Lipid nanoparticles based on multivalent molecules for nucleic acid delivery

Abstract

The present invention relates to nanoparticles, in particular nanoparticles suitable for delivery of nucleic acids to cells. The nanoparticles comprise multivalent molecules to stabilize nucleic acid molecules in the nanoparticles. In particular, the multivalent molecule has a dendrimer-like structure. The invention also relates to the manufacture of nanoparticles and the use of such nanoparticles in the treatment of disease.

Description

Technical Field

[0001] The present invention relates to the field of nucleic acid therapeutics and provides novel and inventive nanoparticles for delivering nucleic acids intracellularly at target sites. Therefore, the present invention relates to nanoparticles comprising nucleic acids. Background Art

[0002] Nucleic acid therapeutic agents such as small antisense oligonucleotides (ASO), small interfering RNA (siRNA), messenger RNA (mRNA) and other types are revolutionary new drugs with the potential to regulate gene expression. In recent years, some nucleic acid-based in vivo application drug products have been approved, including ASO, N-acetylgalactosamine (GalNAc)-siRNA conjugates, lipid nanoparticles (LNP) containing siRNA or mRNA, and many viral vectors containing plasmid DNA (pDNA). In addition, there are also some nucleic acid therapeutic agents in late clinical trials. In addition, some genetically engineered in vitro cell therapy drug products have been approved.

[0003] The therapeutic application of nucleic acids after parenteral administration is challenging. Although the size and physicochemical properties of nucleic acid types vary, their common characteristics include macromolecular size and negative charge. Therefore, after systemic administration, nucleic acids are rapidly removed from the circulation due to renal filtration and nuclease degradation. In addition, nucleic acid therapeutics work intracellularly but cannot easily pass through cell membranes. Finally, the administration of exogenous nucleic acids can trigger an immune response. Although this may be advantageous (e.g., for vaccine development), this usually results in rapid removal of nucleic acids and adverse reactions.

[0004] To overcome these challenges, all nucleic acid therapeutics rely on chemical modifications and / or nanotechnology-based delivery systems. All approved nucleic acid therapeutics rely on chemical modifications and / or nanotechnology platforms to facilitate their intracellular delivery and subsequently induce therapeutic effects after parenteral administration:

[0005] 1) ASOs are extensively chemically modified to increase their stability, reduce immunostimulatory effects, and improve their efficacy. They are administered subcutaneously to target hepatocytes or intrathecally to target cells in the central nervous system.

[0006] 2) GalNAc-siRNA conjugates are modified similarly to ASOs and administered subcutaneously. The GalNAc moiety ensures asialoglycoprotein receptor-mediated uptake in hepatocytes.

[0007] 3) Lipid nanoparticles (LNPs) are about 50-100 nm in diameter and can be administered systemically, intradermally or intramuscularly. After systemic administration, LNPs accumulate efficiently in hepatocytes, providing opportunities 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) and can be used for vaccine purposes. Currently, LNPs are the gold standard for mRNA therapeutics and may also become the standard delivery platform for in vivo gene editing applications. However, LNPs contain synthetic (non-natural) polyethylene glycol (PEG) conjugated lipids, which are associated with hypersensitivity and / or allergic reactions. In addition, after intravenous administration, current LNP systems accumulate mainly in the liver.

[0008] 4) Viral delivery systems, such as adenovirus, lentivirus or adeno-associated virus (AAV) vectors are effective vehicles for delivering DNA. Viral vectors are characterized by their limited payload capacity and immunogenicity. However, in immune privileged tissues such as eyes, viral vectors constitute the gold standard for current nucleic acid therapeutics. Viral vectors are widely used for ex vivo therapeutics (e.g., CAR T) or intravenous administration to target cells in the liver, intravitreal / subretinal administration to target cells in the retina, or intramuscular administration for vaccine purposes.

[0009] In addition to viral vector-based or LNP-mRNA-based vaccines, most approved nucleic acid therapeutics are developed for indications other than immunotherapy. Therefore, delivery of therapeutic nucleic acids to the myeloid compartment remains a challenge. In addition, chemical modification of nucleic acid molecules or viral delivery inherently has the risk of undesirable activation of the immune system, resulting in degradation or clearance of nucleic acid therapeutics, or undesirable immune responses.

[0010] For example, nanoparticles carrying nucleic acids have been described in, for example, WO2009127060A1, which describes the use of a combination of a cationic lipid with a non-cationic lipid and a nucleic acid. The cationic lipid neutralizes the nucleic acid, allowing the formation of nanoparticles that can be used for non-targeted delivery of nucleic acids in a subject. A shortcoming of these nanoparticles is that they are not able to target the myeloid compartment.

[0011] Other systems, such as in WO2019103998A2, describe nanobioformulations that can target the myeloid compartment, comprising phospholipids and apo A1 and small molecule drugs. The disadvantage of these nanobioformulations is that, due to their hydrophobic core, they do not allow the incorporation of polar structures, such as nucleic acids, such as DNA and RNA.

[0012] In WO2017048789A1, dendrimer materials are described with a limited number of ionizable groups per molecule and with non-natural thioether groups that are easily oxidized in vivo. In the preparation of these materials, multifunctional molecules react with bifunctional molecules to give intermediates, which may therefore be difficult to obtain in pure (non-crosslinked) form.

[0013] Therefore, there is a need for improved and alternative delivery systems for therapeutic nucleic acids to the myeloid compartment.

[0014] The above mentioned problems inter alia are solved by the invention as defined in the appended claims. Summary of the invention

[0015] The present invention constitutes a nanoparticle platform technology for targeting and / or delivering nucleic acid therapeutic agents, and more specifically for targeting and / or delivering nucleic acid therapeutic agents to myeloid cell compartments. The nanoparticles taught herein are nanoparticles based on (phosphoric acid) lipids that are stabilized by apolipoprotein stabilizers (or by apolipoprotein derivatives, or by apolipoprotein analogs or analog derivatives). In circulation, stabilizers and (phosphoric acid) lipids shield and / or protect nucleic acid payloads, thereby preventing their degradation and rapid removal. At the same time, the nanoparticles, by limiting the undesirable interactions with components in the blood, such as limiting the undesirable interactions of nucleic acid payloads with components in the blood, reduce the side effects associated with the immunostimulation of nucleic acid therapeutic agents. Apolipoprotein stabilizers also serve as targeting moieties because they can guide nanoparticles to myeloid cell compartments. Therefore, the present invention can achieve efficient nucleic acid therapeutic agents delivered to the myeloid cell compartments in lymphoid organs (such as, for example, bone marrow and spleen) to carry out effective immunotherapy.

[0016] The nanoparticles of this paper's teachings also further comprise multivalent molecules.Multivalent molecules have multiple positively ionizable and / or cationic groups, which can efficiently bind and capture (or compound) nucleic acids.Multivalent molecules of the present invention are more strongly bound to nucleic acids than, for example, monovalent amphiphilic molecules.Therefore, nucleic acids are better bound and / or retained in the nanoparticles of this paper's teachings.In addition, multivalent molecules of the present invention can also interact with apolipoprotein stabilizers (or by a derivative of apolipoprotein, or by a mimetics or mimetics derivatives of apolipoprotein), because the protein has an overall negative charge.As a result, apolipoprotein (or by a derivative of apolipoprotein, or by a mimetics or mimetics derivatives of apolipoprotein) becomes better integrated into the nanoparticles of the present invention.Therefore, in the circulation, for example, in the blood of a subject, the nanoparticles of the present invention will release little apolipoprotein components or will not release apolipoprotein components from the nanoparticles.

[0017] The present invention particularly relates to nanoparticles comprising multivalent molecules such as, but not limited to, dendrimers. In addition, the present invention particularly relates to nanoparticles comprising stabilizer materials such as, but not limited to, apolipoproteins, apolipoprotein derivatives, apolipoprotein mimetics, or apolipoprotein mimetic derivatives. The present invention also relates to therapeutic methods using nanoparticles, such as to treat diseases by stimulating or suppressing innate immune responses. The present invention also relates to in vivo, in vitro, or ex vivo methods of introducing nucleic acids into cells using nanoparticles.

[0018] Thus, in a first aspect, the present invention relates to a nanoparticle comprising a core and an outer layer, wherein the core comprises:

[0019] - nucleic acids;

[0020] - multivalent molecules;

[0021] And wherein the outer layer comprises:

[0022] - an apolipoprotein, an apolipoprotein derivative, an apolipoprotein mimetic and / or an apolipoprotein mimetic derivative;

[0023] - phospholipids,

[0024] - sterols; and optionally,

[0025] - filler molecules,

[0026] Wherein the multivalent molecule has formula (I):

[0027] [nuclear] x –[BU] y –[TU] z (I)

[0028] wherein the core (i.e., the core of the multivalent molecule) is nitrogen or a C1-C ... 18 Straight chain, branched chain or cyclic groups; and

[0029] wherein x represents the number of connections from the core to a branching unit BU (or to a terminal unit TU when y=0), wherein x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 12, and wherein these connections all originate from nitrogen atoms of the core and carbon atoms of the branching unit BU (or the terminal unit TU); and

[0030] The branching unit BU has the formula (IIa), (IIb), (IIc) or (IId):

[0031]

[0032] wherein (IIa) represents an n-alkylene-nitrogen spacer which is connected at the CH2-terminus to the core or to the BU closer to the core and which is connected at the N-terminus to two terminal units (TU) or to two branching units (BU) closer to the TU, wherein the connections are given in the form of a wavy bond, and wherein p is 1, 2, 3 or 4; and

[0033] wherein (IIb) represents an amide containing an n-alkylene-nitrogen spacer, which is connected at the CH2-terminus to the core or to the BU closer to the core, and which is connected at the N-terminus to two terminal units (TU) or to two branching units (BU) closer to the TU, wherein the connections are given in the form of a wavy bond, and wherein q is 1, 2, 3, 4 or 5, and wherein R1 is hydrogen, methyl, ethyl, n-propyl or isopropyl; and

[0034] wherein (IIc) and (IId) are as defined for (IIa) and (IIb), respectively, and wherein R2 is methyl, ethyl, n-propyl, benzyl, acetamide (-CH2-C(O)NH2) or 2-hydroxy-vinyl group, and wherein X - is the counter anion of the quaternary amine cation portion; and

[0035] wherein y represents a specific and discrete generation number of the multivalent molecule, wherein the number represents how many consecutive BU layers are incorporated into the multivalent molecule, as counted proceeding from the inner core to the outer TU groups, and wherein y is 0, 1, 2, 3, 4, or 5; and

[0036] wherein for each individual generation layer, the BU may be independently selected from formula (IIa), (IIb), (IIc) or (IId), i.e., a first BU emanating from the core (representing a first generation layer BU) may be different from a second BU emanating further away from the core (representing a second generation layer BU), and the second BU may be different from a third, fourth and fifth BU emanating further away from the core; and

[0037] wherein the terminal units (TU) are individually and independently selected from hydrogen and C1-C1 optionally containing 1 to 8 heteroatoms. 30 an alkyl, aryl, arylene-alkyl or alkylene-aryl group, wherein the heteroatoms are individually and independently selected from O and N, provided that not all TU in the multivalent molecule are hydrogen; and

[0038] wherein z represents the total number of TU groups attached to the multivalent molecule, wherein z is from 1 to 128; and

[0039] Wherein the multivalent molecule according to formula (I) is multivalent in the positively ionizable groups and / or cationic groups, wherein the cumulative number of positively ionizable groups and cationic groups in the multivalent molecule is 2 or more.

[0040] In a second aspect, the present invention relates to a composition comprising nanoparticles according to the first aspect of the invention and a physiologically acceptable carrier, preferably wherein the composition is a pharmaceutical composition.

[0041] In a third aspect, the present invention relates to a nanoparticle according to the first aspect of the invention or a composition according to the second aspect of the invention for use as a medicament.

[0042] In a fourth aspect, the present invention relates to a nanoparticle according to the first aspect of the invention or a composition according to the second aspect of the invention for use in treating a disease by stimulating or inhibiting an innate immune response, preferably wherein the disease is cancer, cardiovascular disease, an autoimmune disorder or xenograft rejection.

[0043] In a fifth aspect, the present invention relates to a method for producing nanoparticles, comprising the following steps:

[0044] a) mixing, preferably rapidly, a (lipid) component in an organic solvent with nucleic acids in an aqueous buffer to produce nanoparticles, wherein the (lipid) component comprises a phospholipid, a sterol, a multivalent molecule and optionally a filler material (e.g. triglyceride); and wherein the aqueous buffer has a pH of 5.5 or less, preferably 5.0 or less; and

[0045] b) mixing the lipid nanoparticles produced in a with apolipoprotein, an apolipoprotein derivative, an apolipoprotein mimetic and / or an apolipoprotein mimetic derivative, preferably rapidly mixing to produce nanoparticles of the present invention at a pH between 5.5 and 9.0, preferably between 6.0 and 8.0, more preferably between 6.5 and 8.0.

[0046] In a sixth aspect, the present invention relates to an in vitro or ex vivo method for introducing a nucleic acid into a cell, the method comprising contacting the nanoparticle according to the first aspect of the invention or the composition according to the second aspect of the invention with the cell.

[0047] In a seventh aspect, the present invention relates to an in vivo method for introducing a nucleic acid into a cell, the method comprising contacting the nanoparticle according to the first aspect of the invention or the composition according to the second aspect of the invention with the cell.

[0048] In an eighth aspect, the present invention relates to a method for in vivo delivery of a nucleic acid, the method comprising administering to a subject a nanoparticle according to the first aspect of the invention or a composition according to the second aspect of the invention.

[0049] In a ninth aspect, the present invention relates to a method for treating a disease or condition in a subject in need thereof by stimulating or inhibiting an innate immune response, the method comprising administering to the subject a therapeutically effective amount of nanoparticles according to the first aspect of the invention or a composition according to the second aspect of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 .Exemplary multivalent molecular structures and their respective codes.

[0051] Figure 2 .Exemplary multivalent molecular structures and their respective codes.

[0052] Figure 3 .Exemplary multivalent molecular structures and their respective codes.

[0053] Figure 4 .Reference molecular structures of DLin-MC3-DMA and ALC-0315.

[0054] Figure 5 . Representative schematic diagram of the production process of an exemplary RNA containing apolipoprotein nanoparticle (aNP). The lipid component is dissolved in an organic solvent and mixed with RNA (e.g., siRNA) dissolved in buffered water at low pH (e.g., pH 4). The formed particles are dialyzed in PBS at pH 7.4 and then mixed with an apolipoprotein (e.g., apolipoprotein A1). Finally, the particle solution is filtered and centrifuged. The reference LNP nanoparticle contains an ionizable cationic lipid DLin-MC3-DMA and does not contain tricaprylin (TG) or apolipoprotein A1 (for LNP, a second mixing step is not required). The reference aNP (containing DLin-MC3-DMA) and the exemplary aNP (containing a multivalent ionizable molecule) according to the present invention do not contain lipids conjugated to polyethylene glycol (PEG) (aNP is an apolipoprotein nanoparticle). For details, please see Table S3.

[0055] Figure 6 . The siRNA retention value of the prepared nanoparticles was determined by Ribogreen assay. The dark grey column shows the data of reference nanoparticles LNP (containing ionizable cationic lipid DLin-MC3-DMA) and aNP (containing DLin-MC3-DMA), while the light grey column shows exemplary aNPs (comprising multivalent ionizable materials A1, A2, A3, A6, A7, A8, A11, A14, B2, D4, G2, G6, G7, G8, G9, H1 or F1) according to the present invention. Each circle represents a separate repeated experiment.

[0056] Figure 7CryoTEM images of siRNA-aNPs containing A2 (A) and A1 (B). An overview of the particles is shown on the left (white scale bar is 100 nm), and magnification is given on the right. Spherical particles were found for both A2 and A1. For A1, the aNPs appear to have (multiple) outer layers.

[0057] Figure 8 CryoTEM images of siRNA-aNPs containing A6 (C) and A14 (D). An overview of the particles is shown on the left (white scale bar is 100 nm), and magnification is given on the right. For both A6 and A14, spherical particles were found. For A6 and A14, the aNPs appear to have (multiple) outer layers.

[0058] Fig. 9 .The cryoTEM image (A) of the same exemplary apolipoprotein nanoparticle (aNP) according to the present invention containing A6 is compared with the immunogold staining TEM image (B). In both pictures, siRNA-aNPs with a diameter of about 50 nm can be seen. Immunogold staining TEM imaging (IGS-TEM) has been implemented to locate apolipoprotein A1. In this method, a primary antibody is bound to apolipoprotein A1 on the nanoparticle, followed by a bridging antibody (IGg) and protein A coated gold nanoparticles (diameter 10 nm). In (B), the 10 nanometer gold nanoparticles appear as black spheres, where most of them are located on the surface of the aNP.

[0059] Fig.10 Cytotoxicity measurements. MTS assay was performed on RAW 264.7 cell line exposed to siRNA-aNPs taught herein. Increasing amounts of multivalent molecules A1, A2 and A6 did not produce cytotoxicity.

[0060] Fig.11 Dose response curves of siRNA-aNPs (containing A1 or A2 ionizable multivalent molecules) and reference aNPs (containing ionizable cationic lipids of DLin-MC3-DMA or ALC-0315) as taught herein, as performed on RAW 264.7 cell lines. Each point represents a single repetition of a certain concentration. The LNP particles with ionizable cationic lipids of DLin-MC3-DMA show an IC50 value of about 30nM (curve not shown). The aNPs taught herein show higher efficacy than reference aNPs or LNPs containing monovalent ionizable cationic lipids.

[0061] Fig.12.Dual luminescent gene silencing reporter assay. The data show the silencing (grey) and lack of silencing (white) of nanoparticles containing firefly luciferase siRNA and nonspecific siRNA, respectively. Each point represents a separate repetition (average of three technical repetitions). The data show that the exemplary aNPs taught herein are mostly superior to reference LNP and aNP particles.

[0062] Fig.13 . siRNA retention values ​​of A2 aNPs prepared as taught herein as determined by Ribogreen assay. Variations in N / P, tricaprylin (TG) content, cholesterol content, and type of phospholipid used resulted in nanoparticles with high siRNA retention (in most cases). Each circle represents a separate replicate experiment. Thus, aNPs with a variety of compositions can be prepared.

[0063] Fig.14 .Luminescence intensity measured after transfection of RAW 264.7 macrophages with 100 ng of firefly luciferase mRNA per well formulated with nanoparticles. Exemplary aNPs according to the present invention comprise multivalent material A2. Two controls, LNP and aNP, are shown, both containing ALC-0315 as ionizable cationic material. Data are expressed as mean + SD from one experiment with 3 technical replicates. The data show that the exemplary aNPs taught herein outperform the reference LNP and aNP particles.

[0064] Fig.15 Cryo-TEM images of mRNA loaded aNPs containing ionizable material A2. These aNPs have been used for in vivo experiments. Scale bar of 50 nm is given. Spherical particles with what appear to be (multiple) outer layers are found.

[0065] Fig.16 .Luminescence intensity was measured from single cell suspensions prepared from mice injected with 0.5 mg / kg firefly luciferase mRNA (TriLink, CleanCap 5moU) loaded in aNPs containing multivalent material A2. Measurements were taken 16 hours after bolus injection. Data are presented as mean ± SD from two animals. The data show that the exemplary aNPs taught herein efficiently target spleen and bone marrow tissues.

[0066] Fig.17 .Exemplary multivalent molecular structures and their respective codes. DETAILED DESCRIPTION

[0067] For purposes of the present invention, the following terms are defined below.

[0068] As used herein, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, etc.

[0069] As used herein, the term "and / or" refers to a situation where one or more of the stated conditions may occur alone or in combination with at least one of the stated conditions, up to all of the stated conditions.

[0070] As used herein, the term "antigen" refers to a substance to which the binding portion of an antibody can bind. A specific immunoreactive site within an antigen is called an "epitope" (or antigenic determinant). The target of an antibody or its antigen binding portion can include an antigen, such as defined herein.

[0071] As used herein, the term "at least" a particular value means that particular value or higher. 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, ..., etc. 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, etc.

[0072] As used herein, the word "comprise" or variations thereof such as "comprises" or "including" will be understood to mean the inclusion of stated elements, integers or steps, or groups of elements, integers or steps, but not the exclusion of any other elements, integers or steps, or groups of elements, integers or steps. The verb "comprise" encompasses the verbs "consist essentially of" and "consist of."

[0073] As used herein, the term "conventional techniques" refers to situations where the methods of implementing the conventional techniques used in the methods of the present invention are obvious to the skilled person. 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 those skilled in the art and is discussed in, for example, the following references: Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 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.

[0074] As used herein, nanoparticles refer to small particles, such as those ranging in diameter from 10 to 200 nm, that can be used to deliver a payload to a target, such as an organ or a cell within a subject.

[0075] As used herein, the term "identity" refers to a measure of the identity of a nucleotide sequence or an amino acid sequence. In general, the sequences are aligned to obtain the highest order match. "Identity" itself has a meaning recognized in the art and can be calculated using published techniques. See, for example:

[0076] (Computational Molecular Biology, Lesk, AM, ED., Oxford University Press, New York, 1988; Biocomputing: Informatics And Genome Projects, Smith, DW, ED., Academic Press, New York, 1993; Computer Analysis Of Sequence Data, Part I, Griffin, AM, And Griffin, HG, EDS., Humana Press, New Jersey, 1994; Sequence Analysis In Molecular Biology, Von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer; Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). Although there are a variety of methods to measure the identity between two nucleotide sequences or amino acid sequences, 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 the 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. Methods for determining identity and similarity are encoded in computer programs. Preferred computer program methods for determining identity and similarity between two sequences 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, SF et al., J. Molec. Biol. (1990) 215:403).

[0077] As an illustration, a polynucleotide having a nucleotide sequence that is at least, for example, 95% "identical" to a reference nucleotide sequence encoding a polypeptide of a particular sequence means 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 the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted and / or substituted with another nucleotide, and / or up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5' or 3' terminal position of the reference nucleotide sequence, or anywhere between these terminal positions, interspersed individually between nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.

[0078] Similarly, by a polypeptide having an amino acid sequence that is at least, for example, 95% "identical" to a reference amino acid sequence of SEQ ID NO: X, it is meant that the amino acid sequence of the polypeptide is identical to the reference sequence, except that the amino acid sequence may contain up to 5 amino acid changes per 100 amino acids of the reference amino acid sequence of SEQ ID NO: X. In other words, to obtain a polypeptide having an amino acid sequence that is at least 95% identical to a 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 amino acid residues in the reference sequence may be inserted into the reference sequence. These changes to the reference sequence may occur at the amino or carboxyl terminal positions of the reference amino acid sequence or anywhere between these terminal positions, interspersed individually between residues in the reference sequence or in one or more consecutive groups within the reference sequence.

[0079] As used herein, the term "in vitro" refers to experiments or measurements performed using components of an organism that have been isolated from its natural environment.

[0080] As used herein, the term "ex vivo" refers to experiments or measurements performed in or on tissues from an organism in an external environment with minimal alteration from natural conditions.

[0081] 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 (LNA), bridged nucleic acids (BNA), morpholino or peptide nucleic acids (PNA). A nucleic acid (molecule) can be any nucleic acid (molecule), which can be single-stranded or double-stranded.

[0082] As used herein, the term "sequence" refers to the nucleotide sequence of or within a nucleic acid and / or polynucleotide when referring to nucleotides or a "nucleic acid sequence," "nucleotide sequence," or "polynucleotide sequence." In the context of the present invention, a first nucleic acid sequence may be contained within or overlap with another nucleic acid sequence.

[0083] As used herein, the terms "subject" or "individual" or "animal" or "patient" or "mammal" are used interchangeably and refer to any subject, particularly a mammalian subject, for whom diagnosis, prognosis or treatment is desired. Mammalian subjects include humans, livestock, farm animals, and zoo, sports or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, calves, cows, bears, etc. As defined herein, a subject may be alive or dead. Samples may be obtained from a subject post-mortem (i.e., after death), and / or samples may be obtained from a living subject.

[0084] As used herein, the terms "treatment" or "treating", "palliating", "mitigation" or "ameliorating" are used interchangeably and refer to methods for obtaining beneficial or desired results (including but not limited to therapeutic benefit). Therapeutic benefit refers to the eradication or improvement or reduction (or delay) of the progression of the underlying disease being treated. In addition, therapeutic benefit is achieved by eradicating or improving or reducing (or delaying) the progression of one or more physiological symptoms associated with the underlying disease, so that an improvement or slowing or reduction in decline is observed in the patient, although the patient may still suffer from the underlying disease.

[0085] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which the nucleic acid molecule capable of transport has been attached. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be attached. The term "vector" may also refer to a viral particle (i.e., a viral vector) containing a nucleic acid of interest.

[0086] As used herein, the term "payload" generally refers to a substance that is contained in a particle and delivered to a target site. When referring to the nanoparticles of the present invention, the term "payload" refers to a nucleic acid, preferably in combination with a multivalent molecule.

[0087] When used in this article, the term "targeting" should be understood to mean approaching expected cells, organs or tissues, or enriching near expected cells, organs or tissues when referring to targeted cells (e.g., target cells, such as but not limited to myeloid cells) or targeted tissues or organs. This means that when targeting expected cells, organs or tissues, on average more nanoparticles approach expected cells, organs or tissues, as can be expected based on the random or natural distribution of particles. "In proximity" herein refers to being positioned so that nanoparticles can interact with cells (or tissues or organs) to deliver their effective load (nucleic acid).

[0088] As used herein, the term myeloid cell refers to a blood cell derived from a common progenitor cell of megakaryocytes, granulocytes, monocytes, erythrocytes. Myeloid cells are the main cell compartment of the immune system, comprising monocytes, dendritic cells, tissue macrophages and granulocytes. As used herein, the term myeloid compartment refers to the totality of myeloid cells in an organism.

[0089] Alkyl and alkylene groups may be straight chain, branched or cyclic. Alkyl and alkylene groups contain 1 to 36 carbon atoms, preferably 1 to 18 carbon atoms. 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 heteroatoms selected from the group consisting of O, N, P, S and F, preferably 1-8 heteroatoms selected from the group consisting of O and N.

[0090] Aryl and arylene contain 6 to 24 carbon atoms, while alkylenearyl and arylenealkyl contain 7 to 25 carbon atoms. Aryl, arylene, alkylenearyl and arylenealkyl optionally contain heteroatoms selected from the group consisting of O, N, P, S and F, preferably 1-8 heteroatoms selected from the group consisting of O and N.

[0091] Ester, amide, carbamate, urea, carbonate, carboxylic acid, ketone, aldehyde, ether and alcohol groups are defined as follows, wherein R x represents a hydrogen atom or a cyclic, straight-chain or branched alkyl or alkylene group. x In a group of elements, these elements can be selected independently. The ester (functional) groups or moieties indicated in this document are understood to be groups according to the following formula: -C(O)-O-. The amide (functional) groups or moieties indicated in this document are understood to be groups according to the following formula: -NR x -C(O)-. A carbamate (functional) group or moiety referred to in this document is understood to be a group according to the formula: -NR x -C(O)-O-. Urea (functional) groups or moieties referred to in this document are understood to be groups according to the formula: -NRx -C(O)-NR x -. Carbonate (functional) groups or moieties indicated in this document are to be understood as groups according to the following formula: -OC(O)-O-. Carboxylic acid (functional) groups or moieties indicated in this document are to be understood as moieties or groups according to the following formula: -C(O)OH. Ketone (functional) groups or moieties indicated in this document are to be understood as groups according to the following formula: -C(O)-. Aldehyde (functional) groups or moieties indicated in this document are to be understood as groups according to the following formula: -C(O)H. Ether (functional) groups or moieties indicated in this document are to be understood as groups according to the following formula: -O-. Alcohol (or hydroxyl) functional groups or moieties indicated in this document are to be understood as groups according to the following formula: -OH.

[0092] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0093] Various terms related to the methods, compositions, purposes and other aspects of the present invention are used throughout the specification and claims. Unless otherwise indicated, these terms should have their ordinary meanings in the relevant field of the present invention. Other specifically defined terms will be interpreted in a manner consistent with the definitions provided herein. Although any methods and materials similar or equivalent to the methods and materials described herein can be used in the test practice of the present invention, preferred materials and methods are described herein.

[0094] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0095] The present invention constructs a nanoparticle platform technology for targeting and / or delivery of nucleic acid therapeutic agents to myeloid cell compartments. Nanoparticles described herein are by apolipoprotein (or by a derivative of apolipoprotein, or by a mimetics or mimetics derivatives of apolipoprotein), also referred to herein as apolipoprotein stabilizers, stable nanoparticles based on (phosphoric acid) lipids. In circulation, stabilizers and / or (phosphoric acid) lipids shield and / or protect nucleic acid therapeutic agent payloads, thereby preventing their degradation and rapid removal. Meanwhile, the nanoparticles, by limiting the undesirable interactions with components in the blood, such as limiting the undesirable interactions of nucleic acid payloads with components in the blood, reduce the side effects associated with the immunostimulation of nucleic acid therapeutic agents. Apolipoprotein stabilizers also serve as targeting moieties, because they can guide nanoparticles to myeloid cell compartments. Therefore, the present invention can achieve efficient nucleic acid therapeutic agents delivered to the myeloid cell compartments in lymphoid organs (such as, for example, bone marrow and spleen), to carry out effective immunotherapy. In a specific embodiment, the nanoparticles taught herein are used for targeting or delivery to myeloid cells, including all blood cells derived from the progenitor cells of granulocytes, monocytes, erythrocytes or platelets. In a specific embodiment, the nanoparticles taught herein are used for targeting or delivery to monocytes, dendritic cells, tissue macrophages or granulocytes.

[0096] The nanoparticles described herein are lipid-based nano-sized formulations (particle diameter ~ 10-300nm). 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 with incorporated stabilizer proteins (preferably apolipoprotein, apolipoprotein derivatives, apolipoprotein mimics and / or apolipoprotein mimic derivatives). Due to the hydrophobic portion of the multivalent molecule, the core is relatively hydrophobic. The outer surface is mainly composed of a phospholipid layer doped with stabilizer proteins, preferably apolipoprotein stabilizers, and also contains sterols, such as, for example, cholesterol. The hydrophilic head of the phospholipid faces the external water environment, while the hydrophobic tail points inward to the core of the nanoparticle. Possibly, the nanoparticle contains multiple layers, but in any case, the nanoparticle has nucleic acids that are bound and buried inside the particle by hydrophobic multivalent molecules, and has an outer protective surface layer with phospholipids, sterols and stabilizer proteins (preferably apolipoprotein, apolipoprotein derivatives, apolipoprotein mimics and / or apolipoprotein mimic derivatives). Therefore, nanoparticles described herein are different from nanoparticles described in the art, such as vesicle-shaped and nanoparticles comprising lipid bilayers around aqueous cores. The apolipoprotein-modified nanoparticles of the present invention are also different from nanoparticles described in the art, such as nanoparticles that are only stabilized by synthetic (non-natural) hydrophilic polymers (such as, for example, polyethylene glycol (PEG) or derivatives thereof). It is noteworthy that previously reported nanoparticles are generally not allowed to target nucleic acid cargo to specific cells, and the nanoparticles taught herein allow targeting nucleic acid cargo to myeloid cell compartments, as described elsewhere herein.

[0097] The phospholipids in the nanoparticle formulation accumulate at the interface between the hydrophobic core and the aqueous solvent due to their amphiphilic properties, effectively forming a lipid monolayer (or lipid multilayer) surface barrier. For biological uses, single or multiple phospholipid types can be used because of their inherent biocompatibility and net neutral charge. Optionally, a molar percentage of charged (phospho) lipids can be added to give the entire formulation a specific charge characteristic (1-95 mol% or less, such as 1-20 mol% or 1-10 mol% or 1-5 mol%, wherein these molar percentages are relative to the total amount of the phospholipids used).

[0098] Lipid nanoparticles are designed to be compounded in their structural nucleic acids, which are hydrophilic in nature. Therefore, auxiliary molecules are needed to introduce nucleic acids into the hydrophobic nanoparticle core. For this reason, multivalent molecules are included in the nanoparticles. The multivalent molecules contain two or more positively ionizable groups and / or cationic groups. The positively ionizable groups are converted into cationic groups by protonation at lower pH values. The cationic groups can be combined with the anionic phosphate groups in the sugar phosphate backbone of the nucleic acid payload through ionic interactions. The hydrophobic part of the multivalent molecule forms a shell around the hydrophilic nucleic acid molecule. The shell can then interact with the outer layer of the particles consisting of phospholipids, sterols and protein stabilizers (e.g., apolipoprotein stabilizers).

[0099] By using multivalent positively ionizable and / or cationic molecules instead of monovalent amphiphilic molecules, the binding to nucleic acids becomes stronger. As a result, nucleic acids are better combined and retained in the nanoparticles taught herein. In addition, multivalent molecules can also interact with apolipoprotein stabilizers because the protein is generally negatively charged, such as Sparks DL, Lund-Katz S, Phillips MC. The charge and structural stability of apolipoprotein AI indiscoidal and spherical recombinant high density lipoprotein particles. J Biol Chem. 1992 Dec 25; 267 (36): 25839-47. PMID: described in 1464598. As a result, apolipoprotein becomes better connected to the nanoparticles of the present invention and is captured in the nanoparticles of the present invention. Finally, the presence of multivalent molecules may help to transfect nucleic acids into target cells because these molecules can efficiently bind to and interact with a variety of phospholipids at the (myeloid) cell barrier, thereby creating gaps for nucleic acid entry.

[0100] In addition to nucleic acids, multivalent molecules, phospholipids, sterols and stabilizers (which optionally have targeting capabilities, such as the ability to target myeloid cell compartments), additional hydrophobic filler molecules can be included in the nanoparticle formulations taught herein. Their main application is to change the physicochemical properties of the nanoparticles or to improve stability. The filler molecules (mainly) reside in the core of the nanoparticles. The filler molecules are preferably biocompatible, and non-limiting examples include glyceride lipid molecules (lipid diglycerides or triglycerides), sterol esters and fatty acid esters.

[0101] Nanoparticles containing therapeutic nucleic acids are expected to accurately regulate gene expression in myeloid cell compartments, thereby regulating immune response. A major advantage of the nanoparticle platform technology taught herein is that nucleic acid payloads can be exchanged without changing the biological performance and interaction of formulations. Therefore, nanoparticles containing therapeutic nucleic acids taught herein can be used as immunotherapies that promote immune response, such as treating cancer or infectious diseases, or suppressing immune response (such as treating autoimmune disorders or suppressing immune response during organ transplantation).

[0102] Thus, in a first aspect, the present invention relates to a nanoparticle comprising, consisting essentially of or consisting of:

[0103] - nucleic acids;

[0104] - multivalent molecules;

[0105] - a stabilizer, preferably an apolipoprotein, an apolipoprotein derivative, an apolipoprotein mimetic and / or an apolipoprotein mimetic derivative, preferably the stabilizer is also a targeting component, as it is able to direct the nanoparticles to specific cells in the body (i.e. the myeloid compartment);

[0106] - phospholipids,

[0107] - sterols; and optionally

[0108] - filler molecules,

[0109] Wherein the multivalent molecule has formula (I):

[0110] [nuclear] x –[BU] y –[TU] z (I)

[0111] wherein the multivalent molecule has a dendrimer (like) branched architecture consisting of a core connected by x links to an (optional) branching unit (BU) (i.e. the innermost BU) or to a terminal unit (TU) (e.g. in the case where the multivalent molecule does not have any BU), and

[0112] where BUs are stratified into y generations, where y is 0, 1, 2, 3, 4, or 5, and

[0113] wherein the multivalent molecule contains z terminal units (TU), preferably a TU connected to the core (in case y=0) or to the outermost BU unit (in case y=1, 2, 3, 4 or 5), and

[0114] Wherein the multivalent molecule is multivalent in the positively ionizable groups and / or cationic groups, wherein the cumulative number of these groups in the multivalent molecule is at least 2.

[0115] In specific embodiments, the nanoparticle comprises a core and an outer layer, wherein the core comprises, consists essentially of, or consists of:

[0116] - nucleic acids;

[0117] - multivalent molecules;

[0118] and wherein the outer layer comprises, consists essentially of, or consists of:

[0119] - a stabilizer, preferably an apolipoprotein, an apolipoprotein derivative, an apolipoprotein mimetic and / or an apolipoprotein mimetic derivative;

[0120] - phospholipids;

[0121] - sterols; and optionally

[0122] - filler molecules,

[0123] Wherein the multivalent molecule has formula (I):

[0124] [nuclear] x –[BU] y –[TU] z (I)

[0125] wherein the core is nitrogen or a C1-C1-C2-containing 1 to 15 nitrogen heteroatoms and optionally 1 to 4 oxygen heteroatoms. 18 Straight chain, branched chain or cyclic groups; and

[0126] wherein x represents the number of connections from the core to a branching unit BU (i.e., the innermost branching unit BU) or to a terminal unit TU (when y=0), wherein x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 12, and wherein all of these connections are made from nitrogen atoms of the core to carbon atoms of the branching unit BU (i.e., the innermost branching unit BU) or, such as, the terminal unit TU when y=0; and

[0127] The branching unit BU has the formula (IIa), (IIb), (IIc) or (IId):

[0128]

[0129] wherein (IIa) represents an n-alkylene-nitrogen spacer which is connected at the CH2-terminus to the core or to the BU closer to the core and which is connected at the N-terminus to two terminal units (TU) or to two branching units (BU) closer to the TU, wherein the connections are given in the form of wavy bonds, and wherein p is 1, 2, 3 or 4; and

[0130] wherein (IIb) represents an amide containing an n-alkylene-nitrogen spacer, which is connected at the CH2-terminus to the core or to the BU closer to the core, and which is connected at the N-terminus to two terminal units (TU) or to two branching units (BU) closer to the TU, wherein the connections are given in the form of a wavy bond, and wherein q is 1, 2, 3, 4 or 5, and wherein R1 is hydrogen, methyl, ethyl, n-propyl or isopropyl; and

[0131] wherein (IIc) and (IId) are as defined for (IIa) and (IIb), respectively, and wherein R2 is methyl, ethyl, n-propyl, benzyl, acetamide (-CH2-C(O)NH2) or 2-hydroxy-vinyl group, and wherein X - is the counter anion of the quaternary amine cation portion; and

[0132] wherein y represents a specific and discrete generation number of the multivalent molecule, wherein the number represents how many consecutive BU layers are incorporated into the multivalent molecule, as counted going from the inner core to the outer TU groups (e.g., going from the core to the TU groups), and wherein y is 0 or 1 or 2 or 3 or 4 or 5; and

[0133] wherein for each individual generation layer, the BU may be independently selected from formula (IIa), (IIb), (IIc) or (IId), i.e., the first BU emanating from the core (representing the first generation layer BU) may be different from the second BU emanating further away from the core (representing the second generation layer BU), and the second BU may be different from the third, fourth and fifth BU emanating further away from the core; and

[0134] wherein the terminal units (TU) are individually and independently selected from hydrogen and C1-C1 optionally containing 1 to 8 heteroatoms. 30 an alkyl, aryl, arylene-alkyl or alkylene-aryl group, wherein the heteroatoms are individually and independently selected from O and N, provided that not all TU in the multivalent molecule are hydrogen; and

[0135] wherein z represents the total number of TU groups attached to the multivalent molecule, wherein z is from 1 to 128; and

[0136] Wherein the multivalent molecule according to formula (I) is multivalent in the positively ionizable groups and / or cationic groups, wherein the cumulative number of positively ionizable groups and cationic groups in the multivalent molecule is 2 or more.

[0137] Since the branching unit BU branches in two directions, z is equal to x*2 y For example, a second generation multivalent molecule (y=2) with 4 connections (x=4) from the core will have 4*2 2= 16 terminal unit TU. In another example, a third generation multivalent molecule (y = 3) with 4 connections (x = 4) from the core will have 4*2 3 = 32 terminal unit TUs. In another example, a zero-generation multivalent molecule (y = 0) with 5 connections (x = 5) from the core will have 5*2 0 =5 terminal units TU.

[0138] Nanoparticle as herein described has the outer layer that mainly comprises stabilizer, phospholipid and sterol and the core that comprises multivalent molecule and goods (also referred to as effective load herein), and described goods is nucleic acid.Nanoparticle can be used for goods delivery to its expected destination, such as cell, tissue or organ.Preferably, nucleic acid goods are delivered in target cell, tissue or organ by cell.

[0139] The present invention is based on the recognition that the nanoparticles described herein can be successfully loaded with nucleic acids and can be used to deliver the nucleic acids intracellularly, for example in the intended target cells. This is achieved by combining the following features:

[0140] - Use of multivalent molecules to neutralize nucleic acids to allow loading of nucleic acids into the hydrophobic core of the nanoparticles;

[0141] - defining the structural components of the nanoparticles and ranges of their relative amounts, such as the amounts of apolipoproteins, sterols, phospholipids, multivalent molecules, nucleic acids and optionally filler molecules (such as triglycerides).

[0142] In addition, the inventor has developed a method for successfully incorporating nucleic acid into nanoparticles based on stabilizers, preferably based on nanoparticles of apolipoprotein (and / or apolipoprotein mimics), because separate components cannot be simply mixed to obtain nanoparticles as described herein. It is found that it is necessary to carry out a two-step formulation process, wherein nanoparticles containing nucleic acid are formed in the first step, and stabilizers (such as apolipoprotein, apolipoprotein derivatives, apolipoprotein mimics and / or apolipoprotein mimics derivatives) are included in the nanoparticles in the next step. Preferably, the first step is carried out at low pH, and the second step is carried out at physiological pH. This discovery allows nucleic acid to be included in nanoparticles as described herein for the first time, thereby allowing the nucleic acid to be delivered to target cells.

[0143] Therefore, the present invention provides multivalent molecules that allow nucleic acid to be loaded in nanoparticles described herein, meaning nanoparticles with a hydrophobic core. The use of nanoparticles based on hydrophobic cores has previously been described in Jayaraman M et al. (Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo. Angew Chem Int Ed Engl. 2012Aug 20; 51 (34): 8529-33), wherein positively charged amphiphiles (such as Dlin-MC3-DMA) are used to load nucleic acids into the core. Such molecules typically contain amine groups that can be protonated at lower pH. Positively charged amphiphilic molecules are then used to "neutralize" negatively charged nucleic acids to allow them to be loaded into the hydrophobic core. Although this method has its advantages, people worry that the amphiphiles used may be toxic. Therefore, alternative solutions are desired. The inventors describe herein the discovery that multivalent molecules defined herein can be used for this purpose. In addition, and importantly, the inventors surprisingly discovered that using these multivalent molecules, nucleic acids can be more efficiently delivered to target cells to exert their functions. For example, Fig.11 and 12 Described how several formulations according to the invention are more efficient in silencing luciferase reporter genes by delivering specific siRNA to cells compared to aNP or LNP control formulations containing the ionizable cationic lipid DLin-MC3-DMA. In addition, Fig.14 It is described how formulations according to the invention are more efficient in expressing a luciferase reporter gene by delivering specific mRNA to cells compared to aNP or LNP control formulations containing the ionizable cationic lipid ALC-0315. Fig.16 Efficient delivery of formulations according to the invention to spleen and bone marrow tissue in mice is described.

[0144] a. Multivalent molecules

[0145] The multivalent molecule as according to formula (I) is a molecule having a dendrimer branching architecture and is composed of three units: a core unit, a branching unit (BU) and a terminal unit (TU). Alternatively, the BU may be omitted.

[0146] [nuclear] x –[BU] y –[TU] z (I)

[0147] In particular, the multivalent molecule branches from the inner core unit to the outer terminal units (TUs) in a uniform manner. Starting from the core, each branch unit (BU) in the molecule splits in two directions, both of which connect to the next BU or TU.

[0148] In addition, the multivalent molecules described herein are defined as having specific and discrete generations. A multivalent molecule of generation zero (G0, y=0) is a molecule without BUs, wherein the core is directly connected to a TU. A first generation multivalent molecule (G1, y=1) is defined as a molecule having a core connected to 1 layer of BUs, which are connected to a TU; a second generation multivalent molecule (G2, y=2) is defined as a molecule having a core connected to a first layer of BUs, which are connected to a second layer of BUs, wherein these outer second layers of BUs are connected to a TU; a third generation multivalent molecule (G3, y=3) is defined as a molecule having a core connected to a first layer of BUs, which are connected to a second layer of BUs, and which are connected to a third layer of BUs, wherein these outer third layers of BUs are connected to a TU, as is the case for G4 (y=4) and G5 (y=5). Therefore, the generation of a multivalent molecule (represented by the letter y) indicates how many consecutive BU layers are incorporated into the multivalent molecule, as counted from the inner core to the outer TU groups.

[0149] For purposes of illustration and clarity, Scheme 1 below shows a non-limiting schematic example of a multivalent molecule:

[0150] Generation 0 molecules (G0 materials, i.e., y=0) have 5 functional cores (x=5) and 5 TU groups (z=5); and

[0151] Generation 1 molecules (G1 materials, i.e., y=1) have 4 functional cores (x=4) and 8 TU groups (z=8); and

[0152] Generation 2 molecules (G2 materials, i.e. y=2) with 3 functional cores (x=3) and 12 TU groups (z=12); and

[0153] The third generation molecule (G3 material, i.e. y=3) has 2 functional cores (x=2) and 16 TU groups (z=16).

[0154] Wherein, x, y and z refer to x, y and z in formula (I). In scheme 1, the branching units BU of each generation layer are represented by their numbers.

[0155]

[0156] Scheme 1: Examples of structures of dendritic multivalent molecules of the invention. G0, G1, G2 and G3 molecules are shown. For clarity, BU layers of the second generation are shown in bold (for G2 and G3 molecules), as opposed to BU layers of the first or third generation.

[0157] The multivalent molecule can be 0 generation or 1 generation or 2 generation or 3 generation or 4 generation or 5 generation (y=0, 1, 2, 3, 4 or 5). In a specific embodiment, the multivalent molecule is 0 generation (y=0). In one embodiment of the invention, the multivalent molecule is 1 generation (y=1). In one embodiment of the invention, the multivalent molecule is 2 generation (y=2). In one embodiment of the invention, the multivalent molecule is 3 generation (y=3). In one embodiment of the invention, the multivalent molecule is 4 generation (y=4). In one embodiment of the invention, the multivalent molecule is 5 generation (y=5).

[0158] In one embodiment of the invention, the multivalent molecule is of a lower generation, wherein y=0 or y=1, more preferably y=1.

[0159] In one embodiment of the invention, the multivalent molecule is of higher generation, wherein y=2, 3, 4 or 5, preferably y=2 or y=3, more preferably y=2.

[0160] Positively ionizable groups and cationic groups

[0161] Multivalent molecules are multivalent in that the positively ionizable and / or cationic groups are multivalent, enabling them to efficiently bind and capture nucleic acids.

[0162] The positively ionizable group is positively charged, depending on the pH value of the surrounding environment: it can be protonated at a sufficiently low pH value and become positively charged. The positively ionizable group can be any type of amine group. It is worth noting that amides, esters, ethers, carbonates, carbamates and urea groups are not positively ionizable groups. In particular, the positively ionizable group is selected from tertiary amines, secondary amines, primary amines, guanidines and imidazole groups (Scheme 2). Preferred herein are tertiary amines, primary amines, guanidines and imidazole groups. More preferred are tertiary amines and guanidine groups. Most preferred are tertiary amine groups.

[0163] The cationic group is positively charged, regardless of the pH value of the surrounding environment: it cannot deprotonate (lose) or protonate (gain) a positive charge. The cationic group can be any type of cationic group. Preferably, the cationic group contains one nitrogen atom or multiple (up to 3, such as 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 groups and imidazolium groups. More preferred is a quaternary ammonium group.

[0164]

[0165] Scheme 2: Positively ionizable groups A to E (also referred to herein as formulas A to E) and cationic groups F to H (also referred to herein as formulas F to H). aR can be any group (alkyl, alkylene, aryl, arylene, alkylenearyl, alkylenearylene, arylenealkyl or arylenealkylene, optionally containing O or N heteroatoms), and is not hydrogen. b is similarly defined, except that it may also be hydrogen (e.g., in Formula E, not all four R b The first atom directly attached to a group A to H is either carbon or hydrogen and is not a heteroatom.

[0166] The cumulative number of positively ionizable and / or cationic groups in the multivalent molecule is at least 2. Preferably, the cumulative number of positively ionizable 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 and / or cationic groups in the multivalent molecule is a discrete number for the multivalent molecule and is not an average value.

[0167] Herein, preferably only positively ionizable and / or cationic groups are counted which are separated from their adjacent positively ionizable and / or cationic groups by at least 3 atoms, preferably by carbon atoms. For example, the two amines in a piperazine ring are not counted.

[0168] In one embodiment, all positively ionizable and / or cationic groups within the multivalent molecule are separated from each other by at least 3 atoms, wherein the separating atoms are carbon, nitrogen and / or oxygen atoms.

[0169] In one embodiment of the invention, the multivalent molecule has only positively ionizable groups and no cationic groups. Preferably, then the positively ionizable charge resides in the core and BU, rather than in TU. Alternatively, the positively ionizable charge resides in the core, BU, and TU. In another selection, the positively ionizable charge resides in the core and TU, rather than in BU. Finally, in a specific embodiment, the positively ionizable charge resides only in the core. In this embodiment of the invention, the positively ionizable group is preferably a tertiary amine, optionally in combination with a primary amine and / or guanidine, and more preferably the positively ionizable group is a tertiary amine.

[0170] In one embodiment of the invention, the multivalent molecule has a combination of positively ionizable groups and cationic groups. Preferably, the cationic charge then resides only in the core, and not in the BU or TU. Alternatively, the cationic charge then resides in the core and the first generation BU layer, not in other BU layers, nor in the TU. By these means, a multivalent molecule combining shielded cationic groups with external positively ionizable groups is produced. In this embodiment, the cationic group is preferably a quaternary ammonium group; and the positively ionizable group is preferably a tertiary amine, optionally in combination with guanidine, and more preferably these groups are tertiary amines.

[0171] In one embodiment of the invention, the multivalent molecule has only cationic groups and no positively ionizable groups. Preferably, the cationic charge then resides in the core, rather than in the BU or in the TU. Alternatively, the cationic charge then resides in the core and the first generation BU layer, rather than in other BU layers, nor in the TU. In this embodiment, the cationic groups are preferably quaternary ammonium groups, optionally in combination with guanidine, more preferably these groups are only quaternary ammonium groups.

[0172] Multifunctional core (i.e. the center of a multivalent molecule)

[0173] The multivalent molecule has formula (I):

[0174] [nuclear] x –[BU] y –[TU] z (I)

[0175] In formula (I), the core is nitrogen or a C1-C2-containing heteroatom containing 1 to 15 nitrogen atoms and optionally 1 to 4 oxygen atoms. 18 Straight chain, branched chain or cyclic group.

[0176] The core of the multivalent molecule contains 1 to 15 nitrogen atoms, preferably 2 to 10 nitrogen atoms, more preferably 2 to 4 nitrogen atoms, such as 2, 3 or 4 nitrogen atoms. These nitrogen atoms can be part of an amide, carbamate or urea group, or they can be part of a positively ionizable group or a cationic group in the multivalent molecule. Preferably, at least 2 nitrogen atoms in the core are part of a positively ionizable group or a cationic group in the multivalent molecule. Preferably, all nitrogen atoms in the core are part of a positively ionizable group or a cationic group in 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 in the multivalent molecule.

[0177] The nitrogen atoms in the core as part of a positively ionizable group or a cationic group within a multivalent molecule are preferably separated from each other by at least 3 atoms, wherein the separating atoms are carbon, nitrogen and / or oxygen atoms, preferably only carbon atoms.

[0178] The core may contain functional groups such as amide, carbamate, urea or ether groups. Of these functional groups, amides and ethers are preferred. Amides are more preferred. However, most preferably, the core does not contain any of these functional groups.

[0179] The core may contain from 0 (in the case where the core is nitrogen) to 18 carbon atoms, preferably from 2 to 12 carbon atoms, more preferably from 4 to 10 carbon atoms.

[0180] The core may contain 0 to 4 oxygen atoms, preferably 0 to 2 oxygen atoms, and more preferably the core does not have any oxygen atoms.

[0181] The core may be a linear, branched or cyclic group, and is preferably a linear or branched chain, more preferably a linear chain.

[0182] The core has x number of connections from the core to the first generation branching unit BU (or to the terminal unit TU when y=0), where x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 12, and where all of these connections are generated from nitrogen atoms of the core and carbon atoms of the branching unit BU (or the terminal unit TU).

[0183] Preferably, x is 2 or more, more preferably x is 3 or more, even more preferably x is 4 or more. Preferably, x is a discrete number, rather than an average, so that multiple cores are taken into account. Preferably, the core is a single entity (i.e., a single chemical group), rather than a mixture of entities. For example, the core is derived from n-butylene diamine, rather than from both n-butylene diamine and n-propylene diamine.

[0184] In one embodiment of the invention, the nitrogen atom of the core unit is part of a positively ionizable group in a multivalent molecule.

[0185] Schemes 3 and 4 below show preferred core structures. The wavy bonds represent connections to BU or TU of the first generation layer (in the case of y=0). Thus, the number of wavy bonds of a particular core unit represents the x value of that core.

[0186]

[0187] Scheme 3: Examples of nuclear structures where x = 3 (top left), x = 4 (first three rows), x = 5 (bottom two rows; left and center), and x = 6 (bottom two rows; right). All of these nuclear structures are linear, except for those marked with * (nitrogen nuclear structure) or ** (branched nuclear structure).

[0188]

[0189] Scheme 4: Non-limiting example core structures derived from lysine, histidine or arginine amino acid building blocks, where x=2, 4, 8 or 12.

[0190] The core unit can be considered as being derived from amine functional molecules: all x-links of the core to BU (or TU) are replaced with hydrogen atoms and then these amine functional molecules become apparent. Preferably, the core unit is derived from a pure single compound amine, i.e., a purity of about 90% or more, preferably about 97% or more, more preferably about 98% or more, even more preferably about 99% or more.

[0191] Thus, the core unit is preferably derived from ammonia, 1,2-diamino-ethane, 1,3-diamino-propane, 1,4-diamino-butane (or putrescine), 1,5-diamino-pentane, 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-propylenediamine (or norspermidine), N-(4-aminobutyl)-1,4-butanediamine, N-(6-aminohexyl)-1,6-hexanediamine, spermine, spermidine or N,N-bis(2-aminoethyl)-1,2-ethylenediamine. (It is worth noting that 3-(3-aminopropoxy)propylamine and 3,3'-diamino-N-methyldipropylamine can also be regarded as oxygen or methyl nitrogen cores, respectively, with two attached n-propylene nitrogens BU). From this group, the core unit is more preferably derived from ammonia, 1,4-diamino-butane (or putrescine), 1,6-diamino-hexane, 3,3'-diamino-N-methyldipropylamine, N-(3-aminopropyl)-1,3-propylenediamine (or norspermidine), N-(4-aminobutyl)-1,4-butylenediamine, spermine or spermidine. Even more preferred herein is ammonia, 1,4-diamino-butane (or putrescine) or N-(3-aminopropyl)-1,3-propylenediamine (or norspermidine).

[0192] In addition, the core unit can be derived from an amino acid, such as lysine, arginine or histidine, preferably in the form of its naturally occurring L-stereoisomer. Non-limiting examples are core units derived from dimers or trimers of these amino acids, or from the core of a C-amidated amino acid (using ammonia, or a primary or secondary amine), see Scheme 4 for several examples.

[0193] In one embodiment of the present invention, the core unit is derived from a naturally occurring amine functional molecule, such as ammonia, 1,4-diamino-butane (or putrescine), N-(3-aminopropyl)-1,3-propanediamine (or norspermidine), spermine, L-lysinamide or L-argininamide. Preferred herein are ammonia, 1,4-diamino-butane (or putrescine), N-(3-aminopropyl)-1,3-propanediamine (or norspermidine) or spermine. More preferred are 1,4-diamino-butane (or putrescine) or N-(3-aminopropyl)-1,3-propanediamine (or norspermidine).

[0194] In one embodiment of the invention, the nitrogen atoms of the core unit are part of the cationic groups in the multivalent molecule. Preferably, these nitrogen atoms are part of the quaternary ammonium groups. The following scheme 5 shows the preferred core structure of this example. The wavy bond represents the connection with the BU or TU of the first generation layer (the case of y=0). Therefore, the number of wavy bonds of a particular core unit represents the x value of the core. The R3 group is methyl, ethyl, n-propyl, benzyl, acetamide (-CH2-C(O)NH2) or 2-hydroxy-ethylene, preferably methyl, ethyl, benzyl or acetamide groups, more preferably methyl or ethyl groups. For convenience, the counter anion X that neutralizes the cation is not drawn in scheme 5. - The X - The counter anion can be any anion, and can also be a doubly or triply charged anion. Preferably, X - It is the chloride ion Cl - 、Br - 、Iodide ion I - , Tosylate TsO - , carbonate, oxalate, sulfate or phosphate anions. More preferably, X - It is chloride, oxalate, phosphate or bromide, and most preferably chloride or phosphate (including monohydrogen phosphate or dihydrogen phosphate).

[0195]

[0196] Scheme 5: Example of a core structure with a cationic group as part of a multivalent molecule. For convenience, the counter anion X that neutralizes the cation - Not shown in the structure shown in Scheme 5.

[0197] Branch Unit BU

[0198] The branching unit BU has the formula (IIa), (IIb), (IIc) or (IId):

[0199]

[0200] Formula (IIa) represents an n-alkylene-nitrogen spacer, which is connected to the core or to the BU closer to the core at the CH2 end, and which is connected to two terminal units (TU) or to two branch units (BU) closer to the TU at the N end, wherein the connection is given in the form of a wavy bond, and wherein p is 1, 2, 3 or 4.

[0201] Preferably, in (IIa), p is 1 or 2 or 3, preferably 1 or 2, so that (IIa) represents a n-propylene-nitrogen spacer, a n-butylene-nitrogen or a n-pentylene-nitrogen spacer, preferably a n-propylene-nitrogen spacer, or a n-butene-nitrogen. More preferably, p is 1 and (IIa) represents a n-propylene-nitrogen spacer.

[0202] Formula (IIb) represents an amide containing an n-alkylene-nitrogen spacer, which is connected at the CH2 end to the core or to a BU closer to the core, and which is connected at the N-terminus to two terminal units (TU) or to two branch units (BU) closer to the TU, wherein the connection is given in the form of a wavy bond, and wherein q is 1, 2, 3, 4 or 5, and wherein R1 is hydrogen, methyl, ethyl, n-propyl or isopropyl.

[0203] Preferably, q in (IIb) is 1, 2 or 3, more preferably 1 or 2, most preferably 1.

[0204] Preferably, in (IIb), R1 is hydrogen or methyl, more preferably R1 is hydrogen.

[0205] Preferably, in (IIb), q is 1 and R1 is hydrogen, q is 1 and R1 is methyl, q is 2 and R1 is hydrogen, q is 2 and R1 is methyl, or q is 3 and R1 is hydrogen. More preferably, in (IIb), q is 1 and R1 is hydrogen, q is 2 and R1 is hydrogen, or q is 2 and R1 is methyl. Even more preferably, in (IIb), q is 1 and R1 is hydrogen.

[0206] Formula (IIc) is as defined above for (IIa), in addition R2 is methyl, ethyl, n-propyl, benzyl, acetamide (-CH2-C(O)NH2) or 2-hydroxy-ethylene group, and X - It is the counter anion of the quaternary ammonium cation.

[0207] Preferably, in (IIc), R2 is methyl, ethyl or benzyl, more preferably methyl.

[0208] In (IIc), X - The counter anion can be any anion. Preferably, X - It is the chloride ion Cl - 、Br - 、Iodide ion I - , Tosylate TsO - Or dihydrogen phosphate. More preferably, X - It is chloride ion or dihydrogen phosphate, most preferably chloride ion.

[0209] In (IIc), X -The counter anion may also be a doubly or triply charged anion such as carbonate, oxalate, sulfate, monohydrogen phosphate or phosphate anion, more preferably oxalate or monohydrogen phosphate HPO4. 2- In these cases, the counteranion neutralizes multiple branching units (IIc).

[0210] Preferably, in (IIc), R2 is methyl and X - is chloride, or R2 is benzyl and X - It is the chloride ion.

[0211] Preferably, in (IIc), p is 1, R2 is methyl and X - is chloride, p is 1, R2 is benzyl and X - is a chloride ion, p is 2, R2 is a methyl group and X - is chloride, or p is 2, R2 is benzyl and X - More preferably, p is 1, R2 is methyl and X - is chloride, or p is 1, R2 is benzyl and X - It is the chloride ion.

[0212] Formula (IId) is as defined above in (IIb), in addition R2 is methyl, ethyl, n-propyl, benzyl, acetamide (-CH2-C(O)NH2) or 2-hydroxy-ethylene group, and X - It is the counter anion of the quaternary ammonium cation.

[0213] Preferably, in (IId), R2 is methyl, ethyl or benzyl, more preferably methyl.

[0214] In (IId), X - The counter anion can be any anion. Preferably, X - It is the chloride ion Cl - 、Br - 、Iodide ion I - , Tosylate TsO - Or dihydrogen phosphate. More preferably, X - It is chloride ion or dihydrogen phosphate, and most preferably chloride ion.

[0215] In (IId), X - The counter anion may also be a doubly or triply charged anion such as carbonate, oxalate, sulfate, monohydrogen phosphate or phosphate anion, more preferably oxalate or monohydrogen phosphate HPO4. 2- In these cases, the counteranion neutralizes multiple branching units (IId).

[0216] Preferably, in (IId), R2 is methyl and X - is chloride, or R2 is benzyl and X - It is the chloride ion.

[0217] Among the branching units according to formula (II-a), (II-b), (II-c) and (II-d), BU of formula (II-a), (II-b) and (II-c) are preferred. More preferred are BU of formula (II-a) and (II-b), and most preferred is BU of formula (II-a).

[0218] In a multivalent molecule, for each individual generation layer, the BU can be independently selected from formula (IIa), (IIb), (IIc) or (IId), that is, the first BU emanating from the core (representing the first generation layer BU) can be different from the second BU emanating further away from the core (representing the second generation layer BU), and the second BU can be different from the third, fourth and fifth BU emanating further away from the core.

[0219] In one embodiment of the present invention, the BU of the multivalent molecule is the same for each generation. For example, all BU in the multivalent molecule have formula (IIa), for example, where p=1. In another example, all BU in the multivalent molecule have formula (IIa), where p=2. In another example, all BU in the multivalent molecule have formula (IIa), where p=3. In another example, all BU in the multivalent molecule have formula (IIb), where, for example, q=1 and R1=H.

[0220] In one embodiment of the present invention, BUs of formula (IIa) and (IIc) are combined in a multivalent molecule. For example, the BUs of the first generation layer are according to formula (IIc), while the BUs of the other generation layers are of type (IIa). In this example, the multivalent molecule has shielded internal cationic groups and more exposed external positively ionizable groups.

[0221] In one embodiment of the present invention, BU of formula (IIb) and (IId) are combined in a multivalent molecule. For example, the BU of the first generation layer is according to formula (IId), while the BU of the other generation layers are of type (IIb). In this example, the multivalent molecule has shielded internal cationic groups and more exposed external positively ionizable groups.

[0222] In one embodiment of the invention, the multivalent molecule does not have any branching groups BU, and y is then zero.

[0223] Terminal Unit TU

[0224] In formula (I), the terminal units (TU) are individually and independently selected from hydrogen and C1-C1 optionally containing 1 to 8 heteroatoms.30 Alkyl, aryl, arylene-alkyl or alkylene-aryl groups, the heteroatoms being individually and independently selected from O and N, with the proviso that not all TU in the multivalent molecule are hydrogen.

[0225] The terminal unit may be free of functional groups, i.e., only composed of carbon and hydrogen. Alternatively, the terminal unit may contain one or more functional groups, preferably selected from amides, carbamates, ureas, esters, hydroxyls and ether groups. Among these functional groups, amides, esters and hydroxyls are preferred.

[0226] In formula (I), TU can be individually and independently any one selected from the following groups (Scheme 6), such as according to formula (III-a) to (III-j). In each formula, the wavy bond represents the connection to the external BU or the nitrogen atom of the core (in the case of y=0).

[0227]

[0228] Scheme 6: Formulas (III-a) to (III-k) represent possibilities of TU groups. The wavy bond is a connection to BU (preferably external BU) or to the core.

[0229] In formula (III-a), TU is hydrogen.

[0230] In formula (III-b), the R4 group is preferably a straight chain or branched C1-C 18 The alkyl group is or is a (substituted) benzyl group, optionally containing heteroatoms independently selected from O and N. More preferably, R4 is C1-C 12 Straight or branched alkyl group or benzyl group. The alkyl group can be saturated or unsaturated, and is preferably saturated. In some embodiments, the alkyl group is saturated or has one, two or three unsaturations. Optionally, R4 comprises a positively ionizable group or a cationic group, such as a tertiary amine or a quaternary ammonium group.

[0231] In formula (III-c), R5 is preferably hydrogen or a methyl group, more preferably hydrogen. In certain embodiments, for example, when R5 is a methyl group, the stereochemistry in (III-c) is undefined. The R6 group is preferably a straight or branched C1-C 18 An alkyl group optionally containing heteroatoms independently selected from O and N; preferably, R6 is a linear or branched saturated or unsaturated C1-C 17 An alkyl group optionally containing heteroatoms independently selected from O and N. More preferably, R6 is a linear or branched C2-C 14 The alkyl group is even more preferably a linear or branched C4-C 12Alkyl groups. Alkyl groups can be saturated or unsaturated, and are preferably saturated. In some embodiments, the alkyl group is saturated or has one, two or three degrees of unsaturation.

[0232] In formula (III-d), R5 is preferably hydrogen or a methyl group, more preferably hydrogen. In the case where R5 is a methyl group, the stereochemistry in (III-d) is undefined. The R7 group is preferably a straight or branched C1-C 18 An alkyl group optionally containing heteroatoms independently selected from O and N. More preferably, R7 is a linear or branched C2-C 14 The alkyl group is even more preferably a straight chain or branched C4-C 12 The R8 group is preferably hydrogen or a linear or branched C1-C 18 An alkyl group optionally containing heteroatoms independently selected from O and N. In some embodiments, R8 is hydrogen or a linear or branched C1-C 12 An alkyl group optionally containing heteroatoms independently selected from O and N. Preferably, R8 is hydrogen or a linear or branched C1-C 16 alkyl, optionally containing oxygen heteroatoms; preferably, R8 is hydrogen or a linear or branched C1-C 14 An alkyl group optionally containing oxygen heteroatoms; preferably, R8 is hydrogen or a linear or branched C1-C 10 More preferably, R8 is hydrogen or a linear or branched C1-C 12 Alkyl groups, optionally containing oxygen heteroatoms. Even more preferably, R8 is hydrogen, methyl, 2-hydroxyethyl, 3-hydroxypropyl or C2-C8 alkyl groups. The alkyl groups in R7 or R8 can be saturated or unsaturated, and are preferably saturated.

[0233] 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; preferably, r is 0, 1, 5 or 10. In formula (III-e), the R6 group is preferably a linear or branched C1-C 18 An alkyl group optionally containing heteroatoms independently selected from O and N. More preferably, R6 is a linear or branched C2-C 14 The alkyl group is even more preferably a straight chain or branched C4-C 12 Alkyl groups. Alkyl groups can be saturated or unsaturated, and are preferably saturated. In some embodiments, the alkyl group is saturated or has one, two or three degrees of unsaturation.

[0234] 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. The R8 group is preferably hydrogen or a linear or branched C1-C 18 An alkyl group optionally containing heteroatoms independently selected from O and N. Preferably R 10 C1-C 16 A straight or branched alkyl group; preferably, R 10 C1-C 14 More preferably, R8 is hydrogen or a straight or branched C1-C 12 alkyl group, optionally containing oxygen heteroatoms. Even more preferably, R8 is hydrogen, methyl, 2-hydroxyethyl, 3-hydroxypropyl or a C2-C8 alkyl group. The R9 group is preferably hydrogen or a straight or branched C1-C 18 An alkyl group optionally containing heteroatoms independently selected from O and N. More preferably, R9 is hydrogen or a linear or branched C2-C 14 Alkyl groups, even more preferably hydrogen or a linear or branched C4-C 12 Alkyl group: The alkyl group in R8 or R9 may be saturated or unsaturated, and is preferably saturated.

[0235] In formula (III-g), R 10 The group is preferably a straight chain or branched C1-C 18 An alkyl group optionally containing heteroatoms independently selected from O and N. More preferably R 10 C1-C 16 Straight or branched alkyl groups. The alkyl group can be saturated or unsaturated, and is preferably saturated. In a specific embodiment, the stereochemistry in (III-g) is not defined because the hydroxyl group is connected in an undefined manner (racemic). Optionally, the hydroxyl group in (III-g) is acylated to produce an ester group.

[0236] In formula (III-h), R 11 The group is preferably a straight chain or branched C1-C 29 An alkyl group optionally containing heteroatoms independently selected from O and N. Preferably R 11 Straight or branched C1-C 27 An alkyl group, preferably R 11 Straight or branched C1-C 25 An alkyl group; preferably R 11 Straight or branched C1-C 17 An alkyl group optionally containing heteroatoms independently selected from O and N. Preferably R 11 C1-C 15Straight or branched alkyl group; preferably R 11 C1-C 13 Straight or branched alkyl groups, each of which contains heteroatoms independently selected from O and N. More preferably, R 11 C1-C 11 A linear or branched 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 degrees of unsaturation. Optionally, R 11 Contains a positively ionizable group or a cationic group, such as a tertiary amine, guanidine or quaternary ammonium group, preferably a tertiary amine group or a guanidine group.

[0237] In formula (III-i), R 13 The group is preferably a straight chain or branched C1-C 17 An alkyl group optionally containing heteroatoms independently selected from O and N. More preferably R 13 C1-C 17 A straight chain or branched chain alkyl group. The alkyl group may be saturated or unsaturated, and is preferably unsaturated. 12 The group is preferably an amino acid residue, more preferably a residue of lysine, arginine or histidine (preferably in the form of its naturally occurring L-stereoisomer), or a derivative of such a residue. 12 They may contain primary amine, guanidine or imidazole groups, or any derivatives of these groups; thus, these TUs contain positively ionizable or cationic groups.

[0238] In formula (III-j), the terminal unit is an amidine group. However, it is worth noting that this group forms a guanidine group together with the nitrogen atom of the BU unit or core unit to which it is attached. 14 The group can be independently selected for each position and is preferably selected from hydrogen and straight or branched C1-C 18 An alkyl group optionally containing heteroatoms independently selected from O and N. More preferably, R 14 The group is selected from hydrogen and a straight chain C1-C 12 Alkyl group. The alkyl group may be saturated or unsaturated, and is preferably saturated.

[0239] In formula (III-k), R 15 Straight or branched C1-C 18 An alkyl group optionally containing heteroatoms independently selected from O and N; preferably, R 2a It is a straight chain or branched saturated or unsaturated C1-C 15 An alkyl group optionally containing heteroatoms independently selected from O and N. More preferably, R2a A straight or branched C2-C 13 Alkyl groups, even more preferably linear or branched C2-C 11 Alkyl groups. Alkyl groups can be saturated or unsaturated, and are preferably saturated. In some embodiments, the alkyl group is saturated or has one, two or three degrees of unsaturation.

[0240] Among the terminal units TU according to formula (III-a) to (III-k), TU of formula (III-a) to (III-j) is preferred and TU of formula (III-b), (III-c), (III-d), (III-f), (III-g) and (III-j) is more preferred. Even more preferred are TU of formula (III-b), (III-c), (III-d), (III-f) and (III-g) or formula (III-b), (III-c), (III-d) and (III-f). Even more preferred are TU of formula (III-c), (III-d), (III-f) and (III-g) or formula (III-c), (III-d) and (III-f).

[0241] In one embodiment of the present invention, all TUs in the multivalent molecule are the same or are of the same type of formula (III). Preferably, all TUs in the multivalent molecule are the same. For example, all TUs in the multivalent molecule have formula (III-d), for example, for all TUs, R5 is hydrogen, R8 is hydrogen and R7 is n-decyl. In another example, all TUs in the multivalent molecule have formula (III-c), for example, for all TUs, R5 is hydrogen and R6 is n-octyl. In another example, all TUs in the multivalent molecule have formula (III-g), for example, for all TUs, R 10 is n-decyl, preferably with the hydroxyl groups attached in a random (racemic) manner for all TU.

[0242] In one embodiment of the invention, each nitrogen of the external BU unit is connected to two specific TUs selected from formula (III). In a specific embodiment, preferably each nitrogen of the external BU unit is connected to two specific TUs selected from formula (III). Therefore, (approximately or exactly) 50% of the TUs belong to the first specific selection TU-1 and (approximately or exactly 50%) of the TUs belong to the second specific selection TU-2. Suitable combinations for TU-1 and TU-2 are the following specific combinations: (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, please refer to Scheme 7 and these examples below. Other combination examples are the combination of (III-c) and (III-h); (III-d) and (III-h). Still other combination examples are the combination of (III-j) with (III-b), (III-d) or (III-f).

[0243]

[0244] Scheme 7: Examples of TU-1 and TU-2 combinations substituted on each nitrogen of the external branching unit (the nitrogen is shown in bold). Specific examples of these five structures are: for all terminal groups, R 11 is an n-C9 group; for all terminal groups, R 12 is an L-lysine residue and R 13 is an n-C11 group; for all terminal groups, all R 14 The group is n-butyl; for all terminal groups, R4 is methyl, R5 is hydrogen and R6 is an n-octyl group; for all terminal groups, R4 is methyl, R5 is hydrogen, R7 is an n-octyl group and R8 is hydrogen.

[0245] In one embodiment of the present invention, 2 or more TUs selected from formula (III) are used in the multivalent molecule, preferably 2, 3 or 4 TUs, more preferably 2 TUs. In this embodiment, TU-1 and TU-2 (or TU-1, TU-2 and TU-3, etc.) can be randomly distributed on the nitrogen of the external BU unit. TU-1 and TU-2 can be abundant 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%. Suitable combinations for TU-1 and TU-2 are specific combinations of (III-b) with (III-c), (III-d), (III-f), (III-g) or (III-j); or specific combinations of (III-h) with (III-c) or (III-d). Preferably, in this embodiment, the selected TU is a specific TU: for example TU-1 is (III-b) wherein R4 is a methyl group, and TU-2 is (III-d) wherein R5 is hydrogen, R7 is n-octyl and R8 is hydrogen.

[0246] In one embodiment of the present invention, a greater portion of TU is hydrogen. Relative to the total amount of TU, ​​10% or more of TU is hydrogen, or 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, or 70% or more of TU is hydrogen.

[0247] In formula (I), z represents the total number of TU groups attached to the multivalent molecule, wherein 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 one embodiment of the invention, z is 10 or more, 16 or more, or even 20 or more. Preferably, z is less than 129 or less than 128, more preferably less than 80, even more preferably less than 50.

[0248] The multivalent molecule contains multiple hydrophobic groups that reside mostly or exclusively in TU, allowing the molecule to encapsulate the nucleic acid, thereby forming a hydrophobic shell or covering around the assembly of the multivalent molecule and the nucleic acid.

[0249] Therefore, at least a portion (i.e., a certain percentage) of all TUs in the multivalent molecule are hydrophobic in nature, making the multivalent molecule hydrophobic or amphiphilic. The hydrophobic TU preferably comprises C1-C 18 More preferably, these TUs, preferably hydrophobic TUs, contain C4-C 18Aliphatic alkyl chains, wherein the chain may be linear or branched, and wherein it may be saturated or unsaturated in nature. In formula (I), preferably at least 5% of the terminal groups TU are hydrophobic in nature, 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 groups TU are hydrophobic in nature. In one embodiment of the invention, all or nearly all (>97%) of the TU are hydrophobic in nature.

[0250] Aspects of multivalent molecules

[0251] In one embodiment of the invention, the multivalent molecule is a single compound, i.e., not a mixture of compounds. In other words, in one embodiment, all multivalent molecules in the nanoparticles taught herein are identical. The purity of the multivalent molecule / compound is then 60% or higher, preferably 85% or higher, more preferably 90% or higher, most preferably 95% or higher, such as 99% or higher, for example 100%. This is generally applied 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 generally 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, such as GPC measurement or MS-spectroscopy (see, for example, Hummelen et al., Chem. Eur. J. 1997, 3, 9, 1489-1493).

[0252] In one embodiment of the invention, the multivalent molecule is a mixture of compounds. In other words, in one embodiment, the nanoparticle taught herein comprises a mixture of different multivalent molecules. This may be due to the presence of undefined stereocenters in the multivalent molecule. An example is the presence of a branched alkyl chain in a racemic source in the multivalent molecule. Another example is the use of epoxy-alkyl reactants, which results in the formation of stereochemically undefined (racemic) hydroxyls in the multivalent molecule. Therefore, in a specific embodiment, the multivalent molecule in the nanoparticle taught herein may not be pure, but the connectivity of the atoms in all multivalent molecules in the nanoparticle taught herein is the same.

[0253] The multivalent molecule may have a specific core and may have a specific generation (y=0 or 1 or 2 or 3 or 4 or 5), so the multivalent molecule may have a structure with uniformly distributed BU. Preferably, each nitrogen terminal group has the same specific TU connected (or it has the same two specific TUs connected), such as selected from (III-a) to (III-k). Therefore, the multivalent molecule of the present invention preferably has an overall shape that is symmetrical, preferably when the chiral center that may be present in the molecule is ignored, the overall shape is symmetrical.

[0254] In a specific embodiment, the multivalent molecules of the present invention have a molecular weight higher than 400 Daltons, preferably higher than 800 Daltons, more preferably higher than 1200 Daltons, even more preferably higher than 1500 Daltons. In a specific embodiment, the multivalent molecules of the present invention have a molecular weight lower than 80 kilodaltons, preferably lower than 40 kilodaltons, more preferably lower than 20 kilodaltons, even more preferably lower than 12 kilodaltons.

[0255] The molecular weight (MW) of the multivalent molecule of the present invention divided by the total number of positively ionizable and / or cationic groups (#N) 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 and / or cationic groups in the molecule) represents 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 molecules of the present invention may be quite high. For example, the MW / #N values ​​of amphiphilic molecules previously used in the art are 642 (for DLin-MC3-DMA or MC3) and 766 (for ALC-0315).

[0256] Due to the multivalent effect, the multivalent molecules of the present invention bind to nucleic acids more strongly than monovalent amphiphilic molecules. Therefore, nucleic acids are better bound and retained within the nanoparticles of the present invention.

[0257] In addition, the multivalent molecules of the present invention can also interact with apolipoprotein stabilizers (i.e., apolipoprotein, apolipoprotein derivatives, apolipoprotein mimics and / or apolipoprotein mimic derivatives) because the protein is generally negatively charged (such as described in The charge and structural stability of apolipoprotein AI indiscoidal and spherical recombinant high density lipoprotein particles. J Biol Chem. 1992 Dec 25; 267 (36): 25839-47. PMID: 1464598). As a result, apolipoprotein, apolipoprotein derivatives, apolipoprotein mimics and / or apolipoprotein mimic derivatives become better connected to the nanoparticles of the present invention. In the circulation, for example, in the blood, the release of apolipoprotein components (e.g., apolipoprotein, apolipoprotein derivatives, apolipoprotein mimics and / or apolipoprotein mimic derivatives) from the nanoparticles can therefore become less likely.

[0258] The presence of multivalent molecules of the invention may greatly facilitate the transfection of nucleic acids into target cells, since these molecules can efficiently bind to and interact with various phospholipids at the (myeloid) cell barrier, thereby creating a gap for the entry of nucleic acids.

[0259] The multivalent molecules of the present invention can be processed from solution. Therefore, the multivalent molecules are preferably soluble in solvents of different polarity. Therefore, the multivalent molecules are preferably soluble in tricaprylin, ethanol or isopropanol, more preferably soluble in tricaprylin, ethanol and isopropanol. The solubility can be checked in the following way: about 20 mg of the multivalent molecule is stirred in 1 gram of tricaprylin, ethanol or isopropanol, and it is assessed whether all the materials spontaneously dissolve to produce a clear / transparent solution (concentration of about 2 w / w%). The test can be carried out at about 20 degrees Celsius (room temperature) or at about 37 degrees Celsius. Preferably, the multivalent molecules of the present invention are already soluble at room temperature.

[0260] In one embodiment of the invention, the multivalent molecule comprises esters, amides and / or hydroxyls. These groups provide the option of slow in vivo degradation and make the multivalent molecule (more) biocompatible. Preferred groups are amides and / or esters.

[0261] The multivalent molecules of the invention are preferably non-toxic or have limited toxicity either by themselves or when bound to nucleic acids.Cytotoxicity assays can be performed by methods known in the art, such as for example by MTT assay for cell viability, or similar or comparable assays.

[0262] Synthesizing multivalent molecules

[0263] The multivalent molecules can be prepared using any suitable synthetic method or approach. They can be conveniently prepared by taking a multifunctional amine building block and modifying it with a molecule that is reactive toward amines and is hydrophobic in nature. Alternatively, two or more amine-reactive molecules can be used, at least one of which is hydrophobic in nature.

[0264] 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 derivatives of amino acids such as lysine, arginine or histidine, for example dimers or trimers of L-lysine, L-histidine or L-arginine (e.g., see Scheme 4), dimers or trimers of L-lysine or L-arginine, or mixed dimers or trimers of these amino acids.

[0265] Suitable examples of primary amine dendrimers are poly(propyleneimine) (PPI) dendrimers or poly(amido-amine) (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".

[0266] In one embodiment of the present invention, PPI-type dendrimers are used as building blocks to prepare multivalent molecules.

[0267] In another embodiment of the present invention, PAMAM type dendrimers are used as building blocks to prepare multivalent molecules.

[0268] Primary amine PPI dendrimers have n-propyleneamine branching units, and most often they are reported to have a core derived from 1,4-diaminobutane. These PPIs can be described by formula (I), where BU is according to formula (II-a), where p=1, and where all TU are hydrogen. The characteristics of the G1 to G5 (y=1 to 5) PPI building blocks, more specifically the characteristics of the G1 to G5 (y=1 to 5) PPI building blocks with a 1,4-diaminobutane derived core, are summarized in Table 1.

[0269] Table 1: Details of amine-terminated PPI dendrimers with a 1,4-diaminobutane based core.

[0270]

[0271] In addition to using 1,4-diaminobutane based cores, one can also apply other cores to generate PPIs, such as cores derived from ammonia or bis(3-aminopropyl)amine. Table 2 highlights the properties of several PPI building blocks with these two cores.

[0272] Table 2: Details of amine-terminated PPI dendrimers with a core based on ammonia (NH3) or bis(3-aminopropyl)amine.

[0273]

[0274] Primary amine PAMAM dendrimers have N-(2-aminoethyl)propionamide branching units (BU), and in most cases they are reported to 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), where q=1 and R1=H, and where all TU are hydrogen. Table 3 summarizes the characteristics of several G1 and G2 PAMAM building blocks with N-(2-aminoethyl)propionamide BU and different cores.

[0275] Table 3: Characteristics of amine-terminated PAMAM dendrimers derived from 1,4-diaminobutane, bis(3-aminopropyl)amine or 1,2-diaminoethane cores. All BUs are according to (II-b) with q=1 and R1=H.

[0276]

[0277] Dendrimers similar to PPI-dendrimers can also be used, such as poly(butyleneimine) (PBuI), poly(pentyleneimine) (PPeI) or poly(hexyleneimine) (PHel) dendrimers, especially those of lower generation G1 or G2. These dendrimers have BU according to formula (II-a), wherein p is 2, 3 or 4, respectively. Scheme 8 shows an example of the preparation of a G1 poly(butyleneimine) dendrimer with primary amine end groups. Starting from the G1 material, the two reaction sequences are repeated to provide the G2 material. Similarly, poly(pentyleneimine) or poly(hexyleneimine) dendrimers can be prepared.

[0278] In one embodiment of the invention, poly(butyleneimine) dendrimers are used as building blocks to prepare multivalent molecules. In another embodiment, poly(pentadienimine) dendrimers are used as building blocks to prepare multivalent molecules. In another embodiment, poly(hexamethyleneimine) dendrimers are used as building blocks to prepare multivalent molecules.

[0279]

[0280] Scheme 8: Sequence of two reaction steps to generate the G1 polybutylene imine (PBuI) dendrimer building block from putrescine. The bromide reactant is coupled in an alkylation reaction; alternatively, the aldehyde reactant is coupled in a reductive amination. Deprotection of the Boc group generates the amine-functional PBuI of G1.

[0281] Following the PAMAM dendrimer with N-(2-aminoethyl)propionamide BU, similar PAMAM dendrimers with other BUs, especially those of lower generations G1 or G2, can also be used. Scheme 9 shows an example of preparing a G1 PAMAM dendrimer, where BU is as shown in formula (II-b), where q=2 and R1=Me. Repeating the two reaction sequences starting with the G1 material provides the G2 material.

[0282]

[0283] Scheme 9: Two reaction step sequence to generate the G1 PAMAM dendrimer building block from bis(3-aminopropyl)amine. The acrylamide reactant is coupled in a Michael addition reaction. Deprotection of the Boc group generates the amine functional PAMAM of G1.

[0284] Other G1 PAMAM building blocks with different BUs can be prepared by similar synthetic methods, and some examples are highlighted in Table 4 (using a 1,4-diaminobutane-based core) and Table 5 (using a bis(3-aminopropyl)amine-based core).

[0285] Table 4: Characteristics of G1 amine-terminated PAMAM dendrimers derived from a 1,4-diaminobutane core, using the indicated branching units BU.

[0286]

[0287] Table 5: Characteristics of G1 amine-terminated PAMAM dendrimers derived from a bis(3-aminopropyl)amine core, using the indicated branching units BU.

[0288]

[0289] For the preparation of multivalent molecules, lower generation dendrimers are preferred. Thus, G1, G2 or G3 dendrimers are preferred. More preferred are G1 PPI, G1 PBuI, G1 PPeI or G1 PAMAM dendrimers and G2 PPI or G2 PAMAM dendrimers.

[0290] 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, spermidine. These molecules can, for example and preferably, be used for direct attachment to TU.

[0291] Next, the polyfunctional amine building blocks - such as small polyfunctional amine molecules, PPI (or PBuI or PPeI or PHeI) dendrimers or PAMAM dendrimers - are transformed by one or more reactants to obtain multivalent molecules. The following non-limiting examples illustrate how this can be done; see also Scheme 6 for the specified options for formula type (III).

[0292] Introduction of TU of type (III-a). When the amine functional building block does not react with (a small portion of) the primary amine reactive groups, or when the reaction is incomplete, the -NH2 or -NHR hydrogen remains. This is one way to obtain hydrogen TU in a multivalent molecule.

[0293] A TU of type (III-b), (III-e) or (III-f) is introduced. Amine functional building blocks can be alkylated or alkylidene arylated, for example, with alkyl halides, alkyl p-toluenesulfonates or benzyl halides optionally containing O and / or N heteroatoms. Alternatively, alkylation or alkylidene-arylation can be achieved by applying a reductive amination reaction of aldehyde or ketone functional molecules such as, for example, alkylaldehydes or benzylaldehydes.

[0294] TU of type (III-c) is introduced. Amine functional building blocks can be coupled to acrylates or methacrylates, preferably acrylates, by Michael addition. Typically, these esters have alkyl chains that can be linear or branched and saturated or unsaturated. Examples are ethyl acrylate, n-butyl acrylate, n-octyl acrylate, 2-ethylhexyl acrylate, n-decyl acrylate, isodecyl acrylate or citronellyl acrylate, preferably ethyl acrylate, n-butyl acrylate, n-octyl acrylate, n-decyl acrylate, isodecyl acrylate or citronellyl acrylate. Preferred acrylates are C6-C12 acrylates; the C6-C12 alkyl groups can be linear or branched and saturated or unsaturated. We have found that acrylates react (considerably) faster with primary amines than with secondary amines (i.e., selectively). Therefore, this can be used to first react primary amine molecules with 1 molar equivalent of acrylate to convert all primary amines into secondary amines. The remaining secondary amines can be converted by methods such as acylation, alkylation, reductive amination, etc. It is also possible to simply use a molar excess of the acrylate reactant to convert all of the secondary amines with the acrylate. It is also possible to first introduce a first acrylate and then a different second acrylate. Preferably, the amine functional building block reacts with only one acrylate. This reaction can result in complete conversion of the primary and secondary amines with that particular acrylate.

[0295] TU of type (III-d) is introduced. Amine functional building blocks can be coupled to acrylamide or methacrylamide, preferably acrylamide, by Michael addition. Typically, these amides have an alkyl chain, which can be linear or branched and saturated or unsaturated, and which can also have functional groups such as alcohols. 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 generally reacts faster with primary amines than with secondary amines. This selectivity can be used to first react primary amine molecules with acrylamide to convert all primary amines into secondary amines. The remaining secondary amines can then be converted by, for example, acylation, alkylation, reductive amination, etc.

[0296] Alternatively, the amine functional building blocks are first reacted with acrylates (or methacrylates) and these esters are subsequently amidated by reaction with primary or secondary amines. Esters can also be hydrolyzed and then amidated with primary or secondary amines.

[0297] TU of type (III-g) is introduced. The amine-functional building blocks can be alkylated by using 1,2-epoxy-alkanes such as, for example, 1,2-epoxy-hexane, 1,2-epoxy-octane, 1,2-epoxy-decane or 1,2-epoxy-dodecane. The alkanes can be linear or branched and saturated or unsaturated. The reaction of amines with these molecules introduces both alkyl chains and alcohol groups. Preferably, the amine-functional building blocks react with only one 1,2-epoxy alkane, resulting in complete conversion of primary and secondary amines with this specific 1,2-epoxy alkane. Optionally, in a further reaction step, the secondary alcohol produced can be acylated by reaction with a carboxylic acid, an active ester or an anhydride (such as, for example, acetic anhydride).

[0298] TUs of type (III-h), (III-i) and (III-a) are introduced. Amine functional building blocks can be acylated by reaction with, for example, carboxylic acids, active esters or anhydrides. Using this chemical reaction, primary amines are converted into amides, which means that TUs of type (III-h) and type (III-a) (or types (III-i) and (III-a)) are generated simultaneously. The introduced alkanes can be linear or branched and saturated or unsaturated.

[0299] Introduction of type (III-j) and (III-a) TU. Amine functional building blocks can be reacted with guanidinating agents. These reagents react with primary or secondary amines to provide guanidine groups. Examples of such reagents are pyrazole-type guanidinating 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-methyl-isothiourea and N-Boc-S-methyl-isothiourea. Deprotection of the Boc or Cbz groups produces guanidine groups. Using this chemical reaction, type (III-j) and type (III-a) TU can be introduced simultaneously at each primary amine terminal group.

[0300] One or a combination of the above transformation methods may be used to prepare the selected multivalent molecule.

[0301] Cationic groups can be introduced into multivalent molecules by quaternizing tertiary amines, guanidines or imidazoles. In non-limiting examples, all primary amines in PPI (or PBuI or PPeI or PHeI) or PAMAM dendrimer building blocks can first be protected with, for example, Boc groups. This gives molecules such as those shown in Schemes 8 and 9. Next, internal tertiary amines can be quaternized to ammonium groups by reacting with alkylating agents such as, for example, iodomethane or benzyl halides. Boc group deprotection gives PPI (or PBuI or PPeI or PHeI) or PAMAM building blocks with internal quaternary ammonium cationic groups and external primary amines. These external amines can then be converted with amine reactive molecules to obtain multivalent molecules, more particularly multivalent molecules with cationic groups inside the molecule.

[0302] Formulas (IV-A) to (IV-F) in Scheme 10 show various non-limiting embodiments of multivalent molecules, more particularly multivalent molecules according to formula (I). Formulas (IV-A) and (IV-B) show G0 materials (i.e., no BU), formulas (IV-C) and (IV-D) show first generation G1 dendrimers (i.e., 1 BU layer), formula (IV-E) shows second generation G2 dendrimers (2 BU layers) and formula (IV-F) shows third generation (G3) dendrimers (3 BU layers).

[0303]

[0304] Scheme 10: Multivalent molecules according to formula (IV-A) to (IV-F). (IV-A): x = 5, y = 0, z = 5; (IV-B): x = 6, y = 0, z = 6; (IV-C): x = 4, y = 1, z = 8; (IV-D): x = 5, y = 1, z = 10; (IV-E): x = 4, y = 2, z = 16; (IV-F): x = 4, y = 3, z = 32.

[0305] In a further embodiment, the multivalent molecules according to formula (IV-C) to (IV-F) are selected from poly(propyleneimine) (PPI) dendrimers and PAMAM dendrimers, or from modifications thereof. Scheme 11 shows examples of PPI dendrimers of the first generation G1, second generation G2 and third generation G3 with a core derived from 1,4-diaminobutane. Scheme 12 shows examples of PAMAM dendrimers of the first generation G1 and second generation G2 with a core derived from 1,2-diaminoethane. Poly(butyleneimine) (PBuI) dendrimers, poly(pentadienimine) (PPeI) dendrimers, poly(hexamethyleneimine) (PHel) dendrimers can also be used. Scheme 13 shows examples of G1 of PBuI, PPeI and PHel dendrimers with a core derived from 1,4-diaminobutane.

[0306]

[0307] Scheme 11: G1, G2 and G3 PPI dendrimers with an n-butylene diamine-derivatized core (p=1 in Formula IIa) (counterclockwise from top left).

[0308]

[0309] Scheme 12: G1 and G2 PAMAM dendrimers with n-ethylenediamine-derivatized core (q=1 and R1=H in Formula IIb).

[0310]

[0311] Scheme 13: G1 PBuI, G1 PPeI and G1 PHeI dendrimers with n-butylene diamine-derivatized core (p = 2, 3 and 4, respectively, in Formula Ia).

[0312] b. Stabilizer

[0313] Stabilizer has the function of stabilizing nanoparticles, and can be used as, for example, an agent to prevent nanoparticles from aggregating or preventing decay. Stabilizer can be a protein, polysaccharide or other (macro) molecule, or a conjugate (for example, conjugated with phospholipids or lipids) of a protein, polysaccharide or other (macro) molecule and a component of, for example, a nanoparticle. Stabilizer can also be used to control the size of nanoparticles and increase their shelf life. Non-limiting examples of suitable stabilizers are apolipoprotein, apolipoprotein derivatives, apolipoprotein mimics and / or apolipoprotein mimics 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.

[0314] In principle, the stabilizer is present primarily on the outer layer of the particle. However, it will be appreciated that the stabilizer may be a conjugate, such as a conjugate of PEG and a (phospho)lipid, in which case the PEG is primarily present in the aqueous layer immediately surrounding the nanoparticle, while the conjugated lipid is primarily present in the outer layer of the nanoparticle, presumably in close proximity to other (phospho)lipids that are not modified with PEG.

[0315] When the stabilizer contains a non-natural synthetic group, such as, for example, one of the aforementioned polyethylene glycol, polysarcosine or poly(2-oxazoline) polymer chains, this may be less than ideal, as such groups may (often) elicit an immune response upon repeated administration. In addition, such polymer groups generally have antifouling properties, meaning that nanoparticles exposing such groups may encounter barriers when interacting with cells. Therefore, protein or peptide based stabilizers are preferred.

[0316] In a specific embodiment, the stabilizer is an apolipoprotein, an apolipoprotein derivative, an apolipoprotein mimetic and / or an apolipoprotein mimetic derivative. Apolipoprotein is a natural helical protein that has an inherent affinity for (phospho) lipid layers due to its structure and amphipathic properties; however, these materials are not traditional hydrophilic head / hydrophobic tail amphiphiles. There are several types of apolipoproteins, and all can be used as structural components of nanoparticle formulations. Apolipoprotein integration affects the physicochemical properties and shelf life of nanoparticles by providing structural stability. In addition, the presence of apolipoproteins regulates the biological behavior of nanoparticles. For example, apolipoprotein A1 interacts with cells through class B type 1 scavenger receptor (SRB1) and ATP binding cassette transporter ABCA1. This increases the interaction of nanoparticles with myeloid cells in lymphoid organs.

[0317] It has been found that by using apolipoproteins, such as apoA1, nanoparticles can be successfully targeted to specific cells, tissues or organs, such as but not limited to the myeloid compartment. The advantage of this is that the drug can be targeted to specific cells in the subject to stimulate or inhibit a response. For example, immune cells can be targeted to stimulate or inhibit an immune response. There are a variety of therapeutic applications, such as but not limited to cancer, cardiovascular disease, autoimmune disorders and xenograft rejection, where such use is considered beneficial.

[0318] In a specific embodiment, the apolipoprotein, apolipoprotein derivative, apolipoprotein mimetic and / or apolipoprotein mimetic derivative is an apolipoprotein, apolipoprotein derivative, apolipoprotein mimetic and / or apolipoprotein mimetic derivative targeted to the myeloid cell compartment, preferably apoA1 or a mimetic or derivative thereof.

[0319] Because the nanoparticles described herein have the same exterior as HDL particles when apolipoprotein is used as a stabilizer, the nanoparticles do not trigger an immune response that could result in premature degradation or clearance of the nanoparticles by the immune system before reaching their intended target, such as the myeloid compartment.

[0320] The term apolipoprotein used herein refers to a protein that forms lipoprotein, i.e., an assembly of lipids and proteins, together with lipids. Apolipoprotein is generally used for transporting lipids and fat-soluble substances in the blood. The apolipoprotein described includes but is not limited to apo A1, apo A1-Milano, apo A2, apo A4, apo A5, apo B48, apo B100, apo CI, apo C-II, apo C-III, apo C-IV, apo D, apo E, apo F, apo H, apo L and apo M. In a specific embodiment, an apolipoprotein stabilizer or component can be an apolipoprotein derivative. An apolipoprotein derivative can be a simple modification of an apolipoprotein, such as produced by a one-step conversion of a protein. When used in this article, the term apolipoprotein can also refer to an apolipoprotein mimic. An apolipoprotein mimic is a short peptide that simulates the properties of an apolipoprotein. An example of an Apo A1 mimetic peptide is generally referred to as 18A, which is DWLKAF YDKVAE KLKEAF (SEQ ID NO: 1) with unfunctionalized N-terminal and C-terminal ends. Another more convenient and more active mimetic reported is 2F, which 18A (SEQ ID NO: 1) has an acetamide-terminated N-terminal and an amide C-terminal end. More examples of apo A1 peptide mimetics are reported in Leman, LJ et al., J. Med. Chem. 2014, 57, 2169-2196 (10.1021 / jm4005847), especially in Tables 2 and 3. Apo A1 peptide mimetic derivatives, such as dimers, trimers and tetramers of peptides, are described in Zhou et al., J. Am. Chem. Soc. 2013, 135, 13414-13424 (dx.doi.org / 10.1021 / ja404714a). Preferred apo A1 peptide mimetics or peptide mimetic derivatives are 18A, 2F and 4F, and any dimers of these peptides. Preferred apoA1 peptide mimetics are 18A, 2F and 4F, and any dimers of these peptides. More preferred are 2F and any dimers or trimers of the peptide.

[0321] Apolipoprotein is a protein that forms lipoprotein in combination with lipid. They transport lipids and fat-soluble vitamins in blood, cerebrospinal fluid and lymph. The lipid components of lipoprotein are insoluble in water. However, due to its amphipathic properties, apolipoprotein and other amphipathic molecules (such as phospholipids) can surround lipids, produce water-soluble lipoprotein particles themselves, and therefore can be carried through water-based circulation (i.e. blood, extracellular fluid, lymph). In addition to stabilizing lipoprotein structure and dissolving lipid components, apolipoprotein also interacts with lipoprotein receptors and lipid transport proteins, thereby participating in the uptake and removal of lipoprotein. They also serve as enzyme cofactors for specific enzymes involved in lipoprotein metabolism.

[0322] 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. As a component of HDL particles, this protein accepts fat from cells (including macrophages in the arterial wall, which have been overloaded with fat taken up by oxidized LDL particles) and enables fat molecules to be shed for transport (in water outside the cell) elsewhere, including back to the LDL particles or to the liver for excretion.

[0323] It is contemplated that any apolipoprotein may be used in the nanoparticles. Thus, in one embodiment, the apolipoprotein is selected from apo A1, apo A1-Milano, apo A2, apo A4, apo A5, apo B48, apo B100, apo CI, apo C-II, apo C-III, apo C-IV, apo D, apo E, apo F, apo H, apo L, and apo M, and combinations thereof, preferably selected from apo A1, apo A2, apo A4, apo A5, apo B100, apo CI, apo C-II, apo C-III, apo C-IV, and apo E, more preferably selected from apo A1, apo A4, apo A5, apo B100, apo C-III, and apo E, even more preferably selected from apo A1, apoB 100, and apo E. In a particularly preferred embodiment, the apolipoprotein is apo Al, as this allows targeting of the nanoparticles to the myeloid compartment. In an alternative preferred embodiment, the apolipoprotein is apo E, as this allows targeting of the nanoparticles to dendritic cells.

[0324] Apolipoprotein can be produced and purified by methods known in the art, such as expression of recombinant protein from E. coli or other organisms, followed by the steps necessary to isolate apo Al in sufficiently pure form.

[0325] The inventors have found that there are multiple advantages in using apolipoprotein in nanoparticles to deliver nucleic acids to target sites. First, apolipoprotein stabilizes nanoparticles by preventing nanoparticles from aggregating during preparation and storage. In order to keep nanoparticles in a stable emulsion, it is essential that nanoparticles do not aggregate or fuse, and nanoparticle aggregation or fusion may cause particle precipitation. Apolipoprotein helps to stabilize particles and prevent aggregation. In addition, apolipoprotein ensures the in vivo stability of nanoparticles. Because apolipoprotein is naturally present on lipid particles circulating in the bloodstream, such as LDL and HDL, they are not recognized as foreign matter (non-self, non-self) by the immune system, thereby ensuring natural stealth, rather than improving stability with chemical modification or other unnatural methods. Finally, the use of apolipoprotein promotes the interaction with the expectation of immune cells, such as delivering nucleic acid cargo in the myeloid compartment.

[0326] Thus, in one embodiment of the invention, the stabilizer, for example, the apolipoprotein component of the nanoparticle, such as apolipoprotein, an apolipoprotein derivative, an apolipoprotein mimetic and / or an apolipoprotein mimetic derivative, is used to:

[0327] -Prevent aggregation during preparation and storage;

[0328] - Improved in vivo stability;

[0329] - provide natural invisibility; and / or

[0330] - Promote targeting and / or interaction with immune cells.

[0331] In one embodiment, the stabilizer described herein is selected from:

[0332] - an apolipoprotein, an apolipoprotein derivative, an apolipoprotein mimetic and / or an apolipoprotein mimetic derivative;

[0333] - Polyethylene glycol or polyethylene glycol-modified molecules;

[0334] - hydrophilic polymers or hydrophilic polymer-modified molecules;

[0335] - polysaccharides or polysaccharide-modified molecules;

[0336] - polysarcosine or polysarcosine-modified molecules; or,

[0337] - poly(2-oxazoline) or poly(2-oxazoline)-modified molecules,

[0338] or a combination thereof.

[0339] 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.

[0340] In specific embodiments, the apolipoprotein, apolipoprotein derivative, apolipoprotein mimetic and / or apolipoprotein mimetic derivative is located on the outer surface of the outer layer of the nanoparticle.

[0341] In a specific embodiment, an apolipoprotein stabilizer as described elsewhere herein can be fused to a targeting body, which allows the nanoparticles taught herein to be targeted to a target different from the target to which it binds when the apolipoprotein stabilizer is not fused to the targeting body (or in other words, a target different from the target to which it is inherently bound), and / or to bind to its intended target with higher affinity, and may therefore reduce off-target effects. For example, a targeting body can allow 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 bind to myeloid cells with higher affinity.

[0342] The targeting body can be an antibody or an antigen binding fragment thereof, a diversion peptide or a diversion protein, such as a receptor binding peptide, a ligand mimetic peptide, a receptor ligand, a receptor or an interacting protein. It is expected that any type of antigen binding molecule can be used as a targeting body in principle. The non-limiting examples of the targeting body include programmed cell death protein 1 (PD1), signal regulatory protein α (SIRPa). CD40L, GP120, (specifically) an antibody or an antigen binding fragment thereof that binds CD8 (e.g., VHHCD8), a targeting body that can bind to a factor VIII related antigen, such as factor VIII, a targeting body that can bind to CD31 / PECAM-1, such as CD31, a targeting body that can bind to angiotensin converting enzyme (ACE / CD143), such as angiotensin, a targeting body that can bind to CD34, such as L-selectin or a targeting body that can bind to endoglin (CD105).

[0343] In embodiments where the stabilizer is derived from a hydrophilic polymer, polyethylene glycol (PEG), a polysaccharide, polysarcosine or poly(2-oxazoline), then these polymers have been modified with hydrophobic moieties. These may be, for example, hydrophobic polymers, hydrophobic C12-18 alkyl chains or phospholipids with chains derived from C14, C16 or C18 fatty acids. Dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), and distearoylphosphatidylethanolamine (DSPE) phospholipids are particularly suitable for this purpose because they are primary amine functionalized, so they can be linked to oligomeric or polymeric units of PEG, polysaccharides, 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 linked to an oligomeric ethylene glycol chain having 4, 6, 12, or 24 ethylene glycol units. Typically, the oligomeric glycol or PEG is terminated with a methoxy or hydroxyl group.

[0344] c. Nucleic acid

[0345] The purpose of nanoparticles described herein is to deliver nucleic acid to cells.Nucleic acid can be, for example, encoding a peptide of interest to be expressed in a cell or mRNA of a protein, or can include short nucleic acids intended to interfere with gene expression (e.g., gene silencing), such as siRNA, shRNA, or it can include components of CRISPR-Cas or related systems to induce mutations in cell genomes.Therefore, in general, the mode of action of nucleic acid (payload of nanoparticles) is in the nucleus.

[0346] Therefore, the nanoparticles preferably have at least the following characteristics:

[0347] 1) it allows targeting to the intended target cells, and

[0348] 2) It allows the payload to be delivered to where it can exert its effect (thus in most cases in the nucleus of the target cell).

[0349] Without wishing to be bound by theory, it is believed that once the nanoparticles are taken up by target cells, the multivalent molecules described herein play a role in the endosomal escape of the payload, thereby allowing nuclear delivery of the nucleic acid payload.

[0350] Many different types of RNA, DNA or synthetic oligonucleotides have been used as nucleic acid therapeutics. The present invention is not limited to a specific type of nucleic acid, as it is contemplated that the present invention is applicable to any type that can be loaded using a multivalent molecule in a nanoparticle. Thus, in one embodiment, the nucleic acid is RNA, DNA or a nucleic acid analog,

[0351] Preferably, the RNA is microRNA (miRNA), small interfering RNA (siRNA), piwi-interacting RNA (piRNA), small nuclear 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 thereof and / or a modification thereof; or

[0352] Preferably, the DNA is single-stranded or double-stranded DNA; or

[0353] Preferably, the antisense oligonucleotide is a single-stranded DNA or RNA composed of nucleotides or nucleoside analogs containing a phosphodiester backbone or a 2' ribose modification, more preferably, the nucleotides or nucleoside analogs are selected from locked nucleic acids (LNA), bridged nucleic acids (BNA), morpholinos or peptide nucleic acids (PNA).

[0354] d. Sterol

[0355] As used herein, the term sterol refers to compounds derived from sterols (2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17-hexahydro-1H-cyclopenta[a]phenanthrene-3-ol) by replacing some of the hydrogen atoms with other chemical groups, or modifying the bonds in the ring. Sterols and related compounds play an important role in the physiology of eukaryotic organisms. For example, cholesterol forms part of animal cell membranes, where it affects the fluidity of the cell membrane and acts as a second messenger in developmental signaling.

[0356] When used herein, sterol may refer to, for example, cholesterol, ergosterol, hopanes, hydroxysteroids, phytosterols, steroids, hydrogenated cholesterol or zoosterols. In nanoparticles, sterols maintain or regulate fluidity in the membrane, ie, in the phospholipid monolayer surface barrier.

[0357] In one embodiment, the sterol is selected from cholesterol, stigmasterol or β-sitosterol or a combination thereof. In one embodiment, the sterol is selected from cholesterol, ergosterol, hopanes, hydroxysteroids, phytosterols, steroids, zoosterols, stigmasterol or β-sitosterol or a combination thereof. In a preferred embodiment, the sterol is cholesterol or comprises cholesterol.

[0358] e. Phospholipids

[0359] 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, connected by a glycerol molecule. Marine phospholipids usually contain the omega-3 fatty acids EPA and DHA as part of the phospholipid molecule. The phosphate group can be modified with simple organic molecules such as choline, ethanolamine or serine.

[0360] Phospholipids are key components of all cell membranes. Due to their amphiphilic properties, phospholipids can form lipid bilayers. In eukaryotic organisms, cell membranes also contain another class of lipids, namely sterols, which are interspersed among the phospholipids. This combination provides a combined two-dimensional fluidity with mechanical strength against rupture.

[0361] Thus, in one embodiment, the phospholipid is selected from phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylglycerol or a combination thereof.

[0362] The acyl groups in the phospholipids can be derived from medium-chain or long-chain fatty acids individually. In one embodiment, at least one, preferably both, of the acyl groups in the phospholipids are derived from long-chain fatty acids, preferably wherein the long-chain fatty acids are selected from C14, C16 or C18 chains, i.e. selected from myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid and linoleic acid or a combination thereof.

[0363] Lyso-phospholipids are phospholipids in which one acyl group has been removed by hydrolysis, leaving behind an alcohol group. These molecules therefore have one fatty acid chain instead of two. These phospholipids can also be used, for example, to modulate the shape, function and fluidity of the outer layer of the nanoparticle.

[0364] In particularly preferred embodiments, the phospholipid is a neutral phospholipid, meaning that it is zwitterionic (it has a net neutral charge) at physiological pH. Thus, in preferred embodiments, the phospholipid is phosphatidylcholine (PC) or phosphatidylethanolamine (PE).

[0365] Thus, examples of phospholipids that can be used are dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylglycerol (DLPG), daptomycin (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG), dioleoylphosphatidylglycerol (DOPG), dilauroylphosphatidylethanolamine (DLPE), dimethicone (DMPG), dimethicone (DPPG), distearoylphosphatidylglycerol (DSPG), dioleoylphosphatidylglycerol (DOPG), dilauroylphosphatidylethanolamine (DLPE), dimethicone (DMPG), dimethicone (DPPG), distearoylphosphatidylglycerol (DSPG), dimethicone (DOPG), dilauroylphosphatidylethanolamine (DLPE), dimethicone (DMPG), dimethicone (DPPG), distearoylphosphatidylglycerol (DSPG), dioleoylphosphatidylglycerol ...DOPC), dimethicone (DMPG), dimethicone (DPPG), distearoylphosphatidylglycer Myristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), dilauroylphosphatidylserine (DLPS), dimyristoyl phosphate diacylserine (DMPS), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylserine (DSPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and mixtures thereof.

[0366] 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 a mixture thereof can be used.

[0367] f. Filler molecules

[0368] Nanoparticles as described herein may also and optionally include filler molecules. Filler molecules are biocompatible molecules, such as (any kind) esters or amides. Preferably esters, especially those derived from fatty acids or cholesterol, wherein the fatty acids may have saturated or unsaturated chains. Filler molecules are hydrophobic in nature and may be, for example, lipids, such as, but not limited to, triglyceride lipids. Therefore, in one embodiment, the nanoparticles also comprise filler molecules selected from triglycerides, diglycerides, esters derived from fatty acids and cholesterol esters or combinations thereof.

[0369] Nanoparticles as herein described can form nanodiscs or nanospheres, depending on the absence or presence of filler molecules, respectively. Filler can be, for example, triglycerides, which are contained in the core of the particle together with effective load (nucleic acid) and multivalent molecules. Without being bound by theory, it should be understood that comprising more fillers will make the nanoparticles become larger, and the particles may become unstable when reaching a certain degree. Without being bound by theory, comprising a certain amount of filler may help stabilize the nanoparticles, or it may stabilize the inclusion of load, or it may regulate or enhance the delivery of nucleic acid to cells.

[0370] Therefore, in one embodiment, the nanoparticles described herein further comprise filler molecules, preferably wherein the filler molecules are glyceride molecules or cholesterol esters, more preferably wherein the filler molecules are selected from triglycerides and cholesterol esters or combinations thereof. Preferably, triglycerides are derived from C6-C18 fatty acids, preferably C6-C12 fatty acids. Examples of cholesterol esters are cholesterol acetate, cholesterol caprylate and cholesterol oleate. Preferred filler molecules are tricaprylin, cholesterol acetate, cholesterol caprylate and cholesterol oleate, more preferably tricaprylin.

[0371] Nanoparticle characteristics

[0372] The nanoparticles of the present invention comprise nucleic acids, multivalent molecules, phospholipids, sterols, stabilizers and optionally filler molecules, wherein:

[0373] The amount of stabilizer, in particular apolipoprotein, apolipoprotein derivative, apolipoprotein mimetic or apolipoprotein mimetic derivative ranges from 0.1 wt % to 90 wt %; and / or

[0374] The amount of the nucleic acid ranges from 0.01 wt% to 90 wt%; and / or

[0375] The amount of the phospholipids ranges from 0.1 wt % to 95 wt %; and / or

[0376] The amount of the sterol ranges from 0.1 wt % to 95 wt %; and / or

[0377] The amount of the multivalent molecule ranges from 0.1 wt % to 95 wt %,

[0378] Optionally, fillers are present in an amount comprising a range of 0 to 95% by weight,

[0379] These weight percentages are based on the total amount of the five components plus the optional sixth filler component, that is, the five or six components add up to 100% of the weight of the nanoparticles.

[0380] In one embodiment, the amount of stabilizer (preferably apolipoprotein, an apolipoprotein derivative, an apolipoprotein mimetic or an apolipoprotein mimetic derivative) ranges from 0.2 to 50 wt %, more preferably from 0.5 to 30 wt %, more preferably from 1 to 20 wt %, even more preferably from 5 to 20 wt %, such as 5 to 15 wt %.

[0381] In one embodiment, the amount of nucleic acid ranges from 0.02% to 30% by weight, more preferably from 0.05% to 20% by weight, more preferably from 0.1% to 15% by weight, even more preferably from 0.5% to 10% by weight, such as from 0.5% to 5% by weight, from 1% to 5% by weight, or from 0.5% to 4% by weight, such as from 1.2% to 3% by weight.

[0382] In one embodiment, the amount of phospholipids ranges from 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.

[0383] In one embodiment, the amount of sterol ranges from 0.2% to 90% by weight, more preferably from 0.5% to 70% by weight, more preferably from 1% to 50% by weight, even more preferably from 3% to 30% by weight or from 5% to 25% by weight, such as from 8% to 20% by weight.

[0384] In one embodiment, the amount of multivalent molecules ranges from 0.2 wt% to 90 wt%, more preferably from 0.5 wt% to 80 wt%, more preferably from 1 wt% to 70 wt%, even more preferably from 1 wt% to 30 wt%, such as from 5 wt% to 30 wt%, from 5 wt% to 20 wt%, from 10 wt% to 20 wt%, from 5 wt% to 10 wt%, from 15 wt% to 25 wt%.

[0385] In one embodiment, the amount of optional filler molecules ranges from 0 to 90 wt%, more preferably from 0 to 80 wt%, more preferably from 0 to 70 wt%, even more preferably from 0 to 60 wt%.

[0386] The relative amount of the above-mentioned components is controlled by the ratio of their use and mixing in the nanoparticle preparation scheme, because the recovery rate of various components is usually quite high. By adopting methods known in the art, the incorporation level of various components of nanoparticles can be assessed after the particle preparation is completed. Therefore, the above-mentioned incorporation range of components in the nanoparticles can be assessed by weighing the incorporation amount of the components used or by the measured value of the components in the prepared nanoparticle formulation. For example, the level of phospholipids, cholesterol or apo A1 in the nanoparticle sample can be assessed with a commercially available assay kit. In addition, the amount of siRNA loaded and retained in the nanoparticles can be assessed using RiboGreen assay.

[0387] The outer layer of nanoparticles is composed of phospholipids, stabilizers (e.g., apolipoproteins) and sterols. In order to assemble stable nanoparticles, preferably the weight ratio of stabilizer (e.g., apolipoproteins) to phospholipids is 2:1 to 1:10. Therefore, in one embodiment, the weight ratio of apolipoproteins to phospholipids is 2:1 to 1:10, more preferably 1:1 to 1:5, even more preferably 15:1 to 1:4 or 2:3 to 1:4, such as 4:10.

[0388] In the nanoparticles, the multivalent molecules are combined with nucleic acids. The multivalent molecules are multivalent in positively ionizable and / or cationic groups (#N), while the nucleic acids carry multiple phosphate groups (P). For the nanoparticles of the present invention, the ratio (#N:P) between the number of N-groups (#N) and the number of P-groups can vary. The N group (#N): P group (P) ratio (#N:P ratio) can vary between 100:1 and 1:10. However, equimolar or excess #N-groups are preferred, and the #N:P ratio varies from about 50:1 to about 1:1. More preferably, the #N:P ratio is about 20:1 to about 1:1. Even more preferably, the #N:P ratio is about 15:1 to about 1:1.

[0389] Nanoparticles according to the present invention are found to have relatively defined and constant sizes. The average size depends primarily on the core component, i.e. the amount and type of nucleic acid, the amount of multivalent molecules and the amount of sterol and filler molecules. It should be understood that filler molecules are optional and the particle size can be increased by comprising the amount of increased filler molecules. In one embodiment, 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.

[0390] The size of nanoparticles of the present invention can be assessed by methods known in the art. For example, dynamic light scattering (DLS) can be adopted to measure the diameter of nanoparticles. Low temperature TEM measurement can also be used for this purpose. These two technologies can also be used to assess the diameter distribution of the nanoparticle formulations prepared. In addition, low temperature TEM measurement can also be used to assess the shape (round sphere or other shapes) of nanoparticles. It can also assess the further features of nanoparticles (such as the presence of an outer layer or layer; the presence of apolipoprotein; the uniformity of nanoparticle outward appearance).

[0391] The nanoparticles of the present invention may have a certain surface charge, which may be (slightly) negative, (slightly) positive or substantially neutral. Zeta potential analysis or other methods known in the art may be used to assess the charge state of the nanoparticles of the present invention.

[0392] The in vitro activity of the nanoparticles containing siRNA of the present invention can be assessed using dual reporter cells (e.g., RAW264.7 macrophages) transfected with Pmir-Glo plasmids containing firefly luciferase and Renilla luciferase gene expression sequences. In a silencing experiment, macrophages are exposed to nanoparticles loaded with firefly luciferase siRNA or nanoparticles loaded with nonspecific siRNA. Next, one can assess and compare any one of these nanoparticles to low luciferase activity. In a dose-dependent gene knockdown study, one can assess firefly luciferase activity as a function of the firefly luciferase siRNA concentration used. The assay reports the effectiveness of the nanoparticle formulations employed.

[0393] Nanoparticles defined herein comprise a hydrophobic core and a hydrophilic surface (due to the phospholipids and stabilizer components incorporated), and therefore can be dissolved in water or aqueous solutions such as saline solutions or buffers. The inherent properties produced by the components of the nanoparticles defined by the present invention cause the nanoparticles to be stable in suspension for several months. Suitable aqueous buffers are known in the art, such as phosphate buffered saline (PBS), Tris buffered saline (TBS). Suitable saline solutions are known, and non-limiting examples include aqueous solutions of NaCl or KCl. When it is intended that nanoparticles be administered to a subject, for this purpose, the nanoparticles should be suspended in a physiologically acceptable carrier. For example, if the nanoparticles are intended for intravenous delivery, the physiologically acceptable carrier is typically a fluid isotonic with blood. For example, sodium chloride solution, 5% w / v glucose solution, Ringer's solution, Ringer's lactate or Ringer's acetate at a concentration of 0.9% w / v can be used, but other suitable carriers are known.

[0394] Therefore, in one aspect, the present invention relates to a composition comprising a nanoparticle according to the present invention and a physiologically acceptable carrier. In one embodiment, the composition is a pharmaceutical composition. It should be understood that the composition can also include other components, such as but not limited to drugs or biopharmaceuticals. For the combined therapy of nucleic acid (contained in the nanoparticle) and the drug, this may be an attractive option. The drug can be a small compound, an antibody or an antigen binding fragment, a further nanoparticle, etc.

[0395] It is further contemplated that the nanoparticles described herein are used to deliver nucleic acid therapy to a subject.Thus, in one aspect, the present invention relates to a nanoparticle according to the present invention or a composition according to the present invention for use as a medicament.

[0396] It should be understood that the nucleic acid therapy comprising nanoparticles can be applied to a subject in need thereof. Depending on the target cell or tissue, the administration can be parenteral, such as intravenous, intramuscular or subcutaneous. Administration can also be oral, sublingual, topical, rectal, nasal (inhalation) or vaginal. In addition, the targeting of the target tissue or cell is determined by rationally selecting apolipoprotein. In one embodiment, the use of the nanoparticle or composition according to the present invention includes delivering nucleic acid to a myeloid compartment or spleen. This can be achieved, for example, by intravenous parenteral administration. Preferably, apolipoprotein is preferably an apolipoprotein such as apo A1 targeting a myeloid compartment.

[0397] Another aspect provides a nanoparticle as taught herein or a composition as taught herein for use in immunotherapy.

[0398] In one aspect, the present invention relates to nanoparticles according to the present invention or compositions according to the present invention, which are used to treat diseases by stimulating or inhibiting innate immune responses, preferably wherein the disease is a disease that will benefit from stimulating or inhibiting the innate immune response of the subject, such as a disease characterized by a defect in innate immune response, more preferably wherein the disease to be treated is cancer, cardiovascular disease, autoimmune disorder or xenograft rejection. Therefore, the nanoparticles according to the present invention can be used to treat any disease associated with the immune system, such as any immune disorder, or for treating any disease or disorder in which regulating the immune response is considered a viable treatment option. By targeting the myeloid compartment, nucleic acid therapy can be successfully delivered to progenitor cells of different blood cell types, rather than already differentiated cells present in blood and tissues, such as T cells and macrophages. When doing so, the innate immune response can be regulated, for example, stimulated or inhibited by nucleic acid therapy, depending on the desired result. For example, in (prevention) autoimmune disorders, cardiovascular diseases or xenograft rejection, it is desirable to suppress the autoimmune response, and in cancer, it is desirable to stimulate the immune response to the target cancer cells.

[0399] In another aspect, the present invention relates to a method for delivering a nucleic acid in vivo, the method comprising administering to a subject a nanoparticle according to the present invention or a composition according to the present invention.

[0400] On the other hand, the present invention relates to a method for treating a disease or condition in a subject in need thereof by stimulating or inhibiting an innate immune response, the method comprising administering to the subject a therapeutically effective amount of nanoparticles according to the present invention or a composition according to the present invention. In a specific embodiment, the disease or condition is a disease or condition characterized by a defective innate immune response. In one embodiment, the disease or condition is selected from cancer, cardiovascular disease, autoimmune disease, or xenograft rejection.

[0401] The invention provides nanoparticles (aNPs) based on apolipoprotein with nucleic acid. Up to now, it is impossible to include nucleic acid in this type of nanoparticle, because the core of this type of particle is hydrophobic, and therefore due to the hydrophilic nature of nucleic acid and not suitable for incorporation therein. Although it has been described that some multivalent molecules are used together with nucleic acid as a tool for intracellular delivery of nucleic acid, simply multivalent molecules and nucleic acid and other lipid components are combined and can not cause the formation of lipid nanoparticles as described herein. The present invention is around such understanding, that nucleic acid can be incorporated into nanoparticles by using multivalent molecules described herein and by using two-step nanoparticle preparation methods.

[0402] Therefore, in one aspect, the present invention relates to a method for producing nanoparticles comprising the following steps:

[0403] a) mixing a (lipid) component in an organic solvent with nucleic acids in an aqueous buffer, preferably rapidly, to produce nanoparticles, wherein the (lipid) component comprises a phospholipid, a sterol, a multivalent molecule and optionally a filler molecule (e.g. a glyceride, such as a triglyceride); and wherein the aqueous buffer has a pH of 5.5 or less, preferably 5.0 or less; and

[0404] b) mixing the lipid nanoparticles with a stabilizer, preferably an apolipoprotein, an apolipoprotein derivative, an apolipoprotein mimetic and / or an apolipoprotein mimetic derivative, preferably rapidly mixing to produce nanoparticles at a pH between 5.5 and 9.0, preferably between 6.0 and 8.0, more preferably between 6.5 and 8.0.

[0405] Rapid mixing is known in the art and has been described in, for example, Hirota et al. BIOTECHNIQUESVOL. 27, NO. 2, p286-289; Jeffs et al., Pharm Res 22, 362–372 (2005); Kulkarni et al., ACS Nano 2018, 12, 5, 4787–4795.

[0406] The aqueous buffer in step a) has a relatively low pH to ensure that the multivalent molecule is positively charged, thereby allowing the nucleic acid to bind to the multivalent molecule in the particle. For example, the buffer may have a pH of 5.5 or lower, such as 5.4, 5.3, 5.2, 5.1, or the buffer may have a pH of 5.0 or lower, such as 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 may be any buffer that does not damage the nucleic acid. An exemplary buffer is sodium acetate at pH 4.0. Next, the nanoparticle is placed in an aqueous buffer having a pH of about 6 to 8, preferably 7 to 8, more preferably about 7.4. For example, this may be achieved by dialyzing using an aqueous buffer in a specified pH range. A non-limiting example of an aqueous buffer suitable for this step is 150 mM PBS at pH 7.4, but it should be understood that any buffer that does not damage the nucleic acid may be used.

[0407] In step b), the nanoparticles in an aqueous buffer at a pH of 6 to 8, preferably 7 to 8, are rapidly mixed with the apolipoprotein in an aqueous buffer at a pH of 6 to 8, preferably 7 to 8, to obtain the nanoparticles according to the invention.

[0408] In one aspect, the present invention relates to nanoparticles, such as nanoparticles according to the present invention, which are obtained or obtainable by a method for producing nanoparticles as taught herein.

[0409] It should be understood that nanoparticles according to the present invention can deliver nucleic acid to target cells or tissues. Target cells or tissues can be in the subject's body, or can be in vitro or ex vivo. Therefore, in one aspect, the present invention relates to an in vivo, in vitro or ex vivo method for introducing nucleic acid into cells, the method comprising contacting the nanoparticles according to the present invention or the composition according to the present invention with the cells. In one embodiment, the method is an in vitro or ex vivo method. In a specific embodiment, the cell is a cell of a myeloid compartment or a myeloid cell.

[0410] In another aspect, the present invention relates to a method for delivering a nucleic acid in vivo, the method comprising administering to a subject a nanoparticle according to the present invention or a composition according to the present invention.

[0411] On the other hand, the present invention relates to a method for treating a disease or disorder in a subject in need thereof by stimulating or inhibiting an innate immune response, the method comprising administering to the subject a therapeutically effective amount of nanoparticles according to the present invention or a composition according to the present invention. In one embodiment, the disease is selected from cancer, cardiovascular disease, autoimmune disorders or xenograft rejection.

[0412] The present application also provides aspects and embodiments shown in the following statements:

[0413] Statement 1. A nanoparticle comprising a core and an outer layer, wherein the core comprises: - a nucleic acid;

[0414] - multivalent molecules;

[0415] And wherein the outer layer comprises:

[0416] - Stabilizers;

[0417] - phospholipids;

[0418] - sterols; and optionally

[0419] - filler molecules,

[0420] Wherein the multivalent molecule has formula (I):

[0421] [nuclear] x –[BU] y –[TU] z (I)

[0422] wherein the core is nitrogen or a C1-C1-C2-containing 1 to 15 nitrogen heteroatoms and optionally 1 to 4 oxygen heteroatoms. 18 Straight chain, branched chain or cyclic groups; and

[0423] wherein x represents the number of connections from the core to a branching unit BU (or to a terminal unit TU when y=0), wherein x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 12, and wherein these connections all originate from nitrogen atoms of the core and carbon atoms of the branching unit BU (or the terminal unit TU); and

[0424] The branching unit BU has the formula (IIa), (IIb), (IIc) or (IId):

[0425]

[0426] wherein (IIa) represents an n-alkylene-nitrogen spacer which is connected at the CH2-terminus to the core or to the BU closer to the core and which is connected at the N-terminus to two terminal units (TU) or to two branching units (BU) closer to the TU, wherein the connections are given in the form of wavy bonds, and wherein p is 1, 2, 3 or 4; and

[0427] wherein (IIb) represents an amide containing an n-alkylene-nitrogen spacer, which is connected at the CH2-terminus to the core or to the BU closer to the core, and which is connected at the N-terminus to two terminal units (TU) or to two branching units (BU) closer to the TU, wherein the connections are given in the form of a wavy bond, and wherein q is 1, 2, 3, 4 or 5, and wherein R1 is hydrogen, methyl, ethyl, n-propyl or isopropyl; and

[0428] wherein (IIc) and (IId) are as defined for (IIa) and (IIb), respectively, and wherein R2 is methyl, ethyl, n-propyl, benzyl, acetamide (-CH2-C(O)NH2) or 2-hydroxy-vinyl group, and wherein X - is the counter anion of the quaternary amine cation portion; and

[0429] wherein y represents a specific and discrete generation number of the multivalent molecule, wherein the number represents how many consecutive BU layers are incorporated into the multivalent molecule, as counted proceeding from the inner core to the outer TU groups, and wherein y is 0, 1, 2, 3, 4, or 5; and

[0430] wherein for each individual generation layer, the BU may be independently selected from formula (IIa), (IIb), (IIc) or (IId), i.e., the first BU emanating from the core (representing the first generation layer BU) may be different from the second BU emanating further away from the core (representing the second generation layer BU), and the second BU may be different from the third, fourth and fifth BU emanating further away from the core; and

[0431] wherein the terminal units (TU) are individually and independently selected from hydrogen and C1-C1 optionally containing 1 to 8 heteroatoms. 30 an alkyl, aryl, arylene-alkyl or alkylene-aryl group, wherein the heteroatoms are individually and independently selected from O and N, provided that not all TU in the multivalent molecule are hydrogen; and

[0432] wherein z represents the total number of TU groups attached to the multivalent molecule, wherein z is from 1 to 128; and

[0433] wherein the multivalent molecule according to formula (I) is multivalent in the positively ionizable groups and / or cationic groups, wherein N represents the cumulative number of positively ionizable groups and cationic groups in the multivalent molecule, and wherein N is 2 or higher.

[0434] Statement 2. The nanoparticle of statement 1, wherein the multivalent molecule has formula (I):

[0435] [nuclear] x –[BU] y –[TU] z (I)

[0436] Where y=0, 1, 2 or 3.

[0437] Statement 3. A nanoparticle according to any of the preceding statements, wherein the multivalent molecule is a first, second or third generation dendrimer selected from poly(propyleneimine) (PPI) dendrimers or polyamidoamine (PAMAM) dendrimers, most preferably PPI dendrimers, or modifications thereof.

[0438] Statement 4. A nanoparticle according to any of the preceding statements, wherein the multivalent molecule has a structure selected from the group consisting of:

[0439] or

[0440] or

[0441]

[0442] wherein the terminal units (TU) are individually and independently selected from hydrogen and C1-C1 optionally containing 1 to 8 heteroatoms. 30 Alkyl, aryl, arylene-alkyl or alkylene-aryl groups, the heteroatoms being individually and independently selected from O and N, with the proviso that not all TU in the multivalent molecule are hydrogen.

[0443] Statement 5. The nanoparticle according to any of the preceding statements, wherein the stabilizer is:

[0444] - an apolipoprotein, an apolipoprotein derivative, an apolipoprotein mimetic and / or an apolipoprotein mimetic derivative;

[0445] - Polyethylene glycol or polyethylene glycol-modified molecules;

[0446] - hydrophilic polymers;

[0447] - polysaccharides or polysaccharide-modified molecules; or

[0448] - Polysarcosine or polysarcosine-modified molecules.

[0449] Statement 6. A nanoparticle according to any of the preceding statements, wherein the apolipoprotein, apolipoprotein derivative, apolipoprotein mimetic or apolipoprotein mimetic derivative is selected from apo A1, apo A1-Milano, apo A2, apo A4, apo A5, apo B48, apo B100, apo CI, apo C-II, apo C-III, apo C-IV, apo D, apoE, apo F, apo H, apo L and apo M or a mimetic or derivative thereof,

[0450] Preferably, it is selected from apo A1, apo A2, apo A4, apo A5, apo B100, apo CI, apo C-II, apoC-III, apo C-IV and apo E or a mimetic or derivative thereof,

[0451] More preferably, it is selected from apo A1, apo A4, apo A5, apo B100, apo C-III and apo E or a mimetic or derivative thereof,

[0452] Most preferably, it is selected from apo A1, apo B100 and apo E or a mimetic or derivative thereof.

[0453] Statement 7. A nanoparticle according to any of the preceding statements, wherein the nucleic acid is RNA, DNA or a nucleic acid analog,

[0454] Preferably, the RNA is microRNA (miRNA), small interfering RNA (siRNA), piwi-interacting RNA (piRNA), small nuclear 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 modification thereof; or

[0455] Preferably, the DNA is single-stranded or double-stranded DNA; or

[0456] Preferably, the nucleic acid is an antisense oligonucleotide as a single-stranded DNA or RNA, which consists of or comprises nucleotides or nucleoside analogs containing a phosphodiester backbone or a 2' ribose modification, more preferably, wherein the nucleotides or nucleoside analogs are selected from locked nucleic acids (LNA), bridged nucleic acids (BNA), morpholinos or peptide nucleic acids (PNA).

[0457] Statement 8. A nanoparticle according to any of the preceding statements, wherein the sterol is preferably selected from sterol, cholesterol, ergosterol, hopanoid, hydroxysteroid, phytosterol, steroid, zoosterol, stigmasterol or β-sitosterol, or a combination thereof.

[0458] Statement 9. A nanoparticle according to any of the preceding statements, wherein:

[0459] The phospholipid is selected from phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylglycerol or a combination thereof, preferably, wherein at least one, more preferably both, of the acyl groups in the phospholipid are derived from long-chain fatty acids, even more preferably, wherein the long-chain fatty acids are selected from lauric acid, lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid and linoleic acid, or a combination thereof.

[0460] Statement 10. A nanoparticle according to any of the preceding statements, further comprising a filler molecule, preferably wherein the filler molecule is a glyceride molecule, more preferably wherein the filler molecule is selected from triglycerides, modified triglycerides and cholesterol esters, or a combination thereof, preferably wherein the triglyceride is derived from a C6-C18 fatty acid, preferably tricaprylin and / or wherein the cholesterol ester is cholesterol acetate, cholesterol caprylate and / or cholesterol oleate.

[0461] Statement 11. A nanoparticle according to any of the preceding statements, wherein:

[0462] The amount of the apolipoprotein, apolipoprotein derivative, apolipoprotein mimetic or apolipoprotein mimetic derivative ranges from 0.1 wt % to 90 wt %; and / or

[0463] The amount of the nucleic acid ranges from 0.01 wt % to 90 wt %;

[0464] The amount of the phospholipids ranges from 0.1 wt % to 95 wt %; and / or

[0465] The amount of the sterol ranges from 0.1 wt % to 95 wt %; and / or

[0466] The amount of the multivalent molecule ranges from 0.1 wt % to 95 wt %,

[0467] Optionally, fillers are present in an amount comprising a range of 0 to 95% by weight,

[0468] The weight percentages are based on the total amount of the five components plus the optional sixth filler component, that is, the five or six components add up to 100% of the weight of the nanoparticles.

[0469] Statement 12. A composition comprising a nanoparticle according to any one of the preceding statements and a physiologically acceptable carrier, preferably wherein the composition is a pharmaceutical composition.

[0470] Statement 13. The nanoparticle according to any one of statements 1 to 11 or the composition according to statement 12 for use as a medicament.

[0471] Statement 14. The nanoparticle or composition for use according to statement 13, wherein the use comprises delivering a nucleic acid to the myeloid compartment or the spleen.

[0472] Statement 15. A method of producing nanoparticles comprising the steps of:

[0473] a) mixing a (lipid) component in an organic solvent with nucleic acids in an aqueous buffer, preferably rapidly, to produce nanoparticles, wherein the (lipid) component comprises a phospholipid, a sterol, a multivalent molecule and optionally a filler molecule; and wherein the aqueous buffer has a pH of 5.5 or less, preferably 5.0 or less; and

[0474] b) mixing the lipid nanoparticles with a stabilizer, preferably an apolipoprotein, an apolipoprotein derivative, an apolipoprotein mimetic and / or an apolipoprotein mimetic derivative, preferably rapidly mixing to produce nanoparticles at a pH between 5.5 and 9.0, preferably between 6.0 and 8.0, more preferably between 6.5 and 8.0.

[0475] All cited references are incorporated herein by reference in their entirety.

[0476] Example

[0477] Example 1 - Design and synthesis of multivalent molecules.

[0478] An overview of the characteristics of the prepared multivalent molecules is provided in Tables S1 and S2. Figure 1 , 2 and 3 provide molecular structures of a series of multivalent molecules.

[0479] Table S1. Characteristics of multivalent molecules.

[0480]

[0481] MW = molecular weight; #N = number of ionizable and / or cationic groups in the material; BU = branching unit as defined in formula (II); x, y and z are as in formula (I). Unless otherwise indicated, all (IIa) have p = 1. All (IIb) and (IIc) have q = 1 and p = 1, respectively. (IIc) is a methylated dendrimer with a chloride or iodide counter anion, as indicated by MeCl or MeI.

[0482] Table S2. Characteristics of multivalent molecules.

[0483]

[0484]

[0485] MW = molecular weight; #N = number of ionizable and / or cationic groups in the material; BU = branching unit as defined in formula (II); x, y and z are as in formula (I). Unless otherwise indicated, all (IIa) have p = 1. All (IIb) and (IIc) have q = 1 and p = 1, respectively. (IIc) is a methylated dendrimer with a chloride or iodide counter anion, as indicated by MeCl or MeI.

[0486] Materials and methods

[0487] All reagents, chemicals, materials and solvents were obtained from commercial sources and used as received. All solvents were of AR quality. DCM = dichloromethane; CHCl3 = 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 the synthetic procedures, equivalents (eq) are molar equivalents.

[0488] The results were obtained on a Bruker Avance III HD spectrometer at 298 K ( 1 H-NMR and 13 NMR spectra were recorded at 400 MHz and 100 MHz for C-NMR, respectively. Chemical shifts are reported in ppm downfield relative to TMS at room temperature. The abbreviations used for splitting patterns are s=singlet, t=triplet, q=quartet, m=multiplet, and br=broad.

[0489] HPLC-PDA / ESI-MS was performed using a Shimadzu LC-10AD VP series HPLC coupled to a photodiode array detector (Finnigan Surveyor PDA Plus detector, Thermo Electron Corporation) and an ion trap mass spectrometer (LCQ Fleet, Thermo Scientific), using electrospray ionization (ESI). HPLC analysis was performed using an Alltech Alltima HP C18 3μ column, with an injection volume of 1-4μL, a flow rate of 0.2mL / min, and typically, a gradient of MeCN in H2O (both containing 0.1% formic acid) at 298K (5% to 100% in 10 minutes, maintained at 100% for 3min).

[0490] MALDI-TOF-MS was measured 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) matrices.

[0491] Building Blocks

[0492]

[0493] Synthesis of G1 poly(butylene imine) dendrimer. (a) Potassium phthalimide, DMF, 48 h, rt; (b) 1,4-diaminobutane, K2CO3, MeCN, 40 h, reflux; (c) N2H4·H2O, EtOH, 16 h, reflux.

[0494] N-(4-Bromobutyl)phthalimide

[0495] 1,4-dibromobutane (86.4g, 0.4mol, 4eq) and potassium phthalimide (18.5g, 0.1mol, 1eq) were dissolved in DMF (75mL). (Potassium phthalimide was not completely dissolved). The reaction mixture was stirred at room temperature for 48 hours. The reaction mixture was then poured into water (600mL), transferred to an extraction funnel and extracted with ether (2×50mL). The combined ether layer was washed with a saturated KCl solution (2×25mL). The ether layer was dried over Na2SO4, the suspension was filtered, and the filtrate was concentrated in vacuo. The product was purified by silica column chromatography using a mixture of heptane / DCM (0-100% DCM) to give a white solid. The final impurities were removed by stirring the solid in n-pentane for 16 hours at room temperature, filtering, washing, and vacuum drying the residue to give the product (14.9g 52.7mmol, 53%). Alternatively, the crude product can be purified by recrystallization from n-heptane.

[0496] 1 H 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).

[0497] N,N,N',N'-Tetrakis(4-phthalimidobutyl)-1,4-diaminobutane

[0498] N-(4-bromobutyl)phthalimide (1.41 g, 5 mmol, 5 eq), 1,4-diaminobutane (88 mg, 1 mmol, 1 eq) and K2CO3 (691 mg, 5 mmol, 5 eq) were mixed together in MeCN (5 mL) and stirred under reflux for 40 hours. The reaction mixture was cooled, the suspension was filtered, and the filtrate was concentrated in vacuo. The product was purified by silica column chromatography, eluting with a mixture of MeOH / CHCl3 (0-10% MeOH) to give an amber viscous oily product (222 mg, 0.25 mmol, 25%).

[0499] 1 H 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). 13C NMR (100 MHz, CDCl3) 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 calculated for C 52 H 56 N6O8892.42;Obs. 893.58[M+H] + 447.42,[M+H] 2+ .

[0500] N,N,N',N'-Tetrakis(4-aminobutyl)-1,4-diaminobutane(G1-PBuI)

[0501] Hydrazine hydrate (0.96 mL, 19.8 mmol, 20 eq) was added to a stirred suspension of N,N,N',N'-tetrakis(4-phthalimidobutyl)-1,4-diaminobutane (884 mg, 0.99 mmol, 1 eq) in ethanol (30 mL). The reaction mixture was heated to reflux for 16 hours to give a white suspension. The suspension was filtered through a glass sinter funnel and the residue was washed with ethanol. The filtrate was concentrated in vacuo to give a white viscous material (100 mg, 0.27 mmol, 27%), which was a mixture of the product and 2,3-dihydrophthalazine-1,4-dione.

[0502] 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 calculated for C 20 H 48 N6 372.39; Obs.373.50[M+H] + .

[0503]

[0504] Synthesis of G1 poly(pentadienyl imine) dendrimer. (D) 1,4-diaminobutane, K2CO3, MeCN, 40h, reflux; (e) N2H4·H2O, EtOH, 16h, reflux; (f) 2-propanol, 60℃, 112h.

[0505] N,N,N',N'-Tetrakis(5-phthalimidopentyl)-1,4-diaminobutane

[0506] N-(5-bromopentyl)phthalimide (14.8 g, 50 mmol, 5 eq), 1,4-diaminobutane (882 mg, 10 mmol, 1 eq) and K2CO3 (12 mg, 80 mmol, 8 eq) were mixed in MeCN (50 mL) and stirred under reflux for 40 hours. The reaction mixture was cooled, the suspension was filtered, and the filtrate was concentrated in vacuo. The product was purified by silica column chromatography, eluting with a mixture of MeOH / CHCl3 (0-10% MeOH) to give an amber very viscous oily product (3.12 g, 3.28 mmol, 33%).

[0507] 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, CDCl3) 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 calculated for C 56 H 64 N6O8 948.48; Obs.949.58[M+H] + 475.50,[M+H] 2+ .

[0508] N,N,N',N'-Tetrakis(5-aminopentyl)-1,4-diaminobutane(G1-PPeI)

[0509] Hydrazine hydrate (3.08 g, 61.5 mmol, 20 eq) was added to a stirred suspension of N,N,N',N'-tetrakis(5-phthalimidopentyl)-1,4-diaminobutane (2.92 g, 3.07 mmol, 1 eq) in ethanol (50 mL). The reaction mixture was heated at reflux for 16 h to give a white suspension. The suspension was filtered through a glass sinter funnel and washed with ethanol. The filtrate was concentrated in vacuo and redissolved in chloroform to give a suspension. The suspension was filtered and the filtrate was concentrated in vacuo. To remove the last traces of 2,3-dihydrophthalazine-1,4-dione, the residue was redissolved in a mixture of n-pentane / chloroform 1:1, filtered and the filtrate was concentrated in vacuo to give the product as a pale yellow viscous oil (950 mg, 2.22 mmol, 72%).

[0510] 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 calculated for C 24 H 56 N6 428.46; Obs.429.58[M+H] + .

[0511]

[0512] Synthesis of tert-butyl (3-(N-methylacrylamide)propyl) carbamate. (a) (Boc)2O, DCM, 0°C to room temperature, 72h, 95%; (b) 33w / w% methylamine in MeOH, KI, EtOH, 50°C, 20h, 63%; (c) acryloyl chloride, TEA, DCM, 0° to room temperature, 16h, 68%.

[0513] tert-Butyl (3-bromopropyl) carbamate

[0514] Triethylamine (4.2 g, 41.3 mmol, 1.05 eq) was added to an ice-cold solution of 3-bromopropylamine hydrobromide (8.6 g, 39.3, 1 eq) in MeOH (40 mL). Di-tert-butyl carbonate was then added to the reaction mixture in portions. The temperature of the reaction mixture was slowly raised to room temperature and stirred for another 16 hours. The mixture was then concentrated in vacuo, redissolved in a mixture of dichloromethane and water, transferred to a separatory funnel and the aqueous layer was extracted with dichloromethane (3x). The collected dichloromethane layer was washed with 0.5 M citric acid solution (1×25 mL) and brine (1×25 mL). The dichloromethane layer was dried over Na2SO4 and the suspension was filtered. The solvent was evaporated in vacuo to give the product as a colorless oil (8.86 g, 37.2 mmol, 95%).

[0515] tert-Butyl (3-(methylamino)propyl) carbamate

[0516] To a stirred solution of tert-butyl (3-bromopropyl) carbamate (8.86 g, 37.2 mmol, 1 eq) in ethanol (30 mL) was added 40% w / w methylamine in MeOH (45 mL, 446 mmol, 10 eq) and KI (0.74 g, 4.46 mmol, 0.1 eq) and the temperature was raised to 50°C. After 20 hours at 50°C, the reaction mixture was concentrated in vacuo. The residue was redissolved in DCM and water and the aqueous layer was extracted with DCM (3×25 mL). The collected DCM layer was dried over Na2SO4, the suspension was filtered and the filtrate was concentrated in vacuo to give a colorless oily byproduct. The aqueous layer was basified to pH>10 with 6M NaOH solution and extracted with CHCl3 (3×50 mL). The collected CHCl3 layer was dried over Na2SO4 and the suspension was filtered. The filtrate was concentrated in vacuo to give a colorless oil (4.84 g, 25.7 mmol, 58%).

[0517] 1 H 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 calculated for C9H 20 N2O2 188.15. Measured [M+H] + 189.17.

[0518] tert-Butyl (3-(N-methylacrylamido)propyl) carbamate

[0519] A solution of acryloyl chloride (2.36 g, 26.1 mmol, 1.1 eq) in DCM (10 mL) was added dropwise to an ice-cold 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 addition, the reaction temperature was allowed to rise to room temperature. The reaction mixture was stirred at room temperature for 20 hours. The reaction mixture was transferred to an extraction funnel and the DCM layer was washed with water (1×25 mL), 1M KHSO4 solution (1×25 mL), water (1×25 mL), 1M NaHCO3 solution and water (1×25 mL). The DCM layer was dried over Na2SO4, the suspension was filtered and the filtrate was concentrated in vacuo 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 to 5% MeOH in DCM) to afford a light yellow oil (3.85 g, 15.9 mmol, 68%).

[0520] 1 H 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 calculated for C 12 H 22 N2O3 242.16. Measured [M+Na] + 265.17.

[0521]

[0522] Synthetic oil-based amino acid building blocks.

[0523] 2,5-Dioxopyrrolidin-1-yl oleate (Oleic acid-NHS ester)

[0524] N-hydroxy-succinimide (3.37 g, 29.3 mmol, 1.14 eq) and DIPEA (8.0 mL) were dissolved in THF (60 mL) under a nitrogen atmosphere. A solution of oleyl chloride (7.74 g, 25.7 mmol, 1 eq) 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 NaOH aqueous solution (500 mL). The aqueous layer was extracted with chloroform (100 mL). The combined chloroform fractions were washed with brine (200 mL). Purified by silica column chromatography (eluted with chloroform: ethyl acetate 80:20). Yield: 6.0 g (61%) white solid.

[0525] 1 H-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 reflection mode): Obs. m / z = (M+H) + 380.28. Calculated: C 22 H37 NO4 (exact mass 379.27; molecular weight 379.54).

[0526] N 6 -(tert-Butyloxycarbonyl)-N 2 -Oleoyl-L-lysine

[0527] N-ε-tert-Butoxycarbonyl-L-lysine (551 mg, 2.23 mmol, 1.0 eq) was dissolved in DCM (17 mL) in an inert nitrogen atmosphere. 2,5-Dioxopyrrolidine-1-yl oleate (851 mg, 2.21 mmol, 0.99 eq) and triethylamine (0.70 mL; 2 eq) were added and stirring was continued for 19 hours. DCM (50 mL) was added and the organic phase was washed with 1M KHSO4 aqueous solution (3×50 mL), water (3×50 mL) and brine (100 mL). After evaporation to dryness, the crude product was purified by silica column chromatography using chloroform: methanol: acetic acid (10:10:1) eluent. Yield: 813 mg (71%) white solid.

[0528] 1 H 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 reflection mode): Obs. m / z = (M+Na) + 533.39. Calculated: C 29 H 54 N2O5 (exact mass 510.40; molecular weight 510.74).

[0529] 2,3,5,6-Tetrafluorophenyl N 6 -(tert-Butyloxycarbonyl)-N 2 -Oleoyl-L-lysine ester

[0530] N 6 -(tert-Butyloxycarbonyl)-N 2-oleoyl-L-lysine (623 mg, 1.21 mmol, 1.00 eq) and 2,3,5,6-tetrafluorophenol (302 mg, 1.82 mmol, 1.50 eq) were dissolved in DCM (10 mL) and stirred under nitrogen. The clear solution was cooled with an ice / water bath and EDC.HCl (302 mg, 1.57 mmol, 1.3 eq) was added. After 18 hours, chloroform (75 mL) was added and the solution was washed with water (4×50 mL), saturated sodium bicarbonate solution (50 mL), then with water (50 mL) and brine (50 mL). Purification was performed by silica column chromatography, eluting with 9:1 chloroform:ethyl acetate. Yield: 470 mg (59%) white solid.

[0531] 1 H-NMR (400MHz, chloroform-d) δ7.02(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 .6Hz,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=6.9Hz,3H). 19 F-NMR (chloroform-d) δ -138.6 (m, 2F), -152.4 (m, 2F).

[0532] N 2 -(tert-Butyloxycarbonyl)-N 6 -Oleoyl-L-lysine

[0533] (tert-Butoxycarbonyl)-L-lysine (245mg, 0.99mmol) was stirred with dry DCM (6mL) under a nitrogen atmosphere. 2,5-dioxopyrrolidine-1-yl oleate (380mg, 1.0mmol) was added, followed by triethylamine (202mg, 2.0mmol), and the turbid mixture became clear during stirring overnight. Chloroform (50mL) was added, and the mixture was washed with 1M KHSO4 aqueous solution (20mL), water (20mL), brine (50mL), and dried over magnesium sulfate. It was purified by silica column chromatography using chloroform: methanol 20: 1 as eluent. Yield: 342mg (67%) oil.

[0534] 1H 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).

[0535] 2,3,5,6-Tetrafluorophenyl N 2 -(tert-Butyloxycarbonyl)-N 6 -Oleoyl-L-lysine ester

[0536] In a nitrogen atmosphere 2 -(tert-Butyloxycarbonyl)-N 6 -oleoyl-L-lysine (349 mg, 0.67 mmol) and 2,3,5,6-tetrafluorophenol (205 mg, 1.23 mmol, 1.85 eq) in DCM (15 mL) were added EDC.HCl (169 mg, 0.94 mmol, 1.41 eq). After 21 hours, DCM (50 mL) was added and the organic phase was washed with saturated aqueous sodium bicarbonate solution (3×50 mL) and then with brine (50 mL). The solution was evaporated to dryness and the crude product was purified by silica column chromatography using a chloroform: ethyl acetate gradient from 10: 1 to 20: 7. Yield: 295 mg (67%) white powder.

[0537] 1 H NMR(400MHz, CDCl3)δ7.02(m,1H),5.49(t,1H),5.40-5.25(m,2H),5.17(m,1H),4.62and 4.44(two s,1H),3.29(m,2H),2.16(t,J=9.7Hz,2H),2.00(m,4H),1.89(m,2H),1.65-1.15(m,31H),0.88(t,J=6.9Hz,3H).

[0538] Oleoyl-L-arginine

[0539] THF (32 mL) was added to a vigorously stirred aqueous solution (32 mL) of arginine (1.09 g, 6.26 mmol, 1.00 eq). Sodium bicarbonate (896 mg, 10.6 mmol, 1.69 eq) was added and a clear solution was obtained after 15 minutes. A solution of 2,5-dioxopyrrolidine-1-yl oleate (2.64 g, 6.95 mmol, 1.11 eq) in THF (32 mL) was added dropwise over a period of 2 hours. Stirring was continued for 23 hours to obtain a clear solution. THF was removed on a rotary evaporator, and the resulting aqueous phase was transferred to a separatory funnel. KHSO4 aqueous solution (1.49 g, 260 mL) was added and shaken with chloroform (590 mL). The pH of the aqueous layer was 3. The chloroform phase was shaken with brine (300 mL). Chloroform was removed on a rotary evaporator to obtain a crude mixture (3.05 g). By using Büchi 80g HP column on GraceReveleris X2 Flash chromatography system (ELSD detection) with chloroform: methanol 1: 1 elution, its column chromatography purification. The product is dissolved in THF and concentrated (twice: 120mL and 80mL THF) to remove residual methanol (must be treated with HCl before). Yield: 2.0g (TLC, NMR and MALDI-TOF confirmed that the product is pure). Preparation of HCl salt: The product is dissolved with DCM (90mL) and THF (20mL) by stirring in an argon atmosphere, and then cooled on an ice / water bath. 2M HCl in ether (4.5mL) is added dropwise within 5 minutes to obtain a clear solution. The solvent is removed on a rotary evaporator and the residue is vacuum dried. Yield: 2.09g white foam.

[0540] 1 H NMR (400 MHz, chloroform-d+TFA) δ7.13 (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=6.9 Hz, 3H). MALDI-TOF-MS (CHCA matrix, positive reflection mode): Obs. m / z=(M+H)+439.36. Calculated: C 24 H 46 N4O3 (exact mass: 438.36; molecular weight of HCl salt: 475.10).

[0541] A. Functionalization with acrylates (Michael addition)

[0542] General Example A1.G2-ACR-nC8-16

[0543] This general example describes the Michael addition reaction of a G2-PPI-(NH2)8 dendrimer with n-octyl acrylate to produce a fully functionalized PPI dendrimer with 16 octyl ester groups.

[0544] G2 PPI dendrimer (n-butene core; 0.20 g; 0.258 mmol; 2.06 mmol primary amine groups) was dissolved in isopropanol (1 mL). Excess n-octyl acrylate (1.73 mL, 1.53 g, 8.27 mmol; 32 moleq) was added and the reaction mixture was stirred at 55 °C in a sealed vial under an inert nitrogen atmosphere. 1 The reaction progress was monitored by H-NMR. The mixture was stirred for 4 days after which the conversion was complete. The mixture was evaporated to dryness in vacuo and the residue was stirred in MeCN (15 mL) at 4 ° C. After 3-4 hours, the supernatant was decanted (or aspirated) to remove excess acrylate. The MeCN wash was repeated twice more at 4 ° C, with the last wash at -20 ° C (for long-chain acrylates, this may result in precipitation of acrylate). The product was dried in vacuo to give a light yellow oil. Yield: 515 mg (54%). 1 The H-NMR spectrum was consistent with the expected structure.

[0545]

[0546] 1 H-NMR (400MHz, chloroform-d) δ4.04(t,J=6.8Hz,32H,K),2.77(t,J=7.3Hz,32H,I),2.54–2.27(m,84H,B,C,E,F, H,J),1.58(dt,J=22.2,6.9Hz,60H,A,D,G,L),1.30(dd,J=14.0,7.7Hz,160H,M),1.08–0.64(m,48H,N).

[0547] MALDI-TOF-MS (CHCA matrix, positive reflection mode): Obs.m / z=(M+H) + 3722.19. Calculation: C 216 H 416 N 14 O 32 (Exact mass 3719.14; Molecular weight 3721.77).

[0548] Example A2.G1-ACR-nC8-8

[0549] The reaction between G1-PPI-(NH2)4 (n-butene core) and n-octyl acrylate was carried out in a similar manner to Example A1. Yield: 758 mg (67%). 1 The H-NMR spectrum was consistent with the expected structure.

[0550]

[0551] 1 H NMR (400 MHz, CHLOROFORM-d) δ 4.05 (t, J = 6.8 Hz, 16 H, H), 2.77 (t, J = 7.4 Hz, 16 H, F), 2.40 (dt, J = 23.2, 7.5 Hz, 36 H, B, C, E, G), 1.58 (dt, J = 23.6, 6.7 Hz, 28 H, A, D, I), 1.47–1.07 (m, 80 H, J), 1.07–0.57 (m, 24 H, K).

[0552] MALDI-TOF-MS (CHCA matrix, positive reflection mode): Obs.m / z=(M+H) + 1791.52,(M+Na) + 1813.52,(M+K) + 1829.49. Calculation: C 104 H 200 N6O 16 (Exact mass 1789.50; Molecular weight 1790.77).

[0553] Example A3.G1-ACR-nC14-8

[0554] The reaction between G1-PPI-(NH2)4 (n-butene core) and n-tetradecyl acrylate was carried out in a similar manner to Example A1. However, HFIP was used as solvent. Workup: After evaporation of HFIP under vacuum at 50°C, the reaction mixture was co-evaporated 3 times with toluene 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 H-NMR was consistent with the desired structure.

[0555]

[0556] 1H NMR (399MHz, chloroform-d) δ4.04(t,J=6.9Hz,16H,H),2.91–2.70(m,16H,F),2.43(t,J=7.3Hz, 36H,B,C,E,G),1.61(t,J=7.0Hz,28H,A,D,I),1.26(m,176H,J),0.99–0.74(m,24H,K).

[0557] MALDI-TOF-MS (CHCA matrix, positive reflection mode): Obs. m / z (M+H) + 2464.31, (M+Na) + 2489.30. Calculation: C 152 H 296 N6O 16 (Exact mass 2462.25; Molecular weight 2464.07).

[0558] Example A4.G1-ACR-nC4-8

[0559] The reaction between G1-PPI-(NH2)4 (n-butene core) and n-butyl acrylate was carried out in a similar manner to Example A1. Workup: Excess acrylate was removed by vacuum evaporation. Acrylate can also be removed by stirring the reaction mixture with an amine-functionalized silica scavenger (excess butyl acrylate is immobilized by reaction with the amine groups bound to the silica). Yield: 450 mg (53%). 1 H-NMR was consistent with the desired structure.

[0560]

[0561] 1 H NMR (399 MHz, CHLOROFORM-d) δ 4.06 (t, J = 6.7 Hz, 16 H, H), 2.77 (t, J = 7.3 Hz, 16 H, F), 2.55–2.14 (m, 36 H, B, C, E, G), 1.87–1.46 (m, 28 H, A, D, I), 1.46–1.29 (m, 16 H, J), 0.93 (t, J = 7.4 Hz, 24 H, K).

[0562] MALDI-TOF-MS (CHCA matrix, positive reflection mode): Obs.m / z=(M+H) + 1342.02,(M+Na) + 1364.02. Calculation: C 72 H 136 N6O 16 (Exact mass 1341.00; Molecular weight 1341.91).

[0563] Example A5.G2-ACR-nC14-16

[0564] The reaction between G2-PPI-(NH2)8 (n-butene core) and n-tetradecyl acrylate was carried out in a similar manner to Example A3. Yield: 580 mg (60%). 1 H-NMR was consistent with the desired structure.

[0565]

[0566] 1 H NMR (399 MHz, CHLOROFORM-d) δ 4.04 (s, 32H, K), 2.76 (t, J = 7.4 Hz, 32H, I), 2.41 (h, J = 8.0, 7.3 Hz, 84H, B, C, E, F, H, J), 1.60 (q, J = 7.0 Hz, 60H, A, D, G, L), 1.26 (s, 352H, M), 0.88 (s, 48H, N).

[0567] MALDI-TOF-MS (CHCA matrix, positive reflection mode): Obs.m / z=(M+H) + 5068.95. Calculation: C 312 H 608 N 14 O 32 (Exact mass 5064.64; Molecular weight 5068.36).

[0568] Example A6.G1-ACR-isoC10-8

[0569] The reaction between G1-PPI (DAB-Am-4) and isodecyl acrylate was carried out in a similar manner to Example A1. Yield: 0.85 g (67%). 1 The H-NMR spectrum was consistent with the expected structure.

[0570]

[0571] 1 H NMR (399 MHz, chloroform-d) δ 4.23–3.74 (m, 16H, H), 2.77 (t, J = 7.3 Hz, 16H, F), 2.63 (s, 1H), 2.53–2.28 (m, 35H, G, C, E, B), 2.06–1.73 (m, 2H), 1.73–0.97 (m, 90H, A, D, I, J, M, O, L), 0.97–0.54 (m, 75H, K, N, P). MALDI-TOF-MS (CHCA matrix, positive reflection mode): Obs. m / z = (M+H) +2015.17. Small peak, m / z=M+H + ,2029.76 (extra CH2). Calculation: C 120 H 232 N6O 16 (Exact mass 2013.75; Molecular weight 2015.20).

[0572] Example A7.G1(PPeI)-ACR-C8-8

[0573] N,N,N',N'-Tetrakis(5-aminopentyl)-1,4-diaminobutane (G1-PPeI, see above; 200 mg, 0.47 mmol, 1 eq) and n-octyl acrylate (1.6 mL, 1.4 g, 7.6 mmol, 16 eq) were dissolved in 2-propanol (IPA; 1 mL). The mixture was heated to 60 °C for 112 h and heated to 40 °C for 12 h using 1 The reaction was monitored by H-NMR. The mixture was cooled and concentrated in vacuo. The viscous oil was stirred in MeCN, cooled to -20 °C, and the oil was allowed to settle to the bottom of the flask. The supernatant was decanted and the procedure was repeated 3 times to obtain the oily G1(PPeI)-ACR-C8-8 product.

[0574]

[0575] 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 reflection mode): Obs. m / z = (M+H) + 1903.63,(M+Na) + 1925.60Calculated: C 112 H 216 N6O 16 (Exact mass 1901.63; Molecular weight 1902.99).

[0576] Example A8.G2 (PAMAM-C2)-ACR-C8-16

[0577] To a stirred reaction tube containing a solution of G2-PAMAM-C2 (ethylenediamine core; 8 amine end groups; 200 mg, 0.14 mmol) in 2-propanol (IPA; 1 mL) was added n-octyl acrylate (825 mg, 4.48 mmol, 32 eq). The reaction mixture was heated to 60°C and maintained for 304 hours. The reaction mixture was concentrated in vacuo. The residue was stirred in MeCN and cooled to -20°C, allowing the product to settle to the bottom of the flask. The supernatant was decanted and the procedure was repeated four times to give a yellow viscous oil (301 mg, 68.8 μmol, 49%).

[0578]

[0579] 1 H NMR (400 MHz, chloroform-d) δ7.72 (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 reflection mode): Obs. m / z = (M+H) + 4378.32 Calculated: C 238 H 448 N 26 O 44 (Exact mass 4375.36; Molecular weight 4378.34).

[0580] Example A9. Bis(3-aminopropyl)amine-ACR-C8-5

[0581] 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 to 60 °C for 136 hours and then concentrated in vacuo. The residue was stirred in MeCN and cooled to -20 °C to separate the product from the MeCN layer. The supernatant was carefully removed using a pipette and the procedure was repeated five times. The residue was dried in vacuo to give a clear colorless oil (769 mg, 0.73 mmol, 73%).

[0582]

[0583] 1H NMR (400 MHz, CHLOROFORM-d) δ 4.05 (t, J = 6.8 Hz, 10H), 2.76 (td, J = 7.4, 3.8 Hz, 10H), 2.41 (dt, J = 10.7, 6.8 Hz, 18H), 1.74–1.48 (m, 14H), 1.42–1.16 (m, 50H), 0.95–0.80 (m, 15H). 13 C NMR (101 MHz, CDCl3) δ 172.9, 172.7, 64.6, 64.6, 51.8, 51.8, 49.3, 49.2, 32.6, 32.3, 31.8, 29.3, 29.2, 28.7, 28.7, 25.9, 24.8, 22.7, 14.1. MALDI-TOF-MS (CHCA matrix, positive reflection mode): Obs. m / z = (M+H) + 1052.85,(M+Na) + 1074.84,(M+K) + 1090.80.Calculation: C 61 H 117 N3O 10 (Exact mass 1051.87; Molecular weight 1052.62).

[0584] Example A10. Bis(2-aminoethyl)amine-ACR-C8-5

[0585] 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 manner similar to that described in Example A9. Yield: 674 mg (0.66 mmol, 66%).

[0586]

[0587] 1 H NMR (399 MHz, CHLOROFORM-d) δ 4.05 (t, J = 6.8 Hz, 10H), 2.78 (t, J = 7.2 Hz, 10H), 2.51 (s, 8H), 2.43 (t, J = 7.3 Hz, 10H), 1.62 (p, J = 7.0 Hz, 10H), 1.39–1.19 (m, 50H), 1.00–0.76 (m, 15H). 13C NMR (100 MHz, CDCl3) δ 172.7, 172.6, 64.6, 52.9, 52.3, 50.5, 49.8, 32.7, 32.7, 31.8, 29.3, 29.2, 28.7, 28.7, 25.9, 22.7, 14.1. MALDI-TOF-MS (CHCA matrix, positive reflection mode): Obs. m / z = (M+H) + 1024.82,(M+Na) + 1046.80,(M+K) + 1082.78. Calculation: C 59 H 113 N3O 10 (Exact mass 1023.84; Molecular weight 1024.56).

[0588] Example A11. Bis-(3-aminopropyl)methylamine-ACR-C8-4

[0589] 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 manner similar to that described in Example A9. Yield: 552 mg (0.63 mmol, 63%).

[0590]

[0591] 1 H NMR (400MHz, chloroform-d) δ4.05(t,J=6.8Hz,8H),2.77(t,J=7.3Hz,8H),2.43(td,J=7.2,2.8Hz,12H ), 2.29 (t, J = 7.3Hz, 4H), 2.18 (s, 3H), 1.61 (q, 6.7Hz, 12H), 1.30 (m, 40H), 0.99–0.77 (m, 12H). 13 C NMR (101 MHz, CDCl3) 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 reflection mode): Obs. m / z = (M+H) + 882.71,(M+Na) + 904.72. Calculation: C 51 H 99 N3O8 (exact mass 881.74; molecular weight 882.37).

[0592] Example A12. Bis(3-aminopropyl)amine-ACR-(2-ethylhexyl)-5-nitropropene

[0593] 2-Ethylhexyl acrylate (1.4 g, 7.5 mmol, 10 eq) 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 in Example A9. Yield after workup: 278 mg (0.26 mmol, 35%).

[0594]

[0595] 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 reflection mode): Obs. m / z = (M+H) + 1052.88,(M+Na) + 1074.86. Calculation: C 59 H 113 N3O 10 (Exact mass 1051.87; Molecular weight 1052.62).

[0596] Example A13. G1-ACR-(2-ethylhexyl)-8

[0597] The reaction between G1-PPI (DAB-Am-4) and 2-ethylhexyl acrylate was carried out in a similar manner to Example A1. Yield: 0.71 g (0.4 mmol, 73%). 1 The H-NMR spectrum was consistent with the expected structure.

[0598]

[0599] 1H 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 reflection mode): Obs. m / z = (M+H) + 1791.55,(M+Na) + 1813.53. Calculation: C 120 H 232 N6O 16 (Exact mass 1789.50; Molecular weight 1790.77).

[0600] Example A14.G1(PBuI)-ACR-C8-8

[0601] N,N,N',N'-Tetrakis(4-aminobutyl)-1,4-diaminobutane (G1-PBuI) and n-octyl acrylate were dissolved in 2-propanol (IPA; 1 mL). The reaction and workup were similar to those described in Example A7 to provide the G1(PBuI)-ACR-C8-8 product as an oil.

[0602]

[0603] 1 H NMR (400 MHz, chloroform-d) δ 4.05 (t, 16H), 2.76 (t, 16H), 2.41 (q, 36H), 1.61 (p, 16H), 1.43 (p, 20H), 1.37-1.15 (m, 80H), 0.92-0.84 (t, 24H). MALDI-TOF-MS (CHCA matrix, positive reflection mode): Obs. m / z = (M+H) + 1847,(M+Na) + 1869. Calculation: C 108 H 208 N6O 16 (Exact mass 1846; Molecular weight 1846).

[0604] Example A15.G1 (PAMAM-C2)-ACR-(2-ethylhexyl)-8

[0605]

[0606] To a stirred reaction tube containing a solution of G1-PAMAM-C2 (ethylenediamine core; 4 amine end groups; 165 mg, 0.32 mmol) in 2-propanol (IPA; 2 mL) was added 2-ethylhexyl acrylate (589 mg, 3.2 mmol, 10 eq). Subsequently, more 2-ethylhexyl acrylate (600 mg, 3.3 mmol, 10 eq) was added. The reaction mixture was heated to 60° C. and maintained for 408 hours. The reaction mixture was concentrated in vacuo. The residue was further evaporated using an oil pump to remove excess 2-ethylhexyl acrylate (at 90° C. and 0 mbar). The crude product still contained a small amount of acrylate, which was further removed using a short silica column (starting at 2% MeOH / CHCl3 to 10% MeOH / CHCl3) to give a yellow viscous oil (270 mg, 0.13 mol, 41%).

[0607] 1 H NMR (400MHz, chloroform-d) δ7.20 (t, J=5.5Hz, 3H), 4.19-3.78 (m, 16H, I), 3.27 (q, J= 6.0Hz,8H,B),2.78(dt,J=15.0,6.9Hz,22H,F,G),2.57(t,J=6.4Hz,9H,C),2. 52(s,3H,A),2.44(t,J=7.1Hz,15H,H),2.34(t,J=6.4Hz,7H,E),1.63-1.47(m ,8H,J),1.44-1.12(m,64H,K,L,M,O),0.89(td,J=7.5,7.0,3.0Hz,48H,N,P). MALDI-TOF-MS (CHCA matrix, positive reflection mode): Obs.m / z=(M+H) + 1991.56,(M+Na) + 2013.53.Calculation: C 110 H 208 N 10 O 20 (Exact mass 1989.56; Molecular weight 1990.92).

[0608] B. Selective functionalization of primary amines with acrylates (Michael addition) followed by acylation of the remaining secondary amines.

[0609] The basis for the following material was discovered through the observation that the reaction of acrylates with primary amine dendrimers proceeds much faster than the reaction with the secondary amine dendrimers formed. This selectivity can therefore be used to end-cap the formed secondary amine intermediates by acylation.

[0610] General Example B1.G1-ACR-Citro-4 / Ac-4

[0611] This general example describes the Michael addition reaction of a G1-PPI-(NH2)4 dendrimer with (S)-3,7-dimethyloctyl-6-en-1-yl acrylate followed by reaction with acetic anhydride to produce a G1 PPI-dendrimer with 4 (S)-citronellyl ester groups and 4 acylamide groups.

[0612] G1 PPI dendrimer (n-butene core; 0.20 g; 0.631 mmol) was dissolved in isopropanol (1 mL) and (S)-3,7-dimethyloctyl-6-en-1-yl acrylate (0.513 g; 2.44 mmol) was added. The mixture was stirred in a closed vial under an inert nitrogen atmosphere at 55° C. overnight, which resulted in complete conversion of the acrylate. 1 H-NMR, acrylates react much faster with primary amines, resulting in (almost) exclusive monofunctionalization of each primary amine. 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 at 20°C in a sealed vial under a nitrogen atmosphere for 4 hours, then diluted with DCM and subsequently washed with NaHCO3 (aq.) and brine. The organic layer was dried over Na2SO4, filtered, and the filtrate was evaporated to dryness. Coevaporation with DCM was performed to remove traces of acetic anhydride and acetic acid. Yield: 623 mg (74%). 1 The H-NMR spectrum was consistent with the expected structure.

[0613]

[0614] 1 H NMR (399 MHz, chloroform-d) δ5.08 (t, J = 7.0, 1.4 Hz, 4H, N), 4.31–3.87 (m, 8H, H), 3.58 (dt, J = 10.9, 7.2 Hz, 8H, F), 3.31 (q, J = 8.9, 8.3 Hz, 8H, E), 2.70–2.50 (m, 8H, C), 2.4 2(d,J=7.3Hz,12H,B,G),2.18–2.07(m,12H,P),2.07–1.92(m,8H,M),1.89–1.63 (m,24H,O,O”),1.63–1.08(m,32H,A,D,I,J,L),0.91(dd,J=6.6,4.4Hz,12H,K).

[0615] MALDI-TOF-MS (CHCA matrix, positive reflection mode): Obs.m / z=(M+H) + 1326.02,(M+Na) + 1348.00. Calculation: C 76 H 136 N6O 12 (Exact mass 1325.01; Molecular weight 1325.95).

[0616] Example B2. G1-ACR-nC14-4 / Ac-4

[0617] The reaction between G1-PPI-(NH2)4 (n-butene core) and n-tetradecyl acrylate and acetic anhydride was carried out in a similar manner to Example B1. Yield: 689 mg (70%). 1 H-NMR spectrum is consistent with the expected structure. MALDI-TOF-MS (CHCA matrix, positive reflection mode): Obs.m / z = (M+H) + 1558.34,(M+Na) + 1580.33 (main peak). Calculation: C 92 H 176 N6O 12 (Exact mass 1557.33; Molecular weight 1558.45) Also observed: a small peak at m / z = 1786.33 (5 tetradecyl tails and 3 acetyl groups).

[0618] Example B3. G2-ACR-Citro-8 / Ac-8

[0619] The reaction between G2-PPI-(NH2)8 (n-butene core) and (S)-3,7-dimethyloctyl-6-en-1-yl acrylate and acetic anhydride was carried out in a similar manner to Example B1. Yield: 520 mg (72%). 1 H-NMR spectrum is consistent with the expected structure. MALDI-TOF-MS (CHCA matrix, positive reflection mode): Obs.m / z = (M+H) + 2792.18,(M+Na) + 2814.16. Calculation: C 160 H 288 N 14 O 24 (Exact mass 2790.17; Molecular weight 2792.14).

[0620] Example B4. G2-ACR-nC14-8 / Ac-8

[0621] The reaction between G2-PPI-(NH2)8 (n-butene core) and n-tetradecyl acrylate and acetic anhydride was carried out in a similar manner to Example B1. Yield: 631 mg (75%). 1 The H-NMR spectrum was consistent with the expected structure.

[0622]

[0623] 1 H NMR (399MHz, chloroform-d) δ4.06 (dt, J=13.5, 6.8Hz, 16H, K), 3.58 (dt, J=13.9, 6. 7Hz,16H,I),3.31(q,J=9.5,9.0Hz,16H,H),2.60(ddd,J=10.4,6.1,2.4Hz, 16H,J),2.40(p,J=9.9,8.5Hz,36H,B,C,E,F),2.10(d,J=10.7Hz,24H,O),1 .86–1.46(m,44H,A,D,G,L),1.46–1.07(m,176H,M),1.07–0.58(m,24H,N).

[0624] MALDI-TOF-MS (CHCA matrix, positive reflection mode): Obs.m / z=(M+H) + 3257.80,(M+Na) + 3279.79.Calculation: C 192 H 368 N 14 O 24 (Exact mass 3254.80; Molecular weight 3257.13) A small peak at m / z = 3484.01 (9 tetradecyl tails and 7 acetyl groups) was also observed.

[0625] C. Functionalization of primary amines with a first acrylate and then with another second acrylate (Michael addition)

[0626] General Example C1.G3-ACR-nC14-16 / C1-16

[0627] This general example describes G3-PPI-(NH2) 16 Michael addition reaction of the dendrimer with n-tetradecyl acrylate followed by reaction with methyl acrylate produced a PPI dendrimer with 16 n-tetradecyl ester groups and 16 methyl ester groups.

[0628] 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 moleq) was added. The mixture was stirred at 55 °C for 5 hours in a sealed vial under an inert nitrogen atmosphere, which resulted in complete conversion of the primary amine (a small amount of acrylate was still present). 1 H-NMR, acrylates react much faster with primary amines, resulting in almost exclusive monofunctionalization of each primary amine group. Subsequently, methyl acrylate (0.27 mL, 0.244 g, 2.83 mmol, 24 moleq) was added and the mixture was stirred at 55 °C. 1 The progress of the reaction was monitored by H-NMR. After 1 day, an additional amount of methyl acrylate (0.14 mL, 0.122 g, 1.42 mmol, 12 eq) 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 placed at 0 ° C for several hours to induce complete phase separation. Subsequently, the supernatant was decanted. The procedure was repeated three times to remove any unreacted acrylate monomer. Yield: 575 mg (66%). 1 The H-NMR spectrum was consistent with the expected structure.

[0629]

[0630] 1 H NMR (400 MHz, chloroform-d) δ 4.04 (t, J = 6.9 Hz, 32 H, N), 3.65 (s, 48 ​​H, T), 2.76 (t, J = 7.3 Hz, 64 H, L, R), 2.63–2.18 (m, 180 H, B, C, E, F, H, I, K, M, S), 1.80–1.44 (m, 92 H, A, D, G, J, O), 1.26 (s, 352 H, P), 0.88 (t, J = 6.7 Hz, 48 H, Q). MALDI-TOF-MS: MW is too high to measure. Calculation: C 424 H 816 N 30 O 64 (Exact mass 7354.15; Molecular weight 7359.34).

[0631] Example C2.G1-ACR-nC14-4 / C1-4

[0632] The reaction between G1-PPI-(NH2)4 (n-butene core) and n-tetradecyl acrylate and methyl acrylate was carried out in a similar manner to Example C1. Yield: 464 mg (84%). 1 The H-NMR spectrum was consistent with the expected structure.

[0633]

[0634] 1 H NMR (399MHz, chloroform-d) δ4.05(t,J=6.8Hz,8H,H),3.66(s,12H,N),2.77(t,J=7.3Hz,16H,F,L),2.41(ddt,J=22 .6,14.6,7.0Hz,32H,C,E,G,M),1.90-1.50(m,20H,A,D,I),1.50–1.20(m,96H,I,J),1.03–0.72(m,12H,K).

[0635] MALDI-TOF-MS (CHCA matrix, positive reflection mode): Obs.m / z=(M+H) + 1735.44. Small peak, m / z = M + H) + 1917.63 (5 C14 chains and 3 C1 chains). Calculation: C 100 H 192 N6O 16 (Exact mass 1733.44; Molecular weight 1734.66).

[0636] D. Functionalization of amine-terminated PPI dendrimers with acrylates (Michael addition) and quaternization of internal amines

[0637] Example D1.G1-Boc-4

[0638] 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 an amine functionalized silica scavenger overnight to remove excess Boc-anhydride. After filtration, the filtrate was evaporated to dryness to give the product. Yield: 1.16 g (81%). 1 The H-NMR spectrum was consistent with the expected structure.

[0639]

[0640] 1H NMR (400 MHz, CHLOROFORM-d) δ 5.59–4.89 (m, 4H, G), 3.13 (q, J = 6.4 Hz, 8H, E), 2.40 (dt, J = 22.0, 6.6 Hz, 12H, B, C), 1.61 (p, J = 6.7 Hz, 8H, D), 1.40 (s, 40H, A, F).

[0641] Example D2. G1-Boc-4(2MeCl)

[0642] Boc protected G1 PPI dendrimer G1-Boc-4 (0.75 g, 1.04 mmol) was dissolved in DCM (3 mL) and iodomethane (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 give a foam which was dissolved in CHCl3 and precipitated into pentane. The supernatant was decanted and the residue was stirred again with pentane and decanted. The product contained iodide as the counter anion and was exchanged to chloride using an ion exchange column (Lewatit monoplus M600 resin 1.3 meq / mL) with methanol as eluent. The product was checked for the presence of iodide by dissolving about 5 mg in deionized water (1 mL) and adding a few drops of 35% H2O2(aq). No yellow color was observed, indicating the formation of iodine I2; fresh starch solution (1 mL) was added to the mixture and no blue discoloration was observed. Yield: 715 mg (84%). 1 The H-NMR spectrum was consistent with the expected structure.

[0643]

[0644] 1 H NMR (399MHz, chloroform-d) δ5.51(s,4H,G),3.67(s,4H,B),3.39(s,8H,C),3.27(s,8H,E),3.16(s,6H,H),2.10(d,J=14.2Hz,12H,A,D),1.44(s,36H,F).

[0645] Example D3.G1-NH2-4(2MeCl)

[0646] About 800 mg of G1-Boc-4 (2MeCl) was mixed with a 4M HCl (about 5 mL) solution in dioxane to obtain a precipitate. To increase solubility, a 3M HCl solution in methanol (about 5 mL) was added. The reaction mixture was stirred at room temperature overnight. 1H-NMR indicated complete deprotection. The mixture was evaporated to dryness and the residue was co-evaporated with isopropanol to give a hygroscopic solid (the compound was poorly soluble in isopropanol). Yield: 500 mg (122%), due to the inclusion of HCl and / or water. 1 The H-NMR spectrum was consistent with the expected structure.

[0647]

[0648] 1 H NMR (400MHz, methanol-d4) δ3.86–3.63(b,F),3.63–3.44(m,12H,B,C),3.19(s,6H,G),3.10(t,J=7.3Hz,8H,E),2.26(d,J=10.7Hz,8H,D),1.97(s,4H,A).

[0649] Example D4: G1-ACR-nC14-8(2MeCl)

[0650] G1-NH2(2MeCl) (300 mg, 1.77*10 -5 mol) was dissolved in HFIP (2 mL) and n-tetradecyl acrylate (2.04 mL, 2 eq per primary amine) was added. The reaction mixture was a clear solution. The mixture was stirred at 55 °C for one hour, but no reaction occurred. Triethylamine (1.6 mL) was added as a base and the mixture was stirred for 24 hours. 1 H-NMR, reaction is complete. The reaction mixture is evaporated to dryness. The residue is stirred in MeCN, placed at 0 ° C, and the supernatant is decanted. This procedure was performed three times. The residue is then stirred in 1 / 1 EtOAc / hexane. The precipitate is filtered out, which still contains 1% tetradecyl acrylate, which is removed by stirring and filtering in EtOAc. The residue is then placed on an ion exchange resin to exchange the remaining iodide for chloride (1 / 1 CHCl3 / MeOH as eluent). Yield: 468mg (102%). 1 The H-NMR spectrum was consistent with the expected structure.

[0651]

[0652] 1H NMR (399MHz, chloroform-d) δ4.02 (t, J=6.8Hz, 16H, H), 3.84 (d, J=7.5Hz, 4H, B), 3.38 (dd, J= 10.4,6.2Hz,8H,C),3.19(s,6H,L),2.71(td,J=6.7,2.5Hz,16H,F),2.57(t,J=6.0Hz ,8H,E),2.40(t,J=6.6Hz,16H,G),2.19(d,J=7.7Hz,4H,A),2.02(dt,J=13.2,6.4Hz ,8H,D),1.62(q,J=6.9Hz,16H,I),1.52–1.02(m,176H,J),0.88(t,J=6.7Hz,24H,K).

[0653] Example D5.G2-Boc-8

[0654] 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 H-NMR spectrum was consistent with the expected structure.

[0655]

[0656] 1 H NMR (399MHz, methanol-d4) δ7.90 (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).

[0657] Example D6. G2-Boc-8(6MeCl)

[0658] The reaction between G2-Boc-8 and methyl iodide and the subsequent anion exchange was carried out in a similar manner as shown in Example D2. Yield: 699 mg (73%). 1 The H-NMR spectrum was consistent with the expected structure.

[0659]

[0660] 1H NMR(399MHz, methanol-d4)δ3.71(d,J=8.4Hz,4H,I),3.66–3.38(m,34H,B,C,E,F),3.29-3.04(m ,43H,K,H,G),2.43(q,J=8.2Hz,8H,D),1.99(h,J=8.5,6.9Hz,21H,A,G),1.44(s,79H,J).

[0661] Example D7.G2-NH2-8(6MeCl)

[0662] The reaction between G2-Boc-8(6MeCl) and HCl was carried out in a similar manner as shown in Example D3. Yield: 478 mg (93%). 1 The H-NMR spectrum is consistent with the expected structure. This product can be used to react with acrylates, acrylamides or epoxides.

[0663]

[0664] 1 H NMR (399 MHz, deuterium oxide) δ 3.58 (t, J = 8.9 Hz, 36H, B, C, E, F), 3.25 (d, J = 4.4 Hz, 18H) I, J, 3.15 (t, J = 7.7 Hz, 16H, H), 2.42 (s, 8H, D), 2.23 (dd, J = 14.2, 5.5 Hz, 16H, G), 1.98 (s, 4H, A).

[0665] E. Functionalization of amines with acrylamide (Michael addition)

[0666] General Example E1.G1-ACRAMAlc-8

[0667] This general example describes the Michael addition reaction of a G1-PPI-(NH2)4 dendrimer with N-(2-hydroxyethyl)-N-methylacrylamide to produce a fully functionalized PPI-dendrimer with 8 N-(2-hydroxyethyl)-N-methylamide groups.

[0668] 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 moleq per primary amine) was added. The reaction mixture was stirred in a sealed vial under nitrogen atmosphere at 55° C. for 2 days while being heated by 1 H-NMR was used to monitor the conversion. The mixture was evaporated to dryness, the residue was stirred in MeCN, and the mixture was placed at 4°C for several hours. The supernatant was decanted to remove excess acrylamide reactant. The washing with MeCN was repeated twice at 4°C and once at -20°C. The product was dried in vacuo to give a light yellow oil. Yield: 367 mg (86%). 1 The H-NMR spectrum was consistent with the expected structure.

[0669]

[0670] 1 H NMR (399MHz, chloroform-d) δ4.89(s,8H,H),3.71(t,J=5.4Hz,16H,J),3.50(dt,J=10.3,5.1Hz,16H,I),3.12(s,12H,K),2.93(s ,12H,K),2.79(d,J=7.0Hz,16H,F),2.63–2.54(m,8H,E),2.49(d,J=7.5Hz,24H,B,C,G),1.79(s,8H,D),1.46(s,4H,A).

[0671] MALDI-TOF-MS (CHCA matrix, positive reflection mode): Obs.m / z=(M+H) + 1349.98,(M+Na) + 1371.96. Calculation: C 64 H 128 N 14 O 16 (Exact mass 1348.96; Molecular weight 1349.81).

[0672]

[0673] Synthesis of E2 to E5. (a) Isopropanol / HFIP or isopropanol, heating, 64-480h.

[0674] Example E2

[0675] 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-methylacrylamido)propyl)carbamate (1.21 g, 5 mmol, 10 eq) in 2-propanol. The reaction mixture was heated to 60 ° C and maintained for 64 hours. The mixture was then cooled and concentrated in vacuo. The crude viscous oil was purified by silica column chromatography, eluting with a mixture of MeOH / CHCl3 (0-20%) to give E2 as a viscous oil.

[0676] HPLC-ESI-MS: m / z calculated as C 55 H 107 N 11 O 12 1113.81. Measured [M+H] + 1114.83,[M+H] 2+ 558.08,[M+H] 2+ -Boc 508.00[M+H] 2+ -2x Boc 458.00,[M+H] 2+ -3x Boc 408.00,[M+H] 2+ -4xBoc 357.92.

[0677] Example E3

[0678] Di-n-propylenetriamine (65.6 mg, 0.5 mmol, 1 eq) and tert-butyl (3-(N-methylacrylamido) propyl) carbamate (1.21 g, 5 mmol, 10 eq) were dissolved in 2-propanol and HFIP (1 mL). The reaction mixture was heated to 60 ° C and maintained for 480 hours. Although the reaction was not completed after 480 hours, the reaction mixture was cooled and concentrated in vacuo. The crude viscous oil was purified by silica column chromatography and eluted with a mixture of MeOH / CHCl (0-20%) to give E3 in a viscous oily state.

[0679] ESI-MS: m / z calculated as C 66 H 127 N 13 O 15 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+ -2x Boc572.08,[M+H] 2+ -3x Boc 522.08,[M+H] 2+ -4x Boc 472.00,[M+H] 2+ -5x Boc472.00.

[0680] Example E4

[0681] Diethylenetriamine (103 mg, 1 mmol, 1 eq) and tert-butyl (3-(N-methylacrylamido)propyl) carbamate (2.42 g, 10 mmol, 10 eq) were dissolved in 2-propanol and heated to reflux. Although the reaction was not complete after 305 hours of reflux, the reaction was stopped at this time. The reaction mixture was concentrated in vacuo to give a viscous amber oil. The crude product E4 was purified by silica column chromatography using a mixture of MeOH / CHCl (0-20%).

[0682] Example E5

[0683] In a 5 mL flask, 1,4-diaminobutane (88 mg, 1 mmol, 1 eq) and tert-butyl (3-(N-methylacrylamido)propyl) carbamate (1.21 g, 5 mmol, 5 eq) were dissolved in 2-propanol (2 mL). The reaction mixture was heated to 55 ° C. After 350 hours, the reaction was almost complete. The reaction mixture was concentrated in vacuo. The crude product was purified by silica column chromatography, eluting with a mixture of CHCl3 / MeOH (0-20%) to give E5 (756 mg, 0.72 mmol, 72%) as an amber viscous oil.

[0684] 1 H NMR (400MHz, chloroform-d) δ5.81–5.20(m,4H),3.43(t,J=6.4Hz,6H),3.36(t,J=7.8Hz,2H),3.16(q,J=6.4Hz,2H),3.06(q,J=6.3Hz,6 H), 3.02 (s, 9H), 2.91 (s, 3H), 2.81 (t, J = 7.3Hz, 8H), 2.48 (m, 12H), 1.80 (p, J = 6.9Hz, 2H), 1.66 (p, J = 6.4Hz, 6H), 1.43 (m, 40H). 13C NMR (101 MHz, CDCl3) 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 calculated for C 52 H 100 N 10 O 12 1056.75.Measured [M+H] + 1057.92,[M+Na] + 1079.92,[M+H] 2+ 529.58,[M+H] 2+ -Boc 479.50[M+H] 2+ -2x Boc 429.50,[M+H] 2+ -3xBoc 379.50.

[0685] F. Functionalization of primary amines with acrylates and then functionalization of remaining secondary amines with acrylamides (Michael addition). It is also possible to react the amides first and then the esters.

[0686] General Example F1: G1-ACR-nC14-4 / AmAlc-4

[0687] This general example describes the subsequent Michael addition reaction of a G1-PPI-(NH2)4 dendrimer with n-tetradecyl acrylate and N-(2-hydroxyethyl)-N-methylacrylamide to produce a PPI-dendrimer with 4 n-tetradecyl-ester and 4 N-(2-hydroxyethyl)-N-methylamide groups.

[0688] PPI dendrimer G1 (0.10 g, 0.315 mmol) was dissolved in isopropanol (1 mL) and tetradecyl acrylate (0.339 g, 4 moleq per dendrimer) was added. The mixture was stirred at an oil bath temperature of 55°C for 24 hours, which resulted in complete conversion of the primary amine. A slight excess of acrylate reactant was still present. 1H-NMR, acrylate has reacted selectively with primary amine, resulting in monofunctionalization of all primary amine groups. Next, N-(2-hydroxyethyl)-N-methylacrylamide (0.204 g, 1.57 mmol, 5 moleq) was added. The mixture was stirred at 55 °C for a total of 6 days. 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 give an oily product that no longer contained any acrylate or acrylamide. Yield: 344 mg (50%; the lower yield may be due to partial adhesion of the dendrimer to the silica scavenger). 1 The H-NMR spectrum was consistent with the expected structure.

[0689]

[0690] 1 H NMR (399MHz, chloroform-d) δ4.05(t,J=6.8Hz,8H,N),3.72(q,J=5.8Hz,8H,J),3.49(dt,J=17.9,4.7Hz,8H,I),3.12-2.94(singlets,12H,H),2.86(t,J=3.2H z,1H),2.78(q,J=7.5Hz,16H,F,L)),2.68–2.26(m,36H,B,C,E,G,M),1.60( td,J=13.7,7.0Hz,16H,D,O),1.26(s,92H,A,P),0.88(t,J=6.8Hz,12H,K).

[0691] MALDI-TOF-MS (CHCA matrix, positive reflection mode): Obs.m / z=(M+H) + 1907.62,(M+Na) + 1929.6. Calculation: C 108 H 212 N 10 O 16 (Exact mass 1905.61; molecular weight 1906.94) A small peak was also observed at m / z = 2046.78 (5 tetradecyl acrylate and 3 acrylamide groups).

[0692] G. Functionalization of Amines with Epoxides

[0693] General Example G1: G1-EPX-nC8-8

[0694] This general example describes the reaction of a G1-PPI-(NH2)4 dendrimer with (1,2-epoxy)-n-decane to produce a PPI-dendrimer with 2-hydroxy C10 groups.

[0695] PPI dendrimer G1 (0.10 grams 0.315 mmol) was dissolved in isopropanol (1 mL) and 1,2-epoxy-decane (0.43 g, 8.8 moleq) was added. The reaction mixture was stirred at 90 ° C in a sealed vial under an inert nitrogen atmosphere and then evaporated to dryness. The residue was stirred in MeCN and the suspension was left at -20 ° C for several hours. The supernatant was decanted. This procedure was repeated twice, after which the residue no longer contained any remaining 1,2-epoxydecane. The residue was dried in vacuo to give the product. Yield: 390 mg (80%). 1 The H-NMR spectrum was consistent with the expected structure.

[0696]

[0697] 1 H NMR (399 MHz, methanol-d4) δ 3.62 (tt, J = 7.2, 3.8 Hz, 8H, G), 2.68–2.29 (m, 36H, B, C, E, F), 1.63 (p, J = 7.1 Hz, 8H, D), 1.59–1.12 (m, 112H, I, J, K), 1.11–0.67 (m, 24H, L).

[0698] MALDI-TOF-MS (CHCA matrix, positive reflection mode): Obs.m / z=(M+H) + 1567.57,(M+Na) + 1589.56,(M+K) + 1605.53. Calculation: C 96 H 200 N6O8 (exact mass 1565.54; molecular weight 1566.69).

[0699] Example G2.G1-EPX-nC10-8

[0700] The reaction between G1-PPI-(NH2)4 (n-butene core) and 1,2-epoxydodecane was carried out in a similar manner to Example G1. Yield: 447 mg (79%). 1 H-NMR spectrum is consistent with the expected structure. MALDI-TOF-MS (CHCA matrix, positive reflection mode): Obs.m / z = (M+H) + 1791.83,(M+Na) + 1813.82,(M+K) +1829.79.Calculation: C 112 H 232 N6O8 (exact mass 1789.79; molecular weight 1791.12).

[0701] Example G3.G2-EPX-nC10-16

[0702] The reaction between G2-PPI-(NH2)8 (n-butene core) and 1,2-epoxydodecane was carried out in a similar manner to Example G1. Yield: 697 mg (84%). 1 H-NMR spectrum is consistent with the expected structure. MALDI-TOF-MS (CHCA matrix, positive reflection mode): Obs.m / z = (M+H) + 3722.78,(M+Na) + 3744.75,(M+K) + 3760.74. Calculation: C 232 H 480 N 14 O 16 (Exact mass 3719.72; Molecular weight 3722.47).

[0703] Example G4.G3-EPX-nC10-32

[0704] G3-PPI-(NH2) 16 The reaction between (n-butene core) and 1,2-epoxydodecane was carried out in a similar manner to Example G1. Yield: 413 mg (88%). 1 H-NMR was consistent with the desired structure.

[0705]

[0706] 1 H NMR (399 MHz, CHLOROFORM-d) δ 4.97–4.07 (m, 16H, L), 3.59 (s, 16H, J), 2.90–2.20 (m, 84H, B, C, E, F, H, I), 1.79–1.08 (m, 316H, A, D, G, KM, N), 0.88 (t, J = 6.8 Hz, 48H, O).

[0707] Example G5.G1(PPeI)-EPX-nC10-8

[0708] G1-PPeI (100 mg, 0.23 mmol, 1 eq) and 1,2-epoxydodecane (0.81 mL, 684 mg, 3.72 mmol, 16 eq) were dissolved in 2-propanol (IPA; 1 mL). The mixture was heated to 60 °C for 16 h (with 1H-NMR monitoring). The mixture was cooled and concentrated in vacuo. The viscous oil was stirred in MeCN, cooled to -20°C and allowed to settle to the bottom of the flask. The supernatant was decanted and the procedure was repeated 3 times to give the product as a viscous oil (344 mg, 0.20 mol, 88%).

[0709]

[0710] 1 H NMR (399 MHz, chloroform-d) δ 3.60 (m, 8H), 3.19 (m, 8H), 2.64–2.47 (m, 10H), 2.38 (p, J = 7.8, 6.9 Hz, 26H), 1.65–1.16 (m, 172H), 0.88 (t, J = 6.7 Hz, 24H). MALDI-TOF-MS (CHCA matrix, positive reflection mode): Obs. m / z = 1903.99 (M+H) + ,1925.92(M+Na) + Calculated: C 120 H 248 N6O8 (exact mass 1901.92; molecular weight 1903.34).

[0711] Example G6.G2 (PAMAM-C2)-EPX-nC8-16

[0712] 1,2-Epoxydecane (700 mg, 4.38 mmol, 32 eq) was added to a stirred solution of G2-PAMAM-C2 (ethylenediamine core, 8 amine end groups; 200 mg, 0.14 mmol) in 2-propanol (1 mL). The reaction mixture was heated to 60 ° C for 88 hours and then concentrated in vacuo. The residue was stirred in MeCN and cooled to -20 ° C, allowing the product to settle at the bottom of the flask. The supernatant was decanted and the grinding procedure was repeated three times to give a yellow viscous oil (422 mg, 107 μmol, 77%).

[0713]

[0714] 1 H NMR (399 MHz, chloroform-d) δ7.92 (s, 12H), 4.60 (d, J = 29.0 Hz, 16H), 3.59 (s, 16H), 3.31 (t, J = 41.3 Hz, 24H), 2.99–2.09 (m, 108H), 1.62–1.06 (m, 224H), 0.88 (t, J = 6.7 Hz, 48H). MALDI-TOF-MS (DCBT matrix, positive reflection mode): Obs. m / z = (M+H) + 3930.44. Calculation: C222 H 448 N 26 O 28 (Exact mass 3927.44; Molecular weight 3930.18).

[0715] Example G7. Bis(3-aminopropyl)amine-EPX-C8-5

[0716] 1,2-epoxydecane (1.56 g, about 10 mmol, 10 eq) was added to a stirred IPA solution (1 mL) of bis(3-aminopropyl)amine (131 mg, 1 mmol). The reaction mixture was heated to 60 ° C for 40 hours and then concentrated in vacuo. The residue was stirred in MeCN and cooled to -20 ° C to separate the product from the MeCN solvent. The supernatant was carefully removed using a pipette and the grinding procedure was repeated three times. The residue was dried in vacuo to obtain a clear colorless oil (950 mg).

[0717]

[0718] 1 H NMR (399 MHz, methanol-d4) δ7.89 (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 reflection mode): Obs. m / z = (M+H) + 912.87. Calculation: C 56 H 117 N3O5 (exact mass 911.90; molecular weight 912.57).

[0719] Example G8. Bis(2-aminoethyl)amine-EPX-C8-5

[0720] Bis(2-aminoethyl)amine (103 mg, 1 mmol) and 1,2-epoxydecane (1.56 g, about 10 mmol, 10 eq) were reacted in IPA (1 mL). A similar procedure as described in Example G7 material was employed. Yield: 958 mg 1.08 mmol.

[0721]

[0722] 1H NMR (399 MHz, methanol-d4) 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 reflection mode): Obs. m / z = (M+H) + 884.84,[M+Na] + 906.82. Calculation: C 54 H 113 N3O5 (exact mass 883.87; molecular weight 884.51).

[0723] Example G9. Bis(3-aminopropyl)methylamine-EPX-C8-4

[0724] 1,2-Epoxydecane (269 mg, 1.68 mmol, about 6 eq) was added to a stirred IPA solution (1 mL) of N,N-bis(3-aminopropyl)methylamine (42 mg, 0.29 mmol). The reaction mixture was heated to 60 ° C for 16 hours and then concentrated in vacuo. The residue was stirred in MeCN and cooled to -20 ° C to separate the product from the MeCN solvent. The supernatant was carefully removed using a pipette and the grinding procedure was repeated three times. The residue was dried in vacuo to give a clear colorless oil (170 mg, 0.22 mmol, 77%).

[0725]

[0726] 1 H NMR (400 MHz, chloroform-d) δ 4.93–3.84 (m, 3H), 3.84–3.52 (m, 4H), 3.46 (s, 2H), 2.89–2.53 (m, 4H), 2.53–2.20 (m, 11H), 2.20–2.10 (m, 2H), 1.62 (dt, J = 12.7, 6.4 Hz, 4H), 1.53–1.16 (m, 56H), 0.88 (t, J = 6.7 Hz, 12H). MALDI-TOF-MS (CHCA matrix, positive reflection mode): Obs. m / z = (M+H) + 770.73, calculated as: C 47 H 99 N3O4 (exact mass 769.76; molecular weight 770.33).

[0727] H. Functionalization of primary amines with acrylates followed by alkylation of the remaining secondary amines.

[0728] Example H1.G1-ACR-nC14-4 / CH3-4

[0729] This general example describes the Michael addition reaction of a G1-PPI-(NH2)4 dendrimer with n-tetradecyl acrylate followed by reaction with paraformaldehyde to produce a PPI dendrimer with 4 n-tetradecyl ester groups and 4 methyl groups.

[0730] 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 moleq) was added. The mixture was stirred at 55 °C for 24 hours in a closed vial under an inert nitrogen atmosphere, which resulted in complete conversion of the primary amine. Almost complete monofunctionalization of each primary amine group was observed. The reaction mixture was evaporated to remove isopropanol and the residue was co-evaporated 3 times with chloroform. The residue was dissolved in formic acid (2 mL). Paraformaldehyde (121 mg, 4.0 mmol, 6 moleq) and THF (1 mL) were added to dissolve. The mixture was stirred at 60 °C. 1 The progress of the reaction was monitored by H-NMR. After 3.5 hours, 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 0.1M NaOH(aq), followed by washing with saturated NaCl(aq) and drying with Na2SO4. The crude product was stirred in MeCN and placed at 0°C for several hours to induce complete phase separation. Subsequently, the supernatant was decanted. This procedure was repeated twice. The residue was placed on a silica column, initially with 5% MeOH / chloroform to remove impurities. The product was finally eluted from the column by changing the eluent to 5% MeOH and 1% TEA / chloroform. Yield: 560 mg (58%). 1 The H-NMR spectrum was consistent with the expected structure.

[0731]

[0732] 1 H 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 reflection mode): Obs. m / z = (M+H) + 1446.37, small peak m / z=M+H) +,1701.59 (5 C14 chains and 3 CH3). Calculation: C 88 H 176 N6O8 (exact mass 1445.35; molecular weight 1446.41).

[0733] J. Amidation of the amine, optionally followed by alkylation of an internal tertiary amine.

[0734]

[0735] Synthesis of G1 PPI dendrimer amides, also internally quaternized. (a) Toluene, TEA, 90°C, 6h; (b) MeI, chloroform / MeOH, 50°C; (c) Amberlite IRA400 (Cl - ).

[0736] Example J1.G1-Amide-nC16-4

[0737] N,N,N,N-((Butane-1,4-diylbis(azatriyl))tetra(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 sufficient toluene. Then, dissolved in toluene (4 mL), it was added to a solution of phenyl palmitate (6.3 g) in toluene (16 mL). Triethylamine (9 mL) was added and the mixture was stirred at 90 °C for 6 hours. The reaction mixture was concentrated in vacuo and the resulting waxy solid was triturated in acetonitrile (80 mL) for 3 hours. After filtering and washing with acetonitrile, the residue was dried and triturated in hexane. Filtration, washing with hexane and drying the residue gave 3.3 g (82%) of the pure product as an off-white solid. 1 The H-NMR spectrum was consistent with the expected structure.

[0738]

[0739] 1 H 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 reflection mode): Obs. m / z = (M+H) + 1270.31. Small peak, m / z = M + Na +,1292.23. Calculation: C 80 H 160 N6O4 (exact mass 1269.25; molecular weight 1270.20).

[0740] Example J2.G1-Amide-nC16-4(2MeI)

[0741] N,N,N,N-((butane-1,4-diylbis(azatriyl))tetra(propane-3,1-diyl))tetrapalmitamide (2MeI ammonium adduct). Example J1 (4 g) was stirred in a 1:1 chloroform / methanol mixture (80 mL) at 45°C. To this cloudy mixture was added iodomethane (3.14 mL). The oil bath temperature was raised to 50°C and 25 mL of chloroform was added to produce a clear mixture, which was stirred for 16 hours. After adding an additional amount of iodomethane (1 mL), stirring was continued for 4 hours. The mixture was then concentrated in vacuo and co-evaporated twice with methanol and once with chloroform. To obtain complete methylation, the product was redissolved in chloroform / methanol and stirred with 10 equivalents of iodomethane in a capped vial at 90°C under microwave irradiation for 1 hour. The mixture was concentrated in vacuo and co-evaporated with chloroform to give an off-white product quantitatively. 1 The H-NMR spectrum was consistent with the expected structure.

[0742]

[0743] 1 H NMR (400MHz, chloroform-d) δ7.41(t,J=6.1Hz,2H),7.35(s,4H),3.53(s,3H),3.37(p,J=1.6Hz,2H),3.31(p,J=5.7Hz,15H,C,E),3.12(s,9H), 3.04(s,6H,K),2.23(t,J=7.6Hz,8H,F),2.14–1.81(m,12H,A,D),1.61(p,J=7.2Hz,8H,G),1.26(s,99H,H),0.88(t,J=6.7Hz,13H,I).

[0744] Example J3.G1-Amide-nC16-4(2MeCl)

[0745] N,N,N,N-((butane-1,4-diylbis(azatriyl))tetrakis(propane-3,1-diyl))tetrapalmitamide (2MeCl ammonium adduct). Example J2 (3 g) was dissolved in 150 mL of chloroform / methanol mixture (2:1) to obtain a clear solution. Ion exchange resin (50 g) Amberlite IRA 400 (Cl -) was stirred in water and poured onto a 1 cm diameter column. The column was rinsed with water and then with methanol. The amount of chloroform in the rinse eluent slowly rose to 66%. In order to avoid the resin beads from floating, a filler plug was placed on the top of the resin. The solution of J2 was eluted onto the resin with a chloroform / methanol mixture (2:1). All collected eluents were concentrated to obtain the desired product (2.4 g). 1 The H-NMR spectrum was consistent with the expected structure. To show that all iodide ions were replaced, the organic solution of the product was extracted with 1 M NaBr solution. The aqueous layer was then tested for starch, which showed no coloration, demonstrating complete exchange of iodide anions. As a reference benchmark, dendrimer solutions containing iodide were also tested. These dendrimer solutions containing iodide turned blue.

[0746]

[0747] 1 H NMR (400MHz, CDCl3) δ3.45(m,12H,B,C),3.37(t,8H,E),3.06(s,6H,K),2.41(t,8H, F),2.15(m,8H,D),1.96(m,4H,A),1.62(m,8H,G),1.25(m,96H,H),0.88(t,12H,I).

[0748] Example J4. G3-Amide-nC16-16

[0749] This material J4 was obtained by reacting PPI-dendrimer G3 (DAB-Am-16) with phenyl palmitate and was carried out in a similar manner to Example J1. Yield: 5.88 g (90%). 1 The H-NMR spectrum was consistent with the expected structure.

[0750]

[0751] 1 H NMR (400 MHz, chloroform-d) δ 11.04 (d, J = 207.4 Hz, 8H, NH), 7.60–7.16 (m, 9H), 4.06–2.63 (m, 113H, B, C, E, F, H, I, K), 2.46–2.08 (m, 54H, D, G, M), 2.08–1.70 (m, 36H, A, J), 1.57 (p, J = 7.2, 6.7 Hz, 33H, N), 1.26 (d, J = 9.1 Hz, 387H, O), 0.88 (t, J = 6.7 Hz, 48H, P). MALDI-TOF-MS (DCTB matrix, positive reflection mode): Obs. m / z = (M+H) +5503.04. Calculation: C 344 H 688 N 30 O 16 (Exact mass 5497.39; Molecular weight 5501.48).

[0752] Example J5.G3-Amide-nC16-16(14MeI)

[0753] This material J5 was obtained by reacting J4 with MeI and was carried out in a similar manner to Example J2. Yield: 1.32 g (97%). 1 The H-NMR spectrum was consistent with the expected structure.

[0754]

[0755] 1 H NMR (400MHz, CDCl3 / CD3OD) δ7.54(d,J=16.5Hz,1H),7.30(s,9H),3.84(d,J=61.2Hz,48H),3.55(d,J=35.9Hz,41H),3.44(s,5H),3.43–3.12(m,60H),2 .59(s,23H),2.40(s,35H),2.25(t,J=7.8Hz,35H),2.06(d,J=10.4Hz,33H, M), 1.60 (p, J = 7.2Hz, 33H, N), 1.51–1.00 (m, 387H, O), 1.00–0.76 (m, 48H, P).

[0756] Example J6.G3-Amide-nC16-16(14MeCl)

[0757] This material J6 was obtained by eluting J5 on an ion exchange column and was carried out in a similar manner to Example J3. Yield: 2.55 g (99%). 1 The H-NMR spectrum was consistent with the expected structure.

[0758]

[0759] 1H NMR (400MHz, CDCl3 / CD3OD) δ7.87(s,12H),7.30(t,J=7.4Hz,11H),4.16–2.94(m,145H),2.94–2.61(m,52H),2.46(s,22H),2. 20(dq,J=17.3,8.2Hz,30H),1.96(s,33H),1.60(s,28H),1.25(dt,J=20.6,9.1Hz,354H,O),0.88(dt,J=16.5,7.5Hz,48H,p).

[0760]

[0761] Dendrimers G1-dimethylitaconate-4 and G1(PAMAM-C2)-dimethylitaconate-4 were synthesized. Transesterification (by longer chain alcohols) or amidation (by longer chain primary or secondary amines) of the four methyl ester end groups would provide more nonpolar structures.

[0762] Example J7.G1-dimethyl itaconate-4

[0763] Dimethyl itaconate (0.66 g, 4.2 mmol, 4.2 eq) was added to a stirred solution of G1-PPI-(NH2)4 (316 mg, 1 mmol) in IPA (2 mL). The reaction mixture was heated to 60 ° C for 16 hours and then poured into water (15 mL). The aqueous layer was washed with ether (4×5 mL). Traces of solvent were removed in vacuo at room temperature. The aqueous layer was lyophilized to give an amber oil. The product was purified by silica column chromatography using MeOH / CHCl3 (20-30% MeOH) eluent. Yield: colorless oil (414 mg, 0.50 mmol, 50%).

[0764] 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 calculated for C 40 H 64 N6O 12 820.46; Obs.[M+H] 2+ 411.50,[M+H] + 820.50,[M+Na] + 853.50.

[0765] Example J8.G1 (PAMAM-C2)-dimethyl itaconate-4

[0766] Dimethyl itaconate (514 mg, 3.25 mmol, 4.9 eq) was added to a solution of G1-PAMAM-C2 (ethylenediamine core, 4 amine end groups; about 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. The reaction was complete after 184 hours. The mixture was poured into water (15 mL) and the aqueous layer was washed with ether (4×5 mL). Traces of solvent were removed in vacuo and the aqueous layer was lyophilized to give a semi-solid colorless wax (834 mg) still containing some ether.

[0767] 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 calculated for C 46 H 72 N 10 O 16 1021.14; Obs.[M+H] 2+ 511.58,[M+H] + 1021.50,[M+Na] + 1043.50.

[0768] Example J9.G1-Lysine-N 2 -Oleic acid-4

[0769] G1 PPI dendrimer (35 mg; 0.11 mmol; 0.44 mmol primary amine groups) and triethylamine (200 μL) were dissolved in DCM (9 mL) and stirred at room temperature under nitrogen atmosphere. 2,3,5,6-tetrafluorophenyl N 6 -(tert-Butyloxy-carbonyl)-N 2-oleoyl-L-lysine ester (292 mg, 0.44 mmol, 4.00 eq) and the solution was stirred for 24 hours. Chloroform (50 mL) was added and the organic phase was washed with 1N NaOH aqueous solution (20 mL), followed by water (20 mL) and brine (20 mL). The solution was co-evaporated with chloroform (3×20 mL) to remove triethylamine. Yield: 209 mg colorless oil. The oil was dissolved in DCM (10 mL) and TFA (1 mL), and the solution was stirred for 20 hours at room temperature under a nitrogen atmosphere. The mixture was evaporated to dryness, and the residue was dissolved in CHCl3 (70 mL). The solution was washed with 1N NaOH aqueous solution (20 mL), followed by brine (20 mL). The solution was evaporated to dryness to obtain a white waxy product. Yield: 180 mg (86%).

[0770]

[0771] 1 H NMR (400 MHz, chloroform-d) δ7.98 (s, 4H), 7.25-7.00 (s, 4H), 5.38-5.28 (m, 8H), 4.45 (s, 4H), 3.50-2.60 (m, 32H), 2.48 (s, 8H), 2.19 (s, 8H), 2.00 (m, 16H), 1.85-1.20 (m, 120H), 0.88 (t, J = 6.9 Hz, 12H). FTIR (cm -1 )3282(m),2924(s),2854(m),1692(m),1637(vs),1534(m),1455(m),1365(m),1248(m),1171(s). MALDI-TOF-MS (CHCA matrix, positive reflection mode): Obs.m / z=(M+Na)+1909.66(second isotope peak). Calculation: C 112 H 216 N 14 O8 (exact mass 1885.69; molecular weight 1887.00).

[0772] Example J10.G1-Lysine-N 6 -Oleic acid-4

[0773] A solution of G1 PPI dendrimer (31.0 mg, 0.098 mmol; 0.39 mmol primary amine groups) in DCM (1 mL) was added to 2,3,5,6-tetrafluorophenyl N 2 -(tert-Butyloxy-carbonyl)-N 6-oleoyl-L-lysine salt (282 mg, 0.43 mmol) and triethylamine (100 μL) in DCM (3 mL). After 30 hours, 1 H-NMR analysis showed complete conversion. To remove excess TFP-ester, Biotage MP-Trisamine resin (125 mg) was added and stirring was continued for 30 h. DCM (50 mL) was added and the mixture was filtered. The filtrate was washed with 1 N NaOH (20 mL), water (20 mL) and brine (20 mL). After removal of the solvent, the dendrimer was obtained as a white solid. Yield: 198 mg. The dendrimer (195 mg) was dissolved in DCM (4 mL) under nitrogen atmosphere and TFA (1 mL) was added. After 23 h, the reaction mixture was concentrated on a rotary evaporator. The product was co-evaporated 3 times with toluene to remove excess TFA, then dissolved in chloroform (50 mL) and shaken with 1 N NaOH (2 mL) followed by brine (10 mL). Removal of the solvent gave a colorless oil. Yield: 180 mg (97%). 1 The H-NMR spectrum was consistent with the expected structure.

[0774]

[0775] 1 H NMR (400MHz, chloroform-d) δ7.63(s,4H),6.16(t,4H),5.38-5.29(m,8H),4.16(s,4H),3.35-3.20(m,16H),2. 41(t,4H),2.36(t,4H),2.15(t,J=9.7Hz,8H),2.0(m,16H),1.9-1.0(m,136H),0.87(t,J=6.9Hz,12H). FTIR(cm -1 )3309(s),2923(vs),2854(s),1639(vs),1549(s),1466(w). MALDI-TOF-MS (CHCA matrix, positive reflection mode): Obs.m / z=(M+Na)+1909.68(second isotope mass). Calculation: C 112 H 216 N 14 O8 (exact mass: 1885.69; molecular weight 1887.00).

[0776] Example J11.G1-Arginine-Oleic Acid-4

[0777] A solution of G1 PPI dendrimer (69 mg, 0.22 mmol, 0.87 mmol primary amine groups) in THF (2 mL) was added to oleoyl-L-arginine (463 mg, 0.97 mmol) followed by chloroform (2 mL). After stirring for a few minutes, a gel-like mixture formed. HFIP (100 μL) was added and stirring was continued at 40°C to form a clear solution. After cooling to room temperature, DCC (399 mg, 1.93 mmol) was added. The resulting clear solution was heated to 40°C and turned cloudy white (due to the formation of DCU). The progress of the reaction was monitored by analyzing samples by MALDI-TOF-MS. After 6 days of reaction, more oleoyl-L-arginine (220 mg, 0.46 mmol) and DCC (156 mg, 0.76 mmol) were added. After three days, MALDI-TOF-MS showed 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 crude product.

[0778] Water (10mL) is added to part (500mg) of the crude product, and the mixture is sonicated for 30 minutes. After standing overnight, the almost clear aqueous solution is separated from the white precipitate (mainly DCU) and transferred to a dialysis tubing (CE 500-1000 dalton). The solution is dialyzed with water (1L) containing acetic acid (2mL), then dialyzed with water (1L) and acetic acid (1mL), then dialyzed with water (1L) and 1N NaOH (2mL), and finally dialyzed with water (1L). A white fluffy powder is obtained after freeze drying. Yield: 188mg. 1 The H-NMR spectrum was consistent with the expected structure.

[0779]

[0780] 1 H NMR (400MHz, chloroform-d) δ8.20 (s, 4H), 7.60-7.00 (broad peak,20H),5.38-5.28(m,8H),4.40-4.10(m,4H),3.30-3.10(m,16H),2.50-1.10(m,152H),0.88(t,J=6.8Hz,12H). FTIR(cm -1 )3280 (m), 3184 (m), 2984 (vs), 2854 (vs), 1644 (vs), 1549 (vs), 1456 (m), 1403 (m). MALDI-TOF-MS (CHCA matrix, positive reflection mode): Obs.m / z=(M+H)+1999.73 (the second isotope peak). Calculated as C 112 H216 N 22 O8 (1998.72 is the second isotope peak); molecular weight 1999.06.

[0781] K. Functionalization of primary amines with acrylates followed by acylation of the remaining secondary amines (here, the product is a mixture of multivalent molecules)

[0782] Example K1. G1 PPI dendrimer modified with n-octyl acrylate and acetic anhydride

[0783] n-Octyl acrylate (175 mg, 0.95 mmol, 3 eq) was added to a stirred solution of G1-PPI-(NH2)4 dendrimer (100 mg, 0.32 mmol) in IPA (1 mL). The reaction was heated at 60°C for 16 hours, then concentrated in vacuo 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 hours, then diluted with chloroform. The chloroform layer was washed with saturated NaHCO3 solution (1×25 mL) and brine (1×25 mL), dried over Na2SO4 and concentrated in vacuo to give the product (365 mg, 0.35 mmol). The terminal unit groups were a mixture of n-octyl acrylate and acetyl.

[0784] 1 H NMR (400MHz, chloroform-d) δ6.63 (m, 1H), 4.07 (dtd, J = 13.4, 6.8, 2.7Hz, 6H), 3.58 ( dt,J=14.3,5.7Hz,6H),3.30(p,J=7.4Hz,7H),2.76(t,J=7.3Hz,1H),2.67–2 .52(m,6H),2.52–2.29(m,12H),2.17–2.03(m,9H),1.96(d,J=4.8Hz,3H),1 .63(p,J=6.9Hz,14H), 1.30(dd,J=14.1,7.6Hz,34H), 0.88(t,J=6.7Hz,9H). MALDI-TOF-MS (CHCA matrix, positive reflection mode): 3x n-octyl acrylate Obs. m / z = (M+H) + 1037.80,(M+Na) + 1059.77. Calculation: C 57 H 108 N6O 10(Exact mass 1036.81; molecular weight 1037.52). 4x Octyl acrylate Obs. m / z = (M + H) + 1221.95,(M+Na) + 1243.93. Calculation: C 68 H 128 N6O 12 (Exact mass 1220.96; Molecular weight 1221.80).

[0785] Example K2. G2 PPI dendrimer modified with n-octyl acrylate and acetic anhydride

[0786] Analogously to Example K1, G2-PPI-(NH2)8 (102 mg, 0.13 mmol) was reacted with n-octyl acrylate (169 mg, 0.92 mmol, 6.95 eq) in IPA (1 mL) solution in step 1, and DIPEA (231 μL, 172 mg, 1.33 mmol, 10 eq) was used in step 2 to react with acetic anhydride (125 μL, 136 mg, 1.33 mmol, 10 eq) in DCM (1 mL) solution. The terminal unit groups were a mixture of n-octyl acrylate and acetyl.

[0787] 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 reflection mode): 6x n-octyl acrylate Obs. m / z = (M+H) + 2215.75,(M+Na) + 2237.73. Calculation: C 122 H 232 N 14 O 20 (Exact mass 2213.76; molecular weight 2215.28). 7x Octyl acrylate Obs. m / z = (M + H) + 2399.90,(M+Na) + 2421.88. Calculation: C 133 H 252 N14 O 22 (Exact mass 2397.90; Molecular weight 2399.56).

[0788] L. Functionalization of primary amines with acrylamide followed by epoxidation of the remaining secondary amines (here, the product is a mixture of multivalent molecules)

[0789] Example L1. G1 PPI dendrimer modified with n-octylacrylamide and 1,2-epoxydodecane

[0790] n-Octyl-acrylamide (174 mg, 0.95 mmol, 3 eq) was added to a stirred solution of G1-PPI-(NH2)4 (100 mg, 0.32 mmol) in IPA (1 mL). The reaction mixture was stirred at 60 ° C for 160 hours to complete the first step. 1,2-Epoxydodecane (582 mg, 3.16 mmol, 10 eq) and IPA (0.5 mL) were then added and the reaction mixture was stirred at 60 ° C for another 16 hours. The mixture was cooled and concentrated in vacuo. The residue was stirred in MeCN and cooled to -20 ° C to separate the product from the MeCN solvent phase. The supernatant was carefully removed using a pipette, and the grinding procedure was repeated twice to obtain an amber oil (495 mg, 0.28 mmol, 76%). The terminal unit group is a mixture of n-octylacrylamide and epoxide-derived groups.

[0791] 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 reflection mode): Obs. m / z = (M+H) + 1788.71, calculated as: C 109 H 223 N9O8 (exact mass 1786.73; molecular weight 1788.04).

[0792] Example L2. G2 PPI dendrimer modified with n-octylacrylamide and 1,2-epoxydodecane

[0793] Similar to Example L1, G2-PPI-(NH2)8 (100 mg, 0.13 mmol) was reacted with n-octylacrylamide (142 mg, 0.78 mmol, 6 eq) in IPA (1 mL) solution in step 1 and with 1,2-epoxydodecane (477 mg, 2.59 mmol, 20 eq) in step 2. Yield: amber viscous oil (463 mg, 96%). The terminal unit group is a mixture of n-octylacrylamide and epoxide derived groups.

[0794] 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 reflection mode): Obs. m / z = (M+H) + 3717.55, calculated as: C 226 H 462 N 20 O 16 (Exact mass 3713.60; Molecular weight 3716.31).

[0795] Example 2 - Solubility of Multivalent Molecules

[0796] The solubility of various multivalent molecules in ethanol, isopropanol and tricaprylin was tested at a concentration of 20 mg of material per gram of solvent. Spontaneous dissolution upon stirring at room temperature to give a clear solution is an indication of dissolution. 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 was soluble in all three solvents when heated to 37 degrees Celsius. Please note that not all prepared materials were tested.

[0797] These data indicate that the multivalent molecule is well soluble for processing, for example in ethanol or isopropanol. The data also show solubility in tricaprylin, which means that this molecule may have an affinity for the hydrophobic interior of the nanoparticles of the present invention.

[0798] Example 3 - Construction of multivalent molecules containing nanoparticles

[0799] Nanoparticle formulations are self-assembled based on ionic and hydrophobic interactions. These components are prepared in their respective organic solvents (lipids and other structural components) or aqueous buffers (nucleic acid payloads) with desired concentrations. Then the solutions are mixed together by the rapid mixing techniques that encompass microfluidics or T-shaped connections to mix. Excessive aqueous buffer is essential for the formation process. When used herein, excessive aqueous buffer refers to (aqueous buffer): (organic solvent) (based on volume) ratio of at least 2: 1 or higher, such as 2.2: 1, 2.5: 1, 2.8: 1 or 3: 1 or higher. After initial mixing, small fraction organic solvents are removed by, for example, dialysis or centrifugal filtration. These steps produce lipid nanoparticles, which are mixed by rapid mixing techniques such as microfluidics or T-shaped connections or are added thereto apolipoprotein (stabilizer) using, for example, a drip method. After adding apolipoprotein, residual protein needs to be removed by dialysis or centrifugal filtration. Finally, the sample is concentrated to desired concentration.

[0800] The resulting nucleic acid nanoparticles contain:

[0801] (a) Multivalent molecules;

[0802] (b) apolipoprotein stabilizers;

[0803] (c) nucleic acids;

[0804] (d) sterols;

[0805] (e) phospholipids; and

[0806] (f) Optionally, a lipid.

[0807] Therefore, using the indicated multivalent molecules, different compositions as described in Table S3, Table S7 and Table S8 were prepared, combined with the following control formulations:

[0808] The LNP control is a "standard LNP", such as that used in clinically approved LNP-siRNA formulations, as 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 (mol%), thereby ignoring the presence of nucleic acids:

[0809] -DLin-MC3-DMA 50%

[0810] -DSPC 10%

[0811] - Cholesterol 38.5%

[0812] -PEG-DMG 1.5%

[0813] The MC3-based aNP control is an apolipoprotein nanoparticle in which DLin-MC3-DMA is used to complex the nucleic acid instead of the multivalent molecules described herein. The compositions are listed in Tables S3 and S8.

[0814] Table S3. Quantities of components used in the formulation of nanoparticles containing siRNA. Reference nanoparticles are LNPs and aNPs (apolipoprotein nanoparticles) containing ionizable cationic lipid DLin-MC3-DMA. The aNPs of the present invention have multivalent ionizable materials to complex siRNA.

[0815]

[0816]

[0817] The aNPs of the present invention were prepared using the amounts given in the table above 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 multivalent materials. DMG-PEG 2000 is a synthetic lipid and is a polyethylene glycolized myristoyl diglyceride. Tricaprylin is tri-n-octanoin and is a triglyceride (TG).

[0818] Table S7. DLS data of siRNA-aNPs of the present invention containing multivalent ionizable molecule A2. In addition to the single mutations given in the first two columns, the formulation combinations used for the aNPs are shown in Table S3.

[0819] In all cases, nanoparticles with a diameter less than 200 nm were formed.

[0820]

[0821]

[0822] Table S8. Quantities of components used in mRNA loaded nanoparticle formulations. Reference nanoparticles were LNPs and aNPs (apolipoprotein nanoparticles) containing the ionizable cationic lipid ALC-0315.

[0823] The aNPs of the present invention contain a multivalent ionizable material A2.

[0824]

[0825]

[0826] Example 4 - Preparation of Nanoparticles

[0827] 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, USA. Cholesterol and tricaprylin were obtained from Sigma Aldrich. D-Lin-MC3-DMA (MC3) was obtained from the Center for Organic Chemistry, University of British Columbia, Vancouver, Canada. ALC-0315 was synthesized in-house. Recombinant apo A1 was expressed and purified in-house in ClearColi BL21 (DE3) cells. Firefly luciferase and negative control Dicer substrate siRNA were obtained from Integrated DNA Technologies (IDT), Iowa, US. 10.

[0828] Preparation of siRNA-loaded nanoparticles

[0829] The preparation process diagram is Figure 5 The specific formulation compositions of the aNPs of the invention, aNP control particles, and LNP control particles can be found in Table S3. Variations in composition were also evaluated, see Table S7.

[0830] The aNPs loaded with siRNA were prepared by rapid mixing using a T-shaped connector. Lipid molecules (phospholipids, cholesterol, triglycerides and ionizable materials of aNPs; phospholipids, cholesterol, ionizable materials and PEG-lipids of LNPs) were dissolved in ethanol and rapidly mixed with sodium acetate buffer (25mM, pH 4) containing 200μg of anti-firefly luciferase siRNA or scrambled siRNA. The organic phase and aqueous phase were mixed and directly collected on a 12-14kDa MWCO dialysis membrane (Spectra / Por TM). The nanoparticle formulation was dialyzed overnight at 4°C and stirred at 150 rpm against 1x PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 1.8 mM KH2PO4, pH 7.4). The PBS was renewed after approximately 4 hours. The next day, the formulation sample was collected from the dialysis bag and the volume was determined. If the control LNP particles were formulated, the apolipoprotein addition step was skipped. For aNP particles, apolipoprotein A1 was dissolved in PBS and added to the formulation by rapid mixing based on a T-junction.

[0831] The resulting sample was incubated at room temperature for one hour. The siRNA-aNP was filtered through a 0.2 μm filter and concentrated by centrifugal filtration at 1100G in a 100,000 MWCO filter. The sample was concentrated to 1.5 mL and stored at 4°C until further use. For use with cells, the sample was kept sterile after the 0.2 μm filtration step.

[0832] Preparation of mRNA-loaded nanoparticles

[0833] The preparation process diagram is Figure 5 The specific formulation compositions of the aNPs of the present invention, aNP control particles, and LNP control particles can be found in Table S8.

[0834] The mRNA-loaded aNPs were prepared by rapid T-junction mixing in a RNase-free culture hood. 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 100 μg mCherry mRNA (TriLink, CleanCap, 5 moU). The organic phase and aqueous phase were mixed and directly collected on a 12-14 kDa MWCO dialysis membrane (Spectra / Por TM ). The nanoparticle formulation was dialyzed overnight at 4°C and stirred at 150 rpm against 1x PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 1.8 mM KH2PO4, pH 7.4). The PBS was renewed after approximately 4 hours. The next day, the formulation sample was collected from the dialysis bag and the volume was determined. If the control LNP particles were formulated, the apolipoprotein addition step was skipped. For aNP particles, apolipoprotein A1 was dissolved in PBS and added to the formulation through a T-junction with quick mixing.

[0835] The resulting sample was 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 in a 100,000 MWCO filter. The sample was concentrated to 0.5 mL and stored at 4°C until further use. For use with cells, the sample was kept sterile after the 0.2 μm filtration step.

[0836] Example 5 - Characterization of Nanoparticles

[0837] Determination of siRNA encapsulation efficiency

[0838] The Quant-iT Ribo Green assay (Thermo Fisher) was used to quantify the amount of siRNA loaded within the formulated particles. The assay was performed using a black 96-well plate. The formulation samples with a theoretical siRNA concentration of 133.3 μg / mL were mixed in a black 96-well plate with TE buffer (10 mM Tris-HCl in DEPC-treated water, 1 mM EDTA, pH 7.5) and 2% Triton TM Dilute 200-fold in TE buffer containing 2% Triton X-100 to a total volume of 100 μl. Triton detergent destroys lipid-based nanoparticles; therefore, all siRNAs (retained and unretained) can be used in Quant-iT RiboGreen reagent. A siRNA control of known concentration (28.6 μg / mL) was diluted 53.3-fold in both TE buffer and TE buffer containing 2% Triton in a 96-well plate to a total volume of 100 μl. Next, the RiboGreen reagent was diluted 200-fold with TE buffer and TE buffer containing Triton. 100 μl of this dilution was added to each well containing a sample or control to bring the total volume in the plate to 200 μl. Subsequently, 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 a sample or control to bring the total volume in the plate to 200 μl. Subsequently, the RiboGreen reagent was diluted 200-fold in a Tecan The fluorescence of the samples was measured on a microplate reader with an excitation wavelength of 480 nm and an emission wavelength of 520 nm.

[0839] Recovery of siRNA was determined as follows:

[0840]

[0841] And the siRNA cutoff is defined as follows:

[0842]

[0843] siRNA retention = recovery x interception. Figure 6 and Fig.13 Report in.

[0844] Determination of mRNA packaging efficiency

[0845] This was done in a similar manner as described for determining siRNA encapsulation efficiency. The results are reported in Table S9.

[0846] Table S9. Recovery, entrapment, and retention percentages of mRNA in aNPs prepared from the indicated multivalent materials and determined by Ribogreen assay.

[0847] entry experiment Multivalent Materials Recycle(%) Retention (%) reserve(%) 1 in vitro A2 27％ 87％ 23％ 2 in vivo A2 57％ 88％ 50％

[0848] Determination of phospholipid and cholesterol recovery

[0849] The recovery of phospholipids and cholesterol in the prepared nanoparticles was quantified by performing the phospholipid FS and cholesterol FS assay (DiaSYS), which is an enzymatic colorimetric test. Buffer (190 μl) containing the colorimetric 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 Tecan The absorbance was measured at 600 nm by a microplate reader. The results are reported in Table S4.

[0850] Table S4. Recovery of cholesterol, phospholipids and apo-A1 in siRNA-aNPs. The number n indicates the number of individual replicates for each formulation (A1 n=8 means that A1 was formulated 8 different times and the average of the recovery was determined). All particles showed initial stability. * indicates phase separation after two days of storage in a 4°C refrigerator.

[0851]

[0852]

[0853] Determination of apolipoprotein A1 (apo A1) recovery

[0854] Using Apolipoprotein A1 FS Assay Determine the amount of apolipoprotein A1 in the nanoparticle formulation. This is an immunoturbidimetric test based on the interaction between anti-apo A1 antibodies and apo A1 present in the sample. TRIS buffer (200 μl) was added to 10 μl of nanoparticle sample in a clear 96-well plate. After incubation of the plate at 37°C for 5 minutes, the plate was The absorbance was measured by a microplate reader at a wavelength of 580 nm. TRIS (40 μl) containing apolipoprotein A1 antibody was added to the same 96-well plate, the solution was incubated at 37°C for 5 minutes, and then the absorbance was measured under the same settings. The results are reported in Table S4.

[0855] Dynamic Light Scattering (DLS)

[0856] The hydrodynamic diameter of the prepared particles was determined by the number weighted mean diameter provided by the dynamic light scattering (DLS) using a Zetasizer Nano ZS combined with a Malvern Zetasizer NanoSampler (Malvern Instruments, Worcestershire, UK). The size dispersion was measured as a polydispersity index (PDI). For DLS measurements, 100 μl of the prepared nanoparticles were diluted into 700 μl PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 1.8 mM KH2PO4) and balanced at room temperature before analysis. Each sample was measured 5 times at a wavelength of 633 nm, 10 times per 10 seconds, without fixing the attenuator and measurement position. The results are reported in Table S5, Table S7 and Table S10.

[0857] Table S5. DLS (Dynamic Light Scattering) analysis of siRNA-aNPs. The hydrodynamic diameter (number mean) and PDI (polydispersity index) of apolipoprotein nanoparticles (aNPs) are given. * indicates phase separation after two days of storage in a 4°C refrigerator, ** indicates phase separation after overnight dialysis. The standard deviation is measured taking into account the number of replicates (individual replicates) of the same formulation.

[0858]

[0859]

[0860] Table S10. DLS (Dynamic Light Scattering) of mRNA-aNPs. The hydrodynamic diameter (number mean) and PDI (polydispersity index) of the nanoparticles are given.

[0861]

[0862] Cryo-transmission electron microscopy (cryo-TEM)

[0863] The size and shape of the nanoparticles were also evaluated by cryogenic transmission electron microscopy (cryoTEM). The surface of a 200 mesh lace carbon-supported copper grid (Electron Microscopy Sciences) was plasma charged for 40 seconds using a carbon coater (Cressington 208). Subsequently, 3 μl of the nanoparticle sample (~1 mg protein / mL) was pipetted onto the grid and vitrified into a thin film by vitrification in liquid ethane. This step was performed by using an automated robot (FEI Vitrobot Mark IV). Cryogenic TEM images were collected on a cryogenic transmission electron microscope TITAN (ThermoFisher) 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 at 300 kV accelerating voltage, in bright-field TEM mode, with zero-loss energy filtering, 24,000x magnification (dose rate ) and 1 second acquisition time. Images and results are reported in Tables S6, Figure 7 , Figure 8 , Fig. 9 (A) Fig.15 middle.

[0864] Table S6. siRNA-aNP nanoparticle diameters recorded using cryo-TEM. *Indicates phase separation after two days of storage in a 4°C refrigerator. The number of individual particles was taken into account to measure the standard deviation.

[0865]

[0866] Scanning electron microscopy combined with immunogold staining

[0867] Figures and results reported in Fig. 9 (B) Chinese.

[0868] Cytotoxicity measurement of apolipoprotein nanoparticles (aNPs)

[0869] MTS assays were performed on a plurality of apolipoprotein nanoparticles (aNPs) of the present invention. These assays were performed according to protocols known in the art. The results are reported in Fig.10 .

[0870] In vitro silencing (dose-response curve)

[0871] In vitro silencing experiments were performed in the RAW264.7 cell line transfected with the pmirGLO plasmid (containing the gene sequences for Renilla luciferase and firefly luciferase expression). The cells were cultured in T75 cell culture flasks until 80% confluence. The cells were detached, counted, and seeded into 96-well plates at 10,000 cells / well. After standing overnight, the cells were transfected with nanoparticles containing a range of concentrations of anti-firefly luciferase siRNA. After incubation for 48 hours, the old medium was washed off with 1xPBS. Lysate phosphate buffer (Dual- Reporter Assay System, Promega) to lyse the cells. Transfer 10 μl of cell lysate to a white 96-well flat-bottom plate. Then, add 40 μl of ONE-Glo TM Reagents (Dual- Reporter Assay System, Promega) and using Tecan The luminescence was measured by a microplate reader with an integration time of 500 ms and a stabilization time of 1000 ms. 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- Reporter Assay System, Promega), and measure the luminescence again in the same way. Calculate the relative remaining firefly luciferase luminescence. First, the firefly luminescence is normalized with sea renilla luminescence. Subsequently, the normalized sample signal is expressed as a percentage of the untreated sample signal. This produces the following formula:

[0872]

[0873] The results are reported in Fig.11 .

[0874] In vitro silencing (scrambled siRNA and anti-firefly siRNA)

[0875] In vitro silencing experiments were performed in the RAW264.7 cell line transfected with the pmirGLO plasmid (containing the gene sequences for Renilla luciferase and firefly luciferase expression). The cells were cultured in T75 cell culture flasks until 80% confluence. The cells were separated, counted, and seeded into 96-well plates at 10,000 cells / well. After standing overnight, the cells were transfected with nanoparticles containing 100 nM scrambled nonspecific siRNA or anti-firefly luciferase siRNA. After incubation for 48 hours, the old culture medium was washed off with 1xPBS. Lysate phosphate buffer (Dual- Reporter Assay System, Promega) to lyse the cells. Transfer 10 μl of cell lysate to a white 96-well flat-bottom plate. Then, add 40 μl of ONE-Glo TM Reagents (Dual- Reporter Assay System, Promega) and using Tecan The microplate reader measures luminescence with an integration time of 500 ms and a stabilization time of 1000 ms. Luminescence scanning is performed because the luminescence peak should be in the range of 550–570 nm. Then, 40 μl of Stop and Glo reagent (Dual- ReporterAssay System, Promega), and measure luminescence again in the same way. Calculate the relative remaining firefly luciferase luminescence. First, all samples are normalized to firefly luminescence with sea renilla luminescence. Subsequently, the normalized sample signal is expressed as the percentage of the untreated sample signal. This produces the following formula:

[0876]

[0877] The results are reported in Fig.12 .

[0878] In vitro firefly luciferase expression

[0879] To detect firefly luciferase expression in vitro, ONE-Glo TM Luciferase Assay System (Promega). RAW264.7 cells were seeded in 96-well plates at a density of 35 thousand cells per well in 100 μL. Approximately 6 hours later, RAW264.7 cells were transfected with 10 μL of firefly luciferase mRNA (RIBOPRO) using aNPs or LNPs at a dose of 100 ng mRNA per well. 24 hours after transfection, cells were transfected with 10 μL of firefly luciferase mRNA (RIBOPRO) using aNPs or LNPs. Cell viability was determined by AQueous One Solution Cell Proliferation assay (MTS) (Promega). 10 μL of reagent was added to the cells and the absorbance was measured at 495 nm after incubation for 1 hour (37°C, 5% Co2). Subsequently, the cells were washed 3 times with 1xPBS and 40 μL of ONE-Glo was added to each well. TM Luciferase Assay Reagent. After complete cell lysis, transfer the cell lysate to a white flat-bottom 96-well plate. Luminescence is measured using a Tecan Spark plate reader. Luciferase is expressed as luciferase intensity normalized to cell viability and background signal subtracted. Results are reported in Fig.14 .

[0880] In vivo firefly luciferase expression

[0881] To detect firefly luciferase expression in vivo, C57BL / 6 mice (Jackson Laboratory, Germany) were injected with 0.5 mg / kg of firefly luciferase mRNA loaded into aNPs containing multivalent molecule A2 mRNA binders (TriLink, CleanCap, 5moU). After 16 hours, the animals were sacrificed and the liver, spleen, and femur were collected. The organs were processed to produce a single cell suspension. 1 / 10 of the liver was cut into small pieces and incubated with 5 mL of lysis solution for 15 minutes at 37°C, 50RPM. Next, the digested liver was passed through a 70 μm filter and collected in a 50 mL Falcon tube. After washing with 1XPBS, the cells were resuspended in 15 mL 1XPBS. 1 / 10 of the spleen was cut into small pieces, passed through a 70 μm filter and collected in a 50 mL Falcon tube. After washing with 1XPBS, 1 mL of lysis buffer (BD Pharm Lyse TM , catalog number 555899, diluted 10-fold in diH2O) and incubated at room temperature for 4 minutes. Wash the cells with 1XPBS and resuspend in 15mL 1XPBS. Both ends of the femur were broken. Use 10mL 1XPBS to flush the bone marrow cells from the inside of the bone through a 70μm filter and collect in a 50mL falcon tube. Centrifuge the cells and add 1mL lysis buffer (BD Pharm Lyse TM , catalog number 555899, diluted 10-fold in diH2O) to the pellet and incubate at room temperature for 1 minute. Wash cells with 1XPBS and resuspend in 5mL 1XPBS. Count all cell suspensions and seed 500,000 cells into V-bottom plates. Centrifuge the plates and remove the supernatant. Add 40μL ONE-Glo reagent (Promega) and incubate for 5 minutes (432RPM). Transfer 40μL to a white 96-well plate and measure luminescence using the following settings: stabilization time: 1000ms; integration time: 500ms; filter on. Results are reported on Fig.16 in vitro.

Claims

1. A nanoparticle comprising a core and an outer layer, wherein the core comprises: - nucleic acids; - multivalent molecules; and wherein the outer layer comprises: - an apolipoprotein, an apolipoprotein derivative, an apolipoprotein mimetic and / or an apolipoprotein mimetic derivative; - phospholipids; - sterols; and optionally - filler molecules, Wherein the multivalent molecule has formula (I): [core] x –[THIS] y –[TU] z (I) wherein the core is nitrogen or a C1-C1-C2-containing 1 to 15 nitrogen heteroatoms and optionally 1 to 4 oxygen heteroatoms. 18 Straight chain, branched chain or cyclic groups; and wherein x represents the number of connections from the core to a branching unit BU (or to a terminal unit TU when y=0), wherein x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 12, and wherein these connections all originate from nitrogen atoms of the core to carbon atoms of the branching unit BU (or the terminal unit TU); and The branching unit BU has the formula (IIa), (IIb), (IIc) or (IId): wherein (IIa) represents an n-alkylene-nitrogen spacer which is connected at the CH2-terminus to the core or to the BU closer to the core and which is connected at the N-terminus to two terminal units (TU) or to two branching units (BU) closer to the TU, wherein the connections are given in the form of wavy bonds, and wherein p is 1, 2, 3 or 4; and wherein (IIb) represents an amide containing an n-alkylene-nitrogen spacer, which is connected at the CH2-terminus to the core or to the BU closer to the core, and which is connected at the N-terminus to two terminal units (TU) or to two branching units (BU) closer to the TU, wherein the connections are given in the form of a wavy bond, and wherein q is 1, 2, 3, 4 or 5, and wherein R1 is hydrogen, methyl, ethyl, n-propyl or isopropyl; and wherein (IIc) and (IId) are as defined for (IIa) and (IIb), respectively, and wherein R2 is methyl, ethyl, n-propyl, benzyl, acetamide (-CH2-C(O)NH2) or 2-hydroxy-vinyl group, and wherein X - is the counter anion of the quaternary amine cation portion; and wherein y represents a specific and discrete generation number of the multivalent molecule, wherein the number represents how many consecutive BU layers are incorporated into the multivalent molecule, as counted proceeding from the inner core to the outer TU groups, and wherein y is 0, 1, 2, 3, 4, or 5; and wherein for each individual generation layer, the BU may be independently selected from formula (IIa), (IIb), (IIc) or (IId), i.e., the first BU emanating from the core (representing the first generation layer BU) may be different from the second BU emanating further away from the core (representing the second generation layer BU), and the second BU may be different from the third, fourth and fifth BU emanating further away from the core; and wherein the terminal units (TU) are individually and independently selected from hydrogen and C1-C1 optionally containing 1 to 8 heteroatoms. 30 an alkyl, aryl, arylene-alkyl or alkylene-aryl group, wherein the heteroatoms are individually and independently selected from O and N, provided that not all TU in the multivalent molecule are hydrogen; and wherein z represents the total number of TU groups attached to the multivalent molecule, wherein z is from 1 to 128; and wherein the multivalent molecule according to formula (I) is multivalent in the positively ionizable groups and / or cationic groups, wherein the cumulative number of positively ionizable groups and cationic groups in the multivalent molecule is 2 or more.

2. The nanoparticle of claim 1, wherein the multivalent molecule has formula (I): [core] x –[THIS] y –[TU] z (I) Where y=0, 1, 2 or 3.

3. The nanoparticle according to any one of the preceding claims, wherein the multivalent molecule is a first, second or third generation dendrimer selected from poly(propyleneimine) (PPI) dendrimers or polyamidoamine (PAMAM) dendrimers, most preferably a PPI dendrimer, or a modification thereof.

4. The nanoparticle of any one of the preceding claims, wherein the multivalent molecule has a structure selected from the group consisting of: or or wherein the terminal units (TU) are individually and independently selected from hydrogen and C1-C1 optionally containing 1 to 8 heteroatoms. 30 Alkyl, aryl, arylene-alkyl or alkylene-aryl groups, the heteroatoms being individually and independently selected from O and N, with the proviso that not all TU in the multivalent molecule are hydrogen.

5. The nanoparticle according to any one of the preceding claims, wherein the apolipoprotein, apolipoprotein derivative, apolipoprotein mimetic or apolipoprotein mimetic derivative is selected from apo A1, apo A1-Milano, apo A2, apo A4, apo A5, apo B48, apo B100, apo CI, apo C-II, apo C-III, apo C-IV, apo D, apo E, apoF, apo H, apo L and apo M or a mimetic or derivative thereof, Preferably, it is selected from apo A1, apo A2, apo A4, apo A5, apo B100, apo CI, apo C-II, apo C-III, apo C-IV and apo E or a mimetic or derivative thereof, More preferably, it is selected from apo A1, apo A4, apo A5, apo B100, apo C-III and apo E or a mimetic or derivative thereof, Most preferably, it is selected from apo A1, apo B100 and apo E or a mimetic or derivative thereof.

6. The nanoparticle according to any one of the preceding claims, wherein 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 nuclear 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 modification thereof; or Preferably, the DNA is single-stranded or double-stranded DNA; or Preferably, the nucleic acid is an antisense oligonucleotide as a single-stranded DNA or RNA, which consists of or comprises modified nucleotides or nucleoside analogs containing a phosphodiester backbone or a 2' ribose, more preferably, wherein the nucleotides or nucleoside analogs are selected from locked nucleic acids (LNA), bridged nucleic acids (BNA), morpholinos or peptide nucleic acids (PNA).

7. The nanoparticle according to any one of the preceding claims, wherein the sterol is preferably selected from sterols, cholesterol, ergosterol, hopanes, hydroxysteroids, phytosterols, steroids, zoostanols, stigmasterol or β-sitosterol, or a combination thereof.

8. The nanoparticle according to any one of the preceding claims, wherein: The phospholipid is selected from phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylglycerol or a combination thereof, preferably, wherein at least one, more preferably both, of the acyl groups in the phospholipid are derived from long-chain fatty acids, even more preferably, wherein the long-chain fatty acids are selected from lauric acid, lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid and linoleic acid, or a combination thereof.

9. The nanoparticle according to any one of the preceding claims, further comprising a filler molecule, preferably wherein the filler molecule is a glyceride molecule, more preferably wherein the filler molecule is selected from triglycerides, modified triglycerides and cholesterol esters, or a combination thereof, preferably wherein the triglyceride is derived from a C6-C18 fatty acid, preferably tricaprylin and / or wherein the cholesterol ester is cholesteryl acetate, cholesteryl caprylate and / or cholesteryl oleate.

10. The nanoparticle according to any one of the preceding claims, wherein: The amount of the apolipoprotein, apolipoprotein derivative, apolipoprotein mimetic or apolipoprotein mimetic derivative ranges from 0.1 wt % to 90 wt %; and / or The amount of the nucleic acid ranges from 0.01 wt % to 90 wt %; The amount of the phospholipids ranges from 0.1 wt % to 95 wt %; and / or The amount of the sterol ranges from 0.1 wt % to 95 wt %; and / or The amount of the multivalent molecule ranges from 0.1 wt % to 95 wt %, The amount of filler optionally present comprises a range of 0 to 95% by weight, The weight percentages are based on the combined amount of the five components plus the optional sixth filler component, ie, the five or six components add up to 100% of the weight of the nanoparticles.

11. A composition comprising the nanoparticles according to any one of the preceding claims and a physiologically acceptable carrier, preferably wherein the composition is a pharmaceutical composition.

12. The nanoparticle according to any one of claims 1 to 10 or the composition according to claim 11 for use as a medicament.

13. The nanoparticle or composition for use according to claim 12, wherein the use comprises delivering the nucleic acid to the myeloid compartment or the spleen.

14. The nanoparticle according to any one of claims 1 to 10 or the composition according to claim 11, for use in treating a disease by stimulating or inhibiting an innate immune response.

15. The nanoparticle or composition for use according to claim 14, wherein the disease is cancer, cardiovascular disease, an autoimmune disorder or xenograft rejection.

16. A method for producing nanoparticles comprising the steps of: a) mixing, preferably rapidly, a (lipid) component in an organic solvent with nucleic acids in an aqueous buffer to produce nanoparticles, wherein the (lipid) component comprises a phospholipid, a sterol, a multivalent molecule and optionally a filler molecule; and wherein the aqueous buffer has a pH of 5.5 or less, preferably 5.0 or less; and b) mixing the lipid nanoparticles with apolipoprotein, an apolipoprotein derivative, an apolipoprotein mimetic and / or an apolipoprotein mimetic derivative, preferably rapidly, to produce nanoparticles at a pH between 5.5 and 9.0, preferably between 6.0 and 8.0, more preferably between 6.5 and 8.

0.

17. The nanoparticle according to any one of claims 1 to 10, wherein the nanoparticle is obtainable or obtained by the method according to claim 16.

18. An in vitro or ex vivo method for introducing a nucleic acid into a cell, the method comprising contacting the nanoparticle according to any one of claims 1 to 10 or the composition according to claim 11 with the cell.

19. An in vivo method for introducing a nucleic acid into a cell, the method comprising contacting the nanoparticle according to any one of claims 1 to 10 or the composition according to claim 11 with the cell.

20. The nanoparticle of any one of claims 1 to 10 or the composition of claim 11 for use in delivering a nucleic acid to a subject in vivo.

21. A method for delivering a nucleic acid in vivo, the method comprising administering to a subject a nanoparticle according to any one of claims 1 to 10 or a composition according to claim 11.

22. A method for treating a disease or condition in a subject in need thereof by stimulating or inhibiting an innate immune response, the method comprising administering to the subject a therapeutically effective amount of the nanoparticle according to claims 1 to 10 or the composition according to claim 11.

23. The method of claim 22, wherein the disease is selected from cancer, cardiovascular disease, an autoimmune disorder, or xenograft rejection.

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