WO2019110067A1

WO2019110067A1 - Hybrid nanoparticle

Info

Publication number: WO2019110067A1
Application number: PCT/DK2018/050330
Authority: WO
Inventors: Chuanxu YANG; Dang Quang Svend Le
Original assignee: Aarhus Universitet
Priority date: 2017-12-07
Filing date: 2018-12-06
Publication date: 2019-06-13

Abstract

The present invention relates to a nanoparticle comprising at least one amino-rich lipid-like compound and at least one amphiphilic polymer and/or poloxamer. The nanoparticle may be loaded with an active component such as nucleic acids to form a functionalised nanoparticle. The functionalised nanoparticle may be formed by a two-step process by first forming the nanoparticle and hereafter loading it with the active component.

Description

HYBRID NANOPARTICLE

Technical field of the invention

The present invention relates to a nanoparticle comprising at least one amino-rich lipid-like compound and at least one amphiphilic polymer and/or poloxamer. In particular, the present invention relates to a functionalised nanoparticle, where a nanoparticle comprising at least one amino-rich lipid-like compound and at least one amphiphilic polymer and/or poloxamer further comprises an active component such as a nucleic acid. Background of the invention

Specific drug delivery such as nucleic acid drug delivery like RNAi shows great potential as targeted delivery of drugs for treatment of diseases due to their unique target specificity. RNA interference (RNAi) is an important mechanism to regulate gene expression by small RNA molecules via mRNA cleavage or translational inhibition. Small interfering RNA (siRNA), as one of the most potent RNA-based gene regulators, can specifically downregulate virtually any target gene via RNAi.

The application of siRNAs has, however, been hindered by a series of biological barriers, including enzymatic degradation in blood circulation, activation of immune system, limited membrane crossing and endosomal/lysosomal entrapment (Haussecker, D, 2014). Most tissues can only be reached through systemic treatment, which requires siRNAs/miRNAs to overcome a series of barriers before reaching the cytoplasm of target cells to realize its function. Biological barriers play an important role in the defence system to protect organisms from the invasion of pathogens. After intravenous injection, naked small RNA molecules are easily degraded by serum endonucleases, and cleared by kidney filtration. To avoid enzymatic degradation and systemic clearance, appropriate modifications of siRNAs/miRNAs and delivery vectors can be introduced. When delivering with carriers, the delivery systems are usually opsonized, including adsorption of serum proteins such as albumin, lipoproteins, immunoglobulins, laminin and complement components (Tenzer et al, 2013; Owens DE et al. 2006). Depending on which proteins are adsorbed on the surface, opsonization will guide the delivery system to accumulate in the mononuclear phagocytic system (MPS), and phagocytosed by phagocytes in the liver, lung, spleen etc. (Blanco et al, 2015). Accordingly, to deliver siRNAs/miRNAs efficiently, the delivery system should be able to (1) improve the stability of small RNAs against serum nucleases; (2) prolong the circulation time by reducing non-specific interactions with serum proteins to prevent clearance by MPS; (3) avoid activating the immune system; (4) ensure biocompatibility; (5) guarantee effective accumulation in target tissues; (6) promote uptake by target cells and achieve effective endosomal escape to enter the RNAi machinery in the cytoplasm.

In recent years, non-viral nanoparticles for nucleic acid delivery have been created for delivering siRNAs/miRNAs in many diseases, such as various cancers, immune disorders, and infections (Yin, H et al., 2014; Thomas M et al., 2007). Nanoparticles are made of various materials with sizes ranging from 1 ~ 1000 nm (normally 10 nm to 200 nm).

Different types of nanoparticles have been developed in order to be safe, show low toxicity and deliver drugs efficiently. Many of the nanoparticles comprise a lipid-membrane assembled with cholesterol and polyethylene glycol molecules (Dong et al., 2014; Love et al., 2010) However, these nanoparticles show the drawback of introducing cholesterol into the body when used in therapy. In addition, these nanoparticles do only show limited transfection efficiency of cells difficult to transfect such as macrophages. Hence, an improved structure of nanoparticles for safe and efficient delivery of active components such as drugs and in particular, nucleic acids would be advantageous.

Summary of the invention

Thus, an object of the present invention relates to providing a stable and efficient nanoparticle.

In particular, it is an object of the present invention to provide a nanoparticle, which may be prepared prior to loading of the nanoparticle with the drug. Thus, one aspect of the invention relates to a nanoparticle comprising

a) at least one amphiphilic polymer and/or a poloxamer; and

b) at least one amine-rich lipid-like compound as described by formula (I)

where R2, R2', R2- and R₂ are independently selected from H or CH₂-CH(OH)-R3, wherein R₃ is a linear or branched alkyl chain selected from a group having 8-20 carbon atoms; and

where Ri is selected from formula (II)

wherein Xi, X₂ and X3 are independently selected from O, NH, NR₄ or

where R₄ is CI-hCI-hlN RsXRs·) or CH₂-CH(OH)-R3, where R5 and R5- are

independently selected from H or CH2-CH(OH)-R₃;

where m is an integer selected from 0-2, n is an integer selected from 1-4, 0 is an integer selected from 1-4, p is an integer selected from 0- 1, and q is an integer selected from 0- 1.

Another aspect of the present invention relates to a functionalised nanoparticle comprising a nanoparticle as described herein and at least one active component.

Yet another aspect of the present invention is to provide a process for the preparation of a functionalised nanoparticle comprising the steps of

1) mixing :

a) at least one amphiphilic polymer and/or a poloxamer as described herein; and

b) at least one amine-rich lipid-like compound as described herein;

under conditions forming nanoparticles;

2) adding at least one active component as described herein forming functionalised nanoparticles; and

3) purifying said functionalised nanoparticles. Still another aspect of the present invention is to provide a functionalised nanoparticle as described herein for use as a medicament.

Still another aspect of the present invention is to provide a functionalised nanoparticle as described herein for use in the prevention or treatment of a disease selected from the following group of diseases: inflammatory diseases and cancer.

Brief description of the figures

Figure 1 shows in (A) the synthesis of lipid-like materials Al-14 and in (B) the preparation of pluronic polymer/lipid-like material hybrid nanoparticles and their use as RNA delivery system.

Figure 2 shows a characterization of nanoparticles. (A) show the hydrodynamic size and distribution of CHOFREEN (F127/A1-14) and siRNA loaded CHOFREEN (F127/A1-14) nanoparticles measured by dynamic light scattering (DLS). (B) shows transmission electron microscopy (TEM) images of nanoparticles. (C) shows siRNA loading efficiency by CHOFREEN (F127/A1-14) nanoparticles at different ratios (lipids/siRNA, w/w). (D) shows the cell viability of CHOFREEN/siRNA nanoparticle in RAW 264.7 macrophages.

Figure 3 shows an evaluation of the siRNA delivery efficiency by different polymer/lipid-like material hybrid nanoparticles. (A) shows the structure of different amino-rich and lipid-like molecules and their abbreviations. (B) shows a comparison of the GFP silencing efficiency in HeLa-GFP cells by DSPE-PEG/cholesterol/lipid and pluronic F127/lipid nanoparticles. (C) shows an evaluation of the knockdown efficiency of hybrid nanoparticles with different types of pluronic polymers, including F127, P123, F68 and P84.

Figure 4 shows a comparison of the knockdown efficiency of IL-Ib in macrophages, where the cells were transfected with indicated formulations for 48 hrs followed by stimulation with LPS for 6 hrs before measuring the mRNA level of IL-Ib by RT-qPCR. Cells without Lipopolysaccharide (LPS) stimulation (WT) or only stimulated by LPS (LPS only) were included as negative and positive controls, respectively. (A) shows a murine macrophage cell line RAW 264.7 (B) shows knockdown of IL-Ib in primary peritoneal macrophages.

**p<0.01

Figure 5 shows mRNA delivery by nanoparticles. (A) shows a flow cytometry histogram of human adipose-derived stem cells (hADCSs) after transfection with green fluorescent protein mRNA (mGFP) encapsulated nanoparticles for 24 hrs. (B) shows quantification of GFP positive cells from flow cytometry analysis (n = 3). Figure 6 shows co-delivery of Cas9 mRNA and gRNA for genome editing. (A) shows representative images of GFP stably expressing human non-small cell lung carcinoma H1299 cells (H1299-GFP) transfected with Cas9 mRNA (mCas9) and guide RNA targeting GFP (gGFP) using CHOFREEN (F127/A1-14) or lipofectamine. (B) shows a flow cytometry histogram of H1299-GFP cells transfected with indicated formulations for 5 days. (C) shows quantification of genome editing efficiency in H1299-GFP cells from flow cytometry analysis. Figure 7 shows the effect of lyophilization on nanoparticles. (A) shows DLS measurements of DSPE-PEG before and after lyophilization. (B) shows DLS measurements of pluronic F127 formulated nanoparticles before and after lyophilization. (C) shows GFP silencing efficiency in HeLa-GFP cells by nanoparticles before or after lyophilization. Figure 8 shows knockdown of IL-Ib in collagen antibody-induced arthritic (CAIA) mice. (A) shows the biodistribution CHOFREEN/siRNA in CAIA mice at 24 hrs post injection. Mice were injected with (a) CHOFREEN/Cy5.5-siRNA, (b) Cy5.5-siRNA or (c) PBS. (B) shows ex vivo imaging of siRNA distribution in major organs including heart, lung, liver, spleen and kidneys. (C) shows ex vivo imaging of siRNA distribution in arthritic paws. (D) shows mRNA expression of IL-Ib in paws after 9 days as quantified by RT-qPCR. (E) shows the expression of IL-Ib in paw lysates as measured by ELISA. CHOFREEN = F127/A1-14.

Figure 9 shows therapeutic effect of CHOFREEN/siIL-Ib in CAIA mice. (A) shows representative images of paws after 9 days (left: hind paw, right: forepaw). (B) shows the clinical scores of CAIA mice treated with PBS, CHOFREEN/siIL-Ib or CHOFREEN/siNC. (C) shows micro-computed tomographic (micro-CT) images of the paws and knees on day 9. (D) shows straining with H8dº and Goldners Trichrome of the paws. CHOFREEN = F127/A1- 14. Figure 10 shows the cellular uptake mechanism of hybrid nanoparticles being F127/A1-14 hybrid (A) and a lipid system (DSPE-PEG/cholesterol/Al-14) (B), respectively. The siRNA uptake by RAW 264.7 cells at different conditions, including normal transfection, transfection at 4°C, co-incubation with Dynasore or Bafilomycin is demonstrated. Figure 11 shows a comparison of the knockdown efficiency of TNF-a in macrophages by measuring the mRNA level of TNF-a by RT-qPCR after transfection for 48 hrs followed by stimulation with LPS for 6 hrs. RAW 264.7 cells were transfected with siRNA against TNF-a using different transfection agents and different amounts. The present invention will now be described in more detail in the following.

Detailed description of the invention

Definitions

Prior to discussing the present invention in further details, the following terms and conventions will first be defined :

Amine-rich lipid-Hke compound

In the present context, the term "Amine-rich lipid-like compound" is to be understood as a group of hydrophobic organic compounds substituted with one or more nitrogen groups. Lipids are characterized by being insoluble in water but soluble in non-polar organic solvents. In a preferred embodiment of the present invention, the amine-rich lipid-like compounds are cationic in order to electrostatically enhance formation of nucleic acid containing nanoparticles.

Throughout the application "amine-rich lipid-like compound" is used interchangeable with "lipid-like compound".

Alkyl chains

In the present context, the term "alkyl chain" may be a non-cyclic alkyl of general chemical formula C_nH_{2n+ i} or a cyclic alkyl chain of the general chemical formula C_nH_2n-i. Non-cyclic alkyl chains can be linear or branched and may be substituted with functional groups at one or more sites.

Alkyl epoxides

In the present context, the term "alkyl epoxides" is to be understood as an epoxide having at least one alkyl chain attached to the epoxy ring.

Nucleic acid

In the present context, the term "nucleic acid" is to be understood as the conventional DNA and RNA molecules and variants thereof. The nucleic acids may be any type of single stranded or double stranded DNA or RNA. In addition, the typical DNA or RNA nucleotides may be replaced by nucleotide analogues such as 2'-0-Me-RNA monomers, 2'-0-alkyl-RNA monomers, 2'-amino-DNA monomers, 2'-fluoro-DNA monomers, locked nucleic acid (LNA) monomers, arobino nucleic acid (ANA) monomers, 2'-fluoro-ANA monomers, 1,5- anhydrohexitol nucleic acid (HNA) monomers, peptide nucleic acid (PNA), and morpholinos. Oligonucleotide

In the present context, the term "oligonucleotide" is to be understood as a sequence of DNA or RNA nucleotide residues that form an oligomeric molecule. Oligonucleotides can bind their complementary sequences to form duplexes (double-stranded assemblies) or even assemblies of a higher order.

Oligonucleotides can be on a linear form, but may also exist as circular oligonucleotide molecules, such as single— stranded circular RNAs or single-stranded circular DNAs.

When referring to the length of a sequence, reference may be made to the number of nucleotide units or to the number of bases.

Active component

In the present context, the term "active component" is to be understood as a biologically/pharmaceutically active component. For pharmaceutical formulations active components may be used in combination with one or more excipients or in a mixture of compounds typically present in a drug formulation.

Nanoparticle

In the present context, the term "Nanoparticle" is to be understood as a particle of a few nanometers to a few micrometers. The nanoparticle can have a range of sizes and shapes e.g. capsules or spheres, spherical, elongated, cubic etc.

CHOFREEN

In the present context, the term "CHOFREEN" is to be understood as a nanoparticle according to the invention comprising at least one amine-rich lipid-like compound and at least one amphiphilic polymer and/or poloxamer.

Functionalised nanoparticle

In the present context, the term "Functionalised nanoparticle" is to be understood as a nanoparticle, which has been functionalised by combining the nanoparticle with at least one active ingredient such as nucleic acids.

Amphiphilic polymer

In the present context, the term "Amphiphilic polymer" is to be understood as a polymer, where the polymer can be divided into at least two parts having different properties i.e. having both hydrophilic and lipophilic properties. In one embodiment, the amphiphilic polymer comprises a hydrophilic part in one end of the polymer, while the other part of the amphiphilic polymer is hydrophobic. In a different embodiment, the amphiphilic polymer has three parts being a hydrophilic part at each end of the polymer and being separated by a hydrophobic part. Poloxamer

In the present context, the term "Poloxamers" is to be understood as non-ionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene flanked by two hydrophilic chains of polyoxyethylene. The number of propylene oxides is generally between 2-130 and the number of ethylene oxide is generally between 10-100. The lengths of the two hydrophilic chains are not necessarily the same.

Pluronic

Pluronics is a tradename for a poloxamer. In the present context the term "Pluronic", "Pluronics" and "Pluronic polymers" are to be understood as specific poloxamers sold under this tradename. The first letter in the name defines the physical form of the poloxamer at room temperature i.e. L= liquid, P=paste, F=flake (solid) . The first digit or the first two digits in a three-digit number results when multiplied by 300 in the approximate molecular weight of the hydrophobic part of the poloxamer. The last digit multiplied by 10 gives the percentage of the polyoxyethyle content.

Hybrid nanoparticle

The present invention relates to a novel composition of a nanoparticle. This nanoparticle may be used for the delivery of drugs in particular nucleic acids. The nanoparticle is a hybrid nanoparticle as it comprises both a polymeric part and a lipid-like part. The specific components and the combination between the components is chosen in a manner to obtain a stable nanoparticle even without the stabilisation of the lipid-like part of the nanoparticle by addition of cholesterol. In one embodiment, the nanoparticle does not comprise cholesterol.

The specific combination of the components chosen not only forms a very stable nanoparticle but also results in a highly efficient transfectant, which is capable of transfecting even cells, such as macrophages with high efficiency. Macrophages are known in the art to be difficult to transfect.

The nanoparticle comprises

a) at least one amphiphilic polymer and/or a poloxamer; and

b) at least one amine-rich lipid-like compound as described by formula (I)

where Ri is selected from formula (II)

wherein Xi, X₂ and X3 are independently selected from 0, NH, NR4 or

where R₄ is CI-hCI-hlN RsXRs·) or CH₂-CH(OH)-R3, where R5 and R5- are

independently selected from H or CH2-CH(OH)-R₃;

It could be hypothesized that the use of amphiphilic polymers such as poloxamers results in the stability of the nanoparticle by interacting with the lipid-like compounds and hereby stabilises the lipid-membrane formed by the lipid-like compounds as also illustrated in figure IB.

In one embodiment, the nanoparticle is positively charged due to the amino-rich lipid-like compounds being positively charged .

A further aspect of the invention relates to a lyophilized hybrid nanoparticle, which is a hybrid nanoparticle as described herein, which has been lyophilized. Amine-rich lipid-like compound

The nanoparticle may comprise at least one amine-rich lipid-like compound. Hereby is to be understood that the nanoparticle may comprise one amine-rich lipid-like compound alone, two different amine-rich lipid-like compounds, three different amine-rich lipid-like compounds or so forth.

The amine-rich lipid-like compound may be formed by reacting an amine-rich molecule with an epoxide as described in Love K et al., 2010. Alternatively, the amine-rich molecule may be synthesized by other methods known by persons skilled in the art.

In one embodiment, R2=R2 =R2 "=R2 "=H. In a further embodiment, only R₂, R2·, R2" or R₂ - is H. In a further embodiment, only two of R2, R2', R2" or R₂ - is H. In a further embodiment, only R₂- = R2" = H. In a further embodiment, R2 = R2 " = H. In a further embodiment, R₂··· is H. In a further embodiment, R₂- is H. In a further embodiment, R2- is H. In a further embodiment, R2 is H.

In one embodiment, the nanoparticle comprises a mixture of amine-rich lipid-like compounds where Ri and R3 are the same structure for all of the molecules but where the amine-rich lipid-like compounds differ in their number of R2, R2', R2" and R₂··· being different from H.

In one embodiment, Ri is defined by Xi = NH, m=2, n = 3, p=q=0 as shown in formula Bl.

In a further embodiment, Ri is defined by X_I = NR₄, where R₄=CH₂-CH(OH)-R₃; m=2, n=3, p=q = 0.

In a further embodiment, Ri is defined by X_I=X₂=NH, m = 2, n=4, p= l, o=2, q = 0 as shown in formula B2.

In a further embodiment, Ri is defined by Xi=NH, X₂=NR₄, where R₄=CH₂-CH(OH)-R₃; m=2, n=4, p= l, o=2, q = 0. In a further embodiment, Ri is defined by Xi= NR₄, where R4=CH₂-CH(OH)-R3; X₂=NH, m=2, n=4, p= l, o=2, q = 0.

In a further embodiment, Ri is defined by X_I =X₂= N R4, where R4=CH₂-CH(OH)-R3; m=2, n=4, p= l, o=2, q = 0.

In a further embodiment, Ri is defined by X_I= N R4, R4=CH₂CH₂N(R5)(R5-), R5=R5 =H; m = n = l, p=q = 0 as shown in formula B3.

In a further embodiment, Ri is defined by X_I = N R₄, R4=CH₂CH₂N(R5)(R5-), R₅= CH₂-CH(OH)- R₃, R5 =H; m = n = l, p=q = 0

In a further embodiment, Ri is defined by X_I = N R4, R4=CH₂CH₂N(R5)(R5-), R5=H, R₅-=CH₂- CH(OH)-R₃; m = n = l, p=q = 0

In a further embodiment, Ri is defined by X_I= N R₄, R4=CH₂CH₂N(R5)(R5-), R5=R₅-=CH₂- CH(OH)-R₃; m=n = l, p=q=0 In a further embodiment, RI is defined by X_I=X₂=X₃=NH, m = l, n=o=2, p=q = l as shown in formula B4.

In a further embodiment, RI is defined by X_I=X₂=NH, X₃=NR₄, where CH₂-CH(OH)-R₃; m = l, n=o=2, p=q = l.

In a further embodiment, RI is defined by X_I=X₃=NH, X₂=NR₄, where CH₂-CH(OH)-R₃; m = l, n=o=2, p=q = l. In a further embodiment, R1 is defined by X₂=X₃= NH, X_I= NR₄, where CH₂-CH(OH)-R₃; m = l, n=o=2, p=q = l.

In a further embodiment, R1 is defined by X_I=X₂=X₃=NR₄, where CH₂-CH(OH)-R₃; m = l, n=o=2, p=q = l.

In a further embodiment, R1 is defined by X_I=X₂=NR₄, where CH₂-CH(OH)-R_3, X₃=NH; m = l, n=o=2, p=q = l. In a further embodiment, R1 is defined by Xi= NH, X₂=X₃=NR₄, where CH₂-CH(OH)-R₃; m = l, n=o=2, p=q = l.

In a further embodiment, R1 is defined by X₂=NH, X_I=X₃=NR₄, where CH₂-CH(OH)-R₃; m = l, n=o=2, p=q = l.

In a further embodiment, R1 is defined as X=

, m=n = 2, p=q=0 as shown in

B5.

R₃ is a linear or branched alkyl chain selected from a group having 8-20 carbon atoms, such as 10-18 carbon atoms, like 12-16 carbon atoms. In a preferred embodiment, R₃ is a linear alkyl chain. In a more preferred embodiment, R₃ is a linear alkyl chain having 12 or 14 carbon atoms. In one embodiment, R₃ is C_I2H₂₅. In a further embodiment, R₃ is C_I4H₂₉.

In one embodiment, the nanoparticle comprises a mixture of amino-rich lipid-like molecules being a mixture of molecules having R₃ with different number of carbon atoms. The amino-rich lipid-like compounds may be synthesized by means of a ring-opening reaction between alkyl epoxides and amine-rich molecules. In one embodiment, the ratio between the amine-rich molecules and the alkyl epoxides is from 1 : 1 to 1 :8, preferably from 1 :4 to 1 : 5. The average substitution degree would be expected to be similar to the molar feed ratio in a ring-opening reaction due to the efficiency of the reaction. Hereby is to be understood that if the ratio between the amine-rich molecules and the alkyl epoxides is 1 :4, an average of four alkyl epoxides will substitute an H on a nitrogen on the amine-rich molecules forming the following chains CH₂CH(OH)-R₃ on R₂, R₂-, R₂--, R₂ -, R₄, R₅ and/or R₅-.

It is to be understood that since it is an average substitution degree, some of the molecules will be substituted to another degree than four such as for example two, three, five or six. In addition, different positioned H may be substituted such that in one molecule R₂, R_2', R_{2 "}, and R₂ - may be substituted, while in another molecule it could be R₂, R₂ -, R₅ and R_5".

Similarly if the ratio between the amine-rich molecules and the alkyl epoxides is 1 : 5, an average of five alkyl epoxides will substitute an H on a nitrogen on the amine-rich molecules forming the following chains CH₂CH(OH)-R3 on R₂, R₂-, R₂--, R₂ -, R₄, Rs and/or R₅-. It is to be understood that since it is an average substitution degree, some of the molecules will be substituted to another degree than five such as for example three, four, six or seven. In one embodiment, the amine-rich lipid-like compound is a mixture of molecules having a different molecular structure but the same average substitution degree.

In one embodiment, at least three of R_2, R₂-, R₂ - and R₂ - are CH₂-CH(OH)-R₃, R₃ is C_I2H₂₅, Xi is NH, m is 2, n is 3 and p,q is 0.

In one embodiment, the amino-rich lipid-like compound of the nanoparticle is Al-14, where Al-14 is one of the following compounds or a mixture thereof: Ri having Xi=NH, m=2, n=3, p=q=0 or Ri having X_I = NR₄, where R =CH₂-CH(OH)-R₃; m=2, n=3, p=q=0; and where R₃ is C_I2H₂₅; and where an average of four of the following are substituted with CH₂-CH(OH)-R₃: R₂, R₂-, R₂--, R₂ - or R₄, such as R₂, R₂-, R₂ - and R₂ -.

In one embodiment, Al-14 is essentially RI as shown in formula B1 and R₂, R₂-, R₂ - and R₂ - being CH₂-CH(OH)-C_I2H₂₅ In one embodiment, the amino-rich lipid-like compound of the nanoparticle is A2-14, where A2-14 is one of the following compounds or a mixture thereof: Ri having Xi=NH or NR₄, X₂= NH or NR₄, where R₄=CH₂-CH(OH)-R₃; m=2, n=4, p= l, o=2, q=0; and where R₃ is C_I2H₂₅ and where an average of five of the following are substituted with CH₂-CH(OH)- R₃: R2, 2', R2 ", R2 ", R₄,(on Xi) or R₄ (on X₂), such as R₂, R₂·, R2" and R₂ - and either R₄ (on Xi) and R (on X₂) .

In one embodiment, the amino-rich lipid-like compound of the nanoparticle is A3-14, where A3-14 is one of the following compounds or a mixture thereof: Ri having X_I = NR₄, R₄=CH₂CH₂N(R₅)(R₅-), R_S= H or CH₂-CH(OH)-R_3, R₅ = H or CH₂-CH(OH)-R₃; m = n = l, p=q = 0; and where R₃ is C_I2H₂₅ and where an average of five of the following are substituted with CH₂-CH(OH)-R₃: R₂, R₂-, R₂--, R₂--, R₅ or R₅-. In one embodiment, the amino-rich lipid-like compound of the nanoparticle is A4-14, where A4-14 is one of the following compounds or a mixture thereof: Ri having Xi=NH or NR₄, X₂= NH or NR₄, X₃= NH or NR₄, where R₄=CH₂-CH(OH)-R_3; m = l, n=o=2, p=q = l; and where R₃ is C_I2H₂₅ and where an average of five of the following are substituted with CH₂- CH(OH)-R₃: R₂, R_2', R_2", R₂ , R₄ (on Xi), R₄ (on X₂) and R (on X₃), such as R₂, R₂-, R₂--, R₂ - and R₄ on either Xi, X₂ or X .

In one embodiment, the amino-rich lipid-like compound of the nanoparticle is A5-14, one of the following compounds or a mixture thereof: Ri having X=

m = n = 2, p=q=0 and where an average of four of the following are substituted with CH₂-CH(OH)-R₃ and R₃ being C_I2H₂₅: R₂, R₂-, R₂ - and R₂.

Polymer

The nanoparticle comprises at least one amphiphilic polymer and/or a poloxamer. In one embodiment, the nanoparticle comprises at least two different polymers, such as at least three different polymers, like at least four different polymers. In one embodiment, the different polymers are a mixture of poloxamers.

In one embodiment, the amphiphilic polymer is a copolymer i.e. a polymer having a hydrophilic part and a hydrophobic part such as PLGA-PEG, PLA-PEG, PGA-PEG, PEG-PCL, PEG-PE and PEG-PS

In a further embodiment, the copolymer may be a tri-block polymer such as PEG-PPG-PEG, PPG-PEG-PPG, PLA-PEG-PLA, PEG-PLA-PEG, PGA- PEG- PGA, PEG-PGA-PEG, PLGA-PEG- PLGA, PEG-PLGA-PEG. In a further embodiment, the copolymer is PEG-PPG-PEG or PPG- PEG-PPG. In a still further embodiment, the copolymer is PEG-PPG-PEG. In a still further embodiment, the copolymer is PPG-PEG-PPG. In one embodiment, the nanoparticle comprises at least one poloxamer of formula (III) :

wherein Z1 is an integer selected from 2 to 130, preferably from 10 to 100, Y1 is an integer selected from 15 to 90, preferably from 20 to 80 and Z2 is an integer selected from 2 to 130, preferably from 10 to 100. In one embodiment, the poloxamer is a pluronic polymer known as Pluronics and commercially available. As an example the pluronic polymer may be selected from a group comprising F127, P123, F68, P84, L35, L61, L64, L92, L121, F87, F108, P65, P84, P103, P104 and P105. In a further embodiment, the pluronic polymer is selected from a group comprising F127, P123, F68 and P84.

In a further embodiment, the pluronic polymer may be selected from a group comprising Pluronic^®25R4, Pluronic^®31Rl, Pluronic^®F 108 Cast Solid Surfacta, Pluronic^®F 108 Pastille, Pluronic^®F 127, Pluronic^®F 127 NF Prill Poloxamer 407, Pluronic^®F 38, Pluronic^®F 38 Pastille, Pluronic^®F 68 LF Pastille, Pluronic^®F 68 Pastille, Pluronic^®F 77, Pluronic^®F 87, Pluronic^®F 88, Pluronic^®FT L 61, Pluronic^®L 10, Pluronic^®L 35, Pluronic^®L 62 LF, Pluronic^®L 92, Pluronic^®N 3, Pluronic^®P 103, Pluronic^®P 104, Pluronic^®P 105, Pluronic^®P 123 Surfactant, Pluronic^®P 65 and Pluronic^®P 84 .

F127 is characterised by having Zl = 100, Yl = 65, Z2= 100 and a molecular weight of 12.6 kDa. F127 is a solid at room temperature.

F68 is characterised by having Zl = 76, Yl = 29, Z2=75 and a molecular weight of 8.4 kDa. F68 is a solid at room temperature. P123 is characterised by having Zl = 20, Yl =70, Z2=20 and a molecular weight of 5.8 kDa. P123 is a paste at room temperature.

P84 is characterised by having Zl = 19, Yl =43, Z2= 19 and a molecular weight of 4.2 kDa. P84 is a paste at room temperature. In a further embodiment, said at least one amphiphilic polymer and/or poloxamer has a molecular weight in the range of 1-25 kDa, such as between 2-20 kDa, like 4-15 kDa, such as between 5-13 kDa.

In one embodiment, the nanoparticle comprises at least Al-14 and P123. In a further embodiment, the nanoparticle comprises at least A2-14 and P123. In a further embodiment, the nanoparticle comprises at least A3-14 and P123. In a further embodiment, the nanoparticle comprises at least A4-14 and P123. In a further embodiment, the nanoparticle comprises at least A5-14 and P123.

In one embodiment, the nanoparticle comprises at least Al-14 and F68. In a further embodiment, the nanoparticle comprises at least A2-14 and F68. In a further embodiment, the nanoparticle comprises at least A3-14 and F68. In a further embodiment, the nanoparticle comprises at least A4-14 and F68. In a further embodiment, the nanoparticle comprises at least A5-14 and F68.

In one embodiment, the nanoparticle comprises at least Al-14 and P84. In a further embodiment, the nanoparticle comprises at least A2-14 and P84. In a further embodiment, the nanoparticle comprises at least A3-14 and P84. In a further embodiment, the nanoparticle comprises at least A4-14 and P84. In a further embodiment, the nanoparticle comprises at least A5-14 and P84.

In one embodiment, the nanoparticle comprises at least Al-14 and F127. In a further embodiment, the nanoparticle comprises at least A2-14 and F127. In a further embodiment, the nanoparticle comprises at least A3-14 and F127. In a further embodiment, the nanoparticle comprises at least A4-14 and F127. In a further embodiment, the nanoparticle comprises at least A5-14 and F127.

In one embodiment, the nanoparticle comprises said at least one amphiphilic polymer and/or poloxamer being a poloxamer according to formula III, wherein Z1 is around 100,

Y1 is around 65 and Z2 is around 100, and

said at least one amino-rich lipid-like compound of the nanoparticle being defined by R2, R2-, R₂-- and R₂ - being CH₂-CH(OH)-Ci2H25, Xi is NH, m is 2, n is 3 and p,q is 0.

Active component and functionalised nanoparticle

In another aspect of the invention, the hybrid nanoparticles as described above can be functionalised by adding at least one active component to the hybrid nanoparticles forming a functionalised nanoparticle. The at least one active component is selected from a group consisting of nucleic acids, peptides, proteins, small-molecular drugs, imaging agents such as radioisotopes and dyes; and combinations thereof.

In a further embodiment, the nucleic acids are selected from the group consisting of siRNA, miRNA, antisense RNA, mRNA, gRNA, DNA, oligonucleotides, aptamers, plasmids, and combinations thereof.

In a still further embodiment, said nucleic acids are siRNA, microRNA, anti-miRs, mRNA, IncRNA, CircRNA and combinations thereof.

In one embodiment, said nucleic acids are siRNA. In a further embodiment, the siRNA is IL-lp siRNA, GFP siRNA, TNF-a siRNA, COX-2 siRNA and/or NF-kB siRNA. In a further embodiment, the siRNA is IL-Ib siRNA.

In another embodiment, said nucleic acids are mRNA. In a further embodiment, the mRNA is GFP mRNA, luciferase mRNA and/or erythropoietin (EPO) mRNA

In yet another embodiment, said nucleic acids are Cas9 mRNA and gRNA. The functionalised nanoparticles may either be loaded with both Cas9 mRNA and gRNA on the same nanoparticle or two separate types of functionalised nanoparticles may be created - one functionalised with Cas9 mRNA and the other functionalised with gRNA. If two types of functionalised nanoparticles are created, the cells need to be transfected with both types for the system to work properly. This may be performed either by mixing the two types of functionalised nanoparticles prior to transfection or by making a double transfection.

In another embodiment, the hybrid nanoparticle maybe loaded with Cas9 protein/gRNA complexes. Cas9 protein could be mixed with gRNA to form Cas9/gRNA prior to be loaded to nanoparticles for cell transfection and genome editing.

In a further embodiment, the hybrid nanoparticle may be functionalised with a dye, which may find use as a diagnostic tool for tracking therapeutic responses to medical treatment.

The imaging agent may be selected from the group consisting of iodinated imaging agents such as diatriozates, iothalamates, iopromides, iohexol, ioxaglate and iodixanol, gold- based imaging agents, lanthanide-based imaging agents, and heavy metal-based imaging agents, such as tantalum and bismuth. Other relevant radioisotopes could be medical radioisotopes such as Chromium-51, Iodine- 131, Iridium-192, Molybdenum-99, Phosphorus-32, Samarium-153, Technetium-99m, Yttrium-90; Cyclotron-produced medical radioisotopes such as Copper-64, Gallium-67, Iodine-123, Thallium-201, Carbon-11, Nitrogen-13, Oxygen-15, Fluorine-18; Naturally occurring radioisotopes such as Carbon-14, Chlorine-36, Lead-210, Hydrogen-3 (tritium); Artificially produces radioisotopes such as Americium-241, Cobalt-60, Caesium-137, Gold- 198, Gold-198 Technetium-99m, Iridium-192, Tritiated water, Ytterbium-169, Zinc-65 Manganese-54, Iridium-192 Gold-198 Chromium-51.

The imaging agents may be chosen from imaging agents suitable for use with techniques such as optical imaging, spectroscopy, microscopy, mass spectrometry, photoacoustic imaging, MRI, NIR, SPECT and PET.

In a further embodiment, the functionalised nanoparticles may be modified with a targeting ligand for directing the functionalised nanoparticles to specific tissues. For example, the hydroxyl groups on pluronic polymers may be converted to carboxyl group and covalently conjugated with targeting ligands such as antibodies, peptide, cell-surface specific receptors and aptamers. Modifying the functionalised nanoparticles with a targeting ligand may increase the targeted delivery even with a lower does. Thus, side effects in non-target tissues may be minimized.

The obtained functionalised nanoparticles show a well-dispersed distribution in size both with and without the active component.

Furthermore, the functionalised nanoparticles does not exhibit toxicity towards cells.

A further aspect of the invention relates to a lyophilized functionalised nanoparticle, which is a functional nanoparticle as described herein, which has been lyophilized.

Medical use

In a further aspect of the invention, the functionalised nanoparticle as described above is used as a medicament. The functionalised nanoparticle may be used either directly in medicinal compositions or more preferably as a delivery system for drugs such as particularly nucleic acids like siRNA and mRNA.

More specifically, the functionalised nanoparticle can be used in the prevention or treatment of a disease selected from the following group of diseases: inflammatory diseases and cancer. The inflammatory diseases may be diseases such as arthritis, liver inflammation/ fibrosis, kidney inflammation, ocular inflammation, amyloidosis, acne vulgaris, asthma, autoimmune diseases, autoinflammatory diseases, celiac disease, chronic prostatitis, colitis, diverticulitis, glomerulonephritis, hidradenitis suppurativa, hypersensitivities, inflammatory bowel diseases, interstitial cystitis, Mast Cell Activation Syndrome, mastocytosis, otitis, pelvic inflammatory disease, reperfusion injury, rheumatic fever, rheumatoid arthritis, rhinitis, sarcoidosis, transplant rejection, vasculitis.

In one embodiment, said inflammatory disease is arthritis. In a further embodiment, the disease is osteoarthritis also known as degenerative joint disease. In a further embodiment, the disease is rheumatoid arthritis.

A number of routes can be used to administer the functionalised nanoparticle as the functionalised nanoparticle according to this invention is rather stable. The route of administration of the functionalised nanoparticle is selected from the group consisting of inhalation, oral administration, rectally administration, intravaginal administration, nasal administration, intraocular administration, intradermal administration and injection such as intra-articular, intramuscular, subcutaneous, intraosseous, intraperioneal or intravenous injection. In a further embodiment, the functionalised nanoparticle is administered by topical administration.

The functionalised nanoparticle may be formulated into a composition as known by the skilled person in the art whereby the functionalised nanoparticle becomes suitable for administration according to any of the routes mentioned above.

Process of preparation

In a further aspect of the invention, a process for the preparation of a functionalised nanoparticle is described comprising the steps of

1) mixing :

a) at least one amphiphilic polymer and/or a poloxamer as described above; and

b) at least one amine-rich lipid-like compound as described above;

under conditions forming nanoparticles;

2) adding at least one active component as described above forming functionalised nanoparticles; and

3) purifying said functionalised nanoparticles.

In a preferred embodiment, the nanoparticles are prepared by nanoprecipitation. In one embodiment, the nanoparticles may be formed by mixing a solution comprising the at least one amino-rich lipid-like compound e.g. dissolved in an alcohol such as ethanol with a solution comprising the at least one amphiphilic compound and/or poloxamer e.g. dissolved in a mixture of alcohol/DMSO such as ethanol/DMSO. Other water miscible protic or aprotic organic solvents such as ethyl acetate, acetonitrile, formic acid, isopropanol, acetic acid, acetone or tetrahydrofuran may also be utilized.

The nanoparticles may be obtained from the mixed solution by nanoprecipitation. As an example, this may be achieved by injecting the mixed solution into a hydrophilic liquid such as water under vigorous stirring. Hereby, the nanoparticles are self-assembling.

In one embodiment, the concentration of the at least one amine-rich lipid-like compound is from 50 mg/ml to 150 mg/ml, such as 100 mg/ml. In a further embodiment, the total concentration of the amine-rich lipid-like compound(s) is from 50 mg/ml to 150 mg/ml, such as 100 mg/ml.

In a further embodiment, the concentration of the at least one amphiphilic compound and/or poloxamer is from 50 mg/ml to 150 mg/ml, such as 100 mg/ml. In a further embodiment, the total concentration of the amphiphilic compound(s) and/or poloxamer(s) is from 50 mg/ml to 150 mg/ml, such as 100 mg/ml.

In yet a further embodiment, the amine-rich lipid-like compound and the amphiphilic compound and/or poloxamer is mixed in a weight ratio of from 5 : 1 to 1 : 5, like from 2: 1 to 1 :2, preferably 1 :2.

The hybrid nanoparticles could also be prepared by microfluidics, film-hydration or emulsification-evaporation and spray-drying methods. Microfluidics method is similar to the precipitation method but controlling the mixing ratio and speed to generate homogenous nanoparticles. Film-hydration method could be used to prepare nanoparticles with preferred sized by extrusion through the membrane with specific pore size. Emulsification-evaporation could be used if water-immiscible solvents is used for dissolving polymer and lipids such as dichloromethane. Spray-drying methods could be used for large scale synthesis hybrid nanoparticles in water-free form.

The active component may be added to the nanoparticle in a mixture and incubated for at least for 15 min, such as 30 min, like 45 min, preferably at 37°C for loading of the active component to the nanoparticle. In one embodiment, the active component is siRNA and is added in a weight ratio of from 1 to 10 (amino-rich lipid-like compound/siRNA), preferably at a weight ratio of from 1.5 to 7.5, more preferred at a weight ratio of from 5 to 7.5.

The active component could also be loaded by microfluidics method by controlling the mixing ratio and speed to generate homogenous loading.

Conditions for purifying said functionalised nanoparticles can be set and performed according to the knowledge of the persons skilled in the art. As an example, it could be done by washing the solution with PBS after loading of active components, using a centrifugal device with molecular weight cut off (MWCO) of 10 kDa (Pall Corporation) for buffer exchange. Alternatively, the solution could be dialysed against PBS using commercial dialysis cassettes or tubing.

In contrast to other known preparations of functionalised nanoparticles, the functionalised nanoparticles according to the present invention may be formed in a two-step process. This is particularly advantageous as the nanoparticles can be formed before the actual required use and stored until needed. In contrast to only lipid-based nanoparticles the hybrid nanoparticles according to the invention have been shown to be stable during lyophilisation.

In one embodiment, the hybrid nanoparticles are stored before being mixed with the active component to form the functionalised nanoparticle. In a further embodiment, the hybrid nanoparticles are purified before being stored.

In a further embodiment, the hybrid nanoparticles are lyophilized. In a still further embodiment, the hybrid nanoparticle is stored as a lyophilized hybrid nanoparticle.

In a further embodiment, the functionalised nanoparticles are lyophilized. In a still further embodiment, the functionalised nanoparticle is stored as a lyophilized functionalised nanoparticle.

In one embodiment, the nanoparticles may be bought for direct mixing with the active component.

The functionalised nanoparticles may also be prepared in a one-step process, where the at least one active component is mixed together with the at least one amphiphilic polymer and/or poloxamer and the at least one amine-rich lipid-like compound in one step to directly form the functionalised nanoparticles. In one embodiment, the process further comprises a step 1A between step 1) and step 2), purifying said nanoparticles. The nanoparticles may be purified according to methods known to persons skilled in the art. In one embodiment, the nanoparticles may be purified by dialysing the solution against sterilised PBS or other preferred buffers overnight using commercial dialysis cassettes or tubing. It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.

All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.

The invention will now be described in further details in the following non-limiting examples.

Examples

Statistics

The data in the experiments below is presented as mean ± SD of at least 3 independent culture experiments unless stated otherwise. One-way analysis of variance (ANOVA) among groups is used with post hoc tests Dunnett's test for statistics analysis. Statistical significance was established when p < 0.05.

Example 1: Synthesis of the amino-rich lipid-like compounds of the nanoparticle

The amino-rich lipid-like compounds of the hybrid nanoparticles were synthesized by ring opening reaction between alkyl epoxides and amine-rich molecules. Figure 1A illustrates the preparation of the lipid-like compound Al-14 having an average substitution degree of four, as an example.

The amine-rich molecules and alkyl epoxides were combined into a 2 mL glass vial at predetermined molar ratio as defined in Table 1. The structures A1-A5 are illustrated in Figure 3A.

Figure 3A also illustrates that y= 14 when the alkyl on the epoxide is R= C12H25 (1,2- Epoxytetradecane) Table 1: Lipid-like compounds

Al- 14 would be a mixture with an average substitution degree of four i.e. on average four of the H-atoms of compound A1 in Figure 3A would be substituted by CH₂CH(OH)- C12H25.

A2- 14 would be a mixture with an average substitution degree of five i.e. on average five of the H-atoms of compound A2 in Figure 3A would be substituted by CH₂CH(OH)- C12H25.

A3- 14 would be a mixture with an average substitution degree of five i.e. on average five of the H-atoms of compound A3 in Figure 3A would be substituted by CH₂CH(OH)- C12H25.

A4- 14 would be a mixture with an average substitution degree of five i.e. on average five of the H-atoms of compound A4 in Figure 3A would be substituted by CH₂CH(OH)- C12H25.

A5- 14 would be a mixture with an average substitution degree of four i.e. on average four of the H-atoms of compound A5 in Figure 3A would be substituted by CH₂CH(OH)- C12H25. The mixture was heated to 90°C stirring for 2 days. Hereafter, the product was purified by means of chromatography on a silica gel with CH2CI2 as a developing solvent and a mixture of CH₂Cl2/MeOH/NH₄OH (v/v/v, 75 : 22 : 3) for gradient elution . The obtained product was a transparent pale yellow oil and was kept at -20°C before use. Example 2: Preparation of pluronic/lipid-like compound hybrid nanoparticles (CHOFREEN)

The pluronic/lipid-like compound hybrid nanoparticles were prepared by nanoprecipitation. The lipid-like material as prepared in Example 1 was dissolved in 100% ethanol at a concentration of 100 mg/mL.

The pluronic polymers were dissolved in ethanol/DMSO (v/v, 1 : 1) at a concentration of 100 mg/ml. Following, a lipid-polymer mixture was prepared mixing the pluronic polymers with the lipid-like material (lipid/polymer= l : 2, w/w) . The following pluronic polymers were used in the experiments: F127, P123, F68 and P84. These pluronic polymers are commercially available and the formula is provided in Table 2 below.

Table 2: Pluronic polymers

EO: CH CH O; PO; CH₂CH{CH₃)0

The lipid-polymer mixture was injected quickly into autoclaved MilliQ water with vigorous stirring to assemble hybrid nanoparticles spontaneously. The assembled nanoparticles was purified from the water by a centrifugal device with molecular weight cut off (MWCO) of 10 kDa (Pall Corporation) for buffer exchange. This preparation is also illustrated in Figure IB. The isolated nanoparticles were freeze dried or stored at 4°C for later use or used directly in the following experiments for delivering of RNA.

Table 3 shows an overview of the combinations of nanoparticles used in the following experiments.

Table 3: Overview of nanoparticle combinations

Example 3: Encapsulating siRNA in the nanoparticles

To encapsulate siRNA, 20 mM of siRNA solution was mixed with nanoparticles obtained as described in Example 2. The siRNA solution was mixed with the nanoparticles at various weight ratios and the mixture was incubated at 37 °C for 30 min. The encapsulation efficiency for siRNA/miRNA by the nanoparticles was quantified by using RiboGreen reagent (Invitrogen, Copenhagen) according to manufacturer's instructions siRNA loaded nanoparticle solution was mixed with a diluted RiboGreen solution (1 :200 in TE buffer). The fluorescence emission at 520 nm was measured when excited at 480 nm using a FLUOstar OPTIMA (BMG labtechnologies). Free siRNA was included as a reference.

Figure 2C illustrates the %loading efficacy for weight ratio of lipids/siRNA (i.e. lipid-like compound/siRNA) of 1.5, 3, 5 and 7.5, respectfully. The siRNA encapsulation efficiency was calculated by comparing the fluorescence from nanocomplexes to free siRNA and was around 80% at the weight ratios of 5 and 7.5.

If the siRNA loaded nanoparticle was to be used for in vivo studies, the nanoparticle/siRNA was washed with PBS using a centrifugal device with molecular weight cut off (MWCO) of 10 kDa (Pall Corporation) for buffer exchange.

Example 4: Characterisation of nanoparticles

The size and zeta potential of the nanoparticles with and without siRNA were determined by dynamic light scattering (DLS) at 25 °C using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). In these experiments, GFP siRNA was used, however, the charge of siRNA is independent of the sequence.

Figure 2A shows the hydrodynamic size and distribution of F127/A1-14 nanoparticles with and without siRNA as measured by dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern Instruments, Malvern, U.K.).

The morphology of the nanoparticles with and without siRNA was tested using transmission electron microscope (TEM, Technai G2 Spirit) operated at 120 kV. The nanoparticles were loaded on a cooper grid with carbon film and stained with uranyl formate.

Figure 2B shows the transmission electron microscopy (TEM) images of F127/A1-14 nanoparticles with and without siRNA. The nanoparticle F127/A1-14 without or with siRNA showed a well-dispersed distribution and spherical shape from TEM with diameter of approximately 81 nm and 138 nm, respectively.

As shown in Figure 2A and 2B, the size of F127/A1-14 was 81.5+2.07 nm in diameter with a polydispersity (PDI) of 0.25+0.01, and it was positively charged at approximately +22 mV. After complexation with siRNA, the size of F127/Al-14/siRNA complex increased to 131.2±3.22 nm with PDI of 0.34±0.01.

Example 5: Cell viability studies

The effect of the nanoparticles on the cell viability was tested to further characterise the nanoparticles.

Murine macrophage cell line RAW 264.7 cells (ATCC, Manassas, VA, USA) were maintained in DMEM media supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin at 37 °C in 5% CO2 and 100% humidity.

The cell viability was evaluated by using AlamarBlue assay (Molecular Probes, Life Technologies) according to the manufacturer's protocol and performed as follows: RAW 264.7 were seeded in a 96-well plate (5* 10³ cells/well) and incubated overnight. The medium was replaced with 100 pi fresh medium containing nanoparticle (F127/A1- 14)/siRNA at the final concentration of 50 nM siRNA at different weight ratios of lipid-like compound/siRNA (1.5, 3, 5 and 7.5). The cells were rinsed with PBS after 24 hrs' incubation, and incubated with AlamarBlue reagent (10% in medium) for 2 hrs at 37°C. The fluorescent intensity of the supernatant was measured using a plate reader (FLUOstar OPTIMA, Moritex BioScience) at an excitation wavelength of 540 nm and an emission wavelength of 590 nm.

Figure 2D shows the cell viability of the F127/A1-14 nanoparticle loaded with siRNA in RAW 264.7 macrophages. The viability studies in the RAW 264.7 did not show any obvious toxicity.

Example 6: siRNA delivery in vitro

siRNA sequence:

IL- 1b specific siRNA (siIL-Ib) and scrambled negative control siNC were supplied by Integrated DNA Technologies (Coralville, USA). siRNA against GFP (siGFP) was obtained from Ribotask (Odense, Denmark). The sequences are shown in Table 4.

Table 4: siRNA sequences

Knockdown of GFP in HeLa cells:

Green fluorescent protein stably expressing HeLa (HeLa-GFP) cells were obtained by transfection of HeLa cells with pEGFP-Cl (Clontech Laboratories, USA) and were maintained in RPMI 1640 media supplemented with 10% fetal bovine serum, 1% penicillin- streptomycin and 500 pg/mL geneticin (antibiotic G418, Themo Fisher Scientific).

HeLa-GFP cells were plated on 24-well plates (1 x 10⁵ cells/well) in growth medium before transfection. Nanoparticle/siGFP were added at 25 nM final siRNA concentration. After transfection for 24 hrs, the cells were then detached by trypsin-EDTA solution trypsin-EDTA (0.05% trypsin, 0.02% EDTA, GIBCO), washed with PBS and resuspended in PBS containing 1% BSA. The GFP expression level of each sample was quantified by flow cytometry (Becton Dickenson FACSCalibur). A histogram plot with log green fluorescence intensity (FL-1) on the x-axis and cell number on the y-axis was used to define median fluorescence intensity of the main cell population.

DSPE-PEG/cholesterol/lipid-like materials were prepared similar to the method described in Love et al. 2010, using lipid-like materials as described in Example 1 (100 mg/ml), cholesterol (25 mg/ml) and DSPE-PEG (25 mg/ml) in ethanol. The lipid-like materials, cholesterol and DSPE-PEG were mixed at weight ratio of 4: 1 :2. The ethanol mixture was added to 200 mM sodium acetate buffer (pH 5.5) while stirring to spontaneously form the lipid-based system.

Figure 3B illustrates the silencing efficiency in Hela-GFP cells. Five different F127/lipid hybrid nanoparticles were synthesized and compared to a traditional lipid-based system (formulated with DSPE-PEG/cholesterol/lipid-like materials). The F127/lipid hybrid nanoparticles showed a significantly higher silencing effect as compared to the lipid-based system.

Figure 3C illustrates eight different hybrid nanoparticles formulated as combinations of four different pluronic polymers (F127, P123, F68, P84) and two different lipid-like materials (Al-14, A2-14) compared to a traditional lipid-based system (formulated with DSPE- PEG/choleste ro l/lipid-like materials). The hybrid nanoparticles according to the invention achieved better GFP knockdown in HeLa-GFP cells than the traditional lipid-system (DSPE- PEG/choleste ro l/lipid-like molecules).

Knockdown of IL-1B in macrophages

Murine primary macrophages were isolated from peritoneal cavity. Briefly, 5 ml of ice-cold PBS was injected into the peritoneal cavity of the mice after sacrifice. The peritoneal fluid was collected by a syringe after gently shaking. Then the cells were maintained on ice before cell culture. The cells were cultured in DMEM media supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin at 37°C in 5% CO2 and 100% humidity.

RAW 264.7 cells were cultured as described in Example 5.

The silencing efficiency was evaluated on RAW 264.7 cells and primary macrophages. RAW 264.7 cells (5x l0⁴ cells/well) or primary macrophages (2x l0⁵ cells/well) were seeded in a 24-well plate and incubated overnight. F127/Al-14/siIL-ip or F127/Al-14/siNC were added to the cells at the final concentration of 50 nM and compared to transfection with IL- 1b using the commercialized transfection reagent Mirus TKO (Mirus Bio Corp. Madison, WI). F127/Al-14/siIL-ip or F127/Al-14/siNC were prepared as described in the previous examples. Transfection with the transfection reagent Mirus TKO was performed according to the protocol provided by the manufacturer.

After overnight incubation, the culture media was replaced with 800 mΐ. fresh media and incubated for another 24 hrs. The cells were then activated with fresh media containing 100 ng/ml LPS for 6 hrs.

The IL-Ib expression was measured by RT-qPCR. Total RNA was isolated from cells by TRIzol reagent (Invitrogen) and cDNA was prepared using Revert Aid RT Reverse Transcription Kit (Thermo Scientific) performed by SYBR Green kit (Thermo Scientific) on a LightCycler 480 Real-Time PCR system (Roche) using primers as presented in Table 6.

Figure 4A illustrates the testing of hybrid nanoparticles F127/A1-14 and P123/A1-14 for the knockdown of IL-Ib in RAW 264.7 macrophage cells. Both F127/A1-14 and P123/A1- 14 efficiently delivered siIL-Ib and achieved a strong inhibitory effect (~ 80% knockdown by F127/A1-14) on IL-Ib expression. In comparison, the lipid-system (DSPE- PEG/cholesterol/Al-14) did not show any knockdown effect in RAW 264.7 cells.

Figure 4B illustrates that F127/A1-14 hybrid nanoparticles also showed strong knockdown of IL-Ib in primary peritoneal macrophages. This knockdown was significantly better than what was obtained by the Mirus-TKO transfection reagent. Example 7: mRNA delivery in hADSCs

Primary human adipose-derived stem cells (hADSCs, ATCC, Manassas, VA, USA) were cultured in MEM-alpha medium supplemented with 10% fetal bovine serum, 1% penicillin- streptomycin at 37°C in 5% C0₂ and 100% humidity. hADSCs were seeded in a 24-well plate (4x l0⁴ cells/well) and incubated overnight. F127/Al-14/mGFP hybrid nanoparticles were prepared similarly as siRNA complexation (see Example 3). mGFP was obtained from Tebu-Bio (Le Perray-en-Yvelines, France).

A commercial transfection reagent lipofectamine 2000 (ThemoFisher) and lipid-based system (DSPE-PEG/cholesterol/Al-14) were included for comparison. Transfection with lipofectamine was performed according to the manufacturer's instructions. The lipid-based nanoparticle system was prepared as described in Example 6.

The hybrid nanoparticles were added to the cells at a dose of 0.2 pg/well. After transfection for 24 hrs, the cells were then detached by trypsin-EDTA solution trypsin- EDTA (0.05% trypsin, 0.02% EDTA, GIBCO), washed with PBS and resuspended in PBS containing 1% BSA. The GFP expression level of each sample was quantified by flow cytometry (Becton Dickenson FACSCalibur).

Figure 5 illustrates flow cytometry histogram of the human adipose-derived stem cells and quantification of GFP positive cells. mRNAs are much larger RNA molecules compared to siRNA and thus, more challenging to deliver. These results showed that the hybrid nanoparticles according to the invention was also capable of delivering mRNA as well as siRNA.

The hybrid nanoparticles achieved a significant higher level of transfection (higher than 95%) as compared to ~70% by lipofectamine and ~40% by lipid-based system (DSPE- PEG/cholesterol/Al-14) (figure 5B). In addition, the hybrid nanoparticles showed a more homogeneous gene expression as compared to lipofectamine as revealed by flow cytometry analysis (figure 5A). This could be advantageous in relation to achieve homogeneous cell populations for sensitive assays or therapeutic applications.

Example 8: Cas9/gRNA delivery

Green fluorescent protein stably expressing H1299 (H1299-GFP) cells were maintained in RPMI 1640 media supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin and 500 pg/mL geneticin (antibiotic G418, Themo Fisher Scientific). mCas9 was obtained from Tebu-Bio (Le Perray-en-Yvelines, France) and guide RNA targeting GFP (gGFP) was synthesized by AmpliScribe™ T7-Flash™ Transcription Kit (Epicentre). The DNA template for the synthesis of gGFP was obtained from Integrated DNA Technologies, Inc. having a forward strand and reverse strand as described in Table 5. The two strands were annealed by heating to 90°C for 2 min and slowly cooled down to room temperature. The gRNA was synthesized similar to the method provided in Sun et al. 2015.

Table 5: DNA template for gGFP

H1299-GFP cells were seeded in a 24-well plate (5x l0⁴ cells/well) and incubated overnight. F127/Al-14/mCas9+gGFP was prepared similarly as described in Example 3 with a slight modification. First, mCas9 and gGFP were mixed at weight ratio (5/1, mCas9/gGFP) before complexed with F127/A1-14.

F127/Al-14/mCas9+gGFP was added to the cells at dose of 0.2 pg/well. After transfection for 5 days, the cells were then detached by trypsin-EDTA solution trypsin-EDTA (0.05% trypsin, 0.02% EDTA, GIBCO), washed with PBS and resuspended in PBS containing 1% BSA. The GFP expression level of each sample was quantified by flow cytometry (Becton Dickenson FACSCalibur). A commercial transfection reagent lipofectamine 2000 (ThemoFisher) and lipid-based system (DSPE-PEG) were included for comparison. The lipid-based system (DSPE-PEG) was prepared as described in Example 6.

Figure 6 illustrates the co-delivery of Cas9 mRNA and gRNA for genome editing. Figure 6A shows representative images of GFP stably expressing human non-small cell lung carcinoma H1299 cells (H1299-GFP) transfected with Cas9 mRNA (mCas9) and guide RNA targeting GFP (gGFP) using CHOFREEN (F127/A1-14) or lipofectamine. Figure 6B shows flow cytometry histogram of H1299-GFP cells transfected with indicated formulations for 5 days. Figure 6C shows quantification of genome editing efficiency in H1299-GFP cells from flow cytometry analysis. The CRISPR-Cas9 system is known as a powerful, fast and accurate tool for genome editing. However, the non-viral delivery of CRISPR-Cas9 system is challenging. These results show that the hybrid nanoparticles according to this invention can effectively delivery Cas9 mRNA and gRNA simultaneously. The H1299-GFP cells transfected with the hybrid nanoparticles achieved superior genome editing (higher than 60%) comparing to ~40% by lipofectamine and lower than 5% by the lipids system (DSPE- PEG/cholesterol/Al- 14) .

In addition, transfection with only mCas9 or gGFP did not show obvious GFP knockout, indicating both components were essential to achieve genome editing.

Example 9: Lyophilization experiment

F127/A1-14 (w/w, 2/1) nanoparticles were prepared at final F127 concentration of 8 mg/ml. 300 mΐ. hybrid nanoparticle solutions were snap-frozen using liquid nitrogen and freeze-dried in a lyophilizer for two days. The lyophilized nanoparticles were resuspended in autoclaved water before further analysis.

Figure 7 illustrates the effect of lyophilization on nanoparticles. DLS measurements (performed as described in Example 2) of DSPE-PEG and F127/A1-14 hybrid nanoparticles before and after lyophilisation showed that hybrid nanoparticles (F127/A1-14) survived lyophilization with a slight size increase (Figure 7A), while the lipids-based system (DSPE- PEG/cholesterol/Al-14) was severely destroyed with obvious aggregation after lyophilization (Figure 7B). In addition, Figure 7C shows the GFP silencing efficiency in HeLa-GFP cells by nanoparticles before or after lyophilization. This experiment was carried out similarly to that described in Example 6. The results showed that the hybrid nanoparticles were still capable of transfecting the cells with siRNA in contrast to the DSPE-PEG system. This effect could be due to the protecting effect from the pluronic polymers by it's hypothesized interaction in the lipid-like membrane.

Example 10: Arthritis experiment

CAIA mice induction :

Arthritis was induced through collagen antibody-induced arthritis model (CAIA) with Arthrogen-CIA arthrogenic monoclonal antibody 5-clone cocktail (Chondrex). Six to eight weeks old male DBA/1J mice were injected i.v. with 1.5 mg/mouse of 5-clone antibody cocktail on day 0 and i.p. injected with 50 pg/mouse LPS on day 3. To evaluate the biodistribution of nanoparticles in CAIA mice, the mice were intravenously injected with nanoparticles containing Cy5.5-labeled scrambled siRNA or naked Cy5.5- siRNA at the dose of 0.5 mg/kg or PBS as control at day 7 of induction. The mice were subsequently scanned at 30 min, 2 hrs, 4 hrs and 24 hrs using an IVIS® 200 imaging system (Xenogen, Caliper Life Sciences, Hopkinton, MA, USA) under anaesthesia with 2.5% isoflu rane.

The Cy5.5-labeled scrambled siRNA and the naked Cy5.5-siRNA were labelled according a standard NHS coupling reaction as described in Kim et al., 2008. The nanoparticles were prepared as described in the previous Examples and functionalised as described in Example 3.

CAIA mice treatment:

The mice were induced with arthritis (CAIA) and were intravenously injected with nanoparticle/siIL-Ib or nanoparticle with equivalent dose of negative control siRNA (siNC) (1 mg/kg i.v. by tail vein) on day 2, 4, 5, 7. PBS injection was used as the control.

Clinical score of arthritis:

The arthritis condition was evaluated daily after a scoring system with a theoretical maximum of 24 points on the requirement of the Animal Inspectorate, in which 1 point for each swollen toe, 1 point for tarsal/carpal impact and 1 point for metatarsal/metacarpal impact. The animal were killed no later than reaching the score of 10 or 20 percent weight loss. An average change in ankle thickness (2 hind paws) were determined daily by dial calipers. Mice were weighed every other day and the percentage of weight loss was calculated. On day 9, mice were sacrificed, and their paws, blood and organs were harvested for further analysis.

Micro-CT and histology studies:

The paws were fixed in 10% buffered formalin for 48 hrs and washed with PBS. The bone mineral content (BMC) of the isolated paws (n = 6) were detected by micro-CT (pCT-40, ScancoMedical, Bassersdorf, Switzerland; http://www.microct.com) with high resolution of 15 pm at the voltage of 45 kV with an intensity of 88 pA and exposure time of 800 ms. The knees from each group were also scanned at the same parameters. Three-dimensional reconstruction of the paws were obtained and the clinical score of erosion of the paws were evaluated by a double-blind randomized manner by two people separately. Subsequently, the paws were decalcified in EDTA solution, embedded in paraffin and sectioned at 4 pm. Histologic study including standard H8dº stain and Goldners Trichrome stain are carried out to further validate the inflammation and cartilage integrity following a procedure as described in Sullivan-Brown et al. 2011.

Quantification of IL-Ib by RT-gPCR and ELISA:

Arthritic mice were intravenously injected with nanoparticle/siIL-Ib or nanoparticle with equivalent dose of negative control siRNA (siNC) (1 mg/kg i.v. by tail vein) on day 2, 4, 5, 7. Paws were isolated on day 9 and homogenized and the mRNA expression of IL-Ib was quantified by RT-qPCR. Total RNA was extracted from paws, liver, spleen and kidney with Trizol reagent (Invitrogen), according to the manufacturer's protocol. cDNA was prepared using Revert Aid RT Reverse Transcription Kit (Catalog no. K1691) and used as the template for real time PCR using SYBR® Green kit (Invitrogen) running on a LightCycler® 480 Real-Time PCR System (Roche) using the primers as indicated in Table 6. The quantitative data presented is an average of mRNA expression of target gene relative to GAPDH (n=6).

Table 6: Primers used for qPCR

(F) : forward primer; (R) : reverse primer Paws were homogenized in 1 ml PBS and the lysates were cleared by centrifugation. The expression of IL-Ib was measured by enzyme-linked immunoassay ELISA (R8iD Systems) according to the manufacturer's protocol and as described in Zhou et al. (2014).

Figure 8 shows the knockdown of IL-Ib in collagen antibody-induced arthritic (CAIA) mice. Figure 8A shows the biodistribution of F127/Al-14/siRNA in CAIA mice at 24 hrs post injection. The mice was injected with (a) F127/Al-14/Cy5.5-siRNA, (b) Cy5.5-siRNA or (c) PBS. As is evident from the figure, F127/Al-14/Cy5.5-siRNA showed much stronger accumulation in the paws and arthritic joints as compared to naked Cy5.5-siRNA. Ex vivo imaging of siRNA distribution in major organs including heart, lung, liver, spleen and kidneys (Figure 8B) and arthritic paws (Figure 8C) showed that F127/A1-14 was strongly accumulated in the liver while free Cy5.5-siRNA was only detected in the kidneys. In the paws, much stronger fluorescent signals were demonstrated from F127/A1- 14/Cy5.5-siRNA than from free Cy5.5-siRNA. The expression of IL-Ib was quantified both at the mRNA level by RT-qPCR (Figure 8D) and at the protein level as measured by ELISA (Figure 8E). *p<0.05, ***p<0.001. F127/Al-14/siIL-ip (CHOFREEN/siIL-Ib) treatment achieved significant downregulation of IL-Ib in the paws of CAIA mice both at the mRNA and protein level compared to PBS and CHOFREEN/siNC treated mice.

Figure 9 illustrates the therapeutic effect of F127/Al-14/siIL-^ (siIL-Ib) in CAIA mice. Figure 9A shows that F127/Al-14/siIL-^ treatment efficiently suppressed the ankle swelling of CAIA mice including both hind paws and forepaws compared to mice treatment with PBS and F127/Al-14/siNC on day 9 (left: hind paw, right: forepaw). Clinic scoring also showed significant suppression of arthritis development for the F127/Al-14/siIL-^ treatment as compared to PBS and F127/A1-14 (Figure 9B). The therapeutic effect of F127/Al-14/siIL-^ for arthritis was also indicated by attenuation of bone loss in both paws and the knees from micro-CT images of the paws and knees from the CAIA mice on day 9 (Figure 9C). Arrows indicate bone loss.

H8iE and Trichrome stainings were used to assess the extent of inflammation and cartilage integrity as illustrated in Figure 9D. The influx of inflammatory cells and cartilage damage are indicated by arrows. From the illustrations it is evident that the paw from F127/A1- 14/siIL- 1b treated mice appeared similar to un-induced mice having a smooth edge for bone and cartilage structure and no sign of leukocytes influx. In contrast, the sections from PBS and F127/Al-14/siNC treatments showed a dramatic infiltration of inflammatory cells and destruction of cartilage.

Example 10: Cellular uptake mechanism of hybrid nanoparticles

The purpose of this experiment is to investigate the cellular uptake mechanism behind the hybrid nanoparticles and more specifically the energy dependence of the nanoparticle uptake.

The experiments were performed by seeding the macrophage cell line RAW 264.7 cells at a concentration of 5x l0⁴ cells/well in a 24-well plate and incubating overnight. F127/A1- 14/Cy5-siRNA or DSPE-PEG/cholesterol/Al-14/Cy5-siRNA prepared as previously described was used for transfection of the cells. Cy5-siRNA is to be understood as Cy5 labelled siRNA. The siRNA uptake was measured using flow cytometry analysis. A histogram plot with log Cy5 intensity on the x axis and cell number on the y axis was used to define median fluorescence intensity of the main cell population. Different set-ups of transfections were tested. In a first set-up the macrophages were transfected with F127/Al-14/Cy5-siRNA or DSPE-PEG/cholesterol/Al-14/Cy5-siRNA at a concentration of 50 nM at 37°C for 2 hrs before measurement with flow cytometry analysis. This is indicated with the bar "F127/siRNA" and "DSPE-PEG/siRNA" in Fig. A+B.

In a second set-up, the macrophages were transfected with F127/Al-14/Cy5-siRNA or DSPE-PEG/cholesterol/Al-14/Cy5-siRNA at a concentration of 50 nM at 4°C for 2 hrs before measurement with flow cytometry. This is indicated with the bar "4°C" in Fig. A+B. In a third set-up, the macrophages were pre-incubated with small molecule inhibitor Dynasore (Sigma-Aldrich) at a concentration of 50 mM for 1 hr before being transfected with F127/Al-14/Cy5-siRNA or DSPE-PEG/cholesterol/Al-14/Cy5-siRNA at a concentration of 80 nM at 37°C for 2 hrs before measurement with flow cytometry analysis. This is indicated with the bar "Dynasore" in Fig. A+B.

In a fourth set-up, the macrophages were pre-incubated with Bafilomycin (Sigma-Aldrich) at a concentration of 2 mM before being transfected with F127/Al-14/Cy5-siRNA or DSPE- PEG/cholesterol/Al-14/Cy5-siRNA at a concentration of 50 nM at 37°C for 2 hrs before measurement with flow cytometry analysis. This is indicated with the bar "Bafilomycin" in Fig. A+B.

The experiments also included a negative control, where RAW 264.7 cells were cultured as described above but no hybrid nanoparticle was added during the transfection process. This is indicated with the bar "Untreated" in Fig. A+B.

Fig. 10A shows the cellular uptake of Cy5-siRNA as measured by flow cytometry for the different set-ups using a F127/A1-14 hybrid nanoparticle functionalised with Cy5-siRNA. Fig. 10B shows the cellular uptake of Cy5-siRNA as measured by flow cytometry for the different set-ups using a DSPE-PEG/cholesterol/Al-14 lipid nanoparticle.

Figs 10A-B demonstrate that incubation at 4°C or pre-incubated with Dynasore

significantly blocks the uptake of the lipid-based system (DSPE-PEG/cholesterol/Al-14), however, the uptake of the hybrid nanoparticles (F127/A1-14) is less affected. This indicates that uptake of hybrid nanoparticles is less dependent on energy or Dynamin- dependent pathway compared to the lipid-based system.

Conclusively, it is shown by the results in Figs. 10 that the hybrid system exhibits a different uptake mechanism compared to the lipids system. It may be hypothesised that this could be at least part of the reason as to why the hybrid system is observed, as demonstrated in the previous experiments, to be more effective than other transfection systems. Example 11: Comparison of knockdown efficiency of TNF-a in macrophages

The purpose of this experiment is to demonstrate the ability of the hybrid nanoparticle to be able to knockdown TNFa successfully.

TNF-a specific siRNA (siTNFa) were supplied by Integrated DNA Technologies (IDT, Coralville, USA).. The sequences are shown in Table 7.

Table 7: siRNA sequences

The silencing efficiency was evaluated on RAW 264.7 cells. RAW 264.7 cells (5x l0⁴ cells/well) were seeded in a 24-well plate and incubated overnight. F127/Al-14/siTNFa or DSPE-PEG/cholesterol/Al-14/siTNFa were added to the cells at the final concentration of 12.5 nM, 25 nM or 50 nM and compared to the commercialized transfection reagent Mirus TKO (Mirus Bio Corp. Madison, WI). After overnight incubation, the media was replaced with 800 mI_ fresh media and incubated for another 24 h. The cells were then activated with fresh media containing 100 ng/ml LPS for 6 hrs.

The TNFa expression was measured by RT-qPCR as comparable to the method described in Example 6 using primers as presented in Table 8.

Table 8: Primers used for RT-qPCR

(F) : forward primer; (R) : reverse primer Figure 11 illustrates the testing of hybrid nanoparticle F127/A1-14 and DSPE-

PEG/cholesterol/Al-14 for the knockdown of TNFa in RAW 264.7 macrophage cells.

F127/A1-14 efficiently delivered siTNF-a, which showed concentration-dependent inhibitory effect on TNFa expression as measured by RT-qPCT and achieved a strong inhibitory effect (~ 70% knockdown) on TNF-a expression at 50 nM. In contrast, the lipid-based system (DSPE-PEG/cholesterol/Al-14) did not show significant knockdown effect on RAW 264.7 cells. Figure 11 further illustrates that the knockdown of TNFa was inhibited to a similar or larger degree than the level obtained when using the commercially available transfection system TKO. Thus, the hybrid nanoparticle is knockdown was better than the TKO transfection reagent.

Conclusively, the experiment demonstrates that the hybrid nanoparticle is an efficient transfectant compared to lipid-based systems and commercially available systems.

References

Blanco, E., Shen, H. &. Ferrari, M. Principles of nanoparticle design for overcoming biological barriers to drug delivery. Nat. Biotechnol. 33, 941-951 (2015). Dong et al. Lipopeptide nanoparticles for potent and selective siRNA delivery in rodents and nonhuman primates. PNAS 111, 3955-3960 (2014).

Haussecker, D. Current Issues of RNAi Therapeutics Delivery and Development. J. Control. Release Off. J. Control. Release Soc. 2014, 195, 49-54.

Kim et al. Local and systemic delivery of VEGF siRNA using polyelectrolyte complex micelles for effective treatment of cancer. Journal of Controlled Release

129(2), 107-116 (2008). Love, K.T. et al. Lipid-like materials for low-dose, in vivo gene silencing. PNAS 107, 1864- 69 (2010).

Owens, D. E. & Peppas, N. A. Opsonization, biodistribution, and pharmacokinetics of polymeric nanoparticles. Int. J. Pharm. 307, 93-102 (2006).

Sullivan-Brown et al. Embedding, serial sectioning and staining of zebrafish embryos using JB-4 resin. Nature Protecols 6, 46-55 (2011).

Sun et al. Self-Assembled DNA Nanoclews for the Efficient Delivery of CRISPR-Cas9 for Genome Editing. Angewandte Chemie 54(41), 12029-12033 (2015).

Tenzer, S. et al. Rapid formation of plasma protein corona critically affects nanoparticle pathophysiology. Nat. Nanotechnol. 8, 772-781 (2013). Thomas, M., Lu, J. J., Chen, J. & Klibanov, A. M. Non-viral siRNA delivery to the lung. Adv. Drug Deliv. Rev. 59, 124-133 (2007).

Yin, H. et al. Non-viral vectors for gene-based therapy. Nat. Rev. Genet. 15, 541-555 (2014).

Zhou et al. Peptide-siRNA nanocomplexes targeting NF-kB subunit p65 suppress nascent experimental arthritis. J Clin Invest. 124(10), 4363-4374 (2014)

Sequence listing

SEQ NO. 1 : DsiRNA against IL- Ib (sense) (See Table 4) SEQ NO. 2 : DsiRNA against IL- Ib (antisense) (See Table 4) SEQ NO. 3 : DsiRNA, Negative control (sense) (See Table 4) SEQ NO. 4: DsiRNA, Negative control (antisense) (See Table 4) SEQ NO. 5 : siRNA against GFP (siGFP) (sense) (See Table 4) SEQ NO. 6: siRNA against GFP (siGFP) (antisense) (See Table 4) SEQ NO. 7 : Forward strand for gGFP (See Table 5)

SEQ NO. 8 : Reverse strand for gGFP (See Table 5)

SEQ NO. 9 : Forward primer for qPCR for IL- Ib (See Table 6) SEQ NO. 10 : Reverse primer for qPCR for IL- Ib (See Table 6) SEQ NO. 11 : Forward primer for qPCR for GAPDH (See Table 6) SEQ NO. 12 : Reverse primer for qPCR for GAPDH (See Table 6) SEQ NO. 13 : DsiRNA against TNFa (sense) (See Table 7) SEQ NO. 14: DsiRNA against TNFa (antisense) (See Table 7) SEQ NO. 15 : Forward primer for qPCR for TNFa (See Table 8) SEQ NO. 16: Reverse primer for qPCR for TNFa (See Table 8)

Claims

1. A nanoparticle comprising

a) at least one amphiphilic polymer and/or a poloxamer; and

b) at least one amine-rich lipid-like compound as described by formula (I)

where R2, R2-, I and R₂ are independently selected from H or CH₂-CH(OH)-R3, wherein R₃ is a linear or branched alkyl chain selected from a group having 8-20 carbon atoms; and

where Ri is selected from formula (II)

wherein Xi, X₂ and X3 are independently selected from O, NH, NR4 or

where R₄ is CI-hCI-hlN RsXRs·) or CH₂-CH(OH)-R3, where R5 and R5- are

independently selected from H or CH2-CH(OH)-R₃; and

2. The nanoparticle according to claim 1, wherein a) comprises a poloxamer of the formula (III) :

wherein Z1 is an integer selected from 10- 100, Y1 is an integer selected from 20-80 and Z2 is an integer selected from 10- 100.

3. The nanoparticle according to any of the preceding claims, wherein at least three of R2, R2-, R2 - and R₂ - are CH2-CH(OH)-R₃, R3 is C12H25, Xi is NH, m is 2, n is 3 and p,q is 0.

4. The nanoparticle according to any of the preceding claims, wherein said at least one amphiphilic polymer and/or poloxamer have at molecular weight in the range of 1-25 kDa, such as between 2-20 kDa, like 4- 15 kDa, such as between 5- 13 kDa .

5. The nanoparticle according to any one of the claims 2-4, wherein said at least one amphiphilic polymer and/or poloxamer is a poloxamer according to formula III, wherein Z1 is around 100, Y1 is around 65 and Z2 is around 100.

6. The nanoparticle according to any of the preceding claims wherein said at least one amphiphilic polymer and/or poloxamer is a poloxamer according to formula III, wherein Z1 is around 100, Y1 is around 65 and Z2 is around 100, and

said at least one amino-rich lipid-like compound of the nanoparticle is defined by R2, R2', R2" and R_{2 "} being CH₂-CH(OH)-Ci2H25, Xi is NH, m is 2, n is 3 and p,q is 0.

7. A functionalised nanoparticle comprising a nanoparticle as described in any of the claims 1-6 and at least one active component.

8. The functionalised nanoparticle according to claim 7, wherein said at least one active component is selected from a group consisting of nucleic acids, peptides, proteins, small- molecular drugs, imaging agents such as radioisotopes and dyes; and combinations thereof.

9. The functionalised nanoparticle according to claim 8, wherein said nucleic acids are selected from the group consisting of siRNA, microRNA, antisense RNA, mRNA, IncRNA, CircRNA, gRNA, DNA, oligonucleotides, aptamers, plasmids, and combinations thereof.

10. Process for the preparation of a functionalised nanoparticle comprising the steps

1) mixing : a) at least one amphiphilic polymer and/or a poloxamer as described in any of the claims 1-6; and

b) at least one amine-rich lipid-like compound as described in any of the claims 1-6;

under conditions forming nanoparticles;

2) adding at least one active component as described in any of the claims 7-9 forming functionalised nanoparticles; and

3) purifying said functionalised nanoparticles.

11. The process according to claim 10, further comprising the step of purifying the nanoparticles before step 2).

12. The process according to any of the claims 10-11, wherein the nanoparticles are stored before being functionalised.

13. The process according to claim 12, wherein the nanoparticles and/or functionalised nanoparticles are lyophilized.

14. A functionalised nanoparticle as described in any of the claims 7-9 for use as a medicament.

15. A functionalised nanoparticle according to any of the claims 7-9 for use in the prevention or treatment of a disease selected from the following group of diseases: cancer and inflammatory diseases such as arthritis.