Lipid formulations

Abstract

A method of making hybrid lipid particles suitable for delivery of brittle active ingredients, such as nucleic acids, the method comprising the steps of preparing hybrid lipid particles and then "spraying" their surfaces with particles of an inorganic material. The invention also relates to hybrid lipid particles, uses and products, characterized in that the inorganic material particles are present on and in the lipid particles instead of being encapsulated by the lipid particles, and the active ingredient does not need to be encapsulated.

Description

Technical Field

[0001] The present invention relates to improved lipid particles for delivery of nucleic acids. More specifically, the present invention relates to the use of hydrolyzable elemental silicon to improve the stability of lipid particles for delivery of nucleic acids. Background Art

[0002] Lipid particles, including lipid nanoparticles (LNPs), show great promise as vehicles for delivering therapeutic nucleic acids, particularly therapeutic RNA.

[0003] Many compounds with potential application value, for example as active pharmaceutical ingredients, are easily degraded. Nucleic acids such as RNA are particularly prone to degradation during preparation and storage. Various pharmaceutical compositions that attempt to limit the degree of RNA degradation have been proposed. One way to limit RNA degradation is to attempt to encapsulate RNA in a lipid protective organic envelope. For example, EP3677567A1 discloses lipid particles with mRNA molecules encapsulated in the particles.

[0004] The present invention is based in part on the recognition and avoidance of some technical challenges of encapsulating nucleic acids in lipid particles. Chief among these is the necessity to form lipid particles at elevated temperatures. The precise temperature required depends on the lipid used, but generally a temperature of about 60° C. is required. Such elevated temperatures lead to significant degradation of RNA and other fragile active ingredients. This can be counteracted to some extent by using modified active ingredients, such as modified RNA. Notably, US 9504651B2 discloses a method of forming lipid particles "around" mRNA, wherein at least 70% of the mRNA is encapsulated. Of the 30% that is not encapsulated, many appear to be degraded.

[0005] There is also relevant challenge in keeping lipid granules "stable".Improving "stability" includes resisting the tendency of smaller lipid granules to coalesce into larger granules and resisting the tendency of the electric charge of the charged lipids that they lose as a part of lipid granules.In this field, a large amount of work has been carried out to the preparation of novel lipids with favorable characteristics and the development of multiple lipids, and these preparations have favorable characteristics in their ability to form stable particles.An advantage of the present invention is to improve the stability of lipid granules.Another advantage of the present invention is to provide a method for improving particle stability, which methods are not too dependent on the use of specific novel lipids, because the use of these lipids may be subject to technical challenges, supply restrictions and intellectual property restrictions.By using the method and product of the present invention, sufficiently stable lipid granules can be formed, these lipid granules have a wider range of lipids, including lipids that are lower in cost and more easily available than some special lipids, and these special lipids may need to be used in prior art methods and products to obtain lipid granules with sufficient performance.On the contrary, the present invention can also be used together with favorable prior art "high performance" special lipids, to achieve even more excellent performance.

[0006] The lipids in the lipid particles can be constructed in various ways to form liposome particles or to form micellar particles. Liposomal particles are essentially composed of a double layer of lipid molecules. Micellar particles generally have lipids in a non-double layer configuration. The particles of the present invention are referred to herein as "hybrid lipid particles", meaning that they may have regions of micellar lipid configurations and regions of liposomal lipid configurations. Depending on pH, hybrid lipid particles may adopt a liposomal configuration, for example due to deprotonation at neutral pH. According to a preferred embodiment, less than 20% (by weight) of the total lipids exist in a liposomal (double layer) configuration, which means that 80% to 100% (by weight) of the total lipids exist in a non-liposomal (e.g. micellar) configuration. According to certain embodiments, the hybrid lipid particles are micellar lipid particles.

[0007] The present invention is based on such recognition that organic particles, particularly silicon particles (and particles containing hydrolyzable silicon) can be used for stabilizing hybrid lipid particles, suppressing their aggregation, assisting cationic lipids and / or ionizable lipids to maintain their electric charge, and not by encapsulation but by stabilizing the nucleic acid on the surface of the hybrid lipid particles to promote the ability of hybrid lipid particles to protect nucleic acids. Such arrangement advantageously allows hybrid lipid particles to be prepared in the absence of nucleic acids, and only after these particles have been fully formed and any process involving elevated temperature has been completed, nucleic acids are added to the particles.

[0008] The background technology section discloses a variety of lipid particles used in various methods. For example, CN106177892 discloses hyaluronic acid-modified lipid particles having a silica mesoporous core. These particles are disclosed as being compounded with drugs by mixing the silica core with the drug and then adding lipids in a subsequent step. CN114146188 discloses a preparation of CoQ10 as a medically useful compound. It discloses the manufacture of a carrier from silica to silicon, lip encapsulation, and then adding CoQ10. US2022 / 183989, WO2020 / 193999 and Baran-Rachwalska et al. (2020), J.Cont.Rel:326:192-202 all disclose various particles including silicon or silica particles coated with lipids.

[0009] It is noteworthy that these particles are essentially configured as lipids surrounding a silica / silica core. Obviously, this configuration will generally produce particles that are overall larger than the core, but only slightly larger than the core. Summary of the invention

[0010] According to a first aspect of the present invention, there is provided a method for producing a hybrid lipid particle suitable for delivering a fragile active ingredient (e.g., for delivering a nucleic acid), the method comprising the following steps:

[0011] A. Blending one or more lipid components, then

[0012] B. forming the lipid blend into a plurality of hybrid particles having an average diameter of 50 nm to 400 nm, and contacting the plurality of lipid particles with inorganic material particles having an average diameter of 10 nm to 100 nm;

[0013] The average diameter of the hybrid lipid particles is at least 150% of the average diameter of the inorganic material particles.

[0014] According to a second aspect of the present invention, a plurality of hybrid lipid particles are provided, which comprise a blend of one or more lipid components, wherein the hybrid lipid particles have an average diameter of 50 nm to 400 nm and have a coating of inorganic material particles, the inorganic material particles having an average diameter of 10 nm to 100 nm, the plurality of hybrid lipid particles having the ability to bind molecules of active ingredients (such as nucleic acid molecules) to their surfaces and optionally comprising molecules of the active ingredients (such as nucleic acid molecules) bound to their surfaces, wherein the average diameter of the hybrid lipid particles is at least 150% of the average diameter of the inorganic material particles.

[0015] In a further aspect, the present invention provides:

[0016] A pharmaceutical composition comprising a plurality of hybrid lipid particles according to the first aspect of the present invention and a pharmaceutically acceptable carrier.

[0017] A method of providing vaccination to an individual in need thereof, the method comprising administering a pharmaceutical preparation of the present invention, wherein the active ingredient is a nucleic acid molecule, the nucleic acid molecule is an mRNA molecule encoding an antigen or a portion of an antigen of a pathogenic organism or virus, or wherein the active ingredient is a protein or peptide antigen of a pathogenic organism or virus.

[0018] A method of providing vaccination to an individual in need thereof, the method comprising administering a pharmaceutical formulation of the invention, wherein the active ingredient is a nucleic acid molecule, the nucleic acid molecule is an mRNA molecule encoding a tumor antigen or a portion of a tumor antigen, or wherein the active ingredient is a protein or peptide tumor antigen or a portion thereof.

[0019] A method for treating a disease associated with the expression of a gene in an individual, by administering the pharmaceutical preparation of the present invention to the individual, wherein the nucleic acid molecule is siRNA capable of silencing the gene.

[0020] The plurality of hybrid lipid particles of the present invention or the pharmaceutical composition of the present invention is used as a medicament.

[0021] The plurality of hybrid lipid particles or pharmaceutical compositions of the present invention are used as vaccines.

[0022] Use of the multiple hybrid lipid particles of the present invention or the pharmaceutical composition of the present invention in the manufacture of a medicament.

[0023] Use of the multiple hybrid lipid particles of the present invention or the pharmaceutical composition of the present invention in the manufacture of a vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is a graphic illustration of the proposed mechanism of action of the present invention.

[0025] Figure 2 Data related to RNA stability when incorporated into the hybrid lipid particles of the present invention are presented.

[0026] Figure 3 A schematic diagram showing the manufacturing of certain products of the present invention is shown.

[0027] Figure 4 is a TEM photograph of the hybrid lipid particles of the present invention.

[0028] As used herein, a hybrid lipid particle refers to a particle comprising at least some lipids (as defined herein), wherein the lipid molecules present (at least 80% by weight in a preferred embodiment) are arranged to form a hybrid structure that is not a lipid bilayer (i.e., a structure having at least some micellar characteristics). A hybrid lipid particle is different from a liposome comprising a lipid bilayer, but the term "hybrid lipid particle" as used herein allows for the presence of at least some liposomal-type structures within the particle.

[0029] The term "hybrid" is intended to describe lipid particles containing at least some lipids that are different in configuration from the lipid bilayer. The amount of lipids in this configuration may range from 80% to 0% (preferably less than 20%) as a percentage and by weight of the total lipids. DETAILED DESCRIPTION

[0030] According to a first aspect of the present invention, there is provided a method for producing a hybrid lipid particle suitable for delivering an active ingredient such as a fragile active ingredient (e.g., for nucleic acid delivery), the method comprising the steps of:

[0031] A. Blending one or more lipid components, then

[0032] B. forming the lipid blend into a plurality of hybrid particles having an average diameter of 40 nm to 400 nm, and contacting a plurality of organic materials such as lipid particles with inorganic material particles having an average diameter of 10 nm to 100 nm;

[0033] The average diameter of the hybrid lipid particles is at least 150% of the average diameter of the inorganic material particles.

[0034] According to a preferred embodiment of the first aspect of the present invention, there is provided a method for producing a hybrid lipid particle suitable for delivering an active ingredient such as a fragile active ingredient (e.g., for nucleic acid delivery), the method comprising the following steps:

[0035] A. blending one or more cationic lipids or ionizable lipids with one or more other lipids selected from neutral lipids and polar lipids and optionally one or more additional lipid components, and then

[0036] B. forming the lipid blend into a plurality of lipid particles having an average diameter of 40 nm to 400 nm, and contacting the plurality of lipid particles with particles containing hydrolyzable silicon having an average diameter of 10 nm to 100 nm, and then

[0037] C. contacting the hybrid lipid particle with a solution of the active ingredient (e.g., a solution of nucleic acid molecules) so that the molecules of the active ingredient (e.g., nucleic acid molecules) are electrostatically bound to the cationic charges of the cationic lipid and the ionizable lipid, thereby electrostatically binding to the surface of the hybrid lipid particle.

[0038] According to a second aspect of the present invention, a plurality of hybrid lipid particles are provided, which comprise a blend of one or more lipid components, wherein the hybrid lipid particles have an average diameter of 50 nm to 400 nm and have a coating of inorganic material particles, the inorganic material particles having an average diameter of 10 nm to 100 nm, such multiple hybrid lipid particles have the ability to bind molecules of active ingredients, such as nucleic acid molecules, to their surfaces and optionally comprise molecules of the active ingredients, such as nucleic acid molecules, bound to their surfaces, wherein the average diameter of the hybrid lipid particles is at least 150% of the average diameter of the inorganic material particles.

[0039] Without wishing to be bound by theory, it is suggested that the hybrid lipid granules of the present invention are kept together by a combination of electrostatic force, covalent force and van der Waals force. The organic part of the particle is considered to be to stabilize the inorganic part by forming a resonance band (resonance band), and the positive charge in the carboxylic acid group is delocalized between these resonance bands. When active ingredients such as nucleic acids are combined with this delocalized charge, the active ingredient condenses. Condensation increases as the charge increases. The presence of inorganic material particles increases hydrogen aggregation (hydrogen banding) and promotes the combination of active ingredients, thereby allowing better condensation of nucleic acids.

[0040] According to a preferred embodiment of the second aspect of the present invention, a plurality of hybrid lipid particles are provided, which comprise a blend of one or more cationic lipids or ionizable lipids with one or more polar lipids and optionally one or more additional lipid components, wherein the hybrid lipid particles have an average diameter of 50 nm to 400 nm and preferably 80 nm to 400 nm, and have coating particles containing hydrolyzable silicon, which have an average diameter of 10 nm to 60 nm and preferably 10 nm to 60 nm, such multiple hybrid lipid particles have the ability to bind nucleic acid molecules to their surface and optionally contain molecules of active ingredients such as nucleic acid molecules bound to their surface, wherein the average diameter of the hybrid lipid particles is at least 150% of the average diameter of the inorganic material particles.

[0041] In a further aspect, the present invention is as described above.

[0042] In the art, lipids are generally understood to include fatty acids and fatty acid derivatives, glycerolipids, glycerophospholipids, sphingolipids, glycolipids and polyketides.

[0043] As used in this application, the term "lipid" also encompasses lipidated oligopeptides (which term is used interchangeably herein with the term lipopeptide) in which a short peptide sequence (such as a peptide sequence having 3 to 20 amino acid residues, such as 5 to 15 amino acid residues, particularly 3, 4 or 5 amino acid residues and most particularly 5 amino acid residues) is conjugated to one or more fatty acid chains (particularly a fatty acid chain having a carbon chain length of 10 to 24, preferably a fatty acid chain having a carbon chain length of 12 to 18; for example, a fatty acid chain having a carbon chain length of 14, 15 or 16; for example, the peptide portion may be optionally lipidated with a palmitoyl, cetyl or myristoyl moiety).

[0044] The lipidated oligopeptide may optionally be a lipidated tetrapeptide, a lipidated pentapeptide or a lipidated hexapeptide. Preferably, the amino acid residues include at least one amino acid residue (e.g., 2 or 3 amino acid residues) that is cationic at pH 7.4 (physiological pH), such as lysine or arginine. For example, the lipidated oligopeptide may include one or more (e.g., 2) lysine residues. Thus, a particular example is palmitoyl-pentapeptide-4 (CAS No. 214047-00-4):

[0045]

[0046] Therefore, in certain preferred embodiments according to all aspects of the present invention, the one or more lipids are or include one or more lipidated oligopeptides, particularly those lipidated oligopeptides having one or more amino acids (examples of which include lysine and arginine) residues that are cationic at pH 7.4 (physiological pH). Depending on the properties of the peptide component of the lipopeptide, the lipopeptide can be a cationic lipid, an ionizable lipid, a neutral lipid or a polar lipid. According to various embodiments of the present invention, the demand for one or more of a cationic lipid, an ionizable lipid, a neutral lipid or a polar lipid can be met by a suitable lipopeptide. Alternatively or additionally, one or more additional lipid components of the product of the method of the present invention can be provided by a suitable lipopeptide.

[0047] Lipidated oligopeptides can be used in combination with one or more phospholipids such as DOPE or DPPC. It is believed that the alkyl chains of the lipopeptide can be advantageously assimilated by the phospholipid bilayer, and the surface of the bilayer is modified with peptide moieties. It is believed that in this way, the peptide can provide tissue and / or cell targeting; and, for example, when the peptide carries a cationic charge at physiological pH, it can stabilize negatively charged active ingredients, such as nucleic acids, such as mRNA.

[0048] However, it has been found that DOTAP is not always necessary for the formulation of the present invention, and therefore, in certain preferred embodiments, the one or more lipids are or include one or more of a phospholipid (such as DPPC and / or DOPE) and a lipidated oligopeptide having one or more amino acid residues that are cationic at pH 7.4 (physiological pH) (examples of which include lysine and arginine). Optionally, one or more sugars (particularly trehalose) and / or one or more amino acids (particularly glycine) are also present.

[0049] Alternatively, in other preferred embodiments, the one or more lipids are or include one or more of the phospholipids (such as DPPC and / or DOPE) and are formulated with one or more coenzymes (e.g. NAD), one or more flavanols (e.g. quercetin) and / or one or more amino acids (e.g. glycine, tyrosine). Optionally, one or more sugars (particularly trehalose) and / or one or more amino acids (particularly glycine) are also present.

[0050] According to all aspects of the present invention, the blend of one or more cationic lipids or ionizable lipids and one or more neutral lipids or polar lipids comprises at least one cationic lipid or ionizable lipid.Preferably, total cationic lipid or ionizable lipid (by molar ratio) is 20% to 70% of total lipids, for example 30% to 60%, or 40% to 60%.

[0051] According to certain embodiments, the cationic lipid or ionizable lipid is a cationic lipid. In addition to suitable lipopeptides, the cationic lipid can be selected from DOTAP (dioleoyl-3-trimethylammonium propane, 18:1TAP), DODAC (dimethyldioctadecyl-ammonium chloride), SA (stearylamine, octadecylamine) and DOTMA (9-(trimethyl[2,3-(dioleyloxy)propyl]ammonium chloride), and mixtures thereof. Mixtures comprising DOTAP are particularly preferred. According to certain embodiments, at least half or all of the cationic lipids are DOTAP. According to certain embodiments, at least half or all of the cationic lipids are cationic lipopeptides.

[0052] According to other embodiments, the cationic lipid or ionizable lipid is an ionizable lipid. The ionizable lipid may be selected from [(4-hydroxybutyl) azanediyl] di(hexane-6,1-diyl) bis(2-hexyldecanoate, heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate, 7-[(2-hydroxyethyl)[8-(nonyloxy)-8-oxooctyl]amino]heptyl 2-octyldecanoate, (6Z, 9Z, 28Z, 31Z)-heptatriacontane-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butyrate and DODMA, and any mixture thereof.

[0053] According to certain embodiments, the cationic lipid or ionizable lipid component can be a mixture of one or more cationic lipids (e.g., one or more of the cationic lipids listed above) and one or more ionizable lipids (e.g., one or more of the ionizable lipids listed above).

[0054] The lipid blend may also optionally include one or more neutral or polar lipids. The neutral phospholipids DOPE (dioleoylphosphatidylethanolamine), PC (phosphatidylcholine) and phosphatidylcholine (a mixture dominated by PC) are all examples of non-cationic phospholipids that can be used as neutral or polar lipids according to the present invention.

[0055] According to certain preferred embodiments, the lipid blend is all or mainly cationic lipids and phospholipids. For example, the lipid blend can be composed of approximately equal amounts of DOTAP and DOPE. In another example, the lipid blend can be composed of cationic lipopeptides.

[0056] The lipid blend may also optionally and additionally comprise a conjugated lipid (conjugatedlipid) (such as PEGylated lipids, PEI, Brij58 (surfactant)) and / or a steroid / sterol component such as cholesterol as an additional lipid component. According to certain preferred embodiments, the lipid blend does not contain a material amount of conjugated lipids or a material amount of steroid / sterol. In certain embodiments, the additional lipid may include or consist of an additional lipopeptide. The steroid / sterol according to the present invention may optionally be of animal origin and / or may optionally be of non-animal origin (e.g., plant-derived or microbial-derived).

[0057] The lipid blend may also optionally or additionally comprise mitoxantrone (MTO) or a derivative thereof, such as dipalmitoyl MTO or monopalmitoyl MTO.

[0058] granularity

[0059] According to all embodiments, the mean diameter of hybrid lipid granules of the present invention is significantly greater than the mean diameter of inorganic material particles. According to preferred embodiments, the mean diameter of hybrid lipid granules is 80nm to 400nm, and the mean diameter of inorganic material particles is 10nm to 60nm. In these embodiments (and wherein one or both of the mean diameters are more narrowly limited other embodiments), the optional feature of hybrid lipid granules is that its mean diameter is at least 150% of the mean diameter of inorganic material particles.

[0060] Fabrication of hybrid lipid particles

[0061] The hybrid lipid particles of the second aspect of the present invention can be manufactured by any suitable method. They can optionally be manufactured by the method of the first aspect of the present invention. For example, lipid blends as described above can be prepared. In certain embodiments, the lipid blends can be prepared by blending lipids in a suitable solvent. Optionally, any suitable method can be used to dry the lipid blends in the solvent by evaporating the solvent. A rotary evaporator can be optionally used to produce a thin layer of anhydrous lipid blends, or any other method known in the field of lipid particle preparation, such as microfluidization or tangential flow filtration (TFF). Hybrid lipid particles can be produced by hydrating the anhydrous lipid blend (for example, by contacting it with an aqueous solution). These steps are carried out before introducing active ingredients such as nucleic acids, so it is safe and optionally related to the temperature and chemical conditions of the increase that are unfavorable for the stability of active ingredients (for example, the temperature and chemical conditions of the increase that are unfavorable for RNA stability and will tend to cause RNA molecule degradation and loss).

[0062] After forming hybrid lipid particles, the method of the present invention and related methods are then stabilized by treating the newly formed hybrid lipid particles with inorganic material particles (preferably hydrolyzable silicon particles). This treatment can be described as "coating". "Coating" should be understood as inorganic material particles (preferably hydrolyzable silicon particles) will be mainly present on the surface of hybrid lipid particles or towards the surface of the hybrid lipid particles. They may be partially embedded in lipid particles, but at least partially contact the surface of hybrid lipid particles. Preferably, the diameter of inorganic material particles (hydrolyzable silicon particles) is significantly smaller than hybrid lipid particles. For example, inorganic material particles (hydrolyzable silicon particles) can be at least half or at least one-third or at least one-quarter of the average diameter of hybrid lipid particles, or at least one-sixth, one-eighth or one-tenth. Inorganic material particles (hydrolyzable silicon particles) can be mixed with hybrid lipid particles in a wider range of ratios. For example, the amount of inorganic material particles (hydrolyzable silicon particles) can be about 0.1 times to 10 times of the amount of lipid.

[0063] filter

[0064] Inorganic material particles (e.g., hydrolyzable silicon particles) can be added to the lipid particles subsequently, and the resulting particles comprising the hybrid lipids with inorganic particles (preferably hydrolyzable silicon particles) coating can be optionally filtered. According to a first aspect of the present invention, a filtering step can be inserted between step B and C. Although any suitable filtering method can be used, it is preferred to filter using tangential flow filtration. One purpose of filtering is to filter by means of a membrane having a cut-off size in a desired particle size (e.g., 100 nm), to increase the size uniformity of the hybrid lipid particles.

[0065] After filtration, the total proportion of the weight of the hybrid lipid particles composed of inorganic particles (hydrolyzable silica) may be reduced.

[0066] A note on granularity

[0067] The specification describes particle size as mean diameter. Particle diameter may be measured by any suitable method, including electron microscopy and size exclusion. Preferably, the particles have a distribution of diameters around the mean diameter such that 80% of the particles have a diameter within 25% ± the mean diameter. Monodispersity may be improved, particularly after filtration.

[0068] Hydrolyzable Silicon

[0069] As used herein, "hydrolyzable silicon" encompasses pure elemental silicon. However, complete purity is not required. On the contrary, the present invention is not intended to cover pure silicon dioxide (including sand, quartz, silica gel). The key requirement is that the material is hydrolyzable, that is, it will tend to decompose into soluble products, such as orthosilicic acid (OSA) under physiological conditions. According to certain embodiments, the definition "particles containing hydrolyzable silicon" is met if at least half of the mass of the material is hydrolyzed into soluble products within one month after injection into a subject (e.g., after intramuscular or subcutaneous injection).

[0070] The hydrolyzable silicon according to the invention is preferably mesoporous, that is to say it has pores with a diameter of 2 nm to 50 nm.

[0071] The particles containing hydrolyzable silicon may be commercially available or may be produced by any suitable method. For example, bulk silicon may be reduced by grinding or electro-etching. Silicon particles or the desired size may be obtained by any suitable method, such as by using an air classifier.

[0072] Doped Silicon

[0073] Preferably, the hydrolysable silicon used in all aspects of the invention comprises (or consists of) doped silicon. The silicon may be n-doped or p-doped. Most preferably, the silicon is p-doped. Most preferably, the silicon is p-doped with boron. Most preferably, the doping is heavy doping, which is understood to mean the addition of one or more dopant atoms per 10,000 silicon atoms, or even "superdoping", e.g. doping with 10 20 Boron atoms / cm 3 , which depends on the need to generate different zeta charges inside the crystal matrix and / or on the surface of the hybrid system.

[0074] The semiconductor industry provides a ready source of suitably doped silicon as well as doping techniques.

[0075] Active ingredients

[0076] In a preferred embodiment, the active ingredient of the present invention is an active pharmaceutical ingredient. A variety of active ingredients may be present in a single formulation according to the present invention, but more commonly, each embodiment of the present invention relates to a single API. According to certain preferred embodiments, the active ingredient is an API as a nucleic acid. Most preferably, RNA, such as mRNA, siRNA, haRNA or saRNA. In a preferred embodiment, the active ingredient is negatively charged (i.e., having a net negative charge at physiological pH). The active ingredient (API) according to the present invention may preferably be a brittle active ingredient. Brittle active ingredients may be understood as active ingredients with poor stability. For example, active ingredients with poor stability at 20°C and pH 7.4, such as those with a half-life of less than 6 days or less than 1 day.

[0077] Binding of nucleic acids to silica lipid particles

[0078] Contrary to the prior art lipid particles that attempt to encapsulate active ingredients such as nucleic acids in lipid structures, the hybrid lipid particles of the present invention bind active ingredients such as nucleic acids to their surfaces. Due to electrostatic forces and the concentration gradient of the resulting organic-inorganic hybrid system, active ingredients tend to be incorporated into the matrix of silicon and on the surface. Since nucleic acids themselves can act as charge stabilizers, whether ionized drugs are hydrophobic or hydrophilic, they tend to be distributed along the surface of mesoporous silicon. Due to the electronic resonance generated on the surface-polar molecules interact through dipole-dipole intermolecular forces and hydrogen bonds, the mesoporous silicon regions functionalized with nucleic acids will tend to associate with hydrophilic ionized drugs.

[0079] Active ingredients such as nucleic acids can be bound to the surface due to electrostatic attraction between negatively charged active ingredients such as negatively charged nucleic acids and positive charges proximal to the cationic lipid head groups and / or within the silicon matrix constituting the hybrid lipid particle.

[0080] Although the prior art lipid particles containing cationic lipids but not containing inorganic materials (preferably, hydrolyzable silicon) can be used to achieve this electrostatic coordination, it is difficult to maintain the long-term attraction to active ingredients (such as nucleic acids) using prior art particles because cationic lipids have a tendency to lose charge during storage. This process is referred to as lipid aging. The presence of an inorganic material (preferably, hydrolyzable silicon) according to the present invention allows cationic lipids or charged lipids (including ionizable lipids) to stabilize their charge by electrostatic coordination, thereby maintaining their charge. In order to produce stable charge and complex, the inventor further points out that the importance of the degree of binding of nucleic acids on the surface of silicon particles will also depend on the surface contact angle. The surface contact angle and the dissolution behavior that follows depend on the "ratio" of the hydroxide and oxide capping fragments on the surface of the silicon particles. Therefore, it is necessary to carry out careful functionalization of the inorganic silicon particle surface because the combination of silicon porous size and chemical surface is necessary for controlling dissolution rate, stable charge and final release kinetics.

[0081] Nucleic acids used in the present invention

[0082] The present invention is suitable for use with any active ingredient. Preferably, the active ingredient is a nucleic acid. The present invention is particularly suitable for use with RNA, because RNA is particularly susceptible to degradation without the protection provided by the present invention. Therefore, according to certain preferred embodiments of all aspects of the present invention, the nucleic acid is RNA. The RNA may optionally be siRNA. It may optionally be mRNA. For example, it may be an mRNA encoding a vaccine antigen. The RNA may optionally be chemically modified or sequence modified to increase its stability and prevent its degradation. According to certain embodiments of the present invention, the RNA is chemically modified to increase its stability or prevent its degradation. However, in certain preferred embodiments, the RNA is not chemically modified because it has been found that such treatment is unnecessary.

[0083] According to certain embodiments of all aspects of the present invention, nucleic acid is DNA. According to other preferred embodiments, nucleic acid is RNA. It can be siRNA or mRNA. It can have any suitable length, but typically, it can be 10 to 30 nucleotides long for siRNA, or it can be 200 to 2000 nucleotides long for mRNA. It can be double-stranded or single-stranded, or, particularly in the case of siRNA, it can be chemically single-stranded, but with one or more base pairing regions (and with optional unpaired overhangs). It can optionally be chemically modified (e.g., replaced by using N1-methyl pseudouridine) or its sequence can be modified (e.g., shortened by UTR). Preferably, nucleic acid (i.e., RNA) can be unmodified (particularly unchemically modified), because this may be unnecessary for providing stability.

[0084] Particle configuration

[0085] Hybrid lipid particles of the present invention, in all its aspects, except hybrid lipid structure, preferably have the following configuration.Hybrid lipid particles have both integrated in lipid bilayer and partially or completely exposed on the surface of the particle inorganic material particles (preferably, hydrolyzable silicon particles), so that can be used to interact with nucleic acid.Preferably, at least 50% of total inorganic material particles (preferably, hydrolyzable silicon particles) can contact with the surface of lipid particles and are not completely encapsulated in micellar structure.When present, active ingredients such as nucleic acids are mainly located at the particle surface in an electrostatically bound manner.For example, the total active ingredients such as nucleic acids more than 90% of the presence will be bound to the particle surface, and before fully condensing and producing complexes, the total active ingredients less than 10% of the presence will be encapsulated in lipid structure.In some embodiments, the total active ingredients such as nucleic acids of zero or almost zero (for example, less than 0.5%) of the presence will be encapsulated in structure.

[0086] Additional components

[0087] It has been found that in the presence of one or more amino acids and / or one or more non-reducing disaccharides, the stability of the hybrid lipid particles can be further increased. Therefore, the method of the present invention can be optionally carried out in the presence of one or more amino acids and / or one or more non-reducing disaccharides. The hybrid lipid particles of all aspects of the present invention may also include non-reducing disaccharides and / or amino acids. Preferred non-reducing disaccharides are trehalose. Preferred amino acids are glycerol. In certain preferred embodiments, both glycine and trehalose are preferably used. These components can coordinate with inorganic material particles (preferably hydrolyzable silicon) electrostatically and increase the overall stability of the system.

[0088] Particle stability

[0089] Compared with the corresponding particles not containing inorganic material particles according to the present invention (preferably, hydrolyzable silicon), the hybrid lipid particles of the present invention show enhanced dimensional stability. This enhanced dimensional stability is manifested as the resistance of particles to agglomeration into larger particles. According to certain embodiments, the agglomeration rate at 5°C is at least half of the equivalent corresponding particles (having the same composition but not presenting inorganic material particles according to the present invention (preferably, hydrolyzable silicon particles)). According to certain embodiments, after storage in an aqueous solution for 3 months at 5°C and physiological pH, at least 90% of the particles are not agglomerated and retain their original size.

[0090] Charge stability

[0091] The surface charge of hybrid lipid particles can be estimated using zeta potential (electrokinetic potential) parameters. According to experience, the suspension of particles with low zeta potential (0mV to ± 5mV) is unstable and can quickly aggregate. ± 30mV to ± 40mV values ​​correspond to reasonable stability, and ± 40mV to ± 60mV values ​​correspond to good stability, and ± 60mV and above values ​​correspond to excellent stability.

[0092] According to certain embodiments, the hybrid lipid particles of the present invention have a value of> ± 40mV, more preferably> ± 45mV,> ± 50mV or> ± 60mV. Preferably, due to the presence of hydrolyzable silicon, the zeta potential is increased by at least ± 10mV (that is, the zeta potential is at least ± 10mV, greater than ± 10mV, of the zeta potential of lipid particles having the same composition as those of the present invention but not having inorganic material particles according to the present invention (preferably, hydrolyzable silicon particles)).

[0093] The presence of the inorganic material (preferably silicon) also inhibits the loss of the positive charge of the cationic lipids. This is known as lipid aging and is preferably slowed down (at 5°C) by at least 2, 4, 8 or 16 times.

[0094] Stability of active ingredients such as nucleic acids

[0095] The hybrid lipid particles of the present invention are used to protect active ingredients such as nucleic acids (particularly RNA) that are electrostatically complexed with the surface of the hybrid lipid particles. The present invention makes therapeutic preparations such as vaccines more convenient for storage. For example, storage at 5°C or room temperature rather than storage at sub-zero temperatures. It also increases stability and reduces degradation of active ingredients such as nucleic acids during freeze drying, rehydration, transportation and storage. According to certain embodiments of the present invention, compared with corresponding active ingredients such as mRNA that is not complexed with the hybrid lipid particles of the present invention, the half-life of active ingredients such as mRNA is extended by at least 100 times, at least 1000 times or at least 10000 times. According to certain embodiments, compared with corresponding active ingredients such as mRNA complexed with equivalent hybrid lipid particles lacking inorganic particles (preferably, hydrolyzable silicon) components according to the present invention, the half-life of active ingredients such as mRNA is extended by at least 10 times, at least 100 times or at least 1000 times. The half-life can be measured in an aqueous physiological solution (e.g., in a phosphate buffered saline solution) at a pH of 7, 5°C.

[0096] Formulated into drug products

[0097] The present invention also contemplates the use of hybrid lipid granules of the present invention to prepare drug products, which also fall within the scope of the present invention. Such drug products include injections (such as injectable vaccines), topical creams, capsules, tablets and ointments. They also include prodrugs or products, such as dehydration (freeze-drying) products and concentrated products that must be diluted and / or rehydrated before use.

[0098] Treatments and treatment-related products

[0099] The product of the invention may be used in a method of treatment, or may be a product for use in a method of treatment. The method of the invention may also include subsequent steps that constitute a method of treatment.

[0100] Therapeutic methods include treating or preventing diseases. In some embodiments, therapeutic methods may include downregulating gene expression by siRNA. In other embodiments, therapeutic methods may include vaccination, for example, vaccination against cancer or vaccination against infectious diseases by delivering mRNA encoding antigens (or fragments thereof) or infectious disease pathogens (e.g., spike proteins of SARS-CoV-2). Alternatively, peptides or protein antigens corresponding to tumor antigens (or fragments thereof) may be administered as vaccines.

[0101] Optionally, in certain preferred embodiments of all aspects of the invention, the open reading frame of the mRNA encodes a tumor-specific antigen. As used herein, the term tumor-specific antigen may refer to an antigen produced by a non-synonymous somatic mutation (producing a new antigen) or a viral integration mutation (producing an oncovirus antigen) in one or more malignant cancer cells. Therefore, a tumor-specific antigen may refer to an antigen that is completely absent (not expressed) in non-cancerous (healthy, normal) cells.

[0102] Optionally, the open reading frame of mRNA encodes tumor-associated antigen. As used herein, the term tumor-associated antigen may refer to an antigen overexpressed in malignant cancer cells, such as due to genetic amplification or post-translational modification, compared to noncancerous (healthy, normal) cells. The term tumor-associated antigen may encompass overexpressed antigens (the term may refer to proteins moderately expressed in noncancerous (healthy, normal) cells but expressed in large quantities in malignant cancer cells); differentiation antigens (the term may refer to proteins selectively expressed by cell lineages evolved from malignant cells, an example being prostate-specific antigen); and cancer germline antigens (the term may refer to antigens that are usually limited to reproductive tissues but abnormally expressed in malignant cancer cells; for example, melanoma antigen family A3 (MAGE-A3); New York esophageal squamous cell carcinoma-1 antigen (NY-ESO-1); and antigens preferentially expressed in melanoma (PRAME)).

[0103] When the open reading frame of the mRNA encodes a cancer-associated antigen or a cancer-specific antigen, the nucleic acid product of the present invention may be suitable for use in a preventive or therapeutic vaccine composition.

[0104] Optionally, the open reading frame of the mRNA encodes an allergen (including but not limited to one or more nut allergens; the one or more nut allergens include but are not limited to: one or more seed storage proteins, such as vicilin, legumin, albumin; one or more plant defense-related proteins; and one or more assembly inhibitory proteins).

[0105] In some embodiments, a method of treatment may include the delivery of a biologically active compound.

[0106] Proposed mechanism of action.

[0107] Without wishing to be bound by theory and using a formulation containing hydrolyzable silicon to illustrate a broader principle, it is noted that Figure 1 , which illustrates the proposed mechanism of action of the present invention from various aspects. The center of the figure schematically shows particles containing hydrolyzable silicon. Selected Si atoms and certain O atoms are highlighted. It should be noted that even nominally "pure" elemental silicon is unlikely to have surface Si atoms bound to other chemical moieties (such as O and -OH). The figure shows possible modes of electrostatic coordination between glycine and trehalose and the silicon and boron atoms of the hydrolyzable silicon particles. The figure also shows the electrostatic coordination between the atoms of the hydrolyzable silicon particles and phospholipids, and the electrostatic coordination between the atoms of the hydrolyzable silicon particles and DOTAP as an example of a cationic lipid. The figure shows a segment of RNA (as an example of an active agent), which of course has negative charges along its length, and shows a plausible mechanism by which those negative charges and the RNA molecule itself become stabilized by coordination with the atoms of the hydrolyzable silicon particles and with the charges on the lipids (the lipids themselves become stabilized by coordination with the atoms of the hydrolyzable silicon particles).

[0108] The following non-limiting examples illustrate various aspects of the invention.

[0109] Example

[0110] Example 1. Exemplary methods of making the products of the invention.

[0111] The manufacturing process of the product of the present invention is as follows Figure 3As shown, the selected lipids DOTAP, DOPE and mPEG2000-DSPE are first dissolved in methanol. At the same time, the porous silicon nanoparticles are activated by exposure to methanol. Subsequently, the solvent is evaporated in a slow evaporation process to produce activated SiNPs. This activation step is intended to make SiNPs easily dispersed in water. The activated SiNPs are then dispersed in (nuclease-free) water in the presence of trehalose (THR) and glycine (GLY). An appropriate volume of lipids is continuously transferred to a round-bottom flask and methanol is evaporated by rotary evaporation. As a result of this evaporation step, a lipid film is generated on the wall of the flask. The suspension containing dispersed silicon, THR and GLY is then added to the flask containing the lipid film for lipid rehydration. Subsequently, the membrane is extruded through 0.4μm and 0.1μm at 60°C. The extruded sample is then stored under refrigerated conditions. The amount of each component is as follows:

[0112]

[0113] Example 2 .It is demonstrated that the presence of silicon prevents particle agglomeration and promotes the retention of surface charge.

[0114] The particles prepared according to the present invention (as well as comparative particles) were kept at 5°C and regularly evaluated for size (diameter in nm), size dispersity (polydispersity index - PDI) and zeta potential (ZP, which indicates surface charge). Sample SIS0012 was prepared with undoped silicon, SIS0013 was prepared with boron-doped silicon, and NoSiNP was prepared without silicon, and the results are shown below. Both size and surface charge were characterized using a Malvern Zetasizer Advanced Pro series instrument. Samples were prepared using 20 μl of the particles of the present invention and 980 μl of nuclease-free water, with a total of 1000 μl, which were placed in a disposable cuvette. The measurement was performed by performing 3 scans to reduce signal-to-noise interference. The average of the 3 scans was calculated together with the polydispersity index (PDI) as the final size measurement.

[0115] Zeta potential measurements were performed by mixing 200 μl of the particles of the invention with 800 μl of nuclease-free water (for silicon-containing SIS0012) or 1 mM KCl (for NoSiNPs to ensure good conductivity). Five scans were averaged to determine the zeta potentials given in the table below.

[0116]

[0117] It can be seen that the size of particles formulated in the absence of silicon tends to increase over time as the particles agglomerate, and this trend is not observed when silicon is present. Similarly, a loss of surface charge is observed in particles formulated in the absence of silicon, but not in those particles in the presence of silicon.

[0118] Example 3 . Demonstrated ability to stabilize RNA.

[0119] The mRNA (DasherGFP mRNA (lot number: 88103F, Aldevron)) stock solution was prepared by dissolving the mRNA powder in nuclease-free water to a concentration of 0.5 μg / μL. The mRNA was compounded with the hybrid lipid particles of the present invention (prepared as in Example 1, using undoped silicon to prepare samples encoding SIS0012, and using boron-doped silicon to prepare samples encoding SIS0013) by mixing 50 μL (25 μg) of the mRNA solution with 200 μL of the hybrid lipid particle suspension (the weight ratio of hybrid lipid particles / RNA was 12:1). The mixture was incubated at room temperature for 40 minutes to allow it to be fully compounded, and then used in liquid form or freeze-dried. The freeze-dried complex was then embedded in a sodium hyaluronate hydrogel (the freeze-dried complex was mixed directly with the hydrogel, while the liquid complex was analyzed as is). The final preparation was prepared in 30 μL aliquots to minimize the risk of cross-contamination during storage and analysis, and stored at room temperature or 40°C.

[0120] After 7 days of storage, samples were subjected to gel electrophoresis. Naked mRNA stored under similar conditions was used as a control in all cases, and a DNA ladder was used as a size guide. Samples were loaded onto E-Gel TM E-Gel in Power Snap electrophoresis device TM Agarose gel (1%). After 3 and 7 minutes of electrophoresis, the samples were separated using E-Gel TM The gel was illuminated and imaged using a Power Snap electrophoresis camera. The total amount of mRNA for the different samples loaded onto the gel is provided in the table below:

[0121]

[0122] from Figure 2 As can be seen, the degraded mRNA is highly mobile, and the degraded fragment produces tailing on gel. This is observed with naked mRNA. The mRNA compounded with hybrid lipid granule of the present invention is not degraded and keeps its original length, which means that its electrophoretic mobility is much poorer, and (at 3 minutes and 7 minutes) has not yet obviously migrated out from the loading well of gel.

[0123] Example 4 - Use of protein (alkaline phosphatase) in the preparation of the present invention

[0124] Alkaline phosphatase is an enzyme that exists in various forms, catalyzes the degradation of a wide variety of proteins, and is found in all tissues of the human body. It is concentrated primarily in the bones, kidneys, liver, intestines, and placenta. It contributes in particular to: protection of the intestine from bacteria; digestive function; degradation of fats and vitamin B; and bone formation. Alkaline phosphatase exhibits a loss of activity at low pH and high temperatures.

[0125] Alkaline phosphatase activity can be monitored by measuring changes in the concentration of one or more of its substrates or products in an in vitro assay, where the concentration is represented by ultraviolet visible (UV-Vis) absorbance. For example, the concentration of the substrate 4-nitrophenyl phosphatase (PNPP) (whose structure is shown below) can be monitored to track the following reaction:

[0126]

[0127] Materials, methods, and results

[0128] Alkaline phosphatase was isolated from calf intestine and provided as a 56 kD recombinant enzyme, expressed in yeast Pichia Pastoris obtained from Sigma Aldrich / Merck (The Old Brickyard, New Rd, Gillingham, Dorset, SP8 4XT). An aqueous stock solution of alkaline phosphatase (ALP) was prepared at a concentration of 1 U / ml. 1 U (μmol / min) is defined as the amount of ALP required to catalyze the conversion of one micromole of PNPP per minute at 37°C and pH 7.4.

[0129] A 20 mM solution of 4-nitrophenyl phosphatase (PNPP) was prepared using Tris buffer (100 mM / L) at pH 7.4.

[0130] In a 15 ml tube, prepare ALP solutions of the following concentrations from the 1 U / ml stock solution:

[0131] 0.1mU / ml, 0.5mU / ml, 1mU / ml, 5mU / ml, 10mU / ml, 50mU / ml and 100mU / ml.

[0132] Then they were mixed with the prepared 20 mM PNPP Tris buffer solution in an Eppendorf tube.

[0133] The tubes were incubated in a 37°C water bath for 30 minutes and then UV-vis absorbance measurements were taken at 405 nm as shown in FIG. 22 .

[0134] ALP was loaded onto the hybrid lipid particles of the present invention as follows:

[0135] 1. Prepare 3 sets of 8 Eppendorf tubes:

[0136] A Prepare 8 Eppendorf tubes, each containing 50 μL (50 mU / ml) of ALP and 500 μL of hybrid lipid particles (containing undoped Si). After adding these components to these tubes, they are mixed, vortexed and refrigerated overnight.

[0137] B Prepare 8 Eppendorf tubes, each containing 50 μL (50 mU / ml) of ALP and 500 μL of hybrid lipid particles (containing boron-doped Si). After adding these components to these tubes, they are mixed, vortexed and refrigerated overnight.

[0138] C. Prepare 8 Eppendorf tubes, each containing 50 μL (50 mU / ml) of ALP and 500 μL of Tris buffer. After adding the components to the tubes, mix them, vortex and refrigerate overnight.

[0139] 2. After preparation, place all Eppendorf tubes in a 50°C water bath.

[0140] After 2 minutes, 5 minutes, 10 minutes, 20 minutes, 40 minutes, or 60 minutes, each of the eight tubes in each of the three groups of tubes A to C was removed from the water bath.

[0141] 3. Then add 300 μL of PNPP to all Eppendorf tubes in all groups A to C. Mix the tubes and vortex them. Then place them in a 37°C water bath for 30 minutes, during which time dephosphorylation of PNPP occurs.

[0142] 4. Afterwards, all samples in all groups A to C can be subjected to UV-visible analysis (at 405 nm) to assess the ability to stabilize the active protein component (ALP).

[0143] Imaging of particles

[0144] To better understand the configuration of the hybrid lipid particles of the present invention, TEM images were obtained.

[0145] Figure 4is an example of one of the images and shows the hybrid lipid particles before loading with active ingredients. The large spheres with a diameter of about 120 nm are lipid particles and the smaller particles are silicon. It can be seen that the silicon is irregular and the particles are significantly smaller. It can also be seen that while some silicon particles may be embedded in the lipid particles, some are associated with the surface of the lipid particles. It is worth noting that this image was obtained after filtration, which means that any silicon present is sufficiently well associated with the lipid particles to survive the filtration process.

Claims

1. A method for making a hybrid lipid particle suitable for delivering a fragile active ingredient (e.g., for delivering a nucleic acid), the method comprising the following steps: A. Blending one or more lipid components, then B. forming the lipid blend into a plurality of lipid particles having an average diameter of 50 nm to 400 nm, and contacting the plurality of hybrid lipid particles with inorganic material particles having an average diameter of 10 nm to 100 nm; The average diameter of the hybrid lipid particles is at least 150% of the average diameter of the inorganic material particles.

2. The method of claim 1, wherein the inorganic material particles are less than one quarter of the diameter of the hybrid lipid particles, and wherein they are not encapsulated by lipids or are not completely encapsulated by lipids.

3. The method according to claim 1 or claim 2, wherein step A comprises blending one or more cationic lipids or ionizable lipids with one or more other lipids selected from neutral lipids and polar lipids and optionally one or more additional lipid components.

4. The method according to claim 1 or claim 2 or claim 3, wherein the method comprises the following steps as an additional step after step B: C. contacting the hybrid lipid particle with a solution of an active ingredient (e.g., a nucleic acid molecule) having a net negative charge at pH 7.4, so that the active ingredient (e.g., a nucleic acid molecule) is electrostatically bound to the cationic charge of the cationic lipid and / or ionizable lipid, thereby binding to the surface of the hybrid lipid particle.

5. The method according to claim 1, 2, 3 or 4, wherein the inorganic material particles are particles containing hydrolyzable silicon.

6. A method according to claim 5, wherein the hydrolysable silicon-containing particles comprise at least 90% (by weight) elemental silicon, and the elemental silicon is optionally doped with boron.

7. according to claim 3 or according to any one of claims 4 to 6 method dependent on claim 3, wherein the one or more cationic lipids or ionizable lipids are cationic lipids selected from the following items: DOTAP, DODAC, SA or DOTMA or their mixture.

8. The method of claim 7, wherein the cationic lipid or ionizable lipid is DOTAP.

9. A method according to claim 3 or according to any one of claims 4 to 8 when dependent on claim 2, wherein the one or more other lipids are polar phospholipids selected from the group consisting of DOPE, PC and phosphatidylcholine or mixtures thereof.

10. The method of claim 9, wherein the polar phospholipid is DOPE.

11. A method according to claim 3 or according to any one of claims 4 to 10 when dependent on claim 2, wherein the one or more additional lipid components are selected from steroids / sterols (optionally of animal or non-animal origin) and conjugated lipids.

12. A method according to claim 3 or according to any one of claims 4 to 11 as appended to claim 32, wherein the one or more additional lipid components are absent or present at less than 0.1% (by molar ratio) of the total lipids present.

13. A method according to claim 45 or according to any one of claims 6 to 12 when dependent on claim 45, wherein the particles containing hydrolysable silicon are particles comprising mesoporous silicon (eg at least 90 wt% mesoporous silicon). The method of claim 13 , wherein the particles comprising mesoporous silicon comprise boron-doped mesoporous silicon.

15. The method according to any one of claims 1 to 14, wherein the hybrid lipid particle consists of 40% to 60% DOPE and 40% to 60% DOTAP expressed as a molar ratio of the total lipid content.

16. A method according to claim 3 or according to any one of claims 4 to 14 when dependent on claim 3, wherein the active ingredient is DNA.

17. A method according to claim 4 or according to any one of claims 5 to 15 when dependent on claim 4, wherein the active ingredient is mRNA.

18. The method of claim 17, wherein the mRNA encodes an antigen or a portion of an antigen (eg, a tumor-associated antigen or a tumor-specific antigen) for an infectious disease or cancer.

19. The method of claim 17, wherein the mRNA encodes an antigen or a portion of an antigen of a pathogen, such as a viral pathogen, such as a spike protein antigen or a portion thereof of SARS-CoV-2.

20. The method of claim 4 or any one of claims 5 to 15 as dependent upon claim 4, wherein the nucleic acid is siRNA or saRNA or hairpin RNA.

21. The method of claim 20, wherein the siRNA inhibits the expression of a gene associated with a disease in a subject.

22. A method according to claim 34 or according to any one of claims 5 to 21 as dependent on claim 4, wherein more than 90% of the active ingredients (e.g., nucleic acids) present are bound to the outer surface of the hybrid lipid particles and less than 10% of the total active ingredients (e.g., nucleic acids) present are encapsulated within the hybrid lipid particles.

23. according to the method described in any one in claim 1 to 22, described method comprises the other step of freeze-drying described hybrid lipid particle after step A and B or when step C exists after step A to C.

24. according to the method described in any one of claims 1 to 19, described method comprises after step A to C and when exist, optionally after any freeze drying step other step D, wherein step D comprises described hybrid lipid particle is formulated into pharmaceutical composition (it can optionally comprise freeze dried hybrid lipid particle is suspended in hydrogel).

25. The method of claim 23, comprising the further step of administering the pharmaceutical composition to a subject in need thereof after steps A to D.

26. The method according to any one of claims 1 to 25, wherein one or more of steps A to D are performed in the presence of one or more amino acid molecules and / or one or more non-reducing disaccharide molecules.

27. The method of claim 26, wherein the one or more amino acid molecules comprise glycine and the one or more non-reducing disaccharide molecules comprise trehalose.

28. according to the method described in any one of the preceding claims, wherein the lipid granules have a mean diameter of 80nm to 200nm, and the inorganic material particles (preferably containing particles of hydrolyzable silicon) have a mean diameter of 5nm to 50nm.

29. according to the method for claim 27, wherein said hybrid lipid particles have an average diameter of 150nm to 250nm, and said inorganic material particles (preferably particles containing hydrolyzable silicon) have an average diameter of 10 to 50nm, for example 20nm to 45nm.

30. A plurality of hybrid lipid particles, comprising a blend of one or more cationic lipids or ionizable lipids with one or more polar lipids and optionally one or more additional lipid components, wherein the hybrid lipid particles have an average diameter of 50 nm to 400 nm and have a coating of particles containing hydrolyzable silicon, the particles containing hydrolyzable silicon having an average diameter of 10 nm to 100 nm, the plurality of hybrid lipid particles having the ability to bind nucleic acid molecules to their surface and optionally comprising nucleic acid molecules bound to their surface, wherein the average diameter of the hybrid lipid particles is at least 150% of the average diameter of the inorganic material particles.

31. according to the multiple hybrid lipid particles described in claim 30, described multiple hybrid lipid particles also comprise active pharmaceutical ingredient bonded to its surface.

32. according to the multiple hybrid lipid particles described in claim 30, wherein the active ingredient comprises a nucleic acid molecule.

33. A plurality of hybrid lipid particles according to claim 32, wherein the nucleic acid molecule is as defined in any one of claims 15 to 20.

34. according to a plurality of hybrid lipid particles described in any one of claims 30 to 31, wherein the lipid and relative amount thereof are as defined in any one of claims 7 to 12.

35. according to a plurality of hybrid lipid particles described in any one of claims 30 to 34, the plurality of hybrid lipid particles have an average diameter of 80nm to 200nm, and wherein the hydrolyzable silicon particles have an average diameter of 5nm to 50nm.

36. according to the multiple hybrid lipid particles described in claim 35, described multiple hybrid lipid particles have the average diameter of 150nm to 250nm, and wherein said hydrolyzable silicon particles have the average diameter of 10nm to 50nm, for example 20nm to 45nm.

37. A pharmaceutical composition comprising a plurality of hybrid lipid particles according to any one of claims 30 to 36 and a pharmaceutically acceptable carrier.

38. A method of providing vaccination to an individual in need thereof, the method comprising administering a pharmaceutical formulation according to claim 31, wherein the active ingredient is an mRNA molecule encoding an antigen or a portion of an antigen of a pathogenic organism or virus, or wherein the active ingredient is a peptide or protein antigen or a portion of an antigen.

39. A method of providing vaccination to an individual in need thereof, the method comprising administering a pharmaceutical formulation according to claim 36, wherein the active ingredient is an mRNA molecule encoding a tumor antigen or a portion of a tumor antigen, or the active ingredient is a peptide or protein tumor antigen or a portion thereof.

40. A method for treating a disease associated with the expression of a gene in an individual, the method comprising administering to the individual the pharmaceutical preparation according to claim 37, wherein the active ingredient is siRNA capable of silencing the gene.

41. Use of a plurality of hybrid lipid particles according to any one of claims 30 to 36 or a pharmaceutical composition according to claim 37 as a medicament.

42. Use of a plurality of hybrid lipid particles or a pharmaceutical composition according to claim 41, for use as a vaccine.

43. Use of a plurality of hybrid lipid particles according to any one of claims 30 to 31 or a pharmaceutical composition according to claim 37 in the manufacture of a medicament.

44. Use of a plurality of hybrid lipid particles according to any one of claims 30 to 31 or a pharmaceutical composition according to claim 37 in the manufacture of a vaccine.

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