WO2017158093A1

WO2017158093A1 - Nanoparticle compositions comprising plga derivatives and a lipid

Info

Publication number: WO2017158093A1
Application number: PCT/EP2017/056238
Authority: WO
Inventors: Camilla FOGED; Henrik FRANZYK; Xianghui Zeng; Hanne Mørck NIELSEN; Kaushik THANKI
Original assignee: Københavns Universitet
Priority date: 2016-03-17
Filing date: 2017-03-16
Publication date: 2017-09-21

Abstract

The present invention relates to a nanoparticle composition comprising PLGA derivatives and a lipid. The composition is suitable for medical use. Particularly, the present invention relates to a delivery system for delivery of drugs, in particular nucleic acids, with enhanced efficacy and reduced adverse effects, such as reduced toxicity and immunogenicity, on healthy tissue. More precisely, the present invention relates to a delivery system comprising hybrid nanoparticles comprising PLGA and lipidoids for the delivery of siRNA.

Description

NANOPARTICLE COMPOSITIONS COMPRISING PLGA DERIVATIVES AND A LIPID

Technical field of the invention

Background of the invention

The use of nucleic acids as drugs for a number of therapeutic applications has great perspectives due to their unprecedented target specificity. An example is the opportunity for mediating a highly specific cellular gene silencing via the RNA interference (RNAi) machinery by introducing exogenous small interfering RNA (siRNA) molecules into the cytoplasm, thereby in principle enabling the silencing of any gene in the organism. Nucleic acid-based drugs are thus covering a wide clinical area, but they have yet to reach the market. Although nucleic acids, such as siRNA, have the potential to be effective therapeutic drugs, efficient intracellular delivery still remains a major hurdle. Due to their size and charge, nucleic acids cannot pass cellular membranes, and delivery vehicles are thus needed to facilitate their delivery to their intracellular

pharmacological target. A series of nanocarriers have been designed and explored for their potential in efficient delivery of siRNA. Primarily, these nanocarriers may be divided into two major classes i.e. particulate (polymer-based) and vesicular/fluidic (lipid- based). The principle advantages associated with particulate nanocarriers include biocompatibility, capability for sustained drug release, colloidal stability and structural integrity, while advantages associated with vesicular

nanocarriers include better encapsulation and high permeation. In contrast, polymeric nanoparticles usually suffer from poor loading of bioactives, while vesicular nanocarriers often are associated with leakage of bioactives and rapid clearance from the plasma or site of injection. Hence, a novel delivery platform comprising the advantages of both particulate and vesicular properties has been developed, referred to as lipid polymer hybrid nanoparticles (LPNs). Conventionally, a variety of lipids have been used for preparation of LPNs such as fatty acids, tristearin, tripalmitin, lecithin, phosphatidyl choline, 1,2- dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-didodecanoyl-sn- glycero-3-phosphocholine (DLPC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP), to mention a few. Among these, promising results have been obtained with cationic lipids, which may be attributed to the following reasons; (i) higher electrostatic interaction with siRNA mediated by opposite charge, (ii) endosomal escape capabilities, and (iii) self-assembling properties thereby forming uniform layers over polymeric cores. However, the commonly used cationic lipids have only one quaternary ammonium headgroup to interact with the siRNA, and this often leads to problems such as excessive charge, nonspecific protein binding, colloidal instability, drug leakage, and toxicity concerns. Recently, a newer generation of lipid-like materials, referred to as lipidoids, have been discovered as novel gene delivery vectors. Unlike conventional cationic lipids, these lipidoids have a series of secondary and tertiary amines allowing for more efficient interactions with siRNA without remarkably increasing the overall charge of the delivery system.

Interestingly, these lipidoids have been formulated till date as stable nucleic acid lipid particles (SNALPs) or also referred to as lipid nanoparticles comprising lipidoid, cholesterol and polyethylene glycol (PEG)ylated phospholipid, and is actually a vesicular system (Akinc et al., Mol. Ther., 2009). However, to date, these lipidoids have not been combined with polymers in nanoparticles.

Furthermore, it has been noted that the efficacy of these SNALPs is often limited by endocytic recycling. This limitation could be addressed by using improved delivery vehicles. However, for particulate nanocarriers an

intracellular depot formation occurs resulting in enhanced therapeutic effects.

On the other hand, nanoparticles composed of biodegradable polymers, such as the commercially available and FDA-approved poly(lactic-co-glycolic acid) (PLGA), are attractive siRNA carriers because of their low toxicity, good colloidal stability, and the possibility of obtaining a sustained release of their payload. However, the siRNA delivery efficiency of PLGA nanoparticles is generally poor, as compared to that observed with lipid-based carriers. Therefore, incorporation of certain cationic excipients, such as poly(ethyleneimine) or lipids into PLGA nanoparticles is a widely used strategy to improve their transfection capability.

Cationic lipids, e.g. commercially available dioleoyltrimethylammonium propane (DOTAP), have previously been combined successfully with PLGA, formulating siRNA as lipid— polymer hybrid nanoparticles (LPNs) by using various preparation procedures. Among these, LPNs prepared at a DOTAP: PLGA weight ratio of

15 :85 by using a double emulsion solvent evaporation method resulted in nano- sized carriers demonstrating : i) high siRNA loading, ii) sustained release, iii) enhanced transfection efficiency in vitro, and iv) encouraging therapeutic effect in vivo. However, an inherent problem is that DOTAP is not very well tolerated, and dose-limiting adverse effects of the carrier system remains a challenge.

Hence, an improved delivery system for delivery of drugs, in particular nucleic acids, would be advantageous. Especially, a delivery system with enhanced transfection efficiency and reduced adverse effects, such as reduced toxicity and immunogenicity, would be advantageous.

Enhanced efficacy may allow for dose reduction, which eventually will decrease i) undesired side effects, and ii) costs of goods (COGs), which is important for the exploitation of costly biopharmaceuticals.

Summary of the invention

The present invention relates to a new delivery system for the delivery of drugs, in particular nucleic acids, comprising hybrid nanoparticles comprising PLGA and lipids. The choice of lipids was performed with the aim of utilizing an efficient transfection agent so that the dose of the transfection agent could be reduced, resulting in a lower toxicity to healthy tissue. Thus, the invention lies in the fact that it consists of siRNA-loaded lipid/PLGA hybrid nanoparticles that show good transfection efficiency in vitro in cell culture in the presence of serum (most cationic delivery systems lose their transfection efficiency in the presence of serum), while displaying red uced adverse effects to non-target tissue. Thus, an object of the present invention relates to a provision of an improved delivery system that efficiently delivers a drug, with the delivery system having reduced adverse effects to healthy tissue.

In particular, it is an object of the present invention to provide siRNA-loaded lipid polymer hybrid nanoparticles, comprising different types of lipidoids (lipidoid 4, lipidoid 5, lipidoid 6 and combinations thereof) as the lipid(s) and poly(lactic-co- glycolic) acid (PLGA) as the polymer(s), which have enhanced efficacy and reduced toxicity as compared to known delivery systems such as lipoplexes and SNALPs.

Thus, a first aspect of the invention relates to a composition comprising one or more lipids and one or more variants of poly[lactic-co-glycolic acid] (PLGA), wherein said one or more lipids are selected from the group represented by formula (I) :

wherein m and p are integers independently selected from 1-3,

each Ri is represented by formula (II),

each R₂ is independently selected from the group consisting of hydrogen and formulas (II)-(V),

wherein R3 is selected from the group consisting of optionally substituted alkyl chains, fatty acid alkyl derivatives and polyethylene glycols having a chain length of at least 8 carbon atoms, and

I is an integer selected from the group consisting of 1-4,

wherein q is an integer selected from 1-4,

wherein i is an integer selected from 1-17, and

j is an integer selected from 1-4.

One preferred embodiment of the present invention relates to the composition according to said first aspect, wherein the lipid is a compound of formula (VII)

Another preferred embodiment of the present invention relates to the composition according to said first aspect, wherein the composition comprises :

i . the lipid represented by formula (VII),

ii . poly[lactic-co-glycolic acid] (PLGA) having a lactic acid : glycolic acid ratio of 72 : 25, and

iii . siRNA,

wherein the lipid to poly[lactic-co-glycolic acid] (PLGA) weight ratio is 10 : 1 to 10 : 3, and the siRNA : lipid weight ratio is from 1 : 15 to 1 : 30.

A second aspect of the present invention is to provide a method for preparing a composition according to said first aspect, wherein the composition is prod uced by an emulsion-based method, preferably a double emulsion-based method .

Another aspect of the present invention is to provide a composition accord ing to said first aspect obtained by a method according to said second aspect.

Yet another aspect of the present invention relates to a composition accord ing to said first aspect for use as a medicament.

Still another aspect of the present invention relates to a composition accord ing to said first aspect for use in the prevention or treatment of a d isease selected from the group consisting of diseases with an inflammatory component, diseases associated with a condition related to the lungs or the joints, and carcinomas. Brief description of the figures

Figure 1 shows the reaction scheme for synthesis of a lipidoid mixture (Lmix) primarily comprising lipidoid 4 (L₄), lipidoid 5 (Ls) and lipidoid 6 (l_6) .

Figure 2 confirms the presence of lipidoid 4 (U), lipidoid 5 (Ls) and lipidoid 6 (L6) by thin layer chromatography eluted over a TLC Silica gel 60.

Figure 3 shows authentication of individual lipidoid L₄ using nuclear magnetic resonance (NMR) spectroscopy.

Figure 4 shows authentication of individual lipidoid Ls using nuclear magnetic resonance (NMR) spectroscopy. Figure 5 shows authentication of individual lipidoid L6 using nuclear magnetic resonance (NMR) spectroscopy.

Figure 6 shows a HPLC method employing evaporative electron light scattering detector (ELSD) for simultaneous estimation of individual lipidoids.

Figure 7 shows a representative cryo-TEM image of lipidoid 5/PLGA hydrid nanoparticles. Inset image depicts the zoomed in version of one particle revealing the surface coating of lipidoid. Figure 8 shows the efficiency with which siRNA-loaded lipoplexes, of varying lipid constituent, silence EGFP expression. The most efficient lipoplexes contain lipidoid 5 (Ls) .

Figure 9 shows that siRNA-loaded lipid/PLGA hybrid nanoparticles, of varying lipid constituent, silence EGFP expression significantly more efficient than the corresponding lipoplexes of Figure 8. The most efficient lipid/PLGA hybrid nanoparticles are the ones containing lipidoid 5 (Ls) .

Figure 10 shows a direct comparison of the efficiency with which siRNA-loaded and L₄-(top) or Ls (bottom)-containing lipoplexes, lipid/PLGA hybrid nanoparticles and SNALPs silence EGFP expression. The lipid/PLGA hybrid nanoparticles are significantly more efficient than the corresponding lipoplexes and SNALPs.

Figure 11 shows the effect on cell viability of the non-formulated lipidoids against H1299 EGFP cells incubated for 24 h at varying concentrations.

Figure 12 shows the effect on cell viability of the various LPNs against H1299 EGFP cells incubated for 24 h at varying concentrations. Figure 13 shows luciferase splice correction in HeLa cells by delivery of a splice- corrcting antisense oligonucleotide loaded into LPNs (sample 153).

Figure 14 shows TLR4 activation tested by stimulation of a reporter cell line, HEKBlue human TLR4, with (A) bulk lipidoids, (B) lipoplexes, (C) SNALPs and (D) LPNs. Professional antigen presenting cell maturation was tested by stimulating pAPC differentiated with GM-CSF from murine bone marrow (B-C).

Figure 15 shows the effect of Ls-LPNs loaded with therapeutic siRNA directed against tumor necrosis factor (TNF)-a in BALB/c mice with proteoglycan-induced arthritis. (A) shows the experimental design. (B) shows three representative images of arthritic hindlimbs with severity scores of 4 (left), 3 (middle) and 2 (right) on a scale from 0-4, with 0 being normal and 4 signalling severe swelling of whole paw and digits. (C) shows the efficacy of Ls-LPNs versus DOTAP-LPNs. 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 :

Lipid

In the present context, the term "lipid" defines any of a group of hydrophobic organic compounds, including fats, oils, waxes, sterols, triglycerides and other substances of similar properties, such as lipidoids. Lipids are characterized by being insoluble in water but soluble in non-polar organic solvents. In a preferred embodiment of the present invention, lipids are cationic in order to

electrostatically enhance formation of nucleic acid containing nanoparticles. A typical cationic lipid contains a single quaternary ammonium group. Lipids may participate in the formation of vesicles in aqueous solution.

Lipidoid

In the present context, the term "lipidoid" refers to any compound having the characteristics of a lipid. Lipidoids may have a series of secondary and tertiary amines, which increases the net positive charge of the compound.

PLGA

In the present context, the term "poly(lactic-co-glycolic acid) (PLGA)" relates to a co-polymer comprised of cyclic dimers of lactic acid and glycolic acid. Depending on the ratio of lactic acid to glycolic acid used for the polymerization, different variants of PLGA can be obtained . These variants are herein identified in regard to the molar ratio of the monomers used (e.g . PLGA 75 : 25 identifies a copolymer whose composition is 75% lactic acid and 25% glycolic acid). In embodiments of the present invention, variants of PLGA covers the entire spectrum of PLGA 100 :0 to 0 : 100, thus also including PLA and PGA polymers.

Lipid-polymer hybrid nanoparticles (LPNs)

In the present context, the term "lipid-polymer hybrid nanoparticles (LPNs)" relates to nanoparticle structures comprising polymers and lipids. The LPNs may be loaded with nucleic acid material.

Lipoplexes

In the present context, the term "lipoplex" relates to a complex of nucleic acid material with bulk lipid. Stable nucleic acid lipid particles (SNALPs)

In the present context, the term "stable nucleic acid lipid particle (SNALP)" relates to a particle of nano- or micrometer diameter, wherein the nucleic acid material is enclosed in a lipid bilayer comprising a mixture of cationic lipids and PEG- conjugated lipids.

Alkyl chains

In the present context, the term "alkyl chain" may be a non-cyclic alkyl of the general chemical formula C_nH2n+i or a cyclic alkyl chain of the general chemical formula Cnhten-i . Non-cyclic alkyl chains can be linear or branched, and may be substituted with functional groups at one or more sites.

Fatty acid alkyl derivative

In the present context, the term "fatty acid alkyl derivative" refers to the alkyl chain of any fatty acid that may be substituted with a functional group at one or more positions. A fatty acid is a non-aromatic alkyl chain containing a carboxylic acid. The non-aromatic chain may be saturated or unsaturated and can be either linear or branched. Polyethylene glycols

In the present context, the term "polyethylene glycol (PEG)" describes a polyether compound of the general chemical formula H-(0-CH2-CH2)n-OH. PEG is a hydrophilic molecule which is available in molecular weights from 300 g/mol to as much as 10,000,000 g/mol. In drug delivery systems, PEG is typically

incorporated to increase blood circulation times by sterically preventing the in vivo clearance from the bloodstream.

Nucleic acid

In the present context, the term "nucleic acid" refers to the conventional DNA and RNA molecules and variants thereof. In embodiments of the present invention, therapeutic versions of nucleic acids, such as siRNA, miRNA, antisense

oligonucleotides or antisense RNA, may be incorporated in nanoparticle

compositions. In another embodiment of the present invention, the nucleic acids may be any type of single stranded or double stranded DNA or RNA. Furthermore, 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, arabino 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" relates to 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 ingredient

In the present context, the term "active ingredient" relates to the biologically active ingredient, component, or mixture of ingredients typically present in a drug formulation. The phrase active ingredient may be used interchangeably with the similar phrase active pharmaceutical ingredient (API). For pharmaceutical formulations active ingredients may be used in combination with one or more excipients.

Excipient

In the present context, the term "excipient" refers to a diluent, adjuvant, carrier, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in

"Remington's Pharmaceutical Sciences" by E. W. Martin. In the present invention, the nanoparticles as well as single molecular constituents may be regarded as an excipient.

Imaging agent

In the present context, the term "imaging agent" relates to a chemical compound that may be identified using any imaging technique. Examples of imaging agents include, but are not limited to, radioisotopes, variants of metals, magnetic compounds, and fluorescent molecules. Examples of imaging techniques include, but are not limited to, optical imaging, spectroscopy, microscopy, mass

spectrometry, MRI, NIR, SPECT, and PET.

Imaging agents allow visualization of specific organs and tissues, and can therefore be utilized to diagnose and/or monitor disease progression.

Alternatively, imaging agents are used for tracking therapeutic responses to medical treatment.

Antibody

In the present context, the term "antibody" relates to a protein that specifically binds a corresponding antigen. Antibodies may particularly stem from the immune system of e.g. mammals, and may be directed towards antigens related to foreign bodies. An antibody is an intact immunoglobulin having two light and two heavy chains. Thus, a single isolated antibody or fragment may be originating from the non-limiting list of a polyclonal antibody, a monoclonal antibody, a synthetic antibody, a recombinant antibody, a chimeric antibody, a heterochimeric antibody, or a humanized antibody. The term antibody is used both to refer to a well-defined molecular mixture, or a mixture such as a serum product made up of a plurality of different molecular entities.

Nanoparticle

In the present context, the term "nanoparticle" defines an entity of a few nanometers to a few micrometers that can have a range of sizes and shapes, e.g. capsules or spheres, spherical, elongated, cubic etc. A nanoparticle may comprise several constituents, preferably PLGA and a lipidoid and can additionally comprise a drug, such as a nucleic acid.

Double emulsion-based method

In the present context, the term "double emulsion-based method" relates to any method comprising the steps of primary emulsification, re-emulsification and purification to obtain nanoparticles comprising an active ingredient or imaging agent. A primary emulsion is of the form water/oil (W/O), whereas the secondary emulsion can be of either the form W/O/W or the form 0/W/O.

In one embodiment, the primary emulsion (W/O) may be formed by dissolving the active ingredient or imaging agent in the aqueous phase and dispersing it into the polymer lipid solution pre-dissolved in a non-aqueous vehicle such as, but not limited to, dichloromethane, chloroform, acetone, acetonitrile, and ethyl acetate.

The secondary emulsion (W/O/W) may then be formed by addition of excess of aqueous stabilizer solution to the primary emulsion for phase inversion, resulting in the formation of a water/oil/water secondary emulsion. The aqueous stabilizer include, but are not limited to, any routinely employed surfactant, such as polyvinyl alcohol, polyethylene oxides, polypropylene oxides, and polysorbates.

Purification of the double emulsion particles may include evaporation of volatile organic solvent, removal of unentrapped siRNA, and excess stabilizer. This could be achieved using various techniques such as centrifugation i.e. sedimentation of the particles followed by subsequent resuspension, dialysis using appropriate molecular cut-off membrane (e.g. 100 kDa) against aqueous solutions, and filtration using either size exclusion chromatography (e.g. Sephadex^® columns) or cationic filter cartridges (e.g. Mustang Q™, Pall Corporation). Lyoprotectant

In the present context, the term "lyoprotectant" relates to a compound used in lyophilisation {i.e. freeze drying) of a composition in order to protect that composition from taking damage or changing morphology when subjected to lyophilisation. Lyophilisation is performed to increase the shelf life of a

composition. Lyoprotectants may include, but are not limited to, polyhydroxy compounds, such as sugars and polyalcohols. Examples of sugars include, but are not limited to, trehalose, mannitol, dextrose and sucrose.

Lipid-polymer hybrid nanoparticles The present invention relates to a novel delivery system e.g . for the delivery of drugs, in particular nucleic acids, comprising hybrid nanoparticles

comprising PLGA and lipids. The choice of lipids is performed with the aim of utilizing an efficient transfection agent so that the dose of transfection agent can be reduced, resulting in reduced adverse effects, such as toxicity, on healthy tissue.

In particular, it is an object of the present invention to provide siRNA-loaded lipid polymer hybrid nanoparticles using different types of lipidoids (lipidoid 4, lipidoid 5, lipidoid 6 and combination thereof) as lipid and poly(lactic-co-glycolic) acid (PLGA) as polymer, which have enhanced efficacy and reduced toxicity compared to known delivery systems such as lipoplexes and SNALPs.

Thus, a first aspect of the present invention relates to a composition comprising one or more lipids and one or more variants of poly[lactic-co-glycolic acid] (PLGA), wherein said one or more lipids are selected from the group represented by formula (I) :

wherein m and p are integers independently selected from 1-3,

each Ri is represented by formula (II),

wherein R3 is selected from the g roup consisting of optionally substituted alkyl chains, fatty acid alkyl derivatives and polyethylene g lycols having a chain length of at least 8 carbon atoms, and

I is an integer selected from the group consisting of 1-4,

wherein q is an integer selected from 1-4,

wherein i is an integer selected from 1- 17, and

j is an integer selected from 1-4.

Preferably the PLGA variant and the lipid of formula (I) forms nanoparticles, and thus in a preferred embodiment the present composition may be a nanoparticle or a composition comprising a nanoparticle. Lipid variants

The purpose of the lipid constituent of the l ipid-polymer hybrid nanoparticles is to function as an efficient transfection agent. The approach of the present invention revolves around enhancing the packing of active ingredient and thereby facilitate a red uction in the amount of lipid needed . The aim of this approach is to reduce adverse effects, such as toxicity, of the lipid-polymer hybrid nanoparticles as compared to similar technologies, such as lipid systems based on DOTAP, while maintaining or increasing the transfection efficiency of the composition .

The overall strategy to achieve this goal is to utilize a lipid where the driving force for packing of the active ing redient is enhanced . While the main d riving force for packing of the active ing red ient is the presence of charged nitrogen-containing groups, several embodiments of the present invention also include other variations.

Thus, one embod iment of the present invention relates to the composition as described herein, wherein the optional substituent on R3 is an alcohol . The lipids may be specifically designed to be of a more symmetric nature.

Therefore, a preferred embodiment of the present invention relates to the composition as described herein, wherein each R2 is independently selected from the g roup consisting of hydrogen and formula (II) . Other parts of the design of appropriate lipids are focused on the particular structure of formula (II) . Thus, an embodiment of the present invention relates to the composition as described herein, wherein R3 is represented by formula -

(CH₂)nX,

n is an integer selected from the g roup consisting of 8-20, and

X is selected from the g roup consisting of CH3 and CH₂OH .

Another embodiment of the present invention relates to the composition as described herein, wherein I is 1. Yet another embod iment of the present invention relates to the composition as described herein, wherein n is 10.

Still another embodiment of the present invention relates to the composition as described herein, wherein X is Chb.

The lipids of formulas (VI)-(VIII) as described herein belong to the g roup of compounds known as lipidoids. This group of compounds has shown promise in the formulation of drugs and especially negatively charged compounds, d ue to their increased net positive charge.

Thus, an especially preferred embodiment of the present invention relates to the composition as described herein, wherein said one or more lipids are selected from the group consisting compounds of formulas (VI)-(VIII) :

Herein, prod uction, verification and characterization of lipidoid-polymer hybrid nanoparticles as described above is shown in the examples section .

Specifically, it is demonstrated how the typical route of synthesis of lipidoid 98N 12 was shown to represent a mixture of three variants of lipidoids; lipidoid 4 [L₄, formula (VI)], lipidoid 5 [l_₅, formula (VII)] and lipidoid 6 [L₆, formula (VIII)], correspond ing to lipidoids with 4, 5 and 6 chains on the tetraamine. Herein, these three fractions of distinct lipidoids were purified and isolated . Thus, the distinct variants of lipidoids (U, Ls and l_6) were utilized separately as the lipid constituent in the lipid-polymer hybrid nanoparticles of the present invention .

It was demonstrated that all three distinct variants of lipidoids (U, Ls and l_6) can be used in the formulation of LPNs that effectively entrap nucleic acids (example 7) .

A preferred embod iment of the present invention relates to the composition as described herein, wherein the lipid is a compound of formula (VII) :

The lipids of the present invention may be in ionic form depending on the surrounding environment, preferably the lipid is a cationic lipid . For example the tertiary amine may be protonated to form ammonium variants of said lipids, includ ing positively charged ammonium ions, or salts with suitable counter anions.

PLGA variants

In addition to the lipid constituent, the second major constituent of the lipid- polymer hybrid nanoparticles is PLGA. Since PLGA is a co-polymer comprising units of lactic acid and glycolic acid, the properties of PLGA can be tuned by changing the molar ratio of the two units.

Therefore, a preferred embodiment of the present invention relates to the composition as described herein, wherein said one or more variants of poly[lactic- co-glycolic acid ] (PLGA) have a lactic acid : glycolic acid ratio selected from the group consisting of 45 : 55, 50 : 50, 65 : 35, 75 : 25, 85 : 15, 90 : 10 and 99 : 1.

Furthermore, the length of the PLGA co-polymer may be varied to yield different variants of PLGA. Thus, an embodiment of the present invention relates to the composition as described herein, wherein said one or more variants of poly[lactic- co-glycolic acid ] (PLGA) have a molecular weight in the range of 1- 100 kDa, such as 2-50 kDa, or such as 5-20 kDa . It was demonstrated that a wide variability of PLGA types could be used in formulation of LPNs capable of trapping nucleic acids (example 8) . Independently of the above modifications, it is also possible to affect the properties of the PLGA by functionalizing the co-polymer with a chemically functional group. Therefore, an embodiment of the present invention relates to the composition as described herein, wherein said one or more variants of PLGA are end-functionalized with a functional moiety selected from the group consisting of amine, carboxyl and hydroxyl or derivatives thereof.

In the examples section it is shown that the incorporation of PLGA in the composition effectively enhances the silencing capabilities as compared to lipoplexes and SNALPs. Thus, the lipidoid-PLGA hybrid nanoparticles reduced gene expression more than twice as efficiently as the corresponding lipoplexes and SNALPs.

Pharmaceutically active agent

The lipid-polymer hybrid nanoparticles may be formulated without the inclusion of any active ingredients. However, for many applications it will be beneficial also to include one or more active ingredient(s) in the composition. Therefore, a preferred embodiment of the present invention relates to the composition as described herein, further comprising one or more active ingredients.

Preferably the active ingredient is enclosed and/or embedded in the composition inside a nanoparticle formed at least by the PLGA variant and the lipid . Thus, preferably the composition or nanoparticle is a carrier of the active ingredient. Preferably the composition stabilizes the active ingredient in vivo, and allows the active ingredient to reach the relevant site or cells in the body prior to release of the active ingredient from the nanoparticle.

For therapeutic applications, such an active ingredient should have some biological activity that inflict an effect at a target site, such as a site of disease. Thus, an embodiment of the present invention relates to the composition as described herein, wherein said one or more active ingredients are selected from the group consisting of nucleic acids, peptides, proteins, lipids, small-molecule drugs, radioisotopes and combinations thereof, preferably nucleic acids. Nucleic acids are a popular choice of therapeutic molecule because they theoretically hold the potential to target any disease as long as the genetic origin of the disease is known. Therapeutic nucleic acids may excert their function via a number of mechanisms that include, but are not limited to, antisense therapy or induction of RNA interference. Therefore, a preferred embodiment of the present invention relates to the composition as described herein, wherein said nucleic acids are selected from the group consisting of siRNA, miRNA, antisense RNA, DNA, oligonucleotides, aptamers, plasmids, and combinations thereof. An especially preferred embodiment of the present invention relates to the composition as described herein, wherein said nucleic acid is siRNA.

An alternative use of the lipid-polymer hybrid nanoparticles is as carrier for imaging agents that may find use as a diagnostic tool or for tracking therapeutic responses to medical treatment. Imaging agents may be incorporated into the lipid-polymer hybrid nanoparticles in a similar fashion as for the active ingredients. Imaging agents may also be included in the lipid-polymer hybrid nanoparticles in combination with one or more active ingredients. Thus, an embodiment of the present invention relates to the composition as described herein, wherein the composition furthermore comprises an imaging agent.

Another embodiment of the present invention relates to the composition as described herein, wherein said imaging agent is 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.

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

Another possible modification of the lipid-polymer hybrid nanoparticles is to functionalize them with a targeting moiety. Many diseases are primarily localized to specific tissues characterized by specific antigens being expressed in that tissue. Furthermore, in the course of a disease, antigens may be abnormally overexpressed . In either case, antigens may be used to target specific types of diseases and guide therapeutic or diagnostic particles to their intended site of action . Therefore, an embodiment of the present invention relates to the composition as described herein, wherein the composition furthermore comprises an antibody.

Another embodiment of the present invention relates to the composition as described herein, wherein the antibody is coupled to the lipid of formula (I).

Optimization parameters

The chemical and physical properties of the lipid-polymer hybrid nanoparticles may be optimized to suit a specific application . Two important parameters are the lipid to polymer ratio and the nucleic acid to lipid ratio. For the resulting lipid- polymer hybrid nanoparticle, these parameters may influence the efficiency and/or toxicity of the nanoparticles.

The LPNs were shown to effectively entrap nucleic acids at a wide range of lipid to PLGA ratios (example 9, therein measured as the percentage of lipidoid content versus the total solid content) . Specifically, it was shown that low contents of lipidoids was sufficient (e.g. below 20% lipidoid content) to ensure efficient entrapment of nucleic acid content.

The entrapment efficiency (EE) was calculated as a percentage of the ratio of entrapped siRNA to total amount of added siRNA. Thus, EE= [(amount of entrapped siRNA)/(total amount of siRNA added)] x 100.

The practical loading was calculated as the ratio of entrapped siRNA to total weight of nanopraticles. Thus, practical loading = [(amount of entrapped siRNA)/(total weight of nanoparticles)] .

Thus, an embodiment of the present invention relates to the composition as described herein, wherein the lipid to poly[lactic-co-glycolic acid] (PLGA) weight ratio is in the range from 100 : 1 to 1 : 1, such as 20 : 1 to 5 : 2, or such as 10 : 1 to 10 : 3. The effect of different ratios of siRNA to lipidoid was tested experimentally and revealed that a range of of different siRNA to lipidoid ratios yielded high

entrapment efficiencies (example 10).

Another embodiment of the present invention relates to the composition as described herein, wherein the nucleic acid: lipid weight ratio is in the range from 1:2 to 1:100, such as 1:10 to 1:50, preferably 1:15 to 1:30. For some intended use it may be of importance that the nanoparticles is of a certain size, whereas for other applications the morphology may be of importance.

Thus, an embodiment of the present invention relates to the composition as described herein, wherein the one or more lipids, one or more variants of poly[lactic-co-glycolic acid] (PLGA), and optionally one or more pharmaceutically active agents are in the form of nanoparticles.

Another embodiment of the present invention relates to the composition as described herein, wherein the composition is in the form of an aqueous

nanoparticle suspension.

Yet another embodiment of the present invention relates to the composition as described herein, wherein said nanoparticles have an average size of 50-400 nm, such as 100-300 nm, such as 150-275 nm, preferably 175-250 nm.

An especially preferred embodiment of the present invention relates to the composition as described herein, wherein the composition comprises:

i. the lipid represented by formula (VII),

ii. poly[lactic-co-glycolic acid] (PLGA) having a lactic acid:glycolic acid

ratio of 72:25, and

iii. siRNA,

wherein the lipid to poly[lactic-co-glycolic acid] (PLGA) weight ratio is 10:1 to 10:3, and the siRNA: lipid weight ratio is from 1:15 to 1:30. Immunoqenicity (TLR4)

The lipidoids are highly efficient in interacting with anionic siRNA molecules as described herein . Till date, lipidoids have mainly been formulated as long- circulating SNALPs, comprised of cholesterol and PEGylated phospholipids as functional excipients, for intravenous injection . However, the applicability of SNALPs may be limited because of unduly immune activation causing

safety/toxicity hazards for therapeutic applications.

Nucleic acids generally activate the innate immune system via binding to pattern- recognition receptors (PRRs), among them are the Toll-like receptors (TLRs) . Chemical modification of nucleic acids is commonly used to reduce undesired immune activation caused by the active ingred ient. However, excipients used for nucleic acid delivery systems, may also be recognized by the immune system because they adopt a structure similar to components of pathogens and hence stimulate a cascade of deleterious immune effects, which may lead to failure of therapy.

Therefore, the immunogenicity of the lipidoid-modified LPNs was assayed and benchmarked versus the correspond ing bulk lipidoids, lipoplexes and SNALPs (figure 14, example 12) . Overall, the experiments showed that lipidoid-mod ified LPNs do not mediate TLR4 activation, whereas the corresponding bulk lipidoids, lipoplexes and SNALPs do activate TLR4 receptors. This demonstrates the therapeutic applicability of the lipidoid-modified LPNs. The experiments were carried out using directly comparable concentrations of lipidoid-mod ified LPNs, bulk lipidoids, lipoplexes and SNALPs. However, as described herein, the superior loading of LPNs facilitates the use of smaller lipid doses than for other formulations, thereby further reducing potential adverse effects from administration of the LPN composition .

Medical use

The composition of the present invention may be used directly in medicinal application or more preferably as delivery system for drugs and in particular for nucleic acids. Thus, an aspect of the present invention relates to a composition as described herein for use as a med icament. An additional aspect of the present invention relates to a method of delivering a drug and/or active ingredient, such as siRNA, to a target cell and/or tissue by administrering said drug and/or active ingredient incorporated in a composition comprising one or more lipids and one or more variants of poly[lactic-co-glycolic acid] (PLGA), wherein said one or more lipids are selected from the group represented by formula (I) :

wherein m and p are integers independently selected from 1-3,

each Ri is represented by formula (II),

(II) wherein R3 is selected from the group consisting of optionally substituted alkyl chains, fatty acid alkyl derivatives and polyethylene glycols having a chain length of at least 8 carbon atoms, and

I is an integer selected from the group consisting of 1-4,

wherein q is an integer selected from 1-4,

wherein k is an integer selected from 1- 17,

wherein i is an integer selected from 1- 17, and

j is an integer selected from 1-4.

Another aspect of the present invention relates to a composition as described herein for use in the prevention or treatment of a disease selected from the g roup consisting of diseases with an inflammatory component, diseases associated with a condition related to the lungs or the joints, neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and carcinomas.

An embodiment of the present invention relates to the composition as described herein, wherein said disease with an inflammatory component is selected from metabolic syndrome, ulcerative colitis, inflammatory bowel disease, allerg ies, gingivitis, cardio vascular d isease, eye disorders, joint pain, colon cancer, breast cancer and lung cancer.

An embodiment of the present invention relates to the composition as described herein, wherein said disease associated with a condition related to the lungs is selected from the g roup consisting of chronic obstructive pulmonary disease (COPD) and asthma .

Another embodiment of the present invention relates to the composition as described herein, wherein said d isease associated with a cond ition related to the joints is arthritis. The applicability of the lipidoid-LPNs as therapeutic particles for the treatment or amelioration of arthritis was demonstrated by in vivo therapeutic efficacy studies in a murine arthritis model (example 13). The results show that administration of l_5-LPNs loaded with an siRNA directed against tumor necrosis factor (TNF)-a efficiently reduces arthritis symptoms of proteoglycan-induced arthritis in BALB/c mice as compared to administration DOTAP-LPNs (figure 15C).

Yet another embodiment of the present invention relates to the composition as described herein, wherein the route of administration of the composition is selected from the group consisting of inhalation, and injection, such as intraarticular, intramuscular, subcutaneous or intravenous injection.

A further embodiment of the present invention relates to the composition as described herein, wherein the composition is for topical administration.

Another aspect of the invention is the use of the composition as described above as a drug delivery system. Another aspect of the present invention is the composition of the present invention further comprising an active ingredient for use as a medicament, preferably for use in the treatment of an inflammatory condition.

Method of preparation

The lipid-polymer hybrid nanoparticles of the present invention may be prepared by using a double emulsion-based method . This method comprises the steps of primary emulsification, re-emulsification and purification to obtain nanoparticles comprising an active ingredient or imaging agent. Thus, an aspect of the present invention relates to a method for preparing a composition as described herein, wherein the composition is produced by an emulsion-based method, preferably a double emulsion-based method.

Pharmaceutical compositions are typically stored before their intended use and a prerequisite of such compositions are consequently that they can be kept in a form suitable for long-term storage. Furthermore, it is important that the pharmaceutical composition is chemically and physically during storage, such that the efficacy and safety of the composition is maintained upon redispersion and use.

Therefore, an embodiment of the present invention relates to the method as described herein, wherein the composition is lyophilized using a drying protocol in the presence of one or more lyoprotectants, such as polyhydroxy compounds including, but not limited to, sugars (mono-, di-, and polysaccharides), polyalcohols, and their derivatives. Another embodiment of the present invention relates to the method as described herein, wherein the lyoprotectant is selected from the group consisting of trehalose, mannitol, sucrose, and dextrose.

Yet another embodiment of the present invention relates to the method as described herein, wherein the composition is freeze-dried in a trehalose concentration of 2-30%, such as 5-20%.

A final aspect of the present invention relates to a composition as described herein obtained by a method as described herein.

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

Example 1: Synthesis, purification and characterization of lipidoids

N-dodecylprop-2-enamide (Mw 239.40, 3 mmol), synthesized and purified as previously reported (Akinc et al, Nat. Biotechnol., 2008), was mixed with triethylenetetramine (Mw 145.23, 15 mmol) in a thick walled 25 ml glass vial. The vial was then placed in oil bath (90°C), and the mixture was melted. A small magnet bead was added to the vial, which was screw-capped and the mixture was stirred for 6.5 days at 90°C, resulting in the formation of a lipidoid mixture (Lmix) primarily comprising lipidoid 4 (L₄), lipidoid 5 (Ls) and lipidoid 6 (l_6) (Figure 1). The obtained lipidoid mixture was confirmed by thin layer chromatography eluted by using dichloromethane: methanol : ammonia (100 : 10 : 1 volume ratio) as the eluent system over TLC Silica gel 60 plates (Merck Millipore, Merck KGaA,

Darmstadt, Germany) (Figure 2). The U, Ls and l_6 fractions of lipidoids were further purified by using vacuum liquid chromatography on a column packed with Silica gel 60H (Merck Millipore, Merck KGaA, Darmstadt, Germany), and fractions were eluted with different proportions of dichloromethane, methanol and ammonia starting from 350 : 10 : 1 and then increasing polarity. The purity of each fraction was assessed by TLC and pure fractions were concentrated by evaporating the solvents under vacuum. Individual lipidoids were then authenticated using nuclear magnetic resonance (NMR) spectroscopy (Figures 3-5). Further, an HPLC method employing evaporative light scattering detector (ELSD) was developed for simultaneous estimation of the amount, identity and purity of the individual lipidoids (Figure 6) (Riehl et al, Eur. J. Pharm. Biopharm., 2015).

The example demonstrate that the synthesis of lipidoids performed according to Akinc et al. results in a lipidoid mixture (Lmix) containing at least lipidoid 4 (L₄), lipidoid 5 (Ls) and lipidoid 6 (L6) . In the present invention, the presence of each specific lipidoid was verified and each type of lipidoid was purified and quantified independently.

Example 2: Formulation development and optimization of lipidoid PLGA nanoparticles

siRNA-loaded lipid-polymer hybrid nanoparticles were prepared by using a double emulsion solvent evaporation method with slight modification from the procedure reported earlier (Colombo et al., J. Control. Release, 2015). Briefly, the requisite amount of siRNA was dissolved in 125 μΙ of HEPES buffer (pH 7.4) and added to 250 μΙ of dichloromethane containing lipidoid and PLGA (total solid content 60 mg/ml), resulting in the formation of a primary emulsion. The lipidoid content was varied as per experimental design and subsequently adjusted with PLGA to keep the total solid content constant. The formed primary emulsion was probe- sonicated on an ice bath for 90 s at an amplitude of 50 (Misonix, Qsonica LLC, CT, USA), phase-inversed by addition of 1 ml 2 % (w/v) PVA and vortexed vigorously for 1 min resulting in formation of the secondary W/O/W emulsion. The secondary emulsion was again probe-sonicated on an ice bath for 60 s at an amplitude of 50 to reduce the droplet size, and was subsequently transferred to a 25 ml beaker containing a magnet and stirred for 45 min. Additional 5 ml of 2 % w/v PVA solution was added to facilitate the stabilization process. The prepared LPNs were then recovered and subjected to a washing step for removing any unentrapped siRNA and excess PVA. The washing step comprised centrifugation of the nanoparticles at 4°C at varying speeds i.e. 6,000 g for 5 min, 12,000 g for 5 min, 21,000 g for 5 min, 34,000 g for 5 min and 48,000 g for 10 min; a total of 30 min, and resuspension in fresh medium. The prepared formulations were then lyophilized using trehalose (5% w/v) as protectant, and subsequently subjected to physicochemical evaluation of particle size, polydispersity index (PDI), zeta potential, encapsulation efficiency and total siRNA loading. The optimization parameters comprised varying the type of lipidoid (L₄, Ls, Le and Lmix), the lipidoid content (0-20% w/w) and the ratio of siRNA: lipidoid (1 : 10-1 : 50, weight ratio). For comparative purposes, lipoplexes, DOTAP/PLGA nanoparticles and stabilized nucleic acid lipid particles (SNALPs) were also prepared as reported previously (Akinc et al., Nat. Biotechnol., 2008 & Colombo et al., J. Control. Release, 2015).

The mean particle hydrodynamic diameter (Z-average) and polydispersity index (PDI) of the LPNs were determined by using dynamic light scattering employing a Malvern NanoZS (Malvern Instruments, Worcestershire, UK) equipped with a 633 nm laser and 173° detection optics. The zeta potential of the LPNs was measured by using laser doppler micro-electrophoresis and analysed by using the laser interferometric technique called M3-PALS (Phase analysis Light Scattering) equipped by the instrument. The measurements were performed on three independent batches diluted appropriately in water.

The surface morphology of the prepared nanoparticles was examined using cryo- TEM (Tecnai G2 20 TWIN transmission electron microscope, FEI, Hillsboro, OR, USA). A FEI Vitrobot Mark IV, under controlled temperature and humidity conditions within an environmental vitrification system was used to prepare the sample for imaging. Briefly, a small droplet (5 μΙ) was deposited onto a Pelco Lacey carbon-filmed grid and pressed using automation to remove excess fluid vis-a-vis and form a thin film (10-500 nm). The vitrified samples were then transferred in liquid nitrogen to an Oxford CT3500 cryo holder connected to the electron microscope. The sample temperature was maintained below - 180 °C throughout experiment. All observations were made in bright field mode at an acceleration voltage of 120 kV. Digital images were recorded with a Gatan

Imaging Filter 100 CCD camera (Gatan, Pleasanton, CA, USA). Figure 7 shows a representative cryo-TEM image of lipidoid 5/PLGA hydrid nanoparticles. Insert image depicts the magnification of one particle revealing the surface coating of lipidoid.

DLS and cryo-TEM analysis confirmed that LPNs of defined size and morphology were prepared by using the double emulsion-based method of the present invention.

Example 3: Encapsulation efficiency and siRNA loading of lipidoid PLGA nanoparticles

A small aliquot (25 μΙ) of the prepared LPNs was centrifuged (22,000 g) at 4 °C, and then resuspended in 200 μΙ chloroform by resuspension of the pellet by transient vortexing. Additional 475 μΙ of a HD solution, composed of 100 μΜ octyl- β-D glucopyranoside and 1 mg/ml heparin in 10 mM Tris-EDTA buffer, pH 7.5 (TE buffer), was added to the chloroform mixture, which again was vortexed for 1 min. The resulting mixture was rotated end-over-end for 5 min for efficient extraction of siRNA into the aqueous phase. Subsequently, the two phases were separated by centrifugation at 4 °C and 22,000 g for 10 min. The supernatant (aqueous phase) was isolated, suitably diluted with TE buffer (pH 7.4) and incubated at 37°C for removal of any residual organic phase. The siRNA

concentration in the prepared samples was analysed using RiboGreen® RNA reagent as per the manufacturer's instructions employing a fluorescence plate reader (FLUOstar OPTIMA, BMG Labtech, Germany). The excitation and emission wavelengths were set at 485 nm and 520 nm, respectively. Each sample was assayed in triplicate. Example 4: Gene silencing efficiency of lipidoid PLGA nanoparticles

The human non-small lung carcinoma cell line H1299 stably expressing enhanced green fluorescent protein (EGFP, EGFP-H1299) was employed for gene silencing studies. The cells were maintained in RPMI 1640 medium containing 10% fetal bovine serum (FBS, v/v) (Gibco, USA) at 37°C and 5% CO2. Upon confluency, the cells were seeded in 24-well tissue culture plates (Corning, NY, USA) at a density of 1 x 10⁵ cells/well and allowed to adhere overnight. Subsequently, the culture medium was aspirated and mixed with fresh medium comprising varying concentrations of lipoplexes, LPNs and SNALPs. The said formulations were loaded with siRNA directed against EGFP (Colombo et al., J. Control. Release, 2015). The cells were incubated with formulations for 24 h and then washed with phosphate buffer saline (pH 7.4, PBS, Sigma-Aldrich), and reincubated with fresh culture medium for additional 24 h. Post-incubation, cells were washed again with 1 ml PBS and trypsinized by treatment with 300 μΙ trypsin EDTA solution (lOx, Sigma- Aldrich). The gene silencing was measured as a function of residual EGFP within cells upon transfection, measured by flow cytometry using a Gallios flow

cytometer (Beckman Coulter, Brea, CA, USA). Data was analyzed using FlowJo 7.6.5 (Three Star, Ashland, OR, USA). The resulting reduction in EGFP expression caused by lipidoids formulated into lipoplexes and PLGA nanoparticles containing siRNA is displayed in figures 8 and 9, respectively. For both lipoplexes and lipid-PLGA hybrid nanoparticles, the formulations comprising L5 performed best in silencing EGFP expression.

Furthermore, the lipid-PLGA hybrid nanoparticles silence the EGFP expression significantly more efficiently than the corresponding lipoplexes.

Example 5: Comparison of gene silencing efficiency of LPNs, lipoplexes and SNALPs

Lipid-polymer hybrid nanoparticles, lipoplexes and SNALPs comprising either U (figure 10, top) or L5 (figure 10, bottom) and loaded with siRNA for silencing EGFP expression was produced as described in example 2 (Akinc et al., Nat. Biotechnol., 2008, and Colombo et al., J. Control. Release, 2015.) Gene silencing experiments were performed as described in example 4. For both U- and Ls-containing formulations, the lipid-polymer hybrid nanoparticles reduced EGFP expression significantly more efficiently than their lipoplex and SNALP counterparts. Example 6: Effect of lipidoid PLGA nanoparticles on cell viability

For cell viability studies, H1299 mEGFP cells were seeded in 96-well plates at a density of 10,000 cells/well and allowed to adhere overnight. On the next day, formulations (lipidoids and LPNs) in varying concentrations were exposed to cells for 24 h. Post-incubation, the cell culture medium including formulation was aspirated, mixed with freshly prepared 500 g/ml 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide in PBS, 150 μΙ/well (MTT, Sigma-Aldrich) and reincubated for 4 h at 37°C and 5% CO2 for formation of insoluble MTT formazan. Subsequently, the excess MTT solution was carefully removed and 200 μΙ/well dimethyl sulfoxide (DMSO) was added for dissolving the MTT formazan. The cell viability was assessed by measuring the absorbance of dissolved formazan at 550 nm and normalized against absorbance of formazan formed by control cells. Two independent experiments were performed in quadruplicates.

A concentration-dependent effect on cell viability was observed for both the lipidoids (Figure 11) and the LPNs (Figure 12). However, in case of the LPNs, the formulations equivalent to dosing 20 nM siRNA were reasonably safe and more than 70% gene silencing was observed at these concentrations.

Conclusions:

The developed formulation technology offers unique advantages in terms of increased stability and uptake of siRNA across the cellular barriers into cells to reach the target site. Furthermore, efficient gene silencing could be achieved at concentrations, which are anticipated to be safe to healthy tissues, and with better performance as compared to that of classical formulations.

Example 7: The effect of cationic moiety on encapsulation efficiency of siRNA

An array of different types of lipidoids, i.e. L₄, L5 and L6, were synthesized, purified and employed for the preparation of LPNs. The lipidoids did not interfere with the nanoparticle formation process, because only statistically insignificant differences (p-value > 0.05) in the particle size and PDI were noted, as compared to non-modified PLGA nanoparticles (Table 1). Interestingly, a significant increase in the encapsulation efficiency was measured, from 3.1% in case of PLGA NPs and 28.2% for DOTAP/PLGA NPs to >65% for all types of lipidoid-modified LPNs.

Lipid Zeta Entrapment

Particle size

Formulation content PDI potential efficiency

(nm)

(%, w/w)^a (mV) (°/o)

Non-loaded PLGA NPs - 181.7 ± 7.7 0.070 ± 0.041 -24.7 ± 7.8 -

PLGA NPs - 180.2 ± 5.2 0.085 ± 0.019 -33.7 ± 6.8 3.1 ± 0.3

DOTAP/PLGA NPs 10 184.8 ± 10.4 0.100 ± 0.023 20.2 ± 6.43 28.19 ± 2.83

L₄ LPNs 10 202.4 ± 7.1 0.127 ± 0.048 13.2 ± 6.3*** 73.1 ± 4.4***

L₅ LPNs 10 202.6 ± 11.6 0.095 ± 0.058 -11.7 ± 11.4* 74.6 ± 8.1***

L₆ LPNs 10 199.8 ± 7.9 0.123 ± 0.023 -6.5 ± 6.0** 67.7 ± 1.8***

Table 1: Effect of different types of lipid on the physicochemical properties of LPNs. Data represents mean values ± SD (n=3). Statistically significant differences from PLGA NPs are indicated : *p < 0.05, **p < 0.01 and ***p < 0.001. ^a Lipid content relative to the total solid content. The following variables were kept constant: Ratio of siRNA : lipid was 1 : 30 (w/w), PLGA type with lactide to glycolide ratio of 75 : 25, PLGA molecular weight of 20,000 Da.

Conclusion :

The choice of cationic moiety significantly influences the encapsulation of siRNA, with formulations comprising lipidoids yielding significantly higher entrapment efficiency.

Example 8: The effect of different types of PLGA on encapsulation efficiency of siRNA

A number of PLGA polymer types with different properties, i.e., molecular weight, and lactide/glycolide molar ratio, were tested to assess the influence of PLGA type on the siRNA encapsulation efficiency at constant lipidoid content and ratio of lipidoid :siRNA. Significantly higher encapsulation efficiency (74.6%) was measured for LPNs prepared with high molecular weight PLGA (20 kD), as compared to that of LPNs prepared using low molecular weight PLGA (10 kD, 32.5%) at a constant lactide/glycolide molar ratio (75/25) (Table 2). Furthermore, at constant molecular weight (20 kD), a two-fold increase in the encapsulation efficiency was measured for LPNs prepared with PLGA having a glycolide content higher than 25 mol%, as compared to LPNs prepared with PLGA having a glycolide content below 15%.

Zeta Entrapment

PLGA Mw Particle

Formulation PDI potential efficiency

type (kD) size (nm)

(mV) (°/o)

L LPNs 50,50 20 178.4 ± 6.7 0.099 ± 0.003 -6.5 ± 5.3 67.6 ± 9.7

L LPNs 75,25 20 183.0 ± 5.4 0.087 ± 0.021 -6.4 ± 4.5 74.6 ± 8.1

L₅ LPNs 85,15 20 195.4 ± 9.1 0.077 ± 0.026 27.9 ± 10.9** 32.1 ± 1.7***

L₅ LPNs 90,10 5 183.0 ± 6.9 0.083 ± 0.012 31.6 ± 7.8** 41.1 ± 7.1***

L₅ LPNs 75,25 10 170.8 ± 6.0 0.079 ± 0.019 -17.1 ± 7.1 32.5 ± 6.7***

Table 2 : Effect of PLGA type on the physicochemical properties of LPNs. Data represents mean values ± SD (n=3). Statistically significant differences from Ls LPNs (prepared with 75/25 and 20 kD PLGA type) are indicated : *p < 0.05, **p < 0.01 and ***p < 0.001.

The following variables were kept constant: Lipidoid content 10% (w/w) of the total solid content, the ratio of siRNA: lipidoid (w/w) was 1 :30.

Conclusion :

The combination of high molecular weight PLGA (20 kD) with a lactide/glycolide molar ratio of (75/25) yielded the highest entrapment efficiency, although it is noted that also the other formulations of Table 2 resulted in LPNs that may be utilized for nucleic acid delivery.

Example 9: The effect of different ratios of cationic moiety to PLGA on encapsulation efficiency of siRNA

At a constant ratio of siRNA: lipidoid, significantly higher (p-value < 0.001) practical loading of siRNA was achieved, which was proportional to the lipidoid content (Table 3). An approximately five-fold increase in the practical loading was measured when the lipidoid content was increased from 5 to 20% (w/w), although no significant differences in the entrapment efficiency was found .

Practical

Zeta Entrapment

FormuLipid Particle size loading

PDI potential efficiency

lation content³ (nm) (pg siRNA/mg

(mV) (°/o) NPs)

L LPNs 5 189.5 ± 7.3 0.086 ± -20.9 ± 5.0 73.7 ± 10.5 1.2 ± 0.2

0.027

L LPNs 10 202.6 ± 11.6 0.095 ± -11.7 ± 11.4 74.6 ± 8.1 2.5 ± 0.3**

0.058

L₅ LPNs 15 194.1 ± 5.8 0.110 ± 21.7 ± 5.8** 77.3 ± 3.4 3.9 ± 0.2***

0.007

L₅ LPNs 20 192.3 ± 7.2 0.129 ± 20.0 ± 14.4** 77.5 ± 1.5 5.2 ± 0.1***

0.020 Table 3 : Effect of lipidoid content on the physicochemical properties of LPNs. Data represents mean values ± SD (n=3). Statistically significant differences from L5 LPNs (prepared with 5% w/w lipidoid content) are indicated : *p < 0.05, **p < 0.01 and ***p < 0.001. ^a Lipidoid content (% w/w) relative to the total solid content. The following variables were kept constant: Ratio of siRNA: lipid was 1 : 30 (w/w).

Conclusion :

The entrapment efficiency was high for formulations comprising down to as low as a lipidoid content of 5% (w/w). Higher practical loading was achieved with formulations comprising higher contents of lipidoids (up to 20% (w/w)). It is worth noting, that even at a lipidoid content of 20% (w/w), these LPN

formulations comprise less lipid content than typical formulations such as lipoplexes and SNALPs which often comprise approximately 50% lipid content.

Example 10: The effect of different ratios of siRNA to lipidoid on

encapsulation efficiency of siRNA

At a constant lipidoid content of 15% (w/w), significantly higher loading of siRNA was measured upon increasing the siRNA: lipidoid (w/w). The siRNA practical loading was increased from 3.9 Mg/mg nanoparticles at an siRNA: lipidoid weight ratio of 1 : 30, to as high as 10.6 Mg/mg nanoparticles at a ratio of 1 : 10, with significant difference in entrapment efficiency (Table 4). At an siRNA: lipidoid weight ratio of 1 : 30, the PDI increased significantly.

Practical siRNA: Zeta Entrapment

FormuParticle size loading

lipidoid PDI potential efficiency

lation (nm) (pg siRNA/mg

(w/w)^a (mV) (°/o) NPs)

L LPNs 01:30 194.1 ± 5.8 0.110 ± 21.7 ± 5.8 77.3 ± 3.4 3.9 ± 0.2***

0.007

L LPNs 01:20 193.6 ± 9.6 0.102 ± 20.3 ± 7.7 72.5 ± 3.8* 5.4 ± 0.3***

0.007*

L₅ LPNs 01:15 205.4 ± 8.3 0.137 ± 14.8 ± 6.8 82.7 ± 2.7 8.3 ± 0.3

0.017

L₅ LPNs 01:10 276.6 ± 0.465 ± 4.6 ± 10.9 70.9 ± 0.6** 10.6 ± 0.1***

0.010***

Table 4: Effect of siRNA: lipidoid wt. ratio on the physicochemical properties of LPNs. Data represents mean values ± SD (n = 3). Statistically significant differences from Ls LPNs (prepared with siRNA: lipidoid ratio as 1 : 15) are indicated : *p < 0.05, **p < 0.01 and ***p < 0.001. ^a Lipidoid content of 15 % (w/w) relative to the total solid content was used.

Conclusion :

High entrapment efficiencies were obtained at all siRNA: lipidoid weight ratios and it is worth noting that even at low siRNA: lipidoid weight ratio (1 : 30), the lipidoid- modified LPNs is a viable option for delivery of nucleic acids.

Example 11: Luciferase splice correction by delivery of antisense oligonucleotides (ASOs)

Lipidoid-modified LPNs (153) that were loaded with an ASO directed for splice correction of luciferase mRNA exhibited better therapeutic efficacy as compared to other nanoparticulate systems such as chitosan-coated PLGA nanoparticles (CS- PLGA), in this case when tested in a cell-based model of splice correction, i.e. HeLa 705 cells. Zeta potential Entrapment Loading of ASO

Formulation z-Average PDI

(mV) efficiency (%) (pg/mg NPs)

L₅-LPNs (153) 230.7 ± 23.7 0.179 ±

7.3 ± 6.1 105.3 ± 3.9 10.5 ± 0.4 0.024

Conclusion :

The experiment shows that Ls-LPNs can also be used for efficient delivery of single-stranded ASOs.

Example 12: Immunogenicity assessment

Immunogenicity of the lipidoid-modified LPNs was assayed and benchmarked against the corresponding bulk lipidoids, lipoplexes and SNALPs. Lipoplexes, DOTAP-modified PLGA nanoparticles and SNALPs were prepared as reported previously (Akinc et al., Nat. Biotechnol., 2008 & Colombo et al., J. Control.

Release, 2015).

Human TLR reporter cell lines (HEK-Blue hTLR4 reporter cells) were cultured according to the manufacturer ^'s instructions (Invivogen, Toulouse, FR). In brief, cells were seeded in flat 96-well plates and stimulated with different dilutions of fully dispersed lipids (figure 14A), lipoplexes (figure 14B, left), SNALPs (figure 14C, left) and LPNs (figure 14D, left), respectively, for 16 h at 37°C. Absorption was measured using Biorad microplate reader 550 at OD650 nm. Control value (solvent stimulated) was subtracted from sample value. The relative alkaline phosphatase levels were than defined relative to the maximum TLR activation by the corresponding agonist.

Murine bone marrow-derived antigen-presenting cells (BM-APCs) were

differentiated with granulocyte macrophage colony-stimulating factor (GM-CSF) and cultured. BM-APCs were stimulated with PBS (1 : 1000), LPS (10 ng/ml) or with a concentration series of lipoplexes (figure 14B, right), SNALPs (figure 14C, right) or LPNs (figure 14D, right), respectively, for 16 h at 37°C. Maturation was measured by antibody staining of the maturation markers CD40 and CD86, and the samples were analysed using flow cytometry. All lipidoids showed interference with the maximum TLR4 activation by

concentrations of 1 ug/ml of the agonist to the reporter cell line (Figure 14A). To test whether lipolexes have similar immune activation properties as the lipidoids themselves, TLR4 activation and pAPC maturation were assessed. hTLR4 cells were stimulated with a concentration range of lipoplexes, and TLR4 activation was achieved from 0.2 nM siRNA (Figure 14B, left). Moreover, when BM-APCs were stimulated with the lipoplexes, pAPCs maturation was induced, as shown by upregulation of the maturation marker CD40 in a concentration-dependent matter (Figure 14B right). A second delivery system for siRNA is the SNALP containing Ls. To elucidate the immune activation of this delivery system, SNALPs were tested in the TLR4 reporter cell line. Both Ls based SNALPs and Lmix based SNALP showed TLR4 activation from 2.5 nM lipidoid (Figure 14C, left). Furthermore, when looking at pAPC maturation, the lipidoid containing SNALPs did indeed show some upregulation of maturation marker CD86 on the BM-APCs (Figure 15C, right).

In contrast, for LPNs modified with lipidoid L₄, Ls, L6 or DOTAP, no TLR4 activation could be measured for formulations up to siRNA concentrations of 100 nM (Figure 14D, left). This observation was independent of of the lipidoid used for

modification. Furthermore, these LPNs, did not show decrease in viability of BM- APCs (Figure 14D, middle) and also did not show upregulation of maturation markers, indicating that they did not induce pAPC maturation (Figure 14D, right).

Conclusions:

Bulk lipidoids, lipoplexes and SNALPs do activate TLR4, whereas lipidoid-modified LPNs do not mediate TLR4 activation. Thus, as LPNs lack detectable immune activating properties, the LPNs with L₄, Ls or L6 and PLGA appear to be good candidates for therapeutic siRNA delivery.

Example 13: In vivo therapeutic efficacy studies in arthritis model

The in vivo applicability of the lipidoid-LPNs was tested in an murine arthritis model. Compositions of Ls-LPNs and DOTAP-LPNs loaded with siRNA direct against TNF-alpha were assayed versus dexamethasone as a control. Dexamethasone is a type of corticosteroid medication. It is used in the treatment of many conditions, including rheumatic problems, such as rheumatoid arthritis. Briefly, a murine model was established by proteoglycan-induced arthritis in BALB/c mice. Each mouse received an intra-articular injection (10 μΙ) of

proteoglycan (PG)/dimethyl dioctadecyl ammonium bromide (DDA) in one ankle of hind limb on days 0 and 21 (figure 15A). The mice were subsequently treated with formulations of Ls-LPNs, DOTAP-LPNs or dexamethasone on days 25-27 and the severity of arthritis of the paws was scored of the following scale:

0 = normal,

1 = one digit inflamed (red and thickened)

2 = two digits inflamed (red and thickened) and moderate swelling of foot

3 = redness and increased swelling of foot

4 = severe swelling of whole paw and digits

Figure 15B shows representative images of arthritic hindlimbs with severity scores of 4 (left), 3 (middle) and 2 (right).

Conclusion :

The results are displayed in Figure 15C and demonstrate that Ls-LPNs loaded with siRNA directed against TNF-alpha efficiently reduce the arthritis symptoms of the mice compared to DOTAP-LPNs {i.e. the arthritis score is reduced).

References

• Akinc et al., Development of lipidoid-siRNA formulations for systemic

delivery to the liver, Mol. Ther. (2009), 872-879

• Akinc et al., A combinatorial library of lipid-like materials for delivery of RNAi therapeutics, Nat. Biotechnol. (2008), 561-569

• Riehl et al ., Investigation of the stabilizer elimination during the washing step of charged PLGA microparticles utilizing a novel HPLC-UV-ELSD method, Eur.

J. Pharm. Biopharm. (2015), 468-472

• Colombo et al., Mechanistic profiling of the siRNA delivery dynamics of lipid- polymer hybrid nanoparticles, J. Control. Release (2015), 22-31 The work leading to this invention has received support from the Innovative Medicines Initiative Joint Undertaking under grant agreement no. 115363, resources of which are composed of financial contribution from the European Union's Seventh Framework Programme (FP7/2007-2013) and EFPIA companies' in kind contribution.

Claims

1. A composition comprising one or more lipids and one or more variants of poly[lactic-co-glycolic acid ] (PLGA), wherein said one or more lipids are selected from the group represented by formula (I) :

wherein m and p are integers independently selected from 1-3,

each Ri is represented by formula (II),

wherein R3 is selected from the group consisting of optionally substituted alkyl chains, fatty acid alkyl derivatives and polyethylene g lycols having a chain length of at least 8 carbon atoms, and

I is an integer selected from the g roup consisting of 1-4,

wherein q is an integer selected from 1-4,

wherein k is an integer selected from 1-17,

wherein i is an integer selected from 1-17, and

j is an integer selected from 1-4.

2. The composition according to claim 1, wherein the optional substituent on R3 is an alcohol.

3. The composition according to any one of the preceding claims, wherein each R2 is independently selected from the group consisting of hydrogen and formula (II).

4. The composition according to any one of the preceding claims, wherein R3 is represented by formula -(CH2)_nX,

n is an integer selected from the group consisting of 8-20, and

X is selected from the group consisting of CH3 and CH2OH .

5. The composition according to claim 4, wherein I is 1.

6. The composition according to any one of claims 4-5, wherein n is 10.

7. The composition according to any one of claims 4-6, wherein X is CH3.

8. The composition according to claim 1, wherein said one or more lipids are selected from the group consisting compounds of formulas (VI)-(VIII) :

9. The composition according to claim 1, wherein the lipid is a compound of formula (VII) :

10. The composition according to any one of the preced ing claims, wherein said one or more variants of poly[lactic-co-glycolic acid] (PLGA) have a lactic acid : g lycolic acid ratio selected from the group consisting of 45 : 55, 50 : 50, 65 : 35, 75 : 25, 85 : 15, 90 : 10 and 99 : 1.

11. The composition according to any one of the preced ing claims, wherein said one or more variants of poly[lactic-co-glycolic acid] (PLGA) have a molecular weight in the range of 1- 100 kDa, such as 2-50 kDa, or such as 5-20 kDa .

12. The composition according to any one of the preced ing claims, wherein said one or more variants of PLGA are end-functionalized with a functional moiety selected from the group consisting of amine, carboxyl and hydroxyl or derivates thereof.

13. The composition according to any one of the preceding claims, further comprising one or more active ing red ients.

14. The composition according to claim 13, wherein said one or more active ingredients are selected from the g roup consisting of nucleic acids, peptides, proteins, lipids, small-molecule drugs, radioisotopes and combinations thereof, preferably nucleic acids.

15. The composition according to claim 14, wherein said nucleic acids are selected from the group consisting of siRNA, miRNA, antisense RNA, DNA, oligonucleotides, aptamers, plasmids, and combinations thereof.

16. The composition according to any one of claims 14-15, wherein said nucleic acid is siRNA.

17. The composition according to any one of the preceding claims, wherein the composition furthermore comprises an imaging agent.

18. The composition according to claim 17, wherein said imaging agent is 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.

19. The composition according to any one of the preceding claims, wherein the composition furthermore comprises an antibody.

20. The composition according to claim 19, wherein the antibody is coupled to the lipid of formula (I).

21. The composition according to any one of the preceding claims, wherein the lipid to poly[lactic-co-glycolic acid] (PLGA) weight ratio is in the range from 100 : 1 to 1 : 1, such as 20 : 1 to 5 : 2, or such as 10 : 1 to 10 : 3.

22. The composition according to any one of claims 14-21, wherein the nucleic acid : lipid weight ratio is in the range from 1 : 2 to 1 : 100, such as 1 : 10 to 1 : 50, preferably 1 : 15 to 1 : 30.

23. The composition according to any one of the preceding claims, wherein the one or more lipids, one or more variants of poly[lactic-co-glycolic acid] (PLGA), and optionally one or more pharmaceutically active agents are in the form of nanoparticles.

24. The composition according to any one of the preceding claims, wherein the composition is in the form of an aqueous nanoparticle suspension.

25. The composition according to any one of claims 23-24, wherein said nanoparticles have an average size of 50-400 nm, such as 100-300 nm, such as 150-275 nm, preferably 175-250 nm.

5 26. The composition according to any one of the preceding claims, wherein the composition comprises:

i. the lipid represented by formula (VII),

ii. poly[lactic-co-glycolic acid] (PLGA) having a lactic acid :glycolic acid ratio of 72 : 25, and

10 iii. siRNA,

wherein the lipid to poly[lactic-co-glycolic acid] (PLGA) weight ratio is 10 : 1 to 10 : 3, and the siRNA: lipid weight ratio is from 1 : 15 to 1 :30.

27. A composition according to any one of the preceding claims for use as a 15 medicament.

28. A composition according to any one of the preceding claims for use in the prevention or treatment of a disease selected from the group consisting of diseases with an inflammatory component, diseases associated with a condition

20 related to the lungs or the joints, neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and carcinomas.

29. The composition according to claim 28, wherein said disease associated with a condition related to the lungs is selected from the group consisting of chronic

25 obstructive pulmonary disease (COPD) and asthma.

30. The composition according to claim 28, wherein said disease associated with a condition related to the joints is arthritis.

30 31. The composition according to any one of claims 27-30, wherein the route of administration of the composition is selected from the group consisting of inhalation, and injection, such as intra-articular, intramuscular, subcutaneous or intravenous injection.

32. A method for preparing a composition according to any one of the preceding claims 1-26, wherein the composition is produced by an emulsion-based method, preferably a double emulsion-based method.

5 33. The method according to claim 32, wherein the composition is lyophilized using a drying protocol in the presence of one or more lyoprotectants, such as polyhydroxy compounds including, but not limited to, sugars (mono-, di-, and polysaccharides), polyalcohols, and their derivatives.

10 34. The method according to claim 33, wherein the lyoprotectant is selected from the group consisting of trehalose, mannitol, sucrose, and dextrose.

35. The method according to any one of claims 32-34, wherein the composition is freeze-dried in a trehalose concentration of 2-30%, such as 5-20%.

15

36. A composition according to any one of claims 1-26 obtained by a method according to any of claims 32-35.