4)；

RKRRRRCYPKRGCGRC(SEQ ID NO:5)；

RRRRWFCGKCCGRC(SEQ ID NO:6)；

RRRRRWCRGCGRC(SEQ ID NO:7)；

RRRRWFRCLGLPC(SEQ ID NO:8)；

RRRRWFRCLGTLC(SEQ ID NO:9)；

RRRRKFRGSSGRC(SEQ ID NO:10)；

RKRRKWRGCGRC(SEQ ID NO:11)；

RRFKRC(SEQ ID NO:12)；

RRRRRRRRRRWFRCHKCKCVRRCKLKRC(SEQ ID NO:13)；

RRRRHFCPQKKKRC(SEQ ID NO:14)；

RRHFCKRLKRC (SEQ ID NO: 15); or

RRRRWFRCKLKRC(SEQ ID NO:16)。

6. The polypeptide of claim 1, comprising a peptide sequence that is at least 75% similar to SEQ ID No.1 to SEQ ID No.16 of claim 5 and wherein the delivery of the bioactive molecule into the cell is improved by greater than 50%, or greater than 100% or about 200%.

7. A polypeptide nanoparticle composition at least comprises the polypeptide of the general formula I and a PEG high molecular material.

8. The polypeptide nanoparticle composition of claim 7, wherein the PEGylated polymeric material is a lipid-conjugated PEG derivative, poloxamer and/or poloxamine or a poloxamine derivative.

9. The polypeptide nanoparticle composition of claim 8, wherein the lipid-conjugated PEG derivative is mPEG ₂₀₀₀ -C-DMG、mPEG ₁₀₀₀₀ -C-CLS and DSPE-PEG ₂₀₀₀ -Mal、DSPE-PEG ₅₀₀₀ -NH ₂ 、DOPE-PEG ₂₀₀₀ -NH ₂ 、mPEG ₅₀₀₀ -DSPE、mPEG ₂₀₀₀ -DPPE、mPEG ₂₀₀₀ -DMPE。

10. The polypeptide nanoparticle composition according to claim 8, wherein the poloxamine is a polymer of 1, 2-ethylenediamine tetraacetic acid and ethylene oxide and methyl ethylene oxide or a polymer of 1, 2-ethylenediamine tetraacetic acid and methyl ethylene oxide and ethylene oxide, and the structural formula of the poloxamine is shown as formula (II) or formula (III):

11. the polypeptide nanoparticle composition of claim 10, wherein the poloxamine is selected from the group consisting of poloxamines

304、

701、

704、

707、

803、

901、

904、

908、

1107、

1301、

1304、

1307、

90R4 or

150R1 or more.

12. The polypeptide nanoparticle composition of claim 10, wherein the pegylated, polymeric lipid 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):

13. the polypeptide nanoparticle composition of claim 12, wherein the poloxamer is selected from the group consisting of poloxamers

17R4、

P-31R1、

L-121、

L-31、

L-64、

L-81、

P-10R5、

L-35、

L-61、

P-123、

F127 or

One or more of P-85.

14. The polypeptide nanoparticle composition of claim 8, wherein the poloxamine derivative has the formula:

in the formula: x is 4.3, y is 3.8,

in the formula: x is 4.3, y is 3.8,

in the formula: x is 4.3, y is 3.8,

in the formula: x is 4.3, y is 3.8,

in the formula: x is 14, y is 2.1,

in the formula: x is 18.2, y is 2.7,

in the formula: x is 15, y is 11,

in the formula: x is 17 and y is 15,

in the formula: x is 17 and y is 15,

in the formula: x is 17 and y is 15,

in the formula: x is 17 and y is 19,

in the formula: x is 17 and y is 19,

in the formula: x is 14, y is 13,

in the formula: x is 14, y is 13,

in the formula: x is 14 and y is 13.

15. The polypeptide nanoparticle composition of any of claims 7-14, further comprising a natural or synthetic lipid selected from the group consisting of: one or more of PC, DMPC, DPPC, DSPC, DMPE, DOTAP, DOPE, DOTMA, DOPC, DPPE or CLS.

16. A transfection complex comprising the polypeptide nanoparticle composition of claim 14, a nucleic acid.

17. A nucleic acid vaccine composition comprising the polypeptide nanoparticle composition of claim 15 or 16, a nucleic acid.

18. The transfection complex of claim 16 or the vaccine of claim 17, wherein said nucleic acid is RNA, preferably mRNA.

19. The nucleic acid vaccine composition of claim 17 or 18 for use in the prevention or treatment of:

(i) Infectious diseases;

(ii) Allergies or allergic diseases;

(iii) Autoimmune diseases; or

(iv) Cancer or neoplastic disease.