Drug delivery systems

Abstract

The present invention provides a drug delivery system, specifically provides a pharmaceutical carrier lipid conjugate, a pharmaceutical composition containing the lipid conjugate, and its preparation and application, which relates to the field of drug delivery. The lipid conjugate proposed in the present invention can bind to tumor cell surface receptors to improve cell uptake efficiency through structural improvement of the lipid conjugate itself and targeting peptides on the conjugate, thereby improving the expression level.

Description

Technical Field

[0001] The present invention relates to the field of drug delivery, and relates to a drug delivery system, and in particular to a pharmaceutical carrier lipid conjugate, a pharmaceutical composition comprising the lipid conjugate, and a preparation and application thereof. Background Art

[0002] Lipid nanoparticles (LNPs) are nanoparticles used in drug delivery systems, which are tiny particles wrapped by a lipid layer. The core of these nanoparticles is a preferred lipid collection, which is an amphiphilic molecule containing three domains: a polar head group, a hydrophobic tail region, and a connector between the two domains. The lipids of LNPs generally include cholesterol, phospholipids, ionizable / cationic lipids, and PEGylated lipids. Among them, cholesterol ensures the stability and fluidity of the membrane. Phospholipids provide the amphiphilicity necessary to form a lipid bilayer in nanoparticles. Ionizable lipids are protonated at low pH values, which makes them positively charged, but they remain neutral at physiological pH values. The pH sensitivity of ionizable lipids is conducive to the delivery of mRNA in vivo because neutral lipids interact less with the anionic membrane of blood cells, thereby improving the biocompatibility of lipid nanoparticles. PEGylated lipids are usually incorporated into the outer layer of lipid nanoparticles, which helps to improve stability, prolong circulation time, and reduce recognition by the immune system.

[0003] The lipids form a multifunctional structure that encapsulates and shields the payload, providing protection against challenging biological environments. At the same time, the unique composition of these lipids not only facilitates controlled release and targeted delivery, but also enhances biocompatibility, enabling stable drug loading and targeted delivery.

[0004] However, although LNPs preparations have the advantages mentioned above, the lack of selectivity for lesion tissues such as cancer cells is still a major problem. Therefore, there is still an urgent need for nanoparticle drug delivery systems that can effectively target lesions such as tumors and cancer cells to avoid the toxic side effects caused by the non-selective effects of drugs. Summary of the invention

[0005] 1. Technical issues to be resolved

[0006] In view of the deficiencies of the prior art, the present invention provides a lipid conjugate. Applying the lipid conjugate to a lipid nanoparticle system can achieve drug targeting to lesions such as tumors and cancer cells, thereby reducing or avoiding toxic side effects caused by the drugs.

[0007] (II) Technical solution

[0008] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0009] In one aspect, the present invention provides a lipid conjugate, wherein the lipid conjugate is a compound of formula (I):

[0010]

[0011] Ⅰ

[0012] The P (i.e., peptide) is a targeting peptide composed of 3 to about 50 amino acids connected;

[0013] The m is any natural number selected from 1 to 40;

[0014] The n is any natural number selected from 1 to 10;

[0015] Said q is any natural number selected from 1-40;

[0016] The R1 is an R group selected from amino acids;

[0017] R2 and R3 are independently selected from C 10 -C 23 The alkyl group is selected from a linear alkyl group, a branched alkyl group, a saturated alkyl group or a partially unsaturated alkyl group having one to three double bonds.

[0018] In one embodiment, the targeting peptide comprises RGD peptide, iRGD peptide (CRGDKGPDC), NGR peptide, Lyp-1 peptide or GE11 peptide.

[0019] In one embodiment, the peptide is selected from RGD, wherein RGD is a compound of formula (II):

[0020]

[0021] Ⅱ

[0022] Wherein, the connection point between the RGD peptide and formula (I) is .

[0023] In one embodiment, m is selected from any natural number within the range of 1-10.

[0024] In one embodiment, m is selected from 2.

[0025] In one embodiment, n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Preferably, n is 1, 2, 3, 4 or 5.

[0026] In one embodiment, q is any natural number selected from 1-30.

[0027] In one embodiment, q is any natural number selected from 5-20.

[0028] In one embodiment, q is selected from 6, 7, 8, 9 or 10. Preferably, q is selected from 8.

[0029] In one embodiment, R1 is selected from the R group of histidine (His), phenylalanine (Phe), tyrosine (Tyr) or tryptophan (Trp). The R structure of the amino acid refers to the side chain of the amino acid, which determines the characteristics and functions of the amino acid.

[0030] In one embodiment, R2 and R3 are selected from C 12 -C 18 A straight chain alkyl or branched chain alkyl, preferably C 15 Straight chain alkyl.

[0031] In one embodiment, the lipid conjugate is selected from any one of the following structural formulas:

[0032]

[0033] RGD005

[0034]

[0035] RGD006

[0036]

[0037] RGD007

[0038]

[0039] RGD008

[0040]

[0041] RGD032

[0042]

[0043] RGD035

[0044]

[0045] RGD038

[0046]

[0047] RGD041

[0048]

[0049] RGD044

[0050]

[0051] RGD0047

[0052] In one aspect, the present invention provides a lipid nanoparticle, wherein the lipid nanoparticle comprises the lipid conjugate described in any one of the above embodiments.

[0053] In one embodiment, the lipid nanoparticles further include one or more active ingredients, including chemical drugs, biological macromolecules, nucleic acid drugs, etc.

[0054] In one embodiment, the nucleic acid drug is selected from RNA and DNA.

[0055] In one embodiment, the nucleic acid drug is selected from any one of antisense nucleic acid, plasmid, mRNA, interfering nucleic acid, aptamer, antagomir, miRNA, ribozyme and siRNA; preferably any one of DNA, mRNA, miRNA and siRNA.

[0056] In one embodiment, the lipid nanoparticles include steroid lipids, phospholipids, ionizable / cationic lipids, pegylated lipids, and lipid conjugates described in any one of the above embodiments.

[0057] In one embodiment, the lipid nanoparticles include cholesterol, DSPC, any one of Compounds 1-4, DMG-PEG2000, and the lipid conjugate described in any one of the above embodiments.

[0058] In one embodiment, the molar ratio of the components in the lipid nanoparticles is steroid lipid: phospholipid: ionizable / cationic lipid: PEGylated lipid: lipid conjugate described in any of the above embodiments = (30~50): (5~15): (40~55): (1~3): (0.1~1.0), preferably 38:10:50:1.5:0.5.

[0059] In one embodiment, the lipid nanoparticle has a diameter of about 30 nm to about 200 nm.

[0060] In one embodiment, the lipid nanoparticle has a diameter of about 70 nm to about 150 nm.

[0061] In one embodiment, the lipid nanoparticle has a diameter of about 90 nm to about 110 nm.

[0062] In one embodiment, the phospholipid comprises phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine or sphingomyelin. In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0063] In one aspect, the present invention provides a pharmaceutical composition comprising any one of the lipid nanoparticles described above and a pharmaceutically acceptable carrier.

[0064] In one aspect, the present invention provides a method for delivering a drug to cancer cells in a patient or treating cancer, the method comprising administering an effective amount of the above-mentioned pharmaceutical composition or the above-mentioned lipid nanoparticles to cells or subjects in need of cancer treatment.

[0065] In one aspect, the present invention provides use of the above-mentioned pharmaceutical composition or the above-mentioned lipid nanoparticles in the preparation of drugs for preventing and / or inhibiting and / or treating cancer.

[0066] In one aspect, the present invention provides use of the above-mentioned pharmaceutical composition or the above-mentioned lipid nanoparticles in preparing a targeting carrier.

[0067] (III) Beneficial effects

[0068] The present invention provides a lipid conjugate and a lipid nanoparticle system comprising the lipid conjugate. Compared with the prior art, the present invention has the following beneficial effects:

[0069] The lipid conjugate proposed in the present invention can improve the cell uptake efficiency by binding to tumor cell surface receptors through the improvement of the lipid conjugate structure itself and the targeting peptide on the conjugate, thereby improving the in vitro expression level. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0071] Figure 1 High performance liquid chromatogram of compound RGD001; the ordinate of the figure represents mAU, and the abscissa represents time (unit: min);

[0072] Figure 2 High performance liquid chromatogram of compound RGD005; the ordinate of the figure represents AU, and the abscissa represents time (unit: min);

[0073] Figure 3High performance liquid chromatogram of compound RGD006; the ordinate of the figure represents AU, and the abscissa represents time (unit: min);

[0074] Figure 4 High performance liquid chromatogram of compound RGD007; the ordinate of the figure represents AU, and the abscissa represents time (unit: min);

[0075] Figure 5 High performance liquid chromatogram of compound RGD008; the ordinate of the figure represents AU, and the abscissa represents time (unit: min);

[0076] Figure 6 High performance liquid chromatogram of compound RGD047; the ordinate of the figure represents mAU, and the abscissa represents time (unit: min);

[0077] Figure 7 High performance liquid chromatogram of compound RGD032; the ordinate of the figure represents mAU, and the abscissa represents time (unit: min);

[0078] Figure 8 High performance liquid chromatogram of compound RGD035; the ordinate of the figure represents mAU, and the abscissa represents time (unit: min);

[0079] Fig. 9 High performance liquid chromatogram of compound RGD038; the ordinate of the figure represents mAU, and the abscissa represents time (unit: min);

[0080] Fig.10 High performance liquid chromatogram of compound RGD041; the ordinate of the figure represents mAU, and the abscissa represents time (unit: min);

[0081] Fig.11 High performance liquid chromatogram of compound RGD044; the ordinate of the figure represents mAU, and the abscissa represents time (unit: min);

[0082] Fig.12 Effects of LNPs prepared from different lipids on the expression level of cellular luciferase; *** indicates p<0.001, **** indicates p<0.0001;

[0083] Fig.13 The effect of LNPs prepared by modifying RGD peptide on the expression level of cellular luciferase. DETAILED DESCRIPTION

[0084] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0085] The technical solution in the embodiment of the present invention is to solve the above technical problems, and the overall idea is as follows:

[0086] The embodiment of the present invention provides a lipid conjugate, and applies the lipid conjugate to a lipid nanoparticle system, thereby achieving drug targeting to lesions such as tumors and cancer cells, and reducing or avoiding toxic side effects caused by the drug.

[0087] Application The present invention provides a lipid nanoparticle delivery system that exhibits enhanced cellular uptake compared to commonly used nanoparticles having longer PEG chains in their nanoparticles. At the same time, a lipid nanoparticle delivery system containing the lipid conjugate of the present invention also exhibits enhanced cellular uptake compared to other lipid nanoparticle delivery systems prepared from lipids containing targeting peptides.

[0088] Application The lipid nanoparticle delivery system of the present invention can be used to deliver a variety of therapeutic agents, including chemical drugs, macromolecular drugs, nucleic acid drugs, such as mRNA, etc.

[0089] definition:

[0090] The following definitions are included to provide a clear and consistent understanding of the specification and claims. As used herein, the referenced terms have the following meanings. All other terms and phrases used in this specification have their ordinary meanings as understood by those skilled in the art.

[0091] References in the specification to "one embodiment" or "an embodiment" etc. indicate that the described embodiment may include a particular aspect, feature, structure, part or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, part or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment mentioned in other parts of the specification. Moreover, when a particular aspect, feature, structure, part or characteristic is described in conjunction with an embodiment, it is within the knowledge of those skilled in the art to affect or relate such aspect, feature, structure, part or characteristic with other embodiments, whether or not explicitly described.

[0092] Unless the context clearly dictates otherwise, the singular forms "a," "an," and "the" include plural referents. Thus, for example, reference to "a compound" includes a plurality of such compounds, such that a compound X includes a plurality of compounds X. Note also that the claims may be drafted to exclude any optional elements. As such, this statement is intended to serve as antecedent basis for use of exclusive terminology, such as "solely," "only," and the like in connection with any element described herein, and / or for use of claim element invocation or "negative" limitations.

[0093] The term "and / or" means any one of the items, any combination of items, or all of the items associated with the term. The phrase "one or more" is readily understood by those skilled in the art, especially when read in the context of its use. For example, one or more substituents on a benzene ring refers to one to five, or one to four substituents (e.g., if the benzene ring is disubstituted).

[0094] The term "about" may refer to a change of ±5%, ±10%, ±20%, or ±25% of a specified value. For example, "about 50%" may have a change of 45 to 55% in some embodiments. For integer ranges, the term "about" may include one or two integers greater than and / or less than the integers quoted at each end of the range. Unless otherwise specified herein, the term "about" is intended to include values, such as weight percentages, that are equivalent to the functional aspects of a single component, composition, or embodiment near the quoted range. The term about may also modify the endpoints of the quoted range as discussed above in this paragraph.

[0095] As will be appreciated by those skilled in the art, all numerical values ​​(including those expressing amounts of ingredients, properties such as molecular weight, reaction conditions, etc.) are approximate and are to be understood as being optionally modified in all instances by the term "about". These values ​​may vary depending on the desired properties sought to be obtained by one skilled in the art utilizing the teachings of this specification. It is also to be understood that such values ​​inherently contain the variations necessarily resulting from the standard deviation present in their respective testing measurements.

[0096] Each operation or step may be described as multiple separate operations in turn in a manner that may be helpful for understanding the embodiments. However, the order of description should not be interpreted as implying that these operations are order-dependent.

[0097] The term "one embodiment" or "some embodiments" may be used in the specification, which may each refer to one or more identical or different embodiments. In addition, the terms "comprising", "including", "having" and the like are synonymous when used in relation to the embodiments.

[0098] As understood by those skilled in the art, for any and all purposes, especially in providing written descriptions, all ranges quoted herein also encompass any and all possible sub-ranges and combinations of their sub-ranges, as well as individual values, particularly integer values, constituting the ranges. The quoted ranges (e.g., weight percentages or carbon groups) include each specific value, integer, decimal, or entity within the range. Any listed range can be easily considered to fully describe and achieve the same range of being decomposed into at least equal two, three, four, five, or ten points. As a non-limiting example, each range discussed herein can be easily decomposed into the lower third, the middle third, and the upper third, etc. As also understood by those skilled in the art, all languages, such as "up to", "at least", "greater than", "less than", "exceed", "or more", etc., include quoted values, and such terms refer to the ranges that can be subsequently decomposed into sub-ranges as discussed above. In the same manner, all ratios quoted herein also include all sub-ratios falling within the wider ratios. Therefore, the specific values ​​quoted for free radicals, substituents, and ranges are only for illustration; they do not exclude other defined values ​​or other values ​​within the defined ranges of free radicals and substituents.

[0099] It is also readily understood by those skilled in the art that when members are grouped together in a common manner, such as in Markush groups, the present invention encompasses not only the complete group listed as a whole, but also each member of the group and all possible subgroups of the main group individually. In addition, for all purposes, the present invention encompasses not only the main group, but also the main group lacking one or more group members. Therefore, the present invention contemplates the explicit exclusion of any one or more members of the cited group. Therefore, this condition can be applied to any of the disclosed classes or embodiments, whereby any one or more of the cited elements, species, or embodiments can be excluded from the class or embodiment, for example, for explicit negative limitations.

[0100] "Effective amount" refers to an amount effective to treat a disease, disorder, and / or condition, or to bring about the stated effect. For example, an effective amount can be an amount effective to reduce the progression or severity of the disorder or symptom being treated. The determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, particularly in light of the disclosure provided herein. The term "effective amount" is intended to include an amount of a compound described herein or a combined amount of compounds described herein, for example, that is effective to treat or prevent a disease or disorder in a host, or to treat a symptom of the disease or disorder. Thus, "effective amount" generally refers to an amount that provides a desired effect.

[0101] The term "treat" includes (i) preventing a disease, pathology, or medical condition from occurring (e.g., prophylaxis); (ii) inhibiting a disease, pathology, or medical condition, or arresting its development; (iii) alleviating a disease, pathology, or medical condition; and / or (iv) reducing symptoms associated with the disease, pathology, or medical condition. Thus, the term "treat" may extend to prevention, and may include preventing, reducing, stopping, or reversing the progression or severity of the condition or symptom being treated. Thus, the term "treat" may include, as the case may be, medical, therapeutic, and / or prophylactic administration.

[0102] The term "inhibit" refers to slowing, arresting or reversing the growth or progression of a disease, infection, disorder or cell group. Inhibition can be, for example, greater than about 20%, 40%, 60%, 80%, 90%, 95% or 99% compared to growth or progression that occurs in the absence of treatment or contact.

[0103] "Alkyl" means a straight or branched, non-cyclic or cyclic saturated aliphatic hydrocarbon containing a certain number of carbon atoms. Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, etc.; while saturated branched chain alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, etc. Representative saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.; while unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, etc.

[0104] Targeting peptides are a class of peptides that have the ability to bind to specific receptors on the surface of tumor cells and can be used for targeted treatment, precision drug delivery, and the like. The targeting peptides include RGD peptides, iRGD peptides (CRGDKGPDC), NGR peptides, Lyp-1 peptides, or GE11 peptides. Among them, RGD peptides can bind to integrin receptors on the surface of tumor cells to achieve targeted targeted delivery. iRGD peptide (CRGDKGPDC): iRGD can recognize αvβ3 and αvβ5 receptors on the surface of tumor cells, enter tumor cells around blood vessels, stimulate translocation, and increase drug penetration. NGR peptide: NGR can bind to plasminogen activator receptors and is another commonly used targeting peptide suitable for tumor treatment. Lyp-1 peptide: Lyp-1 binds to receptors that selectively activate tumor cell membranes using pulse voltage, and has the potential for targeted anti-tumor drugs. GE11 peptide: GE11 can bind to EGFR receptors overexpressed on the surface of tumor cells for EGFR targeted therapy. Targeting peptides similar to RGD have specific receptor binding abilities and are suitable for different types of targeted therapies and drug delivery applications, providing new possibilities for personalized treatment. By selecting appropriate targeting peptides, precise drug delivery, reduced side effects, and improved treatment effects can be achieved.

[0105] The R group of an amino acid refers to the side chain of the amino acid, which determines the characteristics and functions of the amino acid. The basic structure of an amino acid is shown in Formula III below. Different amino acids have different R groups, for example, the R group of glycine is a hydrogen, the R group of lysine is a butylamine group (-CH2-CH2-CH2-CH2-NH2), and so on. The R group of an amino acid determines its position and function in the protein, and plays a key role in the structure and function of the protein.

[0106]

[0107] III

[0108] "Cationic lipid" refers to a lipid that contains a positive charge or is ionizable as a whole. Cationic lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 3-(didodecylamino)-N1,N1,4-tri-dodecyl-1-piperazineethylamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tri-dodecyl-1,4-piperazinediethylamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-triacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 4-(dimethylamino)butyric acid triheptadecyl-6,9,28,31-tetraen-19-yl ester (DLin-MC3-DMA) and 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), any one of Compound 1, Compound 2, Compound 3, Compound 4 and mixtures thereof.

[0109] The structure of compound 1 is as follows:

[0110] ;

[0111] The structure of compound 2 is as follows:

[0112] ;

[0113] The structure of compound 3 is as follows:

[0114] ;

[0115] The structure of compound 4 is as follows:

[0116] .

[0117] The "steroid lipid" is selected from any one of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol and mixtures thereof.

[0118] Phospholipids include, but are not limited to, 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-3-phosphocholine (DOPC ... sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0DietherPC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-arachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, Any one of oleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dioleoylphosphatidylserine (DOPS), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine and lysophosphatidylethanolamine (LPE) and combinations thereof. The neutral lipid may be of synthetic or natural origin.

[0119] "RNA" refers to ribonucleic acid that may be naturally occurring or non-naturally occurring. For example, RNA may include modified and / or non-naturally occurring components, such as one or more nucleobases, nucleosides, nucleotides, or linkers. RNA may include a cap structure, a chain-terminating nucleoside, a stem-loop, a polyadenylic acid sequence, and / or a polyadenylation signal. RNA may have a nucleotide sequence encoding a polypeptide of interest. For example, RNA may be a messenger RNA (mRNA). Translation of an mRNA encoding a specific polypeptide, such as in vivo translation of an mRNA inside a mammalian cell, may produce an encoded polypeptide. RNA may be selected from a non-limiting group consisting of small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), mRNA, single-stranded guide RNA (sgRNA), cas9mRNA, antisense oligonucleotides, and mixtures thereof. Antisense oligonucleotides or small interfering RNA (siRNA) may inhibit the expression of target genes and target proteins in vitro or in vivo.

[0120] "Delivery" refers to providing an entity to a target. For example, delivering a drug and / or therapeutic agent and / or prophylactic agent to a subject, which is a tissue and / or cell of a human and / or other animal.

[0121] "Pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient or vehicle administered together with the therapeutic agent, and is suitable for contacting the tissues of humans and / or other animals without excessive toxicity, irritation, allergic reaction or other problems or complications corresponding to a reasonable benefit / risk ratio within the scope of reasonable medical judgment. Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention include, but are not limited to, sterile liquids, such as water and oils, including those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is an exemplary carrier. Physiological saline and glucose and glycerol aqueous solutions can also be used as liquid carriers, particularly for injection. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, maltose, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, etc. The composition can also contain a small amount of wetting agent, emulsifier or pH buffer as needed. Oral formulations may contain standard carriers, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Specifically, for example, excipients include, but are not limited to, anti-adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (pigments), demulcents, emulsifiers, fillers (diluents), film formers or coatings, flavorings, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, adsorbents, suspending agents or dispersing agents, sweeteners, and hydration water. More specifically, excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, dicalcium phosphate, calcium stearate, cross-linked carboxymethylcellulose sodium, cross-linked polyvinyl pyrrolidone, citric acid, cross-linked polyvinyl pyrrolidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, phenylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (α-tocopherol), vitamin C, and xylitol.

[0122] The pharmaceutical composition of the present invention can act systemically and / or locally. For this purpose, they can be administered by suitable routes, for example, by injection (such as intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular injection, including instillation) or transdermal administration; or by oral, buccal, nasal, transmucosal, local, in the form of ophthalmic preparations or by inhalation administration. For these routes of administration, the pharmaceutical composition of the present invention can be administered in suitable dosage forms. The dosage form includes but is not limited to tablets, capsules, lozenges, hard candies, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs, syrups.

[0123] In order to better understand the above technical solution, the above technical solution will be described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0124] Example 1 Synthesis of Compound RGD001

[0125] Compound RGD001

[0126] .

[0127] The synthetic route of compound RGD001 is as follows:

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138] The synthesis experiment of compound RGD001 is as follows:

[0139] (1) Synthesis of RGD001-2

[0140] 1) Resin swelling

[0141] SiberResin (20.00 g, 12.00 mmol) and DMF (200 mL) were added to a 1000 ml solid phase synthesis reactor, and the DMF was dried after ventilation for 30 min.

[0142] 2) Coupling Fmoc-Asp(OtBu)-OH

[0143] Add 200 mL of Deblock solution (20% piperidine in DMF solution, V / V), react for 20 min under air pump blowing conditions, then drain the solution. Ninhydrin detection showed that the Fmoc group was completely removed. Drain the reaction solution and wash the resin with DMF (200 mL*5).

[0144] Fmoc-Asp(OtBu)-OH (9.85 g, 24.00 mmol), HOBt (3.24 g, 24.00 mmol), and DIC (3.03 g, 24.00 mmol) were dissolved in DMF (160 mL). After activation for 3 min, the mixture was added into the solid phase reaction reactor and reacted for 1 h under air pump blowing. The reaction was complete when tested with ninhydrin. The reaction solution was drained and the resin was washed with DMF (200 mL*3).

[0145] 3) Coupling Fmoc-Gly-OH

[0146] Add 200 mL of Deblock solution (20% piperidine in DMF solution, V / V) and react for 20 min under air pump conditions. Ninhydrin detection shows that the Fmoc group is completely removed. Drain the reaction solution and wash the resin with DMF (200 mL*5).

[0147] Fmoc-Gly-OH (7.14 g, 24.00 mmol), HOBt (3.24 g, 24.00 mmol), and DIC (3.03 g, 24.00 mmol) were dissolved in DMF (160 mL). After activation for 3 min, the mixture was added into the solid phase reaction reactor and reacted for 1 h under air pump blowing. The reaction was complete when tested with ninhydrin. The reaction solution was drained and the resin was washed with DMF (200 mL*3).

[0148] 4) Coupling Fmoc-Arg(Pbf)-OH

[0149] Add 200 mL of Deblock solution (20% piperidine in DMF solution, V / V) and react for 20 min under air pump conditions. Ninhydrin detection shows that the Fmoc group is completely removed. Drain the reaction solution and wash the resin with DMF (200 mL*5).

[0150] Fmoc-Arg(Pbf)-OH (15.57 g, 24.00 mmol), HOBt (3.24 g, 24.00 mmol), and DIC (3.03 g, 24.00 mmol) were dissolved in DMF (160 mL). After activation for 3 min, the mixture was added into the solid phase reaction reactor and reacted for 1 h under air pump blowing. The reaction was complete when tested with ninhydrin. The reaction solution was drained and the resin was washed with DMF (200 mL*3).

[0151] 5) Coupling Fmoc-Gly-OH

[0152] Add 200 mL of Deblock solution (20% piperidine in DMF solution, V / V) and react for 20 min under air pump conditions. Ninhydrin detection shows that the Fmoc group is completely removed. Drain the reaction solution and wash the resin with DMF (200 mL*5).

[0153] Fmoc-Gly-OH (7.14 g, 24.00 mmol), HOBt (3.24 g, 24.00 mmol), and DIC (3.03 g, 24.00 mmol) were dissolved in DMF (160 mL). After activation for 3 min, the mixture was added into the solid phase reaction reactor and reacted for 1 h under air pump blowing. The reaction was complete when tested with ninhydrin. The reaction solution was drained and the resin was washed with DMF (200 mL*3).

[0154] 6) Coupling Fmoc-Lys(Boc)-OH

[0155] Add 200 mL of Deblock solution (20% piperidine in DMF solution, V / V) and react for 20 min under air pump conditions. Ninhydrin detection shows that the Fmoc group is completely removed. Drain the reaction solution and wash the resin with DMF (200 mL*5).

[0156] Fmoc-Lys(Boc)-OH (11.25 g, 24.00 mmol), HOBt (3.24 g, 24.00 mmol), and DIC (3.03 g, 24.000 mmol) were dissolved in DMF (160 mL). After activation for 3 min, the mixture was added into the solid phase reaction reactor and reacted for 1 h under air pump blowing. The reaction was complete when tested with ninhydrin. The reaction solution was drained and the resin was washed with DMF (200 mL*3).

[0157] 7) Removal of Fmoc

[0158] Add 200 mL of Deblock solution (20% piperidine in DMF solution, V / V) and react for 20 min under air pump blowing. Ninhydrin detection showed that the Fmoc group was completely removed. Drain the reaction solution and wash the resin with DMF (200 mL*5), DCM (200 mL*2), and MeOH (200 mL*2) in sequence. Dry and weigh to obtain resin RGD001-2 (32.38 g, yield: 103.55%).

[0159] (2) Synthesis of RGD001-10

[0160] RGD001-2 (32.38 g, 12 mmol) was added to a 1000 mL single-mouth bottle, and 500 mL of 1% trifluoroacetic acid in dichloromethane solution was added. The reaction was allowed to react for 1 h. The reaction solution was filtered, and the filter cake was washed with DCM once (50 mL*1). The filter cake was cracked again with 500 mL of 1% trifluoroacetic acid in dichloromethane solution for 1 h, filtered, and the filtrate was combined and concentrated to dryness. 400 mL of MTBE was added for slurrying for 30 min, filtered, and the solid was dried to obtain RGD001-10 (11.80 g, yield: 93.35%).

[0161] (3) Synthesis of RGD001-4

[0162] 1) Resin swelling

[0163] 2-CTCResin (10.00 g, 10.30 mmol) and DMF (100 mL) were added into a 250 ml solid phase synthesis reactor and ventilated for 30 min.

[0164] 2) Coupling of Fmoc-9-amino-4,7-dioxanoic acid

[0165] Fmoc-9-amino-4,7-dioxonanoic acid (12.35 g, 30.92 mmol) and DIEA (8.02 g, 62.05 mmol) were added, and the reaction was continued for 5 h under air pump blowing. Then 10 ml of methanol was added, and the reaction was continued for 1 h. The reaction solution was drained and the resin was washed with DMF (100 mL*3).

[0166] 3) Coupling Fmoc-Lys(Boc)-OH

[0167] Add 80 mL of Deblock solution (20% piperidine in DMF solution, V / V) and react for 20 min under air pump blowing conditions. Ninhydrin detection shows that the Fmoc group is completely removed. Drain the reaction solution and wash the resin with DMF (100 mL*5).

[0168] Dissolve Fmoc-Lys(Boc)-OH(8.69 g, 18.55 mmol), HBTU(7.03 g, 18.55 mmol), and DIEA(4.79 g, 37.06 mmol) in DMF (60 mL). After activation for 3 min, add the mixture into the solid phase reaction reactor and react for 1 h under the condition of air pump blowing. The reaction was complete after ninhydrin detection. Drain the reaction solution and wash the resin with DMF (100 mL*3). Repeat three times to couple 3 Fmoc-Lys(Boc)-OH on the resin. Wash the resin with DCM (100 ml*2), MeOH (100 ml*2), and dry to obtain resin RGD001-4 (16.52 g, yield: 60.43%).

[0169] (4) Synthesis of RGD001-7

[0170] 1) Resin swelling

[0171] RGD001-4 (2.00 g, 0.75 mmol) and DMF (20 mL) were added into a 50 ml solid phase synthesis reactor, and the DMF was dried after ventilation for 30 min.

[0172] 2) Coupling 1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16,19,22,25,28-nonaoxa-4-azahexadecane-31-carboxylic acid

[0173] Add 20 mL of Deblock solution (20% piperidine in DMF solution, V / V) and react for 20 min under air pump blowing conditions. The ninhydrin test showed that the Fmoc group was completely removed. Drain the reaction solution and wash the resin with DMF (20 mL*5).

[0174] 1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16,19,22,25,28-nonaoxa-4-azatriacontane-31-carboxylic acid (1.0000 g, 1.51 mmol), HBTU (0.8510 g, 2.24 mmol), and DIEA (0.5803 g, 4.49 mmol) were dissolved in DMF (10 mL). After activation for 3 min, the mixture was added into the solid phase reaction reactor and reacted for 1 h under air pump blowing. The reaction was completed when tested with ninhydrin. The reaction solution was drained and the resin was washed with DMF (20 mL*3).

[0175] 3) Coupling Fmoc-Trp(Boc)-OH

[0176] Add 20 mL of Deblock solution (20% piperidine in DMF solution, V / V) and react for 20 min under air pump blowing conditions. The ninhydrin test showed that the Fmoc group was completely removed. Drain the reaction solution and wash the resin with DMF (20 mL*5).

[0177] Fmoc-Trp(Boc)-OH (1.1819 g, 2.24 mmol), HBTU (0.8510 g, 2.24 mmol), and DIEA (0.5803 g, 4.49 mmol) were dissolved in DMF (10 mL). After activation for 3 min, the mixture was added into the solid phase reaction reactor and reacted for 1 h under air pump blowing. The reaction was complete when tested with ninhydrin. The reaction solution was drained and the resin was washed with DMF (20 mL*3).

[0178] 4) Coupling Fmoc-Lys(Fmoc)-OH

[0179] Add 20 mL of Deblock solution (20% piperidine in DMF solution, V / V) and react for 20 min under air pump blowing conditions. The ninhydrin test showed that the Fmoc group was completely removed. Drain the reaction solution and wash the resin with DMF (20 mL*5).

[0180] Fmoc-Lys(Fmoc)-OH (1.3258 g, 2.24 mmol), HBTU (0.8510 g, 2.24 mmol), and DIEA (0.5803 g, 4.49 mmol) were dissolved in DMF (10 mL) and activated for 3 min before adding them into the solid phase reaction reactor. The reaction was carried out under the condition of air pump blowing for 1 h. The ninhydrin test showed that the reaction was complete. The reaction solution was drained and the resin was washed with DMF (20 mL*3).

[0181] 5) Coupling with palmitic acid

[0182] Add 20 mL of Deblock solution (20% piperidine in DMF solution, V / V) and react for 20 min under air pump blowing conditions. The ninhydrin test showed that the Fmoc group was completely removed. Drain the reaction solution and wash the resin with DMF (20 mL*5).

[0183] Palmitic acid (1.1511 g, 4.49 mmol), HBTU (1.7025 g, 4.49 mmol), and DIEA (0.5803 g, 4.49 mmol) were dissolved in DCM (10 mL) together. After activation for 3 min, they were added into the solid phase reaction synthesis reactor and reacted for 1 h under air pump blowing conditions. The ninhydrin detection showed that the reaction was complete. The reaction liquid was drained and the resin was washed with DMF (20 mL*3), DCM (20 mL*2), and MeOH (20 mL*2) in turn and dried to obtain resin RGD001-7 (2.38 g).

[0184] (5) Synthesis of RGD001-8

[0185] RGD001-7 (2.38 g, 0.75 mmol) was added to a 25 ml single-mouth bottle, and a 1% trifluoroacetic acid solution in dichloromethane was added, and the reaction was continued for 2 h. The reaction solution was concentrated to remove DCM, and then 20 mL of MTBE was added to precipitate a white solid, which was centrifuged and dried to obtain RGD001-8 (1.0028 g, yield: 71.63%).

[0186] (6) Synthesis of RGD001-11

[0187] In a 25mL single-mouth bottle, RGD001-8 (201.5mg, 0.09mmol) and HATU (53.2mg, 0.14mmol) were dissolved in DMF (5mL), and DIEA (30.0mg, 0.23mmol) was added, and finally RGD001-10 (104.3mg, 0.11mmol) was added. After 4h of reaction, the reaction solution was added dropwise to MTBE (50ml), and a white solid was precipitated. The solid was centrifuged and dried to obtain RGD001-11 (252.0mg, yield: 87.94%).

[0188] (7) Synthesis of RGD001

[0189] RGD001-11 (252.0 mg, 0.81 mmol) was added to a 25 mL single-mouth bottle, and 3 mL of the prepared cleavage reagent (TFA: Tis: H2O = 95: 2.5: 2.5) was added. After reacting for 3 hours, the reaction solution was added dropwise to pre-cooled MTBE, and the solid was precipitated, centrifuged, and dried to obtain RGD001 crude product (152.0 mg). The pure product of RGD001 (116.0 mg, purity: 96.51%, yield: 62.37%) was obtained by preparation and purification. MS-ESI (m / z): 764.1 (M+3H) 3+ ,573.3(M+4H) 4+ ,458.8(M+5H) 5+ . HPLC chromatogram Figure 1 shown.

[0190] Example 2 Synthesis of Compound RGD005

[0191] Compound RGD005 is shown below:

[0192] .

[0193] The synthetic route of compound RGD005 is as follows:

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205] .

[0206] The synthesis experiment of compound RGD005 is as follows:

[0207] (1) Synthesis of RGD001-2

[0208] 1) Resin swelling

[0209] SiberResin (20.00 g, 12.00 mmol) and DMF (200 mL) were added to a 1000 ml solid phase synthesis reactor, and the DMF was dried after ventilation for 30 min.

[0210] 2) Coupling Fmoc-Asp(OtBu)-OH

[0211] Add 200 mL of Deblock solution (20% piperidine in DMF solution, V / V), react for 20 min under air pump blowing conditions, then drain the solution. Ninhydrin detection showed that the Fmoc group was completely removed. Drain the reaction solution and wash the resin with DMF (200 mL*5).

[0212] Fmoc-Asp(OtBu)-OH (9.85 g, 24.00 mmol), HOBt (3.24 g, 24.00 mmol), and DIC (3.03 g, 24.00 mmol) were dissolved in DMF (160 mL). After activation for 3 min, the mixture was added into the solid phase reaction reactor and reacted for 1 h under air pump blowing. The reaction was complete when tested with ninhydrin. The reaction solution was drained and the resin was washed with DMF (200 mL*3).

[0213] 3) Coupling Fmoc-Gly-OH

[0214] Add 200 mL of Deblock solution (20% piperidine in DMF solution, V / V) and react for 20 min under air pump conditions. Ninhydrin detection shows that the Fmoc group is completely removed. Drain the reaction solution and wash the resin with DMF (200 mL*5).

[0215] Fmoc-Gly-OH (7.14 g, 24.00 mmol), HOBt (3.24 g, 24.00 mmol), and DIC (3.03 g, 24.00 mmol) were dissolved in DMF (160 mL). After activation for 3 min, the mixture was added into the solid phase reaction reactor and reacted for 1 h under air pump blowing. The reaction was complete when tested with ninhydrin. The reaction solution was drained and the resin was washed with DMF (200 mL*3).

[0216] 4) Coupling Fmoc-Arg(Pbf)-OH

[0217] Add 200 mL of Deblock solution (20% piperidine in DMF solution, V / V) and react for 20 min under air pump conditions. Ninhydrin detection shows that the Fmoc group is completely removed. Drain the reaction solution and wash the resin with DMF (200 mL*5).

[0218] Fmoc-Arg(Pbf)-OH (15.57 g, 24.00 mmol), HOBt (3.24 g, 24.00 mmol), and DIC (3.03 g, 24.00 mmol) were dissolved in DMF (160 mL). After activation for 3 min, the mixture was added into the solid phase reaction reactor and reacted for 1 h under air pump blowing. The reaction was complete when tested with ninhydrin. The reaction solution was drained and the resin was washed with DMF (200 mL*3).

[0219] 5) Coupling Fmoc-Gly-OH

[0220] Add 200 mL of Deblock solution (20% piperidine in DMF solution, V / V) and react for 20 min under air pump conditions. Ninhydrin detection shows that the Fmoc group is completely removed. Drain the reaction solution and wash the resin with DMF (200 mL*5).

[0221] Fmoc-Gly-OH (7.14 g, 24.00 mmol), HOBt (3.24 g, 24.00 mmol), and DIC (3.03 g, 24.00 mmol) were dissolved in DMF (160 mL). After activation for 3 min, the mixture was added into the solid phase reaction reactor and reacted for 1 h under air pump blowing. The reaction was complete when tested with ninhydrin. The reaction solution was drained and the resin was washed with DMF (200 mL*3).

[0222] 6) Coupling Fmoc-Lys(Boc)-OH

[0223] Add 200 mL of Deblock solution (20% piperidine in DMF solution, V / V) and react for 20 min under air pump conditions. Ninhydrin detection shows that the Fmoc group is completely removed. Drain the reaction solution and wash the resin with DMF (200 mL*5).

[0224] Fmoc-Lys(Boc)-OH (11.25 g, 24.00 mmol), HOBt (3.24 g, 24.00 mmol), and DIC (3.03 g, 24.000 mmol) were dissolved in DMF (160 mL). After activation for 3 min, the mixture was added into the solid phase reaction reactor and reacted for 1 h under air pump blowing. The reaction was complete when tested with ninhydrin. The reaction solution was drained and the resin was washed with DMF (200 mL*3).

[0225] 7) Removal of Fmoc

[0226] Add 200 mL of Deblock solution (20% piperidine in DMF solution, V / V) and react for 20 min under air pump blowing. Ninhydrin detection showed that the Fmoc group was completely removed. Drain the reaction solution and wash the resin with DMF (200 mL*5), DCM (200 mL*2), and MeOH (200 mL*2) in sequence. Dry and weigh to obtain resin RGD001-2 (32.38 g, yield: 103.55%).

[0227] (2) Synthesis of RGD001-10

[0228] RGD001-2 (32.38 g, 12 mmol) was added to a 1000 mL single-mouth bottle, and 500 mL of 1% trifluoroacetic acid in dichloromethane solution was added. The reaction was allowed to react for 1 h. The reaction solution was filtered, and the filter cake was washed with DCM once (50 mL*1). The filter cake was cracked again with 500 mL of 1% trifluoroacetic acid in dichloromethane solution for 1 h, filtered, the filtrate was combined, concentrated to dryness, 400 mL of MTBE was added for slurrying for 30 min, filtered, and dried to obtain RGD001-10 (11.80 g, yield: 93.35%).

[0229] (3) Synthesis of RGD002-1

[0230] 1) Resin swelling

[0231] 2-CTCResin (10.00 g, 10.30 mmol) and DMF (100 mL) were added into a 250 ml solid phase synthesis reactor and ventilated for 30 min.

[0232] 2) Coupling of Fmoc-9-amino-4,7-dioxanoic acid

[0233] Fmoc-9-amino-4,7-dioxonanoic acid (12.35 g, 30.92 mmol) and DIEA (8.02 g, 62.05 mmol) were added, and the reaction was continued for 5 h under air pump blowing. Then 10 ml of methanol was added, and the reaction was continued for 1 h. Then the reaction solution was drained, and the resin was washed with DMF (100 mL*3), DCM (100 mL*2), and MeOH (100 mL*2) in sequence and dried to obtain resin RGD002-1 (14.85 g).

[0234] (4) Synthesis of RGD002-2

[0235] 1) Resin swelling

[0236] Resin RGD002-1 (4.95 g, 3.43 mmol) and DMF (50 mL) were added to a 100 mL solid phase synthesis reactor, and the DMF was dried after ventilation for 30 min.

[0237] 2) Coupling Fmoc-Arg(pbf)-OH

[0238] Add 50 mL of Deblock solution (20% piperidine in DMF solution, V / V) and react for 20 min under air pump blowing conditions. Ninhydrin detection shows that the Fmoc group is completely removed. Drain the reaction solution and wash the resin with DMF (50 mL*5).

[0239] Dissolve Fmoc-Arg(pbf)-OH (13.37 g, 20.60 mmol), HBTU (7.81 g, 20.60 mmol), and DIEA (5.33 g, 41.23 mmol) in DMF (50 mL). After activation for 3 min, add them into the solid phase reaction reactor and react for 1 h under the condition of air pump blowing. The reaction is complete when tested with ninhydrin. Drain the reaction liquid and wash the resin with DMF (80 mL*3). Repeat three times to couple 3 Fmoc-Arg(pbf)-OH on the resin.

[0240] 3) Coupling 1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16,19,22,25,28-nonaoxa-4-azahexadecane-31-carboxylic acid

[0241] Add 50 mL of Deblock solution (20% piperidine in DMF solution, V / V) and react for 20 min under air pump blowing conditions. Ninhydrin detection shows that the Fmoc group is completely removed. Drain the reaction solution and wash the resin with DMF (50 mL*5).

[0242] 1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16,19,22,25,28-nonaoxa-4-azahexanedioneconane-31-carboxylic acid (2.46 g, 3.71 mmol), HBTU (2.34 g, 6.17 mmol), and DIEA (1.61 g, 12.46 mmol) were dissolved in DMF (30 mL) and activated for 3 min before adding the mixture into a solid phase reaction reactor. The mixture was reacted for 1 h under air pump blowing conditions. The reaction was complete when tested with ninhydrin. The reaction solution was drained and the resin was washed with DMF (50 mL*3), DCM (50 mL*2), and MeOH (50 mL*2) in sequence and dried to obtain resin RGD002-2 (7.98 g).

[0243] (5) Synthesis of RGD002-3

[0244] 1) Resin swelling

[0245] RGD002-2 (2.00 g, 0.86 mmol) and DMF (20 mL) were added to a 50 mL solid phase synthesis reactor, and the DMF was dried after ventilation for 30 min.

[0246] 2) Coupling Fmoc-Trp(Boc)-OH

[0247] Add 20 mL of Deblock solution (20% piperidine in DMF solution, V / V) and react for 20 min under air pump blowing conditions. The ninhydrin test showed that the Fmoc group was completely removed. Drain the reaction solution and wash the resin with DMF (20 mL*5).

[0248] Fmoc-Trp(Boc)-OH (0.9056 g, 1.72 mmol), HBTU (0.6523 g, 1.72 mmol), and DIEA (0.4446 g, 3.44 mmol) were dissolved in DMF (8 mL) and activated for 3 min before adding them into the solid phase reaction reactor. The reaction was carried out for 1 h under air pump blowing. The ninhydrin test showed that the reaction was complete. The reaction solution was drained and the resin was washed with DMF (20 mL*3).

[0249] 3) Coupling Fmoc-Lys(Fmoc)-OH

[0250] Add 20 mL of Deblock solution (20% piperidine in DMF solution, V / V) and react for 20 min under air pump blowing conditions. The ninhydrin test showed that the Fmoc group was completely removed. Drain the reaction solution and wash the resin with DMF (20 mL*5).

[0251] Fmoc-Lys(Fmoc)-OH (1.0159 g, 1.72 mmol), HBTU (0.6523 g, 1.72 mmol), and DIEA (0.4446 g, 3.44 mmol) were dissolved in DMF (8 mL) and activated for 3 min before adding them into the solid phase reaction reactor. The reaction was carried out for 1 h under air pump blowing. The ninhydrin test showed that the reaction was complete. The reaction solution was drained and the resin was washed with DMF (20 mL*3).

[0252] 4) Coupling with palmitic acid

[0253] Add 20 mL of Deblock solution (20% piperidine in DMF solution, V / V) and react for 20 min under air pump blowing conditions. The ninhydrin test showed that the Fmoc group was completely removed. Drain the reaction solution and wash the resin with DMF (20 mL*5).

[0254] Palmitic acid (0.8821 g, 3.44 mmol), HBTU (1.3046 g, 3.44 mmol), (0.4446 g, 3.44 mmol) were dissolved in DCM (8 mL) together, activated for 3 min, and added into the solid phase reaction synthesis reactor. The reaction was carried out for 1 h under air pump blowing conditions. The ninhydrin detection showed that the reaction was complete. The reaction liquid was drained, and the resin was washed with DMF (20 mL*3), DCM (20 mL*2), and MeOH (20 mL*2) in turn. After drying, resin RGD002-3 (2.12 g) was obtained.

[0255] (6) Synthesis of RGD002-5

[0256] RGD002-3 (2.12 g, 0.86 mmol) was added to a 25 ml single-mouth bottle, and a 1% trifluoroacetic acid solution in dichloromethane was added, and the reaction was continued for 2 h. The reaction solution was concentrated to remove DCM, and then 20 mL of MTBE was added to precipitate a large amount of white solid, which was centrifuged and dried to obtain RGD002-5 (1.2154 g, yield: 58.71%).

[0257] (7) Synthesis of RGD002-7

[0258] In a 25mL single-mouth bottle, RGD002-5 (377.3mg, 0.14mmol) and HATU (79.2mg, 0.21mol) were dissolved in DMF (5mL), and DIEA (35.9mg, 0.28mmol) was added, and finally RGD001-10 (130.4mg, 0.14mmol) was added. After reacting for 2h, the reaction solution was added dropwise to MTBE (50ml), and a white solid was precipitated. The solid was centrifuged and dried to obtain RGD002-7 (490.0mg, yield: 76.86%).

[0259] (8) Synthesis of RGD005

[0260] RGD002-7 (490.0 mg, 0.14 mmol) was added to a 25 mL single-mouth bottle, and 10 mL of the prepared cleavage reagent (TFA: Tis: H2O = 95: 2.5: 2.5) was added. After reacting for 3 hours, the reaction solution was added dropwise to pre-cooled MTBE, solids were precipitated, centrifuged, and dried to obtain crude RGD005, which was prepared and purified to obtain pure RGD001 (78.1 mg, purity: 99.14%, yield: 24.30%). MS-ESI (m / z): 792.1 (M+3H) 3+ ,594.2(M+4H) 4+ ,475.6(M+5H) 5+ . HPLC chromatogram Figure 2 shown.

[0261] Example 3 Synthesis of Compound RGD006

[0262] Compound RGD006

[0263]

[0264] Experimental operation

[0265] The synthetic route is the same as RGD005, and Fmoc-Trp(Boc)-OH is replaced by Fmoc-Tyr(tbu)-OH to obtain compound RGD006 (107.0 mg, purity: 99.75%, yield: 23.20%). MS-ESI (m / z): 784.2 (M+3H) 3+ ,588.4(M+4H) 4+ ,471.0(M+5H) 5+ . HPLC chromatogram Figure 3 shown.

[0266] Example 4 Synthesis of Compound RGD007

[0267] Compound RGD007

[0268]

[0269] Experimental operation

[0270] The synthetic route is the same as RGD005, and Fmoc-Trp(Boc)-OH is replaced by Fmoc-Phe-OH to obtain compound RGD007 (232.6 mg, purity: 98.32%, yield: 43.98%). MS-ESI (m / z): 1168.3 (M+2H) 2+ ,779.3(M+3H) 3+ ,584.8(M+4H) 4+ ,468.1(M+5H) 5+ . HPLC chromatogram Figure 4 shown.

[0271] Example 5 Synthesis of Compound RGD008

[0272] Compound RGD008

[0273]

[0274] Experimental operation

[0275] The synthetic route is the same as RGD005, but Fmoc-Trp(Boc)-OH is replaced by Fmoc-His(trt)-OH to obtain compound RGD008 (60.2 mg, purity: 99.07%, yield: 24.85%). MS-ESI (m / z): 776.0 (M+3H) 3+ ,582.3(M+4H) 4+ ,466.0(M+5H) 5+ . HPLC chromatogram Figure 5 shown.

[0276] Example 6 Synthesis of Compound RGD047

[0277] Compound RGD0047

[0278]

[0279] Experimental operation

[0280] The synthetic route is the same as RGD005, but without coupling with Fmoc-Trp(Boc)-OH, after coupling with 1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16,19,22,25,28-nonaoxa-4-azahexanedecan-31-carboxylic acid, directly coupling with Fmoc-Lys(Fmoc)-OH can produce compound RGD047 (55.4 mg, purity: 92.41%, yield: 13.08%). MS-ESI (m / z): 1092.7 (M+2H) 2+ . HPLC chromatogram Figure 6 shown.

[0281] Example 7 Synthesis of Compound RGD032

[0282] Compound RGD032

[0283]

[0284] Experimental operation

[0285] The synthetic route is the same as RGD005, but without coupling with Fmoc-Arg(pbf)-OH, after coupling with Fmoc-9-amino-4,7-dioxonanoic acid, directly coupling with 1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16,19,22,25,28-nonaoxa-4-azahexanedicosane-31-carboxylic acid can produce compound RGD032 (21.6 mg, purity: 94.25%, yield: 4.88%). MS-ESI (m / z): 953.4 (M+2H) 2+ ,635.9(M+3H) 3+ . HPLC chromatogram Figure 7 shown.

[0286] Example 8 Synthesis of Compound RGD035

[0287] Compound RGD035

[0288]

[0289] Experimental operation

[0290] The synthetic route is the same as RGD005. The number of coupled Fmoc-Arg(pbf)-OH in the synthetic route of intermediate RGD002-5 is changed to 1, and compound RGD035 (128.1 mg, purity: 91.12%, yield: 27.54%) can be obtained. MS-ESI (m / z): 688.1 (M+3H) 3+ ,516.1(M+4H) 4+ . HPLC chromatogram Figure 8 shown.

[0291] Example 9 Synthesis of Compound RGD038

[0292] Compound RGD038

[0293]

[0294] Experimental operation

[0295] The synthetic route is the same as RGD005. The number of coupled Fmoc-Arg(pbf)-OH in the synthetic route of intermediate RGD002-5 is changed to 2, and compound RGD038 (78.1 mg, purity: 95.89%, yield: 17.50%) can be obtained. MS-ESI (m / z): 740.0 (M+3H) 3+ ,555.2(M+4H) 4+ . HPLC chromatogram Fig. 9 shown.

[0296] Example 10 Synthesis of Compound RGD041

[0297] Compound RGD041

[0298]

[0299] Experimental operation

[0300] The synthetic route is the same as RGD005. The number of coupled Fmoc-Arg(pbf)-OH in the synthetic route of intermediate RGD002-5 is changed to 4, and compound RGD041 (82.6 mg, purity: 95.09%, yield: 18.88%) can be obtained. MS-ESI (m / z): 633.3 (M+4H) 4+ ,506.9(M+5H) 5+ . HPLC chromatogram Fig.10 shown.

[0301] Example 11 Synthesis of Compound RGD044

[0302] Compound RGD044

[0303]

[0304] Experimental operation

[0305] The synthetic route is the same as RGD005. The number of coupled Fmoc-Arg(pbf)-OH in the synthetic route of intermediate RGD002-5 is changed to 5, and compound RGD044 (84.6 mg, purity: 99.81%, yield: 20.85%) can be obtained. MS-ESI (m / z): 896.1 (M+3H) 3+ ,672.2(M+4H) 4+ ,538.1(M+5H) 5+ ,448.6(M+6H) 6+ . HPLC chromatogram Fig.11 shown.

[0306] Example 12 Preparation of LNP

[0307] The basic preparation method of LNP is as follows:

[0308] Luciferase mRNA was prepared into an mRNA solution (aqueous phase) with a concentration of 200 μg / ml using 20 mM acetic acid. The cationic lipid (compound 2): DSPC: cholesterol: DMG-PEG2000: RGD compound = 50:10: 38:1.5:0.5 was dissolved in ethanol to prepare a mixed lipid solution (organic phase), and the aqueous phase and the organic phase were mixed by T flow to prepare LNP-encapsulated mRNA. The buffer was replaced with a 2 mM acetic acid-250 mM sucrose solution to reduce the ethanol concentration in the solution, and 500 mMTris was added to adjust the sample pH to 7.2-8.0. Finally, the sample was filtered through a 0.22 μm PES filter for use.

[0309] Finally, the total and free mRNA content in LNP was detected using the Quant-iTRibogreen kit and 10% OTG as a demulsifier, and the encapsulation efficiency of LNP was calculated. The final LNP product was diluted with diluent, added to 1 ml of the particle size pool, and placed on the Malvern ZetaSizer instrument. The particle size and polydispersity index (PDI) of LNP were detected by dynamic light scattering. The results are shown in Table 1.

[0310] The results showed that the preparations obtained by adding different RGD compounds to LNPs were different. According to the use requirements of LNP preparations, these RGD compound-modified LNPs can ultimately meet the actual use requirements.

[0311] Table 1

[0312]

[0313] Example 13 Biological Activity Test of Nucleic Acid Pharmaceutical Compositions Containing Lipid Conjugates of the Present Invention

[0314] 1. Test of cell expression levels in LNP transfected with different RGD compounds

[0315] In order to verify whether LNPs coupled with different RGD compounds can improve the transfection efficiency of tumor cells, the tumor cell line A549 was transfected with RGD-LNPs loaded with luciferase. The transfection efficiency of different RGD-LNPs was compared by detecting the luciferase activity 24 hours after transfection.

[0316] Specific experimental process:

[0317] A549 cells were cultured in black flat-bottom 96-well plates, with 5×10 cells per well. 4 The culture conditions were 37°C and 5% CO2. After 16 hours of culture, 200 ng of LNP-mRNA was added to each well for transfection. Each RGD-LNP was transfected in 3 replicate wells. After 4 hours of transfection, fresh culture medium was replaced and culture was continued. After 24 hours of transfection, the cell culture medium was removed and 20 l of cell lysis solution was added. After the cells were fully lysed, 100 l of luciferase substrate solution was added, and the fluorescence value was detected in a fluorescence detector after mixing. For results, see Fig.12 Statistical method: One-way ANOVA.

[0318] The results show:

[0319] The in vitro expression levels of RGD005, RGD008, and RGD038 were significantly higher than those of RGD001. The in vitro expression levels of RGD006, RGD041, and RGD047 were not significantly different from those of RGD001. The expression levels of RGD007, RGD032, RGD035, and RGD044 were significantly lower than those of RGD001.

[0320] In addition, after the amino acid in front of EG8 was changed from Lys to Arg (the corresponding sample was changed from RGD001 to RGD005), the in vitro expression level was significantly improved.

[0321] After the amino acid after EG8 was changed from Trp to His (corresponding to the sample from RGD005 to RGD008), the in vitro expression level remained basically unchanged; after Trp was changed to Tyr or Phe (corresponding to the sample from RGD005 to RGD006 and RGD007) or after Trp was removed (corresponding to the sample from RGD005 to RGD047), the expression level decreased. Therefore, His≥Trp>Tyr>Phe.

[0322] The effect of the number of Arg in front of EG8 on the expression level is: 2>3>4>5>1>0, and the expression levels of the corresponding samples are RGD038>RGD005>RGD041>RGD044>RGD035>RGD032.

[0323] 2. Verification of the mechanism of action of RGD compound-coupled LNP

[0324] Some RGD compounds (RGD001, RGD005, RGD008, and RGD038) were selected to couple LNPs, and competition experiments were performed to confirm the mechanism by which RGD-LNP affects transfection efficiency.

[0325] The specific experimental process is the same as that of Part 1 of this Example, except that in the competition experiment, a pretreatment control group was set up: A549 cells were first incubated with RGD compound at a final concentration of 0.1 mg / ml for 1 hour, and then LNP-mRNA was added for transfection. Fig.13 Statistical method: One-way ANOVA.

[0326] The results show:

[0327] Whether it is the RGD001 group or the RGD005, RGD008, and RGD038 groups, they all have very significant differences in fluorescence expression compared with their respective groups that were pre-incubated with RGD compounds.

[0328] The above results indicate that the interaction between RGD compounds and aRGD receptors on the surface of tumor cells promotes the efficiency of LNP uptake by cells.

[0329] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A lipid conjugate, wherein the lipid conjugate is a compound of formula (I): Ⅰ The P is a targeting peptide composed of 3 to 50 amino acids; The m is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Said n is selected from 2, 3 or 4; Said q is selected from 6, 7, 8, 9 or 10; The R1 is selected from the R group of histidine or tryptophan; R2 and R3 are independently selected from C 12 -C 18 A straight chain alkyl group or a branched chain alkyl group; The targeting peptide is selected from RGD peptide, and the RGD peptide is a compound of formula (II): Ⅱ in, The connection point between the RGD peptide and formula (I) is .

2. The lipid conjugate according to claim 1, characterized in that R2, R3 are C 15 Straight chain alkyl.

3. The lipid conjugate according to claim 1, characterized in that The lipid conjugate is selected from any one of the following structural formulas: RGD005 RGD008 RGD038 RGD041 4. A lipid nanoparticle, characterized in that: The lipid nanoparticles include the lipid conjugate according to any one of claims 1 to 3.

5. The lipid nanoparticle according to claim 4, characterized in that The lipid nanoparticles also include one or more active ingredients, which include chemical drugs or nucleic acid drugs, and the nucleic acid drugs are selected from any one of antisense nucleic acids, plasmids, mRNA, aptamers, antagomirs, miRNAs, ribozymes and siRNAs.

6. The lipid nanoparticle according to claim 4, characterized in that The lipid nanoparticles further comprise one or more active ingredients, which are selected from biological macromolecules or interfering nucleic acids.

7. The lipid nanoparticle according to claim 4, characterized in that The lipid nanoparticles also include steroid lipids, phospholipids, ionizable / cationic lipids and pegylated lipids.

8. The lipid nanoparticle according to claim 4, characterized in that The lipid nanoparticles further include cholesterol, DSPC, any one of compounds 1-4, and DMG-PEG2000; The structural formula of the compound 1-4 is as follows: The structure of compound 1 is as follows: ； The structure of compound 2 is as follows: ； The structure of compound 3 is as follows: ； The structure of compound 4 is as follows: 。 9. The lipid nanoparticle according to claim 7 or 8, characterized in that The molar ratio of each component in the lipid nanoparticles is steroid lipid: phospholipid: ionizable / cationic lipid: PEGylated lipid: lipid conjugate = (30-50): (5-15): (40-55): (1-3): (0.1-1.0).

10. The lipid nanoparticle according to claim 9, characterized in that The molar ratio of each component in the lipid nanoparticles is steroid lipid: phospholipid: ionizable / cationic lipid: PEGylated lipid: lipid conjugate = 38:10:50:1.5:0.

5.

11. The lipid nanoparticle according to claim 9, characterized in that The diameter of the lipid nanoparticles is 70 nm to 150 nm.

12. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the lipid nanoparticles according to any one of claims 4 to 11 and a pharmaceutically acceptable carrier.

13. Use of the lipid nanoparticles according to any one of claims 4 to 11 or the pharmaceutical composition according to claim 12 in the preparation of drugs for preventing and / or inhibiting and / or treating cancer.

14. Use of the lipid conjugate according to any one of claims 1 to 3 in preparing a targeting vector.

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