Lipid preparations

Abstract

A method for producing hybrid lipid particles suitable for the delivery of fragile active ingredients such as nucleic acids, comprising the steps of creating hybrid lipid particles and then "dusting" their surfaces with particles of inorganic material, as well as related hybrid lipid particles, uses, and products, the important feature of which is that the particles of inorganic material are present on and within the lipid particles rather than being encapsulated by them, and the active ingredient does not need to be encapsulated either.

Description

[Technical Field]

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

[0002] Lipid particles, including lipid nanoparticles (LNPs), hold great promise as vectors for the delivery of therapeutic nucleic acids, and in particular for the delivery of therapeutic RNA.

[0003] For example, many compounds that may be useful as active pharmaceutical ingredients are prone to degradation. Nucleic acids such as RNA are particularly prone to degradation during formulation and storage. Various pharmaceutical compositions have been proposed to limit the extent of RNA degradation. One way to limit RNA degradation is to encapsulate RNA in a protective organic envelope of lipids. For example, European Patent No. 3677567 (A1) discloses lipid particles in which mRNA molecules are encapsulated within the particles.

[0004] The present invention is based in part on recognizing and avoiding some of the technical challenges of encapsulating nucleic acids in lipid particles. Chief among these challenges is the need to form the lipid particles at elevated temperatures. The exact temperature required depends on the lipid used, but temperatures of about 60°C are typically required. At such elevated temperatures, significant degradation of RNA and other sensitive active ingredients occurs. This can be countered to some extent by the use of modified active ingredients, e.g., modified RNA. It is noteworthy that U.S. Patent No. 9,504,651 (B2) discloses a method for forming lipid particles "around" mRNA, in which at least 70% of the mRNA is encapsulated. Of the unencapsulated 30%, much appears to be degraded.

[0005] Maintaining lipid particle "stability" also presents challenges. Improving "stability" involves both countering the tendency of smaller lipid particles to coalesce into larger particles and countering the tendency of charged lipids that are part of the lipid particle to lose their charge. Much research has been conducted in the field of producing new lipids with advantageous properties and developing formulations of multiple lipids with advantageous properties in their ability to form stable particles. One advantage of the present invention is the improvement of lipid particle stability. Another advantage of the present invention is that it provides a method for improving particle stability that is less dependent on the use of specific novel lipids, whose use may be subject to technical challenges, supply constraints, and intellectual property restrictions. Using the methods and products of the present invention, it is possible to form sufficiently stable lipid particles using a wider range of lipids, including lipids that are more readily available at lower cost than some of the specialized lipids that prior art methods and products may require to obtain lipid particles with sufficient performance. Conversely, the present invention may also be used with advantageous prior art "high-performance" specialized lipids to achieve even better performance.

[0006] The lipids in the lipid particles can be organized in various ways to form liposomal or micelle particles. Liposomal particles consist essentially of a bilayer of lipid molecules. Micellar particles generally have lipids in a non-bilayer configuration. The particles of the present invention are referred to herein as "hybrid lipid particles," meaning that they may have regions of micellar lipid configuration and regions of liposomal lipid configuration. Depending on the 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 are present in a liposomal (bilayer) configuration, meaning that 80% to 100% (by weight) of the total lipids are present 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 the idea that organic particles, particularly silicon particles (and particles containing hydrolyzable silicon), can be used to stabilize hybrid lipid particles, inhibit their coalescence, help cationic lipids and / or ionizable lipids maintain their charge, and promote the ability of this hybrid lipid particle to protect nucleic acid by stabilizing nucleic acid on the surface of this hybrid lipid particle, not by encapsulation.This arrangement advantageously allows this hybrid lipid particle to be prepared in the absence of nucleic acid, and nucleic acid can be added to the particle only after they are completely formed and any process involving elevated temperature is completed.

[0008] The background art discloses various lipid particles for use in various methods. For example, Chinese Patent No. 106177892 discloses hyaluronic acid-modified lipid particles with a silica mesoporous core. These particles are disclosed as being complexed with drugs by mixing the silica core with the drug and then adding lipids in a subsequent step. Chinese Patent No. 114146188 discloses the preparation of CoQ10 as a medically useful compound. It discloses the preparation of a silica-to-silicon carrier, liposome encapsulation, and then the addition of CoQ10. U.S. Patent Application Publication No. 2022 / 183989, International Publication No. 2020 / 193999, and Baran-Rachwalska et al. (2020) J. Cont. Rel: 326: 192-202 all disclose various particles, including lipid-coated silicon or silica particles.

[0009] It may be noted that these particles are essentially composed of lipid surrounding a silica / silicon core, and apparently, this composition results in the particles being typically only slightly larger overall than the core. Summary of the Invention

[0010] According to a first aspect of the present invention, there is provided a method of producing hybrid lipid particles suitable for delivery of fragile active ingredients (e.g., delivery of nucleic acids), comprising the steps of: A. Blending one or more lipid components, then B. forming the lipid blend into a plurality of hybrid lipid particles having an average diameter of between 50 nm and 400 nm, and contacting the plurality of lipid particles with particles of an inorganic material having an average diameter of between 10 nm and 100 nm; wherein the hybrid lipid particles have an average diameter that is at least 150% of the average diameter of the particles of the inorganic material.

[0011] According to a second aspect of the present invention, there is provided a plurality of hybrid lipid particles comprising a blend of one or more lipid components, the hybrid lipid particles having an average diameter of 50 to 400 nm and a coating of particles of inorganic material having an average diameter of 10 to 100 nm, the plurality of hybrid lipid particles being capable of binding molecules of an active ingredient (such as nucleic acid molecules) to their surface, and optionally comprising molecules of an active ingredient (such as nucleic acid molecules) bound to their surface, and the hybrid lipid particles having an average diameter that is at least 150% of the average diameter of the particles of inorganic material.

[0012] In further aspects, the present invention provides: A pharmaceutical composition comprising a plurality of hybrid lipid particles according to the first aspect of the present invention and a pharmaceutically acceptable carrier.

[0013] A method of providing vaccination to an individual in need thereof, comprising administering the pharmaceutical formulation of the invention, wherein the active ingredient is a nucleic acid that is an mRNA molecule that encodes an antigen or part of an antigen of a pathogenic organism or virus, or the active ingredient is a peptide or protein antigen of a pathogenic organism or virus.

[0014] A method of providing vaccination to an individual in need thereof, comprising administering the pharmaceutical formulation of the invention, wherein the active ingredient is a nucleic acid that is an mRNA molecule encoding a tumor antigen or a part of a tumor antigen, or wherein the active ingredient is a protein or peptide tumor antigen or a part thereof.

[0015] A method of treating a disease associated with the expression of a gene in an individual by administering to the individual the pharmaceutical formulation of the present invention, wherein the nucleic acid molecule is an siRNA capable of silencing the gene.

[0016] A plurality of hybrid lipid particles of the present invention, or a pharmaceutical composition of the present invention, for use as a medicament.

[0017] A plurality of hybrid lipid particles or pharmaceutical compositions of the present invention for use as a vaccine.

[0018] Use of a plurality of hybrid lipid particles of the present invention or a pharmaceutical composition of the present invention in the manufacture of a medicament.

[0019] Use of a plurality of hybrid lipid particles of the present invention or a pharmaceutical composition of the present invention in the manufacture of a vaccine. [Brief explanation of the drawings]

[0020] [Figure 1] FIG. 1 is a schematic diagram of the proposed mechanism of action of the present invention. [Figure 2] 1 shows data regarding RNA stability when RNA is incorporated into hybrid lipid particles of the present invention. [Figure 3] 1 shows a schematic diagram of the manufacture of certain products of the present invention. [Figure 4] 1 is a TEM photograph of a hybrid lipid particle of the present invention.

[0021] As used herein, a hybrid lipid particle refers to a particle comprising at least some lipid (as defined herein), wherein the lipid molecules (in preferred embodiments, at least 80% by weight) are present so as to arrange themselves in a hybrid structure that is not a lipid bilayer (i.e., a structure that has at least some micellar properties). Hybrid lipid particles differ from liposomes, which contain lipid bilayers, although the term "hybrid lipid particle," as used herein, allows for at least some liposome-type structure within the particle.

[0022] The term "hybrid" is intended to describe lipid particles in which at least some lipid is in a configuration other than a lipid bilayer. The amount of lipid in such a configuration may be 80-0% (preferably less than 20%) of the total lipid by percentage and weight. DETAILED DESCRIPTION OF THE INVENTION

[0023] According to a first aspect of the present invention, there is provided a method of making hybrid lipid particles suitable for delivery of an active ingredient, such as a fragile active ingredient (e.g., nucleic acid delivery), comprising: A. Blending one or more lipid components, then B. forming the lipid blend into a plurality of lipid particles having an average diameter of 40 nm to 400 nm; and contacting a plurality of inorganic material, such as lipid particles, with particles of inorganic material having an average diameter of 10 nm to 100 nm; Methods are provided wherein the hybrid lipid particles have an average diameter that is at least 150% of the average diameter of the particles of the inorganic material.

[0024] According to a preferred embodiment of the first aspect of the present invention, there is provided a method of producing hybrid lipid particles suitable for delivery of an active ingredient, such as a fragile active ingredient (e.g., nucleic acid delivery), comprising: A. blending one or more cationic or ionizable lipids with one or more further lipids selected from neutral lipids and polar lipids, and optionally one or more additional lipid components; B. forming the lipid blend into a plurality of lipid particles having an average diameter of between 40 nm and 400 nm, and contacting the plurality of lipid particles with particles comprising hydrolyzable silicon having an average diameter of between 10 nm and 100 nm; and C. Contacting the hybrid lipid particles with a solution of an active ingredient (e.g., a solution of nucleic acid molecules) such that the molecules of the active ingredient (e.g., nucleic acid molecules) electrostatically bind to the cationic charges of the cationic lipids and ionizable lipids, and thus to the surface of the hybrid lipid particles.

[0025] According to a second aspect of the present invention, there is provided a plurality of hybrid lipid particles comprising a blend of one or more lipid components, the hybrid lipid particles having an average diameter of 50 to 400 nm and a coating of particles of inorganic material having an average diameter of 10 to 100 nm, the plurality of hybrid lipid particles having the ability to bind molecules of an active ingredient, such as nucleic acid molecules, to their surface, and optionally comprising molecules of an active ingredient, such as nucleic acid molecules, bound to their surface, and the hybrid lipid particles having an average diameter that is at least 150% of the average diameter of the particles of inorganic material.

[0026] Without wishing to be bound by theory, it is suggested that the hybrid lipid particles of the present invention are held together by a combination of electrostatic force, covalent bond force, and van der Waals force.It is believed that the organic part of the particle stabilizes the inorganic part through the formation of resonance bands, for example, carboxylic acid groups, where positive charges are delocalized between bands.When an active ingredient such as nucleic acid binds to this delocalized charge, it condenses.As charge increases, condensation increases.The presence of inorganic material particles increases hydrogen banding, promoting active ingredient binding and allowing better condensation of nucleic acid.

[0027] According to a preferred embodiment of the second aspect of the present invention, there is provided a plurality of hybrid lipid particles comprising a blend of one or more cationic or ionizable lipids, one or more polar lipids, and optionally one or more additional lipid components, wherein the hybrid lipid particles have an average diameter of 50 to 400 nm, preferably 80 nm to 400 nm, and coating particles comprising hydrolyzable silicon having an average diameter of 10 nm to 60 nm, preferably 10 nm to 60 nm, wherein the plurality of hybrid lipid particles are capable of binding nucleic acid molecules to their surface and optionally comprise molecules of an active ingredient, such as nucleic acid molecules, bound to their surface, and wherein the hybrid lipid particles have an average diameter that is at least 150% of the average diameter of the particles of the inorganic material.

[0028] In a further aspect, the present invention is as described above.

[0029] Lipids are generally understood in the art to include fatty acids and fatty acid derivatives, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, and polyketides.

[0030] As used in the present application, the term "lipid" also covers lipidated oligopeptides (a term used interchangeably herein with the term lipopeptide) in which a short peptide sequence (e.g., a peptide sequence having 3 to 20 amino acid residues, e.g., 5 to 15 amino acid residues, particularly 3, 4, or 5 amino acid residues, most particularly 5 amino acid residues) is conjugated to one or more fatty acid chains (particularly fatty acid chains having a carbon chain length of 10 to 24, preferably 12 to 18 carbon chain lengths; e.g., 14, 15, or 16 carbon chain lengths; e.g., the peptide moiety may optionally be lipidated with a palmitoyl, cetyl, or myristoyl moiety).

[0031] 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., two or three 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., two) lysine residues. Thus, a particular example is palmitoyl-pentapeptide-4 (CAS No. 214047-00-4). [ka]

[0032] Thus, 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 having one or more amino acid residues that are cationic at a pH of 7.4 (physiological pH; examples include lysine and arginine). Depending on the characteristics of the peptide component of the lipopeptide, the lipopeptide may be a cationic lipid, an ionizable lipid, a neutral lipid, or a polar lipid. According to various embodiments of the present invention, the requirement for one or more of a cationic lipid, an ionizable lipid, a neutral lipid, or a polar lipid may be met by a suitable lipopeptide. Alternatively, or in addition, one or more additional lipid components of the methods and products of the present invention may be provided by a suitable lipopeptide.

[0033] Lipidated oligopeptides may be used in combination with one or more phospholipids, such as DOPE or DPPC. The alkyl chains of the lipopeptides may be advantageously incorporated into the phospholipid bilayer, while the surface of the bilayer may be decorated with peptide moieties. In this way, the peptides may provide tissue and / or cell targeting, and may stabilize negatively charged active ingredients, such as nucleic acids, such as mRNA, if the peptides carry a cationic charge at physiological pH.

[0034] However, it has been found that DOTAP is not necessary for the present formulations, 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 a pH of 7.4 (physiological pH; examples include lysine and arginine). Optionally, one or more sugars (particularly trehalose) and / or one or more amino acids (particularly glycine) are also present.

[0035] Alternatively, in other preferred embodiments, the one or more lipids are or include one or more phospholipids (such as DPPC and / or DOPE), 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 (especially trehalose) and / or one or more amino acids (especially glycine) are also present.

[0036] In accordance with all aspects of the present invention, the blend of one or more cationic or ionizable lipids with one or more neutral or polar lipids comprises at least one cationic or ionizable lipid. Preferably, the total cationic or ionizable lipids (as a molar ratio) is 20-70% of the total lipids, e.g., 30-60%, or 40-60%.

[0037] According to certain embodiments, the cationic lipid or ionizable lipid is a cationic lipid. In addition to suitable lipopeptides, the cationic lipid may be selected from the group consisting of DOTAP (dioleoyl-3-trimethylammonium propane, 18:1 TAP), DODAC (dimethyldioctadecylammonium chloride):SA (stearylamine, octadecylamine) and DOTMA (9-trimethyl[2,3-(dioleyloxy)propyl]ammonium chloride), and any mixture thereof. A mixture containing DOTAP is 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.

[0038] According to another embodiment, the cationic lipid or ionizable lipid is an ionizable lipid. The ionizable lipid may be selected from the group consisting of [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate), 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoic acid heptadecan-9-yl, 2-octyldecanoic acid 7-[(2-hydroxyethyl)[8-(nonyloxy)-8-oxooctyl]amino]heptyl, 4-(dimethylamino)butanoic acid (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl, and DODMA, and any mixture thereof.

[0039] According to certain embodiments, the cationic lipid or ionizable lipid component may 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).

[0040] The lipid blend may optionally further comprise one or more neutral or polar lipids. The neutral phospholipids DOPE (dioleoyl phophatrolylethanolamine), PC (phosphatrdyl chlorine), and lecithin (a mixture predominantly consisting of PC) are all examples of non-cationic phospholipids that may be used as neutral or polar lipids according to the present invention.

[0041] According to certain preferred embodiments, the lipid blend is entirely or primarily cationic lipids and phospholipids. For example, the lipid blend may consist of approximately equal amounts of DOTAP and DOPE. In another example, the lipid blend may consist of cationic lipopeptides.

[0042] The lipid blend may optionally further comprise, as an additional lipid component, a conjugated lipid, e.g., a PEGylated lipid, PEI, Brij58 (a surfactant), and / or a steroid / sterol component such as cholesterol. According to certain preferred embodiments, the lipid blend does not contain a substantial amount of conjugated lipid or a substantial amount of steroid / sterol. In certain embodiments, the additional lipid may comprise 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 or microbial origin).

[0043] The lipid flexure may optionally or additionally further comprise mitoxantrone (MTO) or a derivative thereof such as dipalmitoyl MTO or monopalmitoyl MTO.

[0044] particle size In accordance with all embodiments, the average diameter of the hybrid lipid particles of the present invention is significantly larger than the average diameter of the particles of inorganic material. According to preferred embodiments, the average diameter of the hybrid lipid particles is between 80 nm and 400 nm, and the average diameter of the particles of inorganic material is between 10 nm and 60 nm. In these embodiments (and other embodiments in which one or both of the average diameters are more narrowly defined), the characteristic of the hybrid lipid particles having an average diameter that is at least 150% of the average diameter of the particles of inorganic material is optional.

[0045] Preparation of hybrid lipid particles The hybrid lipid particles of the second aspect of the present invention can be produced by any suitable method. They can optionally be produced by the method of the first aspect of the present invention. For example, the lipid blend described above can be prepared. In certain embodiments, this can be prepared by blending lipids in a suitable solvent. Optionally, the lipid blend in the solvent can be dried by evaporating the solvent using any suitable method. A rotary evaporator can optionally be used to produce a thin layer of the anhydrous lipid blend, or this can be any other method known in the field of lipid particle production, such as microfluidizer filtration or tangential flow filtration (TFF). The 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 the introduction of an active ingredient such as a nucleic acid, and therefore can be carried out safely and optionally with elevated temperatures and chemical conditions that are not conducive to the stability of the active ingredient (e.g., elevated temperatures and chemical conditions that are not conducive to RNA stability and tend to cause degradation and loss of RNA molecules).

[0046] Following the formation of the hybrid lipid particles, the present invention and related methods provide for stabilizing the newly formed hybrid lipid particles by treating them with inorganic particles, preferably hydrolyzable silicon particles. Such treatment may be described as "coating." By "coating," it is understood that the inorganic particles (preferably hydrolyzable silicon particles) are primarily present at or toward the surface of the hybrid lipid particles. They may be partially embedded within the lipid particles, but are at least partially accessible on the surface of the hybrid lipid particles. Preferably, the inorganic particles (hydrolyzable silicon particles) have a diameter significantly smaller than that of the hybrid lipid particles. For example, the inorganic particles (hydrolyzable silicon particles) may be at least half, at least one-third, or at least one-quarter, or even at least one-sixth, one-eighth, or one-tenth the average diameter of the hybrid lipid particles. The inorganic particles (hydrolyzable silicon particles) can be mixed with the hybrid lipid particles in a wider range of ratios. For example, the inorganic particles (hydrolyzable silicon particles) may be present in an amount of about 0.1 to 10 times the amount of lipid.

[0047] filtration After adding inorganic particles (for example, hydrolyzable silicon particles) to lipid particles, the resulting particles containing inorganic particles (preferably, hydrolyzable silicon particles) coating can be optionally filtered.According to the first aspect of the present invention, a filtration step can be inserted between step B and step C.Preferably, this is carried out by using tangential flow filtration, but any suitable filtration process can be used.One purpose of filtration is to increase the size uniformity of hybrid lipid particles by means of filtering through a membrane with a size cutoff at desired particle size (for example, 100 nm).

[0048] After filtration, the total proportion of the weight of the hybrid lipid particles that is composed of inorganic particles (hydrolyzable silicon) may be somewhat reduced.

[0049] Note on particle size In this specification, particle size is described as average diameter.Particle size can be measured by any suitable method, including electron microscopy and size exclusion.Preferably, particle has a diameter distribution around the average diameter, that is, 80% of particles have a diameter within ±25% of the average diameter.This is especially true after filtration, which is known to increase monodispersity.

[0050] Hydrolyzable silicon As used herein, "hydrolyzable silicon" includes pure elemental silicon. However, complete purity is not required. Rather, the present invention is not intended to encompass pure silica (including sand, quartz, and silica gel). The key requirement is that the material be hydrolyzable, i.e., tend to decompose into soluble products such as orthosilicic acid (OSA) under physiological conditions. According to certain embodiments, the definition of "particles comprising hydrolyzable silicon" is met when at least half of the mass of the material is hydrolyzed into soluble products within one month of injection into a subject (e.g., after intramuscular or subcutaneous injection).

[0051] The hydrolyzable silicon according to the invention is preferably mesoporous, i.e. it contains pores with a diameter of between 2 and 50 nm.

[0052] Particles containing hydrolyzable silicon can be purchased commercially or produced by any suitable method. For example, bulk silicon can be reduced by milling or electroetching. Silicon particles or desired size can be obtained by any suitable method, for example, by using an air classifier.

[0053] Doped Silicon Preferably, hydrolyzable silicon for use in all aspects of the present 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 highly concentrated, which is understood to mean that one or more dopant atoms are added per 10,000 silicon atoms, or even "super-doped", e.g., 10 20 atoms / cm 3 The boron doping of the hybrid systems relies on the need to generate different zeta charges inside the crystalline matrix and / or on the surface of the hybrid systems.

[0054] The semiconductor industry provides a ready source of appropriately doped silicon as well as the techniques for doping.

[0055] Active ingredient In a preferred embodiment, the active ingredient of the present invention is an active pharmaceutical ingredient. While multiple active ingredients may be present in a single formulation according to the present invention, each embodiment of the present invention more typically relates to a single API. According to certain preferred embodiments, the active ingredient is a nucleic acid API. Most preferably, this is RNA, such as mRNA, siRNA, haRNA, or saRNA. In a preferred embodiment, the active ingredient is negatively charged (i.e., has a net negative charge at physiological pH). The active ingredient (API) according to the present invention may preferably be a fragile active ingredient. A fragile active ingredient may be understood to be an active ingredient with poor stability. For example, the active ingredient has poor stability at 20°C and pH 7.4, such as a half-life of less than 6 days or less than 1 day.

[0056] Binding of nucleic acids to silicon lipid particles In contrast to prior art lipid particles that attempt to encapsulate active ingredients such as nucleic acids within the lipid structure, the hybrid lipid particles of the present invention bind active ingredients such as nucleic acids to their surface.Due to the electrostatic forces and concentration gradients of the resulting organic-inorganic hybrid system, the active ingredients tend to be incorporated into the silicon matrix and surface.Since nucleic acids can act as charge stabilizers themselves, ionized drugs, whether hydrophobic or hydrophilic, tend to distribute themselves along the surface of mesoporous silicon.The regions of mesoporous silicon functionalized with nucleic acids tend to associate with hydrophilic ionized drugs due to the resonance of electrons generated on the surface, and polar molecules interact through dipole-dipole intermolecular forces and hydrogen bonds.

[0057] Binding of active ingredients, such as nucleic acids, to the surface is possible due to electrostatic attraction between negatively charged active ingredients, such as negatively charged nucleic acids, and the positive charges adjacent to the head groups of the cationic lipids that make up the hybrid lipid particles and / or within the silicon matrix.

[0058] While it may be possible to achieve a certain level of electrostatic coordination using prior art lipid particles containing cationic lipids but no inorganic material (preferably hydrolyzable silicon), it is difficult to maintain long-term attraction of active ingredients such as nucleic acids with these particles because cationic lipids tend to lose their charge during storage. This is a process known as lipid aging. The presence of an inorganic material (preferably hydrolyzable silicon) according to the present invention allows charged lipids, including cationic lipids or ionizable lipids, to maintain their charge by stabilizing it through electrostatic coordination. To create stable charges and complexes, the inventors further note that the importance of the degree of nucleic acid binding on the silicon particle surface also depends on the surface contact angle. The surface contact angle and the resulting dissolution behavior depend on the "ratio" of hydroxide and oxide terminal fragments on the silicon particle surface. Therefore, careful functionalization of the inorganic silicon particle surface is required to control the dissolution rate, stabilize the charge, and ultimately release kinetics, as a result of a combination of both the size and surface chemistry of the Si pores.

[0059] Nucleic acids for use in the present invention The present invention is suitable for use with any active ingredient. Preferably, the active ingredient is a nucleic acid. The present invention is particularly useful for use with RNA, as RNA is particularly susceptible to degradation without the protection provided by the present invention. Thus, 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 also optionally be mRNA. For example, it may be mRNA encoding a vaccine antigen. The RNA may optionally be chemically 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, as such treatment has been found to be unnecessary.

[0060] According to certain embodiments of all aspects of the present invention, the nucleic acid is DNA. According to other preferred embodiments, the nucleic acid is RNA. It may be siRNA or mRNA. It may be of any suitable length, but typically 10-30 nucleotides for siRNA or 200-2000 nucleotides for mRNA. It may be double-stranded or single-stranded, or, particularly in the case of siRNA, it may be chemically single-stranded but have one or more regions of base pairing (and optional unpaired overhangs). It may optionally be chemically modified (e.g., by using N1-methylpseudouridine substitutions) or its sequence may be modified (e.g., by UTR truncation). Preferably, the nucleic acid (i.e., RNA) may be unmodified (particularly chemically unmodified), as this may be unnecessary for providing stability.

[0061] Particle composition In all aspects, the hybrid lipid particles of the present invention preferably have the following components in addition to the hybrid lipid structure: The hybrid lipid particles have inorganic material particles (preferably hydrolyzable silicon particles) that are incorporated into the lipid bilayer and are partially or entirely exposed on the particle surface so as to be accessible for interaction with nucleic acids. Preferably, at least 50% of the total inorganic material particles (preferably hydrolyzable silicon particles) are accessible on the surface of the lipid particle and are not completely encapsulated within the micellar structure. If present, active ingredients such as nucleic acids are primarily located in an electrostatically bound state on the surface of the particle. For example, more than 90% of the total active ingredients such as nucleic acids present are bound to the surface of the particle, and less than 10% are encapsulated within the lipid structure before fully condensing to form complexes. In some embodiments, 0 or substantially 0 (e.g., less than 0.5%) of the total active ingredients such as nucleic acids present are encapsulated within the structure.

[0062] Additional ingredients It has been found that the stability of hybrid lipid particles can be further increased in the presence of one or more amino acids and / or one or more non-reducing disaccharides.Therefore, the method of the present invention can optionally be 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 can further comprise non-reducing disaccharides and / or amino acids.A preferred non-reducing disaccharide is trehalose.A preferred amino acid is glycerin.In certain preferred embodiments, the use of both glycine and trehalose is preferred.These components can be electrostatically coordinated with particles of inorganic material (preferably hydrolyzable silicon), which may increase the overall stability of the system.

[0063] Particle Stability The hybrid lipid particles of the present invention show enhanced dimensional stability compared with the corresponding particles that do not have inorganic material (preferably hydrolyzable silicon) particles according to the present invention.This enhanced dimensional stability is manifested in the resistance of particles to coalescence into larger particles.According to a certain embodiment, the coalescence rate at 5 ℃ is at least half of the coalescence rate of the corresponding particles (with the same composition but without inorganic material (preferably hydrolyzable silicon particles) according to the present invention).According to a certain embodiment, at least 90% of particles do not coalesce and maintain their original size after being stored in aqueous solution at 5 ℃ and physiological pH for 3 months.

[0064] Charge Stability The surface charge of hybrid lipid particles can be estimated using the parameter of zeta potential (electrokinetic potential). As a rule of thumb, suspensions of particles with low zeta potential (0 to ±5 mV) are unstable and rapidly coalesce. Values of ±30 mV to ±40 mV correspond to reasonable stability, values of ±40 mV to ±60 mV correspond to good stability, and values above ±60 mV correspond to excellent stability.

[0065] According to certain embodiments, the hybrid lipid particles of the present invention have a value of more than ±40 mV, more preferably more than ±45 mV, more than ±50 mV, or more than ±60 mV. Preferably, the zeta potential is increased by at least ±10 mV due to the presence of hydrolyzable silicon (i.e., the zeta potential is at least ±10 mV, more than ±10 mV, greater than the zeta potential of lipid particles identical to that of the present invention but in the absence of particles of inorganic material (preferably hydrolyzable silicon particles) according to the present invention).

[0066] The presence of inorganic material (preferably silicon) also inhibits the loss of positive charge of cationic lipids, known as lipid aging, and preferably slows it down by at least 2, 4, 8, or 16 times (at 5° C.).

[0067] Stability of active ingredients such as nucleic acids The hybrid lipid particles of the present invention act to protect active ingredients, such as nucleic acids (especially RNA), electrostatically complexed to the surface of the hybrid lipid particles. The present invention allows for easier storage of therapeutic formulations, such as vaccines, for example, at 5°C or room temperature, as opposed to subzero temperatures. This also increases stability and reduces nucleic acid degradation during lyophilization, rehydration, transportation, and storage. According to certain embodiments of the present invention, the half-life of an active ingredient, such as mRNA, is extended by at least 100-fold, at least 1,000-fold, or at least 10,000-fold compared to the corresponding active ingredient, such as mRNA, not complexed with the hybrid lipid particles of the present invention. According to certain embodiments, the half-life of an active ingredient, such as mRNA, is extended by at least 10-fold, at least 100-fold, or at least 1,000-fold compared to the corresponding active ingredient, such as mRNA, complexed with equivalent hybrid lipid particles lacking the inorganic particle (preferably, hydrolyzable silicon) component of the present invention. The half-life can be measured in aqueous physiological solution (e.g., phosphate-buffered saline), pH 7, and 5°C.

[0068] Pharmaceutical product formulation The present invention further contemplates the use of the hybrid lipid particles of the present invention for formulating pharmaceutical products, which are also included within the scope of the present invention.Such pharmaceutical products include injections (such as injectable vaccines), topical creams, capsules, tablets, and ointments.These also include pharmaceutical precursors or products, such as dehydrated (lyophilized) and concentrated products that need to be diluted and / or rehydrated before use.

[0069] Treatment methods and treatment-related products The products of the invention may be products for use in a method of treatment or for use in a method of sample. The methods of the invention may further comprise subsequent steps that constitute a method of treatment.

[0070] Methods of treatment include treatment or prevention of disease. In some embodiments, methods of treatment may include downregulation of gene expression by siRNA. In other embodiments, methods of treatment may include vaccination, e.g., vaccination against cancer or vaccination against infectious disease by delivery of mRNA encoding an antigen (or fragment thereof) or a causative agent of the infectious disease (e.g., the spike protein of SARS-CoV-2). Alternatively, a peptide or protein antigen corresponding to a tumor antigen (or fragment thereof) may be administered as a vaccine.

[0071] Optionally, in certain preferred embodiments of all aspects of the present 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 that arises in one or more malignant cancer cells from a non-synonymous somatic mutation (resulting in a neoantigen) or a viral integration mutation (resulting in an oncoviral antigen). Thus, a tumor-specific antigen may refer to an antigen that is completely absent (not expressed) in non-cancerous (healthy, normal) cells.

[0072] Optionally, the open reading frame of the mRNA encodes a tumor-associated antigen. As used herein, the term tumor-associated antigen may refer to an antigen that is overexpressed in malignant cancer cells compared to non-cancerous (healthy, normal) cells, for example, due to gene amplification or post-translational modification. The term tumor-associated antigen may encompass overexpressed antigens (this term may refer to proteins that are moderately expressed in non-cancerous (healthy, normal) cells but abundantly expressed in malignant cancer cells), differentiation antigens (this term may refer to proteins preferentially expressed by the cell lineage from which malignant cells evolved; an example is prostate-specific antigen), and cancer-germline antigens (this term may refer to antigens that are normally restricted to reproductive tissues but are aberrantly 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 preferentially expressed antigen in melanoma (PRAME)).

[0073] If the open reading frame of the mRNA encodes a cancer-associated or cancer-specific antigen, the nucleic acid product of the invention may be suitable for use in a prophylactic or therapeutic vaccine composition.

[0074] Optionally, the open reading frame of the mRNA encodes an allergen (including but not limited to one or more nut allergens; which in turn include but are not limited to one or more seed storage proteins, e.g., vicilin, legumin, albumin; one or more plant defense-related proteins; and one or more profilins).

[0075] In some embodiments, methods of treatment may include the delivery of biologically active compounds.

[0076] Proposed mechanism of action Without wishing to be bound by theory, attention is drawn to Figure 1, which illustrates the proposed mechanism of action of the present invention in various embodiments using a formulation containing hydrolyzable silicon to explain the broader principle. The center of the figure shows a schematic representation of a particle containing hydrolyzable silicon. Selected Si atoms are highlighted, as are certain O atoms. Note that even nominally "pure" silicon elements are unlikely to have surface Si atoms bound to other chemical moieties such as O and -OH. The figure illustrates possible modes of electrostatic coordination between glycine and trehalose and the silicon and boron atoms of the hydrolyzable silicon particles. The figure also illustrates the electrostatic coordination between atoms of the hydrolyzable silicon particles and phospholipids, and between atoms of the hydrolyzable silicon particles and DOTAP as an example of a cationic lipid. The diagram illustrates a length of RNA (as an example of an active agent), which naturally carries negative charges along its length, and shows a plausible mechanism by which these negative charges, and therefore the RNA molecule itself, are stabilized by coordination with atoms of the hydrolyzable silicon particles and charges on the lipid (which are themselves stabilized by coordination with atoms of the hydrolyzable silicon particles).

[0077] Various aspects of the present invention are illustrated in the following non-limiting examples. [Example]

[0078] Example 1. Exemplary methods for making products of the present invention.

[0079] The manufacturing process of the present invention is shown schematically in Figure 3 and begins with dissolving the selected lipids DOTAP, DOPE, and mPEG2000-DSPE in methanol. At the same time, porous silicon nanoparticles are activated by exposure to methanol. The solvent is then evaporated in a slow evaporation process to produce activated SiNPs. This activation step aims to make the SiNPs suitable for dispersion in water. The activated SiNPs are then dispersed in (nuclease-free) water in the presence of trehalose (THR) and glycine (GLY). Appropriate volumes of lipids are successively transferred to a round-bottom flask, and the methanol is evaporated by rotary evaporation. As a result of this evaporation step, a lipid film is formed on the walls of the flask. The suspension containing the dispersed silicon, THR, and GLY is then added to the flask containing the lipid thin film for lipid rehydration. The mixture is then passed through 0.4 μm and 0.1 μm extrusion membranes at 60 °C. The extruded sample is then stored under refrigerated conditions. The amounts of each component are listed below. [Table 1]

[0080] Example 2. Demonstration that the presence of silicon prevents particle coalescence and promotes surface charge retention.

[0081] The particles produced according to the present invention (and comparative particles) were kept at 5°C and periodically evaluated for size (diameter in nm), size dispersity (polydispersity index - PDI), and zeta potential (ZP, indicating surface charge). Sample SIS0012 was produced using undoped silicon, SIS0013 was produced using boron-doped silicon, and NoSiNP was produced without silicon, and the results are shown below. Characterization of both size and surface charge was performed using a Malvern Zetasizer Advanced Pro series instrument. Samples were prepared using 20 μL of the particles of the present invention with 980 μL of nuclease-free water, totaling 1000 μL, and placed inside a disposable cuvette. Measurements were performed by performing three scans to reduce signal-to-noise interference. The average of the three scans was calculated as the final size measurement, along with the polydispersity index (PDI).

[0082] Zeta potential measurements were performed by mixing 200 μL of the particles of the present invention with 800 μL of nuclease-free water (for silicon-based 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. [Table 2]

[0083] It can be seen that the size of particles formulated without silicon tends to increase over time as the particles coalesce, a trend that is not seen when silicon is present.Similarly, a loss of surface charge is seen in particles formulated without silicon, which is not seen in particles with silicon present.

[0084] Example 3. Demonstration of the ability to stabilize RNA.

[0085] A stock solution of mRNA (Dasher GFP) mRNA (Lot No. 88103F, Aldevron) was prepared by dissolving mRNA powder in nuclease-free water to a concentration of 0.5 μg / μL. The mRNA was complexed with hybrid lipid particles of the present invention (prepared as in Example 1; the sample coded SIS0012 used undoped silicon, and the sample coded SIS0013 used boron-doped silicon) by mixing 50 μL (25 μg) of the mRNA solution with 200 μL of a hybrid lipid particle suspension (hybrid lipid particle / RNA weight ratio of 12:1). The mixture was incubated at room temperature for 40 minutes to allow complete complexation, and then either used in liquid form or lyophilized. The lyophilized complexes were then embedded in sodium hyaluronate hydrogel (lyophilized complexes were mixed directly with the hydrogel, while liquid complexes were analyzed as is). The final preparation was prepared as 30 μL aliquots to minimize the risk of cross-contamination during storage and analysis and was stored at either room temperature or 40°C.

[0086] After 7 days of storage, the 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. The samples were loaded onto an E-Gel™ agarose gel (1%) in an E-Gel™ Power Snap Electrophoresis Device. After 3 and 7 minutes of electrophoresis, the gel was transilluminated and imaged using an E-Gel™ Power Snap Electrophoresis camera. The total amount of mRNA loaded onto the gel for the different samples is provided in the table below. [Table 3]

[0087] As can be seen in Figure 2, the degraded mRNA is highly mobile, and the degraded fragments produce a smear on the gel, as seen with naked mRNA. The mRNA complexed with the hybrid lipid particles of the present invention fails to degrade and retains its original length, meaning that it has a much lower electrophoretic mobility and does not significantly migrate away from the loading well of the gel (at 3 and 7 minutes).

[0088] Example 4 - Use of a protein (alkaline phosphatase) in formulations of the present invention Alkaline phosphatase exists in various forms and is an enzyme that catalyzes the breakdown of various proteins and can be found in all tissues of the human body. It is mostly concentrated in bone, kidney, liver, intestine, and placenta. It contributes to, among other things, intestinal protection against bacteria, digestive function, fat and vitamin B breakdown, and bone formation. Alkaline phosphatase loses activity at low pH and high temperature.

[0089] Alkaline phosphatase activity can be monitored in an in vitro assay by measuring changes in the concentration of one or more of its substrates or products, as surrogates for UV-Vis absorbance. For example, the concentration of the substrate 4-nitrophenyl phosphatase (PNPP), whose structure is shown below, can be monitored to follow the following reaction: [ka]

[0090] Materials, methods, and results Alkaline phosphatase, isolated from bovine intestine and supplied as a 56 kD recombinant enzyme expressed in the yeast Pichia Pastoris, was obtained from Sigma-Aldrich / Merck (The Old Brickyard, New Road, Gillingham, Dorset, SP8 4XT). A stock solution of alkaline phosphatase (ALP) in water was prepared at a concentration of 1 U / ml. 1 U (μmol / min) is defined as the amount of ALP that catalyzes the conversion of 1 μmole of PNPP per minute at 37°C and pH 7.4.

[0091] A 20 mM solution of 4-nitrophenylphosphatase (PNPP) was prepared using Tris buffer (100 mM / L) at pH 7.4.

[0092] ALP solutions were prepared in 15 ml test tubes from a 1 U / ml stock solution at concentrations of 0.1, 0.5, 1, 5, 10, 50, and 100 mU / ml.

[0093] These were then mixed with the prepared 20 mM solution of PNPP in Tris buffer in an Eppendorf tube.

[0094] The tubes were incubated in a water bath at 37° C. for 30 minutes, after which UV-Vis absorbance measurements were taken at 405 nm, as shown in FIG.

[0095] ALP was loaded onto the hybrid lipid particles of the present invention as follows.

[0096] 1. Three sets of eight Eppendorf tubes were prepared. A. Eight Eppendorf tubes were prepared, each containing 50 μL of ALP (50 mU / ml) and 500 μL of hybrid lipid particles (containing undoped Si). After adding these components to the tubes, they were mixed, vortexed, and refrigerated overnight. B. Eight Eppendorf tubes were prepared, each containing 50 μL of ALP (50 mU / ml) and 500 μL of hybrid lipid particles (containing boron-doped Si). After adding these components to the tubes, they were mixed, vortexed, and refrigerated overnight. C Eight Eppendorf tubes were prepared, each containing 50 μL of ALP (50 mU / ml) and 500 μL of Tris buffer. After adding these components to the tubes, they were mixed, vortexed, and refrigerated overnight.

[0097] 2. After these were prepared, all Eppendorf tubes were placed in a water bath at 50° C. Each of the eight tubes in each of the three sets of tubes A to C was removed from the water bath after 1, 2, 5, 10, 20, 40, or 60 minutes.

[0098] 3. 300 μL of PNPP was then added to all Eppendorf tubes in all sets A to C. The tubes were mixed and vortexed. They were then placed in a 37°C water bath for 30 minutes, during which time dephosphorylation of PNPP occurred.

[0099] 4. Following this, UV-Vis analysis may be performed (at 405 nm) on all samples in all sets A-C to assess their ability to stabilize the protein active principle (ALP).

[0100] Particle Imaging To better understand the organization of the hybrid lipid particles of the present invention, TEM images were obtained.

[0101] Figure 4 is an example of one of these images, showing 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.As can be seen, the silicon is irregular and is a fairly small particle.Some silicon particles may be embedded in the lipid particles, but it can also be seen that some silicon particles are associated with the surface of the lipid particles.Please note that this image was obtained after filtration.This means that any silicon present is sufficiently well bound to the lipid particles to survive the filtration process.

Claims

1. A method for producing hybrid lipid particles suitable for the delivery of fragile active ingredients, A. A step of blending one or more lipid components, then B. A step of forming the obtained lipid blend into a plurality of lipid 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 to form a plurality of hybrid lipid particles. A method comprising the above, wherein the hybrid lipid particles have an average diameter of at least 150% of the average diameter of the particles of the inorganic material.

2. The method according to claim 1, wherein the inorganic material particles are less than one-quarter the diameter of the hybrid lipid particles, and are not encapsulated or are incompletely encapsulated by the lipid.

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

4. The above method, as an additional step after step B, C. The method according to claim 1 or 2, comprising the step of contacting the hybrid lipid particles with a solution of an active ingredient having a net negative charge at pH 7.4, so that the active ingredient electrostatically binds to the cationic charge of the cationic lipid and / or ionizable lipid, and therefore to the surface of the hybrid lipid particles.

5. The method according to claim 4, wherein the active ingredient is nucleic acid.

6. The method according to claim 1 or 2, wherein the particles of the inorganic material are particles containing hydrolyzable silicon.

7. The method according to claim 6, wherein the hydrolyzable silicon-containing particles contain at least 90% (by weight) of silicon element and are optionally doped with boron.

8. The method according to claim 3, wherein the one or more cationic lipids or ionizable lipids are cationic lipids selected from the group consisting of DOTAP, DODAC, SA, or DOTMA, or mixtures thereof.

9. The method according to claim 8, wherein the cationic lipid or ionizable lipid is DOTAP.

10. The method according to claim 3, wherein the one or more further lipids are polar phospholipids selected from DOPE, PC, and lecithin, or mixtures thereof.

11. The method according to claim 10, wherein the polar phospholipid is DOPE.

12. The method according to claim 3, wherein the one or more additional lipid components are selected from the group consisting of steroids / sterols (optionally of animal or non-animal origin) and conjugate lipids.

13. The method according to claim 3, wherein the one or more additional lipid components are absent or present in an amount less than 0.1% (molar ratio) of the total lipids present.

14. The method according to claim 6, wherein the hydrolyzable silicon-containing particles are mesoporous silicon-containing particles.

15. The method according to claim 14, wherein the particles containing the mesoporous silicon contain boron-doped mesoporous silicon.

16. The method according to claim 1 or 2, wherein the hybrid lipid particles consist of 40-60% DOPE and 40-60% DOTAP, expressed as a molar ratio of the total lipid content.

17. The method according to claim 1, wherein the active ingredient is DNA.

18. The method according to claim 4, wherein the active ingredient is mRNA.

19. The method according to claim 18, wherein the mRNA codes for an antigen or a part of an antigen of an infectious disease or cancer.

20. The method according to claim 19, wherein the cancer antigen is a tumor-associated antigen or a tumor-specific antigen.

21. The method according to claim 18, wherein the mRNA encodes an antigen or a part of an antigen of a pathogen.

22. The method according to claim 21, wherein the antigen or a part of the antigen of the pathogen is the spoke protein antigen of SARS-CoV-2 or a part thereof.

23. The method according to claim 5, wherein the nucleic acid is siRNA, saRNA, or hairpin RNA.

24. The method according to claim 23, wherein the siRNA inhibits the expression of a gene associated with the disease in the target.

25. The method according to claim 4, wherein more than 90% of the present active ingredient is bound to the outer surface of the hybrid lipid particles, and less than 10% of the total present active ingredient is encapsulated within the hybrid lipid particles.

26. The method according to claim 1 or 2, further comprising a step of freeze-drying the hybrid lipid particles following steps A and B, or, if step C is present, following steps A to C.

27. The method according to claim 4, further comprising a step D following steps A to C, and, if any, optionally following any freeze-drying step, wherein step D comprises formulating the hybrid lipid particles into a pharmaceutical composition, and optionally suspending the freeze-dried hybrid lipid particles in a hydrogel.

28. The method according to claim 27, wherein one or more of steps A to D are carried out in the presence of one or more amino acid molecules and / or one or more non-reducing disaccharide molecules.

29. The method according to claim 28, wherein the one or more amino acid molecules include glycine, and the one or more molecules of the non-reducing disaccharide include trehalose.

30. The method according to claim 1 or 2, wherein the hybrid lipid particles have an average diameter of 80 nm to 200 nm, and the inorganic material particles have an average diameter of 5 nm to 50 nm.

31. The method according to claim 29, wherein the hybrid lipid particles have an average diameter of 150 nm to 250 nm, and the inorganic material particles have an average diameter of 10 to 50 nm.

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

33. A plurality of hybrid lipid particles according to claim 32, further comprising an active pharmaceutical ingredient bound to its surface.

34. The plurality of hybrid lipid particles according to claim 32, wherein the nucleic acid molecule is siRNA, saRNA, or hairpin RNA.

35. The plurality of hybrid lipid particles according to claim 32, wherein the lipid is a cationic lipid selected from the group consisting of DOTAP, DODAC, SA, or DOTMA, or a mixture thereof.

36. A plurality of hybrid lipid particles according to claim 32, having an average diameter of 80 nm to 200 nm, wherein the particles containing hydrolyzable silicon have an average diameter of 5 nm to 50 nm.

37. A plurality of hybrid lipid particles according to claim 36, having an average diameter of 150 nm to 250 nm, wherein the particles containing the hydrolyzable silicon have an average diameter of 10 to 50 nm.

38. A pharmaceutical composition comprising a plurality of hybrid lipid particles according to claim 32 and a pharmaceutically acceptable carrier.

39. A plurality of hybrid lipid particles according to claim 32, or a pharmaceutical composition according to claim 38, for use as a pharmaceutical.

40. A plurality of hybrid lipid particles or pharmaceutical composition for use as a vaccine, according to claim 39.

41. Use of a plurality of hybrid lipid particles according to claim 32 or the pharmaceutical composition according to claim 38 in the manufacture of a pharmaceutical product.

42. Use of a plurality of hybrid lipid particles according to claim 32 or the pharmaceutical composition according to claim 38 in the manufacture of a vaccine.

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