WO2014057432A2 - Multicomponent lipid nanoparticles and processes for the preparation thereof - Google Patents

Multicomponent lipid nanoparticles and processes for the preparation thereof Download PDF

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WO2014057432A2
WO2014057432A2 PCT/IB2013/059230 IB2013059230W WO2014057432A2 WO 2014057432 A2 WO2014057432 A2 WO 2014057432A2 IB 2013059230 W IB2013059230 W IB 2013059230W WO 2014057432 A2 WO2014057432 A2 WO 2014057432A2
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dope
lipid
choi
peg
dotap
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WO2014057432A3 (en
WO2014057432A9 (en
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Giulio CARACCIOLO
Daniela POZZI
Simona MARCHINI
Augusto Amici
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Universita' Degli Studi Di Roma "La Sapienza"
Universita' Degli Studi Di Camerino
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle

Definitions

  • the present invention relates to the field of gene therapy, and in particular relates to novel multicomponent lipid nanoparticles for the non-viral transport of nucleic acids, and to processes for the preparation thereof and to the use thereof in gene therapy and anti-tumoral therapy.
  • Gene therapy consists of the transfer of genetic material within a cell for the purpose of altering the phenotype thereof temporarily or permanently.
  • This therapy offers new treatment possibilities for numerous pathologies, both hereditary and acquired, which conventional clinical procedures are unable to treat effectively. It is therefore considered to be the therapy of the future, and in recent years has been the subject of intense study and development for potential use on a large scale in a clinical environment ("Clinical development of liposome-based drugs: formulation, characterization, and therapeutic efficacy". Chang, H.; Yeh, M. Int. J. Nanomed. 2012, 7, 49-60).
  • the first step lies in identifying the single gene or the various genes responsible for the genetic disorder.
  • Cancerogenesis is the transformation by means of which somatic cells become tumoral. This may occur due to environmental factors, physical factors, chemical factors or biological factors (what are known as carcinogenic or oncogenic agents).
  • the objective of anti-tumoral therapy is the selective toxicity against the tumoral cells, saving the healthy cells of the host.
  • Anti- tumoral drugs can be divided into three categories: hormones and hormonal antagonists, immunostimulants, and cytotoxic drugs.
  • gene therapy and of anti-tumoral therapy does not depend exclusively on the use of gene material (gene therapy) or on the most suitable active ingredient (anti-tumoral therapy), but also on the ability to reach the specific site of action with an appropriate concentration and for a period of time that is suitable for establishing the therapeutic effect.
  • a key component of gene therapy and of anti-tumoral therapy is the transport of the therapeutic molecule (DNA, active ingredient) within the target cell by means of vectors which protect it against degradation and ensure the transcription thereof.
  • the vectors used are presently separated into viral and non-viral vectors. Viruses have an optimal tendency to infect cells and to insert their own DNA there, both by integrating it and in the form of an episome.
  • viral vectors present some disadvantages which limit the use thereof, such as activation of the immune system, toxicity, and, in the case of gene therapy, the reduced size of the transportable genetic material (normally ⁇ 40,000 base pairs). This last characteristic is very limiting considering that many important human genes have a length greater than 40,000 base pairs, considering also the regulatory sequences and the non-coding introns.
  • cationic liposomes and lipid bilayer vesicles formed by cationic lipids and neutral lipids constitute an attractive opportunity for molecular engineering since they are formed as out-and-out auto-assembling biomaterials on a micrometric/nanometric scale (Elsabahy et al.
  • cationic liposome-DNA complexes constitute the most widespread lipid-based materials for gene therapy.
  • Lipoplexes are prepared by mixing a solution of binary liposomes (formed of a cationic lipid and a neutral lipid) with a solution of nucleic acids at the desired concentrations. The electrostatic interaction between cationic liposomes and nucleic acids of negative charge determines the formation of the lipoplexes.
  • Lipoplexes are compact nanoparticles with an inner multi-lamellar structure ("onion-like"), the repetitive unit of which is formed by a lipid bilayer and an aqueous monolayer occupied by the nucleic acids. Lipoplexes have some considerable advantages compared to viral vectors, such as ease of preparation on a large scale, low toxicity, and virtually no limit of the size of the transportable DNA. This last aspect is significant in the hypothesis of transfer within the nucleus of the inner chromosomal cell (the average size of human chromosomes is between 50 and 280 million base pairs).
  • Multicomponent lipoplexes in which the lipid bilayer is formed by a mixture of cationic lipids and neutral phospholipids are mentioned among the most effective lipoplexes as yet reported in the literature (Caracciolo et al., Biochim Biophys Acta, 1768, 2007, 2280).
  • the main limitation of lipoplexes lies in the low efficiency of transfer of the genetic material (transfection efficiency, TE).
  • This limitation also concerns the aforementioned multicomponent lipoplexes which, although they have a level of efficiency that is, on average, greater than that of binary liposomes, cannot compete in terms of efficiency with the more efficient commercial reagents (Lipofectamines).
  • the primary approach to increase the transfection efficiency of lipoplexes is the synthesis of new amphipathic molecules or the use of adjuvant non-cationic lipid species.
  • lipid nanoparticles have emerged as a potential alternative to lipoplexes, since they have chemical and physical properties (dimensions, surface charge, ability to condense genetic material, etc.) that can be controlled more easily.
  • Such particles are formed of a DNA nucleus (or short-interfering RNA, siRNA) condensed by means of divalent cations (Ca2+, Mg2+, Mn2+; Fe2+), polycations (spermine, spermidine), cationic amino acids (arginine, lysine, histidine) and cationic proteins (protamine, histones) coated by a lipid shell formed by one or more lipid bilayers.
  • lipid nanoparticles are substantially different from lipoplexes.
  • the nucleic acids are pre- condensed by means of cationic condensing agents.
  • the interaction between nucleic acids and condensing agents determines the formation of negatively charged spherical globules (the charge transported from the nucleic acids is in excess compared to that of the condensing agent) on which a number of cationic lipid bilayers (typically 1 -3) are adsorbed by electrostatic interaction.
  • the lipoplex is a compact multi-lamellar structure formed of lipid bilayers alternated with aqueous layers occupied by the nucleic acids. The phenomenon of formation is shown in figure 6, taken from (Caracciolo et al., J. Med. Chem, 54, 201 1 , 4160).
  • the electrostatic interaction between cationic liposomes and nucleic acids determines the formation of multilamellar lipoplexes (to the right, typically 20-30 layers). If, vice versa, the nucleic acids are precondensed with a cationic condensing agent (such as the protamine in the diagram of figure 6), the interaction determines the formation of lipid nanoparticles formed by an aqueous core formed by the nucleic acid/condensing agent complex on which a number of lipid layers (typically 1 -3) are adsorbed.
  • nucleic acids such as plasmid DNA in the diagram of figure 6
  • the preparation method is consolidated and consists of two successive steps: (i) the formation of the DNA/condensing agent nucleus; (ii) the subsequent covering of the DNA/condensing agent nucleus with a lipid shell.
  • the DNA is condensed following the electrostatic interaction between the negative charges of the DNA and the positive charges of the cationic condensing agent.
  • By varying the mixing ratio between the DNA and the condensing agent it is possible to obtain DNA/condensing agent nuclei having different charges. This makes it possible to cover the DNA/condensing agent nucleus with lipid shells of positive charge (cationic liposomes) or negative charge (anionic liposomes) depending on the net charge of the aforementioned nucleus (negative or positive respectively).
  • the active ingredient can be inserted indiscriminately within the aqueous core in the case of hydrophilic active ingredient (such as polyinosinic-polycytidylic (poly(l:C)) acid or within the hydrophobic lipid bilayer of the liposome if the anti-tumoral active ingredient is a hydrophobic molecule (such as doxorubicin, cisplatin, docetaxel, paclitaxel, etc.).
  • hydrophilic active ingredient such as polyinosinic-polycytidylic (poly(l:C)
  • the anti-tumoral active ingredient is a hydrophobic molecule (such as doxorubicin, cisplatin, docetaxel, paclitaxel, etc.).
  • the net charge of the surface of the nanoparticles allows non-covalent functionalization by means of electrostatic interaction with ligands of opposite charge.
  • PEG polyethylene glycol
  • a nanovector In this sector, which is subject to on-going development, but still liable to vast improvements, the main problem encountered by researchers lies in the individualization of a nanovector able to efficiently overcome the extra-cellular and intra-cellular barriers. From the perspective of commercialization and of a medical use that can be approved and standardized, a nanovector must have the following characteristics: (i) controlled dimensions and charge; (ii) improved residence time in the bloodstream; (iii) effective cellular internalization; (iii) cytoplasmic release of the complete genetic and/or therapeutic charge; (iv) effective entry into the nucleus; (v) increased transfection efficiency; (vi) increased cell vitality.
  • the inventors have analyzed numerous formulations of nanoparticles and have identified a specific formulation of nanoparticles that is more efficient than the reagents generally used for lipid-based transfection currently on the market.
  • a multicomponent lipid formulation has been produced that incorporates the advantages of each lipid species.
  • the present invention relates to a multicomponent lipid nanoparticle consisting of a hydrophilic nucleus and an outer monolayer shell consisting of 1 ,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 3 ⁇ -[ ⁇ -( ⁇ ', ⁇ '- dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Choi) and dioleoylphosphatidylethanolamine (DOPE), furthermore at least one of cholesterol hydrochloride (DC-Choi), DOPE functionalized with PEG (DOPE-PEG), and dioleoylphosphocholine (DOPC), and optionally one or more lipophilic anti-tumoral active ingredients; a process for the preparation of multicomponent lipid nanoparticles for gene transport and/or for the transport of anti-
  • DOTAP 1,2-dioleoyl-3-trimethylammonium-propane
  • DC-Choi 3 ⁇ -[ ⁇ -(
  • Said process for the preparation of multicomponent lipid nanoparticles for gene transport and/or for the transport of anti-tumoral drugs comprises the following steps:
  • the process for the preparation of multicomponent lipid nanoparticles for the transport of hydrophobic anti-tumoral drugs comprises the following steps:
  • DOTAP 1,2-dioleoyl-3-trimethylammonium-propane
  • DC-Chol dioleoylphosphatidylethanolamine
  • Choi cholesterol
  • DOPE-PEG DOPE functionalized with PEG
  • DOPC dioleoylphosphocholine
  • a lipophilic anti-tumoral active ingredient in a suitable organic solvent and mixing desired quantities of each lipid and optionally the active ingredient thus dissolved so as to obtain a homogenous mixture
  • step b completely removing the organic solvent from the mixture obtained in step a. thus obtaining a lipid film
  • step b. hydrating the lipid film obtained in step b. with a suitable buffer at physiological cell pH so as to obtain a desired final concentration;
  • step d sonicating the lipid solution obtained in step c. so as to obtain a clear solution (which indicates the formation of uni-lamellar vesicles, also defined here as cationic liposomes).
  • the lipid solution obtained in step c. is extruded by means of a commercial extruder with pore dimensions equal to 200 nm (Avanti Polar Lipidis, USA).
  • step o allowing to equilibrate, for a period of between 30 and 180 minutes at room temperature, a desired volume of the complex obtained in step o;
  • the invention lastly relates to the above-described nanoparticles for use in gene therapy and/or anti-tumoral therapy, or a gene therapy and/or anti-tumoral therapy method comprising a step of administration of said nanoparticles.
  • the inventors have demonstrated (data reported in the experimental section) that, compared to the more widespread reagents for cellular transfection and for the transport of anti-tumoral drugs currently on the market (Lipofectamine, Lipofectamine 2000, Lipofectamine Plus), the multicomponent lipid nanoparticles of the present invention demonstrate numerous advantages in relation to the above- indicated problems.
  • the main advantages are as follows:
  • Transfection efficiency of the nanoparticles is greater than that of the reagents generally used in lipotransfection (such as Lipofectamine, Lipofectamine 2000, Lipofectamine Plus) ( 10 10 RLU/mg protein) in various cell lines (NIH 3T3; HeLa; CHO; A17).
  • lipotransfection such as Lipofectamine, Lipofectamine 2000, Lipofectamine Plus
  • 10 10 RLU/mg protein 10 10 RLU/mg protein
  • the anti-tumoral activity of the multicomponent lipid nanoparticles charged with the active ingredient (poly(l:C), paclitaxel, docetaxel), (quantified by means of measurements of cell vitality and expressed as a percentage compared to untreated cells) is greater than that of the more widespread reagents (Lipofectamine, Lipofectamine 2000, Lipofectamine Plus) in various tumoral cell lines (HeLa; A17).
  • lipid nanoparticles means particles formed by a hydrophilic nucleus coated by a single lipid outer shell (lipid shell), suitable for use in gene therapy and/or anti-tumoral therapy, in which the active ingredient of interest (nucleic acid and/or anti-tumoral acid) will be in the hydrophilic nucleus if hydrophilic or in the lipid shell if hydrophobic.
  • nucleic acid condensing agent means positively charged molecules generally known for their ability to condense nucleic acids, provided with a positive charge, this category containing various classes of substances known in the literature as described in the prior art.
  • lipids means both lipids as generally defined in scientific dictionaries and derivatives thereof, such as derivatives of cholesterol and phospholipids.
  • Nucleic acids means: dsDNA (double strand DNA), ssDNA (single strand DNA), RNA, siRNA (small interfering RNA), shRNA (short hairpin RNA), mRNA, dsRNA (double strand RNA), tRNA, miRNA (microRNA) or a mixture thereof.
  • Such nucleic acids can be single strand and/or double strand, in the form of vectors that can be integrated into the genome or that can be transposed, mini-chromosomes (or equipped with centromere and telomeres or more generally elements that allow autonomous replication), provided with an origin of replication, etc.
  • Gene transport means those methods which make it possible to transfer an external nucleic acid to the inside of a target cell so as to alter temporarily or permanently the phenotype thereof, such as transfection techniques which make it possible to express or silence specific genes in the transformed cells.
  • gene silencing is used in the present description in accordance with the prior art.
  • Gene therapy means the insertion of genetic material, as defined above (nucleic acids) inside cells for the purpose of being able to treat pathologies.
  • This insertion procedure known as transfection, makes it possible to transfer one or more healthy genes into a sick cell for the purpose of treating a pathology caused by the absence or by the defect of one or more (mutated) genes, or one or more nucleic acids able to silence genes that are overexpressed or expressed in the sick cell.
  • Anti-tumoral therapy means a treatment able to block the progression of tumoral cells and cause regression thereof, where possible.
  • Anti-tumoral drug means an active ingredient able to damage the tumoral cells by blocking proliferation thereof and/or promoting the death thereof.
  • Various tumoral drugs are known (apoptotic drugs, immunostimulants, cytotoxic drugs, etc.). The effect of an apoptotic anti-tumoral drug administered to a cell culture in vitro is the death of the tumoral cells (apoptosis).
  • hormone means a molecule able to modify the conditions of the tissues in which neoplasm has developed.
  • immunostimulant means a class of anti-tumoral drugs which tend to develop the immune response.
  • cytotoxic drugs means molecules that intervene in the biosynthesis of ribonucleotides and deoxyribonucleotides in the processes of replication, transcription and translation of DNA and that intervene in the process of formation of the mitotic spindle.
  • DOTAP denotes the cationic lipid 1 ,2-dioleoyl-3- trimethylammonium-propane.
  • DC-Choi denotes the cationic lipid derived from the cholesterol 3B-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride.
  • DOPE denotes the phospholipid dioleoylphosphatidylethanolamine.
  • DOPC denotes the phospholipid dioleoylphosphocholine.
  • PEG polyethylene glycol
  • Figure 1 in 1 (A) shows the transfection efficiency of the fibroblasts NIH 3T3 of various nanoparticles according to the invention (the relative ratios between the cationic lipids 1 ,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 3 ⁇ -[ ⁇ -( ⁇ ', ⁇ '- dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Choi), cholesterol (Choi) and the phospholipids dioleoylphosphocholine (DOPC), dioleoylphosphatidylethanolamine (DOPE) and DOPE functionalized with PEG (DOPE-PEG) are indicated beneath each bar) and of Lipofectamine, which is one of the more common reagents for cellular transfection.
  • DOTAP cationic lipids 1 ,2-dioleoyl-3-trimethylammonium-propane
  • DC-Choi 3 ⁇ -[ ⁇ -( ⁇ ',
  • the efficiency of the multicomponent lipid nanoparticles is also compared with that of the more effective multicomponent lipoplexes (MC lipoplexes) prepared as described in (Caracciolo et al., Biochim Biophys Acta, 1768, 2007, 2280).
  • the bars in grey relate to experiments carried out using 5 ⁇ g of sample per well (the term sample means the solution of cationic liposomes described under step d. of the method described above), the bars in black using 10 ⁇ g of sample per well.
  • the figure shows the transfection efficiency in CHO ovary cells of various nanoparticles according to the invention (the relative ratios between the cationic lipids 1 ,2-dioleoyl-3- trimethylammonium-propane (DOTAP), 3B-[N-(N',N'-dimethylaminoethane)- carbamoyl]cholesterol hydrochloride (DC-Choi), cholesterol (Choi) and the phospholipids dioleoylphosphocholine (DOPC), dioleoylphosphatidylethanolamine (DOPE) and DOPE functionalized with PEG (DOPE-PEG) are indicated beneath each bar).
  • DOTAP cationic lipids 1 ,2-dioleoyl-3- trimethylammonium-propane
  • DC-Choi 3B-[N-(N',N'-dimethylaminoethane)- carbamoyl]cholesterol hydrochloride
  • DOPC diole
  • the efficiency is compared with that of Lipofectamine and with that of the more efficient multicomponent lipoplexes prepared as described in (Caracciolo et al., Biochim Biophys Acta, 1768, 2007, 2280).
  • the bars in grey relate to experiments carried out using 5 ⁇ g of sample per well, the bars in black using 10 ⁇ g of sample per well.
  • Figure 2 shows the results of a cell vitality assay conducted on the CHO ovary cells as described in the experimental section (the relative ratios between the cationic lipids 1 ,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 3 ⁇ -[ ⁇ -( ⁇ ', ⁇ '- dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Choi), cholesterol (Choi) and the phospholipids dioleoylphosphocholine (DOPC), dioleoylphosphatidylethanolamine (DOPE) and DOPE functionalized with PEG (DOPE-PEG) are indicated beneath each bar).
  • DOTAP cationic lipids 1 ,2-dioleoyl-3-trimethylammonium-propane
  • DC-Choi 3 ⁇ -[ ⁇ -( ⁇ ', ⁇ '- dimethylaminoethane)-carbamoyl]cholesterol hydrochloride
  • Figure 3 shows representative superimpositions of fluorescence images and Nomarski images in the CHI cells.
  • the white signal is caused by the presence of fluorescent DNA.
  • a scan along the x-axis was carried out for each image.
  • the DNA is uniformly diffused in the cytoplasm and in the nucleus of the cell.
  • perinuclear aggregates with confined DNA are not visible.
  • Figure 4 shows the results of a cell vitality assay conducted on the A17 cells treated with multicomponent nanoparticles charged with paclitaxel as described in the experimental section (the relative ratios between the cationic lipids 1 ,2-dioleoyl- 3-trimethylammonium-propane (DOTAP), 3B-[N-(N',N'-dimethylaminoethane)- carbamoyl]cholesterol hydrochloride (DC-Choi), cholesterol (Choi) and the phospholipids dioleoylphosphocholine (DOPC), dioleoylphosphatidylethanolamine (DOPE) and DOPE functionalized with PEG (DOPE-PEG) are indicated beneath each bar).
  • DOTAP cationic lipids 1 ,2-dioleoyl- 3-trimethylammonium-propane
  • DC-Choi 3B-[N-(N',N'-dimethylaminoethane)- carbamo
  • Figure 5 shows the results of a cell vitality assay conducted on the A17 cells treated with multicomponent nanoparticles charged with docetaxel as described in the experimental section (the relative ratios between the cationic lipids 1 ,2-dioleoyl- 3-trimethylammonium-propane (DOTAP), 3B-[N-(N',N'-dimethylaminoethane)- carbamoyl]cholesterol hydrochloride (DC-Choi), cholesterol (Choi) and the phospholipids dioleoylphosphocholine (DOPC), dioleoylphosphatidylethanolamine (DOPE) and DOPE functionalized with PEG (DOPE-PEG) are indicated beneath each bar).
  • DOTAP cationic lipids 1 ,2-dioleoyl- 3-trimethylammonium-propane
  • DC-Choi 3B-[N-(N',N'-dimethylaminoethane)- carbamoy
  • the present invention therefore relates to multicomponent lipid nanoparticles consisting of a hydrophilic nucleus and a single lipid outer shell formed by 1 ,2- dioleoyl-3-trimethylammonium-propane (DOTAP), 3 ⁇ -[ ⁇ -( ⁇ ', ⁇ '- dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Choi) and dioleoylphosphatidylethanolamine (DOPE), furthermore at least one of cholesterol (Choi), DOPE functionalized with PEG (DOPE-PEG), and dioleoylphosphocholine (DOPC), and optionally one or more lipophilic anti-tumoral active ingredients.
  • the hydrophilic nucleus may be formed by one or more nucleic acid molecules and by one or more nucleic acid condensing agents.
  • the hydrophilic nucleus will therefore be formed as indicated above and the nucleic acid molecules can be selected from dsDNA (double strand DNA), ssDNA (single strand DNA) or double-strand or single-strand DNA, RNA, siRNA (small interfering RNA), shRNA (short hairpin RNA), mRNA, dsRNA (double strand RNA), tRNA, miRNA (microRNA) or a mixture thereof.
  • the nucleic acid molecules can be inserted into suitable vectors which allow them to become incorporated in the host genome (for example transposable vectors) or which allow them to carry out replication and expression or transcription in the host cell (eukaryotic cell), such as suitable cloning vectors, expression vectors, mini- chromosomes, etc.
  • the nucleic acids also defined herein as "genetic material” in general, can be genes for substitutive gene therapy (that is to say gene therapy in which the disease is caused by a lack of expression of a specific gene or in which a non-functional gene is expressed) or can be sequences with the aim of interfering with the expression of one or more genes expressed and overexpressed in sick cells, such as in tumoral cells, and normally not expressed in healthy cells.
  • the inserted genetic material may also consist of tRNA where the dysfunction is linked to the absence or to the malfunctioning of a particular tRNA.
  • nucleic acids are also defined in the present description as "sequences of interest" since, in the embodiment of the invention as taught here, it is irrelevant, for the purposes of obtaining a particle having the above-listed technical advantages, which sequences are inserted in the nucleic acid/nucleic acid condensing agent nucleus: any sequence can be inserted without changing the chemical and physical characteristics of the nanoparticles.
  • the ratio between the volume of said one or more nucleic acid molecules and said nucleic acid condensing agents in the nanoparticles according to the invention can be comprised between about 1 :1 and about 1 :0.5, and in one embodiment such ratio will be about 1 :0.75.
  • nucleic acid condensing agents generally known in the field can be used.
  • such agents may be selected from polyamines (for example spermine or spermidine), positively charged amino acids (for example arginine and lysine), positively charged proteins, and multivalent metal cations (for example Ca 2+ , Mg 2+ , Mn 2+ , Fe 2+ , Fe 3+ ).
  • nucleus of the nanoparticle will be produced using protamine as condensing agent.
  • the particle can be used for anti-tumoral therapy by means of hydrophilic active ingredients, and the nucleus will therefore be formed by hydrophilic anti-tumoral active ingredients and one or more suitable condensing agents.
  • the active ingredient can be polyinosinic- polycytidylic (poly(l:C)) acid together with one or more condensing agents, as described above for the nucleic acids.
  • this will always comprise 1 ,2- dioleoyl-3-trimethylammonium-propane (DOTAP), 3 ⁇ -[ ⁇ -( ⁇ ', ⁇ '- dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Choi) and dioleoylphosphatidylethanolamine (DOPE), and furthermore at least one of cholesterol (Choi), DOPE functionalized with PEG (DOPE-PEG) and dioleoylphosphocholine (DOPC).
  • DOTAP 1,2- dioleoyl-3-trimethylammonium-propane
  • DC-Choi 3 ⁇ -[ ⁇ -( ⁇ ', ⁇ '- dimethylaminoethane)-carbamoyl]cholesterol hydrochloride
  • DOPE dioleoylphosphatidylethanolamine
  • DOPC dioleoylphosphocholine
  • the above-listed lipids can be present in variable mutual ratios, in accordance with some embodiments 1 ,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 3 ⁇ - [N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Choi), cholesterol (Choi), dioleoylphosphocholine (DOPC), dioleoylphosphatidylethanolamine (DOPE) and DOPE functionalized with PEG (DOPE-PEG) will be present in a mutual ratio selected from: a) 0.5:1 .5:0.5:0:1 .5:0 or b) 1 :1 :0:1 :1 :0 or c) 0.5:1 .5:0:0.5:1 .5:0 or d) 0.5:1 .5:0.5:0:1 .2:0.3 or e) 1 :1 :0:1
  • DOTAP 1,2-dioleoyl-3-trimethylammonium-propane
  • DC-Choi 3 ⁇ - [N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride
  • DOPE dioleoylphosphatidylethanolamine
  • the surface charge of the nanoparticle is positive, for example in the embodiment in which the mutual ratio between the components 1 ,2-dioleoyl-3- trimethylammonium-propane (DOTAP), 3B-[N-(N',N'-dimethylaminoethane)- carbamoyl]cholesterol hydrochloride (DC-Choi), cholesterol (Choi), dioleoylphosphocholine (DOPC), dioleoylphosphatidylethanolamine (DOPE) and DOPE functionalized with PEG (DOPE-PEG) is 0.5:1 .5:0:0.5:1 .5:0 (c), the surface charge of the nanoparticle will have a potential z comprised between about 40 and 45 mV, a non-limiting example being represented by a potential z of about + 42mV.
  • the lipid nanoparticle produced as described above will have a hydrodynamic diameter comprised between 200 and 250
  • the diameter D of the particle will be about 210 ⁇ 12 nm and the polydispersity index will be ⁇ 0.2.
  • the above-mentioned nanoparticles will have the following characteristics:
  • the nanoparticles have controlled and extremely monodisperse dimensions (polydispersity index ⁇ 0.2)
  • the transfection efficiency of the nanoparticles is greater than that of the reagents generally used for lipotransfection, such as lipofectamines ( 10 10 RLU/mg protein) in various cell lines (NIH 3T3; HeLa; CHO; A17).
  • lipofectamines 10 10 RLU/mg protein
  • the nanoparticles are not toxic (cell vitality > 80%).
  • the process for the preparation of multicomponent lipid nanoparticles according to the present invention comprises the following steps:
  • DOTAP 1,2-dioleoyl-3-trimethylammonium-propane
  • DC-Chol dioleoylphosphatidylethanolamine
  • Choi cholesterol
  • DOPE-PEG DOPE functionalized with PEG
  • DOPC dioleoylphosphocholine
  • a lipophilic anti-tumoral active ingredient in a suitable organic solvent and mixing desired quantities of each lipid and one or more lipophilic anti-tumoral active ingredients thus dissolved so as to obtain a homogenous mixture
  • step b completely removing the organic solvent from the mixture obtained in step a. thus obtaining a lipid film
  • step b. hydrating the lipid film obtained in step b. with a suitable buffer at physiological cell pH so as to obtain a desired final concentration;
  • step d sonicating the lipid solution obtained in step c. so as to obtain a clear solution (which indicates the formation of uni-lamellar vesicles, also defined here as cationic liposomes).
  • Anti-tumoral active ingredients can be, for example, paclitaxel, docetaxel, doxorubicin, and derivatives thereof.
  • the active ingredient not entrapped in said nanoparticles is removed by means of centrifugation (for example centrifugation at about 1000 rpm for 10 minutes);
  • the supernatant containing said nanoparticles with the active ingredient encapsulated is centrifuged (50,000 rpm for 30 minutes) to precipitate said nanoparticles;
  • the supernatant is discarded and the pellet of said nanoparticles is washed twice with saline buffer (PBS, pH 7.4).
  • the process may optionally also comprise the following steps:
  • step a allowing to equilibrate, for a period of between 30 and 180 minutes at room temperature, a desired volume of the complex obtained in step a;
  • the application relates to a process for the preparation of multicomponent lipid nanoparticles according to the present invention comprising the following steps:
  • DOTAP 1,2-dioleoyl-3-trimethylammonium-propane
  • DC-Choi 3B-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride
  • DOPE dioleoylphosphatidylethanolamine
  • Choi cholesterol
  • DOPE-PEG DOPE functionalized with PEG
  • DOPC dioleoylphosphocholine
  • step b completely removing the organic solvent from the mixture obtained in step a. thus obtaining a lipid film
  • step b. hydrating the lipid film obtained in step b. with a suitable buffer at physiological cell pH so as to obtain a desired final concentration;
  • step d sonicating the lipid solution obtained in step c. so as to obtain a clear solution
  • step o allowing to equilibrate, for a period of between 30 and 180 minutes at room temperature, a desired volume of the complex obtained in step o;
  • the lipid solution obtained in step c. is extruded by means of a commercial extruder with pore dimensions equal to 200 nm (Avanti Polar Lipidis, USA).
  • step d. will be substituted by the following step:
  • step c. the lipid solution obtained in step c. is extruded by means of a commercial extruder with pore dimensions equal to 200 nm.
  • the nanoparticles obtained by the processes described above can be stored in a refrigerator at 4°C.
  • the nanoparticles can be used to transport lipophilic anti- tumoral drugs, and this occurs when said drugs are inserted in step a.
  • Paclitaxel, docetaxel, doxorubicin and derivatives thereof can be used as lipophilic anti-tumoral active ingredients according to the invention.
  • said active ingredients are inserted in the outer shell of the nanoparticles of the invention, they will be inserted as indicated insteps a., b. and c. of the process in a molar ratio of moles of active ingredient/moles of total lipid comprised between 1 and 4.
  • the nanoparticles according to the invention may comprise, in the hydrophilic nucleus, nucleic acids for gene therapy or hydrophilic anti-tumoral drugs.
  • the outer shell can also be charged with lipophilic anti-tumoral drugs, but also may not comprise such drugs.
  • the outer shell may comprise lipophilic anti-tumoral drugs (active ingredients) or may be formed solely by 1 ,2-dioleoyl-3-trimethylammonium- propane (DOTAP), 3B-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Choi) and dioleoylphosphatidylethanolamine (DOPE) and at least one of cholesterol (Choi), DOPE functionalized with PEF (DOPE-PEG), and dioleoylphosphocholine (DOPC).
  • DOTAP 1,2-dioleoyl-3-trimethylammonium- propane
  • DC-Choi 3B-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride
  • DOPE dioleoylphosphatidylethanolamine
  • DOPC dioleoylphosphocholine
  • the concentration of the solution of nucleic acids and of the solution containing the nucleic acid condensing agent or agents may have a concentration selected according to the user requirements, such concentration, which will be equal for the two different solutions, possibly being between 0.5 and 1 .5 mg/ml for example, that is to say about 0.5; about 0.6; about 0.7; about 0.8; about 0.9; about 1 ; about 1 .1 ; about 1 .2; about 1 .3; about 1 .4; about 1 .5 mg/ml by way of example, although the present invention does not rule out the fact that a person skilled in the art may select different concentrations.
  • the ratio of volume between the solution of nucleic acids and the solution of nucleic acid condensing agent in point a. will be, as described, in a range between 1 :1 and 0.5:1 .
  • this ratio may be, as indicated above (volume of the solution of nucleic acids/volume of the solution of nucleic acid condensing agent), about 0.5:1 : 0.55:1 ; 0.6:1 ; 0.65:1 ; 0.7:1 ; 0.75:1 ; 0.8:1 ; 0.85:1 ; 0.9:1 ; 0.95:1 ; 1 :1 .
  • nucleic acid molecules that can be used for the preparation of the nucleus of the nanoparticles of the invention are those indicated previously in the part relating to the description of the nanoparticles themselves and can therefore be selected from DNA, RNA, siRNA, shRNA, mRNA; miRNA; dsRNA.
  • the nucleic acid condensing agents are selected from polyamines, cationic peptides, cationic lipids, cationic surfactants, positively charged amino acids, positively charged proteins, and polyvalent metal cations.
  • suitable molecules are provided here in the part relating to the description of the nanoparticles themselves and may therefore be as follows: for the polyamines, selected from spermine and spermidine; for the positively charged amino acids, selected from arginine and lysine; for the positively charged proteins, selected from protamine and isotones; for the polyvalent metal cations, selected from Ca 2+ , Mg 2+ , Mn 2+ , Fe 2+ , Fe 3+ .
  • protamine is selected as nucleic acid condensing agent.
  • a desired volume of the complex obtained in a. (which corresponds to the "nucleus" of the nanoparticles) is allowed to equilibrate for a period comprised between about 30 and 180 minutes, this period possibly being, for example, about 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150, 160, 170 or 180 minutes. In general, a period comprised between about 30 and 60 minutes, including the extremes, is also sufficient.
  • the nuclei thus obtained can be stored for a period from a few hours to about 24 hours at about +4°C.
  • nucleus obtained in p. containing nucleic acids is more delicate compared to the uni-lamellar vesicles obtained in step q., which can be stored in a refrigerator (at about +4°C) for about one week or two, it is advisable to prepare the vesicles first, or to prepare the nucleus and the vesicles at the same time so as to use the nucleus obtained in p. as early as possible and to avoid possible degradation thereof so as to obtain multicomponent nanoparticles having the greatest efficacy.
  • the above-indicated lipids (cationic and phospholipids) will be used in the desired mutual ratios. It has been demonstrated that the various assayed ratios are all effective for producing vesicles having good transfection properties and low toxicity.
  • the cationic lipids 1 ,2-dioleoyl-3-trimethylammonium- propane (DOTAP), 3B-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Choi), cholesterol (chol) and the phospholipids dioleoylphosphocholine (DOPC), dioleoylphosphatidylethanolamine (DOPE) and DOPE-PEG can be mixed in a mutual ratio selected from: a) 0.5:1 .5:0.5:0:1 .5:0 or b) 1 :1 :0:1 :1 :0 or c) 0.5:1 .5:0:0.5:1 .5:0 or d) 0.5:1 .5:0.5:0:1 .2:0.3 or e) 1 :1 :0:1 :0.8:0.2 or f) 0.5:1 .5
  • DOTAP 1 .5
  • DC-Choi 0.5 Chol:0
  • DOPC 1 DOPE; 0 DOPE-PEG; in case b, b) 1 :1 :0:1 :1 :0
  • DOTAP 1,2-dioleoyl-3-trimethylammonium-propane
  • DC-Choi 3 ⁇ -[ ⁇ - (N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride
  • DOPE dioleoylphosphatidylethanolamine
  • the above-mentioned lipids can be mixed, and therefore present in the coating of the nucleic acid/condensing agent nucleus of the nanoparticles described here, in the mutual ratio (in which the order of the lipids is that indicated in the previous paragraph) of 0.5:1 .5:0.5:0:1 .5:0.
  • Each lipid will be dissolved in a suitable organic solvent and then mixed in the desired ratios or in the ratios indicated above.
  • suitable organic solvent can be used, for example suitable organic solvents generally used by a person skilled in the art may be chloroform or ethanol.
  • the reagents will be mixed so as to obtain a homogenous solution, at ambient temperature for a period comprised between 1 and 5 minutes, for example about 1 - 2 minutes.
  • the lipids dissolve in the suitable solvent, for example chloroform, and are mixed so as to obtain a homogenous mixture of the four lipid species (in organic solvent the mixture of lipid molecules is a homogenous mixture).
  • the amount of solvent to be used depends on the solubility of the lipid species used, for example, the solutions can be prepared with about 10/20 mg of lipid/ml of chloroform.
  • step b The organic solvent is then removed in step b., which can be achieved using any suitable means known to a person skilled in the art, such as by using a conventional rotary evaporator.
  • This process leads to the formation of a thin lipid film at the base of the container used.
  • the removal of the organic solvent must be complete; in order to ensure the complete removal of the organic solvent, the lipid film can also be kept under vacuum for 24 hours, for example.
  • the lipid film in step b. is then hydrated with a suitable buffer at physiological cell pH so as to obtain a final desired concentration.
  • Any buffer generally suitable for use with vital cells which stabilizes the pH at physiological levels such as the buffer Tris-HCI at physiological pH, can be used.
  • Tris-HCI (10 mM, pH 7.4) necessary to obtain a final concentration of 1 mg/ml, can be used.
  • the lipid solution obtained in step c. will be sonicated so as to obtain a clear solution, the clarity of the solution indicating the formation of lamellar vesicles which are formed by the above-mentioned lipids in the ratios selected by the user and which will be used to coat the nucleus formed in step p.
  • Any suitable sonicator conventionally used by a person skilled in the art can be used, such as a titanium-tip sonicator, for a period of about 10 minutes.
  • a person skilled in the art will choose the most effective sequence of on-off cycles with no need for further teachings.
  • the lipid solution obtained in step c. will be extruded by means of a commercial extruder with pore dimensions equal to 200 nm so as to obtain a clear solution (number of steps in the extruder >20), the clarity of the solution indicating the formation of lamellar vesicles which are formed by the above-mentioned lipids in the ratios selected by the user and which will be used to coat the nucleus formed in step p.
  • the lipophilic anti-tumoral active ingredients as indicated above will be charged in the lipid shell of the nanoparticle in a molar ratio (moles of active ingredient/moles of total lipid) between 1 and 4.
  • the nucleus formed in step p can be constituted similarly by nucleic acid molecules or polyinosinic-polycytidylic (poly(l:C)) acid molecules and a pool of one or more nucleic acid condensing agents which are dissolved separately, at equal concentration in distilled H 2 0, and are then mixed together at a ratio of between 1 :1 and 1 :0.5, thus obtaining a negatively charged complex (with light stirring).
  • the vesicles thus prepared can be stored at approximately +4°C for a period up to 1 or 2 weeks, and can then be used directly for step d. of the process for preparing the nanoparticles of the invention.
  • a suitable volume of said vesicles is mixed with a suitable volume of the balanced complex obtained in step p.
  • about 5 microliters of vesicles can be mixed with 2 microliters of condensing agent/nucleic acid at a charge ratio (moles of cationic lipid/nucleic acid bases ) comprised between about 2 and 3, including the extremes.
  • This ratio in a non-limiting exemplary embodiment, can be about 2.5, for example.
  • the charge ratio can be calculated as follows (the example relates to an embodiment in which the nucleic acid is DNA):
  • rho number of moles of cationic lipid/number of nucleotides (or bases).
  • Incubation can be implemented for a period between about 30 minutes and 3 hours, for example a period of about 30, 40, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150, 160, 170 or 180 minutes.
  • DOTAP-DC-Chol-Chol-DOPC-DOPE-DOPE/PEG (0.5:1 .5:0.5:0:1 .5:0)
  • DOTAP-DC-Chol-Chol-DOPC-DOPE-DOPE/PEG (1 :1 :0:1 :1 :0)
  • DOTAP-DC-Chol-Chol-DOPC-DOPE-DOPE/PEG 0.5:1 .5:0:0.5:1 .5:0.
  • DOTAP-DC-Chol-Chol-DOPC-DOPE-DOPE/PEG 0.5:1 .5:0.5:0:1 .2:0.3
  • DOTAP-DC-Chol-Chol-DOPC-DOPE-DOPE/PEG (1 :1 :0:1 :0.9:0,1 )
  • DOTAP-DC-Chol-Chol-DOPC-DOPE-DOPE/PEG 0.5:1 .5:0:0.5:1 .2:0.3.
  • the solution containing the nucleus and that containing the multicomponent vesicles obtained as described above are mixed in such a way that the charge ratio (moles of cationic lipid/bases of nucleic acid or poly(l:C) acid) is equal to about 2.5.
  • the nanoparticles obtained have the composition in lipids in the coating with mutual ratios equal to those used in mixing step a.
  • nanoparticles defined as “nanoparticles obtainable by means of the process of the invention as defined in the description and in the claims relating to said process".
  • the present invention relates to nanoparticles as described here for use in gene therapy and in anti-tumoral therapy.
  • the invention relates to a method of gene therapy and/or anti- tumoral therapy which comprises the step of administering the nanoparticles of the invention to a patient having a need therefor.
  • nanoparticles will be formed using appropriate nucleic acids selected in accordance with the desired gene therapy or using appropriate anti- tumoral drugs in accordance with the desired anti-tumoral therapy.
  • the more significant aspect is the extraordinary capability exhibited by the multicomponent lipid nanoparticles to release DNA or poly(l:C) or lipophilic drugs in the cytoplasm and in the cell nucleus (see figure 3).
  • This property makes the multicomponent lipid nanoparticles unique and is the most likely explanation of their increased efficiency (figure 1 ) and also of their ability to induce apoptosis in A17 tumoral cells (figure 4 and figure 5).
  • the anti-tumoral drugs can be selected from poly(l:C), active ingredients such as paclitaxel, docetaxel, doxorubicin or derivatives thereof, or from a mixture thereof.
  • active ingredients such as paclitaxel, docetaxel, doxorubicin or derivatives thereof, or from a mixture thereof.
  • the DNA/protamine shell is left to equilibrate for one hour at ambient temperature.
  • a volume of DNA/protamine complex thus prepared is interacted for 20 minutes with a solution of cationic liposomes formed by a mixture of the commercial cationic lipid DOTAP, the cationic derivative of cholesterol DC-Choi and by two neutral phospholipids, such as DOPE and DOPC.
  • the four above-mentioned molecular species are mixed in a ratio of 0.5:1 .5:0.5:1 .5 (c). After 2 hours of incubation, the formation of the nanoparticles is complete.
  • the surface charge is positive with the DNA/protamine nucleus covered by a lipid shell formed by one or more lipid bilayers.
  • the lipid formulation used in the present invention has never been used before for the production of nanoparticles having a DNA/protamine nucleus. The same process has been used to produce the nanoparticles described as a, b, d, e and f.
  • DOTAP cationic lipids 1 ,2-dioleoyl-3-trimethylammonium-propane
  • DC-Choi 3 ⁇ -[ ⁇ - (N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride
  • DOPC phospholipids dioleoylphosphocholine
  • DOPE dioleoylphosphatidylethanolamine
  • DOPE-PEG DOPE-PEG
  • the fluorescent neutral lipids DOPC-NBD and DOPE-NBD acquired from Avanti Polar Lipids were used for the fluorescence microscopy measurements.
  • the lipids were dissolved in chloroform and mixed so as to obtain a homogenous mixture of the lipid species.
  • the amount of chloroform to be used depends on the solubility of the lipid species used; generally the solutions were prepared with about 10/20 mg of lipid/ml of chloroform.
  • the organic solvent was removed by using a rotary evaporator, which led to the formation of a thin lipid film at the base of an ampoule.
  • the lipid film was kept under vacuum for 24 hours.
  • the lipid film was then hydrated by adding the volume of Tris-HCI buffer (10 mM, pH 7.4) necessary to obtain a final concentration of 1 mg/ml.
  • the lipid solutions used were prepared by varying the ratio between the cationic lipid species. a) 0.5:1 .5:0.5:0:1 .5:0
  • DOTAP 1 .5
  • DC-Choi 0.5 Chol:0
  • DOPC 1 DOPE; 0 DOPE-PEG; in case b, b) 1 :1 :0:1 :1 :0
  • Plasmid DNA (pGL3 which codes for luciferases), acquired from Promega (Madison, Wl) and dissolved in bi-distilled water (Carlo Erba Reagenti, Milano, Italia) at a concentration of 1 mg/ml, was used for the transfection experiments. Plasmid DNA 2.7-kbp labeled with Cy3 (Mirus Bio Corporation, Madison, Wl) was used for the fluorescence microscopy experiments.
  • the nucleus of the nanoparticles was obtained by mixing the DNA (linear, plasmid and fluorescent) and the solution of protamine at a ratio by volume of 1 :0.75 so as to obtain a negatively charged complex.
  • the lipophilic drugs (docetaxel, paclitaxel) were acquired from Sigma Aldrich. In the case of preparation of nanoparticles with lipophilic drugs charged in the shell, the lipids were dissolved in chloroform and mixed so as to obtain a homogenous mixture. The amount of chloroform to be used is dependent on the solubility of the lipid species used; generally the solutions were prepared at about 10/20 mg of lipid/ml of chloroform and 0.3/0.7 mg of drug/ml of chloroform. The organic solvent was removed using a rotary evaporator, which led to the formation of a thin lipid film at the base of an ampoule. To ensure complete removal of the organic solvent, the lipid film was kept under vacuum for 24 hours.
  • the lipid film was then hydrated by adding the volume of Tris-HCI buffer (10 mM, pH 7.4) necessary to obtain a final concentration of 1 mg/ml.
  • Tris-HCI buffer 10 mM, pH 7.4
  • the lipid solutions used were prepared by varying the ratio between the cationic lipid species:
  • DOTAP 1 .5
  • DC-Choi 0.5 Chol:0
  • DOPC 1 DOPE; 0 DOPE-PEG; in case b, b) 1 :1 :0:1 :1 :0
  • the lipids were dissolved in chloroform and mixed so as to obtain a homogenous mixture of the lipid species.
  • the amount of chloroform to be used is dependent on the solubility of the lipid species used; generally the solutions were prepared at about 10/20 mg of lipid/ml of chloroform.
  • the organic solvent was removed using a rotary evaporator, which led to the formation of a thin lipid film at the base of an ampoule. To ensure complete removal of the organic solvent, the lipid film was kept under vacuum for 24 hours. The lipid film was then hydrated by adding the volume of Tris-HCI buffer (10 mM, pH 7.4) necessary to obtain a final concentration of 1 mg/ml.
  • the lipid solutions used were prepared by varying the ratio between the cationic lipid species: a) 0.5:1 .5:0.5:0:1 .5:0
  • DOTAP 1 .5
  • DC-Choi 0.5 Chol:0
  • DOPC 1 DOPE; 0 DOPE-PEG; in case b, b) 1 :1 :0:1 :1 :0
  • the polyinosinic-polycytidylic acid was acquired from Invivogen.
  • the protamine sulphate was dissolved in distilled water at a concentration of 1 mg/ml.
  • the nucleus of the nanoparticles was obtained by mixing the polyinosinic-polycytidylic acid (1 mg/ml) and the solution of protamine at a ratio by volume of 1 :0.75 so as to obtain a negatively charged complex.
  • PROTOCOL FOR A TRANSFECTION EXPERIMENT CONDITIONS BASED ON A WELL OF A 24-WELL PLATE
  • lysis buffer Promega
  • each tube was mixed with vortex (about 10 seconds) and centrifuged (2 minutes, 12000 g, 4° C); the supernatant was then aliquoted into two new tubes and stored at - 80° C.
  • the cell lysates were assayed by luminometer (Berthold AutoLumat luminometer LB-953). To this end, 100 microliters of substrate (luciferin) were added to 20 microliters of each sample and subjected to readings for 10 seconds (2 sec delay), following the protocol accompanying Promega's Luciferase Assay System kit. The reaction which occurs converts into light the chemical energy freed from the oxidation of luciferin into oxyluciferin, catalyzed by the luciferase.
  • the luminous intensity, remaining constant for at least one minute, is acquired as a numerical value corresponding to the area subtended by the emission curve over the set period of time. Each sample was measured twice. To standardize the values obtained with respect to the total proteins present in the lysates, the same samples were then analyzed by spectrophotometer.
  • Spectrophotometer readings The luminometer readings were standardized for the milligrams of total cell proteins present in the lysates using the Bio-Rad Protein Assay Dye Reagent (Bio-Rad), in accordance with Bradford's method.
  • a calibration curve was formed, starting from a mother solution of BSA (bovine serum albumin) at known concentration (2 mg/ml) and diluting 100 microliters thereof in 2 ml of final H 2 0. Two series from 6 cuvettes containing scalar amounts of BSA, H 2 0 and Bio-Rad were then assayed:
  • BSA bovine serum albumin
  • each cuvette has to be stirred with vortex and left in the dark for a period between 5 and 60 minutes.
  • the statistical analysis of the data obtained from the measurements was carried out using the program Microsoft Excel.
  • the transfection efficiency (TE) values TE were calculated as an average of at least four experimental examinations.
  • the standard deviation and the S.E.M. standard error of the mean were also determined for the same samples.
  • the treatments were carried out in 100 microliters of final Optimem per well of a 96-well plate, that is to say 80 microliters of Optimem + 20 microliters of multicomponent lipid nanoparticles (generated using 0.1 micrograms of DNA per well and maintaining the same ratios between DNA and protamine and between DNA/protamine and cationic liposomes described in section 3: transfection protocol).
  • the treatments were carried out in 100 microliters of final Optimem per well in a 96- well plate, that is to say 80 microliters of Optimem + 20 microliters of multicomponent lipid nanoparticles incorporating the active ingredient.
  • the MTT final concentration of 0.5 mg/ml
  • the plate is placed in the dark at 37°C for 4 hours.
  • the medium is then removed from the wells by being suctioned off delicately.
  • 100 microliters of DMSO are added to each well and the plate is stirred for 15 minutes at ambient temperature in the dark for the purpose of dissolving the crystals of formazan salt. If the cells are living and metabolically active, they reduce the MTT and the formation of a purple color is observed when the DMSO is added.
  • Vitality is quantified by means of a reading of the absorbance by means of an ELISA plate reader at a wavelength of 540 nm. The data is expressed as a percentage compared to the control represented by cells not treated with the lipid mixtures.

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Abstract

The present invention relates to novel multicomponent lipid nanoparticles for the non-viral transport of nucleic acids and/or anti-tumoral drugs and to processes for the preparation thereof and their use in gene therapy and/or anti-tumoral therapy.

Description

MULTICOMPONENT LIPID NANOPARTICLES AND PROCESSES FOR THE
PREPARATION THEREOF
DESCRIPTION
The present invention relates to the field of gene therapy, and in particular relates to novel multicomponent lipid nanoparticles for the non-viral transport of nucleic acids, and to processes for the preparation thereof and to the use thereof in gene therapy and anti-tumoral therapy.
PRIOR ART
Gene therapy consists of the transfer of genetic material within a cell for the purpose of altering the phenotype thereof temporarily or permanently. This therapy offers new treatment possibilities for numerous pathologies, both hereditary and acquired, which conventional clinical procedures are unable to treat effectively. It is therefore considered to be the therapy of the future, and in recent years has been the subject of intense study and development for potential use on a large scale in a clinical environment ("Clinical development of liposome-based drugs: formulation, characterization, and therapeutic efficacy". Chang, H.; Yeh, M. Int. J. Nanomed. 2012, 7, 49-60). The first step lies in identifying the single gene or the various genes responsible for the genetic disorder. This is possible thanks to the significant progress in the methods based on molecular biology, which have been developed since the 1980s. Such techniques enable cloning and sequencing of various genes. This has involved the precise identification of many gene alterations in various pathologies and the ability, thanks to DNA recombinant techniques, to modify microorganisms (such as bacteria or fungi), so as to allow them to express molecules of interest. The next step lies in the evaluation of the possibility to transfect the somatic cells of an individual suffering from a genetic disorder with a segment of DNA containing the healthy allele. This approach was then also extended to pathologies such as tumors and HIV infections, and other pathologies in which a defective gene is not substituted, but a gene that is able to trigger a therapeutically useful phenomenon is added.
Due to the reduction in deaths caused by infectious diseases over recent decades, neoplastic diseases have become one of the main causes of death in industrialized societies. In the United States, cancer is the second cause of death after cardiovascular diseases. Cancerogenesis is the transformation by means of which somatic cells become tumoral. This may occur due to environmental factors, physical factors, chemical factors or biological factors (what are known as carcinogenic or oncogenic agents). The objective of anti-tumoral therapy is the selective toxicity against the tumoral cells, saving the healthy cells of the host. Anti- tumoral drugs can be divided into three categories: hormones and hormonal antagonists, immunostimulants, and cytotoxic drugs.
The success of gene therapy and of anti-tumoral therapy does not depend exclusively on the use of gene material (gene therapy) or on the most suitable active ingredient (anti-tumoral therapy), but also on the ability to reach the specific site of action with an appropriate concentration and for a period of time that is suitable for establishing the therapeutic effect. A key component of gene therapy and of anti-tumoral therapy is the transport of the therapeutic molecule (DNA, active ingredient) within the target cell by means of vectors which protect it against degradation and ensure the transcription thereof. The vectors used are presently separated into viral and non-viral vectors. Viruses have an optimal tendency to infect cells and to insert their own DNA there, both by integrating it and in the form of an episome. By contrast, viral vectors present some disadvantages which limit the use thereof, such as activation of the immune system, toxicity, and, in the case of gene therapy, the reduced size of the transportable genetic material (normally < 40,000 base pairs). This last characteristic is very limiting considering that many important human genes have a length greater than 40,000 base pairs, considering also the regulatory sequences and the non-coding introns. Among the non-viral vectors, cationic liposomes and lipid bilayer vesicles formed by cationic lipids and neutral lipids constitute an attractive opportunity for molecular engineering since they are formed as out-and-out auto-assembling biomaterials on a micrometric/nanometric scale (Elsabahy et al. "Non-Viral Nucleic Acid Delivery: Key Challenges and Future Directions". Current Drug Delivery, 8, 201 1 , 235-244). In particular, cationic liposome-DNA complexes (lipoplexes) constitute the most widespread lipid-based materials for gene therapy. Lipoplexes are prepared by mixing a solution of binary liposomes (formed of a cationic lipid and a neutral lipid) with a solution of nucleic acids at the desired concentrations. The electrostatic interaction between cationic liposomes and nucleic acids of negative charge determines the formation of the lipoplexes. Measurements taken by means of transmission electron microscopy have demonstrated that the lipoplexes are compact nanoparticles with an inner multi-lamellar structure ("onion-like"), the repetitive unit of which is formed by a lipid bilayer and an aqueous monolayer occupied by the nucleic acids. Lipoplexes have some considerable advantages compared to viral vectors, such as ease of preparation on a large scale, low toxicity, and virtually no limit of the size of the transportable DNA. This last aspect is significant in the hypothesis of transfer within the nucleus of the inner chromosomal cell (the average size of human chromosomes is between 50 and 280 million base pairs). Multicomponent lipoplexes in which the lipid bilayer is formed by a mixture of cationic lipids and neutral phospholipids are mentioned among the most effective lipoplexes as yet reported in the literature (Caracciolo et al., Biochim Biophys Acta, 1768, 2007, 2280). Despite the aforementioned advantages, the main limitation of lipoplexes lies in the low efficiency of transfer of the genetic material (transfection efficiency, TE). This limitation also concerns the aforementioned multicomponent lipoplexes which, although they have a level of efficiency that is, on average, greater than that of binary liposomes, cannot compete in terms of efficiency with the more efficient commercial reagents (Lipofectamines). To this day, the primary approach to increase the transfection efficiency of lipoplexes is the synthesis of new amphipathic molecules or the use of adjuvant non-cationic lipid species.
Recently, lipid nanoparticles have emerged as a potential alternative to lipoplexes, since they have chemical and physical properties (dimensions, surface charge, ability to condense genetic material, etc.) that can be controlled more easily. Such particles are formed of a DNA nucleus (or short-interfering RNA, siRNA) condensed by means of divalent cations (Ca2+, Mg2+, Mn2+; Fe2+), polycations (spermine, spermidine), cationic amino acids (arginine, lysine, histidine) and cationic proteins (protamine, histones) coated by a lipid shell formed by one or more lipid bilayers. From a structural viewpoint, lipid nanoparticles are substantially different from lipoplexes. In the case of nanoparticles, the nucleic acids are pre- condensed by means of cationic condensing agents. The interaction between nucleic acids and condensing agents determines the formation of negatively charged spherical globules (the charge transported from the nucleic acids is in excess compared to that of the condensing agent) on which a number of cationic lipid bilayers (typically 1 -3) are adsorbed by electrostatic interaction. By contrast, the lipoplex is a compact multi-lamellar structure formed of lipid bilayers alternated with aqueous layers occupied by the nucleic acids. The phenomenon of formation is shown in figure 6, taken from (Caracciolo et al., J. Med. Chem, 54, 201 1 , 4160).
The electrostatic interaction between cationic liposomes and nucleic acids (such as plasmid DNA in the diagram of figure 6) determines the formation of multilamellar lipoplexes (to the right, typically 20-30 layers). If, vice versa, the nucleic acids are precondensed with a cationic condensing agent (such as the protamine in the diagram of figure 6), the interaction determines the formation of lipid nanoparticles formed by an aqueous core formed by the nucleic acid/condensing agent complex on which a number of lipid layers (typically 1 -3) are adsorbed.
The preparation method is consolidated and consists of two successive steps: (i) the formation of the DNA/condensing agent nucleus; (ii) the subsequent covering of the DNA/condensing agent nucleus with a lipid shell. The DNA is condensed following the electrostatic interaction between the negative charges of the DNA and the positive charges of the cationic condensing agent. By varying the mixing ratio between the DNA and the condensing agent, it is possible to obtain DNA/condensing agent nuclei having different charges. This makes it possible to cover the DNA/condensing agent nucleus with lipid shells of positive charge (cationic liposomes) or negative charge (anionic liposomes) depending on the net charge of the aforementioned nucleus (negative or positive respectively).
In the case of anti-tumoral therapy, the active ingredient can be inserted indiscriminately within the aqueous core in the case of hydrophilic active ingredient (such as polyinosinic-polycytidylic (poly(l:C)) acid or within the hydrophobic lipid bilayer of the liposome if the anti-tumoral active ingredient is a hydrophobic molecule (such as doxorubicin, cisplatin, docetaxel, paclitaxel, etc.).
The net charge of the surface of the nanoparticles allows non-covalent functionalization by means of electrostatic interaction with ligands of opposite charge. In addition, in the lipid formulation of the nanoparticles, it is possible to introduce a percentage of functionalized lipid molecules, by means of covalent bond, with a polymeric chain of polyethylene glycol (PEG). The activity of the lipid nanoparticles is closely linked to the specific chemical and physical properties of the adopted formulation.
There are numerous publications in the field of lipid nanoparticles, such as the publication by the group of Dr. L. Zhang of the University of California in San Diego (UCSD).
In the publication "Programmed packaging of multicomponent envelope-type nanoparticle system for gene delivery" Daniela Pozzi, Carlotta Marianecci, Maria Carafa, Cristina Marchini, Maura Montani, Augusto Amici and Giulio Caracciolo. Appl. Phys. Lett. 96, 183702 (2010); doi: 10.1063/1 .3427354, it is demonstrated that a procedure of "programmed packaging" makes it possible to obtain lipid nanoparticles of controlled dimensions and charge. The lipids used are anionic lipids.
In the publication "Factors Determining the Superior Performance of Lipid/DNA/Protammine Nanoparticles over Lipoplexes" Giulio Caracciolo, Daniela Pozzi, Anna Laura Capriotti, Carlotta Marianecci, Maria Carafa, Cristina Marchini, Maura Montani, Augusto Amici, Heinz Amenitsch, Michelle A. Digman, Enrico Gratton, Susana S. Sanchez, and Aldo Lagana. J. Med. Chem. 54 (12), pp 4160- 4171 (201 1 ), nanoparticles formed of a single cationic lipid species (DOTAP/protamine/DNA) are described. In this same publication, the factors that determine greater efficiency of the lipid nanoparticles of DOTAP compared to DOTAP/DNA lipoplexes are discussed.
In this sector, which is subject to on-going development, but still liable to vast improvements, the main problem encountered by researchers lies in the individualization of a nanovector able to efficiently overcome the extra-cellular and intra-cellular barriers. From the perspective of commercialization and of a medical use that can be approved and standardized, a nanovector must have the following characteristics: (i) controlled dimensions and charge; (ii) improved residence time in the bloodstream; (iii) effective cellular internalization; (iii) cytoplasmic release of the complete genetic and/or therapeutic charge; (iv) effective entry into the nucleus; (v) increased transfection efficiency; (vi) increased cell vitality.
SUMMARY OF THE INVENTION
In order to solve the above-indicated technical problem, the inventors have analyzed numerous formulations of nanoparticles and have identified a specific formulation of nanoparticles that is more efficient than the reagents generally used for lipid-based transfection currently on the market. In fact, a multicomponent lipid formulation has been produced that incorporates the advantages of each lipid species.
This formulation has proven to be particularly versatile, efficient both for genetic transport (gene therapy) and for the transport of anti-tumoral drugs (anti- tumoral therapy). In fact, the present invention relates to a multicomponent lipid nanoparticle consisting of a hydrophilic nucleus and an outer monolayer shell consisting of 1 ,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 3β-[Ν-(Ν',Ν'- dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Choi) and dioleoylphosphatidylethanolamine (DOPE), furthermore at least one of cholesterol hydrochloride (DC-Choi), DOPE functionalized with PEG (DOPE-PEG), and dioleoylphosphocholine (DOPC), and optionally one or more lipophilic anti-tumoral active ingredients; a process for the preparation of multicomponent lipid nanoparticles for gene transport and/or for the transport of anti-tumoral drugs in which said anti-tumoral drugs can be hydrophilic (polyinosinic-polycytidylic (poly(l:C)) acid); or lipophilic (paclitaxel, docetaxel, doxorubicin).
Said process for the preparation of multicomponent lipid nanoparticles for gene transport and/or for the transport of anti-tumoral drugs comprises the following steps:
The process for the preparation of multicomponent lipid nanoparticles for the transport of hydrophobic anti-tumoral drugs comprises the following steps:
a. dissolving individually 1 ,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 3B-[N-(N\N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Chol) and dioleoylphosphatidylethanolamine (DOPE) and at least one of cholesterol (Choi), DOPE functionalized with PEG (DOPE-PEG), dioleoylphosphocholine (DOPC), and optionally a lipophilic anti-tumoral active ingredient in a suitable organic solvent and mixing desired quantities of each lipid and optionally the active ingredient thus dissolved so as to obtain a homogenous mixture;
b. completely removing the organic solvent from the mixture obtained in step a. thus obtaining a lipid film;
c. hydrating the lipid film obtained in step b. with a suitable buffer at physiological cell pH so as to obtain a desired final concentration;
d. sonicating the lipid solution obtained in step c. so as to obtain a clear solution (which indicates the formation of uni-lamellar vesicles, also defined here as cationic liposomes).
Alternatively, the lipid solution obtained in step c. is extruded by means of a commercial extruder with pore dimensions equal to 200 nm (Avanti Polar Lipidis, USA).
The process indicated above may comprise further steps as follows:
o. dissolving separately, at equal concentration in distilled H20, nucleic acid molecules or polyinosinic-polycytidylic acid (poly(l:C) acid) molecules and a pool of one or more nucleic acid condensing agents and then mixing the same together at a ratio between 1 :1 and 1 :0.5, thus obtaining a negatively charged complex (with light stirring)
p. allowing to equilibrate, for a period of between 30 and 180 minutes at room temperature, a desired volume of the complex obtained in step o;
q. interacting the balanced complex obtained in step o. with a suitable volume of the solution obtained in step d. at a charge ratio rho ( ) ( = moles of cationic lipid/nucleic acid bases) comprised between 2 and 3, including the extremes, and incubating at about 37 °C until formation of the nanoparticles.
The invention lastly relates to the above-described nanoparticles for use in gene therapy and/or anti-tumoral therapy, or a gene therapy and/or anti-tumoral therapy method comprising a step of administration of said nanoparticles.
The inventors have demonstrated (data reported in the experimental section) that, compared to the more widespread reagents for cellular transfection and for the transport of anti-tumoral drugs currently on the market (Lipofectamine, Lipofectamine 2000, Lipofectamine Plus), the multicomponent lipid nanoparticles of the present invention demonstrate numerous advantages in relation to the above- indicated problems. The main advantages are as follows:
(i) The nanoparticles have controlled and extremely monodisperse dimensions;
(ii) The surface charge of the nanoparticles is positive and extremely reproducible;
(iii) Cellular internalization is quick and extremely effective;
(iv) If charged with nucleic acids, the multicomponent lipid nanoparticles completely release the genetic charge in the cytoplasm of the cells;
(v) The released genetic material enters the nucleus effectively;
(vi) Transfection efficiency of the nanoparticles is greater than that of the reagents generally used in lipotransfection (such as Lipofectamine, Lipofectamine 2000, Lipofectamine Plus) ( 1010 RLU/mg protein) in various cell lines (NIH 3T3; HeLa; CHO; A17).
(vii) the anti-tumoral activity of the multicomponent lipid nanoparticles charged with the active ingredient (poly(l:C), paclitaxel, docetaxel), (quantified by means of measurements of cell vitality and expressed as a percentage compared to untreated cells) is greater than that of the more widespread reagents (Lipofectamine, Lipofectamine 2000, Lipofectamine Plus) in various tumoral cell lines (HeLa; A17).
(vii) In the absence of anti-tumoral drug, the nanoparticles are not toxic (cell vitality > 70%).
GLOSSARY
For the purposes of the present invention, lipid nanoparticles means particles formed by a hydrophilic nucleus coated by a single lipid outer shell (lipid shell), suitable for use in gene therapy and/or anti-tumoral therapy, in which the active ingredient of interest (nucleic acid and/or anti-tumoral acid) will be in the hydrophilic nucleus if hydrophilic or in the lipid shell if hydrophobic.
The term nucleic acid condensing agent means positively charged molecules generally known for their ability to condense nucleic acids, provided with a positive charge, this category containing various classes of substances known in the literature as described in the prior art.
For the purposes of the present invention, lipids means both lipids as generally defined in scientific dictionaries and derivatives thereof, such as derivatives of cholesterol and phospholipids.
Nucleic acids means: dsDNA (double strand DNA), ssDNA (single strand DNA), RNA, siRNA (small interfering RNA), shRNA (short hairpin RNA), mRNA, dsRNA (double strand RNA), tRNA, miRNA (microRNA) or a mixture thereof. Such nucleic acids can be single strand and/or double strand, in the form of vectors that can be integrated into the genome or that can be transposed, mini-chromosomes (or equipped with centromere and telomeres or more generally elements that allow autonomous replication), provided with an origin of replication, etc.
Gene transport means those methods which make it possible to transfer an external nucleic acid to the inside of a target cell so as to alter temporarily or permanently the phenotype thereof, such as transfection techniques which make it possible to express or silence specific genes in the transformed cells.
The term gene silencing is used in the present description in accordance with the prior art.
Gene therapy means the insertion of genetic material, as defined above (nucleic acids) inside cells for the purpose of being able to treat pathologies. This insertion procedure, known as transfection, makes it possible to transfer one or more healthy genes into a sick cell for the purpose of treating a pathology caused by the absence or by the defect of one or more (mutated) genes, or one or more nucleic acids able to silence genes that are overexpressed or expressed in the sick cell.
Anti-tumoral therapy means a treatment able to block the progression of tumoral cells and cause regression thereof, where possible.
Anti-tumoral drug means an active ingredient able to damage the tumoral cells by blocking proliferation thereof and/or promoting the death thereof. Various tumoral drugs are known (apoptotic drugs, immunostimulants, cytotoxic drugs, etc.). The effect of an apoptotic anti-tumoral drug administered to a cell culture in vitro is the death of the tumoral cells (apoptosis).
The term hormone means a molecule able to modify the conditions of the tissues in which neoplasm has developed.
The term immunostimulant means a class of anti-tumoral drugs which tend to develop the immune response.
The term cytotoxic drugs means molecules that intervene in the biosynthesis of ribonucleotides and deoxyribonucleotides in the processes of replication, transcription and translation of DNA and that intervene in the process of formation of the mitotic spindle.
The acronym DOTAP denotes the cationic lipid 1 ,2-dioleoyl-3- trimethylammonium-propane.
The acronym DC-Choi denotes the cationic lipid derived from the cholesterol 3B-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride.
The acronym Choi denotes cholesterol
The acronym DOPE denotes the phospholipid dioleoylphosphatidylethanolamine.
The acronym DOPC denotes the phospholipid dioleoylphosphocholine.
The acronym PEG denotes polyethylene glycol
DETAILED DESCRIPTION OF THE DRAWINGS
Figure 1 in 1 (A) shows the transfection efficiency of the fibroblasts NIH 3T3 of various nanoparticles according to the invention (the relative ratios between the cationic lipids 1 ,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 3β-[Ν-(Ν',Ν'- dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Choi), cholesterol (Choi) and the phospholipids dioleoylphosphocholine (DOPC), dioleoylphosphatidylethanolamine (DOPE) and DOPE functionalized with PEG (DOPE-PEG) are indicated beneath each bar) and of Lipofectamine, which is one of the more common reagents for cellular transfection. The efficiency of the multicomponent lipid nanoparticles is also compared with that of the more effective multicomponent lipoplexes (MC lipoplexes) prepared as described in (Caracciolo et al., Biochim Biophys Acta, 1768, 2007, 2280). The bars in grey relate to experiments carried out using 5 μg of sample per well (the term sample means the solution of cationic liposomes described under step d. of the method described above), the bars in black using 10 μg of sample per well. In 1 (B), the figure shows the transfection efficiency in CHO ovary cells of various nanoparticles according to the invention (the relative ratios between the cationic lipids 1 ,2-dioleoyl-3- trimethylammonium-propane (DOTAP), 3B-[N-(N',N'-dimethylaminoethane)- carbamoyl]cholesterol hydrochloride (DC-Choi), cholesterol (Choi) and the phospholipids dioleoylphosphocholine (DOPC), dioleoylphosphatidylethanolamine (DOPE) and DOPE functionalized with PEG (DOPE-PEG) are indicated beneath each bar). The efficiency is compared with that of Lipofectamine and with that of the more efficient multicomponent lipoplexes prepared as described in (Caracciolo et al., Biochim Biophys Acta, 1768, 2007, 2280). The bars in grey relate to experiments carried out using 5 μg of sample per well, the bars in black using 10 μg of sample per well.
Figure 2 shows the results of a cell vitality assay conducted on the CHO ovary cells as described in the experimental section (the relative ratios between the cationic lipids 1 ,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 3β-[Ν-(Ν',Ν'- dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Choi), cholesterol (Choi) and the phospholipids dioleoylphosphocholine (DOPC), dioleoylphosphatidylethanolamine (DOPE) and DOPE functionalized with PEG (DOPE-PEG) are indicated beneath each bar).
Figure 3 shows representative superimpositions of fluorescence images and Nomarski images in the CHI cells. The white signal is caused by the presence of fluorescent DNA. A scan along the x-axis was carried out for each image. As can be seen in the two representative images, the DNA is uniformly diffused in the cytoplasm and in the nucleus of the cell. With regard to that which can be observed with the more widespread lipid-based reagents for transfection, perinuclear aggregates with confined DNA are not visible.
Figure 4 shows the results of a cell vitality assay conducted on the A17 cells treated with multicomponent nanoparticles charged with paclitaxel as described in the experimental section (the relative ratios between the cationic lipids 1 ,2-dioleoyl- 3-trimethylammonium-propane (DOTAP), 3B-[N-(N',N'-dimethylaminoethane)- carbamoyl]cholesterol hydrochloride (DC-Choi), cholesterol (Choi) and the phospholipids dioleoylphosphocholine (DOPC), dioleoylphosphatidylethanolamine (DOPE) and DOPE functionalized with PEG (DOPE-PEG) are indicated beneath each bar).
Figure 5 shows the results of a cell vitality assay conducted on the A17 cells treated with multicomponent nanoparticles charged with docetaxel as described in the experimental section (the relative ratios between the cationic lipids 1 ,2-dioleoyl- 3-trimethylammonium-propane (DOTAP), 3B-[N-(N',N'-dimethylaminoethane)- carbamoyl]cholesterol hydrochloride (DC-Choi), cholesterol (Choi) and the phospholipids dioleoylphosphocholine (DOPC), dioleoylphosphatidylethanolamine (DOPE) and DOPE functionalized with PEG (DOPE-PEG) are indicated beneath each bar).
DETAILED DESCRIPTION OF THE INVENTION
The present invention therefore relates to multicomponent lipid nanoparticles consisting of a hydrophilic nucleus and a single lipid outer shell formed by 1 ,2- dioleoyl-3-trimethylammonium-propane (DOTAP), 3β-[Ν-(Ν',Ν'- dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Choi) and dioleoylphosphatidylethanolamine (DOPE), furthermore at least one of cholesterol (Choi), DOPE functionalized with PEG (DOPE-PEG), and dioleoylphosphocholine (DOPC), and optionally one or more lipophilic anti-tumoral active ingredients. In accordance with an embodiment of the invention, the hydrophilic nucleus may be formed by one or more nucleic acid molecules and by one or more nucleic acid condensing agents.
In the case of use of the nanoparticle in gene therapy, the hydrophilic nucleus will therefore be formed as indicated above and the nucleic acid molecules can be selected from dsDNA (double strand DNA), ssDNA (single strand DNA) or double-strand or single-strand DNA, RNA, siRNA (small interfering RNA), shRNA (short hairpin RNA), mRNA, dsRNA (double strand RNA), tRNA, miRNA (microRNA) or a mixture thereof.
The nucleic acid molecules can be inserted into suitable vectors which allow them to become incorporated in the host genome (for example transposable vectors) or which allow them to carry out replication and expression or transcription in the host cell (eukaryotic cell), such as suitable cloning vectors, expression vectors, mini- chromosomes, etc. The nucleic acids (also defined herein as "genetic material" in general), can be genes for substitutive gene therapy (that is to say gene therapy in which the disease is caused by a lack of expression of a specific gene or in which a non-functional gene is expressed) or can be sequences with the aim of interfering with the expression of one or more genes expressed and overexpressed in sick cells, such as in tumoral cells, and normally not expressed in healthy cells. The inserted genetic material may also consist of tRNA where the dysfunction is linked to the absence or to the malfunctioning of a particular tRNA.
Such nucleic acids are also defined in the present description as "sequences of interest" since, in the embodiment of the invention as taught here, it is irrelevant, for the purposes of obtaining a particle having the above-listed technical advantages, which sequences are inserted in the nucleic acid/nucleic acid condensing agent nucleus: any sequence can be inserted without changing the chemical and physical characteristics of the nanoparticles.
It is clear that in the preferred embodiment such sequences will have a recognized function in gene therapy.
A person skilled in the art will know "to charge" the nanoparticles of the invention with the nucleic acids suitable for the medical objective to be pursued without the need for further teachings in the present description, which provides effective "transport means" for such nucleic acids and detailed processes for producing the same.
The ratio between the volume of said one or more nucleic acid molecules and said nucleic acid condensing agents in the nanoparticles according to the invention can be comprised between about 1 :1 and about 1 :0.5, and in one embodiment such ratio will be about 1 :0.75.
To implement the present invention, one or more nucleic acid condensing agents generally known in the field can be used. For example, such agents may be selected from polyamines (for example spermine or spermidine), positively charged amino acids (for example arginine and lysine), positively charged proteins, and multivalent metal cations (for example Ca2+, Mg2+, Mn2+, Fe2+, Fe3+).
The above-indicated examples do not limit the implementation of the present invention, but are intended to provide the reader of the present invention with specific examples of suitable molecules among those known to a person skilled in the art. ln a specific embodiment, the nucleus of the nanoparticle will be produced using protamine as condensing agent.
In this embodiment or also in the case in which the nucleus contains poly(l:C) as described above, the nucleus will have a hydrodynamic diameter, D = 230 + 18 nm; and a surface charge = - 19.5 + 2.5 mV.
In accordance with another embodiment, the particle can be used for anti-tumoral therapy by means of hydrophilic active ingredients, and the nucleus will therefore be formed by hydrophilic anti-tumoral active ingredients and one or more suitable condensing agents. In this embodiment, the active ingredient can be polyinosinic- polycytidylic (poly(l:C)) acid together with one or more condensing agents, as described above for the nucleic acids.
With regard to the outer shell of the nanoparticle, this will always comprise 1 ,2- dioleoyl-3-trimethylammonium-propane (DOTAP), 3β-[Ν-(Ν',Ν'- dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Choi) and dioleoylphosphatidylethanolamine (DOPE), and furthermore at least one of cholesterol (Choi), DOPE functionalized with PEG (DOPE-PEG) and dioleoylphosphocholine (DOPC). As already mentioned, in the case in which it is desired to use the nanoparticle to transport lipophilic anti-tumoral drugs, such drugs will be charged directly in the lipid shell.
The above-listed lipids can be present in variable mutual ratios, in accordance with some embodiments 1 ,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 3β- [N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Choi), cholesterol (Choi), dioleoylphosphocholine (DOPC), dioleoylphosphatidylethanolamine (DOPE) and DOPE functionalized with PEG (DOPE-PEG) will be present in a mutual ratio selected from: a) 0.5:1 .5:0.5:0:1 .5:0 or b) 1 :1 :0:1 :1 :0 or c) 0.5:1 .5:0:0.5:1 .5:0 or d) 0.5:1 .5:0.5:0:1 .2:0.3 or e) 1 :1 :0:1 :0.8:0.2 or f) 0.5:1 .5:0:0.5:1 .2:0.3.
Therefore, in case a, the mutual ratios will be
a) 0.5:1 .5:0.5:0:1 .5:0
0.5 DOTAP: 1 .5 DC-Choi: 0.5 Chol:0 DOPC: 1 DOPE; 0 DOPE-PEG; in case b, the mutual ratios will be
b) 1 :1 :0:1 :1 :0
1 DOTAP: 1 DC-Choi: 0 Choi: 1 DOPC: 1 DOPE; 0 DOPE-PEG
in case c, the mutual ratios will be
c) 0.5:1 .5:0:0.5:1 .5:0
0.5 DOTAP: 1 .5 DC-Choi: 0 Chol:0.5 DOPC: 1 .5 DOPE; 0 DOPE-PEG
in case d, the mutual ratios will be d) 0.5:1 .5:0.5:0:1 .2:0.3
0.5 DOTAP: 1 .5 DC-Choi: 0.5 Chol:0 DOPC: 1 .2 DOPE; 0.3 DOPE-PEG
in case e, the mutual ratios will be
e) 1 :1 :0:1 :0.8:0.2
1 DOTAP: 1 DC-Choi: 0 Chol:1 DOPC: 0,8 DOPE; 0.2 DOPE-PEG
in case f, the mutual ratios will be
f) 0.5:1 .5:0:0.5:1 .2:0.3
0.5 DOTAP: 1 .5 DC-Choi: 0 Chol:0.5 DOPC: 1 .2 DOPE; 0.3 DOPE-PEG.
Therefore, in all cases, 1 ,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 3β- [N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Choi) and dioleoylphosphatidylethanolamine (DOPE) will be present in a mutual ratio always comprised between 0.5 and 1 .5 as well as at least one of the other components.
In the embodiment of the present invention, given the selection of the coating lipids, the surface charge of the nanoparticle is positive, for example in the embodiment in which the mutual ratio between the components 1 ,2-dioleoyl-3- trimethylammonium-propane (DOTAP), 3B-[N-(N',N'-dimethylaminoethane)- carbamoyl]cholesterol hydrochloride (DC-Choi), cholesterol (Choi), dioleoylphosphocholine (DOPC), dioleoylphosphatidylethanolamine (DOPE) and DOPE functionalized with PEG (DOPE-PEG) is 0.5:1 .5:0:0.5:1 .5:0 (c), the surface charge of the nanoparticle will have a potential z comprised between about 40 and 45 mV, a non-limiting example being represented by a potential z of about + 42mV. The lipid nanoparticle produced as described above will have a hydrodynamic diameter comprised between 200 and 250nm and may have a polydispersity index <0.2.
In the embodiment in which the mutual ratio between the components 1 ,2-dioleoyl- 3-trimethylammonium-propane (DOTAP), 3B-[N-(N',N'-dimethylaminoethane)- carbamoyl]cholesterol hydrochloride (DC-Choi), cholesterol (Choi), dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphocholine (DOPC) is 0.5:1 .5:0:0.5:1 .5:0 (c), the diameter D of the particle will be about 210 ± 12 nm and the polydispersity index will be <0.2. The above-mentioned nanoparticles will have the following characteristics:
(i) The nanoparticles have controlled and extremely monodisperse dimensions (polydispersity index < 0.2)
(ii) They have a positive and extremely reproducible surface charge;
(iii) Cellular internalization is quick and extremely effective;
(iv) The cytoplasmic release of the genetic charge is complete;
(v) The genetic material released enters the nucleus effectively; (vi) The transfection efficiency of the nanoparticles is greater than that of the reagents generally used for lipotransfection, such as lipofectamines ( 1010 RLU/mg protein) in various cell lines (NIH 3T3; HeLa; CHO; A17).
(vii) The nanoparticles are not toxic (cell vitality > 80%).
The process for the preparation of multicomponent lipid nanoparticles according to the present invention comprises the following steps:
a. dissolving individually 1 ,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 3B-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Chol) and dioleoylphosphatidylethanolamine (DOPE) and at least one of cholesterol (Choi), DOPE functionalized with PEG (DOPE-PEG), dioleoylphosphocholine (DOPC), and optionally a lipophilic anti-tumoral active ingredient in a suitable organic solvent and mixing desired quantities of each lipid and one or more lipophilic anti-tumoral active ingredients thus dissolved so as to obtain a homogenous mixture;
b. completely removing the organic solvent from the mixture obtained in step a. thus obtaining a lipid film;
c. hydrating the lipid film obtained in step b. with a suitable buffer at physiological cell pH so as to obtain a desired final concentration;
d. sonicating the lipid solution obtained in step c. so as to obtain a clear solution (which indicates the formation of uni-lamellar vesicles, also defined here as cationic liposomes).
In this case therefore, the nanoparticles obtained will make it possible to transport anti-tumoral active ingredients of the lipophilic type. Anti-tumoral active ingredients can be, for example, paclitaxel, docetaxel, doxorubicin, and derivatives thereof.
The process described above may comprise further steps as follows:
e. the active ingredient not entrapped in said nanoparticles is removed by means of centrifugation (for example centrifugation at about 1000 rpm for 10 minutes);
f. the supernatant containing said nanoparticles with the active ingredient encapsulated is centrifuged (50,000 rpm for 30 minutes) to precipitate said nanoparticles;
g. the supernatant is discarded and the pellet of said nanoparticles is washed twice with saline buffer (PBS, pH 7.4).
h. the pellet is suspended in distilled H20 containing sucrose (molar ratio sugar:lipid = 2.5).
In a further embodiment, the process may optionally also comprise the following steps:
o. dissolving separately, at equal concentration in distilled H20, nucleic acid molecules or polyinosinic-polycytidylic acid (poly(l:C)) acid molecules and a pool of one or more nucleic acid condensing agents and then mixing the same together at a ratio between 1 :1 and 1 :0.5, thus obtaining a negatively charged complex (with light stirring)
p. allowing to equilibrate, for a period of between 30 and 180 minutes at room temperature, a desired volume of the complex obtained in step a;
q. interacting the balanced complex obtained in step o. with a suitable volume of the solution obtained in step d. at a charge ratio rho ( ) ( = moles of cationic lipid/nucleic acid bases) comprised between 2 and 3, including the extremes, and incubating at about 37 °C until formation of the nanoparticles.
Lastly, again in a further embodiment, the application relates to a process for the preparation of multicomponent lipid nanoparticles according to the present invention comprising the following steps:
a. dissolving individually 1 ,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 3B-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Choi) and dioleoylphosphatidylethanolamine (DOPE) and at least one of cholesterol (Choi), DOPE functionalized with PEG (DOPE-PEG), dioleoylphosphocholine (DOPC) and mixing desired quantities of each lipid thus dissolved so as to obtain a homogenous mixture;
b. completely removing the organic solvent from the mixture obtained in step a. thus obtaining a lipid film;
c. hydrating the lipid film obtained in step b. with a suitable buffer at physiological cell pH so as to obtain a desired final concentration;
d. sonicating the lipid solution obtained in step c. so as to obtain a clear solution;
o. dissolving separately, at equal concentration in distilled H20, nucleic acid molecules or polyinosinic-polycytidylic acid (poly(l:C)) acid molecules and a pool of one or more condensing agents and then mixing the same together at a ratio between 1 :1 and 1 :0.5, thus obtaining a negatively charged complex (with light stirring)
p. allowing to equilibrate, for a period of between 30 and 180 minutes at room temperature, a desired volume of the complex obtained in step o;
q. interacting the balanced complex obtained in step o. with a suitable volume of the solution obtained in step d. at a charge ratio rho ( ) ( = moles of cationic lipid/nucleic acid bases) comprised between 2 and 3, including the extremes, and incubating at about 37 °C until formation of the nanoparticles.
Alternatively, the lipid solution obtained in step c. is extruded by means of a commercial extruder with pore dimensions equal to 200 nm (Avanti Polar Lipidis, USA).
In this case, step d. will be substituted by the following step:
d'. the lipid solution obtained in step c. is extruded by means of a commercial extruder with pore dimensions equal to 200 nm.
The nanoparticles obtained by the processes described above can be stored in a refrigerator at 4°C.
In one embodiment, the nanoparticles can be used to transport lipophilic anti- tumoral drugs, and this occurs when said drugs are inserted in step a. Paclitaxel, docetaxel, doxorubicin and derivatives thereof can be used as lipophilic anti-tumoral active ingredients according to the invention.
In the case in which said active ingredients are inserted in the outer shell of the nanoparticles of the invention, they will be inserted as indicated insteps a., b. and c. of the process in a molar ratio of moles of active ingredient/moles of total lipid comprised between 1 and 4.
The nanoparticles according to the invention may comprise, in the hydrophilic nucleus, nucleic acids for gene therapy or hydrophilic anti-tumoral drugs. In this case, the outer shell can also be charged with lipophilic anti-tumoral drugs, but also may not comprise such drugs.
According to the present invention therefore, if the hydrophilic nucleus comprises nucleic acids of drugs, the outer shell may comprise lipophilic anti-tumoral drugs (active ingredients) or may be formed solely by 1 ,2-dioleoyl-3-trimethylammonium- propane (DOTAP), 3B-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Choi) and dioleoylphosphatidylethanolamine (DOPE) and at least one of cholesterol (Choi), DOPE functionalized with PEF (DOPE-PEG), and dioleoylphosphocholine (DOPC).
In the process described here, the concentration of the solution of nucleic acids and of the solution containing the nucleic acid condensing agent or agents may have a concentration selected according to the user requirements, such concentration, which will be equal for the two different solutions, possibly being between 0.5 and 1 .5 mg/ml for example, that is to say about 0.5; about 0.6; about 0.7; about 0.8; about 0.9; about 1 ; about 1 .1 ; about 1 .2; about 1 .3; about 1 .4; about 1 .5 mg/ml by way of example, although the present invention does not rule out the fact that a person skilled in the art may select different concentrations.
The ratio of volume between the solution of nucleic acids and the solution of nucleic acid condensing agent in point a. will be, as described, in a range between 1 :1 and 0.5:1 . By way of non-limiting example, this ratio may be, as indicated above (volume of the solution of nucleic acids/volume of the solution of nucleic acid condensing agent), about 0.5:1 : 0.55:1 ; 0.6:1 ; 0.65:1 ; 0.7:1 ; 0.75:1 ; 0.8:1 ; 0.85:1 ; 0.9:1 ; 0.95:1 ; 1 :1 .
The nucleic acid molecules that can be used for the preparation of the nucleus of the nanoparticles of the invention are those indicated previously in the part relating to the description of the nanoparticles themselves and can therefore be selected from DNA, RNA, siRNA, shRNA, mRNA; miRNA; dsRNA.
In the process described above, the nucleic acid condensing agents are selected from polyamines, cationic peptides, cationic lipids, cationic surfactants, positively charged amino acids, positively charged proteins, and polyvalent metal cations. Non-limiting examples of suitable molecules are provided here in the part relating to the description of the nanoparticles themselves and may therefore be as follows: for the polyamines, selected from spermine and spermidine; for the positively charged amino acids, selected from arginine and lysine; for the positively charged proteins, selected from protamine and isotones; for the polyvalent metal cations, selected from Ca2+, Mg2+, Mn2+, Fe2+, Fe3+.
In a specific embodiment, protamine is selected as nucleic acid condensing agent. In the process described above, a desired volume of the complex obtained in a. (which corresponds to the "nucleus" of the nanoparticles) is allowed to equilibrate for a period comprised between about 30 and 180 minutes, this period possibly being, for example, about 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150, 160, 170 or 180 minutes. In general, a period comprised between about 30 and 60 minutes, including the extremes, is also sufficient.
The nuclei thus obtained can be stored for a period from a few hours to about 24 hours at about +4°C.
In the case in which nanoparticles with a nucleus comprising nucleic acids are produced, since the nucleus obtained in p. containing nucleic acids is more delicate compared to the uni-lamellar vesicles obtained in step q., which can be stored in a refrigerator (at about +4°C) for about one week or two, it is advisable to prepare the vesicles first, or to prepare the nucleus and the vesicles at the same time so as to use the nucleus obtained in p. as early as possible and to avoid possible degradation thereof so as to obtain multicomponent nanoparticles having the greatest efficacy.
In the process described, for the preparation of the vesicles to be used in order to produce the coating of the nucleus (shell), the above-indicated lipids (cationic and phospholipids) will be used in the desired mutual ratios. It has been demonstrated that the various assayed ratios are all effective for producing vesicles having good transfection properties and low toxicity.
In particular, in step a., the cationic lipids 1 ,2-dioleoyl-3-trimethylammonium- propane (DOTAP), 3B-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Choi), cholesterol (chol) and the phospholipids dioleoylphosphocholine (DOPC), dioleoylphosphatidylethanolamine (DOPE) and DOPE-PEG can be mixed in a mutual ratio selected from: a) 0.5:1 .5:0.5:0:1 .5:0 or b) 1 :1 :0:1 :1 :0 or c) 0.5:1 .5:0:0.5:1 .5:0 or d) 0.5:1 .5:0.5:0:1 .2:0.3 or e) 1 :1 :0:1 :0.8:0.2 or f) 0.5:1 .5:0:0.5:1 .2:0.3.
That is to say
a) 0.5:1 .5:0.5:0:1 .5:0
0.5 DOTAP: 1 .5 DC-Choi: 0.5 Chol:0 DOPC: 1 DOPE; 0 DOPE-PEG; in case b, b) 1 :1 :0:1 :1 :0
1 DOTAP: 1 DC-Choi: 0 Choi: 1 DOPC: 1 DOPE; 0 DOPE-PEG
c) 0.5:1 .5:0:0.5:1 .5:0
0.5 DOTAP: 1 .5 DC-Choi: 0 Chol:0.5 DOPC: 1 .5 DOPE; 0 DOPE-PEG
d) 0.5:1 .5:0.5:0:1 .2:0.3
0.5 DOTAP: 1 .5 DC-Choi: 0.5 Chol:0 DOPC: 1 .2 DOPE; 0.3 DOPE-PEG
e) 1 :1 :0:1 :0.8:0.2
1 DOTAP: 1 DC-Choi: 0 Chol:1 DOPC: 0.8 DOPE; 0.2 DOPE-PEG
f) 0.5:1 .5:0:0.5:1 .2:0.3
0.5 DOTAP: 1 .5 DC-Choi: 0 Chol:0.5 DOPC: 1 .2 DOPE; 0.3 DOPE-PEG.
In all cases therefore, 1 ,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 3β-[Ν- (N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Choi) and dioleoylphosphatidylethanolamine (DOPE) will be present in a mutual ratio always comprised between 0.5 and 1 .5 as well as at least one of the other components.
In a particularly interesting embodiment, the above-mentioned lipids can be mixed, and therefore present in the coating of the nucleic acid/condensing agent nucleus of the nanoparticles described here, in the mutual ratio (in which the order of the lipids is that indicated in the previous paragraph) of 0.5:1 .5:0.5:0:1 .5:0.
Each lipid will be dissolved in a suitable organic solvent and then mixed in the desired ratios or in the ratios indicated above.
Any suitable organic solvent can be used, for example suitable organic solvents generally used by a person skilled in the art may be chloroform or ethanol.
The reagents will be mixed so as to obtain a homogenous solution, at ambient temperature for a period comprised between 1 and 5 minutes, for example about 1 - 2 minutes.
In an embodiment of the invention, the multicomponent cationic liposomes formed by the four lipid species obtained in step d. can be prepared with a molar fraction of neutral lipid in the bilayer = (neutral lipid/total lipid) (mol/mol) = between 0.4 and 0.6, for example 0.5. The lipids dissolve in the suitable solvent, for example chloroform, and are mixed so as to obtain a homogenous mixture of the four lipid species (in organic solvent the mixture of lipid molecules is a homogenous mixture). The amount of solvent to be used depends on the solubility of the lipid species used, for example, the solutions can be prepared with about 10/20 mg of lipid/ml of chloroform.
The organic solvent is then removed in step b., which can be achieved using any suitable means known to a person skilled in the art, such as by using a conventional rotary evaporator. This process leads to the formation of a thin lipid film at the base of the container used. The removal of the organic solvent must be complete; in order to ensure the complete removal of the organic solvent, the lipid film can also be kept under vacuum for 24 hours, for example.
The lipid film in step b. is then hydrated with a suitable buffer at physiological cell pH so as to obtain a final desired concentration.
Any buffer generally suitable for use with vital cells which stabilizes the pH at physiological levels, such as the buffer Tris-HCI at physiological pH, can be used.
For example, Tris-HCI (10 mM, pH 7.4) necessary to obtain a final concentration of 1 mg/ml, can be used.
At this point, the lipid solution obtained in step c. will be sonicated so as to obtain a clear solution, the clarity of the solution indicating the formation of lamellar vesicles which are formed by the above-mentioned lipids in the ratios selected by the user and which will be used to coat the nucleus formed in step p.
Any suitable sonicator conventionally used by a person skilled in the art can be used, such as a titanium-tip sonicator, for a period of about 10 minutes. A person skilled in the art will choose the most effective sequence of on-off cycles with no need for further teachings. By way of non-limiting example, a sequence of cycles 8 s on - 6 s off can be used with t= 10 min.
Alternatively, the lipid solution obtained in step c. will be extruded by means of a commercial extruder with pore dimensions equal to 200 nm so as to obtain a clear solution (number of steps in the extruder >20), the clarity of the solution indicating the formation of lamellar vesicles which are formed by the above-mentioned lipids in the ratios selected by the user and which will be used to coat the nucleus formed in step p. The lipophilic anti-tumoral active ingredients as indicated above will be charged in the lipid shell of the nanoparticle in a molar ratio (moles of active ingredient/moles of total lipid) between 1 and 4.
The nucleus formed in step p can be constituted similarly by nucleic acid molecules or polyinosinic-polycytidylic (poly(l:C)) acid molecules and a pool of one or more nucleic acid condensing agents which are dissolved separately, at equal concentration in distilled H20, and are then mixed together at a ratio of between 1 :1 and 1 :0.5, thus obtaining a negatively charged complex (with light stirring).
The vesicles thus prepared can be stored at approximately +4°C for a period up to 1 or 2 weeks, and can then be used directly for step d. of the process for preparing the nanoparticles of the invention.
In this step, a suitable volume of said vesicles is mixed with a suitable volume of the balanced complex obtained in step p.
In an exemplary embodiment, obviously not limiting the invention, about 5 microliters of vesicles can be mixed with 2 microliters of condensing agent/nucleic acid at a charge ratio (moles of cationic lipid/nucleic acid bases ) comprised between about 2 and 3, including the extremes. This ratio, in a non-limiting exemplary embodiment, can be about 2.5, for example.
The charge ratio can be calculated as follows (the example relates to an embodiment in which the nucleic acid is DNA):
the number of moles of cationic lipid is given by the following calculation:
cationic lipid mass/molecular weight of the cationic lipid;
the number of nucleotides is given by the following calculation:
mass of DNA/324.5 (molecular weight of a nucleotide which contains a negative charge).
From this, rho=number of moles of cationic lipid/number of nucleotides (or bases). Incubation can be implemented for a period between about 30 minutes and 3 hours, for example a period of about 30, 40, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150, 160, 170 or 180 minutes.
In a very effective embodiment, multicomponent nanoparticles are prepared in which the nucleus is formed by nucleic acid or poly(l:C) acid and protamine in a ratio by volume of nucleic acid or poly(l:C) acid/protamine equal to about 1 :0.5m starting from initial solutions having a concentration c=1 mg/ml. The nucleus has dimensions with diameter D=230±18 nm and surface charge =-19.5 ± 2.5 mV.
The solutions of DOTAP, DC-Choi, Choi, DOPC, DOPE and DOPE-PEG are prepared in chloroform at a molar fraction of neutral lipid in the bilayer = (neutral lipid/total lipid) (mol/mol) equal to 0.5 and the mutual ratio at which they are mixed in step a. (in the above-indicated order) is selected from:
DOTAP-DC-Chol-Chol-DOPC-DOPE-DOPE/PEG (0.5:1 .5:0.5:0:1 .5:0) DOTAP-DC-Chol-Chol-DOPC-DOPE-DOPE/PEG (1 :1 :0:1 :1 :0)
DOTAP-DC-Chol-Chol-DOPC-DOPE-DOPE/PEG (0.5:1 .5:0:0.5:1 .5:0).
· DOTAP-DC-Chol-Chol-DOPC-DOPE-DOPE/PEG (0.5:1 .5:0.5:0:1 .2:0.3) DOTAP-DC-Chol-Chol-DOPC-DOPE-DOPE/PEG (1 :1 :0:1 :0.9:0,1 )
DOTAP-DC-Chol-Chol-DOPC-DOPE-DOPE/PEG (0.5:1 .5:0:0.5:1 .2:0.3). The solution containing the nucleus and that containing the multicomponent vesicles obtained as described above are mixed in such a way that the charge ratio (moles of cationic lipid/bases of nucleic acid or poly(l:C) acid) is equal to about 2.5. The nanoparticles obtained have the composition in lipids in the coating with mutual ratios equal to those used in mixing step a.
The invention clearly also relates to nanoparticles defined as "nanoparticles obtainable by means of the process of the invention as defined in the description and in the claims relating to said process".
As already indicated, the present invention relates to nanoparticles as described here for use in gene therapy and in anti-tumoral therapy.
In other words, the invention relates to a method of gene therapy and/or anti- tumoral therapy which comprises the step of administering the nanoparticles of the invention to a patient having a need therefor.
It is clear that the nanoparticles will be formed using appropriate nucleic acids selected in accordance with the desired gene therapy or using appropriate anti- tumoral drugs in accordance with the desired anti-tumoral therapy.
Without being bound to specific theories, the inventors of the present invention have formulated the following hypotheses.
The electrostatic interactions are not sufficient to describe the formation and thermodynamic stability of the DNA or poly(l:C)/condensing agent nucleus, and it is necessary to refer to the release mechanism of the counterions. Condensation in accordance with the Poisson-Boltzmann equation requires the charged macromolecules to be surrounded in solution by a diffuse layer of counterions. Beneath formation of the complex, the macromolecules release their own counterions in solution with a significant entropy gain of the system (about 1 KT for each released counterion). The same mechanism of action is able to justify the formation of the nanoparticle. The more significant aspect is the extraordinary capability exhibited by the multicomponent lipid nanoparticles to release DNA or poly(l:C) or lipophilic drugs in the cytoplasm and in the cell nucleus (see figure 3). This property makes the multicomponent lipid nanoparticles unique and is the most likely explanation of their increased efficiency (figure 1 ) and also of their ability to induce apoptosis in A17 tumoral cells (figure 4 and figure 5). This intrinsic ability to exit from the endosomes and release DNA and/or the anti-tumoral drug is likely caused, among other factors, by the number, type and ratio of the lipid species involved that are able to maximize the entropy of lipid mixing, making the mixing with the lipid species of the endosomal membranes favored in terms of energy (Caracciolo et al. The Journal of Physical Chemistry B 1 10, 20829-20835 (2006)). This mixing appears to be necessary for the formation of the structural intermediates that lead to the fusion of the lipid bilayer of the nanoparticles and that of the membranes of the endosomes. Moreover, the presence of a single lipid shell compared to the multiple layers formed in the lipoplexes of said composition is compatible with easier disassembly of the nanoparticles compared to multi-lamellar lipoplexes.
In accordance with the present invention, in the case of use of the nanoparticle in anti-tumoral therapy, the anti-tumoral drugs can be selected from poly(l:C), active ingredients such as paclitaxel, docetaxel, doxorubicin or derivatives thereof, or from a mixture thereof. A person skilled in the art will know "to charge" the nanoparticles of the invention with the above-mentioned hydrophobic anti- tumoral drugs suitable for the medical objective to be pursued (or with a mixture thereof) without the need for further teachings in the present description, which provides effective "transport means" for such nucleic acids and detailed processes for producing the same.
The aim of the following examples is to illustrate the invention, without limiting the content thereof however, so as to provide a person skilled in the art with specific examples of embodiments of the invention.
EXAMPLES and EXPERIMENTAL PART
The transfection experiments reported below were carried out on commercial cell lines and
NIH 3T3 ATCC number CRL-1658
HeLa ATCC number CCL-2
CHO (ATCC number CCL-61 )
on murine cells referred to here as A17 isolated by the inventors.
1. PROCESS FOR PREPARING LIPID NANOPARTICLES CONTAINING
NUCLEIC ACID IN THE HYDROPHILIC NUCLEUS
The nanoparticles are formed by a DNA/protamine nucleus obtained by mixing two solutions of DNA and protamine in a ratio by volume of DNA/protamine of 1 :0.75 (the starting solutions of DNA and protamine have the same concentration, c=1 mg/ml). The DNA/protamine shell is left to equilibrate for one hour at ambient temperature. At the end of this process, the DNA/protamine shell is negatively charged (zeta potential = -19.5 ± 2.5 mV) and has characteristic dimensions equal to about 230 nm (hydrodynamic diameter, D = 230 ± 18 nm). A volume of DNA/protamine complex thus prepared is interacted for 20 minutes with a solution of cationic liposomes formed by a mixture of the commercial cationic lipid DOTAP, the cationic derivative of cholesterol DC-Choi and by two neutral phospholipids, such as DOPE and DOPC. The four above-mentioned molecular species are mixed in a ratio of 0.5:1 .5:0.5:1 .5 (c). After 2 hours of incubation, the formation of the nanoparticles is complete. The surface charge is positive with the DNA/protamine nucleus covered by a lipid shell formed by one or more lipid bilayers. The lipid formulation used in the present invention has never been used before for the production of nanoparticles having a DNA/protamine nucleus. The same process has been used to produce the nanoparticles described as a, b, d, e and f.
2. DETAILED PROCESS FOR PREPARING THE NANOPARTICLES USED IN THE EXPERIMENTS REPORTED IN THE FIGURES
The cationic lipids 1 ,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 3β-[Ν- (N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Choi) and the phospholipids dioleoylphosphocholine (DOPC), dioleoylphosphatidylethanolamine (DOPE) and DOPE-PEG were acquired from Avanti Polar Lipids (Alabaster, AL) and used without further purification. The cholesterol (Choi) was acquired from Sigma Aldrich. The fluorescent neutral lipids DOPC-NBD and DOPE-NBD acquired from Avanti Polar Lipids were used for the fluorescence microscopy measurements. The multicomponent cationic liposomes were prepared with a molar fraction of neutral lipid in the bilayer = (neutral lipid/total lipid) (mol/mol) = 0.5. The lipids were dissolved in chloroform and mixed so as to obtain a homogenous mixture of the lipid species. The amount of chloroform to be used depends on the solubility of the lipid species used; generally the solutions were prepared with about 10/20 mg of lipid/ml of chloroform. The organic solvent was removed by using a rotary evaporator, which led to the formation of a thin lipid film at the base of an ampoule. To ensure complete removal of the organic solvent, the lipid film was kept under vacuum for 24 hours. The lipid film was then hydrated by adding the volume of Tris-HCI buffer (10 mM, pH 7.4) necessary to obtain a final concentration of 1 mg/ml. To obtain uni-lamellar vesicles, the lipid solutions were sonicated using a titanium-tip sonicator with a sequence of cycles (8 s on - 6 s off) for t=10 min. The lipid solutions used were prepared by varying the ratio between the cationic lipid species. a) 0.5:1 .5:0.5:0:1 .5:0
0.5 DOTAP: 1 .5 DC-Choi: 0.5 Chol:0 DOPC: 1 DOPE; 0 DOPE-PEG; in case b, b) 1 :1 :0:1 :1 :0
1 DOTAP: 1 DC-Choi: 0 Choi: 1 DOPC: 1 DOPE; 0 DOPE-PEG
c) 0.5:1 .5:0:0.5:1 .5:0
0.5 DOTAP: 1 .5 DC-Choi: 0 Chol:0.5 DOPC: 1 .5 DOPE; 0 DOPE-PEG
d) 0.5:1 .5:0.5:0:1 .2:0.3
0.5 DOTAP: 1 .5 DC-Choi: 0.5 Chol:0 DOPC: 1 .2 DOPE; 0.3 DOPE-PEG
e) 1 :1 :0:1 :0.8:0.2
1 DOTAP: 1 DC-Choi: 0 Chol:1 DOPC: 0.8 DOPE; 0.2 DOPE-PEG
f) 0.5:1 .5:0:0.5:1 .2:0.3
0.5 DOTAP: 1 .5 DC-Choi: 0 Chol:0.5 DOPC: 1 .2 DOPE; 0.3 DOPE-PEG.
Double-strand calf thymus DNA was acquired from Sigma (St. Louis, MO) and used without subsequent purification. It was dissolved in distilled water at a concentration of 1 mg/ml and sonicated using a tip sonicator (t=5 min., 8 s on - 6 s off) to obtain a solution homogenous in length (DNA length from 500 to 1000 base pairs). The linear DNA was used for all the nanoparticle chemical/physical characterization experiments.
Plasmid DNA (pGL3 which codes for luciferases), acquired from Promega (Madison, Wl) and dissolved in bi-distilled water (Carlo Erba Reagenti, Milano, Italia) at a concentration of 1 mg/ml, was used for the transfection experiments. Plasmid DNA 2.7-kbp labeled with Cy3 (Mirus Bio Corporation, Madison, Wl) was used for the fluorescence microscopy experiments.
Salmon protamine sulphate (MW = 5.1 kDa), acquired from Sigma, was dissolved in distilled water at a concentration of 1 mg/ml. The nucleus of the nanoparticles was obtained by mixing the DNA (linear, plasmid and fluorescent) and the solution of protamine at a ratio by volume of 1 :0.75 so as to obtain a negatively charged complex. A desired volume of DNA/protamine was allowed to equilibrate for one hour at ambient temperature and was then interacted with an appropriate volume of the solution of cationic liposomes at a charge ratio (moles of cationic lipid/DNA bases) = 2.5. After 2 hours of incubation, the formation of the nanoparticles was complete.
3. PREPARATION OF NANOPARTICLES CHARGED WITH LIPOPHILIC ANTI- TUMORAL DRUGS
The lipophilic drugs (docetaxel, paclitaxel) were acquired from Sigma Aldrich. In the case of preparation of nanoparticles with lipophilic drugs charged in the shell, the lipids were dissolved in chloroform and mixed so as to obtain a homogenous mixture. The amount of chloroform to be used is dependent on the solubility of the lipid species used; generally the solutions were prepared at about 10/20 mg of lipid/ml of chloroform and 0.3/0.7 mg of drug/ml of chloroform. The organic solvent was removed using a rotary evaporator, which led to the formation of a thin lipid film at the base of an ampoule. To ensure complete removal of the organic solvent, the lipid film was kept under vacuum for 24 hours. The lipid film was then hydrated by adding the volume of Tris-HCI buffer (10 mM, pH 7.4) necessary to obtain a final concentration of 1 mg/ml. To obtain uni-lamellar vesicles, the lipid solutions were sonicated using a titanium-tip sonicator with a sequence of cycles (8 s on - 6 s off) for t=10 min. The lipid solutions used were prepared by varying the ratio between the cationic lipid species:
a) 0.5:1 .5:0.5:0:1 .5:0
0.5 DOTAP: 1 .5 DC-Choi: 0.5 Chol:0 DOPC: 1 DOPE; 0 DOPE-PEG; in case b, b) 1 :1 :0:1 :1 :0
1 DOTAP: 1 DC-Choi: 0 Choi: 1 DOPC: 1 DOPE; 0 DOPE-PEG
c) 0.5:1 .5:0:0.5:1 .5:0
0.5 DOTAP: 1 .5 DC-Choi: 0 Chol:0.5 DOPC: 1 .5 DOPE; 0 DOPE-PEG
d) 0.5:1 .5:0.5:0:1 .2:0.3
0.5 DOTAP: 1 .5 DC-Choi: 0.5 Chol:0 DOPC: 1 .2 DOPE; 0.3 DOPE-PEG
e) 1 :1 :0:1 :0.8:0.2
1 DOTAP: 1 DC-Choi: 0 Chol:1 DOPC: 0.8 DOPE; 0.2 DOPE-PEG
f) 0.5:1 .5:0:0.5:1 .2:0.3
0.5 DOTAP: 1 .5 DC-Choi: 0 Chol:0.5 DOPC: 1 .2 DOPE; 0.3 DOPE-PEG.
4. PROCESS FOR PREPARAING LIPID NANOPARTICLES CONTAINING POLYINOSINIC-POLYCYTIDYLIC ACID IN THE HYDROPHILIC NUCLEUS
The lipids were dissolved in chloroform and mixed so as to obtain a homogenous mixture of the lipid species. The amount of chloroform to be used is dependent on the solubility of the lipid species used; generally the solutions were prepared at about 10/20 mg of lipid/ml of chloroform. The organic solvent was removed using a rotary evaporator, which led to the formation of a thin lipid film at the base of an ampoule. To ensure complete removal of the organic solvent, the lipid film was kept under vacuum for 24 hours. The lipid film was then hydrated by adding the volume of Tris-HCI buffer (10 mM, pH 7.4) necessary to obtain a final concentration of 1 mg/ml. To obtain uni-lamellar vesicles, the lipid solutions were sonicated using a titanium-tip sonicator with a sequence of cycles (8 s on - 6 s off) for t=10 min. The lipid solutions used were prepared by varying the ratio between the cationic lipid species: a) 0.5:1 .5:0.5:0:1 .5:0
0.5 DOTAP: 1 .5 DC-Choi: 0.5 Chol:0 DOPC: 1 DOPE; 0 DOPE-PEG; in case b, b) 1 :1 :0:1 :1 :0
1 DOTAP: 1 DC-Choi: 0 Choi: 1 DOPC: 1 DOPE; 0 DOPE-PEG
c) 0.5:1 .5:0:0.5:1 .5:0
0.5 DOTAP: 1 .5 DC-Choi: 0 Chol:0.5 DOPC: 1 .5 DOPE; 0 DOPE-PEG
d) 0.5:1 .5:0.5:0:1 .2:0.3
0.5 DOTAP: 1 .5 DC-Choi: 0.5 Chol:0 DOPC: 1 .2 DOPE; 0.3 DOPE-PEG
e) 1 :1 :0:1 :0.8:0.2
1 DOTAP: 1 DC-Choi: 0 Chol:1 DOPC: 0.8 DOPE; 0.2 DOPE-PEG
f) 0.5:1 .5:0:0.5:1 .2:0.3
0.5 DOTAP: 1 .5 DC-Choi: 0 Chol:0.5 DOPC: 1 .2 DOPE; 0.3 DOPE-PEG.
The polyinosinic-polycytidylic acid was acquired from Invivogen. The protamine sulphate was dissolved in distilled water at a concentration of 1 mg/ml. The nucleus of the nanoparticles was obtained by mixing the polyinosinic-polycytidylic acid (1 mg/ml) and the solution of protamine at a ratio by volume of 1 :0.75 so as to obtain a negatively charged complex. A desired volume of polyinosinic-polycytidylic acid/protamine was allowed to equilibrate for one hour at ambient temperature and was then interacted with an appropriate volume of the solution of cationic liposomes at a charge ratio (moles of cationic lipid/moles of polyinosinic-polycytidylic acid) = 2.5. After 2 hours of incubation, the formation of the nanoparticles was complete. 5. PROTOCOL FOR A TRANSFECTION EXPERIMENT (CONDITIONS BASED ON A WELL OF A 24-WELL PLATE)*
1 . 0.5 microliters of DNA (concentration 1 mg/ml) were mixed with 0.35 microliters of protamine (concentration 1 mg/ml) and allowed to incubate for 10 minutes.
2. 49.1 microliters of Opti-MEM® (Life Technologies) were added to the DNA/protamine complex formed.
3. 5 microliters of the solution of cationic liposomes were diluted in 45 microliters of Opti-MEM®.
4. 50 microliters of the DNA/protamine solution were mixed with 50 microliters of the solution of cationic liposomes. The nanoparticles thus formed were allowed to incubate for 2 hours at ambient temperature.
5. 100 microliters of the solution of nanoparticles were diluted in 400 microliters of Opti-MEM® (volume of 500 x well) and transferred to a well of a 24-well plate
(cells at 70% confluency).
6. After 4 hours, the 500 microliters were removed from the well and 1 ml of culture medium (DMEM), complemented with serum, was added.
* In this case, the charge ratio of the nanoparticles is p=2.5. To double the charge ratio, it is necessary to use 10 microliters of the solution of cationic liposomes (point 6. EVALUATION OF TRANSFECTION EFFICACY (FIGURE 3)
QUANTIFICATION OF TRANSFECTION
The data relating to transfection efficiency are illustrated in figure 1 in the form of a bar chart and in figure 3 as fluorescence images.
a. Sampling of the cell lysates
48 hours after transfection, the cell lysates to be used for luminometer and spectrophotometer analysis were sampled. In each well of the culture plate:
- the medium was suctioned off and washed with PBS at ambient temperature (1 ml/well);
- 200 microliters of lysis buffer (Promega) were added, and the cell lysate was recovered and transferred into a suitably labeled Eppendorf tube and stored in ice.
At this point, each tube was mixed with vortex (about 10 seconds) and centrifuged (2 minutes, 12000 g, 4° C); the supernatant was then aliquoted into two new tubes and stored at - 80° C.
b. Luminometer readings
For quantitative analysis of the activity of the luciferase, which indicates transfection efficiency, the cell lysates were assayed by luminometer (Berthold AutoLumat luminometer LB-953). To this end, 100 microliters of substrate (luciferin) were added to 20 microliters of each sample and subjected to readings for 10 seconds (2 sec delay), following the protocol accompanying Promega's Luciferase Assay System kit. The reaction which occurs converts into light the chemical energy freed from the oxidation of luciferin into oxyluciferin, catalyzed by the luciferase. The luminous intensity, remaining constant for at least one minute, is acquired as a numerical value corresponding to the area subtended by the emission curve over the set period of time. Each sample was measured twice. To standardize the values obtained with respect to the total proteins present in the lysates, the same samples were then analyzed by spectrophotometer.
Figure imgf000029_0001
Bioluminescent reaction catalyzed by firefly luciferase.
Spectrophotometer readings The luminometer readings were standardized for the milligrams of total cell proteins present in the lysates using the Bio-Rad Protein Assay Dye Reagent (Bio-Rad), in accordance with Bradford's method.
For the purpose of obtaining reference values with which the experimental data can be compared, a calibration curve was formed, starting from a mother solution of BSA (bovine serum albumin) at known concentration (2 mg/ml) and diluting 100 microliters thereof in 2 ml of final H20. Two series from 6 cuvettes containing scalar amounts of BSA, H20 and Bio-Rad were then assayed:
- 800 microliters of H20 + 200 microliters of Bio-Rad
- 20 microliters of BSA + 780 microliters of H20 + 200 microliters of Bio-Rad
- 50 microliters of BSA + 750 microliters of H20 + 200 microliters of Bio-Rad
- 100 microliters of BSA + 700 microliters of H20 + 200 microliters of Bio-Rad
- 150 microliters of BSA + 650 microliters of H20 + 200 microliters of Bio-Rad
- 200 microliters of BSA + 600 microliters of H20 + 200 microliters of Bio-Rad Once the curve had been sketched, two cuvettes for each transfected cell lysate were then provided for the reading, said cuvettes containing:
- 5 microliters of sample
- 795 microliters of H20
- 200 microliters of Bio-Rad
Subjected first to a reading at 595 nm, the content of each cuvette has to be stirred with vortex and left in the dark for a period between 5 and 60 minutes.
Analysis of the data
The statistical analysis of the data obtained from the measurements was carried out using the program Microsoft Excel. The transfection efficiency (TE) values TE (relative light unit/mg of protein) were calculated as an average of at least four experimental examinations. The standard deviation and the S.E.M. (standard error of the mean) were also determined for the same samples.
7 SYSTEMATIC COMPARISON
The transfection efficacy of the multicomponent lipid nanoparticles, evaluated as specified for point 6 above "EVALUATION OF THE TRANSFECTION EFFICACY" is shown in figure 3, and was compared systematically with that of the multicomponent lipoplexes prepared as described in (Caracciolo et al., Biochim Biophys Acta, 1768, 2007, 2280). The greater transfection efficiency of the multicomponent nanoparticles compared to that of the multicomponent lipoplexes is clear from the systematic comparison, both in NIH 3T3 murine fibroblasts and in the CHO ovary cell line.
8. EVALUATION OF CYTOTOXICITY Bar charts of the results obtained from the assay reported below are presented in figures 2, 4 and 5. The living cells are able to chemically reduce the compound MTT (a tetrazole salt soluble in water) to formazan salt insoluble in water and purple in color (Mosmann, T., Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods, 1983. 65(1 - 2): p. 55-63). The toxicity test of the multicomponent lipid nanoparticles was carried out on CHO and 3T3 ovary cells seeded on a 96-well plate (8000 cells per well) so as to have 16 wells per experimental condition. The treatments were carried out in 100 microliters of final Optimem per well of a 96-well plate, that is to say 80 microliters of Optimem + 20 microliters of multicomponent lipid nanoparticles (generated using 0.1 micrograms of DNA per well and maintaining the same ratios between DNA and protamine and between DNA/protamine and cationic liposomes described in section 3: transfection protocol). In the case of anti-tumoral therapy, the treatments were carried out in 100 microliters of final Optimem per well in a 96- well plate, that is to say 80 microliters of Optimem + 20 microliters of multicomponent lipid nanoparticles incorporating the active ingredient.
After incubation with the nanoparticles (19 hours), the MTT (final concentration of 0.5 mg/ml) is added to the culture medium and the plate is placed in the dark at 37°C for 4 hours. The medium is then removed from the wells by being suctioned off delicately. After having verified that no traces of solution are remaining in the wells, 100 microliters of DMSO are added to each well and the plate is stirred for 15 minutes at ambient temperature in the dark for the purpose of dissolving the crystals of formazan salt. If the cells are living and metabolically active, they reduce the MTT and the formation of a purple color is observed when the DMSO is added. Vitality is quantified by means of a reading of the absorbance by means of an ELISA plate reader at a wavelength of 540 nm. The data is expressed as a percentage compared to the control represented by cells not treated with the lipid mixtures.
BIBLIOGRAPHY
- Akita H., et al., Multi-layered nanoparticles for penetrating, Biomaterials 30 (2009). 2940-2949.
- Caracciolo G. et al "Factors Determining the Superior Performance of Lipid/DNA/Protammine Nanoparticles over Lipoplexes" J. Med. Chem. 54 (12), pp 4160-4171 (201 1 )
- Chang, H.; Yeh, M. "Clinical development of liposome-based drugs: formulation, characterization, and therapeutic efficacy". Int. J. Nanomed. 2012, 7, 49-60"
- Elsabahy et al. "Non-Viral Nucleic Acid Delivery: Key Challenges and Future Directions". Current Drug Delivery, 8, 201 1 , 235-244
- Pozzi D. et al "Programmed packaging of multicomponent envelope-type nanoparticle system for gene delivery". Appl. Phys. Lett. 96, 183702 (2010); doi: 10.1063/1 .3427354

Claims

1 . A multicomponent lipid nanoparticle consisting of a hydrophilic nucleus and a single outer lipid shell formed by 1 ,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 3B-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Choi), and dioleoylphosphatidylethanolamine (DOPE), furthermore at least one of cholesterol (Choi), DOPE functionalized with PEG (DOPE-PEG), and dioleoylphosphocholine (DOPC) and optionally one or more lipophilic anti-tumoral active ingredients.
2. The lipid nanoparticle according to claim 1 , wherein said 1 ,2-dioleoyl-3- trimethylammonium-propane (DOTAP), 3B-[N-(N',N'-dimethylaminoethane)- carbamoyl]cholesterol hydrochloride (DC-Choi), and dioleoylphosphatidylethanolamine (DOPE), furthermore at least one of cholesterol (Choi), DOPE functionalized with PEG (DOPE-PEG), and dioleoylphosphocholine (DOPC) are present in the following mutual ratios:
a) 0.5:1 .5:0.5:0:1 .5:0
0.5 DOTAP: 1 .5 DC-Choi: 0.5 Chol:0 DOPC: 1 DOPE; 0 DOPE-PEG; in case b, b) 1 :1 :0:1 :1 :0
1 DOTAP: 1 DC-Choi: 0 Choi: 1 DOPC: 1 DOPE; 0 DOPE-PEG
c) 0.5:1 .5:0:0.5:1 .5:0
0.5 DOTAP: 1 .5 DC-Choi: 0 Chol:0.5 DOPC: 1 .5 DOPE; 0 DOPE-PEG
d) 0.5:1 .5:0.5:0:1 .2:0.3
0.5 DOTAP: 1 .5 DC-Choi: 0.5 Chol:0 DOPC: 1 .2 DOPE; 0.3 DOPE-PEG
e) 1 :1 :0:1 :0.8:0.2
1 DOTAP: 1 DC-Choi: 0 Chol:1 DOPC: 0.8 DOPE; 0.2 DOPE-PEG
f) 0.5:1 .5:0:0.5:1 .2:0.3
0.5 DOTAP: 1 .5 DC-Choi: 0 Chol:0.5 DOPC: 1 .2 DOPE; 0.3 DOPE-PEG.
3. The lipid nanoparticle according to any one of claims 1 or 2, wherein said hydrophilic nucleus comprises water and said outer shell comprises one or more lipophilic anti-tumoral active ingredients.
4. The lipid nanoparticle according to claim 3, wherein said lipophilic anti- tumoral active ingredients are charged in said lipid shell in a molar ratio expressed as moles of active ingredient/moles of total lipid comprised between 1 and 4.
5. The lipid nanoparticle according to claim 5, wherein said anti-tumoral active ingredients are selected from paclitaxel, docetaxel and doxorubicin or derivatives thereof.
6. The lipid nanoparticle according to any one of claims 1 to 5, wherein said hydrophilic nucleus comprises one or more nucleic acid molecules or one or more polyinosinic-polycytidylic (poly(l:C)) acid molecules together with one or more suitable condensing agents.
7. The lipid nanoparticle according to claim 6, wherein said nucleic acid molecules are selected from dsDNA (double strand DNA), ssDNA (single strand DNA), RNA, siRNA (small interfering RNA), shRNA (short hairpin RNA), mRNA, dsRNA (double strand RNA), tRNA, miRNA (microRNA) or a mixture thereof.
8. The lipid nanoparticle according to claim 7, wherein said nucleic acid molecules are mini-chromosomes comprising sequences of interest, expression vectors comprising sequences of interest, transposable elements comprising sequences of interest.
9. The lipid nanoparticle according to any one of claims 6 to 8, wherein the ratio between the volume of said one or more nucleic acid molecules and said one or more polyinosinic-polycytidylic (poly(l:C)) acid molecules and said condensing agents is about 1 :0.75.
10. The lipid nanoparticle according to any of claims 6 to 9, wherein said one or more condensing agents are selected from polyamines, cationic peptides, cationic lipids, cationic surfactants, positively charged amino acids, positively charged proteins, multivalent metal cations.
1 1 . The lipid nanoparticle according to claim 10, wherein said polyamines are selected from spermine and spermidine, said positively charged amino acids are selected from arginine and lysine, said positively charged proteins are selected from protamine and histones, said metal multivalent cations are selected from Ca2+, Mg2+, Mn2+; Fe2+; Fe3+.
12. The lipid nanoparticle according to any one of claims 6 to 1 1 , wherein said nucleus consists of nucleic acid and protamine, having a hydrodynamic diameter, D = 230 + 18 nm; and a surface charge = - 19.5 + 2.5 mV.
13. The lipid nanoparticle according to claim 12, wherein said nucleic acid condensing agents are protamine.
14. The lipid nanoparticle according to any one of claims 1 to 13, wherein the surface charge of said nanoparticle is positive and has a potential z of about +
42mV.
15. The lipid nanoparticle according to any of claims 1 to 14 having a diameter between 200 and 250 nm and a polydispersity index of <0.2.
16. A process for the preparation of multicomponent lipid nanoparticles comprising the following steps:
a. dissolving individually 1 ,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 3B-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Chol) and dioleoylphosphatidylethanolamine (DOPE) and at least one of cholesterol (Choi), DOPE functionalized with PEG (DOPE-PEG), dioleoylphosphocholine (DOPC), and optionally a lipophilic anti-tumoral active ingredient in a suitable organic solvent and mixing desired quantities of each lipid and one or more lipophilic anti-tumoral active ingredients thus dissolved so as to obtain a homogenous mixture;
b. completely removing the organic solvent from the mixture obtained in step a. thus obtaining a lipid film;
c. hydrating the lipid film obtained in step b. with a suitable buffer at physiological cell pH so as to obtain a desired final concentration;
d. sonicating the lipid solution obtained in step c. so as to obtain a clear solution.
17. The process according to claim 16, further comprising the following steps: o. dissolving separately, at equal concentration in distilled H20, nucleic acid molecules or polyinosinic-polycytidylic (poly(l:C)) acid molecules and a pool of one or more suitable condensing agents and then mixing the same together at a ratio between 1 :1 and 1 :0.5, thus obtaining a negatively charged complex;
p. allowing to equilibrate, for a period of between 30 and 180 minutes at room temperature, a desired volume of the complex obtained in step a;
q. interacting the balanced complex obtained in step o. with a suitable volume of the solution obtained in step d. at a charge ratio rho ( ) comprised between 2 and 3, including the extremes, and incubating at about 37 °C until formation of the nanoparticles.
18. A process for the preparation of multicomponent lipid nanoparticles comprising the following steps:
a. dissolving individually 1 ,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 3B-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Chol) and dioleoylphosphatidylethanolamine (DOPE) and at least one of cholesterol (Choi), DOPE functionalized with PEG (DOPE-PEG), dioleoylphosphocholine (DOPC), and mixing desired quantities of each lipid thus dissolved so as to obtain a homogenous mixture;
b. completely removing the organic solvent from the mixture obtained in step a. thus obtaining a lipid film;
c. hydrating the lipid film obtained in step b. with a suitable buffer at physiological cell pH so as to obtain a desired final concentration;
d. sonicating the lipid solution obtained in step c. so as to obtain a clear solution; o. dissolving separately, at equal concentration in distilled H20, nucleic acid molecules or polyinosinic-polycytidylic (poly(l:C)) acid molecules and a pool of one or more suitable condensing agents and then mixing the same together at a ratio between 1 :1 and 1 :0.5, thus obtaining a negatively charged complex;
p. allowing to equilibrate, for a period of between 30 and 180 minutes at room temperature, a desired volume of the complex obtained in step a;
q. interacting the balanced complex obtained in step o. with a suitable volume of the solution obtained in step d. at a charge ratio rho ( ) ( = moles of cationic lipid/nucleic acid bases) comprised between 2 and 3, including the extremes, and incubating at about 37 °C until formation of the nanoparticles.
19. The process according to any one of claims 16 to 18, further comprising the following steps:
e. the active ingredient not entrapped in said nanoparticles is removed by means of centrifugation;
f. the supernatant containing said nanoparticles with the active ingredient encapsulated is centrifuged to precipitate said nanoparticles;
g. the supernatant is discarded and the pellet of said nanoparticles is washed twice with saline buffer.
h. the pellet is suspended in distilled H20 containing sucrose.
20. The process according to any one of claims 16 to 19, step d. being substituted by the following steps:
d'. the lipid solution obtained in step c. is extruded by means of a commercial extruder with pore dimensions equal to 200 nm;
21 . The process according to any one of claims 16, 17, 19 or 20, wherein said anti-tumoral active ingredients are selected from paclitaxel, docetaxel and doxorubicin.
22. The process according to any one of claims 17 to 21 , wherein said solutions of nucleic acid or polyinosinic-polycytidylic (poly(l:C)) acid together with one or more condensing agents have a concentration of about 1 mg/ml.
23. The process according to claim 22, wherein said ratio in step o. is about
1 :0.75.
24. The process according to any one of claims 17 to 23, wherein said nucleic acid molecules are selected from dsDNA (double strand DNA), ssDNA (single strand DNA), RNA, siRNA (small interfering RNA), shRNA (short hairpin RNA), mRNA, dsRNA (double strand RNA), tRNA, miRNA (microRNA) or a mixture thereof.
25. The process according to any one of claims 17 to 24 wherein said one or more condensing agents are selected from polyamines, cationic peptides, cationic lipids, cationic surfactants, positively charged amino acids, positively charged proteins, multivalent metal cations.
26. The process according to claim 25, wherein said polyamines are selected from spermine and spermidine, said positively charged amino acids are selected from arginine and lysine, said positively charged proteins are selected from protamine and histones, said multivalent metal cations are selected from Ca2+, Mg2+, Mn2+; Fe2+; Fe3+.
27. The process according to any one of claims 16 to 26, wherein said cationic lipids 1 ,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 3β-[Ν-(Ν',Ν'- dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Choi) and said phospholipids dioleoylphosphocholine (DOPC), dioleoylphosphatidylethanolamine (DOPE) and DOPE-PEG are mixed in step a. in a mutual ratio selected from:
a) 0.5:1 .5:0.5:0:1 .5:0
0.5 DOTAP: 1 .5 DC-Choi: 0.5 Chol:0 DOPC: 1 DOPE; 0 DOPE-PEG; in case b b) 1 :1 :0:1 :1 :0
1 DOTAP: 1 DC-Choi: 0 Choi: 1 DOPC: 1 DOPE; 0 DOPE-PEG
c) 0.5:1 .5:0:0.5:1 .5:0
0.5 DOTAP: 1 .5 DC-Choi: 0 Chol:0.5 DOPC: 1 .5 DOPE; 0 DOPE-PEG
d) 0.5:1 .5:0.5:0:1 .2:0.3
0.5 DOTAP: 1 .5 DC-Choi: 0.5 Chol:0 DOPC: 1 .2 DOPE; 0.3 DOPE-PEG
e) 1 :1 :0:1 :0.8:0.2
1 DOTAP: 1 DC-Choi: 0 Chol:1 DOPC: 0.8 DOPE; 0.2 DOPE-PEG
f) 0.5:1 .5:0:0.5:1 .2:0.3
0.5 DOTAP: 1 .5 DC-Choi: 0 Chol:0.5 DOPC: 1 .2 DOPE; 0.3 DOPE-PEG.
28. The process according to any one of claims 16 to 27, wherein said organic solvent in step a. is chloroform or ethanol.
29. The process according to any one of claims 17 to 28, wherein said incubation in step q. is carried out for a period comprised between about 30 minutes and 3 hours.
30. Nanoparticles according to any one of claims 1 to 15 for use in gene therapy and/or anti-tumoral therapy.
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