Lipid nanoparticles based on multivalent molecules for nucleic acid delivery

Abstract

The present invention relates to nanoparticles, specifically, nanoparticles suitable for the delivery of nucleic acids to cells. The nanoparticles contain a multivalent molecule that stabilizes nucleic acid molecules in the nanoparticles. Specifically, the multivalent molecule has a dendrimer-like structure. The present invention further relates to the production of the nanoparticles and the use of such nanoparticles in the treatment of diseases.

Description

Technical Field

[0001] The present invention relates to the field of nucleic acid therapeutics and provides novel and inventive nanoparticles for intracellular delivery of nucleic acids to a target site. The present invention thus relates to nanoparticles containing nucleic acids.

Background Art

[0002] Nucleic acid therapeutics, such as small molecule antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), messenger RNAs (mRNAs), and other types, are an innovative new class of drugs with the ability to regulate gene expression. In recent years, several nucleic acid-based drug products for in vivo applications, including ASOs, N-acetylgalactosamine (GalNAc)-siRNA conjugates, lipid nanoparticles (LNPs) containing siRNA or mRNA, and some viral vectors containing plasmid DNA (pDNA), have been approved. In addition, several nucleic acid therapeutics are in late-stage clinical trials. Furthermore, several genetically engineered ex vivo cell therapy drug products have been approved.

[0003] The therapeutic application of nucleic acids by parenteral administration is difficult. Although the size and physicochemical properties vary depending on the type of nucleic acid, common properties include a large size, high molecular weight, and negative charge. As a result, when administered systemically, nucleic acids are rapidly cleared from the circulation due to renal filtration and nuclease degradation. In addition, nucleic acid therapeutics act intracellularly but cannot easily cross the cell membrane. Finally, the administration of exogenous nucleic acids elicits an immune response. This can be beneficial (e.g., for vaccine development), but usually, this contributes to the rapid clearance and harmful effects of nucleic acids.

[0004] To overcome these challenges, all nucleic acid therapeutics rely on chemical modifications and / or delivery systems based on nanotechnology. All approved nucleic acid therapeutics rely on chemical modifications and / or nanotechnology platforms to facilitate their intracellular delivery and subsequently induce a therapeutic effect after parenteral administration: 1) ASOs are highly chemically modified to increase their stability, reduce immunostimulatory effects, and increase efficacy. They are administered subcutaneously to target hepatocytes or intrathecally to target cells in the central nervous system. 2) GalNAc-siRNA conjugates are modified similarly to ASOs and are administered subcutaneously. The GalNAc moiety ensures asialoglycoprotein receptor-mediated uptake in hepatocytes. 3) Lipid nanoparticles (LNPs) are approximately 50 - 100 nm in diameter and can be administered systemically, intradermally, or intramuscularly. After systemic administration, LNPs efficiently accumulate in hepatocytes, providing an opportunity for gene silencing (siRNA) or protein production (mRNA). After intradermal or intramuscular administration, LNPs are taken up by immune cells, such as antigen-presenting cells that can be utilized for vaccine purposes. LNPs are the current gold standard for mRNA therapy and are likely to become the standard delivery platform for in vivo gene editing applications. However, LNPs contain synthetic (non-natural) polyethylene glycol (PEG)-conjugated lipids, which have been associated with hypersensitivity reactions and / or anaphylaxis. In addition, current LNP systems predominantly accumulate in the liver after intravenous administration. 4) Virus delivery systems, such as adenovirus, lentivirus, or adeno-associated virus (AAV) vectors, are effective means of transporting DNA. Viral vectors are characterized by their limited payload capacity and immunogenicity. However, in immunoprivileged tissues such as the eye, viral vectors are currently the optimal standard for nucleic acid therapeutics. Viral vectors are widely used in ex vivo therapy (e.g., CAR T), or are administered intravenously to target cells in the liver, intravitreally / subretinally to target cells in the retina, or intramuscularly for vaccine purposes.

[0005] With the exception of viral vector- or LNP-mRNA-based vaccines, the majority of approved nucleic acid therapeutics have been developed for indications other than immunotherapy. Delivering therapeutic nucleic acids to the myeloid compartment thus remains an issue. Furthermore, chemical modification of nucleic acid molecules or viral delivery inherently poses a risk of unwanted activation of the immune system, leading to degradation or clearance of the nucleic acid therapeutic, or an undesired immune response.

[0006] For example, nanoparticles for transporting nucleic acids are described in WO 2009127060 A1 pamphlet, which describes the use of cationic lipids combined with non-cationic lipids and nucleic acids. Cationic lipids neutralize nucleic acids and enable the formation of nanoparticles that can be used for non-targeted delivery of nucleic acids in a subject. The drawback of these nanoparticles is that they cannot target the myeloid compartment.

[0007] Other systems, such as WO 2019103998 A2 pamphlet, describe nanobiologics that can target the myeloid compartment, which contain phospholipids and apoA1, as well as small molecule drugs. The drawback of these nanobiologics is that due to their hydrophobic core, incorporation of polar constructs such as nucleic acids, e.g., DNA and RNA, is not possible.

[0008] In International Publication No. WO 2017 / 048789 A1 pamphlet, dendrimer materials are described that have a limited number of ionizable groups per molecule and have unnatural sulfide groups that tend to oxidize in vivo. In the preparation of these materials, polyfunctional molecules react with bifunctional molecules to form intermediates, which may thus be difficult to obtain in pure (non-crosslinked) form.

[0009] Therefore, there is a need for an improved alternative delivery system for therapeutic nucleic acids to the myeloid compartment.

[0010] The above problems are solved, inter alia, by the present invention as defined in the appended claims. SUMMARY OF THE INVENTION

[0011] The present invention constructs a nanoparticle platform technology for the targeting and / or delivery of nucleic acid therapeutics, more specifically for the targeting and / or delivery of nucleic acid therapeutics to the myeloid cell compartment. The nanoparticles taught herein are (phospho)lipid-based nanoparticles stabilized by apolipoprotein stabilizers (or derivatives of apolipoproteins or mimics or mimic derivatives of apolipoproteins). In circulation, the stabilizer and (phospho)lipid encapsulate and / or protect the nucleic acid payload, thereby preventing degradation and rapid clearance. At the same time, the nanoparticles reduce the immune-stimulatory related adverse effects of nucleic acid therapeutics by limiting undesirable interactions with components in the blood, for example, limiting undesirable interactions between the nucleic acid payload and components in the blood. The apolipoprotein stabilizer can also direct the nanoparticles to the myeloid cell compartment and thus acts as a targeting moiety. Thus, the present invention enables efficient delivery of nucleic acid therapeutics to the myeloid cell compartment in lymphoid organs, such as the bone marrow and spleen, for effective immunotherapy.

[0012] The nanoparticles taught in this specification further include multivalent molecules. The multivalent molecules have multiple positively ionizable groups and / or cationic groups that can efficiently bind to nucleic acids and capture (or complex with) them. The multivalent molecules of the present invention bind to nucleic acids more strongly than, for example, monovalent amphiphilic molecules. Thus, nucleic acids are better bound and / or retained within the nanoparticles taught in this specification. In addition, the multivalent molecules of the present invention can also interact with apolipoprotein stabilizers (or by derivatives of apolipoprotein, or mimetics or mimetic derivatives of apolipoprotein) because apolipoproteins as a whole have a negative charge. As a result, apolipoproteins (or by derivatives of apolipoprotein, or mimetics or mimetic derivatives of apolipoprotein) will be better incorporated into the nanoparticles of the present invention. Thus, in circulation, for example, in the blood of a subject, the nanoparticles of the present invention release little or no apolipoprotein components from the nanoparticles.

[0013] The present invention specifically relates to nanoparticles containing multivalent molecules such as, but not limited to, dendrimers. Further, the present invention specifically relates to nanoparticles containing stabilizer materials such as, but not limited to, apolipoproteins, apolipoprotein derivatives, apolipoprotein mimetics, or apolipoprotein mimetic derivatives. The present invention further relates to methods of treatment using nanoparticles in the treatment of diseases, for example, by stimulating or inhibiting the innate immune response. The present invention further relates to in vivo, in vitro, or ex vivo methods for introducing nucleic acids into cells using nanoparticles.

[0014] Thus, in a first aspect, the present invention is a nanoparticle comprising a core and an outer layer, wherein the core comprises - nucleic acid - multivalent molecule and the outer layer comprises - apolipoprotein, apolipoprotein derivative, apolipoprotein mimetic, and / or apolipoprotein mimetic derivative, - phospholipids, - sterols, and optionally - filler molecules, comprising a polyvalent molecule having the formula (I): [Core] x -[BU] y -[TU] z (I) wherein the core (i.e., the core of the polyvalent molecule) is nitrogen or a C 1 ~C 18 linear, branched, or cyclic group containing 1 to 15 nitrogen heteroatoms and optionally 1 to 4 oxygen heteroatoms, x represents the number of connections from the core to the branching unit BU (or to the terminal unit TU when y = 0), and x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 12, and all of these connections constitute a connection from a nitrogen atom of the core to a carbon atom of the branching unit BU (or terminal unit TU), the branching unit BU has the formula (IIa), (IIb), (IIc), or (IId):

[0015]

Chemical formula

[0016] In a second aspect, the invention relates to a composition comprising the nanoparticles according to the first aspect of the invention and a physiologically acceptable carrier, preferably a pharmaceutical composition.

[0017] In a third aspect, the present invention relates to the nanoparticles according to the first aspect of the present invention or the composition according to the second aspect of the present invention for use as a medicament.

[0018] In a fourth aspect, the present invention is a nanoparticle or composition according to the first aspect of the present invention or the second aspect of the present invention for use in the treatment of a disease by stimulating or inhibiting the innate immune response, preferably wherein the disease is cancer, cardiovascular disease, autoimmune disorder, or xenograft rejection.

[0019] In a fifth aspect, the present invention is a method for producing nanoparticles, a) mixing a (lipid) component in an organic solvent with nucleic acid in an aqueous buffer, preferably by rapid mixing, to produce nanoparticles, wherein the (lipid) component comprises phospholipids, sterols, multivalent molecules, and optionally a filler material (e.g., triglyceride), and the aqueous buffer has a pH of 5.5 or less, preferably 5.0 or less; b) mixing the lipid nanoparticles produced in a) with an apolipoprotein, apolipoprotein derivative, apolipoprotein mimetic, and / or apolipoprotein mimetic derivative, preferably by rapid mixing, to produce the nanoparticles of the present invention at a pH of 5.5 to 9.0, preferably 6.0 to 8.0, more preferably 6.5 to 8.0; and the method comprises the above steps.

[0020] In a sixth aspect, the present invention is an in vitro or ex vivo method for introducing nucleic acid into cells, the method comprising contacting the cells with the nanoparticles according to the first aspect of the present invention or the composition according to the second aspect of the present invention.

[0021] In a seventh aspect, the present invention is an in vivo method for introducing nucleic acid into cells, the method comprising contacting the cells with the nanoparticles according to the first aspect of the present invention or the composition according to the second aspect of the present invention.

[0022] In an eighth aspect, the invention relates to a method for in vivo delivery of nucleic acids, the method comprising administering to a subject a nanoparticle according to a first aspect of the invention or a composition according to a second aspect of the invention.

[0023] In a ninth aspect, the invention relates to a method for treating a disease or disorder in a subject in need thereof by stimulating or inhibiting the innate immune response, the method comprising administering to the subject a therapeutically effective amount of a nanoparticle according to a first aspect of the invention or a composition according to a second aspect of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0024]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

DETAILED DESCRIPTION OF THE INVENTION

[0025] For the purposes of the present invention, the following terms are defined below.

[0026] As used herein, the terms "a", "an", and "the" in the singular form include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes combinations of two or more cells and the like.

[0027] As used herein, the term "and / or" refers to a situation where one or more of the recited instances may occur alone, or in combination with at least one of the recited instances, up to and including all of the recited instances.

[0028] As used herein, the term "antigen" refers to a substance to which the binding portion of an antibody can bind. The specific immunoreactive site within an antigen is known as an "epitope" (or antigenic determinant). The target of an antibody or its antigen-binding portion can include an antigen, for example, as defined herein.

[0029] As used herein, the term "at least" a particular value means that particular value or more. For example, "at least 2" is understood to be the same as "2 or more", i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and so on. As used herein, the term "at most" a particular value means that particular value or less. For example, "at most 5" is understood to be the same as "5 or less", i.e., 5, 4, 3, …, -10, -11, and so on.

[0030] As used herein, the word "comprise" or variations thereof, such as "comprises" or "comprising", are understood to mean that the recited element, integer, or step, or group of elements, integers, or steps is included, but does not exclude any other element, integer, or step, or group of elements, integers, or steps. The verb "comprise" includes the verbs "consist essentially of" and "consist of".

[0031] As used herein, the term "conventional techniques" refers to situations where the methods for performing conventional techniques used in the methods of the present invention would be apparent to one of ordinary skill in the art. The practice of conventional techniques in molecular biology, biochemistry, computational chemistry, cell culture, recombinant DNA, bioinformatics, genomics, sequencing, and related fields is well known to one of ordinary skill in the art and is discussed, for example, in the following references: Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987 and periodic updates, and the series Methods in Enzymology, Academic Press, San Diego.

[0032] As used herein, nanoparticles refer to small particles, for example, in the range of 10 to 200 nm in diameter, which can be used to deliver a payload to a target, for example, an organ or cell in a subject.

[0033] As used herein, the term "identity" refers to a measure of nucleotide or amino acid sequence identity. Generally, sequences are aligned so as to obtain the highest degree of match. "Identity" itself has the meaning recognized in the art and can be calculated using published techniques. See, for example, (Computational Molecular Biology, Lesk, A. M., ED., Oxford University Press, New York, 1988; Biocomputing: Informatics And Genome Projects, Smith, D. W., ED., Academic Press, New York, 1993, Computer Analysis Of Sequence Data, Part I, Griffin, A. M., And Griffin, H. G., EDS., Humana Press, New Jersey, 1994, Sequence Analysis In Molecular Biology, Von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer; Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). There are several methods for measuring identity between two nucleotide or amino acid sequences, but the term "identity" is well known to those skilled in the art (Carillo, H., and Lipton, D., SIAM J. Applied Math (1988) 48:1073). Methods commonly used to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Guide To Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994 and Carillo, H., and Lipton, D., Siam J. Applied Math (1988) 48:1073).The method for determining identity and similarity is programmed by a computer program. Preferred computer program methods for determining identity and similarity between two arrays include, but are not limited to, the GCS program package (Devereux, J., et al., Nucleic Acids Research (1984) 12(1):387), BLASTP, BLASTN, FASTA (Atschul, S. F. et al., J. Molec. Biol. (1990) 215:403).

[0034] By way of example, a polynucleotide having a nucleotide sequence that has at least, for example, 95% "identity" to a reference nucleotide sequence encoding a polypeptide of a particular sequence is intended to mean that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may contain up to 5 point mutations per 100 nucleotides of the reference amino acid sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted and / or replaced with another nucleotide, and / or up to 5% of the number of all nucleotides in the reference sequence may be inserted into the reference sequence. These mutations in the reference sequence can occur at positions at the 5' or 3' ends of the reference nucleotide sequence, or at any position between the end positions, either individually among the nucleotides of the reference sequence or in one or more consecutive groups within the reference sequence.

[0035] Similarly, a polypeptide having an amino acid sequence that has at least, for example, 95% "identity" to the reference amino acid sequence of SEQ ID NO: X is intended to have an amino acid sequence that is identical to the reference sequence, except that the amino acid sequence can contain up to 5 amino acid changes per 100 amino acids of the reference amino acid of SEQ ID NO: X. In other words, to obtain a polypeptide having an amino acid sequence that is at least 95% identical to the reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or up to 5% of the total number of amino acid residues in the reference sequence may be inserted into the reference sequence. These changes to the reference sequence can occur at the amino or carboxy terminal positions of the reference amino acid sequence, or at any position between the terminal positions, either individually among the residues of the reference sequence or in one or more contiguous groups within the reference sequence.

[0036] As used herein, the term "in vitro" refers to experiments or measurements performed using components of an organism that have been isolated from their natural state.

[0037] As used herein, the term "ex vivo" refers to experiments or measurements performed within or on tissue derived from an organism in an external environment with minimal modification from its natural state.

[0038] As used herein, the terms "nucleic acid", "nucleic acid molecule", and "polynucleotide" are intended to include DNA molecules and RNA molecules, as well as locked nucleic acids (LNAs), bridged nucleic acids (BNAs), morpholinos, or peptide nucleic acids (PNAs). The nucleic acid (molecule) can be any nucleic acid (molecule), which can be single-stranded or double-stranded.

[0039] As used herein, the terms "sequence", or "nucleic acid sequence", "nucleotide sequence", or "polynucleotide sequence" when referring to a nucleotide, refer to the order of nucleotides in a nucleic acid and / or polynucleotide. Within the context of the present invention, a first nucleic acid sequence may be contained within, or overlap with, a further nucleic acid sequence.

[0040] As used herein, the terms "subject", "individual", "animal", "patient", or "mammal", used interchangeably, refer to any subject for which diagnosis, prognosis, or treatment is desired, specifically, a mammalian subject. Mammalian subjects include humans, domestic animals, farm animals, and zoo animals, sport animals, or pets, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, bears, and the like. As defined herein, a subject may be alive or dead. A sample may be taken from a subject postmortem, i.e., after death, and / or a sample may be taken from a living subject.

[0041] As used herein, the terms "treatment", "treating", "alleviating temporarily", "reducing", or "relieving", used interchangeably, refer to an approach for obtaining a beneficial or desired result, including, but not limited to, a therapeutic benefit. A therapeutic benefit means eradication of the underlying disease, or alleviation or reduction (or delay) of its progression. Further, a therapeutic benefit is achieved by eradication of one or more of the physiological symptoms associated with the underlying disease, or alleviation or reduction (or delay) of its progression, such that improvement or slowing or reduction of decline is observed in a patient, even if the patient may still be suffering from the underlying disease.

[0042] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid molecule that is ligated thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop to which additional DNA segments can be ligated. The term "vector" also refers to a viral particle containing the nucleic acid of interest (i.e., a viral vector).

[0043] As used herein, the term "payload" generally refers to a substance contained within a particle and delivered to a target site. When referring to the nanoparticles of the present invention, the term "payload" preferably refers to a nucleic acid in combination with a multivalent molecule.

[0044] As used herein, when referring to targeting a cell (e.g., a target cell such as, but not limited to, a myeloid cell) or targeting a tissue or organ, the term "targeting" is understood to mean being placed in the vicinity of, or concentrated in the vicinity of, the intended cell, organ, or tissue. This indicates that, on average, more nanoparticles are present in the vicinity of the intended cell, organ, or tissue than would be predicted based on the random or natural distribution of the nanoparticles when targeting the intended cell, organ, or tissue. As used herein, "vicinity" means being positioned such that the nanoparticles can interact with the cell (or tissue or organ) to deliver its payload (nucleic acid).

[0045] As used herein, the term "myeloid cell" refers to blood cells derived from a common progenitor cell of megakaryocytes, granulocytes, monocytes, and erythrocytes. Myeloid cells are a major cell compartment of the immune system, including monocytes, dendritic cells, tissue macrophages, and granulocytes. The term "myeloid compartment" as used herein refers to all myeloid cells in an organism.

[0046] Alkyl and alkylene groups may be straight-chain, branched-chain or cyclic. Alkyl and alkylene groups contain from 1 to 36 carbon atoms, preferably from 1 to 18 carbons. Alkyl and alkylene groups optionally contain one or more double bonds (C=C), in which case these groups are unsaturated. Alkyl and alkylene groups optionally contain from 1 to 8 heteroatoms selected from the group consisting of O, N, P, S, and F, preferably from the group consisting of O and N.

[0047] Aryl and arylene groups contain from 6 to 24 carbon atoms, while alkylene aryl and arylene alkyl groups contain from 7 to 25 carbon atoms. Aryl, arylene, alkylene aryl, and arylene alkyl groups optionally contain from 1 to 8 heteroatoms selected from the group consisting of O, N, P, S, and F, preferably from the group consisting of O and N.

[0048] Ester, amide, urethane, urea, carbonate, carboxylic acid, ketone, aldehyde, ether, and alcohol groups are defined below, where R x represents a hydrogen atom, or a cyclic, straight-chain, or branched-chain alkyl or alkylene group. In groups containing more than one R x element, these elements may be selected independently. The ester (functional) group or moiety shown in this document is understood as a group according to the formula: -C(O)-O-. The amide (functional) group or moiety shown in this document is understood as a group according to the formula: -NR x -C(O)-. The urethane (functional) group or moiety shown in this document is understood as a group according to the formula: -NR x -C(O)-O-. The urea (functional) group or moiety shown in this document is understood as a group according to the formula: -NR x -C(O)-NR xis understood as a group represented by. The carbonate (functional) group or moiety shown in this document is understood as a group represented by the formula: -O-C(O)-O-. The carboxylic acid (functional) group or moiety shown in this document is understood as a moiety or group represented by the formula: -C(O)OH. The ketone (functional) group or moiety shown in this document is understood as a group represented by the formula: -C(O)-. The aldehyde (functional) group or moiety shown in this document is understood as a group represented by the formula: -C(O)H. The ether (functional) group or moiety shown in this document is understood as a group represented by the formula: -O-. The alcohol (or hydroxy) functional group or moiety shown in this document is understood as a group represented by the formula: -OH.

[0049] The section headings used in this specification are for organizational purposes only and are not to be considered as limiting the subject matter described.

[0050] Various terms related to the methods, compositions, uses, and other aspects of the present invention are used throughout this specification and the claims. Such terms shall also, unless otherwise indicated, have the meanings as commonly understood in the art to which the present invention pertains. Other specifically defined terms shall be construed in a manner consistent with the definitions provided herein. Although preferred materials and methods are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention.

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0052] The present invention constructs a nanoparticle platform technology for the targeting and / or delivery of nucleic acid therapeutics to the myeloid cell compartment. The nanoparticles described herein are (phospho)lipid-based nanoparticles stabilized by apolipoproteins (or derivatives of apolipoproteins or mimics or mimic derivatives of apolipoproteins), also referred to herein as apolipoprotein stabilizers. In circulation, the stabilizer and / or (phospho)lipid encapsulate and / or protect the nucleic acid therapeutic payload, thereby preventing degradation and rapid clearance. At the same time, the nanoparticles reduce the immune-stimulatory related adverse effects of the nucleic acid therapeutic by limiting undesirable interactions with components in the blood, for example, limiting undesirable interactions between the nucleic acid payload and components in the blood. The apolipoprotein stabilizer can also direct the nanoparticles to the myeloid cell compartment and thus acts as a targeting moiety. Accordingly, the present invention enables efficient delivery of nucleic acid therapeutics to the myeloid cell compartment in lymphoid organs, such as the bone marrow and spleen, for effective immunotherapy. In certain embodiments, the nanoparticles taught herein are used to target or deliver to myeloid cells, including all blood cells derived from progenitor cells of granulocytes, monocytes, erythrocytes, or platelets. In certain embodiments, the nanoparticles taught herein are used to target or deliver to monocytes, dendritic cells, tissue macrophages, or granulocytes.

[0053] The nanoparticles described herein are lipid-based nano-sized formulations (particle diameter about 10 - 300 nm). Without being bound by theory, the inventors believe that the particles have a core composed of nucleic acids that interact with multivalent molecules, and an outer surface incorporating a stabilizing protein, preferably an apolipoprotein, an apolipoprotein derivative, an apolipoprotein mimetic, and / or an apolipoprotein mimetic derivative. The core is relatively hydrophobic due to the hydrophobic portion of the multivalent molecule. The outer surface consists mostly of a layer of phospholipids incorporating a stabilizing protein, preferably an apolipoprotein stabilizer, and further contains a sterol, such as cholesterol. The hydrophilic head of the phospholipid faces the outer aqueous environment, while the hydrophobic tail faces inward towards the core of the nanoparticle. Possibly, the nanoparticles contain multiple layers, but in any case, the nanoparticles have nucleic acids bound and embedded inside the particle by a hydrophobic multivalent molecule, and an outer protective surface layer having phospholipids, sterols, and a stabilizing protein, preferably an apolipoprotein, an apolipoprotein derivative, an apolipoprotein mimetic, and / or an apolipoprotein mimetic derivative. Thus, the nanoparticles described herein are different from the nanoparticles described in the art, for example, vesicles-like nanoparticles containing a lipid bilayer surrounding an aqueous core. The apolipoprotein-decorated nanoparticles of the present invention are also different from the nanoparticles described in the art, for example, nanoparticles simply stabilized by synthetic (non-natural) hydrophilic polymers, such as polyethylene glycol (PEG) or its derivatives. Notably, previously reported nanoparticles typically cannot target nucleic acid cargo to specific cells, whereas the nanoparticles taught herein can target nucleic acid cargo to the myeloid cell compartment, as described elsewhere herein.

[0054] In the nanoparticle formulation, phospholipids accumulate at the interface between the hydrophobic core and the aqueous solvent due to their amphiphilic nature, effectively forming a lipid monolayer (or lipid multilayers) surface barrier. For use in biopharmaceuticals, single or multiple phospholipid types can be used due to their inherent biocompatibility and net neutral charge. Optionally, charged (phosphorus) lipids in molar percentages may be added to impart specific charge characteristics to the overall formulation (1 - 95 mol% or less, e.g., 1 - 20 mol% or 1 - 10 mol% or 1 - 5 mol%, these molar percentages are relative to the total amount of phospholipids utilized).

[0055] Lipid nanoparticles are engineered to form complexes with nucleic acids, which are hydrophilic in nature, within their structure. Therefore, helper molecules are required to draw the nucleic acids into the hydrophobic nanoparticle core. For this purpose, multivalent molecules are included in the nanoparticles. Multivalent molecules contain two or more positively ionizable groups and / or cationic groups. Positively ionizable groups become cationic groups upon protonation at low pH. Cationic groups can bind to anionic phosphate groups in the sugar - phosphate backbone of the nucleic acid payload via ionic interactions. The hydrophobic portion of the multivalent molecule forms a shell around the hydrophilic nucleic acid molecule. This shell can then interact with the outer layer of the particle composed of phospholipids, sterols, and protein stabilizers (e.g., apolipoprotein stabilizers).

[0056] By utilizing multivalent positively ionizable molecules and / or cationic molecules instead of monovalent amphiphilic molecules, the binding to nucleic acids becomes stronger. As a result, the nucleic acids are better bound within and / or retained in the nanoparticles taught herein. Additionally, multivalent molecules can also interact with apolipoprotein stabilizers, as described in Sparks DL, Lund-Katz S, Phillips MC. The charge and structural stability of apolipoprotein A-I in discoidal and spherical recombinant high density lipoprotein particles. J Biol Chem. 1992 Dec 25;267(36):25839-47. PMID: 1464598, since apolipoproteins have an overall negative charge. As a result, apolipoproteins are better bound within and trapped in the nanoparticles of the present invention. Finally, the transfection of nucleic acids into target cells can be assisted by the presence of multivalent molecules, as multivalent molecules can efficiently bind to and interact with multiple phospholipids in the (myeloid) cell barrier, thereby creating a gap for the entry of nucleic acids.

[0057] In addition to nucleic acids, multivalent molecules, phospholipids, sterols, and stabilizers (optionally having targeting ability, e.g., the ability to target the myeloid cell compartment), additional hydrophobic filler molecules may be included in the nanoparticle formulations taught herein. The main uses of these are to alter the physicochemical properties of the nanoparticles or to improve stability. The filler molecules are present (primarily) in the core of the nanoparticles. The filler molecules are preferably biocompatible and non-limiting examples include glyceride lipid particles (lipid diglycerides or triglycerides), sterol esters, and esters of fatty acids.

[0058] Nanoparticles containing therapeutic nucleic acids are predicted to precisely regulate gene expression in the myeloid cell compartment and thereby modulate the immune response. A major advantage of the nanoparticle platform technology taught herein is that the nucleic acid payload can be exchanged without altering the biological behavior and interactions of the formulation. Nanoparticles containing therapeutic nucleic acids taught herein can thus be implemented as immunotherapy, for example, to boost the immune response to treat cancer or infectious diseases, or to reduce the immune response, for example, to treat autoimmune diseases or during organ transplantation.

[0059] Thus, in a first aspect, the invention provides - a nucleic acid, - a multivalent molecule, - a stabilizer, preferably an apolipoprotein, apolipoprotein derivative, apolipoprotein mimetic, and / or apolipoprotein mimetic derivative, which is also a targeting component since the nanoparticles can be directed to specific cells in the body (i.e., the myeloid compartment), - a phospholipid, - a sterol, and optionally - a filler molecule comprising, consisting essentially of, or consisting of nanoparticles, wherein the multivalent molecule has the formula (I): [Core] x -[BU] y -[TU] z (I) and wherein the multivalent molecule has a dendrimer (like) branched chain architecture composed of a core linked by x connections to an (optional) branch unit (BU) (i.e., the innermost BU) or a terminal unit (TU) (e.g., if the multivalent molecule has no BUs), the BUs are stacked in y layers of generations, where y is 0, 1, 2, 3, 4, or 5, The multivalent molecule comprises z terminal units (TU), preferably TU connected to the core (when y = 0) or the outermost BU unit (when y = 1, 2, 3, 4, or 5), Relating to nanoparticles, wherein the multivalent molecule is multivalent with respect to positively ionizable groups and / or cationic groups, and the cumulative number of such groups in the multivalent molecule is at least 2.

[0060] In certain embodiments, the nanoparticle comprises a core and an outer layer, and the core - nucleic acid - multivalent molecule comprises, consists essentially of, or consists of and the outer layer - a stabilizer, preferably an apolipoprotein, apolipoprotein derivative, apolipoprotein mimetic, and / or apolipoprotein mimetic derivative, - a phospholipid, - a sterol, and optionally - a filler molecule comprises, consists essentially of, or consists of The multivalent molecule has the formula (I): [Core] x -[BU] y -[TU] z (I) wherein the core is nitrogen or a C containing 1 to 15 nitrogen heteroatoms and optionally 1 to 4 oxygen heteroatoms 1 ~C 18 linear, branched, or cyclic group, x represents the number of connections from the core to the branching unit BU (i.e., the innermost branching unit BU) or to the terminal unit TU when y = 0, and x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 12, and all of these connections are from a nitrogen atom of the core to a carbon atom of the branching unit BU (i.e., the innermost branching unit BU) or to the terminal unit TU when y = 0, etc., The branching unit BU has the formula (IIa), (IIb), (IIc), or (IId):

[0061]

Chem.

[0062] Since the branching unit BU branches in two directions, z is equal to x×2 y For example, a second-generation polyvalent molecule (y = 2) having 4 connections from the core (x = 4) will have 4×2 2 = 16 terminal units TU. In another example, a third-generation polyvalent molecule (y = 3) having 4 connections from the core (x = 4) will have 4×2 3 = 32 terminal units TU. In another example, a generation 0 polyvalent molecule (y = 0) having 5 connections from the core (x = 5) will have 5×2 0 = 5 terminal units TU.

[0063] The nanoparticles described herein mainly have an outer layer containing a stabilizer, a phospholipid, and a sterol, and a core containing a polyvalent molecule and a cargo which is a nucleic acid (also referred to herein as a payload). The nanoparticles can be used to deliver the cargo to its intended destination, such as a cell, tissue, or organ. Preferably, the nucleic acid cargo is delivered intracellularly in the target cell, tissue, or organ.

[0064] The present invention is based on the recognition that nucleic acids can be successfully loaded into the nanoparticles described herein and that the nanoparticles can be used to deliver the nucleic acids intracellularly, for example, to intended target cells. This has been achieved by a combination of the following properties: - The ability to load nucleic acids into the hydrophobic core of the nanoparticles by neutralizing the nucleic acids using multivalent molecules; - Defining the range of amounts of the structural components of the nanoparticles and their relative amounts, for example, the amounts of apolipoproteins, sterols, phospholipids, multivalent molecules, nucleic acids, and optionally filler molecules (e.g., triglycerides).

[0065] In addition, the inventors have developed a method for successfully incorporating nucleic acids into stabilizer-based nanoparticles, preferably apolipoprotein (and / or apolipoprotein mimetic)-based nanoparticles, since the individual components cannot simply be mixed to obtain the nanoparticles described herein. It has been found that it is essential to perform a two-step formulation process in which, in a first step, nanoparticles containing nucleic acids are formed and, in the next step, a stabilizer (e.g., an apolipoprotein, apolipoprotein derivative, apolipoprotein mimetic, and / or apolipoprotein mimetic derivative) is included in the nanoparticles. Preferably, the first step is carried out at low pH and the second step is carried out at physiological pH. This discovery has made it possible for the first time to include nucleic acids in the nanoparticles described herein, thereby enabling delivery of the nucleic acids to target cells.

[0066] The present invention thus provides multivalent molecules that enable the loading of nucleic acids onto the nanoparticles described herein, i.e., nanoparticles having a hydrophobic core. The use of nanoparticles based on a hydrophobic core has been previously described in Jayaraman M et al. (Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo. Angew Chem Int Ed Engl. 2012 Aug 20;51(34):8529-33), where nucleic acids are loaded into the core using a positively charged amphiphilic substance, such as Dlin-MC3-DMA. Such molecules typically contain amine groups that can be protonated at low pH. A positively charged amphiphilic molecule is then used to "neutralize" the negatively charged nucleic acid, enabling loading into the hydrophobic core. While this method has its advantages, there are some concerns regarding the potential toxicity of the amphiphilic substances applied. Thus, alternative solutions are desired. The inventors herein describe the discovery that the multivalent molecules defined herein can be used for this purpose. Furthermore, and importantly, the inventors have surprisingly found that these multivalent molecules can be used to more efficiently deliver nucleic acids to target cells and enable their function. For example, FIGS. 11 and 12 describe how some formulations according to the present invention are much more efficient at silencing a luciferase reporter gene by delivering a specific siRNA to cells compared to aNP or LNP control formulations containing the ionizable cationic lipid Dlin-MC3-DMA. In addition, FIG. 14 describes how formulations according to the present invention are much more efficient at expressing a luciferase reporter gene by delivering a specific mRNA to cells compared to aNP or LNP control formulations containing the ionizable cationic lipid ALC-0315. FIG. 16 describes the efficient delivery of formulations according to the present invention to spleen and bone marrow tissues in mice. a. Multivalent molecule

[0067] The multivalent molecule according to formula (I) is a molecule having a dendrimer-like branched-chain architecture and is composed of three units: a core unit, a branching unit (BU), and a terminal unit (TU). Optionally, the BU may be omitted. [Core] x -[BU] y -[TU] z (I)

[0068] Specifically, starting from the internal core unit, the multivalent molecule branches in an equal manner towards the external terminal unit (TU). Starting from the core, all the branching units (BU) within the molecule are divided into two directions, both of which are connected to either the next BU or TU.

[0069] Furthermore, the multivalent molecules described herein are defined as having specific discrete generations. A multivalent molecule of generation zero (G0, y = 0) is a molecule without a BU, where the core is directly attached to the TU. A multivalent molecule of the first generation (G1, y = 1) is defined as a molecule in which the core is connected to one layer of BUs, which are in turn connected to the TU. A multivalent molecule of the second generation (G2, y = 2) is defined as a molecule in which the core is connected to the first layer of BUs, which are connected to the second layer of BUs, and these external second-layer BUs are connected to the TU. A multivalent molecule of the third generation (G3, y = 3) is defined as a molecule in which the core is connected to the first layer of BUs, which are connected to the second layer of BUs, which are connected to the third layer of BUs, and these external third-layer BUs are connected to the Tu. The same applies to G4 (y = 4) and G5 (y = 5). Thus, the generation number of the multivalent molecule represented by the letter y indicates how many consecutive layers of BUs are incorporated into the multivalent molecule when counting from the internal core towards the external TU group.

[0070] For purposes of illustration and clarification, the following Scheme 1 shows a non-limiting schematic example of a multivalent molecule: A molecule of generation 0 (G0 material, i.e., y = 0) having a pentafunctional core (x = 5) and five TU groups (z = 5), A molecule of generation 1 (G1 material, i.e., y = 1) having a tetrafunctional core (x = 4) and eight TU groups (z = 8), A molecule of generation 2 (G2 material, i.e., y = 2) having a trifunctional core (x = 3) and twelve TU groups (z = 12), A molecule of generation 3 (G3 material, i.e., y = 3) having a bifunctional core (x = 2) and sixteen TU groups (z = 16), Here, x, y, and z refer to x, y, and z in formula (I). In Scheme 1, the branching unit BU of each generation's layer is indicated by its number.

[0071] [Chemical formula]

[0072] The polyvalent molecule can be of generation 0, or 1, or 2, or 3, or 4, or 5 (y = 0, 1, 2, 3, 4, or 5). In certain embodiments, the polyvalent molecule is of generation 0 (y = 0). In some embodiments of the present invention, the polyvalent molecule is of generation 1 (y = 1). In some embodiments of the present invention, the polyvalent molecule is of generation 2 (y = 2). In some embodiments of the present invention, the polyvalent molecule is of generation 3 (y = 3). In some embodiments of the present invention, the polyvalent molecule is of generation 4 (y = 4). In some embodiments of the present invention, the polyvalent molecule is of generation 5 (y = 5).

[0073] In some embodiments of the present invention, the polyvalent molecule is of a lower generation, y = 0 or y = 1, more preferably y = 1.

[0074] In some embodiments of the present invention, the polyvalent molecule is of a higher generation, y = 2, 3, 4, or 5, preferably y = 2 or y = 3, more preferably y = 2. Positively ionizable groups and cationic groups

[0075] The multivalent molecule is multivalent with respect to positively ionizable groups and / or cationic groups, and thus can efficiently bind to and capture nucleic acids.

[0076] The positively ionizable group can be positively charged depending on the pH of the surrounding environment and can be protonated at a sufficiently low pH to be in a positively charged state. The positively ionizable group can be any kind of amine group. Note that amide, ester, ether, carbonate, urethane, and urea groups are not positively ionizable groups. Specifically, the positively ionizable group is selected from tertiary amines, secondary amines, primary amines, guanidines, and imidazole groups (Scheme 2). Tertiary amines, primary amines, guanidines, and imidazole groups are preferred herein. Tertiary amines and guanidine groups are more preferred. Tertiary amine groups are most preferred.

[0077] The cationic group is positively charged independently of the pH of the surrounding environment and cannot be deprotonated (lose a positive charge) or protonated (gain a positive charge). The cationic group can be any kind of cationic group. Preferably, the cationic group contains one nitrogen atom or multiple (up to 3, e.g., 2 or 3) nitrogen atoms. The cationic group is preferably selected from quaternary ammonium, imidazolium, and guanidinium groups (Scheme 2). Preferred cationic groups are quaternary ammonium and imidazolium groups. Quaternary ammonium is more preferred.

[0078]

Chemical formula

[0079] The cumulative number of positively ionizable groups and / or cationic groups in the multivalent molecule is at least 2. Preferably, the cumulative number of positively ionizable groups and / or cationic groups in the multivalent molecule is 3 or more, 4 or more, 5 or more, 6 or more, 8 or more, 10 or more, 12 or more, or 14 or more. Preferably, the cumulative number of positively ionizable groups and / or cationic groups in the multivalent molecule is a discrete number for each multivalent molecule and not an average value.

[0080] In the present specification, preferably, only positively ionizable groups and / or cationic groups separated by at least 3 atoms, preferably carbon atoms, from adjacent positively ionizable groups and / or cationic groups are counted. For example, the two amines in the piperazine ring are not counted.

[0081] In certain embodiments, all positively ionizable groups and / or cationic groups within the multivalent molecule are separated from each other by at least 3 atoms, and these spacer atoms are carbon, nitrogen, and / or oxygen atoms.

[0082] In certain embodiments of the present invention, the multivalent molecule has only positively ionizable groups and no cationic groups. Preferably, the positively ionizable charges are then present in the core and the BU and not in the TU. Alternatively, the positively ionizable charges are present in the core, the BU, and the TU. In another option, the positively ionizable charges are present in the core and the TU and not in the BU. Finally, in certain embodiments, the positively ionizable charges are present in the core only. In this embodiment of the present invention, the positively ionizable group is preferably a tertiary amine, optionally combined with a primary amine and / or guanidine, and more preferably, the positively ionizable group is a tertiary amine.

[0083] In certain embodiments of the present invention, the multivalent molecule has a combination of a positively ionizable group and a cationic group. Preferably, the cationic charge is then present only on the core and not on either the BU or the TU. Alternatively, the cationic charge is then present on the core and the first generation of BU layers and not on further BU or TU. In these modes, a multivalent molecule is generated in which the encapsulated cationic groups are combined with external positively ionizable groups. In this embodiment, the cationic group is preferably a quaternary ammonium group and the positively ionizable group is preferably a tertiary amine, optionally combined with guanidine, and more preferably these groups are tertiary amines.

[0084] In certain embodiments of the present invention, the multivalent molecule has only cationic groups and no positively ionizable groups. Preferably, the cationic charge is then present on the core and not on either the BU or the TU. Alternatively, the cationic charge is then present on the core and the first generation of BU layers and not on further BU or TU. In this embodiment, the cationic group is preferably a quaternary ammonium group, optionally combined with guanidinium, and more preferably these groups are only quaternary ammonium groups. Multifunctional core (i.e., the center of the multivalent molecule)

[0085] The multivalent molecule has the formula (I): [Core] x -[BU] y -[TU] z and has.

[0086] In formula (I), the core is nitrogen or a C containing 1 to 15 nitrogen heteroatoms and optionally 1 to 4 oxygen heteroatoms 1 ~C 18 which is a linear, branched or cyclic group.

[0087] The core of the multivalent molecule contains from 1 to 15 nitrogen atoms, preferably from 2 to 10 nitrogen atoms, more preferably from 2 to 4 nitrogen atoms, for example, 2, 3, or 4 nitrogen atoms. These nitrogen atoms may be part of an amide, urethane, or urea group, or they may be part of a positively ionizable group or a cationic group within the multivalent molecule. Preferably, at least two nitrogen atoms in the core are part of a positively ionizable group or a cationic group within the multivalent molecule. Preferably, all nitrogen atoms in the core are part of a positively ionizable group or a cationic group within the multivalent molecule or they are part of an amide group. More preferably, all nitrogen atoms in the core are part of a positively ionizable group or a cationic group within the multivalent molecule.

[0088] The nitrogen atoms in the core that are part of a positively ionizable group or a cationic group within the multivalent molecule are preferably separated from each other by at least 3 atoms, and these spacer atoms are carbon, nitrogen, and / or oxygen atoms, preferably only carbon atoms.

[0089] The core may contain functional groups, such as amide, urethane, urea, or ether groups. Among these functional groups, amide and ether are preferred. Amide is more preferred. However, more preferably, the core does not contain any of these functional groups.

[0090] The core may contain from 0 (when the core is nitrogen) to 18 carbon atoms, preferably from 2 to 12 carbon atoms, more preferably from 4 to 10 carbon atoms.

[0091] The core may contain from 0 to 4 oxygen atoms, preferably from 0 to 2 oxygen atoms, and more preferably, the core does not have any oxygen atoms.

[0092] The core can be a linear, branched, or cyclic group, preferably linear or branched, and more preferably linear.

[0093] The core has x connections from the core to the first-generation branching unit BU (or to the terminal unit TU if y = 0), where x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 12, and all of these connections constitute a connection from the nitrogen atom of the core to the carbon atom of the branching unit BU (or terminal unit TU).

[0094] Preferably, x is 2 or more, more preferably, x is 3 or more, and even more preferably, x is 4 or more. Preferably, x is a discrete number and not an average number considering multiple cores. Preferably, the core is a single entity (i.e., a single chemical group) and not a mixture of entities. For example, the core is derived from n-butylenediamine and not from both n-butylenediamine and n-propylenediamine.

[0095] In certain embodiments of the present invention, the nitrogen atom of the core unit is part of a positively ionizable group in a polyvalent molecule.

[0096] The following Schemes 3 and 4 show preferred core structures. The wavy bonds indicate connections to the BU of the first-generation layer or the TU (when y = 0). Thus, the number of wavy bonds of a particular core unit represents the value of x for that core.

[0097]

Chemical formula

[0098]

Chemical formula

[0099] The core unit can be confirmed to be derived from amine-functional molecules: replacing all x connections of the core to BU (or TU) with hydrogen atoms reveals these amine-functional molecules. Preferably, the core unit is derived from a single compound amine having a purity of pure, i.e., about 90% or more, preferably about 97% or more, more preferably about 98% or more, and even more preferably about 99% or more.

[0100] Accordingly, the core unit is preferably derived from ammonia, 1,2-diaminoethane, 1,3-diaminopropane, 1,4-diaminobutane (or putrescine), 1,5-diaminopentane, 1,6-diaminohexane, 1,8-diaminooctane, 1,10-diaminodecane, 2-(2-aminoethoxy)ethylamine, 3-(3-aminopropoxy)propylamine, 3,3'-diamino-N-methyldipropylamine, N-(3-aminopropyl)-1,3-propanediamine (or norspermidine), N-(4-aminobutyl)-1,4-butanediamine, N-(6-aminohexyl)-1,6-hexanediamine, spermine, spermidine, or N,N-bis(2-aminoethyl)-1,2-ethanediamine. (Note that 3-(3-aminopropoxy)propylamine and 3,3'-diamino-N-methyldipropylamine can alternatively be identified as oxygen or methyl-nitrogen cores with two n-propylene-nitrogen BUs bonded respectively). Among this group, the core unit is more preferably derived from ammonia, 1,4-diaminobutane (or putrescine), 1,6-diaminohexane, 3,3'-diamino-N-methyldipropylamine, N-(3-aminopropyl)-1,3-propanediamine (or norspermidine), N-(4-aminobutyl)-1,4-butanediamine, spermine, or spermidine. Ammonia, 1,4-diaminobutane (or putrescine), or N-(3-aminopropyl)-1,3-propanediamine (or norspermidine) are even more preferred herein.

[0101] Furthermore, the core unit is derived from amino acids, such as lysine, arginine, or histidine, preferably those in their naturally occurring L-configuration. Non-limiting examples are core units derived from dimers or trimers of these amino acids, or cores derived from C-amidated amino acids (using ammonia or primary or secondary amines), and for some examples see Scheme 4.

[0102] In certain embodiments of the invention, the core unit is derived from a naturally occurring amine-functional molecule, such as ammonia, 1,4-diaminobutane (or putrescine), N-(3-aminopropyl)-1,3-propanediamine (or norspermidine), spermine, L-lysine amide, or L-arginine amide. Ammonia, 1,4-diaminobutane (or putrescine), N-(3-aminopropyl)-1,3-propanediamine (or norspermidine), or spermine are preferred herein. 1,4-Diaminobutane (or putrescine) or N-(3-aminopropyl)-1,3-propanediamine (or norspermidine) are more preferred.

[0103] In certain embodiments of the invention, the nitrogen atoms of the core unit are part of a cationic group in a polyvalent molecule. Preferably, these nitrogen atoms are, in that case, part of a quaternary ammonium group. The following Scheme 5 shows a preferred core structure for this embodiment. The wavy bond indicates the connection to the BU, or TU (when y = 0) of the first generation layer. Thus, the number of wavy bonds of a particular core unit represents the x value of that core. The R 3 group is methyl, ethyl, n-propyl, benzyl, acetamide (-CH 2 -C(O)NH 2 )), or 2-hydroxyethylene group, preferably methyl, ethyl, benzyl, or acetamide group, more preferably methyl or ethyl group. For convenience, the counter anion X - is not depicted in Scheme 5. This X- The counter anion can be any anion and may also be a divalent or trivalent anion. Preferably, X - is a chloride ion Cl - , a bromide ion Br - , an iodide ion I - , a tosylate ion TsO - , carbonate, oxalate, sulfate, or phosphate anion. More preferably, X - is a chloride ion, oxalate, phosphate, or bromide ion, and most preferably, a chloride ion or phosphate ion (including monohydrogen phosphate or dihydrogen phosphate).

[0104]

Chemical formula

[0105] The branching unit BU has the formula (IIa), (IIb), (IIc), or (IId):

[0106]

Chemical formula

[0107] Formula (IIa) represents an n-alkylene-nitrogen spacer that is connected to the core at the CH 2 terminus or to a BU closer to the core and to two terminal units (TU) at the N-terminus or to two branching units (BU) closer to the TU. The connection is shown by a wavy bond, and p is 1, 2, 3, or 4.

[0108] Preferably, in (IIa), p is 1 or 2 or 3, preferably 1 or 2, and thus, (IIa) represents an n-propylene-nitrogen spacer, an n-butylene-nitrogen, or an n-pentylene-nitrogen spacer, preferably an n-propylene-nitrogen spacer or an n-butylene-nitrogen. More preferably, p is 1, and (IIa) represents an n-propylene-nitrogen spacer.

[0109] Formula (IIb) is CH 2 An amide containing an n-alkylene-nitrogen spacer that is connected at the end to the core or to a BU closer to the core and at the N-terminus to two terminal units (TU) or to two branching units (BU) closer to the TU, the connection being shown by a wavy bond, q is 1, 2, 3, 4, or 5, and R 1 is a hydrogen, methyl, ethyl, n-propyl, or isopropyl group.

[0110] Preferably, in (IIb), q is 1, 2, or 3, more preferably 1 or 2, and most preferably 1.

[0111] Preferably, in (IIb), R 1 is hydrogen or methyl, and more preferably, R 1 is hydrogen.

[0112] Preferably, in (IIb), either q is 1 and R 1 is hydrogen, or q is 1 and R 1 is methyl, or q is 2 and R 1 is hydrogen, or q is 2 and R 1 is methyl, or q is 3 and R 1 is hydrogen. More preferably, in (IIb), either q is 1 and R 1 is hydrogen, or q is 2 and R 1 is hydrogen, or q is 2 and R 1 is methyl. Even more preferably, in (IIb), q is 1 and R 1 is hydrogen.

[0113] Formula (IIc) is defined as above for (IIa), and further, R 2 is methyl, ethyl, n-propyl, benzyl, acetamide (-CH 2 -C(O)NH 2 ), or a 2-hydroxyethylene group, and X -is a counter anion to the quaternary amine cation moiety.

[0114] Preferably, in (IIc), R 2 is methyl, ethyl, or benzyl, and more preferably, methyl.

[0115] In (IIc), X - The counter anion can be any anion. Preferably, X - is chloride ion Cl - bromide ion Br - iodide ion I - tosylate ion TsO - or dihydrogen phosphate. More preferably, X - is chloride ion or dihydrogen phosphate, and most preferably, chloride ion.

[0116] In (IIc), X - The counter anion can also be a divalent or trivalent anion, for example, carbonate, oxalate, sulfate, hydrogen phosphate, or phosphate anion, and more preferably, oxalate or hydrogen phosphate HPO 4 2- anion. In these cases, the counter anion neutralizes a plurality of branched units (IIc).

[0117] Preferably, in (IIc), R 2 is methyl and X - is chloride ion, or R 2 is benzyl and X - is chloride ion.

[0118] Preferably, in (IIc), p is 1 and R 2 is methyl and X - is chloride ion, or p is 1 and R 2 is benzyl and X - is chloride ion, or p is 2 and R 2 is methyl and X - is chloride ion, or p is 2 and R2 is benzyl and X - is a chloride ion. More preferably, p is 1 and R 2 is methyl and X - is a chloride ion, or p is 1 and R 2 is benzyl and X - is a chloride ion.

[0119] Formula (IId) is defined as described above for (IIb), and further, R 2 is methyl, ethyl, n-propyl, benzyl, acetamide (-CH 2 -C(O)NH 2 ), or a 2-hydroxyethylene group, and X - is a counter anion to the quaternary amine cation moiety.

[0120] Preferably, in (IId), R 2 is methyl, ethyl, or benzyl, and more preferably, it is methyl.

[0121] In (IId), X - the counter anion can be any anion. Preferably, X - is a chloride ion Cl - , a bromide ion Br - , an iodide ion I - , a tosylate ion TsO - , or dihydrogen phosphate. More preferably, X - is a chloride ion or dihydrogen phosphate, and most preferably, it is a chloride ion.

[0122] In (IId), X - the counter anion can also be a divalent or trivalent anion, for example, carbonate, oxalate, sulfate, monohydrogen phosphate, or a phosphate anion, and more preferably, it can be an oxalate or monohydrogen phosphate HPO 4 2- anion. In these cases, the counter anion neutralizes a plurality of branched units (IId).

[0123] Preferably, in (IId), R 2 is methyl and X - is chloride ion, or R 2 is benzyl and X - is chloride ion.

[0124] Among the branched units according to formulas (II-a), (II-b), (II-c), and (II-d), the BUs of formulas (II-a), (II-b), and (II-c) are preferred. The BUs of formulas (II-a) and (II-b) are more preferred, and the BU of formula (II-a) is most preferred.

[0125] Within the multivalent molecule, the BUs are independently selected from (IIa), (IIb), (IIc), or (IId) for each separate generation layer; that is, the first BU arising from the core representing the BU of the first generation layer may be different from the second BU (representing the BU of the second generation layer) arising in the direction away from the core, which may be different from the third, fourth, and fifth BUs arising in the direction away from the core.

[0126] In certain embodiments of the present invention, the BUs of the multivalent molecule are the same for each generation layer. For example, all BUs in the multivalent molecule are of formula (IIa), for example, with p = 1. In another example, all BUs in the multivalent molecule are of formula (IIa), with p = 2. In another example, all BUs in the multivalent molecule are of formula (IIa), with p = 3. In another example, all BUs in the multivalent molecule are of formula (IIb), for example, with q = 1 and R 1 = H.

[0127] In certain embodiments of the present invention, the BUs of formulas (IIa) and (IIc) are combined within the multivalent molecule. For example, the BU of the first generation layer is of formula (IIc), while the BUs of the other generation layers are of the (IIa) type. In this example, the multivalent molecule has a sealed internal cationic group and a more exposed external positively ionizable group.

[0128] In certain embodiments of the present invention, the BUs of formula (IIb) and (IId) are combined within the polyvalent molecule. For example, the BU of the first generation layer is of formula (IId), while the BU of the other generation layers is of the (IIb) type. In this example, the polyvalent molecule has a sealed internal cationic group and a more exposed external positively ionizable group.

[0129] In certain embodiments of the present invention, the polyvalent molecule has no branching group BUs, and y is, in that case, 0. Terminal unit TU

[0130] In formula (I), the terminal unit (TU) is hydrogen and C optionally containing 1 to 8 heteroatoms independently selected from the group consisting of O and N, 1 ~C 30 independently selected from alkyl, aryl, arylenealkyl, or alkylenearyl groups, provided that not all TUs in the polyvalent molecule need be hydrogen.

[0131] The terminal unit may have no functional groups, i.e., it may consist of only carbon and hydrogen. Alternatively, the terminal unit may preferably contain one or more functional groups selected from amide, urethane, urea, ester, hydroxy, and ether groups. Among these functional groups, amide, ester, and hydroxy groups are preferred.

[0132] In formula (I), TU can be independently selected from any of the following groups (Scheme 6) of formula (III-a) to (III-j). In each of these formulas, the wavy bond indicates the connection to the nitrogen atom of the external BU or the core (when y = 0).

[0133]

Chemical formula

[0134] In formula (III-a), TU is hydrogen.

[0135] In formula (III-b), R 4 group is preferably a straight-chain or branched-chain C 1 ~C 18 alkyl group or (substituted) benzyl group that independently contains a heteroatom optionally selected from O and N. More preferably, R 4 is a C 1 ~C 12 straight-chain or branched-chain alkyl group or benzyl group. The alkyl group may be saturated or unsaturated, preferably saturated. In some embodiments, the alkyl group is saturated or has one, two, or three unsaturations. Optionally, R 4 contains a positively ionizable group or a cationic group, such as a tertiary amine or a quaternary ammonium group.

[0136] In formula (III-c), R 5 is preferably hydrogen or a methyl group, more preferably hydrogen. In certain embodiments, such as when R 5 is a methyl group, the stereochemistry in (III-c) is not defined. The R 6 group is preferably a straight-chain or branched-chain C 1 ~C 18 alkyl group, preferably R 6 is a straight-chain or branched-chain saturated or unsaturated C 1 ~C 17 alkyl group that independently contains a heteroatom optionally selected from O and N. More preferably, R 6 is a straight-chain or branched-chain C 2 ~C 14 alkyl group, even more preferably a straight-chain or branched-chain C 4 ~C 12 alkyl group. The alkyl group may be saturated or unsaturated, preferably saturated. In some embodiments, the alkyl group is saturated or has one, two, or three unsaturations.

[0137] In formula (III-d), R 5 is preferably hydrogen or a methyl group, more preferably hydrogen. When R 5 is a methyl group, the stereochemistry in (III-d) is not defined. The R 7 group is preferably a straight-chain or branched-chain C 1 ~C 18 alkyl group optionally containing a heteroatom independently selected from O and N. More preferably, R 7 is a straight-chain or branched-chain C 2 ~C 14 alkyl group, and even more preferably a straight-chain or branched-chain C 4 ~C 12 alkyl group. The R 8 group is preferably hydrogen or a straight-chain or branched-chain C 1 ~C 18 alkyl group optionally containing a heteroatom independently selected from O and N. In some embodiments, R 8 is hydrogen or a straight-chain or branched-chain C 1 ~C 12 alkyl group optionally containing a heteroatom independently selected from O and N. Preferably, R 8 is hydrogen or a straight-chain or branched-chain C 1 ~C 16 alkyl group optionally containing an oxygen heteroatom, preferably R 8 is hydrogen or a straight-chain or branched-chain C 1 ~C 14 alkyl group optionally containing an oxygen heteroatom, preferably R 8 is hydrogen or a straight-chain or branched-chain C 1 ~C 10 alkyl group optionally containing an oxygen heteroatom. More preferably, R 8 is hydrogen or a straight-chain or branched-chain C 1 ~C 12 alkyl group optionally containing an oxygen heteroatom. Even more preferably, R 8 is hydrogen, methyl, 2-hydroxyethyl, 3-hydroxypropyl, or C2 ~C 8 is an alkyl group. R 7 or R 8 The alkyl group in may be saturated or unsaturated, and is preferably saturated.

[0138] In formula (III-e), r = 0 or 1, preferably 1, or a number selected from 3 to 10, r is preferably 1, 5, or 10, and preferably r is 0, 1, 5, or 10. In formula (III-e), R 6 group is preferably a linear or branched C 1 ~C 18 alkyl group optionally containing a heteroatom independently selected from O and N. More preferably, R 6 is a linear or branched C 2 ~C 14 alkyl group, and even more preferably a linear or branched C 4 ~C 12 alkyl group. The alkyl group may be saturated or unsaturated, and is preferably saturated. In some embodiments, the alkyl group is saturated or has one, two, or three unsaturations.

[0139] In formula (III-f), s = 0 or 1, preferably 1, or a number selected from 3 to 10, s is preferably 1, 5, or 10. R 8 group is preferably hydrogen, or a linear or branched C 1 ~C 18 alkyl group optionally containing a heteroatom independently selected from O and N. Preferably, R 10 is a C 1 ~C 16 linear or branched alkyl group, and preferably R 10 is a C 1 ~C 14 linear or branched alkyl group. More preferably, R 8 is hydrogen, or a linear or branched C 1 ~C12 is an alkyl group. Even more preferably, R 8 is hydrogen, methyl, 2-hydroxyethyl, 3-hydroxypropyl, or a C 2 -C 8 alkyl group. The R 9 group is preferably hydrogen or a linear or branched C 1 -C 18 alkyl group optionally containing a heteroatom independently selected from O and N. Even more preferably, R 9 is hydrogen or a linear or branched C 2 -C 14 alkyl group, and even more preferably hydrogen or a linear or branched C 4 -C 12 alkyl group. The alkyl group in R 8 or R 9 may be saturated or unsaturated, and is preferably saturated.

[0140] In formula (III-g), the R 10 group is preferably a linear or branched C 1 -C 18 alkyl group optionally containing a heteroatom independently selected from O and N. Even more preferably, R 10 is a C 4 -C 16 linear or branched alkyl group. The alkyl group may be saturated or unsaturated, and is preferably saturated. In certain embodiments, the stereochemistry in (III-g) is not defined because the hydroxy group is connected in a manner such that (racemic) is not defined. Optionally, the hydroxy group in (III-g) is acylated to form an ester group.

[0141] In formula (III-h), the R 11 group is preferably a linear or branched C 1 -C 29 alkyl group optionally containing a heteroatom independently selected from O and N. Preferably, R 11 is a linear or branched C 1 -C27 is an alkyl group, preferably, R 11 is a linear or branched C 1 ~C 25 alkyl group, preferably, R 11 is a linear or branched C containing a heteroatom independently selected from O and N optionally 1 ~C 17 alkyl group. Preferably, R 11 is C 1 ~C 15 linear or branched alkyl group, preferably, R 11 is C 1 ~C 13 linear or branched alkyl group, each of said groups containing a heteroatom independently selected from O and N. More preferably, R 11 is C 1 ~C 11 linear or branched alkyl group. The alkyl group may be saturated or unsaturated, preferably, saturated. In some embodiments, the alkyl group is saturated or has one, two, or three unsaturations. Optionally, R 11 is a positively ionizable group or a cationic group, for example, a tertiary amine, guanidine, or quaternary ammonium group, preferably, a tertiary amine or guanidine group.

[0142] In formula (III-i), the R 13 group is preferably a linear or branched C containing a heteroatom independently selected from O and N optionally 1 ~C 17 alkyl group. More preferably, R 13 is C 1 ~C 17 linear or branched alkyl group. The alkyl group may be saturated or unsaturated, preferably, unsaturated. The R 12 group is preferably a residue of an amino acid, more preferably, a residue of lysine, arginine, or histidine (preferably, in their naturally occurring L-configuration) or a derivative of such a residue. Thus, R12 may include a primary amine, guanidine, or imidazole group, or any derivatized form of these groups, such that these TUs include a positively ionizable group or a cationic group.

[0143] In formula (III-j), the terminal unit is an amidine group. However, it should be noted that this group, together with the nitrogen atom of the BU unit or the core unit to which it is attached, forms a guanidine group. R 14 groups can be independently selected for each position, and preferably are hydrogen and a straight or branched C 1 ~C 18 alkyl group independently selected from O and N. More preferably, R 14 groups are hydrogen and straight-chain C 1 ~C 12 alkyl group selected from. The alkyl group may be saturated or unsaturated, and is preferably saturated.

[0144] In formula (III-k), R 15 is a straight or branched C 1 ~C 18 alkyl group that optionally contains a heteroatom independently selected from O and N, and preferably, R 2a is a straight or branched saturated or unsaturated C 1 ~C 15 alkyl group that optionally contains a heteroatom independently selected from O and N. More preferably, R 2a is a straight or branched C 2 ~C 13 alkyl group, and even more preferably, a straight or branched C 2 ~C 11 alkyl group. The alkyl group may be saturated or unsaturated, and is preferably saturated. In some embodiments, the alkyl group is saturated or has one, two, or three unsaturations.

[0145] Among the terminal units TU according to formulas (III-a) to (III-k), the TUs of formulas (III-a) to (III-j) are preferred, and the TUs of formulas (III-b), (III-c), (III-d), (III-f), (III-g), and (III-j) are more preferred. The TUs of formulas (III-b), (III-c), (III-d), (III-f), and (III-g), or the TUs of formulas (III-b), (III-c), (III-d), and (III-f) are even more preferred. The TUs of formulas (III-c), (III-d), (III-f), and (III-g), or the TUs of formulas (III-c), (III-d), and (III-f) are even more preferred.

[0146] In certain embodiments of the present invention, all TUs of the multivalent molecule are the same or of the same formula (III) type. Preferably, all TUs of the multivalent molecule are the same. For example, all TUs in the multivalent molecule are of formula (III-d), for example, for all TUs, R 5 is hydrogen, R 8 is hydrogen, and R 7 is an n-decyl group. In another example, all TUs in the multivalent molecule are of formula (III-c), for example, for all TUs, R 5 is hydrogen and R 6 is an n-octyl group. In another example, all TUs in the multivalent molecule are of formula (III-g), for example, for all TUs, R 10 is an n-decyl group, and preferably, for all TUs, the hydroxy groups are connected in a random (racemic) pattern.

[0147] In certain embodiments of the present invention, all of the nitrogen of the external BU unit is connected to two specific TUs selected from formula (III). In certain embodiments, it is preferred that all of the nitrogen of the external BU unit is connected to two specific TUs selected from formula (III). Thus, (about or exactly) 50% of the TUs are those of the first specific option TU-1 and (about or exactly 50%) are those of the second specific option TU-2. Suitable combinations of TU-1 and TU-2 are the specific combinations of (III-a) and (III-h), (III-a) and (III-i), (III-a) and (III-j), (III-b) and (III-c), (III-b) and (III-d). For clarity, see Scheme 7 below for these examples. Examples of other combinations are those of (III-c) and (III-h), (III-d) and (III-h). Yet other examples of combinations are those of (III-j) with any of (III-b), (III-d), or (III-f).

[0148]

Chemical formula

[0149] In certain embodiments of the present invention, two or more TUs selected from formula (III), preferably two, three, or four TUs, more preferably two TUs, are applied in the polyvalent molecule. In this embodiment, TU-1 and TU-2 (or TU-1, TU-2, and TU-3, etc.) can be randomly distributed to the nitrogen of the external BU unit. TU-1 and TU-2 can be present in different ratios ranging from about 1% - 99% to about 99% - 1%, preferably from about 10% - 90% to about 90% - 10%, more preferably from about 30% - 70% to about 70% - 30%. Preferred combinations of TU-1 and TU-2 are specific combinations of (III-b) with any of (III-c), (III-d), (III-f), (III-g), or (III-j), or specific combinations of (III-h) with any of (III-c) or (III-d). Preferably, in this embodiment, the selected TUs are specific TUs. For example, TU-1 is (III-b) where R 4 is a methyl group, and TU-2 is where R 5 is hydrogen, R 7 is an n-octyl group, and R 8 is hydrogen (III-d).

[0150] In certain embodiments of the present invention, most of the TUs are hydrogen. With respect to the total amount of TUs, 10% or more, or 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, or 70% or more of the TUs are hydrogen.

[0151] In formula (I), z represents the total number of TU groups attached to the polyvalent molecule, and z is from 1 to 128. Preferably, z is 4 or more, more preferably z is 6 or more, even more preferably z is 8 or more. In certain embodiments of the present invention, z is 10 or more, 16 or more, or even 20 or more. Preferably, z is less than 129 or 128, more preferably less than 80, even more preferably less than 50.

[0152] The multivalent molecule contains a plurality of hydrophobic groups that are mostly or exclusively present in the TU. Therefore, this molecule can encapsulate nucleic acids, thereby creating a hydrophobic shell or cover around the assembly of the multivalent molecule and the nucleic acid.

[0153] Thus, at least a portion (i.e., a certain percentage) of all the TUs in the multivalent molecule is of a hydrophobic nature, thus making the multivalent molecule hydrophobic or amphiphilic. The hydrophobic TUs preferably contain a C 1 ~C 18 alkyl, aryl, arylenealkyl, or alkylenearyl chain. More preferably, these TUs, preferably the hydrophobic TUs, contain a C 4 ~C 18 aliphatic alkyl chain, where this chain may be straight or branched and may be saturated or unsaturated in nature. In formula (I), preferably at least 5%, or at least 10%, or at least 20%, or at least 30%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% of the terminal group TUs are of a hydrophobic nature. In certain embodiments of the present invention, all or substantially all (more than 97%) of the TUs are of a hydrophobic nature. Aspects of the multivalent molecule

[0154] In certain embodiments of the invention, the multivalent molecule is a single compound, i.e., not a mixture of compounds. In other words, in certain embodiments, all of the multivalent molecules within the nanoparticles taught herein are the same. The purity of the multivalent molecule / compound is 60% or greater, preferably 85% or greater, more preferably 90% or greater, most preferably 95% or greater, e.g., 99% or greater, e.g., 100%. This typically applies to G0, G1, and G2 materials (y = 0, 1, or 2). For higher generation G3, G4, and G5 materials (y = 3, 4, or 5), the dispersity D (= Mw / Mn) of the multivalent molecule is typically less than about 1.2, preferably less than 1.1, more preferably less than 1.03. The dispersity D of the material can be evaluated by methods known in the art, e.g., GPC measurement or MS spectroscopy (see, e.g., Hummelen et al., Chem. Eur. J. 1997, 3, 9, 1489-1493).

[0155] In certain embodiments of the invention, the multivalent molecule is a mixture of compounds. In other words, in certain embodiments, the nanoparticles taught herein contain a mixture of different multivalent molecules. This can be due to the presence of undefined stereocenters in the multivalent molecule. An example is the presence of a branched alkyl of racemic origin in the multivalent molecule. Another example is the use of an epoxyalkyl reactant that results in the formation of a (racemic) hydroxy group in the multivalent molecule where the stereochemistry is not defined. Thus, in certain embodiments, the multivalent molecules within the nanoparticles taught herein may not be pure, but the connectivity of the atoms within all of the multivalent molecules within the nanoparticles taught herein is the same.

[0156] The multivalent molecule may have a specific core and may be of a specific generation (y = 0, or 1, or 2, or 3, or 4, or 5), and thus the multivalent molecule may have a structure with evenly distributed BUs. Preferably, all nitrogen terminal groups are bonded to the same specific TU selected from (III-a) to (III-k) (or the same two specific TUs are bonded). Therefore, the multivalent molecule of the present invention preferably has an overall shape that is symmetric, when ignoring chiral centers that may exist in the molecule.

[0157] In certain embodiments, the multivalent molecule of the present invention has a molecular weight higher than 400 Daltons, preferably higher than 800 Daltons, more preferably higher than 1200 Daltons, and even more preferably higher than 1500 Daltons. In certain embodiments, the multivalent molecule of the present invention has a molecular weight lower than 80 kDa, preferably lower than 40 kDa, more preferably lower than 20 kDa, and even more preferably lower than 12 kDa.

[0158] The molecular weight (MW) of the multivalent molecule of the present invention divided by the total number (#N) of positively ionizable groups and / or cationic groups in the molecule (or in other words, the ratio of the molecular weight of the multivalent molecule of the present invention to the total number of positively ionizable groups and / or cationic groups in the molecule) indicates the concentration of these groups in the molecule. MW / #N is preferably higher than 80, more preferably higher than 150, and most preferably higher than 200. MW / #N is preferably lower than 900, more preferably lower than 600, and most preferably lower than 450. These numbers indicate that the concentration of groups capable of binding to nucleic acids in the multivalent molecule of the present invention can be extremely high. For example, amphiphilic molecules previously used in the art have MW / #N values of 642 (DLin-MC3-DMA or MC3) and 766 (ALC-0315).

[0159] Due to their multivalent action, the multivalent molecules of the present invention bind to nucleic acids more strongly than monovalent amphiphilic molecules. Thus, nucleic acids are better bound and retained within the nanoparticles of the present invention.

[0160] In addition, the multivalent molecules of the present invention can also interact with apolipoprotein stabilizers (i.e., apolipoproteins, apolipoprotein derivatives, apolipoprotein mimetics, and / or apolipoprotein mimetic derivatives) because apolipoproteins as a whole have a negative charge (as described, for example, in Sparks DL, Lund-Katz S, Phillips MC. The charge and structural stability of apolipoprotein A-I in discoidal and spherical recombinant high density lipoprotein particles. J Biol Chem. 1992 Dec 25;267(36):25839-47. PMID: 1464598). As a result, apolipoproteins, apolipoprotein derivatives, apolipoprotein mimetics, and / or apolipoprotein mimetic derivatives will bind better to the nanoparticles of the present invention. In circulation, for example, in blood, the release of apolipoprotein components (e.g., apolipoproteins, apolipoprotein derivatives, apolipoprotein mimetics, and / or apolipoprotein mimetic derivatives) from the nanoparticles can thus be less likely.

[0161] Transfection of nucleic acids into target cells can be very well assisted by the presence of the multivalent molecules because the multivalent molecules of the present invention can efficiently bind to and interact with multiple phospholipids in the (myeloid) cell barrier, thereby creating a gap for the entry of nucleic acids.

[0162] The multivalent molecules of the present invention can be processed from solution. Thus, the multivalent molecules are preferably soluble in solvents of various polarities. Thus, the multivalent molecules are preferably soluble in tricaprylin, ethanol, or isopropanol, and more preferably soluble in all three of tricaprylin, ethanol, and isopropanol. Solubility can be investigated by stirring about 20 mg of the multivalent molecule in 1 gram of tricaprylin, ethanol, or isopropanol and evaluating whether all the materials spontaneously dissolve to give a clear / transparent solution (at a concentration of about 2 wt / wt %). The test can be carried out at about 20 degrees Celsius (room temperature) or about 37 degrees Celsius. Preferably, the multivalent molecules of the present invention are already soluble at room temperature.

[0163] In certain embodiments of the present invention, the multivalent molecules contain ester, amide, and / or hydroxy groups. Such groups provide options for slow in vivo degradation and make the multivalent molecules (more) biocompatible. Preferred groups are amide and / or ester.

[0164] The multivalent molecules of the present invention are preferably non-toxic or have limited toxicity, either by themselves or when bound to nucleic acids. Toxicity cell tests can be performed by methods known in the art, such as the cell viability MTT assay or similar or equivalent tests. Synthesis of multivalent molecules

[0165] The multivalent molecules can be prepared by using any suitable synthetic method or route. Conventionally, they can be prepared by obtaining polyfunctional amine building blocks and modifying them with molecules that are reactive towards amines and have hydrophobic properties. Alternatively, two or more amine-reactive molecules, at least one of which has hydrophobic properties, may be applied.

[0166] Particularly useful building blocks are (primary) amine-functional dendrimers. Other useful building blocks are small multifunctional amine molecules such as 1,4-diaminobutane (or putrescine), 3,3'-diamino-N-methyldipropylamine, N-(3-aminopropyl)-1,3-propanediamine (or norspermidine), N-(6-aminohexyl)-1,6-hexanediamine, spermine, spermidine. Still other useful building blocks are amino acids such as lysine, arginine, or derivatives of histidine, such as dimers or trimers of L-lysine, L-histidine, or L-arginine (see, for example, Scheme 4), dimers or trimers of L-lysine or L-arginine, or mixed dimers or trimers of these amino acids.

[0167] Suitable examples of primary amine dendrimers are poly(propyleneimine) (PPI) dendrimers or poly(amidoamine) (PAMAM) dendrimers. These types of dendrimers are known in the art. The synthesis and molecular structure of PPI and PAMAM dendrimers are described in detail in “Materials science and technology series: Synthesis of polymers. Edited by A Dieter Schlueter, Wiley-VCH Verlag GmbH, Weinheim 1999; ISBN 3-527-26831-6; Chapter 12, pp 403-458; HM Janssen, EW Meijer, The synthesis and characterization of dendritic molecules”.

[0168] In certain embodiments of the invention, PPI-type dendrimers are used as building blocks for preparing polyvalent molecules.

[0169] In another embodiment of the invention, PAMAM-type dendrimers are used as building blocks for preparing polyvalent molecules.

[0170] Primary amine PPI dendrimers have been reported to have n-propyleneamine branching units and mostly have cores derived from 1,4-diaminobutane. These PPIs can be described by formula (I), where BU is according to formula (II-a), p = 1, and all TUs are hydrogen. In Table 1, the characteristics of the PPI building blocks G1 to G5 (y = 1 to 5), more specifically the PPI building blocks G1 to G5 (y = 1 to 5) having cores derived from 1,4-diaminobutane, are summarized.

[0171] [Table 1]

[0172] Instead of using a core based on 1,4-diaminobutane, other cores, cores derived from ammonia or bis(3-aminopropyl)amine, can also be applied for the production of PPIs. Table 2 focuses on the characteristics of some PPI building blocks having these two cores.

[0173] [Table 2]

[0174] Primary amine PAMAM dendrimers have been reported to have N-(2-aminoethyl)propionamide branching units (BU) and mostly have cores derived from 1,2-diaminoethane or ammonia. These PAMAMs can be described by formula (I), where BU is according to formula (II-b), q = 1, R 1 = H, and all TUs are hydrogen. The characteristics of some G1 and G2 PAMAM building blocks having N-(2-aminoethyl)propionamide BU and various cores are summarized in Table 3.

[0175] [Table 3]

[0176] Dendrimers similar to PPI dendrimers, such as poly(butyleneimine) (PBuI), poly(pentyleneimine) (PPeI), or poly(hexyleneimine) (PHeI) dendrimers, especially those of lower generations G1 or G2, can also be used. These dendrimers have BU according to formula (II-a), with p = 2, 3, or 4 respectively. Scheme 8 shows an example of the preparation of a G1 poly(butyleneimine) dendrimer with primary amine end groups. By repeating a series of two reactions starting from the G1 material, a G2 material is obtained. Similarly, poly(pentyleneimine) or poly(hexyleneimine) dendrimers can be prepared.

[0177] In certain embodiments of the present invention, poly(butyleneimine) dendrimers are used as building blocks for preparing multivalent molecules. In another embodiment, poly(pentyleneimine) dendrimers are used as building blocks for preparing multivalent molecules. In another embodiment, poly(hexyleneimine) dendrimers are used as building blocks for preparing multivalent molecules.

[0178]

Chemical formula

[0179] Next to the PAMAM dendrimer having N-(2-aminoethyl)propionamide BU, other similar PAMAM dendrimers having BU, especially those of lower generations G1 or G2, can be used. Scheme 9 shows an example of the preparation of a G1 PAMAM dendrimer where BU is according to formula (II-b), q = 2 and R 1 = Me, and a G2 material is obtained by repeating a series of two reactions starting from the G1 material.

[0180]

Chemical formula

[0181] Additional G1 PAMAM building blocks with various BUs can be prepared by a similar synthetic approach, and in Tables 4 (using a core based on 1,4-diaminobutane) and 5 (using a core based on bis(3-aminopropyl)amine), pay attention to some examples.

[0182] [Table 4]

[0183] [Table 5]

[0184] Regarding the preparation of multivalent molecules, lower generation dendrimers are preferred. Thus, G1, G2, or G3 dendrimers are preferred. G1 PPI, G1 PBuI, G1 PPeI, or G1 PAMAM dendrimers, and G2 PPI or G2 PAMAM dendrimers are more preferred.

[0185] Other building blocks for preparing multivalent molecules are small multifunctional amines such as N-(3-aminopropyl)-1,3-propanediamine (or norspermidine), N-(6-aminohexyl)-1,6-hexanediamine, spermine, and spermidine. These molecules can be used, for example, preferably to connect directly to TU.

[0186] Next, multifunctional amine building blocks such as small multifunctional amine molecules, PPI (or PBuI or PPeI or PHeI) dendrimers, or PAMAM dendrimers are converted with one reactant or several reactants to obtain multivalent molecules. The following non-limiting examples illustrate how this can be done, and for the options shown in formula type (III), see Scheme 6.

[0187] Introduction of type (III-a) TU. If the amine-functional building block remains unreacted with respect to the (minor) portion of the primary amine reaction groups, or if the reaction is incomplete, the -NH 2 or -NHR hydrogen will remain untouched. This is one means of obtaining hydrogen TU in a polyvalent molecule.

[0188] Introduction of type (III-b), (III-e), or (III-f) TU. The amine-functional building block can be alkylated or alkylene-arylated, for example, with an alkyl halide, an alkyl tosylate, or a benzyl halide, optionally containing O and / or N heteroatoms. Alternatively, the alkylation or alkylene-arylation can be achieved by a reductive amination reaction applying an aldehyde or ketone-functional molecule, such as an alkyl aldehyde or a benzyl aldehyde.

[0189] Introduction of type (III-c) TUs. The amine-functional building block can couple to an acrylate or methacrylate, preferably an acrylate, in a Michael addition reaction. Typically, these esters have an alkyl chain, which may be straight or branched and may be saturated or unsaturated. Examples are ethyl acrylate, n-butyl acrylate, n-octyl acrylate, 2-ethylhexyl acrylate, n-dodecyl acrylate, isodecyl acrylate, or citronellyl acrylate, preferably ethyl acrylate, n-butyl acrylate, n-octyl acrylate, n-dodecyl acrylate, isodecyl acrylate, or citronellyl acrylate. Preferred acrylates are C6-C12 acrylates, where the C6-C12 alkyl group may be straight or branched and may be saturated or unsaturated. The inventors have found that acrylates react (substantially) more rapidly with primary amines than with secondary amines (i.e., selective reaction). Thus, this can be utilized to first react the primary amine molecules with 1 molar equivalent of acrylate to convert all primary amines to secondary amines. The remaining secondary amines can then be converted, for example, by acylation, alkylation, reductive amination, etc. It is also possible to convert all secondary amines with acrylate simply by using a molar excess of the acrylate reactant. It is also possible to first introduce a first acrylate and then a second different one. Preferably, the amine-functional building block is reacted with only one acrylate. Such a reaction can result in complete conversion of both primary and secondary amines with this particular acrylate.

[0190] Introduction of type (III-d) TUs. The amine-functional building blocks can couple to acrylamide or methacrylamide, preferably acrylamide, in a Michael addition reaction. Typically, these amides have an alkyl chain, which may be linear or branched, saturated or unsaturated, and they may have functional groups such as alcohol. Examples are n-butylacrylamide, n-octylacrylamide, n-decylacrylamide, N,N-diethylacrylamide, N,N-dibutylacrylamide, N-methyl-N-hexylacrylamide, and N-(2-hydroxyethyl)-N-methylacrylamide, preferably n-butylacrylamide, n-octylacrylamide, n-decylacrylamide, N,N-diethylacrylamide, N,N-dibutylacrylamide, and N-methyl-N-hexylacrylamide. Acrylamide typically reacts more rapidly with primary amines than with secondary amines. Utilizing this selectivity, first, the primary amine molecules can be reacted with acrylamide to convert all primary amines to secondary amines. The remaining secondary amines can then be converted, for example, by acylation, alkylation, reductive amination, etc.

[0191] Alternatively, the amine-functional building blocks are first reacted with acrylic esters (or methacrylic esters), and subsequently, these esters are amidated by reaction with a primary or secondary amine. These esters may also be hydrolyzed and then amidated with a primary or secondary amine.

[0192] Introduction of type (III-g) TUs. The amine-functional building block can be alkylated by using a 1,2-epoxyalkane such as 1,2-epoxyhexane, 1,2-epoxyoctane, 1,2-epoxydecane, or 1,2-epoxydodecane. The alkane can be straight-chain or branched-chain and can be saturated or unsaturated. The reaction of these molecules with the amine results in the introduction of both an alkyl chain and an alcohol group. Preferably, the amine-functional building block is reacted with only one 1,2-epoxyalkane, resulting in the complete conversion of both primary and secondary amines by this particular 1,2-epoxyalkane. Optionally, in a further reaction step, the secondary alcohol produced can be acylated by reaction with a carboxylic acid, an activated ester, or an anhydride such as acetic anhydride.

[0193] Introduction of type (III-h), (III-i), and (III-a) TUs. The amine-functional building block can be acylated, for example, by reaction with a carboxylic acid, an activated ester, or an anhydride. This chemical reaction is used to convert a primary amine to an amide, and type (III-h) and type (III-a) TUs (or type (III-i) and (III-a) ones) are simultaneously produced. The introduced alkane can be straight-chain or branched-chain and can be saturated or unsaturated.

[0194] Introduction of TUs of type (III-j) and (III-a). The amine-functional building blocks can be reacted with a guanidinylating agent. These agents react with primary or secondary amines to yield a guanidine group. Examples of such agents are pyrazole-type guanidinylating agents, such as 1-amidinopyrazole hydrochloride, 1-carbamimidoyl-1,2,4-triazole hydrochloride, 1-(N-Boc-amidino)-pyrazole, 1-(N-Cbz-amidino)-pyrazole, or other molecules, such as N,N'-di-Boc-S-methylisothiourea and N-Boc-S-methylisothiourea. Deprotection of the Boc or Cbz group creates the guanidine group. Using this chemical reaction, TUs of type (III-j) and type (III-a) can be introduced simultaneously at all primary amine end groups.

[0195] One of the above-described conversion methods, or a combination of the above-described methods, can be utilized to prepare a selected multivalent molecule.

[0196] The cationic group can be introduced into the multivalent molecule by quaternizing a tertiary amine, guanidine, or imidazole. In a non-limiting example, all primary amines in a PPI (or PBuI or PPeI or PHeI) or PAMAM dendrimer building block can first be protected, for example, with a Boc group. This yields molecules such as those shown in Schemes 8 and 9. The internal tertiary amine is then quaternized by reaction with an alkylating agent, such as methyl iodide or benzyl halide, etc., to form an ammonium group. Deprotection of the Boc group gives a PPI (or PBuI or PPeI or PHeI) or PAMAM building block having an internal quaternary ammonium cationic group as well as external primary amines. These external amines can then be converted with an amine-reactive molecule to obtain a multivalent molecule, more specifically, a multivalent molecule having a cationic group inside the molecule.

[0197] In Scheme 10, formulas (IV-A) to (IV-F) show various non-limiting embodiments of a polyvalent molecule, more specifically, a polyvalent molecule according to formula (I). Formulas (IV-A) and (IV-B) show G0 materials (i.e., without BU), formulas (IV-C) and (IV-D) show first-generation G1 dendrimers (i.e., one BU layer), formula (IV-E) shows second-generation G2 dendrimers (two BU layers), and formula (IV-F) shows third-generation (G3) dendrimers (three BU layers).

[0198]

Chemical formula

[0199] In a further embodiment, the polyvalent molecules according to formulas (IV-C) to (IV-F) are selected from poly(propylene imine) (PPI) dendrimers and PAMAM dendrimers, or modified forms thereof. Scheme 11 shows examples of first-generation G1, second-generation G2, and third-generation G3 PPI dendrimers having a core derived from 1,4-diaminobutane. Scheme 12 shows examples of first-generation G1 and second-generation G2 PAMAM dendrimers having a core derived from 1,2-diaminoethane. Poly(butylene imine) (PBuI) dendrimers, poly(pentylene imine) (PPeI) dendrimers, and poly(hexylene imine) (PHeI) dendrimers can also be used. Scheme 13 shows examples of G1 of PBuI, PPeI, and PHeI dendrimers having a core derived from 1,4-diaminobutane.

[0200]

Chemical formula

[0201]

Chemical formula

[0202]

Chemical formula

[0203] The stabilizer has the function of stabilizing the nanoparticles and can, for example, function as an agent to prevent aggregation or disintegration of the nanoparticles. The stabilizer can be a protein, a polysaccharide, or other (macromolecules), or a conjugate of a protein, a polysaccharide, or other (macromolecules) with the components of the nanoparticles (for example, conjugated to a phospholipid or a lipid). The stabilizer can further be used to control the size of the nanoparticles and increase their shelf life. Non-limiting examples of suitable stabilizers are apolipoproteins, apolipoprotein derivatives, apolipoprotein mimics, and / or apolipoprotein mimic derivatives. Other non-limiting examples of stabilizers are hydrophilic polymers or hydrophilic polymer-modified molecules, such as polyethylene glycol (PEG) or polyethylene glycol-modified molecules, polysaccharides or polysaccharide-modified molecules, polysarcosine or polysarcosine-modified molecules, or poly(2-oxazoline) or poly(2-oxazoline)-modified molecules.

[0204] In principle, the stabilizer is mainly present in the outer layer of the particles. However, the stabilizer can be, for example, a conjugate of PEG and (phospho)lipids, in which case PEG is mainly present in the aqueous layer directly surrounding the nanoparticles, while the conjugated lipid is mainly present in the outer layer of the nanoparticles adjacent to other (phospho)lipids that are presumably not modified with PEG.

[0205] When the stabilizer contains a non-natural synthetic group, such as one of the aforementioned polyethylene glycol, polysarcosine, or poly(2-oxazoline) polymer chains, such a group can (frequently) cause an immune response when repeatedly administered, which may not be very desirable. Furthermore, such polymer groups usually have non-fouling characteristics, and it is suggested that nanoparticles exposing such groups may experience a barrier when interacting with cells. Therefore, a protein- or peptide-based stabilizer is preferred.

[0206] In certain embodiments, the stabilizer is an apolipoprotein, apolipoprotein derivative, apolipoprotein mimetic, and / or apolipoprotein mimetic derivative. Apolipoproteins are natural helical proteins that have an inherent affinity for (phospho)lipid layers due to their architecture and amphiphilic characteristics, however, these materials are not conventional hydrophilic head / hydrophobic tail amphiphilic substances. There are multiple classes of apolipoproteins, any of which can be used as a structural component of nanoparticle formulations. Apolipoprotein incorporation affects the physicochemical properties and shelf life of nanoparticles by providing structural stability. Furthermore, the presence of apolipoproteins modulates the biological behavior of nanoparticles. For example, apolipoprotein A1 interacts with cells via scavenger receptor class B type 1 (SRB1) and ATP-binding cassette transporter ABCA1. This increases the interaction of nanoparticles with myeloid cells in lymphoid organs.

[0207] By using apolipoproteins, such as apoA1, nanoparticles can successfully target specific cells, tissues, or organs, such as, but not limited to, the myeloid compartment. This has the advantage that specific cells in a subject can be targeted with a drug to stimulate or inhibit a response. For example, immune cells can be targeted to stimulate or inhibit an immune response. There are several therapeutic applications, such as, but not limited to, cancer, cardiovascular disease, autoimmune disorders, and xenograft rejection, where such use is considered beneficial.

[0208] In certain embodiments, the apolipoprotein, apolipoprotein derivative, apolipoprotein mimetic, and / or apolipoprotein mimetic derivative is an apolipoprotein, apolipoprotein derivative, apolipoprotein mimetic, and / or apolipoprotein mimetic derivative that targets the myeloid cell compartment, preferably apoA1 or a mimetic or derivative thereof.

[0209] The nanoparticles described herein have an appearance identical to HDL particles when using apolipoproteins as stabilizers, so the nanoparticles do not trigger an immune response that could result in premature degradation or clearance by the immune system before reaching their intended target, such as the myeloid compartment.

[0210] The term "apolipoprotein", as used herein, refers to a protein that, together with lipids, forms lipoproteins, i.e., assemblies of lipids and proteins. Apolipoproteins typically function to transport lipids and fat-soluble substances in the blood. Apolipoproteins have been described and include, but are not limited to, A1, apoA1-Milano, apoA2, apoA4, apoA5, apoB48, apoB100, apoC-I, apoC-II, apoC-III, apoC-IV, apoD, apoE, apoF, apoH, apoL, and apoM. In certain embodiments, an apolipoprotein stabilizer or component can be an apolipoprotein derivative. An apolipoprotein derivative can be a simple modified form of an apolipoprotein, for example, produced by a one-step conversion from the protein. As used herein, the term "apolipoprotein" can further refer to an apolipoprotein mimetic. An apolipoprotein mimetic is a short peptide that mimics the properties of an apolipoprotein. An example of an apoA1 mimetic peptide is commonly referred to as 18A, which is DWLKAF YDKVAE KLKEAF (SEQ ID NO: 1) and has an unfunctionalized N-terminus and C-terminus. Another reported mimetic that is more convenient and more active is 2F, which is 18A (SEQ ID NO: 1) with an acetamide-capped N-terminus and an amide C-terminus. In Leman, L.J. et al., J. Med. Chem. 2014, 57, 2169-2196 (10.1021 / jm4005847), further examples of apoA1 peptide mimetics are reported, particularly in Tables 2 and 3. ApoA1 peptide mimetic derivatives, such as dimeric, trimeric, and tetrameric peptides, are exemplified in Zhou et al., J. Am. Chem. Soc. 2013, 135, 13414-13424 (dx.doi.org / 10.1021 / ja404714a). Preferred apoA1 peptide mimetics or peptide mimetic derivatives are 18A, 2F, and 4F, and any dimers of these peptides.Preferred apolipoprotein A1 peptidomimetics are 18A, 2F, and 4F, and any dimers of these peptides. 2F and any dimers or trimers of this peptide are more preferred.

[0211] Apolipoproteins are proteins that bind to lipids to form lipoproteins. They transport lipids and fat-soluble vitamins in blood, cerebrospinal fluid, and lymph. The lipid components of lipoproteins are insoluble in water. However, due to their amphiphilic properties, apolipoproteins and other amphiphilic molecules, such as phospholipids, can surround lipids and form lipoprotein particles that are themselves water-soluble and can thus be transported through a water-based circulation (i.e., blood, extracellular fluid, lymph). In addition to stabilizing the lipoprotein structure and the lipid components, apolipoproteins interact with lipoprotein receptors and lipid transport proteins and are thereby involved in the uptake and clearance of lipoproteins. They function as enzyme cofactors for certain enzymes involved in lipoprotein metabolism.

[0212] Apolipoprotein A1 is a protein encoded by the APOA1 gene in humans. As a major component of HDL particles, it has a specific role in lipid metabolism. This protein, as a component of HDL particles, enables the excretion of lipid molecules by transporting them (in extracellular water) to other locations, including accepting lipids from within cells (including macrophages within the arterial wall that are in a state of being overloaded with lipids taken up from oxidized LDL particles) and returning them to LDL particles or sending them to the liver for excretion.

[0213] It is contemplated that any apolipoprotein can be used in the nanoparticles. Thus, in certain embodiments, the apolipoprotein is selected from apoA1, apoA1-Milano, apoA2, apoA4, apoA5, apoB48, apoB100, apoC-I, apoC-II, apoC-III, apoC-IV, apoD, apoE, apoF, apoH, apoL, and apoM, and combinations thereof, preferably selected from apoA1, apoA2, apoA4, apoA5, apoB100, apoC-I, apoC-II, apoC-III, apoC-IV, and apoE, more preferably selected from apoA1, apoA4, apoA5, apoB100, apoC-III, and apoE, even more preferably selected from apoA1, apoB100, and apoE. In a particularly preferred embodiment, the apolipoprotein is apoA1, as it enables targeting of the nanoparticles to the myeloid compartment. In an alternative preferred embodiment, the apolipoprotein is apoE, as it enables targeting of the nanoparticles to dendritic cells.

[0214] Apolipoproteins can be produced and purified by methods known in the art, such as recombinant protein expression from E. coli or other organisms, followed by the steps necessary to isolate apoA1 in a sufficiently pure form.

[0215] The inventors have found several advantages associated with the use of apolipoproteins in nanoparticles for delivering nucleic acids to target sites. First, apolipoproteins stabilize the nanoparticles by preventing aggregation during preparation and storage. For the nanoparticles to remain in a stable emulsion, it is essential that the nanoparticles do not undergo aggregation or fusion, which can lead to particle sedimentation. Apolipoproteins stabilize the particles and prevent aggregation. Further, apolipoproteins ensure the in vivo stability of the nanoparticles. Since apolipoproteins are naturally present in lipid particles circulating in the bloodstream, such as LDL and HDL, they are not recognized as non-self by the immune system, thereby ensuring natural stealth, in contrast to improving stability by chemical modification or other non-natural methods. Finally, the use of apolipoproteins promotes desirable interactions with immune cells, for example, in the myeloid compartment, for delivering nucleic acid cargo.

[0216] Accordingly, in certain embodiments of the invention, stabilizers, such as apolipoprotein components, such as apolipoproteins, apolipoprotein derivatives, apolipoprotein mimetics, and / or apolipoprotein mimetic derivatives, are used in nanoparticles to - prevent aggregation during preparation and storage, - improve in vivo stability, - provide natural stealth, and / or - promote targeting and / or interaction with immune cells.

[0217] In certain embodiments, the stabilizers described herein are - apolipoproteins, apolipoprotein derivatives, apolipoprotein mimetics, and / or apolipoprotein mimetic derivatives, - polyethylene glycol or polyethylene glycol-modified molecules, - hydrophilic polymers or hydrophilic polymer-modified molecules, - polysaccharides or polysaccharide-modified molecules, - Polysarcosine or a polysarcosine-modified molecule, or - Poly(2-oxazoline) or a poly(2-oxazoline)-modified molecule, or a combination thereof is selected from.

[0218] Preferably, the stabilizer is an apolipoprotein, an apolipoprotein derivative, an apolipoprotein mimetic, and / or an apolipoprotein mimetic derivative, or a polyethylene glycol-modified molecule, or a combination thereof. More preferably, the stabilizer is an apolipoprotein, an apolipoprotein derivative, an apolipoprotein mimetic, and / or an apolipoprotein mimetic derivative. Most preferably, the stabilizer is an apolipoprotein. In certain embodiments, the apolipoprotein, apolipoprotein derivative, apolipoprotein mimetic, and / or apolipoprotein mimetic derivative is positioned on the outer surface of the outer layer of the nanoparticles.

[0219] In certain embodiments, the apolipoprotein stabilizer described elsewhere herein can be fused to a targeting moiety, whereby the nanoparticles taught herein are targeted to a different target (or put another way, a target different from that to which it would bind in the absence of the apolipoprotein stabilizer being fused to the targeting moiety) and / or can bind to its intended target with a higher affinity, and as a result, off-target effects can be reduced. For example, the targeting moiety can enable the nanoparticles to bind to non-myeloid cells, such as lymphocytes, such as T cells, B cells, or natural killer (NK) cells, or endothelial cells, or to myeloid cells with a higher affinity.

[0220] The targeting entity can be an antibody or its antigen-binding fragment, a rerouting peptide or protein, such as a receptor-binding peptide, a ligand-mimicking peptide, a receptor ligand, a receptor, or an interacting protein. It is contemplated that any type of antigen-binding molecule can in principle be used as a targeting entity. Non-limiting examples of targeting entities include antibodies or their antigen-binding fragments that (specifically) bind to programmed cell death protein 1 (PD1), signal regulatory protein alpha (SIRPa), CD40L, GP120, CD8 (e.g., VHH CD8), targeting entities that can bind to factor VIII-related antigen, such as factor VIII, targeting entities that can bind to CD31 / PECAM-1, such as CD31, targeting entities that can bind to angiotensin-converting enzyme (ACE / CD143), such as angiotensin, targeting entities that can bind to CD34, such as L-selectin, or targeting entities that can bind to endoglin (CD105).

[0221] In embodiments where the stabilizer is derived from a hydrophilic polymer, polyethylene glycol (PEG), a polysaccharide, polysarcosine, or poly(2-oxazoline), these polymers are modified with a hydrophobic moiety. These can be, for example, a hydrophobic polymer, a hydrophobic C12-18 alkyl chain, or a phospholipid having a chain derived from a C14, C16, or C18 fatty acid. Dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), and distearoyl phosphatidylethanolamine (DSPE) phospholipids are primary amine functional and are thus particularly useful for this purpose as they can be connected to oligomeric or polymeric units of PEG, a polysaccharide, polysarcosine, or poly(2-oxazoline). The molecular weight of the oligomer or polymer can be about 200 Daltons or more, about 500 Daltons or more, about 1000 Daltons or more, or about 2000 Daltons or more. At lower molecular weights, the oligomers can be discrete. For example, DPPE or DSPE can be connected to an oligoethylene glycol chain having 4, 6, 12, or 24 ethylene glycol units. Typically, the oligoglycol or PEG is capped with a methoxy or hydroxy group. c. Nucleic acid

[0222] The purpose of the nanoparticles described herein is to deliver nucleic acids to cells. The nucleic acid can be, for example, mRNA encoding a peptide or protein intended to be expressed in the cell, or can include short nucleic acids intended to interfere with gene expression (e.g., gene silencing), such as siRNA, shRNA, or can include components of the CRISPR-Cas or related systems for inducing mutations in the genome of the cell. Thus, generally, the mode of action of the nucleic acid (the payload of the nanoparticle) is nuclear. Thus, the nanoparticles preferably have at least the following properties: 1) Enable targeting of the intended target cells, and 2) enabling delivery of the payload to a location where it can exert its action (and thus, in many cases, into the nucleus of the target cell).

[0223] While not wishing to be bound by theory, the multivalent molecules described herein are thought to play a role in endosomal escape of the payload after the nanoparticle has been taken up by the target cell and thus enable nuclear delivery of the nucleic acid payload.

[0224] A number of different types of RNA, DNA, or synthetic oligonucleotides are being used as nucleic acid therapeutics. Since the present invention is envisioned to function with any type that can be loaded with a multivalent molecule onto the nanoparticle, the present invention is not limited to a particular type of nucleic acid. Thus, in certain embodiments, the nucleic acid is RNA, DNA, or a nucleic acid analog, preferably, the RNA is microRNA (miRNA), small interfering RNA (siRNA), piwi-interacting RNA (piRNA), small nucleolar RNA (snoRNA), transfer RNA (tRNA), tRNA-derived small RNA (tsRNA), small regulatory RNA (srRNA), messenger RNA (mRNA), modified mRNA, ribosomal RNA (rRNA), long non-coding RNA (lncRNA), or guide RNA (gRNA), or a combination and / or modified forms thereof, or preferably, the DNA is single-stranded or double-stranded DNA, or preferably, the antisense oligonucleotide is single-stranded DNA or RNA consisting of nucleotides or nucleoside analogs containing a modification of the phosphodiester backbone or 2'-ribose, and more preferably, the nucleotides or nucleoside analogs are selected from locked nucleic acid (LNA), bridged nucleic acid (BNA), morpholino, or peptide nucleic acid (PNA). d. Sterol

[0225] As used herein, the term "sterol" refers to a compound derived from sterol (2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17 - hexadecahydro - 1H - cyclopenta[a]phenanthren - 3 - ol) by substituting some of the hydrogen atoms with other chemical groups or modifying the bonds within the ring. Sterols and related compounds play an essential role in eukaryotic physiology. For example, cholesterol forms part of the cell membrane in animals, affects the fluidity of the cell membrane, and functions as a secondary messenger in signal transduction during development.

[0226] As used herein, "sterol" can refer to, for example, cholesterol, ergosterol, hopanoid, hydroxysteroid, phytosterol, steroid, hydrogenated cholesterol, or zoosterol. In nanoparticles, sterols maintain or regulate the fluidity in the membrane (i.e., the phospholipid monolayer surface barrier).

[0227] In certain embodiments, the sterol is selected from cholesterol, stigmasterol, or β - sitosterol, or combinations thereof. In certain embodiments, the sterol is selected from cholesterol, ergosterol, hopanoid, hydroxysteroid, phytosterol, steroid, zoosterol, stigmasterol, or β - sitosterol, or combinations thereof. In preferred embodiments, the sterol is cholesterol or includes it. e. Phospholipid

[0228] Phospholipids, also known as phosphatides, are a class of lipids whose molecules have a hydrophilic head containing a phosphate group and two hydrophobic tails derived from fatty acids, linked by a glycerol molecule. Marine phospholipids typically have omega-3 fatty acids EPA and DHA incorporated as part of the phospholipid molecule. The phosphate group can be modified with simple organic molecules such as choline, ethanolamine, or serine.

[0229] Phospholipids are an important component of all cell membranes. Due to their amphiphilic nature, phospholipids can form lipid bilayers. In eukaryotes, cell membranes also contain another class of lipids, sterols, dispersed among the phospholipids. This combination provides a combination of two-dimensional fluidity and mechanical strength against rupture.

[0230] Thus, in certain embodiments, the phospholipid is selected from phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylglycerol, or combinations thereof.

[0231] The acyl groups in phospholipids can each be derived from medium-chain or long-chain fatty acids. In certain embodiments, at least one, preferably both, of the acyl groups in the phospholipid are derived from long-chain fatty acids, preferably the long-chain fatty acids are C14, C16, or C18 chains, i.e., myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, and linoleic acid, or combinations thereof.

[0232] Lysophospholipids are phospholipids in which one of the acyl groups has been removed by hydrolysis, leaving an alcohol group. These molecules thus have one, rather than two, fatty acid chains. These phospholipids can also be applied, for example, to regulate the shape, function, and fluidity of the outer layer of nanoparticles.

[0233] In a particularly preferred embodiment, the phospholipid is a neutral phospholipid, i.e., zwitterionic (having a net neutral charge) at physiological pH. Thus, in a preferred embodiment, the phospholipid is phosphatidylcholine (PC) or phosphatidylethanolamine (PE).

[0234] Thus, examples of phospholipids that can be used are dilauroyl phosphatidylcholine (DLPC), dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dilauroyl phosphatidylglycerol (DLPG), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearoyl phosphatidylglycerol (DSPG), dioleoyl phosphatidylglycerol (DOPG), dilauroyl phosphatidylethanolamine (DLPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), distearoyl phosphatidylethanolamine (DSPE), dilauroyl phosphatidylserine (DLPS), dimyristoyl phosphatidylserine (DMPS), dipalmitoyl phosphatidylserine (DPPS), distearoyl phosphatidylserine (DSPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and mixtures thereof.

[0235] As lysophospholipids, 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine (MHPC), 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (PHPC), and 1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine (SHPC), or mixtures thereof, can be utilized. f. Filler molecules

[0236] The nanoparticles described herein may further and optionally contain filler molecules. The filler molecules are biocompatible molecules, such as esters or amides (of any kind). Esters, in particular those derived from fatty acids or cholesterol, are preferred, and the fatty acids may have saturated or unsaturated chains. The filler molecules are of a hydrophobic nature and can be lipids such as, but not limited to, triglyceride lipids. Thus, in certain embodiments, the nanoparticles further contain filler molecules selected from triglycerides, diglycerides, esters derived from fatty acids, and cholesteryl esters, or combinations thereof.

[0237] The nanoparticles described herein can form nanodisks or nanospheres, respectively, depending on the absence or presence of filler molecules. The filler can be, for example, a triglyceride, which is included in the core of the particle together with the payload (nucleic acid) and the multivalent molecule. Without being bound by theory, it is understood that including more filler will make the nanoparticles larger up to the point where the particles may become unstable. Without being bound by theory, including a certain amount of filler can contribute to the stability of the nanoparticles, or can stabilize the inclusion of the payload, or can modulate or enhance the delivery of the nucleic acid to the cells.

[0238] Thus, in certain embodiments, the nanoparticles described herein further contain filler molecules, preferably the filler molecules are glyceride molecules or cholesteryl esters, more preferably the filler molecules are selected from triglycerides and cholesteryl esters, or combinations thereof. Preferably, the triglyceride is derived from C6-C18 fatty acids, preferably C6-C12 fatty acids. Examples of cholesteryl esters are cholesteryl acetate, cholesteryl caprylate, and cholesteryl oleate. Preferred filler molecules are tricaprylin, cholesteryl acetate, cholesteryl caprylate, and cholesteryl oleate, with tricaprylin being more preferred. Properties of Nanoparticles

[0239] The nanoparticles of the present invention contain nucleic acids, multivalent molecules, phospholipids, sterols, stabilizers, and optionally filler molecules, The amount of the stabilizer, particularly an apolipoprotein, apolipoprotein derivative, apolipoprotein mimetic, or apolipoprotein mimetic derivative, is in the range of 0.1 to 90% by weight, and / or The amount of the nucleic acid is in the range of 0.01 to 90% by weight, and / or The amount of the phospholipid is in the range of 0.1 to 95% by weight, and / or The amount of the sterol is in the range of 0.1 to 95% by weight, and / or The amount of the multivalent molecule is in the range of 0.1 to 95% by weight, The amount of the optionally present filler is in the range of 0 to 95% by weight, These weight percentages are based on the combined amount of these five components and an optional sixth filler component, i.e., adding these five or six components gives a maximum of 100% of the weight of the nanoparticles.

[0240] In certain embodiments, the amount of the stabilizer, preferably an apolipoprotein, apolipoprotein derivative, apolipoprotein mimetic, or apolipoprotein mimetic derivative, is in the range of 0.2 to 50% by weight, more preferably 0.5 to 30% by weight, more preferably 1 to 20% by weight, even more preferably 5 to 20% by weight, for example, 5 to 15% by weight.

[0241] In certain embodiments, the amount of the nucleic acid is in the range of 0.02 to 30% by weight, more preferably 0.05 to 20% by weight, more preferably 0.1 to 15% by weight, even more preferably 0.5 to 10% by weight, for example, 0.5 to 5% by weight, 1 to 5% by weight, or 0.5 to 4% by weight, for example, 1.2 to 3% by weight.

[0242] In certain embodiments, the amount of phospholipid is in the range of 0.2 to 60% by weight, such as 0.2 to 50% by weight, 5 to 50% by weight, 10 to 50% by weight, 0.2 to 45% by weight, 5 to 45% by weight, 10 to 45% by weight, more preferably 1 to 40% by weight, more preferably 3 to 40% by weight, 5 to 40% by weight, 10 to 40% by weight, or 3 to 30% by weight, most preferably 10 to 45% by weight, such as 15 to 40% by weight.

[0243] In certain embodiments, the amount of sterol is in the range of 0.2 to 90% by weight, more preferably 0.5 to 70% by weight, more preferably 1 to 50% by weight, even more preferably 3 to 30% by weight, or 5 to 25% by weight, such as 8 to 20% by weight.

[0244] In certain embodiments, the amount of the polyvalent molecule is in the range of 0.2 to 90% by weight, more preferably 0.5 to 80% by weight, more preferably 1 to 70% by weight, even more preferably 1 to 30% by weight, such as 5 to 30% by weight, 5 to 20% by weight, 10 to 20% by weight, 5 to 10% by weight, or 15 to 25% by weight.

[0245] In certain embodiments, the amount of the optional filler molecule is in the range of 0 to 90% by weight, more preferably 0 to 80% by weight, more preferably 0 to 70% by weight, even more preferably 0 to 60% by weight.

[0246] The above relative amounts of the components are controlled by the ratios at which the components are used and mixed in the nanoparticle preparation protocol, since the recovery rates of the various components are usually very high. By using methods known in the art, the level of incorporation of the various components of the nanoparticles can be evaluated after particle preparation is complete. Thus, the above ranges regarding the incorporation of components in the nanoparticles can be evaluated by assessing the mixing amounts of the components utilized or by the measured values of the components in the prepared nanoparticle formulation. For example, assay kits for evaluating the phospholipid, cholesterol, or apoA1 levels in a nanoparticle sample are commercially available. Additionally, the amount of siRNA loaded and retained inside the nanoparticles can be evaluated by using a RiboGreen assay.

[0247] The outer layer of the nanoparticles is composed of phospholipids, stabilizers (e.g., apolipoproteins), and sterols. To assemble stable nanoparticles, preferably, the ratio of the stabilizer (e.g., apolipoprotein) to the phospholipid based on weight is from 2:1 to 1:10. Thus, in certain embodiments, the ratio of apolipoprotein to phospholipid based on weight used is from 2:1 to 1:10, more preferably from 1:1 to 1:5, even more preferably from 15:1 to 1:4 or from 2:3 to 1:4, for example, 4:10.

[0248] Within the nanoparticles, the multivalent molecule binds to the nucleic acid. The multivalent molecule is multivalent with respect to positively ionizable groups and / or cationic groups (#N), while the nucleic acid has multiple phosphate groups (P). The ratio (#N:P) between the number of N groups (#N) and the number of P groups can vary for the nanoparticles of the present invention. The ratio of N groups (#N):P groups (P) (#N:P ratio) can vary from 100:1 to 1:10. However, equimolar amounts or excess amounts of #N groups are preferred, and the #N:P ratio varies from about 50:1 to about 1:1. A #N:P ratio of from about 20:1 to about 1:1 is more preferred. A #N:P ratio of from about 15:1 to about 1:1 is even more preferred.

[0249] The nanoparticles according to the present invention have been found to have a relatively defined and constant size. The average size is mostly determined by the core components, namely the amount and type of nucleic acid, the amount of multivalent molecules, and the amount of sterol and filler molecules. The filler is optional, and it is understood that the particle size can be increased by including increasing amounts of filler. In certain embodiments, the nanoparticles according to the present invention have an average diameter of about 10 to about 300 nm, preferably about 20 to about 200 nm, more preferably about 30 to about 100 nm.

[0250] The size of the nanoparticles of the present invention can be evaluated by methods known in the art. For example, dynamic light scattering (DLS) can be utilized to measure the diameter of the nanoparticles. Cryo-TEM measurements can also be used for this purpose. Any of these techniques can be used to evaluate the diameter distribution of the prepared nanoparticle formulation. Cryo-TEM measurements can further be used to evaluate the shape of the nanoparticles (circular spheres, or other shapes). This can also evaluate further characteristics of the nanoparticles (e.g., the presence of one or more outer layers, the presence of apolipoproteins, the uniformity of the nanoparticle appearance).

[0251] The nanoparticles of the present invention can have a certain surface charge that can be (slightly) negative, (slightly) positive, or essentially neutral. Zeta potential analysis, or other methods known in the art, can be utilized to evaluate the charged state of the nanoparticles of the present invention.

[0252] The in vitro activity of the nanoparticles containing the siRNA of the present invention can be evaluated using dual reporter cells (e.g., RAW264.7 macrophages) transfected with a Pmir-Glo plasmid containing the firefly luciferase and Renilla luciferase gene expression sequences. In the silencing experiment, macrophages are exposed to either nanoparticles loaded with firefly luciferase siRNA or nanoparticles loaded with non-specific siRNA. Next, the knockdown of luciferase activity by any of these nanoparticles can be evaluated and compared. In the dose-dependent gene knockout study, the activity of firefly luciferase can be evaluated as a function of the concentration of firefly luciferase siRNA used. This assay reports on the efficacy of the nanoparticle formulation utilized.

[0253] The nanoparticles defined herein include a hydrophobic core and a hydrophilic surface (as a result of the incorporation of lipid and stabilizer components), and can thus be dissolved in water, or an aqueous solution, e.g., physiological saline or buffer. The unique properties resulting from the components of the nanoparticles defined by the present invention result in nanoparticles that are stable for several months in suspension. Suitable aqueous buffers such as phosphate buffered saline (PBS), Tris buffered saline (TBS) are known in the art. Suitable saline solutions are known and non-limiting examples include aqueous solutions of NaCl or KCl. When the nanoparticles are intended to be administered to a subject, the nanoparticles shall be suspended in a physiologically acceptable carrier for that purpose. For example, when the nanoparticles are intended for intravenous delivery, the physiologically acceptable carrier is typically a fluid isotonic with blood. For example, a sodium chloride solution at a concentration of 0.9% weight / volume, a dextrose solution at 5% weight / volume, Ringer's solution, lactated Ringer's solution, or acetate Ringer's solution can be used, although other suitable carriers are known.

[0254] Accordingly, in one aspect, the present invention relates to a composition comprising nanoparticles according to the present invention and a physiologically acceptable carrier. In certain embodiments, the composition is a pharmaceutical composition. It is understood that the composition may further comprise additional components such as, but not limited to, pharmaceutical drugs or biopharmaceutical drugs. This can be a promising option for combination therapy of nucleic acids (contained in the nanoparticles) and drugs. The drug can be a small molecule compound, an antibody or antigen-binding fragment, additional nanoparticles, and the like.

[0255] It is further contemplated to deliver nucleic acid therapy to a subject using the nanoparticles described herein. Accordingly, in one aspect, the present invention relates to nanoparticles according to the present invention or a composition according to the present invention for use as a medicament.

[0256] It is understood that a nucleic acid therapy comprising nanoparticles can be administered to a subject in need thereof. Depending on the target cell or tissue, the administration can be parenteral, for example, intravenous, intramuscular, or subcutaneous. The administration can further be oral, sublingual, topical, rectal, intranasal (inhalation), or vaginal. Further, the targeting of the target tissue or cell is determined by the appropriate selection of apolipoprotein. In certain embodiments, the use of the nanoparticles or composition according to the present invention comprises delivering nucleic acids to the myeloid compartment or spleen. This can be achieved, for example, by intravenous parenteral administration. Preferably, the apolipoprotein is preferably a myeloid compartment-targeting apolipoprotein, such as apoA1.

[0257] A further aspect provides nanoparticles taught herein or a composition taught herein for use in immunotherapy.

[0258] In one aspect, the present invention is a nanoparticle according to the present invention or a composition according to the present invention for use in the treatment of a disease by stimulating or inhibiting the innate immune response, preferably, the disease is a disease in which benefit would be obtained by stimulating or inhibiting the innate immune response in a subject, for example, a disease characterized by a defective innate immune response, more preferably, the disease to be treated is cancer, cardiovascular disease, autoimmune disorder, or xenograft rejection. Thus, the nanoparticles according to the present invention can be used for the treatment of any disease related to the immune system, for example, in the treatment of any immune disorder, or for the treatment of any disease or disorder that is considered to be a treatment option capable of modulating the immune response. By targeting the myeloid compartment, nucleic acid therapy can successfully deliver to progenitor cells of different blood cell types, in contrast to already differentiated cells present in the blood and tissues, such as T cells and macrophages. By doing so, the innate immune response can be modulated by nucleic acid therapy, for example, stimulated or inhibited, depending on the desired result. For example, in autoimmune disorders, cardiovascular disease, or xenograft rejection, inhibition (prevention) of the autoimmune response is desirable, while in cancer, stimulation of the immune response against the target cancer cells is desirable.

[0259] In a further aspect, the present invention relates to a method for in vivo delivery of nucleic acids, comprising the step of administering a nanoparticle according to the present invention or a composition according to the present invention to a subject.

[0260] In a further aspect, the present invention relates to a method for treating a disease or disorder in a subject in need thereof by stimulating or inhibiting the innate immune response, comprising the step of administering a therapeutically effective amount of a nanoparticle according to the present invention or a composition according to the present invention to the subject. In certain embodiments, the disease or disorder is a disease or disorder characterized by a defective innate immune response. In one embodiment, the disease or disorder is selected from cancer, cardiovascular disease, autoimmune disorder, or xenograft rejection.

[0261] The present invention provides apolipoprotein-based nanoparticles (aNPs) having nucleic acids. The core of such particles is hydrophobic and thus not suitable for the incorporation of nucleic acids due to the hydrophilic nature of the nucleic acids. Thus, heretofore, it has not been possible to include nucleic acids in such nanoparticles. Although the use of some multivalent molecules together with nucleic acids has been described as a tool for intracellular delivery of nucleic acids, simply combining the multivalent molecules and nucleic acids with other lipid components does not result in the formation of the lipid nanoparticles described herein. The present invention centers on the recognition that nucleic acids can be incorporated into nanoparticles by the use of the multivalent molecules described herein and by using a two-step nanoparticle preparation process.

[0262] Accordingly, in one aspect, the present invention is a method for producing nanoparticles, comprising: a) mixing a (lipid) component in an organic solvent with a nucleic acid in an aqueous buffer, preferably by rapid mixing, to produce nanoparticles, wherein the (lipid) component comprises a phospholipid, a sterol, a multivalent molecule, and optionally a filler molecule (e.g., a glyceride, e.g., a triglyceride), and the aqueous buffer has a pH of 5.5 or less, preferably 5.0 or less; b) mixing the lipid nanoparticles with a stabilizer, preferably an apolipoprotein, an apolipoprotein derivative, an apolipoprotein mimetic, and / or an apolipoprotein mimetic derivative, preferably by rapid mixing, to produce the nanoparticles at a pH of 5.5 to 9.0, preferably 6.0 to 8.0, more preferably 6.5 to 8.0; and relates to a method comprising.

[0263] Rapid mixing is known in the art and is described, for example, in Hirota et al. BIOTECHNIQUES VOL. 27, NO. 2, p286-289, Jeffs et al., Pharm Res 22, 362-372 (2005), Kulkarni et al., ACS Nano 2018, 12, 5, 4787-4795.

[0264] The aqueous buffer in step a) has a low pH to ensure that the multivalent molecules are positively charged and enable the binding of the nucleic acid and the multivalent molecules within the particles. For example, the buffer can have a pH of 5.5 or lower, for example, 5.4, 5.3, 5.2, 5.1, or the buffer can have a pH of 5.0 or lower, for example, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, or lower. The aqueous buffer can be any buffer that does not damage the nucleic acid. An exemplary buffer is sodium acetate at pH 4.0. Next, the nanoparticles are collected in an aqueous buffer having a pH of approximately 6-8, preferably 7-8, more preferably approximately 7.4. This can be achieved, for example, by dialysis using an aqueous buffer in the indicated pH range. A non-limiting example of an aqueous buffer suitable for this step is 150 mM PBS, pH 7.4, but it is understood that any buffer that does not damage the nucleic acid can be used.

[0265] In step b), the nanoparticles in an aqueous buffer having a pH of 6-8, preferably 7-8, are rapidly mixed with apolipoprotein in an aqueous buffer having a pH of 6-8, preferably 7-8, to obtain the nanoparticles according to the present invention.

[0266] In one aspect, the present invention relates to nanoparticles, for example, nanoparticles according to the present invention, obtainable or obtainable by a method of producing the nanoparticles taught herein.

[0267] It is understood that the nanoparticles according to the present invention can deliver nucleic acids to target cells or tissues. The target cells or tissues can be in a subject or can be in vitro or ex vivo. Thus, in one aspect, the present invention relates to an in vivo, in vitro, or ex vivo method for introducing nucleic acids into cells, the method comprising the step of contacting the cells with the nanoparticles according to the present invention or the composition according to the present invention. In certain embodiments, the method is an in vitro or ex vivo method. In specific embodiments, the cells are cells of the myeloid compartment or myeloid cells.

[0268] In a further aspect, the present invention relates to a method for in vivo delivery of nucleic acids, the method comprising the step of administering to a subject the nanoparticles according to the present invention or the composition according to the present invention.

[0269] In a further aspect, the present invention relates to a method for treating a disease or disorder in a subject in need thereof by stimulating or inhibiting the innate immune response, the method comprising the step of administering to the subject a therapeutically effective amount of the nanoparticles according to the present invention or the composition according to the present invention. In certain embodiments, the disease is selected from cancer, cardiovascular disease, autoimmune disorder, or xenograft rejection.

[0270] This application also provides aspects and embodiments described in the following statements. Statement 1. A nanoparticle comprising a core and an outer layer, wherein the core comprises - nucleic acid, - a multivalent molecule and the outer layer comprises - a stabilizer, - a phospholipid, - a sterol, and optionally - a filler molecule, wherein the multivalent molecule has the formula (I): [Core] x -[BU] y -[TU] z (I) and in the formula, the core is nitrogen or a C 1 ~C 18 linear, branched or cyclic group containing 1 to 15 nitrogen heteroatoms and optionally 1 to 4 oxygen heteroatoms, x represents the number of connections from the core to the branching unit BU (or to the terminal unit TU when y = 0), and x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 12, and all of these connections constitute a connection from a nitrogen atom of the core to a carbon atom of the branching unit BU (or terminal unit TU), the branching unit BU has the formula (IIa), (IIb), (IIc) or (IId):

[0271]

Chemical formula

[0272]

Chem.

[0273]

Chem.

[0274] All cited references are incorporated herein by reference in their entirety.

Examples

[0275] [Example 1] Design and synthesis of multivalent molecules. Tables S1 and S2 provide an overview of the properties of the prepared multivalent molecules. Figures 1, 2, and 3 provide the molecular structures of a series of multivalent molecules.

[0276]

Table 6

[0277]

Table 7

[0278] All reagents, chemicals, materials, and solvents were obtained from commercial sources and used as received. All solvents were of AR quality. DCM = dichloromethane, CHCl 3 = chloroform, MeCN = acetonitrile, MeOH is methanol, EtOH = ethanol, IPA = isopropanol, DMSO = dimethyl sulfoxide, THF = tetrahydrofuran, 1,1,1,3,3,3 - hexafluoroisopropanol = HFIP, DMF = dimethylformamide. In synthetic procedures, equivalents (eq) are molar equivalents.

[0279] NMR spectra were recorded at 298K on a Bruker Avance III HD spectrometer ( 1 H - NMR and 13 C - NMR at 400 MHz and 100 MHz, respectively). Chemical shifts are reported in ppm from TMS on the low - field side at room temperature. Abbreviations used for splitting patterns are s = singlet, t = triplet, q = quartet, m = multiplet, and br = broad.

[0280] HPLC of the Shimadzu LC - 10 AD VP series connected to a photodiode array detector (Finnigan Surveyor PDA Plus detector, Thermo Electron Corporation) and an ion trap mass spectrometer (LCQ Fleet, Thermo Scientific) was used, and electrospray ionization (ESI) was applied to perform HPLC - PDA / ESI - MS. An Alltech Alltima HP C18 3μ column was used, with an injection volume of 1 - 4 μL, a flow rate of 0.2 mL min-1, and typically H 2HPLC analysis was performed at 298 K using a gradient of MeCN in O (both containing 0.1% formic acid) from 5% to 100% over 10 minutes and holding at 100% for an additional 3 minutes.

[0281] MALDI-TOF-MS measurements were carried out on an Autoflex Speed (Bruker) spectrometer using α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), or trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene] malononitrile (DCTB) matrix. Building block

[0282] [Chemical formula] N-(4-bromobutyl)phthalimide

[0283] 1,4-Dibromobutane (86.4 g, 0.4 mol, 4 equivalents) and potassium phthalimide (18.5 g, 0.1 mol, 1 equivalent) were dissolved in DMF (75 mL). (Potassium phthalimide did not dissolve completely.) The reaction mixture was stirred at room temperature for 48 h. The reaction mixture was then poured into water (600 mL), transferred to an extraction funnel, and extracted with diethyl ether (2 × 50 mL). The combined ether layers were washed with saturated KCl solution (2 × 25 mL). The ether layer was dried using Na 2 SO 4 and the suspension was filtered, and the filtrate was concentrated under vacuum. The product was purified using silica column chromatography with a mixture of heptane / DCM (0 - 100% DCM) to give a white solid. The last impurities were removed by stirring the solid in n-pentane at room temperature for 16 h, filtering, washing, and drying the residue under vacuum to give the product (14.9 g, 52.7 mmol, 53%). Alternatively, the crude product may be purified by recrystallization from n-heptane. 11H NMR (400 MHz, chloroform-d) δ 7.85 (m, 2H), 7.72 (m, 2H), 3.73 (t, J = 6.6 Hz, 2H), 3.45 (t, J = 6.3 Hz, 2H), 1.97 - 1.80 (m, 4H). N,N,N’,N’-Tetra(4-phthalimidobutyl)-1,4-diaminobutane

[0284] N-(4-Bromobutyl)phthalimide (1.41 g, 5 mmol, 5 equiv), 1,4-diaminobutane (88 mg, 1 mmol, 1 equiv), and K 2 CO 3 (691 mg, 5 mmol, 5 equiv) were mixed together in MeCN (5 mL) and stirred at reflux for 40 h. The reaction mixture was cooled, the suspension was filtered, and the filtrate was concentrated under vacuum. The product was purified using silica column chromatography eluting with a mixture of MeOH / CHCl 3 (0 - 10% MeOH) to give the product as an amber viscous oil (222 mg, 0.25 mmol, 25%). 1 1H NMR (399 MHz, chloroform-d) δ 7.86 - 7.77 (m, 7H), 7.75 - 7.64 (m, 8H), 3.68 (t, J = 7.0 Hz, 8H), 2.69 (sb, 4H), 2.39 (s, 8H), 1.68 (s, 13H), 1.40 (d, J = 45.6 Hz, 10H). 13 13C NMR (100 MHz, CDCl 3 ) δ 168.19, 133.73, 132.03, 123.00, 53.77, 53.25, 37.73, 26.42, 24.74, 24.25. ESI-MS: m / z C 52 H 56 N 6 O 8 calculated for 892.42; found 893.58 [M+H] + 447.42, [M+H] 2+ . N,N,N’,N’-Tetra(4-aminobutyl)-1,4-diaminobutane (G1-PBuI)

[0285] Hydrazine hydrate (0.96 mL, 19.8 mmol, 20 equiv) was added to a stirred suspension of N,N,N’,N’-tetra(4-phthalimidobutyl)-1,4-diaminobutane (884 mg, 0.99 mmol, 1 equiv) in EtOH (30 mL). The reaction mixture was heated to reflux for 16 h to give a white suspension. The suspension was filtered through a glass frit funnel and the residue was washed with ethanol. The filtrate was concentrated under vacuum to give a white sticky material (100 mg, 0.27 mmol, 27%) which was a mixture of the product and 2,3-dihydrophthalazine-1,4-dione. 1 H NMR (399 MHz, chloroform-d) δ 2.75 - 2.65 (m, 8H), 2.46 - 2.34 (m, 12H), 2.24 (s br, 8H), 1.56 - 1.42 (m, J = 4.4 Hz, 16H), 1.42 - 1.32 (m, 4H). ESI-MS: m / z C 20 H 48 N 6 calculated for 372.39; found 373.50 [M+H] + .

[0286]

Chemical Structure

[0287] N-(5-Bromopentyl)phthalimide (14.8 g, 50 mmol, 5 equiv), 1,4-diaminobutane (882 mg, 10 mmol, 1 equiv), and K 2 CO 3 (12 mg, 80 mmol, 8 equiv) were mixed in MeCN (50 mL) and stirred at reflux for 40 h. The reaction mixture was cooled, the suspension was filtered, and the filtrate was concentrated under vacuum. The product was purified by MeOH / CHCl 3Purification was carried out using silica column chromatography eluting with a mixture (0 - 10% MeOH) to give an amber, very highly viscous oily product (3.12 g, 3.28 mmol, 33%). 1 H NMR (400 MHz, chloroform - d) δ 7.82 (dd, J = 5.4, 3.0 Hz, 8H), 7.74 - 7.65 (m, 8H), 3.67 (t, J = 7.2 Hz, 8H), 2.41 (m, 12H), 1.68 (p, J = 7.4 Hz, 8H), 1.48 (m, 8H), 1.39 (m, 4H), 1.37 - 1.20 (m, 8H). 13 C NMR (101 MHz, CDCl 3 ) δ 168.29, 133.81, 132.07, 123.07, 53.62, 53.50, 37.77, 28.36, 25.89, 24.62, 24.24. ESI - MS: m / z C 56 H 64 N 6 O 8 calculated for 948.48; found 949.58 [M + H] + 475.50, [M + H] 2+ . N,N,N’,N’ - tetra(5 - aminopentyl)-1,4 - diaminobutane (G1 - PPeI)

[0288] Hydrazine hydrate (3.08 g, 61.5 mmol, 20 equiv) was added to a stirred suspension of N,N,N’,N’-tetra(5-phthalimidopentyl)-1,4-diaminobutane (2.92 g, 3.07 mmol, 1 equiv) in EtOH (50 mL). The reaction mixture was heated to reflux for 16 h to give a white suspension. The suspension was filtered through a glass frit funnel and washed with ethanol. The filtrate was concentrated under vacuum, redissolved in chloroform to give a suspension, which was filtered and the filtrate concentrated under vacuum. To completely remove traces of 2,3-dihydrophthalazine-1,4-dione, the residue was redissolved in a 1:1 mixture of n-pentane / chloroform, filtered, and the filtrate concentrated under vacuum to give the product as a pale yellow viscous oil (950 mg, 2.22 mmol, 72%). 1 H NMR (400 MHz, chloroform-d) δ 2.69 (t, J = 7.0 Hz, 8H), 2.39 (m, 12H), 1.67 (s br, 8H), 1.45 (m, 20H), 1.37 - 1.26 (m, 8H). ESI-MS: m / z C 24 H 56 N 6 calculated for 428.46; found 429.58 [M+H] + .

[0289]

Chemical Structure

[0290] To an ice-cold solution of 3-bromopropylamine hydrobromide (8.6 g, 39.3 mmol, 1 equiv) in MeOH (40 mL) was added triethylamine (4.2 g, 41.3 mmol, 1.05 equiv). Then, di-tert-butyl carbonate was added portionwise to the reaction mixture. The temperature of the reaction mixture was slowly raised to room temperature and stirred for an additional 16 h. The mixture was then concentrated under vacuum, redissolved in a mixture of dichloromethane and water, transferred to a separatory funnel, and the aqueous layer was extracted with dichloromethane (3×). The combined dichloromethane layers were washed with 0.5 M citric acid solution (1×25 mL) and brine (1×25 mL). The dichloromethane layer was dried using Na 2 SO 4 and the suspension was filtered. The solvent was evaporated under vacuum to afford the product as a colorless oil (8.86 g, 37.2 mmol, 95%). tert-Butyl (3-(methylamino)propyl)carbamate

[0291] To a stirred solution of tert-butyl (3-bromopropyl)carbamate (8.86 g, 37.2 mmol, 1 equiv) in ethanol (30 mL) was added a 40% w / w solution of methylamine in MeOH (45 mL, 446 mmol, 10 equiv) and KI (0.74 g, 4.46 mmol, 0.1 equiv), and the temperature was raised to 50 °C. After 20 h at 50 °C, the reaction mixture was concentrated under vacuum. The residue was redissolved in DCM and water, and the aqueous layer was extracted with DCM (3×25 mL). The combined DCM layers were dried using Na 2 SO 4 and the suspension was filtered. The filtrate was concentrated under vacuum to afford a colorless oily byproduct. The aqueous layer was basified with 6 M NaOH solution until the pH exceeded 10 and extracted with CHCl 3 (3×50 mL). The combined CHCl 3 layers were dried using Na 2 SO 4 and the suspension was filtered. The filtrate was concentrated under vacuum to afford a colorless oil (4.84 g, 25.7 mmol, 58%). 11H NMR (399 MHz, chloroform-d) δ 5.06 (s, 1H), 3.20 (q, J = 6.3 Hz, 2H), 2.63 (t, J = 6.7 Hz, 2H), 2.42 (s, 3H), 1.65 (p, J = 6.7 Hz, 2H), 1.44 (s, 11H), 1.34 (s, 2H). ESI-MS: m / z C 9 H 20 N 2 O 2 calculated value of 188.15. Measured value [M+H] + 189.17. tert-Butyl (3-(N-methylacrylamide)propyl)carbamate

[0292] A solution of acryloyl chloride (2.36 g, 26.1 mmol, 1.1 eq) in DCM (10 mL) was added dropwise to an ice-cooled solution of tert-butyl (3-(methylamino)propyl)carbamate (4.43 g, 23.5 mmol, 1 eq) and triethylamine (3.9 mL, 28.2 mmol, 1.2 eq) in DCM (50 mL). After the addition, the reaction temperature was raised to room temperature. The reaction mixture was stirred at room temperature for 20 h. The reaction mixture was transferred to a separatory funnel and the DCM layer was washed with water (1×25 mL), 1 M KHSO 4 solution (1×25 mL), water (1×25 mL), 1 M NaHCO 3 solution and water (1×25 mL). The DCM layer was dried using Na 2 SO 4 and the suspension was filtered. The filtrate was concentrated under vacuum to give the crude product as a yellow oil. The acrylamide product was further purified using column chromatography eluting with a mixture of MeOH and DCM (0 - 5% MeOH in DCM) to give a pale yellow oil (3.85 g, 15.9 mmol, 68%). 11H NMR (399 MHz, chloroform-d) δ 6.57 (m, 1H), 6.32 (dd, J = 16.7, 2.0 Hz, 1H), 5.70 (m, 1H), 5.42 (s,.74H), 4.63 (s,.26H), 3.51 (t, J = 6.4 Hz, 1.42H), 3.41 (t, J = 7.7 Hz,.65H), 3.11 (m, 4H), 3.06 (s, 2.33H), 3.00 (s,.87H), 1.80 (p, J = 7.2 Hz,.57H), 1.71 (p, J = 6.3 Hz, 1.47H), 1.44 (s, 9H). ESI-MS: m / z C 12 H 22 N 2 O 3 Calculated value for 242.16. Measured value [M+Na] + 265.17.

[0293]

Chemical formula

[0294] N-Hydroxysuccinimide (3.37 g, 29.3 mmol, 1.14 equivalents) and DIPEA (8.0 mL) were dissolved in THF (60 mL) under a nitrogen atmosphere. A solution of oleoyl chloride (7.74 g, 25.7 mmol, 1 equivalent) in THF (50 mL) was added dropwise at 0 °C. After 20 hours at room temperature, the reaction mixture was evaporated to dryness. The crude product was dissolved in chloroform (200 mL) and washed with 0.2 M aqueous NaOH (500 mL). The aqueous layer was extracted with chloroform (100 mL). The combined chloroform fractions were washed with brine (200 mL). Purification was carried out by column chromatography on silica (elution with chloroform:ethyl acetate 80:20). Yield: 6.0 g of a white solid (61%). 1H-NMR (400 MHz, chloroform-d) δ 5.72 - 4.84 (m, 2H), 2.83 (s, 4H), 2.60 (t, J = 7.5 Hz, 2H), 2.16 - 1.89 (m, 4H), 1.75 (m, J = 7.4 Hz, 2H), 1.69 - 1.51 (m, 2H), 1.51 - 1.10 (m, 18H), 0.99 - 0.45 (m, 3H, J = 6.8 Hz). MALDI-TOF-MS (CHCA matrix, positive reflector mode): found m / z = (M+H)+ 380.28. calculated: C 22 H 37 NO 4 (accurate mass 379.27; molecular weight 379.54). N 6 -(tert-Butoxycarbonyl)-N 2 -oleoyl-L-lysine

[0295] N-epsilon-t-butyloxycarbonyl-L-lysine (551 mg, 2.23 mmol, 1.0 equiv) was dissolved in DCM (17 mL) under an inert nitrogen atmosphere. 2,5-Dioxopyrrolidin-1-yl oleate (851 mg, 2.21 mmol, 0.99 equiv) and triethylamine (0.70 mL, 2 equiv) were added and stirring was continued for 19 h. DCM (50 mL) was added and the organic phase was washed with 1 M KHSO 4 aqueous solution (3 × 50 mL), water (3 × 50 mL), and brine (100 mL). After evaporation to dryness, the crude product was purified by column chromatography on silica using chloroform:methanol:acetic acid (10:10:1) eluent. Yield: 813 mg of white solid (71%). 11H NMR (400 MHz, chloroform-d) δ 6.60 - 6.40 (m, 1H), 5.47 - 5.22 (m, 2H), 4.67 (s, 1H), 4.51 (s, 1H), 3.13 (m, 2H), 2.3 - 1.5 (m, 12H), 1.44 (s, 9H), 1.5 - 1.2 (m, 22H), 0.88 (t, J = 6.9 Hz, 3H). MALDI-TOF-MS (CHCA matrix, positive reflector mode): found m / z = (M+Na)+ 533.39. calculated: C 29 H 54 N 2 O 5 (Exact mass 510.40; molecular weight 510.74). 2,3,5,6-Tetrafluorophenyl N 6 -(tert-Butoxycarbonyl)-N 2 -oleoyl-L-lysinate

[0296] N 6 -(tert-Butoxycarbonyl)-N 2 -oleoyl-L-lysine (623 mg, 1.21 mmol, 1.00 equiv) and 2,3,5,6-tetrafluorophenol (302 mg, 1.82 mmol, 1.50 equiv) were dissolved in DCM (10 mL) and stirred under a nitrogen atmosphere. The clear solution was cooled in an ice / water bath and EDC.HCl (302 mg, 1.57 mmol, 1.3 equiv) was added. Chloroform (75 mL) was added after 18 h and the solution was washed with water (4 × 50 mL), saturated sodium bicarbonate solution (50 mL), followed by water (50 mL) and brine (50 mL). Purified by column chromatography on silica eluting with 9:1 chloroform:ethyl acetate. Yield: 470 mg of white solid (59%). 1H-NMR (400 MHz, chloroform-d) δ 7.02 (m, 1H), 6.41 - 5.98 (m, 1H), 5.34 (m, 2H), 4.93 (m, 1H), 4.61 (m, 1H), 3.15 (m, 2H), 2.26 (t, J = 7.6 Hz, 2H), 2.00 (m, 4H), 1.95 (m, 2H), 1.65 (m, 2H), 1.52 (m, 4H), 1.44 (s, 9H), 1.35 - 1.22 (m, 22H), 0.88 (t, J = 6.9 Hz, 3H). 19 F-NMR (chloroform-d) δ -138.6 (m, 2F), -152.4 (m, 2F). N 2 -(tert-Butoxycarbonyl)-N 6 -oleoyl-L-lysine

[0297] (tert-Butoxycarbonyl)-L-lysine (245 mg, 0.99 mmol) was stirred with dry DCM (6 mL) under a nitrogen atmosphere. 2,5-Dioxopyrrolidin-1-yl oleate (380 mg, 1.0 mmol) was added, followed by triethylamine (202 mg, 2.0 mmol). The turbid mixture cleared during overnight stirring. Chloroform (50 mL) was added, and the mixture was washed with 1 M KHSO 4 aqueous solution (20 mL), water (20 mL), brine (50 mL), and dried over magnesium sulfate. This was purified by column chromatography on silica using chloroform:methanol 20:1 as the eluent. Yield: 342 mg (67%) of an oil. 11H NMR (400 MHz, chloroform-d) δ 5.70 (t, 1H), 5.45 - 5.17 (m, 2H), 5.25 (m, 1H), 4.30 - 4.10 (m, two s, 1H), 3.25 (m, 2H), 2.17 (t, J = 9.7 Hz, 2H), 1.99 (m, 4H), 1.86 (m, 1H), 1.73 (m, 1H), 1.65 - 1.15 (m, 31H), 0.87 (t, J = 6.9 Hz, 3H). 2,3,5,6 - Tetrafluorophenyl N 2 -(tert - Butoxycarbonyl)-N 6 -oleoyl - L - lysinate

[0298] In DCM (15 mL), N 2 -(tert - Butoxycarbonyl)-N 6 To a solution of -(tert - butoxycarbonyl)-N 1 -oleoyl - L - lysine (349 mg, 0.67 mmol) and 2,3,5,6 - tetrafluorophenol (205 mg, 1.23 mmol, 1.85 equiv) in DCM (15 mL) under a nitrogen atmosphere, EDC·HCl (169 mg, 0.94 mmol, 1.41 equiv) was added. After 21 h, DCM (50 mL) was added and the organic phase was washed with saturated aqueous sodium bicarbonate (3 × 50 mL), followed by brine (50 mL). The solution was evaporated to dryness and the crude product was purified by column chromatography on silica using a gradient of chloroform:ethyl acetate from 10:1 to 20:7. Yield: 295 mg (67%) of a white powder. 1 1H NMR (400 MHz, CDCl 3 ) δ 7.02 (m, 1H), 5.49 (t, 1H), 5.40 - 5.25 (m, 2H), 5.17 (m, 1H), 4.62 and 4.44 (two s, 1H), 3.29 (m, 2H), 2.16 (t, J = 9.7 Hz, 2H), 2.00 (m, 4H), 1.89 (m, 2H), 1.65 - 1.15 (m, 31H), 0.88 (t, J = 6.9 Hz, 3H). Oleoyl - L - arginine

[0299] THF (32 mL) was added to a vigorously stirred solution of arginine (1.09 g, 6.26 mmol, 1.00 equiv) in water (32 mL). Sodium bicarbonate (896 mg, 10.6 mmol, 1.69 equiv) was added and after 15 minutes, a clear solution was obtained. A solution of 2,5-dioxopyrrolidin-1-yl oleate (2.64 g, 6.95 mmol, 1.11 equiv) in THF (32 mL) was added dropwise over 2 hours. Stirring was continued for 23 hours and a clear solution was obtained. THF was removed by rotary evaporation and the resulting aqueous phase was transferred to a separatory funnel. A solution of KHSO 4 in water (1.49 g in 260 mL) was added and shaken with chloroform (590 mL). The aqueous layer had a pH of 3. The chloroform phase was shaken with brine (300 mL). Chloroform was removed by rotary evaporation to give a crude mixture (3.05 g). This was purified by column chromatography on a Grace Reveleris X2 Flash Chromatography System (ELSD detection) using a Buchi 80 g HP column eluting with chloroform:methanol 1:1. The product was dissolved in THF and concentrated (twice: 120 mL and 80 mL of THF) to remove residual methanol (required before treatment with HCl). Yield: 2.0 g (confirmed by TLC, NMR, and MALDI-TOF that the product was pure). Preparation of the HCl salt: The product was dissolved by stirring with DCM (90 mL) and THF (20 mL) under an argon atmosphere and then cooled in an ice / water bath. A 2 M HCl solution in ether (4.5 mL) was added dropwise over 5 minutes to give a clear solution. The solvent was removed by rotary evaporation and the residue was dried under vacuum. Yield: 2.09 g of a white foam. 11H NMR (400 MHz, chloroform-d + TFA) δ 7.13 (d, J = 7.5 Hz, 1H), 6.54 (s, 1H), 6.13 (s, 4H), 5.42 - 5.29 (m, 2H), 4.69 (m, 1H), 3.31 (m, 2H), 2.1 - 1.55 (m, 12H), 1.48 - 1.08 (m, 20H), 0.87 (t, J = 6.9Hz, 3H). MALDI-TOF-MS (CHCA matrix, positive reflector mode): Observed m / z = (M+H)+ 439.36. Calculated: C 24 H 46 N 4 O 3 (Exact mass: 438.36; molecular weight of HCl salt: 475.10). A. Functionalization with acrylate ester (Michael addition reaction) General Example A1. G2-ACR-nC8-16

[0300] This general example describes obtaining a fully functionalized PPI dendrimer with 16 octyl ester groups by the Michael addition reaction of the G2-PPI-(NH 2 ) 8 dendrimer with n-octyl acrylate.

[0301] The G2 PPI dendrimer (n-butylene core, 0.20 g, 0.258 mmol, 2.06 mmol of primary amine groups) was dissolved in isopropanol (1 mL). An excess of n-octyl acrylate (1.73 mL, 1.53 g, 8.27 mmol, 32 molar equivalents) was added and the reaction mixture was stirred in a sealed vial at 55 °C under an inert nitrogen atmosphere. The progress of the reaction was monitored by 1It was monitored by 1H-NMR. The mixture was stirred for 4 days, after which the conversion was complete. The mixture was evaporated to dryness under vacuum, and the residue was stirred in MeCN (15 mL) at 4 °C. After 3 - 4 hours, the supernatant was decanted (or pipetted) to remove the excess acrylate. Washing with MeCN was repeated 2 more times at 4 °C and finally once at -20 °C (for long-chain acrylates, this may cause precipitation of the acrylate). The product was dried under vacuum to give a slightly yellowish oil. Yield: 515 mg (54%). 1 The 1H-NMR spectrum was consistent with the desired structure.

[0302] [Chemical formula] 1 1H-NMR (400 MHz, chloroform-d) δ 4.04 (t, J = 6.8 Hz, 32H, K), 2.77 (t, J = 7.3 Hz, 32H, I), 2.54 - 2.27 (m, 84H, B, C, E, F, H, J), 1.58 (dt, J = 22.2, 6.9 Hz, 60H, A, D, G, L), 1.30 (dd, J = 14.0, 7.7 Hz, 160H, M), 1.08 - 0.64 (m, 48H, N). MALDI-TOF-MS (CHCA matrix, positive reflector mode): found m / z = (M+H) + 3722.19. Calculated: C 216 H 416 N 14 O 32 (Exact mass 3719.14; molecular weight 3721.77). Example A2. G1-ACR-nC8-8

[0303] G1-PPI-(NH 2 ) 4 The reaction between G1-PPI-(NH) (n-butylene core) and n-octyl acrylate was carried out in a similar manner as in Example A1. Yield: 758 mg (67%). 1The 1H-NMR spectrum was consistent with the desired structure.

[0304]

Chem.

[0305] G1-PPI-(NH 2 ) 4 The reaction between G1-PPI-(NH) (n-butylene core) and n-tetradecyl acrylate was carried out in a similar manner to that in Example A1. However, HFIP was used as the solvent. Post-treatment: After evaporation of HFIP under vacuum at 50 °C, the reaction mixture was co-evaporated with toluene three times at 50 °C. The residue was purified by treatment with MeCN in a similar manner to that described in Example 1. Yield: 615 mg (75%). 1 The 1H-NMR was consistent with the desired structure.

[0306]

Chem.

[0307] G1-PPI-(NH 2 ) 4 (The reaction between (n-butylene core) and n-butyl acrylate was carried out in the same manner as in Example A1. Work-up: The excess acrylate was removed by evaporation under vacuum. The acrylate can also be removed by stirring the reaction mixture with an amine-functionalized silica scavenger (the excess butyl acrylate is immobilized by reaction with the silica-bonded amine groups). Yield: 450 mg (53%). 1 1H-NMR was consistent with the desired structure.

[0308]

Chemical Structure

[0309] G2-PPI-(NH 2 ) 8 (The reaction between (n-butylene core) and n-tetradecyl acrylate was carried out in the same manner as in Example A3. Yield: 580 mg (60%). 1 1H-NMR was consistent with the desired structure.

[0310]

Chemical Structure

[0311] The reaction between G1-PPI(DAB-Am-4) and isodecyl acrylate was carried out in the same manner as in Example A1. Yield: 0.85 g (67%). 1 The 1H-NMR spectrum was consistent with the desired structure.

[0312]

Chemical formula

[0313] N,N,N’,N’-Tetra(5-aminopentyl)-1,4-diaminobutane (G1-PPeI, see above, 200 mg, 0.47 mmol, 1 equivalent) and n-octyl acrylate (1.6 mL, 1.4 g, 7.6 mmol, 16 equivalents) were dissolved in propan-2-ol (IPA, 1 mL). The mixture was heated at 60 °C for 112 h and the reaction was 1 monitored using 1 H-NMR. The mixture was cooled and concentrated under vacuum. The viscous oil was stirred in MeCN, cooled to -20 °C, and the oil was precipitated to the bottom of the flask. The supernatant was decanted and the procedure was repeated three times to obtain the oily G1(PPeI)-ACR-C8-8 product.

[0314] [Chemical formula] 1 H NMR (400 MHz, chloroform-d) δ 4.05 (t, J = 6.8 Hz, 16H), 2.76 (t, J = 7.3 Hz, 16H), 2.41 (q, J = 8.6, 8.1 Hz, 36H), 1.61 (p, J = 6.9 Hz, 16H), 1.43 (p, J = 7.5 Hz, 20H), 1.37 - 1.15 (m, 88H), 0.92 - 0.84 (t, J = 6.8 Hz, 24H). MALDI-TOF-MS (CHCA matrix, positive reflector mode): found m / z = (M+H) + 1903.63, (M+Na) + 1925.60 calculated: C 112 H 216 N 6 O 16 (accurate mass 1901.63; molecular weight 1902.99). Example A8. G2(PAMAM-C2)-ACR-C8-16

[0315] To a reaction tube having a stirred solution of G2-PAMAM-C2 (ethylenediamine core, 8 amine end groups, 200 mg, 0.14 mmol) in propan-2-ol (IPA, 1 mL), n-octyl acrylate (825 mg, 4.48 mmol, 32 equivalents) was added. The reaction mixture was heated at 60 °C for 304 h. The reaction mixture was concentrated under vacuum. The residue was stirred in MeCN and cooled to -20 °C to precipitate the product at the bottom of the flask. The supernatant was decanted and the procedure was repeated 4 times to give a yellow viscous oil (301 mg, 68.8 μmol, 49%).

[0316] [Chemical formula] 1 H NMR (400 MHz, chloroform-d) δ 7.72 (s, 12H), 4.04 (t, J = 6.8 Hz, 32H), 3.27 (q, J = 5.8 Hz, 24H), 2.79 (dt, J = 13.8, 6.8 Hz, 56H), 2.57 (dt, J = 12.1, 5.9 Hz, 28H), 2.42 (t, J = 6.9 Hz, 32H), 2.35 (d, J = 6.7 Hz, 24H), 1.62 (p, J = 6.8 Hz, 32H), 1.41 - 1.18 (m, 160H), 0.95 - 0.82 (m, 48H). MALDI-TOF-MS (DCBT matrix, positive reflector mode): found m / z = (M+H) + 4378.32 calculated: C 238 H 448 N 26 O 44 (accurate mass 4375.36; molecular weight 4378.34). Example A9. Bis(3-aminopropyl)amine-ACR-C8-5

[0317] n-Octyl acrylate (1.84 g, 10 mmol, 10 eq) was added to a stirred solution of bis(3-aminopropyl)amine (131 mg, 1 mmol) in isopropanol (1 mL). The reaction mixture was heated at 60 °C for 136 h and then concentrated under vacuum. The residue was stirred in MeCN and cooled to -20 °C, and the product was phase-separated from the MeCN layer. The supernatant was carefully removed using a pipette, and the procedure was repeated 5 times. The residue was dried under vacuum to give a clear colorless oil (769 mg, 0.73 mmol, 73%).

[0318]

Chemical Structure

[0319] n-Octyl acrylate (1.84 g, 10 mmol, 10 eq) and bis(2-aminoethyl)amine (103 mg, 1 mmol) were reacted in isopropanol in a similar manner as described for Example A9. Yield: 674 mg (0.66 mmol, 66%).

[0320]

Chemical formula

[0321] n-Octyl acrylate (1.47 g, 8 mmol, 8 eq) and bis-N,N-(3-aminopropyl)methylamine (145 mg, 1 mmol) were reacted in isopropanol in a similar manner as described for Example A9. Yield: 552 mg (0.63 mmol, 63%).

[0322] [Chemical Formula] 1 H NMR (400 MHz, chloroform-d) δ 4.05 (t, J = 6.8 Hz, 8H), 2.77 (t, J = 7.3 Hz, 8H), 2.43 (td, J = 7.2, 2.8 Hz, 12H), 2.29 (t, J = 7.3 Hz, 4H), 2.18 (s, 3H), 1.61 (q, 6.7 Hz, 12H), 1.30 (m, 40H), 0.99 - 0.77 (m, 12H). 13 C NMR (101 MHz, CDCl 3 ) δ 172.7, 64.6, 55.8, 51.9, 49.3, 42.1, 32.7, 31.8, 29.3, 29.2, 28.7, 25.9, 25.1, 22.7, 14.1. MALDI-TOF-MS (CHCA matrix, positive reflector mode): found m / z = (M+H) + 882.71, (M+Na) + 904.72. Calculated: C 51 H 99 N 3 O 8 (Exact mass 881.74; molecular weight 882.37). Example A12. Bis(3-aminopropyl)amine-ACR-(2-ethylhexyl)-5

[0323] 2-Ethylhexyl acrylate (1.4 g, 7.5 mmol, 10 equiv) was added to a stirred solution of bis(3-aminopropyl)amine (100 mg, 0.75 mmol) in isopropanol (2 mL). The components were reacted in a similar manner as described for Example A9. Yield after workup: 278 mg (0.26 mmol, 35%).

[0324] [Chemical formula] 1 H NMR (399 MHz, chloroform-d) δ 4.19 - 3.78 (m, 10H), 2.77 (t, J = 7.3 Hz, 10H), 2.56 - 2.25 (m, 17H), 2.01 (s, 3H), 1.67 (s, 1H), 1.64 - 1.48 (m, 9H), 1.48 - 1.11 (m, 40H), 0.89 (td, J = 7.1, 2.8 Hz, 30H). MALDI-TOF-MS (CHCA matrix, positive reflector mode): found m / z = (M+H) + 1052.88, (M+Na) + 1074.86. Calculated: C 59 H 113 N 3 O 10 (Exact mass 1051.87; molecular weight 1052.62). Example A13. G1-ACR-(2-ethylhexyl)-8

[0325] The reaction between G1-PPI(DAB-Am-4) and 2-ethylhexyl acrylate was carried out in a similar manner as in Example A1. Yield: 0.71 g (0.4 mmol, 73%). 1 The H-NMR spectrum was consistent with the desired structure.

[0326] [Chemical formula] 11H NMR (399 MHz, chloroform-d) δ 4.36 - 3.57 (m, 16H, H), 2.78 (t, J = 7.4 Hz, 16H, F), 2.41 (dt, J = 25.0, 7.5 Hz, 35H, G, C, E, B), 1.69 - 1.48 (m, 16H), 1.48 - 1.11 (m, 68H), 0.89 (td, J = 7.1, 2.8 Hz, 48H, M,O). MALDI-TOF-MS (CHCA matrix, positive reflector mode): Measured m / z = (M+H) + 1791.55, (M+Na) + 1813.53. Calculated: C 120 H 232 N 6 O 16 (Exact mass 1789.50; molecular weight 1790.77). Example A14. G1(PBuI)-ACR-C8-8

[0327] N,N,N’,N’-Tetra(4-aminobutyl)-1,4-diaminobutane (G1-PBuI) and n-octyl acrylate were dissolved in propan-2-ol (IPA, 1 mL). The reaction and work-up were carried out as described for Example A7 to give an oily G1(PBuI)-ACR-C8-8 product.

[0328]

Chemical Structure

[0329] [Chemical formula]

[0330] To a reaction tube having a stirred solution of G1-PAMAM-C2 (ethylenediamine core, 4 amine end groups, 165 mg, 0.32 mmol) in propan-2-ol (IPA, 2 mL), 2-ethylhexyl acrylate (589 mg, 3.2 mmol, 10 equivalents) was added. Further 2-ethylhexyl acrylate was added later (600 mg, 3.3 mmol, 10 equivalents). The reaction mixture was heated at 60 °C for 408 h. The reaction mixture was concentrated under vacuum. The residue was further evaporated with an oil pump to remove the excess 2-ethylhexyl acrylate (at 90 °C and 0 mbar). The crude product still contained a small amount of acrylate, which was removed using a short silica column (starting from 2% MeOH / CHCl 3 to 10% MeOH / CHCl 3 ) to give a yellow viscous oil (270 mg, 0.13 mol, 41%). 11H NMR (400 MHz, chloroform-d) δ 7.20 (t, J = 5.5 Hz, 3H), 4.19 - 3.78 (m, 16H, I), 3.27 (q, J = 6.0 Hz, 8H, B), 2.78 (dt, J = 15.0, 6.9 Hz, 22H, F, G), 2.57 (t, J = 6.4 Hz, 9H, C), 2.52 (s, 3H, A), 2.44 (t, J = 7.1 Hz, 15H, H), 2.34 (t, J = 6.4 Hz, 7H, E), 1.63 - 1.47 (m, 8H, J), 1.44 - 1.12 (m, 64H, K, L, M, O), 0.89 (td, J = 7.5, 7.0, 3.0 Hz, 48H, N, P). MALDI-TOF-MS (CHCA matrix, positive reflector mode): found m / z = (M+H) + 1991.56, (M+Na) + 2013.53. Calculated: C 110 H 208 N 10 O 20 (Exact mass 1989.56; molecular weight 1990.92). B. Selective functionalization of primary amines with acrylate esters (Michael addition reaction), followed by acylation of the remaining secondary amines.

[0331] The basis of the following materials was found by the observation that the reaction of acrylate with primary amine dendrimers proceeds much more rapidly than that with the formed secondary amine dendrimers. Therefore, this selectivity can be used to cap the formed secondary amine intermediates by acylation. General Example B1. G1-ACR-Citro-4 / Ac-4

[0332] This general example is for G1-PPI-(NH 2 ) 4The Michael addition reaction of a dendrimer, followed by a reaction with acetic anhydride, to obtain a G1 PPI-dendrimer having four (S)-citronellyl ester groups and four acylamide groups is described.

[0333] G1 PPI dendrimer (n-butylene core, 0.20 g, 0.631 mmol) was dissolved in isopropanol (1 mL), and (S)-3,7-dimethyloct-6-en-1-yl acrylate (0.513 g, 2.44 mmol) was added. The mixture was stirred in a sealed vial at 55 °C overnight under an inert nitrogen atmosphere, thereby obtaining complete conversion of the acrylate. 1 According to 1H-NMR, the acrylate reacted more rapidly with the primary amines, and thus, almost exclusive monofunctionalization of each of the primary amines was obtained. The mixture was evaporated to dryness, and the residue was dissolved in dichloromethane (DCM, 1 mL). Subsequently, triethylamine (0.36 mL, 0.268 g, 2.65 mmol) was added, followed by acetic anhydride (0.271 g, 2.65 mmol). The mixture was stirred in a sealed vial at 20 °C for 4 h under a nitrogen atmosphere, which was then diluted with DCM and subsequently washed with NaHCO 3 (aqueous solution) and brine. The organic layer was dried over Na 2 SO 4 filtered, and the filtrate was evaporated to dryness. Co-evaporation with DCM was performed to remove traces of acetic anhydride and acetic acid. Yield: 623 mg (74%). 1 The 1H-NMR spectrum was consistent with the desired structure.

[0334]

Chemical formula

[0335] G1-PPI-(NH 2 ) 4 (n-butylene core) and the reaction between n-tetradecyl acrylate and acetic anhydride were carried out in a similar manner as in Example B1. Yield: 689 mg (70%). 1 The 1H-NMR spectrum was consistent with the desired structure. MALDI-TOF-MS (CHCA matrix, positive reflector mode): Measured m / z = (M+H) + 1558.34, (M+Na) + 1580.33 (main peak). Calculated: C 92 H 176 N 6 O 12(Precision mass 1557.33; molecular weight 1558.45). A further small peak was observed at m / z = 1786.33 (five tetradecyl tails and three acetyl groups). Example B3. G2-ACR-citro-8 / Ac-8

[0336] G2-PPI-(NH 2 ) 8 (n-butylene core) and the reaction between (S)-3,7-dimethyloct-6-en-1-yl acrylate and acetic anhydride were carried out in a similar manner as in Example B1. Yield: 520 mg (72%). 1 The 1H-NMR spectrum was consistent with the desired structure. MALDI-TOF-MS (CHCA matrix, positive reflector mode): Measured value m / z = (M+H) + 2792.18, (M+Na) + 2814.16. Calculated for C 160 H 288 N 14 O 24 (Precision mass 2790.17; molecular weight 2792.14). Example B4. G2-ACR-nC14-8 / Ac-8

[0337] G2-PPI-(NH 2 ) 8 (n-butylene core) and the reaction between n-tetradecyl acrylate and acetic anhydride were carried out in a similar manner as in Example B1. Yield: 631 mg (75%). 1 The 1H-NMR spectrum was consistent with the desired structure.

[0338]

Chemical Structure

[0339] This general example describes obtaining a PPI dendrimer with 16 n-tetradecyl ester groups and 16 methyl ester groups by Michael addition reaction of the G3-PPI-(NH 2 ) 16 dendrimer with n-tetradecyl acrylate followed by reaction with methyl acrylate.

[0340] The G3 PPI dendrimer (0.20 g, 0.119 mmol) was dissolved in isopropanol (1 mL), and tetradecyl acrylate (0.513 g, 1.91 mmol, 16 molar equivalents) was added. The mixture was stirred in a sealed vial at 55 °C for 5 h under an inert nitrogen atmosphere, resulting in complete conversion of the primary amine (a trace amount of acrylate still remained). 1 According to 1H-NMR, acrylate reacted more rapidly with the primary amine, thus providing almost exclusive monofunctionalization of each of the primary amine groups. Subsequently, methyl acrylate was added (0.27 mL, 0.244 g, 2.83 mmol, 24 molar equivalents), and the mixture was stirred at 55 °C. The progress of the reaction was 1 monitored by 1H-NMR. After 1 day, an excess amount of methyl acrylate (0.14 mL, 0.122 g, 1.42 mmol, 12 equivalents) was added. After stirring at 55 °C for a total of 4 days, the reaction was complete and the mixture was evaporated to dryness. The crude product was stirred in MeCN and left standing at 0 °C for several hours to induce complete phase separation. Subsequently, the supernatant was decanted. This procedure was repeated three times to remove any unreacted acrylate monomer. Yield: 575 mg (66%). 1 The 1H-NMR spectrum was consistent with the desired structure.

[0341]

Chemical formula

[0342] G1-PPI-(NH 2 ) 4 (n-butylene core) and the reaction between n-tetradecyl acrylate and methyl acrylate was carried out in a similar manner to Example C1. Yield: 464 mg (84%). 1 The 1H-NMR spectrum was consistent with the desired structure.

[0343]

Chemical formula

[0344] The G1 PPI dendrimer (0.513 g, 1.62 mmol) was dissolved in DCM (20 mL), and the solution was cooled in an ice-salt bath at -10 °C. Boc anhydride (1.48 g, 6.79 mmol) was added. A precipitate formed almost immediately. The mixture was stirred at room temperature for 4 hours, after which the reaction was complete. The mixture was filtered, and the filtrate was stirred with amine-functionalized silica scavenger overnight to remove the excess Boc anhydride. After filtration, the filtrate was evaporated to dryness to obtain the product. Yield: 1.16 grams (81%). 1 The 1H-NMR spectrum was consistent with the desired structure.

[0345]

Chemical Structure

[0346] The Boc-protected G1 PPI dendrimer G1-Boc-4 (0.75 g, 1.04 mmol) was dissolved in DCM (3 mL), and methyl iodide (1.78 g, 0.78 mL, 12.55 mmol) was added. The mixture was stirred at 45 °C overnight and then evaporated to dryness to obtain a foam, which was dissolved in CHCl 3 and precipitated into pentane. The supernatant was decanted, and the residue was stirred with pentane again and decanted. The product contains iodide as the counter anion, which was exchanged with chloride using an ion exchange column (Lewatit monoplus M600 resin, 1.3 meq / mL) with methanol as the eluent. Approximately 5 mg of the product was dissolved in demi water (1 mL), and 35% H 2O 2 (An aqueous solution) was examined for the presence of iodide by adding a few drops. Iodine I 2 The yellow indicating the formation was not observed. To this mixture, a fresh starch solution (1 mL) was added, but no blue discoloration was observed either. Yield: 715 mg (84%). 1 The 1H-NMR spectrum was consistent with the desired structure.

[0347]

Chemical formula

[0348] About 800 mg of G1-Boc-4(2 MeCl) was mixed with 4 M HCl in dioxane (about 5 mL), thereby obtaining a precipitate. 3 M HCl in methanol was added (about 5 mL) to increase solubility. The reaction mixture was stirred overnight at room temperature. 1 1H-NMR showed complete deprotection. The mixture was evaporated to dryness and the residue was co-evaporated with isopropanol to obtain a hygroscopic solid (the compound had poor solubility in isopropanol). Yield: 500 mg (122%), due to the inclusion of HCl and / or water. 1 The 1H-NMR spectrum was consistent with the desired structure.

[0349]

Chemical formula

[0350] G1-NH obtained from Example D3 2 (2 MeCl) (300 mg, 1.77×10 -5 mol) was dissolved in HFIP (2 mL), and n-tetradecyl acrylate (2.04 mL, 2 equivalents per primary amine) was added. The reaction mixture was a clear solution. The mixture was stirred at 55 °C for 1 hour, but no reaction occurred. Triethylamine (1.6 mL) was added as a base, and the mixture was stirred for 24 hours. The reaction was 1 completed as determined by 1H-NMR. The reaction mixture was evaporated to dryness. The residue was stirred in MeCN, allowed to stand at 0 °C, and the supernatant was decanted. This procedure was carried out 3 times. The residue was then stirred in 1 / 1 EtOAc / hexane. The precipitate was filtered, which still contained 1% of tetradecyl acrylate, and this was removed by stirring and filtration in EtOAc. The residue was then allowed to stand on an ion exchange resin to exchange the residual iodide with chloride (eluent 1 / 1 CHCl 3 / MeOH). Yield: 468 mg (102%). 1 The 1H-NMR spectrum was consistent with the desired structure.

[0351] [Chemical Structure Diagram] 11H NMR (399 MHz, chloroform-d) δ 4.02 (t, J = 6.8 Hz, 16H, H), 3.84 (d, J = 7.5 Hz, 4H, B), 3.38 (dd, J = 10.4, 6.2 Hz, 8H, C), 3.19 (s, 6H, L), 2.71 (td, J = 6.7, 2.5 Hz, 16H, F), 2.57 (t, J = 6.0 Hz, 8H, E), 2.40 (t, J = 6.6 Hz, 16H, G), 2.19 (d, J = 7.7 Hz, 4H, A), 2.02 (dt, J = 13.2, 6.4 Hz, 8H, D), 1.62 (q, J = 6.9 Hz, 16H, I), 1.52 - 1.02 (m, 176H, J), 0.88 (t, J = 6.7 Hz, 24H, K). Example D5. G2-Boc-8

[0352] The reaction between G2 PPI dendrimer (DAB-Am-8) and Boc anhydride was carried out in a similar manner as shown in Example D1. Yield: 796 mg (99%). 1 The 1H-NMR spectrum was consistent with the desired structure.

[0353] [Chemical formula] 1 1H NMR (399 MHz, methanol-d 4 ) δ 7.90 (s, 4H, I), 3.07 (t, J = 6.8 Hz, 16H, H), 2.46 (p, J = 8.5, 7.6 Hz, 35H, B, E, F), 1.63 (p, J = 6.8 Hz, 24H, D, G), 1.43 (s, 77H, A,J). Example D6. G2-Boc-8(6 MeCl)

[0354] The reaction between G2-Boc-8 and methyl iodide, followed by anion exchange, was carried out in a similar manner as shown in Example D2. Yield: 699 mg (73%).1 The 1H-NMR spectrum was consistent with the desired structure.

[0355]

Chemical formula

[0356] The reaction between G2-Boc-8(6 MeCl) and HCl was carried out in the same manner as shown in Example D3. Yield: 478 mg (93%). 1 The 1H-NMR spectrum was consistent with the desired structure. This product can be used to react with, for example, acrylic esters, or acrylamides, or epoxides.

[0357]

Chemical formula

[0358] This general example describes the Michael addition reaction of N-(2-hydroxyethyl)-N-methylacrylamide to G1-PPI-(NH 2 ) 4 to obtain a fully functionalized PPI dendrimer with eight N-(2-hydroxyethyl)-N-methylamide groups.

[0359] N-(2-hydroxyethyl)-N-methylacrylamide was prepared according to Moszner, Macromol. Chem. Phys 2007, 208, 529-540 DOI:10.1002 / MACP.200600513. G1 PPI dendrimer (0.10 g, 0.315 mmol) was dissolved in isopropanol (1 mL), and an excess of N-(2-hydroxyethyl)-N-methylacrylamide (0.515 g, 3.98 mmol, 3 molar equivalents per primary amine) was added. The reaction mixture was stirred in a sealed vial at 55 °C for 2 days under a nitrogen atmosphere, and the conversion was 1 monitored by 1H-NMR. The mixture was evaporated to dryness, the residue was stirred in MeCN, and the mixture was then allowed to stand at 4 °C for several hours. The supernatant was decanted to remove the excess acrylamide reactant. This washing step with MeCN was repeated twice at 4 °C and once at -20 °C. The product was dried under vacuum to obtain a slightly yellowish oil. Yield: 367 mg (86%). 1 The 1H-NMR spectrum was consistent with the desired structure.

[0360]

Chemical formula

[0361]

Chem.

[0362] HFIP (252 mg, 1.5 mmol, 3 eq) was added to a stirred solution of N,N-bis(3-aminopropyl)methylamine (65.6 mg, 0.5 mmol, 1 eq) and tert-butyl (3-(N-methylacrylamide)propyl)carbamate (1.21 g, 5 mmol, 10 eq) in propan-2-ol. The reaction mixture was heated at 60 °C for 64 h. The mixture was then cooled and concentrated under vacuum. The crude viscous oil was purified using silica column chromatography eluting with a mixture of MeOH / CHCl 3 (0 - 20%) to give E2 as a viscous oil. HPLC-ESI-MS: m / z C 55 H 107 N 11 O 12 Calculated value for 1113.81. Measured value [M+H]+ 1114.83, [M+H] 2+ 558.08, [M+H] 2+ -Boc 508.00 [M+H] 2+ -2xBoc 458.00, [M+H] 2+ -3xBoc 408.00, [M+H] 2+ -4xBoc 357.92. Example E3

[0363] Di-n-propylenetriamine (65.6 mg, 0.5 mmol, 1 equiv) and tert-butyl (3-(N-methylacrylamide)propyl)carbamate (1.21 g, 5 mmol, 10 equiv) were dissolved in propan-2-ol and HFIP (1 mL). The reaction mixture was heated at 60 °C for 480 h. The reaction was not complete, but after 480 h the reaction mixture was cooled and concentrated under vacuum. The crude viscous oil was purified using silica column chromatography eluting with a mixture of MeOH / CHCl 3 (0 - 20%) to give E3 as a viscous oil. ESI-MS: m / z C 66 H 127 N 13 O 15 Calculated for 1341.96. Measured [M+H] + 1343.00, [M+Na] + 1364.92, [M+H] 2+ 672.08, [M+H] 2+ -Boc 622.08 [M+H] 2+ -2xBoc 572.08, [M+H] 2+ -3xBoc 522.08, [M+H] 2+ -4xBoc 472.00, [M+H] 2+ -5xBoc 472.00. Example E4

[0364] Diethylenetriamine (103 mg, 1 mmol, 1 equiv) and tert-butyl (3-(N-methylacrylamide)propyl)carbamate (2.42 g, 10 mmol, 10 equiv) were dissolved in propan-2-ol and heated to reflux. After 305 h at reflux, the reaction was still not complete, but the reaction was stopped at that point. The reaction mixture was concentrated under vacuum to give an amber viscous oil. The crude product E4 was purified using silica column chromatography with a mixture of MeOH / CHCl3 (0 - 20%). Example E5

[0365] In a 5 ml flask, 1,4-diaminobutane (88 mg, 1 mmol, 1 equiv) and tert-butyl (3-(N-methylacrylamide)propyl)carbamate (1.21 g, 5 mmol, 5 equiv) were dissolved in propan-2-ol (2 ml). The reaction mixture was heated to 55 °C. After 350 h, the reaction was almost complete. The reaction mixture was concentrated under vacuum. The crude product was purified using silica column chromatography eluting with a mixture of CHCl 3 / MeOH (0 - 20%) to give E5 as an amber viscous oil (756 mg, 0.72 mmol, 72%). 1 H NMR (400 MHz, chloroform-d) δ 5.81 - 5.20 (m, 4H), 3.43 (t, J = 6.4 Hz, 6H), 3.36 (t, J = 7.8 Hz, 2H), 3.16 (q, J = 6.4 Hz, 2H), 3.06 (q, J = 6.3 Hz, 6H), 3.02 (s, 9H), 2.91 (s, 3H), 2.81 (t, J = 7.3 Hz, 8H), 2.48 (m, 12H), 1.80 (p, J = 6.9 Hz, 2H), 1.66 (p, J = 6.4 Hz, 6H), 1.43 (m, 40H). 13 C NMR (101 MHz, CDCl 3) δ 172.4, 171.6, 156.2, 156.1, 79.0, 78.8, 77.5, 77.4, 77.2, 76.8, 54.1, 49.5, 49.4, 47.6, 44.6, 44.5, 38.0, 36.9, 35.4, 35.3, 33.3, 30.9, 30.4, 29.1, 28.4, 28.4, 27.2, 24.8. ESI-MS: m / z C 52 H 100 N 10 O 12 Calculated value of 1056.75. Measured value [M+H] + 1057.92, [M+Na] + 1079.92, [M+H] 2+ 529.58, [M+H] 2+ -Boc 479.50 [M+H] 2+ -2 x Boc 429.50, [M+H] 2+ -3 x Boc 379.50. F. Functionalization of primary amines with acrylate esters, followed by functionalization of the remaining secondary amines with acrylamide (Michael addition). The amide may be reacted first and the ester second. General Example F1: G1-ACR-nC14-4 / AmAlc-4

[0366] This general example describes the Michael addition reaction of G1-PPI-(NH 2 ) 4 to obtain a PPI dendrimer having four n-tetradecyl esters and four N-(2-hydroxyethyl)-N-methylamide groups by Michael addition reaction of the dendrimer.

[0367] PPI dendrimer G1 (0.10 g, 0.315 mmol) was dissolved in isopropanol (1 mL), and tetradecyl acrylate (0.339 g, 4 molar equivalents per dendrimer) was added. The mixture was stirred at an oil bath temperature of 55 °C for 24 h, thereby obtaining complete conversion of the primary amine. A slight excess of the acrylate reactant was still present. 1 According to 1H-NMR, the acrylate reacted selectively with the primary amine, and monofunctionalization of all the primary amine groups was obtained. Next, N-(2-hydroxyethyl)-N-methylacrylamide (0.204 g, 1.57 mmol, 5 molar equivalents) was added. The mixture was stirred at 55 °C for a total of 6 days. The unreacted acrylate or acrylamide was then removed by stirring the reaction mixture with an amine-functionalized silica scavenger. The mixture was filtered, and the silica scavenger was washed with chloroform / MeOH (5%). The filtrate was evaporated to dryness to obtain an oily product that no longer contained any acrylate or acrylamide. Yield: 344 mg (50%, this low yield may be due to partial attachment of the dendrimer to the silica scavenger). 1 The 1H-NMR spectrum was consistent with the desired structure.

[0368] [Chemical formula] 11H NMR (399 MHz, chloroform-d) δ 4.05 (t, J = 6.8 Hz, 8H, N), 3.72 (q, J = 5.8 Hz, 8H, J), 3.49 (dt, J = 17.9, 4.7 Hz, 8H, I), 3.12 - 2.94 (singlet, 12H, H), 2.86 (t, J = 3.2 Hz, 1H), 2.78 (q, J = 7.5 Hz, 16H, F, L), 2.68 - 2.26 (m, 36H, B, C, E, G, M), 1.60 (td, J = 13.7, 7.0 Hz, 16H, D, O), 1.26 (s, 92H, A, P), 0.88 (t, J = 6.8 Hz, 12H, K). MALDI - TOF - MS (CHCA matrix, positive reflector mode): Observed m / z = (M + H) + 1907.62, (M + Na) + 1929.6. Calculated: C 108 H 212 N 10 O 16 (Exact mass 1905.61; molecular weight 1906.94). A small peak was further observed at m / z = 2046.78 (five tetradecyl acetates and three acetylamide groups) G. Functionalization of amines with epoxides General Example G1: G1 - EPX - nC8 - 8

[0369] This general example describes obtaining a PPI dendrimer having 2 - hydroxy C10 groups by the reaction of G1 - PPI - (NH 2 ) 4 dendrimer with (1,2 - epoxy) - n - decane.

[0370] PPI dendrimer G1 (0.10 g, 0.315 mmol) was dissolved in isopropanol (1 mL), and 1,2-epoxydecane (0.43 g, 8.8 molar equivalents) was added. The reaction mixture was stirred in a sealed vial at 90 °C under an inert nitrogen atmosphere and then evaporated to dryness. The residue was stirred in MeCN, and the suspension was left to stand at -20 °C for several hours. The supernatant was decanted. This procedure was repeated twice, after which the residue no longer contained any residual 1,2-epoxydecane. The residue was dried under vacuum to obtain the product. Yield: 390 mg (80%). 1 The 1H-NMR spectrum was consistent with the desired structure.

[0371]

Chemical formula

[0372] G1-PPI-(NH 2 ) 4(n-Butylene core) and 1,2-epoxydodecane were reacted in the same manner as in Example G1. Yield: 447 mg (79%). 1 The 1H-NMR spectrum was consistent with the desired structure. MALDI-TOF-MS (CHCA matrix, positive reflector mode): Observed m / z = (M+H) + 1791.83, (M+Na) + 1813.82, (M+K) + 1829.79. Calculated for C 112 H 232 N 6 O 8 (Exact mass 1789.79; molecular weight 1791.12). Example G3. G2-EPX-nC10-16

[0373] G2-PPI-(NH 2 ) 8 (n-Butylene core) and 1,2-epoxydodecane were reacted in the same manner as in Example G1. Yield: 697 mg (84%). 1 The 1H-NMR spectrum was consistent with the desired structure. MALDI-TOF-MS (CHCA matrix, positive reflector mode): Observed m / z = (M+H) + 3722.78, (M+Na) + 3744.75, (M+K) + 3760.74. Calculated for C 232 H 480 N 14 O 16 (Exact mass 3719.72; molecular weight 3722.47). Example G4. G3-EPX-nC10-32

[0374] G3-PPI-(NH 2 ) 16 (n-Butylene core) and 1,2-epoxydodecane were reacted in the same manner as in Example G1. Yield: 413 mg (88%). 1 1H-NMR was consistent with the desired structure.

[0375]

Chemical Structure

[0376] G1-PPeI (100 mg, 0.23 mmol, 1 equiv) and 1,2-epoxydodecane (0.81 mL, 684 mg, 3.72 mmol, 16 equiv) were dissolved in propan-2-ol (IPA, 1 mL). The mixture was heated at 60 °C for 16 h ( 1 monitored by 1H-NMR). The mixture was cooled and concentrated under vacuum. The viscous oil was stirred in MeCN, cooled to -20 °C, and precipitated at the bottom of the flask. The supernatant was decanted and the procedure was repeated three times to obtain the product as a viscous oil (344 mg, 0.20 mol, 88%).

[0377]

Chemical Structure

[0378] 1,2-Epoxydecane (700 mg, 4.38 mmol, 32 equiv) was added to a stirred solution of G2-PAMAM-C2 (ethylenediamine core, eight amine end groups, 200 mg, 0.14 mmol) in propan-2-ol (1 mL). The reaction mixture was heated at 60 °C for 88 h and then concentrated under vacuum. The residue was stirred in MeCN and cooled to -20 °C, and the product was precipitated at the bottom of the flask. The supernatant was decanted, and this trituration procedure was repeated three times to give a yellow viscous oil (422 mg, 107 μmol, 77%).

[0379]

Chemical formula

[0380] 1,2-Epoxydecane (1.56 g, ~10 mmol, 10 equiv) was added to a stirred solution of bis(3-aminopropyl)amine (131 mg, 1 mmol) in IPA (1 mL). The reaction mixture was heated at 60 °C for 40 h and then concentrated under vacuum. The residue was stirred in MeCN and cooled to -20 °C, and the product was phase-separated from the MeCN solvent. The supernatant was carefully removed using a pipette, and the trituration procedure was repeated three times. The residue was dried under vacuum to afford a clear colorless oil (950 mg).

[0381] [Chemical formula] 1 H NMR (399 MHz, methanol-d 4 ) δ 7.89 (s, 1H), 3.63 (qd, J = 7.2, 4.5, 3.6 Hz, 5H), 2.74 - 2.26 (m, 17H), 1.64 (q, J = 7.3 Hz, 4H), 1.56 - 1.21 (m, 70H), 1.00 - 0.79 (m, 15H). MALDI-TOF-MS (ODCB matrix, positive reflector mode): found m / z = (M+H) + 912.87. calculated: C 56 H 117 N 3 O 5 (accurate mass 911.90; molecular weight 912.57). Example G8. Bis(2-aminoethyl)amine-EPX-C8-5

[0382] Bis(2-aminoethyl)amine (103 mg, 1 mmol) and 1,2-epoxydecane (1.56 g, ~10 mmol, 10 equiv) were reacted in IPA (1 mL). The same procedure was utilized as described for the materials of Example G7. Yield: 958 mg, 1.08 mmol.

[0383] [Chemical formula] 1 H NMR (399 MHz, methanol-d 4 ) δ 3.83 - 3.55 (m, 5H), 2.91 - 2.29 (m, 18H), 1.61 - 1.10 (m, 70H), 1.02 - 0.79 (m, 15H). MALDI-TOF-MS (DCTB matrix, positive reflector mode): found m / z = (M+H) + 884.84, [M+Na] + 906.82. Calculated: C 54 H 113 N 3 O 5 (Exact mass 883.87; molecular weight 884.51). Example G9. Bis(3-aminopropyl)methylamine-EPX-C8-4

[0384] 1,2-Epoxydecane (269 mg, 1.68 mmol, ca. 6 equiv) was added to a stirred solution of N,N-bis(3-aminopropyl)methylamine (42 mg, 0.29 mmol) in IPA (1 mL). The reaction mixture was heated at 60 °C for 16 h and then concentrated under vacuum. The residue was stirred in MeCN and cooled to -20 °C and the product was phase separated from the MeCN solvent. The supernatant was carefully removed using a pipette and the trituration procedure was repeated three times. The residue was dried under vacuum to give a clear colorless oil (170 mg, 0.22 mmol, 77%).

[0385]

Chemical Structure

[0386] This general example describes obtaining a PPI dendrimer with 4 n-tetradecyl ester groups and 4 methyl groups by Michael addition reaction of the G1-PPI-(NH 2 )4 dendrimer with n-tetradecyl acrylate, followed by reaction with paraformaldehyde.

[0387] G1 PPI dendrimer DAB-Am-4 (0.21 g, 0.671 mmol) was dissolved in isopropanol (1 mL), and tetradecyl acrylate (0.72 g, 2.68 mmol, 4 molar equivalents) was added. The mixture was stirred in a sealed vial at 55 °C for 24 h under an inert nitrogen atmosphere, thereby obtaining complete conversion of the primary amine. Almost exclusive monofunctionalization of each of the primary amine groups was observed. The reaction mixture was evaporated to remove isopropanol, and the residue was co-evaporated three times with chloroform. The residue was dissolved in formic acid (2 mL). Paraformaldehyde (121 mg, 4.0 mmol, 6 molar equivalents) was added, and THF (1 mL) was also added for solubility. The mixture was stirred at 60 °C. The progress of the reaction was 1 monitored by 1H-NMR. After 3.5 h, the reaction was complete, and the mixture was evaporated to dryness and co-evaporated twice with chloroform. The crude mixture was dissolved in chloroform and washed with 0.1 M NaOH (aqueous solution), followed by saturated NaCl (aqueous solution), and dried over Na 2 2 4 SO4. The crude product was stirred in MeCN and left to stand at 0 °C for several hours to induce complete phase separation. Subsequently, the supernatant was decanted. This procedure was repeated twice. The residue was applied to a silica column starting with 5% MeOH / chloroform to remove impurities. Finally, the product was eluted from the column by changing the eluent to 5% MeOH and 1% TEA / chloroform. Yield: 560 mg (58%). 1 The 1H-NMR spectrum was consistent with the desired structure.

[0388] [Chemical Structure] 11H NMR (399 MHz, chloroform-d) δ 4.06 (td, J = 6.8, 1.3 Hz, 8H, F), 2.69 (t, J = 7.3 Hz, 8H, K), 2.62 - 2.29 (m, 25H, B, C, E, L), 2.23 (d, J = 1.3 Hz, 12H, J), 1.81 - 1.55 (m, 14H, D, G), 1.55 - 1.01 (m, 90H, H), 0.99 - 0.73 (m, 12H, I). MALDI-TOF-MS (CHCA matrix, positive reflector mode): Observed m / z = (M+H) + 1446.37, small peak m / z = M+H) + , 1701.59 (5 C14 chains and 3 CH 3 ). Calculated: C 88 H 176 N 6 O 8 (Exact mass 1445.35; molecular weight 1446.41). Amidation of J. amine, optionally followed by alkylation of the internal tertiary amine therein.

[0389]

Chemical Structure

[0390] N,N,N,N-((Butane-1,4-diylbis(azanetriyl))tetrakis(propane-3,1-diyl))tetrapalmitamide. N,N’-(Butane-1,4-diyl)bis(N1-(3-aminopropyl)propane-1,3-diamine) (PPI-G1, 1 g) was co-evaporated twice with a sufficient amount of toluene. It was then dissolved in toluene (4 mL), and this was added to phenyl palmitate (6.3 g) in toluene (16 mL). Triethylamine (9 mL) was added, and the mixture was stirred at 90 °C for 6 h. The reaction mixture was concentrated under vacuum, and the resulting waxy solid was triturated in acetonitrile (80 mL) for 3 h. After filtration and washing with acetonitrile, the residue was dried and triturated in hexane. After filtration, washing with hexane, and drying the residue, 3.3 g (82%) of the pure product was obtained as an off-white solid. 1 The 1H-NMR spectrum was consistent with the desired structure.

[0391] [Chemical formula] 1 1H NMR (399 MHz, chloroform-d) δ 6.48 (t, J = 5.7 Hz, 3H, J), 3.29 (q, J = 6.5 Hz, 8H, E), 2.52 - 2.26 (m, 12H, B, C), 2.16 (t, J = 7.7 Hz, 8H, F), 1.61 (dd, J = 15.4, 8.6 Hz, 22H, A, D, G), 1.25 (s, 95H, H), 0.88 (t, J = 6.7 Hz, 12H, I). MALDI-TOF-MS (CHCA matrix, positive reflector mode): found m / z = (M+H) + 1270.31. Minor peak m / z = M+Na + , 1292.23. Calculated: C 80 H 160 N 6 O 4 (Exact mass 1269.25; molecular weight 1270.20). Example J2. G1 - amide - nC16 - 4(2MeI)

[0392] N,N,N,N - ((butane - 1,4 - diylbis(azanetriyl))tetrakis(propane - 3,1 - diyl))tetrapalmitamide (2MeI ammonium adduct). Example J1 (4 g) was stirred at 45 °C in a 1:1 chloroform / methanol mixture (80 mL). Methyl iodide (3.14 mL) was added to this turbid mixture. The temperature of the oil bath was raised to 50 °C and a clear mixture was obtained by adding 25 mL of chloroform, which was stirred for 16 h. After adding an excess amount of methyl iodide (1 mL), stirring was continued for 4 h. The mixture was then concentrated under vacuum and co - evaporated twice with methanol and once with chloroform. To obtain complete methylation, the product was redissolved in chloroform / methanol with 10 equivalents of methyl iodide and stirred in a sealed vial at 90 °C for 1 h under microwave irradiation. The mixture was concentrated under vacuum and co - evaporated with chloroform to obtain a off - white product quantitatively. 1 The 1H - NMR spectrum was consistent with the desired structure.

[0393]

Chem.

[0394] N,N,N,N-((butane-1,4-diylbis(azanetriyl))tetrakis(propane-3,1-diyl))tetrapalmitamide (2 MeCl ammonium adduct). Example J2 (3 g) was dissolved in 150 mL of a chloroform / methanol mixture (2:1) to obtain a clear solution. An ion exchange resin (50 grams) Amberlite IRA 400 (Cl - ) was stirred in water and poured into a column with a diameter of 1 cm. Water was passed through the column, and then methanol was passed through. The amount of chloroform in the eluate was slowly increased to 66%. A plug of packing was placed on top of the resin to avoid floating of the resin beads. The solution of J2 was eluted onto the resin with a chloroform / methanol mixture (2:1). All the collected eluate was concentrated to obtain the desired product (2.4 g). 1 The 1H-NMR spectrum was consistent with the desired structure. To show that all iodide ions had been replaced, the organic solution of the product was extracted with 1 M NaBr solution. The aqueous layer was then used in the starch test, and no coloration was shown, indicating complete exchange of iodide anions. As a reference benchmark, dendrimer solutions containing iodide were tested in the same way. These solutions turned blue.

[0395]

Chemical formula

[0396] This material J4 was obtained by reacting PPI dendrimer G3 (DAB-Am-16) with phenyl palmitate, which was carried out in a similar manner as that in Example J1. Yield: 5.88 g (90%). 1 The 1H-NMR spectrum was consistent with the desired structure.

[0397]

Chemical formula

[0398] This material J5 was obtained by reacting J4 with MeI, which was carried out in a similar manner as that in Example J2. Yield: 1.32 g (97%). 1 The 1H-NMR spectrum was consistent with the desired structure.

[0399]

Chemical formula

[0400] This material J6 was obtained by eluting J5 through an ion - exchange column, which was carried out in a similar manner as that in Example J3. Yield: 2.55 g (99%). 1 The 1H - NMR spectrum was consistent with the desired structure.

[0401]

Chemical Structure

[0402] [Chemical] Example J7. G1-Dimethyl itaconate-4

[0403] Dimethyl itaconate (0.66 g, 4.2 mmol, 4.2 equiv) was added to a stirred solution of G1-PPI-(NH 2 ) 4 (316 mg, 1 mmol) in IPA (2 mL). The reaction mixture was heated at 60 °C for 16 h and then poured into water (15 mL). The aqueous layer was washed with diethyl ether (4 × 5 mL). A trace amount of solvent was removed at room temperature under vacuum. The aqueous layer was lyophilized to give an amber oil. The product was purified by silica column chromatography using MeOH / CHCl 3 (20 - 30% MeOH) eluent. Yield: colorless oil (414 mg, 0.50 mmol, 50%). 1 H NMR (400 MHz, chloroform-d) δ 3.74 (s, 12H), 3.70 - 3.53 (m, 8H), 3.39 - 3.17 (m, 12H), 2.77 - 2.60 (m, 8H), 2.53 - 2.27 (m, 12H), 1.66 (m, 8H), 1.39 (m, 4H). ESI-MS: m / z C 40 H 64 N 6 O 12 calculated for 820.46; found [M+H] 2+ 411.50, [M+H] + 820.50, [M+Na] + 853.50. Example J8. G1(PAMAM-C2)-Dimethyl itaconate-4

[0404] Dimethyl itaconate (514 mg, 3.25 mmol, 4.9 eq) was added to a solution of G1-PAMAM-C2 (ethylene diamine core, four amine end groups, ~340 mg, 0.66 mmol) in MeOH (2 mL). The mixture was stirred at room temperature and the progress of the reaction was monitored using HPLC-MS. After 184 h, the reaction was complete. The mixture was poured into water (15 mL) and the aqueous layer was washed with diethyl ether (4 x 5 mL). Trace amounts of solvent were removed under vacuum and the aqueous layer was lyophilized to give a semi-solid colorless wax (834 mg), which still contained some ether. 1 H NMR (400 MHz, chloroform-d) δ 7.71 (t, J = 5.3 Hz, 4H), 3.74 (s, 16H), 3.66 (dd, J = 9.9, 5.9 Hz, 4H), 3.55 - 3.39 (m, 10H), 3.39 - 3.17 (m, 12H), 2.74 - 2.55 (m, 16H), 2.52 (s, 4H), 2.32 (t, J = 6.5 Hz, 8H). ESI-MS: m / z C 46 H 72 N 10 O 16 calculated for 1021.14; found [M+H] 2+ 511.58, [M+H] + 1021.50, [M+Na] + 1043.50. Example J9. G1-lysine-N 2 -oleate-4

[0405] G1 PPI dendrimer (35 mg, 0.11 mmol, 0.44 mmol of primary amine groups) and triethylamine (200 μL) were dissolved in DCM (9 mL) and stirred at room temperature under a nitrogen atmosphere. 2,3,5,6-Tetrafluorophenyl N 6 -(tert-butoxycarbonyl)-N 2-Oleoyl-L-lysinate (292 mg, 0.44 mmol, 4.00 equiv) was added and the solution was stirred for 24 h. Chloroform (50 mL) was added and the organic phase was washed with 1 N aqueous NaOH (20 mL), followed by water (20 mL) and brine (20 mL). The solution was co-evaporated with chloroform (3 x 20 mL) to remove triethylamine. Yield: 209 mg of a colorless oil. The oil was dissolved in DCM (10 mL) and TFA (1 mL) and the solution was stirred at room temperature for 20 h under a nitrogen atmosphere. The mixture was evaporated to dryness and the residue was dissolved in CHCl 3 (70 mL). The solution was washed with 1 N aqueous NaOH (20 mL), followed by brine (20 mL). The solution was evaporated to dryness to give a white wax product. Yield: 180 mg (86%).

[0406]

Chemical formula

[0407] A solution of G1 PPI dendrimer (31.0 mg, 0.098 mmol, 0.39 mmol of primary amine groups) in DCM (1 mL) was added to a solution of 2,3,5,6-tetrafluorophenyl N 2 -(tert-butoxycarbonyl)-N 6 -oleoyl-L-lysinate (282 mg, 0.43 mmol) and triethylamine (100 μL) in DCM (3 mL) under a nitrogen atmosphere. After 30 hours, 1 completion of the conversion was indicated by 1H-NMR analysis. To remove the excess TFP-ester, Biotage MP-trisamine resin (125 mg) was added and stirring was continued for 30 hours. DCM (50 mL) was added and the mixture was filtered. The filtrate was washed with 1N NaOH (20 mL), water (20 mL), and brine (20 mL). After removing the solvent, the dendrimer was obtained as a white solid. Yield: 198 mg. The dendrimer (195 mg) was dissolved in DCM (4 mL) under a nitrogen atmosphere and TFA (1 mL) was added. After 23 hours, the reaction mixture was concentrated on a rotary evaporator. The product was co-evaporated with toluene three times to remove the excess TFA, which was then dissolved in chloroform (50 mL) and shaken with 1N NaOH (2 mL) followed by brine (10 mL). Removal of the solvent gave a colorless oil. Yield: 180 mg (97%). The 1H-NMR spectrum was consistent with the desired structure.

[0408]

Chemical Structure

[0409] A solution of G1 PPI dendrimer (69 mg, 0.22 mmol, 0.87 mmol of primary amine groups) in THF (2 mL) was added to oleoyl-L-arginine (463 mg, 0.97 mmol), followed by the addition of chloroform (2 mL). After stirring for several minutes, a gel-like mixture was formed. HFIP (100 μL) was added and stirring was continued at 40 °C, resulting in the formation of a clear solution. After cooling to room temperature, DCC (399 mg, 1.93 mmol) was added. The resulting clear solution became turbid (due to DCU formation) when heated to 40 °C. The progress of the reaction was monitored by MALDI-TOF-MS analysis of the samples. After 6 days of reaction, additional oleoyl-L-arginine (220 mg, 0.46 mmol) and DCC (156 mg, 0.76 mmol) were added. After 3 days, MALDI-TOF-MS indicated complete conversion of the dendrimer and the reaction was stopped. Water (0.38 mL) and acetic acid (0.20 mL) were added. Concentration on a rotary evaporator gave 1.10 g of a crude product.

[0410] Water (10 mL) was added to a portion of the crude product (500 mg) and the mixture was sonicated for 30 minutes. After standing overnight, the nearly clear aqueous solution was separated from the white precipitate (mainly DCU) and transferred to dialysis tubing (CE 500 - 1000 Daltons). The solution was dialyzed against water containing acetic acid (2 mL), followed by water containing acetic acid (1 mL), followed by water (1 L) containing 1 N NaOH (2 mL), and finally water (1 L). After lyophilization, a white fluffy powder was obtained. Yield: 188 mg. 1 The 1H-NMR spectrum was consistent with the desired structure.

[0411]

Chemical Structure

[0412] n-Octyl acrylate (175 mg, 0.95 mmol, 3 eq) was added to a stirred solution of G1-PPI-(NH 2 ) 4 dendrimer (100 mg, 0.32 mmol) in IPA (1 mL). The reaction was heated at 60 °C for 16 h, then concentrated under vacuum and dried. The residue was redissolved in DCM (1 mL), and triethylamine (185 μL, 134 mg, 1.33 mmol, 4.2 eq) and acetic anhydride (125 μL, 135 mg, 1.33 mmol, 4.2 eq) were added. The reaction mixture was stirred at room temperature for 2 h and then diluted with chloroform. The chloroform layer was washed with saturated NaHCO 3 solution (1×25 mL) and brine (1×25 mL), and Na 2 SO 4It was dried using [the method] and concentrated under vacuum to obtain the product (365 mg, 0.35 mmol). The terminal unit group is a mixture of n-octyl acrylate and an acetyl group. 1 H NMR (400 MHz, chloroform-d) δ 6.63 (m, 1H), 4.07 (dtd, J = 13.4, 6.8, 2.7 Hz, 6H), 3.58 (dt, J = 14.3, 5.7 Hz, 6H), 3.30 (p, J = 7.4 Hz, 7H), 2.76 (t, J = 7.3 Hz, 1H), 2.67 - 2.52 (m, 6H), 2.52 - 2.29 (m, 12H), 2.17 - 2.03 (m, 9H), 1.96 (d, J = 4.8 Hz, 3H), 1.63 (p, J = 6.9 Hz, 14H), 1.30 (dd, J = 14.1, 7.6 Hz, 34H), 0.88 (t, J = 6.7 Hz, 9H). MALDI-TOF-MS (CHCA matrix, positive reflector mode): For 3 x n-octyl acrylate, measured m / z = (M+H) + 1037.80, (M+Na) + 1059.77. Calculated: C 57 H 108 N 6 O 10 (Exact mass 1036.81; molecular weight 1037.52). For 4 x octyl acrylate, measured m / z = (M+H) + 1221.95, (M+Na) + 1243.93. Calculated: C 68 H 128 N 6 O 12 (Exact mass 1220.96; molecular weight 1221.80). Example K2. G2 PPI Dendrimer Modified with n-Octyl Acrylate and Acetic Anhydride

[0413] Similar to Example K1, G2-PPI-(NH 2 ) 8(102 mg, 0.13 mmol) was reacted with n-octyl acrylate (169 mg, 0.92 mmol, 6.95 eq) in IPA (1 mL) in Step 1 and then reacted with acetic anhydride (125 μL, 136 mg, 1.33 mmol, 10 eq) using DIPEA (231 μL, 172 mg, 1.33 mmol, 10 eq) in DCM (1 mL) in Step 2. The terminal unit groups are a mixture of n-octyl acrylate and acetyl groups. 1 H NMR (400 MHz, chloroform-d) δ 4.06 (dt, J = 13.5, 6.8 Hz, 14H), 3.58 (dt, J = 13.5, 7.5 Hz, 14H), 3.31 (q, J = 9.4, 8.6 Hz, 15H), 2.76 (t, J = 7.3 Hz, 1H), 2.70 - 2.52 (m, 14H), 2.40 (m, 36H), 2.10 (d, J = 11.6 Hz, 21H), 1.96 (s, 3H), 1.85 - 1.44 (m, 38H), 1.45 - 1.10 (m, 74H), 0.88 (t, J = 6.7 Hz, 21H). MALDI-TOF-MS (CHCA matrix, positive reflector mode): 6 x n-octyl acrylate found m / z = (M+H) + 2215.75, (M+Na) + 2237.73. Calculated: C 122 H 232 N 14 O 20 (Exact mass 2213.76; molecular weight 2215.28). 7 x octyl acrylate found m / z = (M+H) + 2399.90, (M+Na) + 2421.88. Calculated: C 133 H 252 N 14 O 22 (Exact mass 2397.90; molecular weight 2399.56). Functionalization of the primary amine with L-acrylamide, followed by epoxidation of the remaining secondary amines (where the product is a mixture of polyvalent molecules) Example L1. G1 PPI Dendrimer Modified with n-Octyl-Acrylamide and 1,2-Epoxydodecane

[0414] n-Octylacrylamide (174 mg, 0.95 mmol, 3 eq) was added to a stirred solution of G1-PPI-(NH 2 ) 4 (100 mg, 0.32 mmol) in IPA (1 mL). The reaction mixture was stirred at 60 °C for 160 h to complete the first step. Then, 1,2-epoxydodecane (582 mg, 3.16 mmol, 10 eq) and IPA (0.5 mL) were added and the reaction mixture was stirred at 60 °C for an additional 16 h. The mixture was cooled and concentrated under vacuum. The residue was stirred in MeCN, cooled to -20 °C, and the product was phase separated from the MeCN solvent. The supernatant was carefully removed using a pipette and the trituration procedure was repeated twice to afford an amber oil (495 mg, 0.28 mmol, 76%). The terminal unit groups are a mixture of groups derived from n-octylacrylamide and epoxide. 1 H NMR (400 MHz, chloroform-d) δ 6.79 (s, 3H), 3.76 (m, 5H), 3.62 (m, 5H), 3.20 (q, J = 7.7, 6H), 3.01 - 2.77 (m, 3H), 2.77 - 2.12 (m, 37H), 1.68 - 1.06 (m, 138H), 0.88 (t, J = 6.8 Hz, 24H). MALDI-TOF-MS (DCTB matrix, positive reflector mode): found m / z = (M+H) + 1788.71, calculated: C 109 H 223 N 9 O 8 (exact mass 1786.73; molecular weight 1788.04). Example L2. G2 PPI Dendrimer Modified with n-Octyl-Acrylamide and 1,2-Epoxydodecane

[0415] Similar to Example L1, G2-PPI-(NH 2 ) 8 (100 mg, 0.13 mmol) was reacted with n-octyl-acrylamide (142 mg, 0.78 mmol, 6 equivalents) in IPA (1 mL) in Step 1 and then with 1,2-epoxydodecane (477 mg, 2.59 mmol, 20 equivalents) in Step 2. Yield: Amber viscous oil (463 mg, 96%). The terminal unit groups are a mixture of groups derived from n-octylacrylamide and the epoxide. 1 H NMR (400 MHz, chloroform-d) δ 6.96 (s, 6H), 3.84 (d, J = 8.1 Hz, 10H), 3.61 (s, 10H), 3.18 (t, J = 7.0 Hz, 12H), 2.99 - 2.77 (m, 6H), 2.77 - 2.09 (m, 90H), 1.27 (d, J = 5.6 Hz, 280H), 0.88 (t, J = 6.7 Hz, 48H). MALDI-TOF-MS (DCTB matrix, positive reflector mode): Found m / z = (M+H) + 3717.55, calculated: C 226 H 462 N 20 O 16 (Exact mass 3713.60; molecular weight 3716.31).

[0416] [Example 2] Solubility of polyvalent molecules The solubility of various multivalent molecules was tested in ethanol, isopropanol, and tricaprylin at a concentration of 20 mg of material per gram of solvent. Spontaneous dissolution, which results in a clear solution when stirred at room temperature, was used as an indicator of solubility. The multivalent molecules of Examples A1, A2, A4, A6, A7, A8, A9, A10, A11, B1, B2, B3, B4, C2, E1, F1, G1, G2, G3, G5, G7, G8, H1, J10, and J11 were found to be soluble in all three solvents at room temperature. Example C1 was soluble in isopropanol and tricaprylin. Example D4 became soluble in the three solvents when heated to 37 °C. Note that not all of the prepared materials were tested.

[0417] These data indicate that the multivalent molecules are sufficiently soluble, for example, with respect to processing in ethanol or isopropanol. The data also show solubility in tricaprylin, suggesting that the molecules likely have an affinity for the hydrophobic interior of the nanoparticles of the present invention.

[0418] [Example 3] Construction of Multivalent Molecules Containing Nanoparticles Nanoparticle formulations self-assemble based on ionic and hydrophobic interactions. The components are prepared at the desired concentrations in their respective organic solvents (lipids and other structural components) or aqueous buffers (nucleic acid payloads). The solutions are then combined together by rapid mixing techniques including microfluidics or T-shaped mixing. An excess amount of aqueous buffer is essential for the formation process. As used herein, an excess amount of aqueous buffer refers to a ratio of at least 2:1 or more, for example, 2.2:1, 2.5:1, 2.8:1, or 3:1 or more of (aqueous buffer):(organic solvent) (volume-based). After the initial mixing, a small fraction of the organic solvent is removed, for example, by dialysis or tangential flow filtration. These steps result in lipid nanoparticles to which apolipoprotein (stabilizer) is added by rapid mixing techniques such as microfluidics or T-shaped mixing, or using, for example, the drip method. After the addition of apolipoprotein, residual protein needs to be removed by dialysis or tangential flow filtration. Finally, the sample is concentrated to the desired concentration.

[0419] The nucleic acid nanoparticles produced in this way (a) multivalent molecules, (b) apolipoprotein stabilizers, (c) nucleic acids, (d) sterols, (e) phospholipids, and (f) optionally lipids are included.

[0420] Accordingly, the various compositions described in Tables S3, S7, and S8 are prepared using the multivalent molecules shown and combined with the following control preparations.

[0421] The LNP control is the "standard LNP" used in the clinically approved LNP-siRNA formulation described in Akinc A, et al. The Onpattro story and the clinical translation of nanomedicines containing nucleic acid-based drugs. Nat Nanotechnol. 2019 Dec;14(12):1084-1087. doi: 10.1038 / s41565-019-0591-y. The composition is as follows (mol %), whereby the presence of nucleic acid is ignored: - DLin-MC3-DMA 50% - DSPC 10% - Cholesterol 38.5% - PEG-DMG 1.5%

[0422] The MC3-based aNP control is an apolipoprotein nanoparticle complexed with nucleic acid using DLin-MC3-DMA instead of the multivalent molecules described herein. The composition is listed in Tables S3 and S8.

[0423]

Table 8

[0424] The aNPs of the present invention were prepared using the amounts provided in the above table in combination with 1.00 (for A1), 1.13 (A2), 1.60 (A3), 1.27 (A6), 1.20 (A7), 1.18 (A8), 1.11 (A11), 1.16 (A14), 0.98 (B2), 1.67 (D4), 1.20 (F1), 1.16 (G2), 1.48 (G6), 1.15 (G7), 1.11 (G8), 0.97 (G9), or 0.91 (H1) mg of the multivalent material. DMG-PEG 2000 is a synthetic lipid and is PEGylated myristoyl diglyceride. Tricaprylin is glyceryl tri-n-octanoate and is a triglyceride (TG).

[0425]

Table 9

[0426]

Table 10-1

[0427]

Table 10-2

[0428] [Example 4] Preparation of Nanoparticles Materials. 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) were purchased from Avanti Polar Lipids New Jersey, US. Cholesterol and tricaprylin were obtained from Sigma Aldrich. D-Lin-MC3-DMA (MC3) was obtained from the University of British Columbia, Centre for Organic Chemistry, Vancouver, Canada. ALC-0315 was synthesized in-house. Recombinant apoA1 was expressed and purified from ClearColi BL21(DE3) cells in-house. Firefly luciferase and negative control Dicer-substrate siRNA were obtained from Integrated DNA Technologies (IDT), Iowa, US. 10 Preparation of siRNA-Loaded Nanoparticles

[0429] The scheme of the formulation process can be found in Figure 5. The specific formulation compositions of the aNP, aNP control particles, and LNP control particles of the present invention can be found in Table S3. Variations in the composition were also evaluated, see Table S7.

[0430] aNPs loaded with siRNA were formulated by rapid mixing using a T-shaped device. Lipid molecules (phospholipids, cholesterol, triglycerides, and ionizable materials for aNPs; phospholipids, cholesterol, ionizable materials, and PEG-lipids for LNPs) were dissolved in ethanol and rapidly mixed with sodium acetate buffer (25 mM, pH 4) containing 200 μg of anti-firefly luciferase siRNA or scrambled siRNA. The organic and aqueous phases were mixed and directly recovered into a 12 - 14 kDa MWCO dialysis membrane (Spectra / Por™). The nanoparticle formulation was dialyzed against 1×PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, pH 7.4) at 4 °C overnight with stirring at 150 rpm. The PBS was refreshed after approximately 4 hours. The next day, the formulation samples were recovered from the dialysis bag and the volume was determined. When formulating control LNP particles, the apolipoprotein addition step was skipped. For aNP particles, apolipoprotein A1 was dissolved in PBS and added to the formulation by rapid T-base mixing.

[0431] The resulting samples were incubated at room temperature for 1 hour. The siRNA-aNPs were filtered through a 0.2 μm filter and concentrated by centrifugal filtration at 1100 G through a 100,000 MWCO filter. The samples were concentrated to 1.5 mL and stored at 4 °C until further use. For use in cells, the samples were kept sterile after the 0.2 μm filtration step. Preparation of Nanoparticles Loaded with mRNA

[0432] The scheme of the formulation process is found in Figure 5. The specific formulation compositions of the aNPs, aNP control particles, and LNP control particles of the present invention can be found in Table S8.

[0433] aNP loaded with mRNA was formulated by rapid T-shaped mixing in an RNase-free culture hood. Lipid molecules (phospholipids, cholesterol, triglycerides, and ionizable materials for aNP; phospholipids, cholesterol, ionizable materials, and PEG-lipids for LNP) were dissolved in ethanol and rapidly mixed with sodium acetate buffer (25 mM, pH 4) containing 100 μg of mCherry mRNA (TriLink, CleanCap, 5moU). The organic and aqueous phases were mixed and directly recovered into a 12 - 14 kDa MWCO dialysis membrane (Spectra / Por™). The nanoparticle formulation was dialyzed against 1×PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, pH 7.4) at 4°C overnight with stirring at 150 rpm. The PBS was refreshed approximately 4 hours later. The next day, the formulation sample was recovered from the dialysis bag and the volume was determined. When formulating control LNP particles, the apolipoprotein addition step was skipped. For aNP particles, apolipoprotein A1 was dissolved in PBS and added to the formulation by rapid T-shaped mixing.

[0434] The resulting samples were incubated at room temperature for 15 minutes. The mRNA-aNP was filtered through a 0.2 μm filter and concentrated by centrifugal filtration at 1100G on a 100,000 MWCO filter. The samples were concentrated to 0.5 mL and stored at 4°C until further use. For use in cells, the samples were kept sterile after the 0.2 μm filtration step.

[0435] [Example 5] Characterization of Nanoparticles Determination of siRNA Encapsulation Efficiency The amount of siRNA loaded inside the formulated particles was quantified using the Quant-iT RiboGreen assay (Thermo Fisher). This assay was performed using a black 96-well plate. Formulation samples with a theoretical siRNA concentration of 133.3 μg / mL were diluted 200-fold in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5 in DEPC-treated water) and TE buffer containing 2% Triton™ X-100 to a total volume of 100 μl in the black 96-well plate. The Triton surfactant disrupts lipid-based nanoparticles, thus making all siRNA (both retained and non-retained) accessible to the Quant-iT RiboGreen reagent. An siRNA control at a known concentration (28.6 μg / mL) was diluted 53.3-fold in both TE buffer and TE buffer containing 2% Triton to a total volume of 100 μl in the 96-well plate. Next, the RiboGreen reagent was diluted 200-fold in TE buffer and TE buffer containing Triton. 100 μl of this dilution was added to each well containing the sample or control to bring the total volume in the plate to 200 μl. Subsequently, the fluorescence of the samples was measured on a Tecan Spark® microplate reader at an excitation wavelength of 480 nm and an emission wavelength of 520 nm.

[0436] The recovery of siRNA was

[0437]

Number

[0438]

Number

[0439] siRNA retention = recovery × encapsulation. The results are reported in Figures 6 and 13. Determination of mRNA encapsulation efficiency

[0440] This was performed in a similar manner as described for the determination of siRNA encapsulation efficiency. The results are reported in Table S9.

[0441] [Table 11] Determination of the recovery of phospholipids and cholesterol

[0442] The recovery of phospholipids and cholesterol in the prepared nanoparticles was quantified by performing the phospholipid FS and cholesterol FS assays (DiaSYS), which are enzymatic colorimetric analysis tests. A buffer (190 μl) containing the coloring reagent was added to 10 μl of the nanoparticle sample in a transparent 96-well plate and incubated at 37 °C for 30 minutes. The absorbance was measured at 600 nm using a Tecan Spark (registered trademark) plate reader. The results are reported in Table S4.

[0443] [Table 12] Determination of the recovery of apolipoprotein A1 (apoA1)

[0444] The amount of apolipoprotein A1 in the nanoparticle formulation was determined using the apolipoprotein A1 FS assay (DiaSYS) (registered trademark). This is an immunoturbidimetry test based on the interaction between an anti-apoA1 antibody and apoA1 present in the sample. Tris buffer (200 μl) was added to 10 μl of the nanoparticle sample in a transparent 96-well plate. After incubating the plate at 37 °C for 5 minutes, the absorbance was measured at a wavelength of 580 nm using a Tecan Spark (registered trademark) microplate reader. Tris (40 μl) containing the apolipoprotein A1 antibody was added to the same 96-well plate, and after incubating the solution at 37 °C for 5 minutes, the absorbance was measured with the same settings. The results are reported in Table S4. Dynamic light scattering (DLS)

[0445] The hydrodynamic diameter of the formulated particles was determined by the number-weighted average diameter obtained by dynamic light scattering (DLS) using a Zetasizer Nano ZS in combination with a Malvern Zetasizer NanoSampler (Malvern Instruments, Worcestershire, UK). The size dispersity was measured as the polydispersity index (PDI). For DLS measurements, 100 μl of the formulated nanoparticles were diluted in 700 μl of PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4), equilibrated at room temperature, and then analyzed. Each sample was measured five times at a wavelength of 633 nm in 10 runs of 10 seconds each without fixing the attenuation device and the measurement position. The results are reported in Tables S5, S7, and S10.

[0446]

Table 13

[0447]

Table 14

[0448] The size and shape of the nanoparticles were also evaluated by cryogenic transmission electron microscopy (cryo-TEM). The surface of a 200-mesh lacey carbon-coated copper grid (Electron Microscopy Sciences) was plasma-charged for 40 s using a carbon coating device (Cressington 208). Subsequently, 3 μl of the nanoparticle sample (approximately 1 mg of protein / ml) was pipetted onto the grid and vitrified into a thin film by plunge vitrification in liquid ethane. This step was performed using an automated robot (FEI Vitrobot Mark IV). Cryo-TEM imaging was acquired using a cryogenic transmission electron microscope TITAN (Thermo Fisher) equipped with a field emission gun (FEG), a post-column Gatan imaging filter (model 2002), and a post-GIF 2k×2k Gatan CCD camera (model 794). Imaging was performed in bright-field TEM mode at an acceleration voltage of 300 kV with zero-loss energy filtering, a magnification of 24,000× (dose rate 11.8 e− / Å2·s), and an acquisition time of 1 s. The pictures and results are reported in Figure S6, Figure 7, Figure 8, Figure 9(A), and Figure 15.

[0449]

Table 15

[0450] The pictures and results are reported in Figure 9(B). Cytotoxicity measurement of apolipoprotein nanoparticles (aNP)

[0451] MTS assays were performed on multiple apolipoprotein nanoparticles (aNP) of the present invention. These assays were performed according to protocols known in the art. The results are reported in Figure 10. In vitro silencing (dose-response curve)

[0452] An in vitro silencing experiment was conducted in a RAW264.7 cell line transfected with the pmirGLO plasmid (containing the gene sequences expressing Renilla luciferase and firefly luciferase). The cells were cultured in a T75 cell culture flask until 80% confluence. The cells were detached, counted, and seeded at 10,000 cells / well in a 96-well plate. After standing overnight, the cells were transfected with nanoparticles containing anti-firefly luciferase siRNA in a certain concentration range. After 48 hours of incubation, the old medium was washed away with 1×PBS. Lysis phosphate buffer (Dual-Luciferase® Reporter Assay System, Promega) was added to lyse the cells. 10 μl of the cell lysate was transferred to a white 96-well flat-bottom plate. Subsequently, 40 μl of ONE-Glo™ reagent (Dual-Luciferase® Reporter Assay System, Promega) was added, and the luminescence was measured with a Tecan Spark® microplate reader at an integration time of 500 milliseconds and a waiting time of 1000 milliseconds. Since the luminescence peak should be in the range of 550 - 570 nm, a luminescence scan was performed to confirm the wavelength. Next, 40 μl of Stop and Glo reagent (Dual-Luciferase® Reporter Assay System, Promega) was added to each well, and the luminescence was measured again in the same manner. The relative residual firefly luciferase luminescence was calculated. First, the luminescence of the firefly was normalized by the luminescence of Renilla. Subsequently, the normalized sample signal was expressed as a percentage relative to the untreated sample signal. Thus, the following formula was obtained:

[0453] [Number]

[0454] The results are reported in Figure 11. In Vitro Silencing (Comparison between Scrambled siRNA and Anti-Firefly siRNA)

[0455] An in vitro silencing experiment was conducted in a RAW264.7 cell line transfected with the pmirGLO plasmid (containing a gene sequence expressing Renilla luciferase and firefly luciferase). The cells were cultured in a T75 cell culture flask until 80% confluence. The cells were detached, counted, and seeded at 10,000 cells / well in a 96-well plate. After standing overnight, the cells were transfected with nanoparticles containing either 100 nM of scrambled non-specific siRNA or anti-firefly luciferase siRNA. After 48 hours of incubation, the old medium was washed away with 1×PBS. Lysis phosphate buffer (Dual-Luciferase® Reporter Assay System, Promega) was added to lyse the cells. 10 μl of the cell lysate was transferred to a white 96-well flat-bottom plate. Subsequently, 40 μl of ONE-Glo™ reagent (Dual-Luciferase® Reporter Assay System, Promega) was added, and luminescence was measured with a Tecan Spark® microplate reader at an integration time of 500 milliseconds and a waiting time of 1000 milliseconds. Since the luminescence peak should be in the range of 550 - 570 nm, a luminescence scan was performed. Then, 40 μl of Stop and Glo reagent (Dual-Luciferase® Reporter Assay System, Promega) was added to each well, and luminescence was measured again in the same manner. The relative residual firefly luciferase luminescence was calculated. First, for all samples, the firefly luminescence was normalized by the Renilla luminescence. Subsequently, the normalized sample signal was expressed as a percentage relative to the untreated sample signal. This gives the following equation:

[0456]

Equation

[0457] The results are reported in Figure 12. In vitro firefly luciferase expression

[0458] For the detection of firefly luciferase expression in vitro, the ONE-Glo™ Luciferase Assay System (Promega) was used. RAW264.7 cells were seeded in 96-well plates at a density of 35,000 cells per well in 100 μL. Approximately 6 hours later, RAW264.7 cells were transfected with 10 μL of firefly luciferase mRNA (RIBOPRO) using aNP or LNP at a dose of 100 ng of mRNA per well. Twenty-four hours after transfection, cell viability was determined by the CellTiter 96® AQueous One Solution Cell Proliferation assay (MTS) (Promega). Ten μL of reagent was added to the cells, and after a 1-hour incubation (37 °C, 5% CO2), the absorbance was measured at 495 nm. Subsequently, the cells were washed three times with 1×PBS and then 40 μL of ONE-Glo™ luciferase assay reagent was added to each well. After complete lysis of the cells, the cell lysate was transferred to a white flat-bottom 96-well plate. Luminescence was measured using a Tecan Spark plate reader. Luciferase expression was expressed as luciferase intensity after normalization to cell viability and subtraction of the background signal. The results are reported in Figure 14. In vivo firefly luciferase expression

[0459] For the detection of in vivo firefly luciferase expression, C57BL / 6 mice (Jackson laboratories Germany) were injected with 0.5 mg / kg of firefly luciferase mRNA (TriLink, CleanCap, 5moU) loaded with aNP containing the multivalent molecule A2 mRNA conjugate. After 16 hours, the animals were sacrificed and the liver, spleen, and femur were harvested. The organs were processed to obtain single cell suspensions. One-tenth of the liver was cut into small pieces and incubated at 37 °C for 15 minutes at 50 RPM with 5 mL of liberase solution. Next, the digested liver was passed through a 70 μm sieve and collected in a 50 mL Falcon tube. After washing with 1×PBS, the cells were resuspended in 15 mL of 1×PBS. One-tenth of the spleen was cut into small pieces, passed through a 70 μm sieve, and collected in a 50 mL Falcon tube. After washing with 1×PBS, 1 mL of lysis buffer (BD Pharm Lyse™, catalog number 555899, 10-fold diluted in diH2O) was added and incubated at room temperature for 4 minutes. The cells were washed with 1×PBS and resuspended in 15 mL of 1×PBS. Both ends of the femur were broken. Bone marrow cells were flushed out from the inside of the bone with 10 mL of 1×PBS, passed through a 70 μm sieve, and collected in a 50 mL Falcon tube. The cells were centrifuged, 1 mL of lysis buffer (BD Pharm Lyse™, catalog number 555899, 10-fold diluted in diH2O) was added to the pellet, and incubated at room temperature for 1 minute. The cells were washed with 1×PBS and resuspended in 5 mL of 1×PBS. All cell suspensions were counted and 500,000 cells were seeded in a V-bottom plate. The plate was centrifuged and the supernatant was removed. 40 μL of ONE-Glo reagent (Promega) was added and 40 μL incubated for 5 minutes (432 RPM) was transferred to a white 96-well plate and luminescence was measured using the following settings: wait time: 1000 milliseconds, integration time: 500 milliseconds, open filter. The results are reported in Figure 16. In vitro

Claims

1. A nanoparticle comprising a core and an outer layer, wherein the core is - Nucleic acids, - Polyvalent molecules The outer layer includes, - Apolipoproteins, apolipoprotein derivatives, apolipoprotein mimes, and / or apolipoprotein mime derivatives, - Phospholipids, - Sterols, and optionally - Filler molecules Includes, The aforementioned polyvalent molecule is given by formula (I): [コア] ｘ -[BU] ｙ -[TU] ｚ (I) The formula comprises a C that is nitrogen or contains 1 to 15 nitrogen heteroatoms and optionally 1 to 4 oxygen heteroatoms. １ ~C １８ It is a linear, branched, or cyclic group. x represents the number of connections from the core to the branch unit BU (or to the terminal unit TU if y=0), where x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 12, and all of these connections extend from the nitrogen atom of the core to the carbon atom of the branch unit BU (or terminal unit TU). The branching unit BU is defined by formula (IIa), (IIb), (IIc), or (IId): 【Chemistry 1】 It has, (IIa) is CH ２ This represents an n-alkylene-nitrogen spacer connected at the terminal to a core or a BU closer to the core, and at the N-terminus to two terminal units (TUs) or two branch units (BUs) closer to the TUs, where the connections are shown as waveform couplings, and p is 1, 2, 3, or 4. (IIb) is CH ２ Represents an amide containing an n-alkylene-nitrogen spacer, connected at the terminal to a core or a BU closer to the core, and at the N-terminus to two terminal units (TUs) or two branch units (BUs) closer to the TUs, wherein the connection is indicated by a waveform coupling, and q is 1, 2, 3, 4, or 5, R １ is a hydrogen, methyl, ethyl, n-propyl, or isopropyl group, (IIc) and (IId) are each defined as (IIa) and (IIb), respectively, and R ２ is methyl, ethyl, n-propyl, benzyl, acetamide (-CH ２ -C(O)NH ２ ), or a 2-hydroxyethylene group, and X － is a counteranion to the quaternary amine cation moiety, y is a specific discrete generation number of the polyvalent molecule, and this number indicates how many consecutive BU layers are incorporated into the polyvalent molecule, counting from the inner core to the outer TU group, where y can be 0, 1, 2, 3, 4, or 5. For each distinct generation of layers, the BU is independently selected from (IIa), (IIb), (IIc), or (IId), that is, the first BU arising from the core representing the BU of the first generation layer may be different from the second BU (representing the BU of the second generation layer) arising away from the core, which may be different from the third, fourth, and fifth BUs arising away from the core. The terminal unit (TU) optionally contains 1 to 8 heteroatoms individually and independently selected from the group consisting of hydrogen, O, and N. １ ~C ３０ A group is independently selected from alkyl, aryl, arylenealkyl, or alkylenearyl groups, provided that not all TUs in the polyvalent molecule are hydrogen. z represents the total number of TU groups attached to the polyvalent molecule, and z is between 1 and 128. Nanoparticles in which the polyvalent molecule according to formula (I) is polyvalent with respect to positively ionizable groups and / or cationic groups, and the cumulative number of positively ionizable groups and cationic groups in the polyvalent molecule is 2 or more.

2. The aforementioned polyvalent molecule is given by formula (I): [コア] ｘ -[BU] ｙ -[TU] ｚ (I) It has, The nanoparticle according to claim 1, wherein y = 0, 1, 2, or 3 in the formula.

3. The nanoparticle according to claim 1, wherein the polyvalent molecule is a first, second, or third-generation dendrimer selected from poly(propyleneimine) (PPI) dendrimers or polyamidoamine (PAMAM) dendrimers.

4. The aforementioned polyvalent molecule 【Chemistry 2-1】 【Chemistry 2-2】 Having a structure selected from, The terminal unit (TU) optionally contains 1 to 8 heteroatoms individually and independently selected from the group consisting of hydrogen, O, and N. １ ~C ３０ The nanoparticle according to claim 1, wherein an alkyl, aryl, arylenealkyl, or alkylenearyl group is independently selected, provided that not all TUs in the polyvalent molecule are hydrogen.

5. The nanoparticle according to claim 1, wherein the apolipoprotein, apolipoprotein derivative, apolipoprotein mimetic, or apolipoprotein mimetic derivative is selected from apoA1, apoA1-Milano, apoA2, apoA4, apoA5, ​​apoB48, apoB100, apoC-I, apoC-II, apoC-III, apoC-IV, apoD, apoE, apoF, apoH, apoL, and apoM, or mimetic or derivative thereof.

6. The nanoparticle according to claim 1, wherein the nucleic acid is RNA, DNA, or a nucleic acid analog.

7. The nanoparticle according to claim 1, wherein the nucleic acid is a microRNA (miRNA), small interfering RNA (siRNA), piwi interfering RNA (piRNA), nuclear small RNA (snoRNA), transfer RNA (tRNA), tRNA-derived small RNA (tsRNA), small regulatory RNA (srRNA), messenger RNA (mRNA), modified mRNA, ribosomal RNA (rRNA), self-amplifying RNA (saRNA), circular RNA (circRNA), long non-coding RNA (lncRNA), or guide RNA (gRNA), or a combination thereof and / or a modified form thereof.

8. The nanoparticle according to claim 1, wherein the nucleic acid is single-stranded or double-stranded DNA.

9. The nanoparticle according to claim 1, wherein the nucleic acid is an antisense oligonucleotide which consists of or contains a nucleotide or nucleoside analog comprising a phosphodiester backbone or a modification of the 2'-ribose, or single-stranded DNA or RNA.

10. The nanoparticles according to claim 1, wherein the sterol is selected from sterols, cholesterol, ergosterol, hopanoids, hydroxysteroids, phytosterols, steroids, zoosterols, stigmasterols, or β-sitosterol, or a combination thereof.

11. The nanoparticles according to claim 1, wherein the phospholipid is selected from phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylglycerol, or a combination thereof.

12. The nanoparticle according to claim 1, further comprising a filler molecule, wherein the filler molecule is a glyceride molecule.

13. The amount of apolipoprotein, apolipoprotein derivative, apolipoprotein mimetic, or apolipoprotein mimetic derivative is in the range of 0.1 to 90% by weight, and / or The amount of nucleic acid is in the range of 0.01 to 90% by weight. The amount of phospholipids is in the range of 0.1 to 95% by weight, and / or The amount of sterols is in the range of 0.1 to 95% by weight, and / or The amount of polyvalent molecules is in the range of 0.1 to 95% by weight. The amount of filler present by choice is in the range of 0 to 95% by weight. The nanoparticle according to claim 1, wherein the weight percentage is based on the sum of these five components plus an optional sixth filler component, i.e., the sum of these five or six components accounts for up to 100% of the weight of the nanoparticle.

14. A composition comprising the nanoparticles described in Claim 1 and a physiologically acceptable carrier.

15. The nanoparticles according to claim 1 for use in treating diseases by stimulating or inhibiting the innate immune response.

16. The composition according to claim 14 for use in treating a disease by stimulating or inhibiting the innate immune response.

17. Nanoparticles for use according to claim 15, wherein the disease is cancer, cardiovascular disease, autoimmune disorder, or xenograft rejection.

18. The composition for use according to claim 16, wherein the disease is cancer, cardiovascular disease, autoimmune disorder, or xenograft rejection.

19. A method for producing nanoparticles, a) A step of producing nanoparticles by mixing a (lipid) component in an organic solvent with nucleic acid in an aqueous buffer, wherein the (lipid) component comprises phospholipids, sterols, polyvalent molecules, and optionally filler molecules, and the aqueous buffer has a pH of 5.5 or less. b) A step of mixing lipid nanoparticles with apolipoproteins, apolipoprotein derivatives, apolipoprotein mimes, and / or apolipoprotein mime derivatives to produce the nanoparticles at a pH of 5.5 to 9.

0. Methods that include...

20. The nanoparticles described in claim 1 can be obtained by the method of claim 19, or are obtained by the method of claim 19.

21. An in vitro or ex vivo method for introducing nucleic acids into cells, comprising the step of bringing the nanoparticles described in claim 1 into contact with the cells.

Images