Patent Application
20240189235
The present application is being filed along with a Sequence Listing XML in electronic format. The Sequence Listing XML is provided as an XML file entitled P4283_SEQ_AF.xml, created Oct. 19, 2023, which is 10 Kb in size. The information in the electronic format of the Sequence Listing XML is incorporated herein by reference in its entirety.
The present disclosure relates to novel cationic lipids and their uses for enhancing the delivery of therapeutic agents. More particularly, the present disclosure relates to cationic lipids and their use for encapsulating and delivering nucleic acids into cells.
Gene therapy is the process of introducing foreign genomic materials into host cells to elicit a therapeutic benefit. Different types of nucleic acids including DNA, plasmids-based interfering nucleic acids, small interfering nucleic acids for use in RNA interference (RNAi), antisense molecules, and aptamers, are currently being developed as therapeutic agents in gene therapy. Meanwhile, two major delivery systems—viral and non-viral systems are also being developed for encapsulating and delivering aforementioned therapeutic agents in a stable and long half-life form.
Although numerous viral and non-viral gene delivery systems are available for decades, each of them is limited in some way. For example, viral delivery systems are advantageous in their high transfection efficiency, yet they have some bio-safety concerns. Non-viral delivery systems (e.g., polymers, lipids, liposomes, micelles, dendrimers, and nanomaterials) are highly versatile in their abilities in encapsulating various sizes of nucleic acids without affecting the recipient's immune system; however, their transfection efficiency is yet to be improved.
Lipoplexes are defined as the complexes among nucleic acids and cationic lipids, and becoming one of the most studied non-viral delivery systems to date. Lipofectamine is a conventionally available cationic lipid, yet one of its components—DOSPA (2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propaniminium trifluoroacetate) was reported to have a high cytotoxicity during gene transfection.
In view of the foregoing, there exists in the related art a need of a novel, safe cationic lipid for effectively delivering therapeutic agents into cells.
The following presents a simplified summary of the disclosure in order to provide a basic understanding to the reader. This summary is not an extensive overview of the disclosure and it does not identify key/critical elements of the present invention or delineate the scope of the present invention. Its sole purpose is to present some concepts disclosed herein in a simplified form as a prelude to the more detailed description that is presented later.
The present disclosure provides novel cationic lipids and compositions thereof for encapsulating and delivering nucleic acids into cells. Also disclosed herein is a treatment of diseases via use of the present cationic lipids and/or compositions.
In one aspect, the present disclosure is directed to a cationic lipid of formula (I) shown below:
In this formula, at least one of X and Y is —(C═O)CH2NH2, and the other X, Y, and Z are independently selected from the group consisting of —(C═O)CH2NH2, —[(C═O)CH2 NH](C═O)R1, —(C═O)R1, and H, wherein R1 is C9-25 alkyl, or C13-21 alkenyl.
According to some embodiments of the present disclosure, in the formula (I), X and Z are independently —(C═O)CH2NH2, Y is —[(C═O)CH2NH](C═O)R1, and R1 is C13-21 alkenyl.
According to other embodiments of the present disclosure, in the formula (I), X is —(C═O)CH2NH2, Y and Z are independently —[(C═O)CH2NH](C═O)R1, and R1 is C13-21 alkenyl.
According to further embodiments of the present disclosure, in the formula (I), Y is —(C═O)CH2NH2, X and Z are independently —(C═O)R1, and R1 is C13-21 alkenyl.
According to still other embodiments of the present disclosure, in the formula (I), X and Z are independently —(C═O)CH2NH2, Y is —(C═O)R1, and R1 is C13-21 alkenyl.
According to further embodiments of the present disclosure, in the formula (I), X is —(C═O)CH2NH2, Y is H, Z is —(C═O)R1, and R1 is C13-21 alkenyl.
Preferably, a subset of the cationic lipid of formula (I) is selected from the group consisting of
Another aspect of the present disclosure is directed to a lipid nanoparticle (LNP) comprising the present cationic lipid, a non-cationic lipid, and an active pharmaceutical ingredient (API).
According to some embodiments of the present disclosure, the API disposed in the present LNP may be a nucleic acid, a peptide, a polypeptide, a protein, a carbohydrate, a proteoglycan, a glycoprotein, or a combination thereof.
Example of the nucleic acid suitable for use in the present LNP includes, but is not limited to, mitochondrial DNA (mtDNA), chloroplast DNA (cpDNA), plasmid, messenger RNA (mRNA), small interfering RNA (siRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), and aptamer. Preferably, the miRNA is a precursor miRNA or a mature miRNA.
In some preferred embodiments of the present disclosure, in the present LNP, the cationic lipid may be selected from the group consisting of
and the API is the miRNA comprising a sequence of SEQ ID NO:1. In working embodiments, the present miRNA has a sequence of SEQ ID NO:2, 3, 4, or 5
In other preferred embodiments of the present disclosure, in the LNP, the cationic lipid is
and the API is the siRNA having a sequence of SEQ ID NO: 6 or 7.
According to some embodiments of the present disclosure, the non-cationic lipids comprised in the present LNP may be a helper lipid, a phospholipid, a PEGylated lipid, a PEGylated phospholipid, or a combination thereof. Examples of the helper lipid suitable for use in the present disclosure include, but are not limited to, dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), and distearoylphosphatidylcholine (or 1,2 -distearoyl-sn-glycero-3-phosphocholine, DSPC). According to embodiments of the present disclosure, the non-cationic lipid comprised in the present LNP is a helper lipid of distearoylphosphatidylcholine (DSPC).
According to some embodiments of the present disclosure, the cationic lipid and the non-cationic lipid are respectively present in the LNP at a molar ratio of 1:5 to 5:1.
In alterative or optional embodiments, the present LNP further comprises a steroid. Examples of steroid suitable for use in the present LNP include, but are not limited to, cholesterol, lanosterol, ergosterol, phytosterol, stigmasterol, and brassicasterol. In one working example, the present LNP further comprises cholesterol.
Another aspect of the present disclosure is directed to a method for treating a disease in a subject in need thereof, comprising administering an effective amount of the LNP set forth above to the subject.
According to embodiments of the present disclosure, the disease may be a cancer, an infectious disease, or an autoimmune disease.
Examples of the cancer treatable by the present method include, but are not limited to, a bladder cancer, a bone cancer, a bone marrow cancer, a brain cancer, a breast cancer, a cholangiocarcinoma, a colon cancer, an esophagus cancer, a gastrointestinal cancer, a gum cancer, a head and neck cancer, a kidney cancer, liver cancer, a lung cancer, a nasopharyngeal carcinoma, a leukemia, a lymphoma, an ovary cancer, a prostate cancer, a skin cancer, a stomach cancer, a testis cancer, a tongue cancer, and a uterus cancer.
In some preferred embodiments of the present disclosure, when the disease is the lung cancer, the present LNP comprises a cationic lipid selected from the group consisting of
and the API is a miRNA comprising a sequence of SEQ ID NO:1. Preferably, the miRNA has a sequence of SEQ ID NO:2, 3, 4, or 5.
In other preferred embodiments, when the disease is the lung cancer, the present LNP comprises the cationic lipid of
and the API is a siRNA having a sequence of SEQ ID NO: 6 or 7.
According to some embodiments of the present disclosure, the subject is a human. Still another aspect of the present disclosure is directed to a method for delivering an API into a cultivated cell, comprising contacting the cultivated cell with an effective amount of the LNP set forth above, wherein the API is a nucleic acid, a polypeptide, a protein, a carbohydrate, a proteoglycan, a glycoprotein, or a combination thereof.
Example of the nucleic acid suitable for use in the present method includes, but is not limited to, mitochondrial DNA (mtDNA), chloroplast DNA (cpDNA), plasmid, messenger RNA (mRNA), small interfering RNA (siRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), and aptamer. Preferably, the miRNA is a precursor miRNA or a mature miRNA.
Example of the cultivated cell suitable for use in the present method includes, but is not limited to, a multipotent stem cell and an immune cell.
Many of the attendant features and advantages of the present disclosure will becomes better understood with reference to the following detailed description considered in connection with the accompanying drawings.
The present description will be better understood from the following detailed description read in light of the accompanying drawings, where:
The detailed description provided below in connection with the appended drawings is intended as a description of the present examples and is not intended to represent the only forms in which the present example may be constructed or utilized. The description sets forth the functions of the example and the sequence of steps for constructing and operating the example. However, the same or equivalent functions and sequences may be accomplished by different examples.
1. Definitions
For convenience, certain terms employed in the specification, examples and appended claims are collected here. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of the ordinary skilled in the art to which this invention belongs.
The singular forms “a”, “and”, and “the” are used herein to include plural referents unless the context clearly dictates otherwise.
The term “alkyl” used herein refers to a straight or branched hydrocarbon group, containing 9-25 carbon atoms. Examples of C9-25 alkyl suitable for use in the present invention include nonyl, decyl, undecyl (also hendecyl), dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, icosyl, heneicosyl (also henicosyl), docosyl, tricosyl, tetracosyl, and pentacosyl.
The term “alkenyl” used refers to a long, straight hydrocarbon group, containing 13-21 carbon atoms and one or more double bonds. Examples of C13-21 alkenyl suitable for used in the present invention include tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, nonadecenyl, icosenyl, and heneicosenyl (also henicosenyl). Generally, the total numbers of carbon atoms (C) and double bonds (D) in alkenes are respectively represented by numerical symbol of “C:D”, such as 14:1, 16:1, 18:1, 18:2, 18:3, 20:4, or 20:5.
The term “active pharmaceutical ingredient (API)” used in this paper refers to an ingredient that is biologically active as a, or a part of the form of medicine, drugs, compositions, vaccines, and/or pharmaceutical products, by which a disease and/or a condition in a subject may be delayed, ameliorated, hindered or prevented. Examples of APIs suitable for use in the present disclosure include, but are not limited to, nucleic acids, polypeptides, proteins, carbohydrates, proteoglycans, glycoproteins, and a combination thereof.
The term “cationic lipid” as used herein refers to those lipids having one or more fatty acid or fatty alkyl chains and attaining positive charge(s) through one or more amines present in the polar head group. The cationic lipid is typically protonated (i.e., positively charged) at a pH below its pKa.
The term “non-cationic lipid” as used herein refers to lipids that do not possess positive charge(s), such as a neutral lipid or an anionic lipid. According to embodiments of the present disclosure, the term “non-cationic lipid” also intend to encompass “helper lipid” known to stabilize the structure of LNP, which in turn enhance the delivery of API carried by the LNPs A helper lipid also helps optimizing the encapsulation of APIs (e.g., nucleic acids), protect them from degradation, and facilitate their transport into target cells. Examples of the helper lipid suitable for use in the present disclosure include, but are not limited to, dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), and distearoylphosphatidylcholine (or 1,2-di stearoyl- sn-glycero-3-pho sphocholine, DSPC).
The term “steroid” as used herein refers to a class of chemicals that derived from a core structure (i.e., steroid nucleus) of gonane, or cyclopentanoperhydrophenanthrene, which is a tetracyclic hydrocarbon with no double bonds. Examples of steroid that play roles in the formation of the present LNPs include, but are not limited to, cholesterol, lanosterol, ergosterol, phytosterol, stigmasterol, and bras sicasterol.
The term “microRNA” or “miRNA” used herein refers to a class of non-coding RNAs that play roles in regulating gene expression. Most miRNAs are transcribed from DNA sequences into primary miRNAs (pri-miRNAs) and processed into precursor miRNAs (pre-miRNAs) and finally mature miRNAs. Accordingly, the term miRNA used herein refers to all non-coding RNAs involved in miRNA processing and maturation, preferably including precursor miRNAs and mature miRNAs.
The term “infectious diseases” used herein refers to disorders caused by pathogens, such as bacteria, viruses, fungi or parasites, which typically cause acute symptoms, such as fever, inflammation, upper respiratory symptoms, diarrhea, and the like.
The term “subject” or “patient” is used interchangeably herein and is intended to mean a mammal including the human species that is treatable by the API within the present cationic lipids. The term “mammal” refers to all members of the class Mammalia, including humans, primates (e.g., monkey, and chimpanzee), domestic and farm animals, such as rabbit, pig, goat, sheep, and cattle; as well as zoo, sports or pet animals (e.g., a horse, a dog, a cat and etc); and rodents, such as mouse, rat, guinea pig, and hamster. In a working example, the subject is a human. Further, the term “subject” or “patient” intended to refer to both the male and female gender unless one gender is specifically indicated.
2. Detail Description of Preferred Embodiments
The present disclosure is based, at least in part, on the discovery that some novel cationic lipids may act as vehicles for the delivery of polynucleotides into cells. Accordingly, the present disclosure provides a novel cationic lipid and a composition thereof that serve as a vehicle for the delivery of therapeutic agents. Also disclosed herein is a treatment of diseases by use of the present cationic lipid and/or composition.
2.1 Cationic Lipids of the Present Disclosure
The present disclosure aims at providing novel cationic lipids and compositions comprising the same for delivering therapeutic agents. In one aspect, the present invention relates to a cationic lipid of formula (I),
wherein, at least one of X and Y is —(C═O)CH2NH2, and the other X, Y, and Z are independently selected from the group consisting of —(C═O)CH2NH2, —[(C═O)CH2NH](C═O)R1, —(C═O)R1, and H, wherein R1 is C9-25 alkyl, or C13-21 alkenyl.
In some embodiments, R1 is C9-25 alkyl; in preferred embodiments, R1 is C11-23 alkyl; in other preferred embodiments, R1 is C13-21 alkyl; and in still other preferred embodiments, R1 is C15-19 alkyl. In some working examples, R1 is C15 alkyl.
In other embodiments, R1 is C13-21 alkenyl; and in preferred embodiments, R1 is C15-19 alkenyl. In some working examples, R1 is C15 alkenyl; in other working examples, R1 is C17 alkenyl.
Exemplary compound of formula (I) is
The cationic lipids of the present disclosure may be prepared in accordance with procedures described in the working examples. Briefly, amino group (NH2) of glycine is firstly protected by protecting groups such as phenacyl (PAc) or tert-butyloxycarbonyl (Boc) groups. Then, at least one PAc- or Boc protected glycine is linked with glycerol via condensation reaction (i.e., dehydration) occurring between at least one hydroxyl group of glycerol and the carboxyl group of glycine. Finally, one or two fatty acid(s) are linked to the rest of hydroxyl group(s) of glycerol to form the present cationic lipid. According to alternative embodiments, a condensation reaction occurs between the carboxyl group of fatty acid and the amino group of glycine gives rise to an amide bond therebetween, thereby produces an intermediate compound having a free carboxyl group for subsequent reacting with hydroxyl groups of glycerol, thus produces the final cationic lipid products of the present invention. The detail reaction conditions and chemical agents for producing the present cationic lipid compounds can be modified by the skill persons in the art without departing from the spirit of the present disclosure.
Examples of fatty acid suitable for use in production of the present compound of formula (I) include, but are not limited to, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, and docosahexaenoic acid. In working examples, the fatty acid used in the production of the present compound of formula (I) is palmitoleic acid or linoleic acid.
All stereoisomers of the present compounds, such as those which may exist due to asymmetric carbons on each X, Y, and Z substituents of the compound of formula (I) including enantiomeric and diastereomeric forms, are contemplated within the scope of this invention.
2.2 Lipid Nanoparticles
Also disclosed herein is a novel lipid nanoparticle (LNP) that facilitates the intracellular delivery of biologically active and therapeutic molecules into cells of patients.
According to embodiments of the present disclosure, the lipid nanoparticle (LNP) comprises a cationic lipid of formula (I) described above, a non-cationic lipid, and an active pharmaceutical ingredient (API). Examples of the non-cationic lipids suitable for constructing the present LNP include, but are not limited to, helper lipids, phospholipids, PEGylated lipids, PEGylated phospholipids, and a combination thereof. Examples of the helper lipid suitable for use in the present disclosure include, but are not limited to, dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), and distearoylphosphatidylcholine (or 1,2-distearoyl- sn-glycero-3-phosphocholine, DSPC). According to embodiments of the present disclosure, the non-cationic lipid comprised in the present LNP is a helper lipid of distearoylphosphatidylcholine (DSPC). Examples of PEGylated lipids suitable for use in the present disclosure include, but are not limited to, PEG conjugated to diethylene glycol (DEG-PEG) (e.g., 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DEG-PEG 2000); PEG conjugated to dialkyloxypropyl (PEG-DAA) (e.g., dioleoylphosphatidylethanolamine (DOPE)); PEG conjugated to diacylglycerol (PEG-DAG) (e.g., 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N[methoxy(polyethylene glycol)-2000] (DOPE-PEG 2000); PEG coupled to phosphatidylethanolamine (PEG-PE) (e.g., 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(Polyethylene Glycol)-2000], DSPE-PEG 2000); and a combination thereof. In one working example, the PEGylated lipid is DEG-PEG 2000.
According to embodiments of the present disclosure, the present cationic lipids and the non-cationic lipid are present in the LNP at a molar ratio of 1:5 to 5:1, such as 1:5, 1:4.5, 1:4, 1:3.5, 1:3, 1:2.5, 1:2, 1:1.5, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1 or 5:1. In some working examples, the present cationic lipid and the non-cationic lipid are present in the LNP at a molar ratio of 1:1. In alternative working examples, the present cationic lipid and the non-cationic lipid are present in the LNP at a molar ratio of about 4.3:1.
Alternatively or in addition, the present LNP may further comprise a steroid to help stabilize the structure of the LNP. Examples of the steroid suitable for constructing the present LNP include, but are not limited to, cholesterol, lanosterol, ergosterol, phytosterol, stigmasterol, and brassicasterol. Preferably, the present LNP further comprises cholesterol.
According to some embodiments of the present disclosure, each LNP has a hydrophilic core enclosed by a lipid bilayer consisting of both cationic (e.g., at least one compounds of formula (I)) and non-cationic lipids, and optionally, the steroid and/or the helper lipids embedded within the lipid bilayer; accordingly, APIs that are hydrophilic in nature may be disposed in the hydrophilic core, while APIs that are hydrophobic in nature are intercalated between the hydrophobic region between the lipid bilayer. In other embodiments, instead of having a hydrophilic core enclosed by a lipid bilayer as indicated above, each LNP is in the structure of a micelle with a plurality of smaller inverse micelles enclosed therein. The micelle has a hydrophobic core enclosed by a single layer of lipids (e.g., the cationic and non-cationic lipids of the present disclosure) with the hydrophobic tails of the lipids extending toward the center, while the hydrophilic head groups of the lipids are facing out. Additionally, multiple inverse micelles that are smaller than the micelle in size may be enclosed within the hydrophobic core of the micelle; note that the term “inverse micelle” refers to a micelle, in which the head groups of the lipids are at the center while the hydrophobic tails of the lips are extending out. Accordingly, the hydrophilic API (e.g., nucleic acids) may be disposed inside the hydrophilic cores of the inverse micelles.
The present LNP can be produced via procedures and/or tools well known in the art, including thin-layer hydration, ethanol injection, microfluidic devices, and the like, in which the cationic lipids (i.e., the compounds of formula (I)), the non-cationic lipids, and optionally, the steroid, are self-assembled to form a lipid bilayer, and the APIs may be directly encapsulated within the LNP, or may be coupled to the cationic lipids before being encapsulated in the LNP, thereby producing a complex in the form of nanoparticles, liposomes, or micelles. The APIs to be delivered by the present LNP, either in the form of a liquid or a solid, may be a nucleic acid, a polypeptide, a protein, an antibody, a carbohydrate, a proteoglycan, a glycoprotein, a small molecule, or a combination thereof.
In some embodiments of the present disclosure, the API is the nucleic acid, which includes but is not limited to, mitochondrial DNA (mtDNA), chloroplast DNA (cpDNA), plasmid, messenger RNA (mRNA), small interfering RNA (siRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), or aptamer. In preferred embodiments, the API is miRNA or siRNA. Examples of miRNA includes, but is not limited to, a precursor miRNA or a mature miRNA. In some working examples, the miRNA to be encapsulated and delivered via the present cationic lipids comprising a sequence of SEQ ID NO:1; more preferably, the miRNA has a sequence of SEQ ID NO:2, 3, 4, or 5. In other working examples, the siRNA used to be delivered via the present cationic lipids has a sequence of SEQ ID NO: 6 or 7. In some alternative embodiments, the API is a plasmid.
2.3 Treatments Via Use of the Present Cationic Lipids
The cationic lipids of formula (I) and lipid nanoparticles thereof are useful for the delivery of biologically active agents, thus they may be used in the treatment of diseases. The present disclosure thus encompasses a method of treating a disease in a subject in need thereof. The method mainly includes, administering an effective amount of the composition (i.e., the lipid nanoparticles (LNPs) composed of the compound of formula (I)) to the subject.
The present lipid nanoparticles comprising the cationic lipids of formula (I) may be formulated into powders, granules, solutions or suspensions, suppositories or patches, and such formulations can be administered or parenterally, to effectively transport the active ingredients to the appropriate or desired site of action and lesions. According to the present disclosure, the present lipid nanoparticle can be parenterally administered to lesions (e.g., the tumor) of the subject. Exemplary suitable parenteral route includes, but is not limited to, transdermal, percutaneous, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, intradermal, subcutaneous, rectal, intravaginal, and intraperitoneal routes. Suitable routes vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the dosage and the nature of active ingredients, genetic factors and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. In general, the most appropriate route of administration depends on a variety of factors including the agent's stability in the environment of the circulatory system, and/or the condition of the subject (e.g., the severity of lung cancer of the subject, or whether the subject is able to tolerate conventional treatment). According to some embodiments of the present disclosure, the present lipid nanoparticle is formulated into injectable formulation for parental administration. According to other embodiments of the present disclosure, the present lipid nanoparticle is formulated into patches for transdermal administration.
The present lipid nanoparticle can be administered at a frequency that effectively prevent, inhibit, suppress, or treat diseases, conditions, or traits in the subject. In some embodiments, the lipid nanoparticle can be administered at a frequency of four times a day to once every three months; for example, at a frequency of four times a day, three times a day, twice a day, once a day, once every other day, once every third day, once every week, once every other week, once monthly, twice monthly, thrice monthly, once every other month, or once every three months. Preferably, the lipid nanoparticle is administered to the subject at a frequency of twice per week (once every third or four day). Optionally, the lipid nanoparticle is administered to the subject at a frequency of once a week. Still optionally, the lipid nanoparticle is administered to the subject at a frequency of once every other week.
According to embodiments of the present disclosure, diseases treatable by the present method may be cancers, infectious diseases (including inflammatory diseases), transplant and/or tissue rejection, autoimmune diseases, and the like.
Examples of cancer treatable by the present method include, but are not limited to, bladder cancer, bone cancer, bone marrow cancer, brain cancer, breast cancer, cholangiocarcinoma, colon cancer, esophagus cancer, gastrointestinal cancer, gum cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal carcinoma, leukemia, lymphoma, ovary cancer, prostate cancer, skin cancer, stomach cancer, testis cancer, tongue cancer, and uterus cancer.
In one preferred embodiment, the lung cancer subject is treated via administering to the subject an effective amount of the present lipid nanoparticle, which comprises the cationic lipid of
and the siRNA having a sequence of SEQ ID NO: 6 or 7.
In another preferred embodiment, the lung cancer subject is treated administering to the subject an effective amount of the present lipid nanoparticle, in which the cationic lipid is selected from the group consisting of
and the miRNA has a sequence selected from the group consisting of SEQ ID NO:2, 3, 4, and 5.
2.4 API Delivery Platform Via Use of the Present Cationic Lipids
The present lipid nanoparticles (LNPs) are useful for the delivery of APIs into cultivated cells, thus they may be used as a platform for the delivery of therapeutic materials in preparation of cellular therapies. The present disclosure thus encompasses a method of delivering an API into a cultivated cell. The method mainly includes, contacting the cultivated cell with an effective amount of the present LNP, wherein the API is a nucleic acid, a polypeptide, a protein, a carbohydrate, a proteoglycan, a glycoprotein, or a combination thereof.
The cultivated cells provided in the present embodiments may be isolated or derived from subjects, and potent to differentiate or develop into cellular transplants with therapeutic benefits by receiving genes or gene products delivered via the present LNP, such that the cellular transplants are useful in and beneficial to cell therapy adaptive to select fields, including regenerative medicine, immune system disorders, and cancer. Generally, the cultivated cells include stem cells and immune cells. Examples of stem cells suitable for use in the present method include, but are not limited to pluripotent stem cells (PSCs; for example, embryonic stem cells (ESCs), epiblast stem cells (EpiSCs), embryonic germ cells (EGCs), and induced pluripotent stem cells (iPSCs)), adult stem cells (ASCs; for example, hematopoietic stem cells (HSCs), skin stem cells (SSCs), neural stem cells (NSCs), and mesenchymal stem cells (MSCs)), and cancer stem cells (CSCs). Examples of immune cells suitable for use in the present method include but are not limited to, T cells, dendritic cells (DCs), natural killer (NK) cells, and macrophages. The immune cells express genetically engineered antigen receptors, including engineered T cell receptors (TCRs) and functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs), including activating, stimulatory, and costimulatory CARs, and combinations thereof.
By the virtue of the above features, the present disclosure provides novel cationic lipids capable of encapsulating and delivering polynucleotides loaded therein to cells, thereby allowing diseases including cancers to be effectively and efficiently treated.
Materials and Methods
Cell Culture
NCI-H460 cells (large cell lung cancer cell line) were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with heat-inactivated 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS) solution. A549 cells (established from human cancerous lung tissues) were cultured in Ham's F-12K (Kaighn's) medium supplemented with heat-inactivated 10% FBS and 1% PS solution. All cells were cultured at 37° C. under an atmosphere containing 5% CO2.
Plasmid Stabilization Via the Present Cationic Lipids
The present cationic lipids (i.e., compounds 6, 9, 11, 14, 18, 22, or 24), lecithin, and cholesterol were fully mixed at a molar ratio of 2:2:1 and dissolved in ethanol completely, and the solution was diluted from 30 to 50 μL by DPBS buffer. The diluted solution was then co-cultivated with 50 μL of sucrose aqueous (5% w/v) containing 1.5 μg of EGFP-plasmid (pUC19, 4564 bp, Addgene) at room temperature for one hour to produce a cationic lipid-DNA complex. The obtained solution was divided into two vials, one of which was added with 1 μL of DNase and 10 μL of 10×DNase buffer and incubated at 37° C. for further 15 minutes. After that, 100 μL of phenol/chloroform (5/1) was added and mixed by centrifugation at 13000×g for 5 minutes to extract DNA. All the samples were subjected to gel electrophoresis (1% agarose gel, 110V).
Preparation of Cationic Lipid-RNA Complexes
To produce the present composition, the present cationic lipids (compound 6 or 22), cholesterol, DEG-PEG 2000, and distearoylphosphatidylcholine (DSPC) were fully mixed in ethanol at a molar ratio of 50:38.5:1.5:10, and the total volume was brought to 500 μL, thereby producing a liquid formulation of lipid nanoparticles (LNPs). As for control group, D-lin-MC3-DMA (MC30), an ionizable amino lipid that conventionally used in LNPs preparation, was mixed with cholesterol, DEG-PEG 2000, and DSPC at the same molar ratio as the present LNPs. 0.268 mM, 265 μL of miRNAs or siRNAs (e.g., the polynucleotides of SEQ ID NO: 2 to 7) were added in 695 μL acidic buffer (composed of 100 mM citrate buffer (pH4.0) and nuclease-free water) to produce a RNA working solution, and the total volume was brought to 960 μL. The RNA working solution and the LNPs (molar ratio was 1:6) were respectively pumped into the two inlets and mixed in the channels of the microfluidics device (NanoAssemblr platform) with flow rate ratio (FRR) set to 3 and total flow rate (TFR) set to 12 mL/min, and the confluent liquid reaching the outlet of the microfluidics was collected with a total volume of 1.2 mL. The collected mixture was then filtrated and centrifuged at 2,000 g for 20 minutes×three times by centrifugal filters (membrane filter with a cutoff molecular weight of 10 kDa), and the filtrate containing lipid nanoparticles having miRNA or siRNA encapsulated therein (i.e., cationic lipid-RNA complexes) were stored at 4° C. until use.
Cationic Lipid-RNA Complexes Characterization
Particle sizes and zeta potentials of cationic lipid-RNA complexes were respectively determined by using a particle size and zeta potential analyzer (DelsaNano C, Beckman Coulter).
The encapsulation ratio of liposomes for encapsulating miRNAs or siRNAs was determined by fluorescence reader (Infinite F200 Pro) with the aid of RNA quantification kit containing a fluorescent dye (RiboGreen). Absorption spectra of miRNAs or siRNAs with known concentrations (i.e. 100 μg/mL) were first acquired for the construction of a standard curve. The cationic lipid-RNA complexes (typically with a total volume of 50 μL for each sample well) were then mixed with the reagent from the RNA quantification kit in each well. Optionally, the cationic lipid-RNA complexes were subjected to lysis buffer, thereby bringing RNA back into suspension. The amount of miRNAs or siRNAs were quantified by interpolating their absorption strengths at 535 nm to that of the standard curve, and the encapsulation efficiency (EE) was calculated by the following formula:
Encapsulation efficiency (EE %)=(Wt/Wi)×100%,
where Wt is the total amount of miRNAs or siRNAs released in the LNPs lysis suspension, and Wi is the total quantity of miRNAs or siRNAs added initially during preparation.
Flow Cytometry Analysis
Cell sorting was conducted via flow cytometry with fluorochrome-conjugated miRNAs or siRNAs, specifically the miRNAs were conjugated with a far-red—fluorescent label Cy5 dye. After a final wash prior to sorting, cells were filtered through a 35 μm nylon cell strainer for removal of cell clumps, and then subjected to sorting using a flow cytometer (BD LSRFortessa X-20) and analysis software (FlowJo, BD Biosciences).
The present cationic lipids were respectively synthesized in accordance with steps as described in schemes I-IV. In general, the amino group (NH2) of glycine was firstly protected by phenacyl (PAc) or tert-butyloxycarbonyl (Boc) groups. After the condensation reaction (i.e., dehydration) between glycerol and glycine, and/or between glycerol and fatty acids, the protective groups were removed and reduced to NH2 group.
The methods for preparing the exemplary compounds, as well as the analytical data for the compounds thus prepared, are set forth below:
1.1 TGG-Linoleic Acid (Compound 6)
Preparation of tent-butyl 2-((9Z,12Z)-octadeca-9,12-dienamido)acetate (1) To a solution of N,N′-Dicyclohexylcarbodiimide (DCC, 0.40 g, 1.96 mmol) dissolved in 10 mL of dichloromethane (DCM), linoleic acid (0.5 g, 1.78 mmol), t-butyl 2-aminoacetate (0.27 mL, 1.96 mmol), and DMAP (0.13, 1.07 mmol) was added. The solution was stirred overnight at room temperature. After fully mixed, 1,3-Dicyclohexyl urea (DCU) was filtered off, the solution was removed by rotary evaporator. The residual solution was purified by column chromatography eluted with ethyl acetare (Hexane:EA, 3:1) to obtain 480 mg of solid (compound 1) (yield 69%). 1H NMR (600 MHz, CDCl3) δ 5.93 (s, 1H), 5.44-5.26 (m, 4H), 3.95 (d, J=4.9 Hz, 2H), 2.78 (t, J=6.9 Hz, 2H), 2.29-2.17 (m, 2H), 2.10-2.02 (m, 4H), 1.66 (dd, J=15.7, 8.8 Hz, 3H), 1.49 (s, 9H), 1.39-1.28 (m, 14H), 0.91 (t, J=7.0 Hz, 3H).
Preparation of 2-((9Z,12Z)-octadeca-9,12-dienamido)acetic acid (2) Trifluoroacetic acid (TFA, 2.80 mL, 36.59 mmol) was lowly added to a solution of compound 1 (480 mg, 1.22 mmol, dissolved in 8 mL of DCM). The solution was stirred and monitored by TLC. After fully reacted, DCM was removed under reduced pressure. The residue was washed by hexane and EA for several times. Remained solvent was removed by rotary evaporator to obtain compound 2 for further use.
Preparation of 2-((tert-butoxycarbonyl)amino)acetic acid (3) To a bottle having solution of 2N NaOH (8.66 mL, 17.32 mmol), glycine (1 g, 13.32 mmol) was added, and the mixture was stirred until all glycine was dissolved, then a solution of Boc2O (3.34 g, 15.32 mmol) dissolved in THF (20 mL) was added into bottle and stirred at room temperature overnight. THF was removed by evaporation. To make basic solution, 2N NaOH solution was added to the residue and washed by EA. The pH value was adjusted to 3-4 by adding 2M HCl. The aqueous phase was extracted with EA. Organic phase was dried over MgSO4, filtered and the solvent was removed by rotary evaporator to obtain 1.38 g of white solid product (compound 3, yield 59%). 1H NMR (600 MHz, CDCl3) δ 5.03 (s, 1H), 3.97 (t, J=20.4 Hz, 2H), 1.48 (s, 9H).
Preparation of 2-hydroxypropane-1,3-diyl bis(2-((tert-butoxycarbonyl)amino)acetate) (4) To a solution of DCC (0.99 g, 4.78 mmol) dissolved in DCM, glycerol (0.2 g, 2.17 mmol), compound 3 (0.80 g, 4.56 mmol) and DMAP (0.27 g, 2.17 mmol) were added and stirred overnight at room temperature. The mixture was subjected to filtration to remove DCU and then evaporated to give a crude product. Crude product was purified by column chromatography (Hexane:EA, 50:5030:70) to obtain 130 mg of clear sticky liquid product (compound 4, yield 15%). LC-MS (ESI); m/z: [M+H]+ Calcd. for C17H30N2O9,406.20. Found 407.41.
Preparation of 2-(2-((9Z,12Z)-octadeca-9,12-dienamido)acetoxy)propane -1,3-diyl bis(2-((tert-butoxycarbonyl)amino)acetate) (5) To a solution of DCC (0.073 g, 0.35 mmol) dissolved in DCM, compound 4 (0.13 g, 0.32 mmol), compound 2 (0.12 g, 0.35 mmol) and DMAP (0.039 g, 0.32 mmol) were added. The solution was stirred overnight at room temperature. After fully mixed, the solution was filtered to remove DCU, and then evaporated under reduced pressure to remove DCM. The residual solution was purified by column chromatography (Hexane:EA, 2:3) to obtain 200 mg of white solid product (compound 5, yield 86%). 1NMR (600 MHz, CDCl3) δ 5.36 (ddd, J=27.2, 14.1, 6.3 Hz, 4H), 5.21 (s, 1H), 4.40 (dt, J=18.0, 9.0 Hz, 2H), 4.34-4.23 (m, 2H), 4.13-3.97 (m, 2H), 3.97-3.82 (m, 4H), 2.79 (t, J=6.8 Hz, 1H), 2.27 (dd, J=14.3, 6.9 Hz, 2H), 2.11 (d, J=5.8 Hz, 1H), 2.06 (dd, J=13.8, 6.8 Hz, 3H), 1.95 (s, 2H), 1.86 (dd, J=9.8, 4.7 Hz, 2H), 1.78 (d, J=13.3 Hz, 2H), 1.70-1.61 (m, 3H), 1.47 (s, 18H), 1.41-1.26 (m, 16H), 0.91 (t, J=6.6 Hz, 3H). LC-MS (ESI); m/z: [M+H]+ Calcd. for C37H63N3O11, 725.45. Found 726.7.
Preparation of 2-(2-((9Z,12Z)-octadeca-9,12-dienamido)acetoxy)propane-1,3-diyl bis(2-aminoacetate) (6) To a solution of compound 5 (0.2 g, 0.28 mmol) dissolved in 6 mL of DCM, TFA (0.42 mL, 5.51 mmol) was added slowly. The solution was stirred until the reactant was consumed. The resulting solution was then evaporated under reduced pressure to remove solvent, and washed with ether and DCM. The residual solvent was removed by evaporation to provide 48 mg of clear sticky product (compound 6, yield 33%). 1NMR (600 MHz, MeOD) δ 5.53-5.44 (m, 1H), 5.41-5.28 (m, 4H), 4.60-4.37 (m, 4H), 3.94-3.84 (m, 6H), 2.79 (t, J=6.7 Hz, 2H), 2.28 (dd, J=9.8, 5.0 Hz, 2H), 2.07 (dd, J=12.2, 6.2 Hz, 4H), 1.67-1.55 (m, 2H), 1.41-1.27 (m, 15H), 0.92 (t, J=6.8 Hz, 3H). LC-MS (ESI); m/z: [M+H]+ Calcd. for C27H47N3O7, 525.34. Found 527.38.
1.2 TGG-di(Linoleic Acid) Compound 9
Procedure 1
Preparation of 2,3-dihydroxypropyl 2-((tert-butoxycarbonyl)amino)acetate (7) Glycerol (0.42 g, 4.57 mmol), compound 3 (0.2 g, 1.14 mmol), EDCI (0.35 g, 2.28 mmol) and DMAP (0.14 g, 1.14 mmol) were mixed and dissolved in DCM. The solution was stirred at room temperature overnight. The resulting solution was evaporated to remove DCM to obtain a crude product. The crude product was purified by column chromatography (EA, 100%) to obtain 120 mg sticky liquid product (compound 7, yield 42%). 1H NMR (600 MHz, MeOD) δ 4.59 (s, 1H), 4.22 (dd, J=11.3, 4.3 Hz, 1H), 4.16-4.10 (m, 1H), 3.88 - 3.81 (m, 2H), 3.57 (dd, J=5.6, 2.1 Hz, 2H), 1.46 (s, 9H).
10 Preparation of 3-(2-((tent-butoxycarbonyl)amino)acetoxy)propane-1,2-diyl bis(2-((9Z,12Z)-octadeca-9,12-dienamido)acetate) (8) Compound 7 (0.1 g, 0.40 mmol), compound 2 (0.30 g, 0.88 mmol), EDCI (0.16 g, 1.00 mmol) and DMAP (0.059 g, 0.48 mmol) were mixed and dissolved in DCM. After stirred at room temperature overnight, the mixture was evaporated to remove DCM. The residual solution was extracted with EA/H2O. Organic phase was collected, dried over MgSO4, and filtered. The resulting filtrate was evaporated to remove EA. Crude product was purified by column chromatography (Hexane:EA, 3:7) to obtain 190 mg of brown sticky liquid product (compound 9, yield 53%). 1H NMR (600 MHz, CDCl3) δ 5.46-5.31 (m, 8H), 4.46 (dd, J=12.0, 3.5 Hz, 1H), 4.41-4.33 (m, 2H), 4.28 (ddd, J=17.9, 12.1, 6.2 Hz, 1H), 4.14 (q, J=7.1 Hz, 1H), 4.07 (ddd, J=19.8, 9.8, 5.1 Hz, 3H), 4.00 (d, J=3.6 Hz, 2H), 3.96-3.83 (m, 2H), 2.79 (t, J=6.8 Hz, 3H), 2.27 (ddd, J=9.9, 7.8, 5.2 Hz, 4H), 2.13-2.00 (m, 8H), 1.76-1.61 (m, 8H), 1.47 (s, 9H), 1.40-1.26 (m, 28H), 0.91 (t, J=7.0 Hz, 6H).
Procedure 2
Preparation of 3-(2-(tritylamino)acetoxy)propane-1,2-diyl bis(2-((9Z,12Z)-octadeca-9,12-dienamido)acetate) (25) Glycerol (0.29 g, 3.15 mmol), trt-glycine (0.2 g, 0.63 mmol), were mixed and dissolved in a solvent of DCM/dioxane to provide a mixture. To the mixture, DCC (0.17 g, 0.82 mmol) and DMAP (0.038 g, 0.32 mmol) were gradually added and stirred overnight at room temperature. The solution was filtrated to remove DCU, and evaporated under reduced pressure to remove solvent. Crude product was purified by column chromatography (Hex:EA=1:3) to obtain 195 mg white solid product (compound 25, yield 79%). 1H NMR (600 MHz, MeOD) δ 7.62-7.38 (m, 6H), 7.30 (dd, J=10.6, 5.0 Hz, 6H), 7.24-7.16 (m, 3H), 4.11 (dd, J=11.3, 4.2 Hz, 1H), 4.02 (dd, J=11.3, 6.4 Hz, 1H), 3.76 (td, J=9.9, 5.7 Hz, 1H), 3.50 (d, J=5.7 Hz, 2H), 3.13 (s, 2H).
Preparation of 3-(2-(tritylamino)acetoxy)propane-1,2-diyl bis(2-((9Z,12Z)-octadeca-9,12-dienamido)acetate) (26) Compound 25 (0.2 g, 0.51 mmol), compound 2 (0.40 g, 1.18 mmol), EDCI (0.18 g, 1.18 mmol), and DMAP (0.062 g, 0.51 mmol) were mixed and dissolved in DCM. The solution was stirred at room temperature overnight, then evaporated and dried under reduced pressure to remove DCM. Crude product was purified by column chromatography (Hexane:EA, 3:2) to obtain 266 mg of white sticky product (compound 26, yield 51%). 1H NMR (600 MHz, CDCl3) δ 7.49 (d, J=7.7 Hz, 6H), 7.36 (d, J=6.4 Hz, 5H), 7.31 (dd, J=6.7, 2.3 Hz, 2H), 5.46-5.29 (m, 6H), 5.27 (d, J=6.0 Hz, 1H), 4.26 (ddd, J=19.0, 11.9, 5.4 Hz, 3H), 4.14 (dd, J=11.8, 5.8 Hz, 1H), 4.09-3.91 (m, 4H), 2.79 (t, J=6.8 Hz, 3H), 2.27 (dd, J=14.0, 6.6 Hz, 4H), 2.07 (dd, J=12.3, 5.3 Hz, 6H), 1.62 (dd, J=27.0, 19.6 Hz, 11H), 1.52-1.20 (m, 28H), 0.97-0.81 (m, 6H).
Preparation of 3-(2-aminoacetoxy)propane-1,2-diyl bis(2-((9Z,12Z)-octadeca-9,12-dienamido)acetate) (9) To a solution of compound 26 (0.27 g, 0.26 mmol) dissolved in DCM, TFA (0.3 mL, 3.93 mmol) was added slowly and stirred until all the reactant was consumed. The solution was evaporated and washed with ether few times to remove solvent. Crude product was purified by column chromatography (Hex:EA=1:1→DCM:MeOH, 95:5) to obtain 131 mg brown gluey product (compound 9, yield 64%). 1H NMR (600 MHz, MeOD) δ 5.48-5.25 (m, 8H), 4.53-4.44 (m, 2H), 4.37 (ddd, J=18.1, 12.1, 5.0 Hz, 2H), 4.06-3.81 (m, 6H), 2.79 (t, J=6.8 Hz, 3H), 2.27 (td, J=7.5, 4.1 Hz, 4H), 2.08 (dt, J=10.9, 5.1 Hz, 7H), 1.72-1.56 (m, 4H), 1.50-1.14 (m, 28H), 0.92 (t, J=7.0 Hz, 6H). LC-MS (ESI); m/z: [M+H]+0 Calcd. for C45H77N3O8,787.57. Found 789.48.
1.3 DGG-Linoleic Acid (Compound 11)
Preparation of 24(9Z,12Z)-octadeca-9,12-dienoyloxy)propane-1,3-diyl bis(2-((tert-butoxycarbonyl)amino)acetate) (10) To a solution of DCC (76 mg, 0.37 mmol) dissolved in 8 mL of DCM, compound 4 (0.15 g, 0.37 mmol), linoleic acid (93 mg, 0.33 mmo), and DMAP (32 mg, 0.26 mmol) were mixed and added. The mixture was stirred overnight at room temperature, and then filtered to remove DCU. The solution was then evaporated to obtain a crude product. Crude product was purified by column chromatography (Hexane:EA, 7:3) to obtain 140 mg of clear liquid product (compound 10, yield 63%). 1H NMR (600 MHz, CDCl3) δ 5.33 (ddt, J=14.9, 10.0, 7.8 Hz, 2H), 5.14-5.01 (m, 1H), 4.47-4.34 (m, 2H), 4.25 (dd, J=11.9, 5.7 Hz, 1H), 4.22-4.11 (m, 1H), 4.11-4.00 (m, 1H), 3.93 (s,4H), 2.79 (t, J=6.8 Hz, 1H), 2.41-2.28 (m, 2H), 2.16-2.01 (m, 2H), 1.92-1.83 (m, 1H), 1.69-1.51 (m, 9H), 1.47 (s, 18H), 1.42-1.20 (m, 14H), 0.91 (dd, J=8.6, 5.3 Hz, 3H).
Preparation of 24(9Z,12Z)-octadeca-9,12-dienoyloxy)propane-1,3-diyl bis(2-aminoacetate) (11) To a solution containing compound 10 (0.14 g, 0.21 mmol) dissolved in 6 mL of DCM, TFA (0.40 mL, 5.23 mmol) was added slowly. The solution was stirred until reactant was consumed, and then evaporated to remove solvent and washed with ether and DCM. The resulting was evaporated to obtain compound 11 (60 mg of clear gluey produce, yield 61%). 1H NMR (600 MHz, MeOD) δ 5.51 (s, 1H), 5.45 (dd, J=9.3, 4.9 Hz, 1H), 5.42-5.29 (m, 4H), 4.57 (dt, J=12.0, 4.1 Hz, 1H), 4.50-4.43 (m, 1H), 4.39 (dd, J=12.0, 6.4 Hz, 1H), 3.96-3.82 (m, 3H), 2.79 (t, J=6.7 Hz, 2), 2.38 (td, J=7.5, 4.9 Hz, 2H), 2.16-1.99 (m, 3H), 1.64 (d, J=5.9 Hz, 2H), 1.44-1.26 (m, 13H), 0.92 (dd, J=9.4, 4.5 Hz, 3H). LC-MS (ESI); m/z: [M+H]+ Calcd. for C25H44N2O6,468.32. Found 470.22.
1.4 MGG-di(Linoleic Acid) (Compound 14)
Preparation of (9Z,9′Z,12Z,12′Z)-2-hydroxypropane-1,3-diylbis(octadeca-9,12-dienoate) (12) Glycerol (0.2 g, 2.17 mmol), linoleic acid (1.28 g, 4.56 mmol), EDCI (0.78 g, 5.00 mmol) and DMAP (0.27 g, 2.17 mmol) were mixed dissolved in 12 mL of DCM in a round bottom bottle. The solution was stirred at room temperature overnight, and then evaporated to remove DCM. Crude product was purified by column chromatography (Hexane:EA, 3:2) to obtain 310 mg of product (compound 12, yield 23%). 1H NMR (600 MHz, CDCl3) δ 5.51-5.24 (m, 7H), 4.33-3.94 (m, 5H), 2.79 (t, J=6.8 Hz, 4H), 2.37 (t, J=7.6 Hz, 4H), 2.07 (q, J=6.9 Hz, 8H), 1.64 (dd, J=14.5, 7.2 Hz, 4H), 1.48-1.19 (m, 28H), 0.91 (t, J=7.0 Hz, 6H).
Preparation of (9Z,9′Z,12Z,12′Z)-2-(2-((tert-butoxycarbonyl)amino)acetoxy)propane-1,3-diyl bis(octadeca-9,12-dienoate) (13) To a solution of DCC (0.12 g, 0.60 mmol) dissolved in 10 mL of DCM, compound 12 (0.31 g, 0.50 mmol), compound 3 (0.11 g, 0.60 mmol), and DMAP (4.3 mg, 0.35 mmol) were added. The solution was stirred overnight at room temperature, and then filtrated to remove DCU. The solution was evaporated to remove solvent and purified by column chromatography (Hexane:EA, 7:3) to obtain 270 mg of the product (compound 13, yield 70%). 1H NMR (600 MHz, CDCl3) δ 5.46-5.21 (m, 8H), 4.99 (s,1H), 4.35 (dd, J=12.1, 4.1 Hz, 2H), 4.17 (dd, J=12.1, 5.9 Hz, 2H), 3.95 (d, J=5.2 Hz, 2H), 2.79 (t, J=6.8 Hz, 4H), 2.33 (t, J=7.6 Hz, 4H), 2.07 (q, J=7.0 Hz, 7H), 1.62 (dd, J=15.9, 8.6 Hz, 5H), 1.47 (s, 9H), 1.43-1.17 (m, 28H), 0.91 (t, J=6.9 Hz, 6H).
Preparation of (9Z,9′Z,12Z,12′Z)-2-(2-aminoacetoxy)propane-1,3-diyl bis(octadeca-9,12-dienoate) (14) To a solution of compound 13 (0.27 g, 0.35 mmol) dissolved in 5 mL of DCM, TFA (0.53 mL, 6.98 mmol) was added slowly and stirred until reactant was consumed. The solution was evaporated to remove solvent. The residue was washed with ether and hexane, and the residual solvent was removed by evaporation under reduced pressure, thereby obtaining about 200 mg of orange gluey product (compound 14, yield 85%). 1H NMR (600 MHz, MeOD) δ 5.43-5.28 (m, 8H), 4.43 (dd, J=12.2, 4.2 Hz, 2H), 4.26 (dd, J=12.2, 5.7 Hz, 2H), 3.88 (s, 2H), 2.79 (t, J=6.6 Hz, 4H), 2.37 (t, J=7.4 Hz, 4H), 2.15-2.03 (m, 8H), 1.68-1.58 (m, 4H), 1.44-1.25 (m, 28H), 0.92 (t, J=7.0 Hz, 6H). LC-MS (ESI); m/z: [M+H]+ Calcd. for C41H71NO6, 673.53. Found 675.91.
1.5 MGG-Linoleic Acid (Compound 18)
Preparation of (9Z,12Z)-3-(2-((tert-butoxycarbonyl)amino)acetoxy)-2-hydroxypropyl octadeca-9,12-dienoate (17) Compound 7 (0.19 g, 0.75 mmol), linoleic acid (0.1 g, 0.36 mmol), 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 0.11 g, 0.73 mmol) and DMAP (44 mg, 0.36 mmol) were mixed and dissolved in 10 mL of DCM in a round bottom bottle. The solution was stirred at room temperature overnight, and then evaporated to remove DCM. Crude product was purified by column chromatography (Hex:EA, 2:3) to obtain 75 mg of sticky liquid product (compound 17, yield 41%). 1H NMR (600 MHz, CDCl3) δ 5.47-5.30 (m, 2H), 4.50-4.01 (m, 5H), 3.96 (d, J=5.4 Hz, 2H), 2.79 (t, J=6.9 Hz, 1H), 2.37 (t, J=7.5 Hz, 2H), 2.07 (dd, J=13.4, 7.2 Hz, 2H), 1.63 (dd, J=23.5, 18.2 Hz, 8H), 1.53-1.38 (m, 9H), 1.45 -1.19 (m, 14H), 0.99-0.85 (m, 3H).
Preparation of (9Z,12Z)-3-(2-aminoacetoxy)-2-hydroxypropyl octadeca-9,12-dienoate (18) To a solution of compound 17 (75 mg, 0.15 mmol) dissolved in 4 mL of DCM, TFA (0.22 mL, 2.93 mmol) was added slowly and stirred until the reactant was consumed, then the solution was evaporated to remove DCM. The residue was washed with ether and DCM. The remained solvent was then evaporated again under reduced pressure, thereby obtaining about 75 mg of light brown gluey product (compound 18, yield 100%). 1H NMR (600 MHz, MeOD) δ 5.43-5.29 (m, 4H), 4.33 (dd, J=11.4, 4.3 Hz, 1H), 4.28 (dd, J=11.4, 5.8 Hz, 1H), 4.15 (dd, J=5.4, 3.8 Hz, 1H), 4.11-4.03 (m, 1H), 3.89 (s, 2H), 2.79 (t, J=6.6 Hz, 2H), 2.38 (t, J=7.5 Hz, 2H), 2.11-2.05 (m, 4H), 1.68-1.59 (m, 2H), 1.41-1.28 (m, 14H), 0.92 (t, J=7.0 Hz, 3H). LC-MS (ESI); m/z: [M+H]+ Calcd. for C23H41NO5,411.30. Found 412.98.
1.6 TGG-di(Palmitoleic Acid) (Compound 22)
Procedure 1
Preparation of (Z)-2-(2-(hexadec-9-enamido)acetoxy)propane-1,3-diyl bis(2-((tert-butoxycarbonyl)amino)acetate) (19)
To a solution of DCC (0.97 g, 4.72 mmol) dissolved in 12 mL of DCM in a round bottom bottle, palmitoleic acid (1 g, 3.93 mmol), t-butyl 2-aminoacetate (0.62 g, 4.72 mmol), and DMAP (0.34 g, 2.75 mmol) were added. The solution was stirred at room temperature overnight, then was filtered to remove DCU and evaporated to remove solvent. Crude product was purified by column chromatography (Hexane:EA, 3:1) to obtain about 1.04 g of clear sticky liquid product (compound 19, yield 72%). 1H NMR (600 MHz, CDCl3) δ 5.92 (s, 1H), 5.36 (ddd, J=7.5, 3.9, 1.9 Hz, 2H), 3.96 (d, J=4.9 Hz, 2H), 2.24 (dd, J=10.1, 5.3 Hz, 2H), 2.03 (q, J=6.5 Hz, 3H), 1.66 (dd, J=14.5, 7.7 Hz, 2H), 1.49 (s, 9H), 1.31 (ddd, J=21.0, 13.6, 7.1 Hz, 16H), 0.90 (dd, J=9.7, 4.4 Hz, 3H).
Preparation of (Z)-2-(hexadec-9-enamido)acetic acid (20) To a solution of compound 19 (1.04 g, 2.83 mmol) dissolved in DCM, TFA (4.33 mL, 56.59 mmol) was added slowly and stirred until the reactant was consumed. The solution was evaporated to remove DCM. The residue was washed with ether and hexane, and the remained solvent was removed again by evaporation under reduced pressure, so as to provide a white powder product (about 870 mg, compound 20) for further use (Yield 99%). 1H NMR (600 MHz, MeOD) δ 5.36 (t, J=4.7 Hz, 2H), 3.90 (s, 2H), 2.26 (t, J=7.5 Hz, 2H), 2.04 (dd, J=11.9, 6.2 Hz, 4H), 1.73-1.60 (m, 2H), 1.33 (dd, J=14.6, 8.6 Hz, 16H), 0.92 (t, J=7.0 Hz, 3H).
Preparation of (Z)-3-(2-((tert-butoxycarbonyl)amino)acetoxy)propane-1,2-diyl bis(2-((Z)-hexadec-9-enamido)acetate) (21) Compound 7 (50 mg, 0.20 mmol), compound 20 (0.14 g, 0.44 mmol), EDCI (70 mg, 0.44 mmol), and DMAP (25 mg, 0.20 mmol) were mixed and added in 10 ml of DCM in a round bottom bottle. The solution was stirred overnight at room temperature overnight, then was evaporated under reduced pressure to remove DCM. Crude product was purified by column chromatography (Hex:EA, 1:4) to obtain 140 mg of orange sticky liquid product (compound 21, yield 84%). 1H NMR (600 MHz, CDCl3) δ 5.43-5.33 (m, 2H), 4.41-4.32 (m, 2H), 4.11-4.03 (m, 4H), 3.95-3.86 (m, 2H), 2.28 (tt, J=13.8, 6.9 Hz, 4H), 2.07-1.99 (m, 4H), 1.92 (tdd, J=20.5, 12.6, 7.8 Hz, 2H), 1.86-1.76 (m, 2H), 1.75-1.51 (m, 12H), 1.48 (s, 9H), 1.44 - 1.21 (m, 32H), 0.91 (q, J=6.9 Hz, 6H).
Preparation of (Z)-3-(2-aminoacetoxy)propane-1,2-diyl bis(2-((Z)-hexadec-9-enamido)acetate) (22) To a solution of compound 21 (0.14 g, 0.17 mmol) dissolved in 4 mL of DCM, TFA (0.26 mL, 3.35 mmol) was added slowly and stirred until reactant was consumed completely. The solution was then evaporated to remove DCM, and the residue was washed with hexane and DCM. The remained solvent was further removed by evaporation under reduced pressure. Thus, about 80 mg of orange gluey product was obtained (compound 22, yield 64%). 1H NMR (600 MHz, MeOD) δ 5.37 (dd, J=19.9, 15.0 Hz, 5H), 4.61-4.24 (m, 4H), 4.04-3.79 (m, 6H), 2.27 (td, J=7.5, 4.0 Hz, 4H), 2.17-1.92 (m, 8H), 1.70-1.55 (m, 4H), 1.49 -1.23 (m, 32H), 0.92 (t, J=7.0 Hz, 6H). LC-MS (ESI); m/z: [M+H]+Calcd. for C41H73N3O8, 735.54. Found 737.33.
Procedure 2
Preparation of (Z)-2-(2-(hexadec-9-enamido)acetoxy)propane-1,3-diyl bis(2-((tert-butoxycarbonyl)amino)acetate) (19) To a solution of DCC (1.16 g, 5.66 mmol) dissolved in 10 ml of DCM in a round bottom bottle, palmitoleic acid (1.2 g, 4.72 mmol), t-butyl 2-5 aminoacetate (0.74 g, 5.66 mmol), and DMAP (0.4 g, 3.3 mmol) were added gradually and stirred at room temperature overnight. The mixture was filtered to remove DCU and evaporated to remove solvent. Crude product was purified by column chromatography (Hexane:EA, 4:1) to obtain about 1.37 g of clear sticky liquid product (compound 19, yield 72%).
Preparation of (Z)-2-(hexadec-9-enamido)acetic acid (20) To a solution of compound 19 (1.37 g, 3.73 mmol) dissolved in DCM, TFA (4.28 mL, 55.91 mmol) was added slowly and stirred until the reactant was consumed. The solution was evaporated to remove DCM. The residue was washed with ether and hexane, and the remained solvent was removed again by evaporation under reduced pressure, so as to provide a white powder product (about 1150 mg, compound 20) for further use (Yield 99%).
Preparation of 3-(2-(tritylamino)acetoxy)propane-1,2-diyl bis(2-((9Z,12Z)-octadeca-9,12-dienamido)acetate) (25) Glycerol (0.29 g, 3.15 mmol), trt-glycine (0.2 g, 0.63 mmol), were placed in a round bottom bottle and the mixture of DCM/dioxane was added as solvent. DCC (0.17 g, 0.82 mmol) and DMAP (38 mg, 0.32 mmol) were added into the round bottom bottle gradually. DCU was removed after the solution was stirred overnight at room temperature. Solvent was removed under reduced pressure. Crude product was purified by column chromatography (Hex:EA=1:3) to obtain 195 mg white solid product (yield 79%).
Preparation of (Z)-3-(2-(tritylamino)acetoxy)propane-1,2-diyl bis(2-((Z)-hexadec-9-enamido)acetate) (27) Compound 25 (0.2 g, 0.51 mmol), compound 20 (0.37 g, 1.18 mmol), EDCI (0.18 g, 1.18 mmol), and DMAP (60 mg, 0.51 mmol) were mixed and dissolved in 10 ml of DCM, and the mixture was stirred at room temperature overnight. The mixture was then evaporated to remove DCM to obtain crude product. The crude product then was purified by column chromatography (Hex:EA, 3:2) to obtain 280 mg of white viscous product (compound 27, yield 56%). 1H NMR (600 MHz, CDCl3) δ 7.49 (d, J=7.6 Hz, 6H), 7.36 (s, 5H), 5.37 (dd, J=9.6, 4.3 Hz, 2H), 5.32-5.20 (m, 1H), 4.31-4.20 (m, 3H), 4.15 (s, 1H), 4.09-4.02 (m, 4H), 3.96 (dd, J=18.1, 5.3 Hz, 1H), 2.27 (td, J=7.7, 3.9 Hz, 4H), 2.08-1.97 (m, 4H), 1.97 -1.87 (m, 2H), 1.86-1.75 (m, 2H), 1.63 (dd, J=23.3, 15.7 Hz, 14H), 1.49-1.20 (m, 30H), 0.91 (q, J=6.9 Hz, 6H).
Preparation of (Z)-3-(2-aminoacetoxy)propane-1,2-diyl bis(2-((Z)-hexadec-9-enamido)acetate) (22) To a solution of compound 27 (0.28 g, 0.29 mmol) dissolved in 8 mL of DCM, TFA (0.33 mL, 4.29 mmol) was added slowly and stirred until reactant was consumed completely. The solution was evaporated to remove solvent, and the residue was purified by column chromatrography (DCM:MeOH, 95:5 to 90:10) to obtain about 200 mg of white to pale yellow viscous solid product (compound 22, yield 94%). 1H NMR (600 MHz, MeOD) δ 5.40 (tt, J=6.3, 4.1 Hz, 1H), 5.37-5.29 (m, 3H), 4.51-4.43 (m, 2H), 4.37 (tt, J=18.1, 6.2 Hz, 2H), 4.05-3.76 (m, 6H), 2.26 (td, J=7.5, 4.1 Hz, 4H), 2.02 (dd, J=20.5, 13.2 Hz, 7H), 1.71-1.55 (m, 5H), 1.46-1.17 (m, 32H), 0.90 (t, J=7.0 Hz, 6H). LC-MS (ESI); m/z: [M+H]+ Calcd. for C41H73N3O8, 735.54. Found 737.18.
1.7 TGG-Palmitoleic Acid (Compound 24)
Preparation of (Z)-2-(2-(hexadec-9-enamido)acetoxy)propane-1,3-diyl bis(2-25 ((tent-butoxycarbonyl)amino)acetate) (23) Compound 4 (0.22 g, 0.54 mmol), compound 20 (0.20 g, 0.65 mmol), EDCI (0.10 g, 0.65 mmol), and DMAP (0.07 g, 0.54 mmol) were mixed and dissolved in 10 ml of DCM in a round bottom bottle. The mixture was stirred at room temperature overnight, and then evaporated to remove DCM. The residue was extracted with EA/H2O, and organic layer was collected, dried over MgSO4, and filtered. The filtrate was evaporated under reduced pressure to remove the solvent. Crude product was purified by column chromatography (Hexane:EA, 2:3) to obtain 290 mg of clear sticky liquid product (compound 23, yield 77%). 1H NMR (600 MHz, CDCl3) δ 5.43-5.31 (m, 2H), 5.21 (s, 1H), 4.50-4.35 (m, 2H), 4.30 (dd, J=15.4, 8.4 Hz, 2H), 4.14-3.96 (m, 3H), 3.98-3.81 (m, 3H), 2.27 (ddd, J=15.2, 7.3, 3.2 Hz, 2H), 2.03 (dd, J=13.1, 6.7 Hz, 2H), 1.92 (ddd, J=19.1, 9.5, 4.8 Hz, 1H), 1.87-1.75 (m, 1H), 1.75-1.51 (m, 8H), 1.47 (s, 15H), 1.44-1.23 (m, 16H), 0.91 (q, J=6.9 Hz, 3H).
Preparation of (Z)-2-(2-(hexadec-9-enamido)acetoxy)propane-1,3-diyl bis(2-aminoacetate) (24) To a solution of compound 23 (0.29 g, 0.41 mmol) dissolved in 5 mL of DCM, TFA (0.95 mL, 12.43 mmol) was added slowly and stirred until all the reactant was consumed. After DCM in solution was removed by rotary evaporator, the residue was washed by hexane and ether. The remained solvent was removed by evaporation under reduced pressure, thereby obtain about 170 mg of clear sticky product (compound 24, yield 83%). 1H NMR (600 MHz, MeOD) δ 5.51-5.45 (m, 1H), 5.36 (dd, J=7.5, 3.4 Hz, 2H), 4.57-4.44 (m, 3H), 3.94-3.88 (m, 5H), 2.33-2.22 (m, 2H), 2.05 (dt, J=12.1, 4.2 Hz, 4H), 1.63 (dd, J=14.4, 7.2 Hz, 2H), 1.45-1.22 (m, 16H), 0.91 (t, J=7.0 Hz, 3H). LC-MS (ESI); m/z: [M+H]+ Calcd. for C25H45N3O7499.33. Found 501.12.
2.1 Encapsulation of Nucleic Acids
In this example, the ability for the present cationic lipids for protecting nucleic acids was investigated. To this purpose, the cationic lipids of Example 1 (i.e., compounds 6, 9, 11, 14, 18, 22, or 24) were independently co-cultivated with plasmids, then subjected to DNA digestion and extraction by following the procedures described in “Materials and Methods” section. Samples were analyzed via agarose gel electrophoresis, and results are provided in
It was evident from the gel electrophoresis that, after digestion, all detectable nucleotides were extracted and derived from the liposomes (i.e., the cationic lipid-DNA complexes), indicating that the present cationic lipids are able to encapsulate and protect plasmids from the action of nucleases (lanes 25-28, and 31-34 of
Further, the encapsulation ratio of the present cationic lipids for encapsulating small RNAs was investigated. To this purpose, the commercial cationic lipids and the present cationic lipids (i.e., compounds 6 (TGG-linoleic acid) and 22 (TGG-di(palmitoleic acid)) were respectively mixed with fluorochrome-conjugated small RNAs (i.e., Cy5-mir302a-siRNA, SEQ ID NO: 6 and 7) by following the preparation procedures described in “Materials and Methods” section, thereby producing lipid nanoparticles (LNPs) containing small RNAs therein. The size, zeta potentials, and encapsulation efficiency (EE) of each LNPs were analyzed in accordance with procedures described in “Materials and Methods” section. Results are summarized in Table 1.
The data in Table 1 revealed that both compounds 6 and 22 successfully formed liposomes and encapsulated siRNAs therein. Specifically, as compared with the LNPs made by commercial cationic lipids MC30, the present LNPs composed of the present compound 22 had a smaller size and a higher encapsulation efficiency.
2. 2 Delivery of Nucleic Acids
In this example, whether the present cationic lipids may promote the delivery of nucleic acids into cells was investigated. To this purpose, A549 and NCI-H460 lung cancer cell lines were respectively co-cultivated with the present or control LNPs with various concentration of siRNAs (0.25, 1.25 or 2.5 ng/μL) therein for 24 hours, and the transfection efficiency was verified by flow cytometry. Results are depicted in
It is evident from the flow cytometry histograms in
The data set forth above collectively indicates that the present novel cationic lipids are capable of encapsulating functional nucleic acids therein, thus, the lipid nanoparticles assembled by the present cationic lipids can serve as a carrier for the delivery of therapeutic agents into cells, thereby achieving effective gene editing, cell engineering, and disease treatment.
It will be understood that the above description of embodiments is given by way of example only and that various modifications may be made by those with ordinary skill in the art. The above specification, examples, and data provide a complete description of the structure and use of exemplary embodiments of the invention. Although various embodiments of the invention have been described above with a certain degree of particularity, or with reference to one or more individual embodiments, those with ordinary skill in the art could make numerous alterations to the disclosed embodiments without departing from the spirit or scope of this invention.
This application claims priority and the benefit of U.S. Provisional Patent Application No. 63/423,506, filed Nov. 8, 2022, the entireties of which is incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 63423506 | Nov 2022 | US |
