Manufacturing process for hybrid lipid particles

Abstract

A method for producing an aqueous suspension of hybrid lipid particles, comprising the steps of: mixing one or more lipids in a solvent or solvent mixture with a suspension of inorganic material particles in a solvent or solvent mixture in an aqueous medium; and then passing the mixture obtained from step A through the pores of an extruded membrane, wherein the hybrid lipid particles have an average diameter at least twice that of the inorganic material particles. Related products, therapeutic methods, and medical applications.

Description

[Technical Field]

[0001] The present invention relates to hybrid lipid particles and methods for producing hybrid lipid particles. In particular, the present invention relates to methods for producing hybrid lipid particles, comprising the use of hydrolyzable silicon particles to stabilize intermediate hybrid lipid particles to which active compounds such as APIs can be added to form hybrid lipid particle carriers for the active compounds. In particular, the present invention relates to expandable methods for producing hybrid lipid particles particularly suitable for the delivery of nucleic acid compounds. The present invention further relates to products produced by or related to the methods of the present invention.

[0002] In certain embodiments, the present invention further relates to a convergent manufacturing method used to produce hybrid lipid particles for use as a carrier for active compounds, wherein an empty carrier is first produced and purified, typically as part of a filling and finishing operation, before the active compound, such as nucleic acid, is loaded in a separate step. [Background technology]

[0003] Lipid nanoparticles (LNPs) have revolutionized the field of nucleic acid therapy by overcoming significant challenges in the cellular delivery of DNA and RNA. They have become particularly widely known as mRNA delivery technology in the Moderna and Pfizer / BioNTech COVID-19 vaccines (Wang et al., 2021) and are now the dominant delivery mechanism in more than 200 ongoing clinical trials of other RNA-based drugs (Curreri et al., 2023). Despite these impressive successes, current LNPs and common liposomal vesicle formulations face several recognized shortcomings that must be overcome to achieve improved clinical transitions of RNA-based therapies (Moss et al., 2019; Verma et al., 2023).

[0004] One significant drawback concerns the inherent chemical and metabolic instability of RNA, particularly its hydrolytic instability (Schoenmaker et al., 2021), which poses a major challenge for large mRNA constructs, the majority of which are products in clinical development (Curreri et al., 2023). As a result, most commercially available nucleic acid therapies include extensive chemical modifications to enhance stability and efficacy (Bege & Borbas, 2022; Egli & Manoharan, 2023). When LNPs are used as delivery vehicles, another problem arises: the need to introduce the RNA payload early in the production process. Effectively, LNPs must form around the RNA, as there is no effective method for encapsulating the RNA later (Nag et al., 2022; Cameau et al., 2022). This also limits batch sizes in commercial manufacturing due to RNA instability (Catignol & Lim, 2022), potentially compromising product quality. Todinamelan, the mRNA-based Pfizer / BioNTech COVID-19 vaccine, is estimated to retain only 70% of its initial mRNA integrity at the end of the manufacturing process (Daniel et al., 2022). Furthermore, currently available mRNA products (todinamelan, elasomelan, and derivatives) require cold chain distribution and storage to make them clinically and commercially viable. This is not ideal for long-term sustainability or global convenience (Andoh & Yu, 2023; Khairi et al., 2022).

[0005] SiSaf has recently developed silicon-stabilized lipid nanoparticles as an alternative nanocarrier for therapeutic RNA. This carrier addresses the aforementioned challenges. The incorporation of hydrolyzable silicon uniquely and advantageously modifies the properties of the resulting nanoparticles, particularly conferring excellent long-term stability (Saffie-Siebert et al., 2023), and allows for easy formulation of drugs for targeted nucleic acid delivery. The present invention is based on the understanding that silicon-stabilized hybrid lipid nanoparticles can be manufactured in an "empty" form, transported at room temperature (e.g., in a liquid suspension or as a lyophilized powder), and that desired therapeutic RNA can be loaded onto the silicon-stabilized hybrid lipid nanoparticles at the time of use.

[0006] Unlike conventional lipid nanoparticles, which require high temperatures (approximately 70-95°C depending on the system), RNA can penetrate the lipid bilayer of vesicles at room temperature. The silicon nanostructure likely facilitates loading at room temperature because the lipid bilayer is discontinuous with silicon traversing the vesicle surface. The silicon particles may act like a Trojan horse to help RNA penetrate the lipid bilayer.

[0007] The successful clinical transition of a technology requires a reliable manufacturing process that can be deployed on a large scale. It is known that small changes in the manufacturing of a carrier for an active compound can affect the physical and chemical properties of both the carrier and the active compound itself, thereby impacting the clinical profile of the active compound. Lipid particles, sometimes called lipid nanoparticles, particularly liposomal lipid particles, are of particular interest for the delivery of active pharmaceutical ingredients (APIs). They are also of interest for the delivery of cosmetics, nutritional products, dietary supplements, animal and plant health products, and other purposes.

[0008] Micelle particles are approximately spherical in shape and are formed from lipids (particularly a mixture of lipids including lipids having hydrophilic "head" groups (e.g., phospholipids) and lipids having hydrophobic head groups), with the hydrophilic tails attracted to each other and packed together in a supramolecular assembly with the hydrophilic heads facing outward. Micelles have been used to encapsulate delicate APIs, thereby protecting them from a harsher external aqueous environment. For example, U.S. Patent Application Publication No. 2018 / 022151 discloses certain lipid particles suitable for encapsulation of APIs. Liposomal lipid particles are also approximately spherical in shape. They are formed from a lipid bilayer and can be visualized as a lipid "bubble" surrounding an internal space that can be a hydrophilic environment. Alternatively, an aqueous environment can exist inside the liposomal lipid particles. Liposomal lipid particles are particularly suitable for encapsulating active compounds.

[0009] Small lipid particles, particularly small liposomal lipid particles, have been found to be useful for the protection of active compounds such as APIs and for the effective delivery of active compounds such as APIs. Small particles in this context mean particles having a diameter less than about 200 nm, less than about 150 nm, less than about 100 nm, or less than about 50 nm. Since the internal space of liposomes can provide an aqueous environment for active compounds protected from the external aqueous environment, liposomes can be particularly useful for the delivery of hydrophilic active compounds. This can be particularly advantageous, for example, when the external environment contains enzymes or other factors that can damage the active compound or API. Summary of the Invention Problems to be Solved by the Invention

[0010] Small lipid particles, both those having liposome characteristics and those having micelle characteristics, have a tendency to coalesce into larger particles, as described in UK Patent Application Publication No. 2210794.0. The inventors have found that inorganic solid particles, which are themselves smaller than the lipid particles, particularly particles of a hydrolyzable silicon-containing material, can be "sprayed" onto the surface of the lipid and / or onto the surface of the lipid particles (i.e., penetrate partially into the lipid particles, but a portion of the particles of the inorganic material is accessible on the surface), thereby not only suppressing the tendency of the lipid particles to coalesce with each other, but also coordinating with any charged lipid component of the lipid particles to protect it and further providing lipid particles with an excellent ability to form a complex with a specific API.

[0011] The inventors have also found that inorganic solid particles, which are themselves smaller than the lipid particles, particularly particles of a hydrolyzable silicon-containing material, can penetrate into the lipid particles, thereby providing a pathway for APIs, particularly nucleic acids, from the outside to the inside of the lipid particles. When the inorganic solid particles are particles of hydrolyzable silicon, they can optionally be present as aggregates, for example, as chains. These aggregates extend from the outside to the inside of the lipid particles, thereby providing an entry pathway for APIs into the lipid particles, as further described herein.

[0012] The lipid particles of the present invention are characterized as lipid particles in which the internal space of the particle remains accessible, unlike conventional liposomal lipid particles. They are referred to herein as “hybrid lipid particles,” the term “hybrid” used to recognize their lipid properties and components, as well as their non-lipid properties and components (particles of inorganic materials, e.g., silicon-containing material particles). Thus, they are in contrast to conventional lipid particles in which silicon-containing material particles are located inside a lipid coating or liposome. In such conventional products, silicon-containing material particles tend to be encapsulated within the lipid. While silicon-containing particles in such products may provide a favorable environment within the encapsulation for the protection of, for example, an active ingredient or API, generally, there is no pathway from or to the interior unless the lipid encapsulation is broken and subsequently reformed, which is a significant drawback, and there is no significant exposure of silicon-containing particles on the surface of conventional particles. This means that any interaction between the active ingredient and the silicon-containing particles occurs mainly within the encapsulation. In some embodiments, the structure of the hybrid lipid particles of the present invention can be visualized as incomplete liposomes having a pathway through which the active compound passes from the external to the internal environment.

[0013] Lipid particles (LPs) similar to those of the present invention are generally disclosed in UK Patent Application Publication No. 2210794.0 (incorporated herein by reference and may be obtained from other patent applications, e.g., at the time of publication of the international patent application claiming priority thereto, or from files generally available at the time of publication). They can be produced by conventional lipid particle manufacturing techniques. Essentially, such techniques can be broadly interpreted as involving the preparation of a suitable mixture of lipids in a solvent, followed by the formation of a thin film of lipid material by evaporating the solvent in, for example, a rotary evaporator, then hydrating it to form lipid particles, to which inorganic material particles and active compounds (particularly APIs) may be added to form the hybrid lipid particles of the present invention. While successful in producing the desired products, such manufacturing processes may be difficult to scale effectively because the evaporation process is limited by the surface area of ​​the evaporator used and does not increase at the same rate as the volume with increasing scale.

[0014] The present invention also relates to improvements to particles disclosed in UK Patent Application Publication No. 2210794.0, primarily concerning the relative dimensions of lipid particles and the inorganic material particles within them, and the discovery that they can be fabricated as hybrid lipid particles. The present invention also relates to improvements in the manufacturing process of these particles and discloses advantageous properties related to the hybrid nature of the particles of the present invention as hybrid lipid particles.

[0015] The hybrid lipid particles according to the present invention are also called “stabilized hybrid lipid particles” (shLPs) because they use inorganic material particles to stabilize them, and in certain preferred embodiments, the inorganic material particles are hydrolyzable silicon particles, resulting in stabilized (particularly silicon-stabilized) hybrid lipid particles (shLPs; in the case of silicon-stabilized hybrid lipid particles, particularly sshLPs). Such particles are novel lipid nanoparticles (LNPs) according to the present invention, particularly suitable for nucleic acid delivery. From a manufacturing standpoint, a key difference between the shLP manufacturing process and the prior art LP manufacturing process is the step in which active compounds, such as nucleic acids, are incorporated into the particles. This difference is made possible by the presence of inorganic material particles, which stabilize the liposomes, which are inherently imperfectly formed. From a product standpoint, key differences between shLPs and prior art LPs are their hybrid nature, which is further described herein, and the difference in relative size between the lipid particles and the inorganic material particles. From a user's perspective, a key difference between shLP and conventional LP is that shLP may be manufactured "empty" and then loaded with an active compound or API, whereas with conventional liposomal lipid particles, it is necessary to form the particles "around" the active compound or API to achieve active compound / API encapsulation, or, if pre-formed liposomal lipid particles are not, to use harsh conditions such as acids, solvents, or surfactants, or high temperatures, to break down their lipid layers and allow the active compound or API to penetrate before the lipid layers are reformed.

[0016] Conventional processes typically employ a sequential manufacturing method in which active compounds, such as nucleic acids, are supported during the initial particle formation process, and the supported LPs are subsequently purified before undergoing packing and finishing operations. Alternative conventional methods may involve forming "empty LPs" and then reforming the LPs around the active ingredient. In contrast, the present invention employs a convergent manufacturing method for the production of shLPs, in which empty shLPs are first generated and purified before the active compound (particularly APIs, and even more particularly nucleic acids such as RNA) is supported in a separate process, usually as part of the packing and finishing operations. There may be a large temporal and spatial gap between the formation and loading processes. This offers several practical advantages.

[0017] Hybrid lipid particles While the use of a convergent manufacturing process in which an empty hybrid lipid carrier is first generated and then an active compound (particularly an API) is added to it has advantages, the present invention also presents challenges that are mitigated by providing hybrid lipid particles and methods for producing hybrid lipid particles. The main challenge of the prior art is that, after formation, liposomes or other lipid carriers exhibit a continuous, unbroken hydrophilic barrier to their internal space. This means that active compounds, such as nucleic acids, cannot be easily accessed into the internal space of the particles, especially if they are hydrophilic (i.e., they cannot be easily encapsulated within the particles). Methods of the prior art that enable access to the internal space of pre-formed liposomal lipid particles include, for example, disrupting the liposomal lipid particles using a solvent, low pH, or high temperature, or using a non-convergent manufacturing process in which the liposomal carrier is first formed around the active compound. The first method has the disadvantage that the solvent may damage the carrier or the active compound, and it may also introduce complexity into the process. The second method has the disadvantage that forming liposome carriers may require the use of conditions such as the presence of a solvent, a low pH, and high temperature, which can damage fragile active compounds as liposomes may need to rupture and reform, and some of the ruptured liposomes may not be able to reform. This can also lead to a lack of control and a decrease in yield, meaning that the process is not only difficult to control but also results in a decrease in the yield and loss of lipid components. The method of the present invention includes a method for an active component / API to access the internal space of pre-formed hybrid lipid particles without using a solvent, low pH, or high temperature to break down the lipid barrier and allow the active component / API to enter. The present invention takes a different approach in that it uses hybrid lipid particles that have a mostly liposome-like shell, which, due to the presence of inorganic material particles such as hydrolyzable silicon and the extrusion method used in their manufacture, imperfectly seal the inside of the particles in the absence of active compounds (particularly APIs). Furthermore, when the active compound or a portion thereof accesses the inside of the particle, the particle "condenses" or "shrinks" around the active compound, thereby sealing the active compound.This has been found to be particularly effective when the active compound is hydrophilic, and more particularly when the active compound is negatively charged, for example, when the active compound is a nucleic acid. The hybrid lipid particles of the present invention may also optionally contain inorganic solid particles, for example, particles of hydrolyzable silicon (or aggregates thereof, such as chains) extending from the inside to the inside of the hybrid lipid particles, thereby providing a pathway for the active compound (particularly API) from the outside to the inside of the hybrid lipid particles.

[0018] This remote drug delivery method of the present invention also eliminates the problem of low drug concentration relative to the carrier, which can become a further issue when the drug needs to be clinically and ultimately marketed as a product.

[0019] Empty shLP according to the present invention can be produced using any of the well-established LP manufacturing processes, including evaporation, microfluidic technology, supercritical fluid technology, or flow injection technology. Many of these techniques can be optimized to produce particles with the desired particle size and low polydispersity. If the initial particles produced are too large or their sizes are too unevenly distributed, membrane extrusion can be used to modify or reshape the particles. It has been found that the combination of the presence of inorganic material particles (such as hydrolyzable silicon particles), which are up to half the size of the hybrid lipid particles of the present invention, and the use of a manufacturing method including membrane extrusion, produces particles according to the present invention, which are hybrid particles that have largely the properties of liposomes, but nevertheless have their internal space imperfectly isolated from the surrounding medium, and can preferably become completely sealed liposome particles when an active compound (particularly an API) is supported on the particles. From a terminology standpoint, when an active compound is added and the internal space is completely sealed, the lipid particles may be strictly called liposome lipid particles rather than hybrid lipid particles.

[0020] Once particles with the correct physical properties are obtained, they are purified and concentrated, for example, by tangential flow filtration, before being loaded with active compounds such as nucleic acids.

[0021] This convergent approach to shLP production offers several advantages compared to the manufacturing processes used for conventional LP products.

[0022] Firstly, the manufacturing of shLP is cost-effective. • Degradation of the active compound during particle formation and purification is avoided. This can be particularly important when the active compound is a fragile compound such as RNA, e.g., mRNA, saRNA, or siRNA. • Loading of the active compound onto shLP should be carried out under mild conditions, i.e., at approximately 20°C for less than 1 hour. This is especially important when the active compound is heat-sensitive, such as when the active compound is a nucleic acid, particularly RNA, and even more particularly single-stranded mRNA.

[0023] Secondly, the shLP manufacturing process is scalable and flexible, operating at output volumes from a few milliliters to several thousand liters, and is therefore suitable for both low-demand and high-demand products. The hybrid lipid particle formation technology can be adapted to the required production scale, for example, by using microfluidics when small quantities are needed and flow injection when large quantities are needed. The process is also robust and reproducible, producing materials with consistent composition and physical properties, including lipid content, particle size (e.g., low polydispersity), and zeta potential. This is highly cost-effective due to reduced waste of lipid compounds by eliminating reforming steps that reduce yield and by maintaining API activity and allowing the use of relatively small initial amounts of API.

[0024] Finally, shLP can increase the availability of both high-demand and low-demand products. • High Demand: Current LP products generally require cryogenic transport and storage to maintain the activity of fragile APIs such as nucleic acids, but empty shLPs can be stored and transported under refrigerated conditions. Empty shLPs can be easily transported worldwide for loading of active compounds, which is carried out as part of localized filling and finishing operations. • Low demand: For example, for personalized medicines—such as patient-specific active compound loading, or patient-specific nucleic acid loading, small amounts of empty shLP can be supplied to clinics.

[0025] Accordingly, the present invention provides a method for producing hybrid lipid particles (and preferably a method for eliminating a solvent evaporation step) using what is called an “extrusion” technique, as described in more detail below, wherein the solvent used to activate the inorganic material particles and to prepare the lipid mixture used optionally is removed from the product by a non-evaporative method after the particles have been formed. The extrusion technique involves passing the lipid and aqueous components of the suspension of hybrid lipid particles through the pores of an extrusion membrane, often multiple times, to aid in the formation of a large number of uniform hybrid lipid particles. The present invention is based on the discovery that including inorganic material particles, particularly hydrolyzable silicon-containing particles according to the present invention, in the mixture passing through the extrusion membrane not only yields hybrid lipid particles having similar advantageous properties to those described in UK Patent Application Publication No. 2210794.0 when supported with an active compound (particularly an API), but also that the presence of inorganic material particles promotes the formation of hybrid lipid particles having desirable properties.

[0026] In such extrusion methods, adjusting the extrusion force and the tensile strength of the lipid membrane can be particularly important for the successful generation of lipid particles. In the prior art, cholesterol is known to improve the stability of the lipid membrane. However, the problem with cholesterol is that it makes the lipid bilayer even more hydrophobic. This can alter the dynamics of lipid particle formation, in that the amount of water available during lipid particle formation in the pores of the extruded membrane may be insufficient, especially when the extruded membrane has a small pore size, unless the extrusion force is significantly increased, and the newly formed lipid particles may rupture prematurely within the extruded pores. Therefore, the products of the present invention typically utilize reduced cholesterol levels, including the complete absence of cholesterol. In some embodiments, cholesterol constitutes less than 10%, less than 5%, or less than 1% of the total lipid content (by weight). In other embodiments, the products of the present invention do not contain significant levels of cholesterol.

[0027] Replacing cholesterol with inorganic material particles (particularly hydrolyzable silicon particles) yielded unexpected results. The introduction of inorganic material particles (especially hydrolyzable silicon particles) in membrane extrusion increased the amount of water available for lipid particle formation in the pores of the extruded membrane. Therefore, lipid particle formation at the pore openings did not indicate premature rupture of the newly formed lipid particles within the extruded pores.

[0028] Such a finding is surprising because it can be inferred that for inorganic material particles (e.g., hydrolyzable silicon particles) to exert a beneficial effect on the stability and performance of hybrid lipid particles, a relatively high level of inorganic material particles (e.g., hydrolyzable silicon particles) must remain as part of the lipid particles. This is found when lipid particles are prepared by conventional evaporation methods, as described in UK Patent Application Publication No. 2210794.0. Surprisingly, this is not the case when hybrid lipid particles are prepared using the extrusion technique according to the present invention. The use of the extrusion technique results in a significant reduction in the level of inorganic material particles remaining as part of the lipid particles, and it has been found that when relatively high levels of inorganic material particles are used to produce hybrid lipid particles by the extrusion technique, the level of inorganic material particles in the final hybrid lipid particle product can be reduced to a relatively low level during extrusion, and yet it can still exhibit the favorable properties normally expected of lipid particles prepared by the “evaporation” method, where a relatively high level of inorganic material particles must be retained in the final lipid particles for the resulting advantages. Accordingly, the present invention also relates to hybrid lipid particles having a relatively low level of inorganic material particles, prepared by extrusion techniques involving a relatively high level of inorganic material particles. Corresponding methods are also included in the present invention.

[0029] Theoretical basis The applicant does not wish to be bound by any particular theoretical explanation on which the present invention is based. For the sole purpose of facilitating understanding, two non-mutually exclusive mechanisms may help support the inventors' unexpected finding that hybrid lipid particles having relatively low levels of inorganic material particles have beneficial properties when produced using relatively high levels of inorganic material particles and then reduced to relatively low levels by extrusion techniques.

[0030] The first theoretical explanation is that, most important for the beneficial properties of hybrid lipid particles, inorganic material particles are present in specific niches or locations within or on the surface of the hybrid lipid particles, where they associate particularly strongly with lipid molecules. Hydrogen bonding in this situation is thought to reduce the barrier to the migration of active compounds (particularly APIs), i.e., increase the permeability of the lipid particles to active compounds. A relatively large number of inorganic material particles may be initially required to ensure that all or most of these specific niches are filled, but once filled, the overall level of inorganic particles can be reduced without compromising their effectiveness.

[0031] An alternative or additional explanation is that the inorganic material (e.g., hydrolyzable silicon) may initially need to be at a relatively high level to assist in the dehydration of the hybrid lipid particles (i.e., by consuming water molecules trapped in the hybrid structure through silicon hydrolysis), and that once this initial dehydration occurs, the high level of inorganic material is no longer required. [Means for solving the problem]

[0032] According to a first aspect of the present invention, A. A step of mixing one or more lipids in a solvent or solvent mixture with a suspension of inorganic material particles in a solvent or solvent mixture in an aqueous medium, then, B. The process of passing the mixture obtained from step A through the pores of an extrusion membrane. A method for producing a suspension of hybrid lipid particles, comprising: A method is provided in which hybrid lipid particles have an average diameter at least twice that of the average diameter of inorganic material particles.

[0033] According to a second aspect of the present invention, an aqueous suspension of hybrid lipid particles having an average diameter of 50 to 150 nm is provided. The hybrid lipid particles comprise one or more types of lipids and inorganic material particles, and preferably, the average diameter of the hybrid lipid particles is at least twice the average diameter of the inorganic material particles.

[0034] According to a third aspect of the present invention, an aqueous suspension of liposomal lipid particles having an average diameter of 50 to 150 nm is provided. The liposomal lipid particles comprise an active compound (particularly an API), one or more lipids, and particles of an inorganic material, wherein the average diameter of the hybrid lipid particles is at least twice the average diameter of the inorganic material particles.

[0035] A fourth aspect of the present invention provides a method for producing an aqueous suspension of liposome lipid particles according to the third aspect of the present invention from an aqueous suspension of hybrid lipid particles according to the second aspect of the present invention, the method comprising the step of contacting the aqueous suspension of hybrid lipid particles with an active compound (particularly an API).

[0036] According to a fifth aspect of the present invention, a freeze-dried powder of liposomal lipid particles having an average diameter of 50 to 150 nm is provided, wherein the liposomal lipid particles comprise a mixture of one or more cationic or ionizable lipids, one or more further lipids selected from neutral and polar lipids, and optionally one or more additional lipid components, wherein the liposomal lipid particles comprise inorganic material particles (preferably hydrolyzable silicon particles) having an average diameter of up to 1 / 2 the average diameter of the liposomal lipid particles, the weight ratio of inorganic material particles to lipids is 1:2 to 1:100 (preferably 1:10 to 1:100, more preferably 1:20 to 1:10), and the liposomal lipid particles further comprise one or more active compounds, for example, one or more active pharmaceutical ingredients (APIs) in which at least a portion is encapsulated inside the liposomal particles.

[0037] According to a sixth aspect of the present invention, a freeze-dried powder of hybrid lipid particles having an average diameter of 50 to 150 nm is provided, wherein the hybrid lipid particles comprise a mixture of one or more cationic lipids or ionizable lipids, one or more further lipids selected from neutral lipids and polar lipids, and optionally one or more additional lipid components, wherein the hybrid lipid particles comprise inorganic material particles (preferably hydrolyzable silicon particles) having an average diameter of up to half the average diameter of the hybrid lipid particles, and the weight ratio of inorganic material particles to lipids is 1:2 to 1:100 (preferably 1:10 to 1:100, more preferably 1:20 to 1:10).

[0038] According to a seventh aspect of the present invention, a pharmaceutical composition comprising an aqueous suspension of the hybrid lipid particles of the present invention, or a freeze-dried powder of the hybrid lipid particles of the present invention, A pharmaceutical composition is provided which comprises an aqueous suspension of the liposomal lipid particles of the present invention, or a lyophilized powder of the liposomal lipid particles of the present invention.

[0039] Further embodiments of the present invention provide the use of the pharmaceutical composition of the present invention as a pharmaceutical, and related methods for medical treatment.

[0040] The present invention also, (a) (i) Mixing one or more lipids in a solvent or solvent mixture with a suspension of inorganic material particles in a solvent or solvent mixture in an aqueous medium. (ii) Passing the mixture obtained from step (i) through the pores of the extruded film, (iii) Optionally, purify and / or sterilize the suspension by tangential flow filtration. To obtain an aqueous suspension of hybrid lipid particles manufactured by, and (b) Provide an aqueous suspension of lipid particles containing a pharmaceutically active ingredient (API), which is produced by a method comprising contacting the hybrid lipid particles with an active compound, in particular an API.

[0041] The present invention also, (a) (i) Mixing one or more lipids in a solvent or solvent mixture with a suspension of inorganic material particles in a solvent or solvent mixture in an aqueous medium. (ii) Passing the mixture obtained from step (i) through the pores of the extruded film. To obtain an aqueous suspension of hybrid lipid particles manufactured by, and (b) Provide an aqueous suspension of hybrid lipid particles containing a pharmaceutically active ingredient (API), which is produced by a method comprising purifying and / or sterilizing the suspension by tangential flow filtration. [Brief explanation of the drawing]

[0042] [Figure 1A] Figure 1A is a schematic diagram of a manufacturing method according to a specific embodiment of the present invention. [Figure 1B] Figure 1B provides a more detailed schematic illustration of the manufacturing method according to a specific embodiment. [Figure 2] Figure 2 outlines a manufacturing strategy for generating a scalable process for producing micelle lipid particles. [Figure 3] Figure 3 shows the manufacturing process of micelle lipid particles by evaporation. [Figure 4] Figure 4 shows the results of a visual inspection of the obtained solution for sample MVI0001. [Figure 5] Figure 5 shows the DLS and zeta potential analysis of sample MVI0001 throughout the extrusion process. [Figure 6] Figure 6 shows a general procedure for generating sample MVI0002. [Figure 7] Figure 7 shows the results of a visual inspection of the obtained solution for sample MVI0002. [Figure 8] Figure 8 shows the DLS and zeta potential analysis of sample MVI0002 throughout the extrusion process. [Figure 9]Figure 9 shows a general procedure for generating sample MVI0003. [Figure 10] Figure 10 shows the results of a visual inspection of the obtained solution for sample MVI0003.

[0043] [Figure 11] Figure 11 shows the DLS and zeta potential analysis of sample MVI0003 throughout the extrusion process. [Figure 12] Figure 12 shows the results of a visual inspection of the obtained solution for sample MVI0004. [Figure 13] Figure 13 shows the DLS and zeta potential analysis of sample MVI0004 throughout the extrusion process. [Figure 14] Figure 14 shows a representative photograph obtained by visually inspecting the solution for sample MVI0005. [Figure 15] Figure 15 shows the DLS and zeta potential analysis of sample MVI0005 throughout the extrusion process. [Figure 16] Figure 16 shows the DLS and zeta potential analysis of sample MVI0006 throughout the extrusion process. [Figure 17] Figure 17 shows the experimental conditions for sample MVI0007 using a 0.8 μm film. [Figure 18] Figure 18 shows the results of a visual inspection of the solutions obtained in each step of Experiment MVI0007. [Figure 19A] Figure 19A shows photographs of the 0.4 μm and 0.1 μm films after extrusion, without using the 0.8 μm film. [Figure 19B] Figure 19B shows photographs of the 0.8 μm, 0.4 μm, and 0.1 μm films after extrusion.

[0044] [Figure 20] Figure 20 shows the DLC and zeta potential analysis of MVI0007 throughout the extrusion process. [Figure 21-1] Figure 21 shows the results of the investigation into the dilution effect on DLS and zeta potential measurements (Figure 21A: 20x dilution factor; Figure 21B: 5x dilution factor). [Figure 21-2] As described in Figure 21-1. [Figure 21-3] As described in Figure 21-1. [Figure 21-4] As described in Figure 21-1. [Figure 22] Figure 22 shows the results of a visual inspection of the solutions obtained in each step of Experiment MVI0008. [Figure 23] Figure 23 shows the DLS and zeta potential analysis of MVI0008 throughout the extrusion process. [Figure 24] Figure 24 shows the experimental conditions for MVI0011. [Figure 25] Figure 25 shows the results of a visual inspection of the solutions obtained in each step of experiment MVI0011. [Figure 26] Figure 26 shows the DLS and zeta potential analysis of MVI001. [Figure 27] Figure 27 shows the results of a visual inspection of the solutions obtained in each step of experiment MVI0012. [Figure 28] Figure 28 shows the film after extrusion. [Figure 29] Figure 29 shows the DLS and zeta potential investigations of MVI0012.

[0045] [Figure 30] Figure 30 shows the pressure during extrusion in experiment MVI0007, using a 25 mm diameter membrane with an extrusion volume of 50 ml. [Figure 31] Figure 31 shows the pressure during extrusion in Experiment MVI0011, using a 25 mm diameter membrane and an extrusion volume of 100 ml. The SiNP solution was filtered through a 0.8 μm syringe filter before extrusion. [Figure 32] Figure 32 shows the pressure during extrusion in experiment MVI0012, using a 47 mm diameter membrane with an extrusion volume of 1 L. [Figure 33-1] Figure 33 shows chromatograms of separate lipid components. [Figure 33-2] As described in Figure 33-1. [Figure 33-3]As described in Figure 33-1. [Figure 34] Figure 34 shows the chromatogram of the lipid mixture. [Figure 35] Figure 35 shows the gradient profiles of the modified and initial HPLC methods. [Figure 36] Figure 36 shows the chromatogram of the lipid mixture at final product concentrations of [DOTAP-Cl]=0.725 mg / ml, [DOPE]=0.73 mg / ml, and [mPEG2000'DSPE]=0.145 mg / ml. [Figure 37] Figure 37 shows the calibration curves for each lipid in MVI0010. [Figure 38] Figure 38 shows the chromatogram of experiment MVI0010. [Figure 39] Figure 39 shows the calibration curve for experiment MVI0017.

[0046] [Figure 40] Figure 40 shows the measurement chromatogram of the undiluted MVI0017 sample. [Figure 41] Figure 41 shows the calibration curve of MVI0017 using a diluted standard lipid solution. [Figure 42] Figure 42 shows the measurement chromatogram of the MVI0017 sample diluted 2-fold. [Figure 43] Figure 43 shows chromatograms of nuclease-free water and THR-GLY solution. [Figure 44] Figure 44 shows chromatograms of samples stored at room temperature at different time points. [Figure 45] Figure 45 shows the time course of lipid concentrations in the sample analyzed at room temperature. [Figure 46] Figure 46 shows chromatograms of samples stored at room temperature at different time points. [Figure 47] Figure 47 shows the time course of lipid concentrations in the sample analyzed at 4°C. [Figure 48] Figure 48 shows the TFF conditions used in the example. [Figure 49] Figure 49 shows the MeOH calibration curve.

[0047] [Figure 50] Figure 50 shows the 1H-NMR signals of MVI0010 before and after TFF. [Figure 51] Figure 51 shows the experimental conditions for MVI0013 using a pre-filtration step. [Figure 52] Figure 52 shows the results of a visual inspection of the solutions obtained in each step of Experiment MVI0013. [Figure 53] Figure 53 shows photographs of the 0.8 μm hydrophilic polyethersulfone syringe filter (A) and extruded membrane (B) after filtration and extrusion, respectively. [Figure 54] Figure 54 shows the DLC and zeta potential analysis of MVI0013 throughout the extrusion process. [Figure 55] Figure 55 shows the extrusion pressure during the investigation of experiment MVI0013. [Figure 56] Figure 56 shows the zeta potential analysis of MVI0013. [Figure 57] Figure 57 shows the experimental conditions for MVI0014. [Figure 58] Figure 58 shows the results of a visual inspection of the solutions obtained in each step of experiment MVI0014. [Figure 59] Figure 59 shows the DLS and zeta potential analysis of MVI0014 throughout the extrusion process.

[0048] [Figure 60] Figure 60 shows the results of the extrusion pressure investigation for MVI0014. [Figure 61] Figure 61 shows the experimental conditions for MVI0015. [Figure 62] Figure 62 shows the results of a visual inspection of the solutions obtained in each step of experiment MVI0015. [Figure 63] Figure 63 shows the DLS and zeta potential analysis of MVI0015 throughout the extrusion process. [Figure 64] Figure 64 shows the experimental procedure used to produce a 2 kg batch. [Figure 65] Figure 65 shows a photograph of the extruded film after the extrusion process. [Figure 66] Figure 66 shows the results of DLS and zeta potential investigations for MVI0022. [Figure 67] Figure 67 shows the trend of size increase with extrusion volume. [Figure 68] Figure 68 shows various possible configurations for rapid mixing according to a particular embodiment of the present invention. [Figure 69] Figure 69 shows the long-term stability of the hybrid lipid particles of the present invention compared to lipid particles formulated without the use of silicon or other inorganic materials.

[0049] [Figure 70] Figure 70 shows that the size stability of the particles of the present invention is maintained after mRNA loading. Figures 71 to 81 show the methods and results for Examples 17 and 18. [Figure 71A] Figure 71 shows (A) the workflow of the original lipid thin film hydration method (Method 1) in which an organic solvent is evaporated from activated silicon nanoparticles, and a revised protocol (Method 2) in which the activated silicon nanoparticle suspension is added directly to the aqueous phase. [Figure 71B] In Figure 71, in Method 2 (B) (lower image), even more precipitate was observed after lipid membrane hydration. [Figure 71C] Figure 71 shows that (C)DLS results indicate that hybrid lipid particles produced using the revised method have lower PDI and higher zeta potential. [Figure 72A] Figure 72 illustrates the optimization of the solvent injection mixing method by schematically eliminating the second evaporation step. (A) Workflow of the initial direct injection mixing method (Method 3). [Figure 72B] Figure 72 shows the optimization of the solvent injection mixing method by schematically eliminating the second evaporation step. (B) Less precipitation was observed than in lipid thin film hydration. [Figure 72C]Figure 72 illustrates the optimization of the solvent injection mixing method by schematically eliminating the second evaporation step. (C) Modified workflow incorporating an additional 0.8 μm extrusion step and other adjustments as shown. [Figure 72D] Figure 72 illustrates the schematic elimination of the second evaporation step and the optimization of the solvent injection mixing method. (D) An additional extrusion step (Method 4) resulted in even smaller hybrid lipid particles with similar PDI and zeta potential. [Figure 72E] Figure 72 illustrates the optimization of the solvent injection mixing method by schematically eliminating the second evaporation step. (E) Pre-filtration before extrusion (Method 6, right) removed insoluble aggregates that would normally accumulate on the 0.8 μm extruded membrane (Method 4, left), while the 0.4 μm membrane appeared to remove additional agglomerating material. [Figure 72F] Figure 72 illustrates the schematic elimination of the second evaporation step and the optimization of the solvent injection mixing method. (F) The introduction of the prefiltration step did not affect the properties of the final hybrid lipid particles. [Figure 73A] Figure 73 shows the purification of the hybrid lipid particles of the present invention by TFF. (A) A schematic diagram of the experimental conditions showing how a second pump (pump 2) was incorporated to enable both ultrafiltration and diafiltration operations. [Figure 73B] Figure 73 shows the purification of the hybrid lipid particles of the present invention by TFF. (B) Representative results showing the successful removal of MeOH from the hybrid lipid particles produced by Method 5, as determined by 1H NMR. (C, D) The TFF process was found to slightly alter the DLS properties (C) and lipid content (D) of the particles, but these variations were within the relevant reference range and therefore not considered problematic. [Figure 74A] Figure 74 shows the initial large-scale (1L) run for generating hybrid lipid particles. (A) Schematic diagram of the workflow used; scaled-up of Method 5. [Figure 74B]Figure 74 shows the first large-scale (1L) run to produce hybrid lipid particles. (B) Representative images of the extruded membrane and sample aliquot (lower right quarter section), showing some insoluble material remaining after the first extrusion step and accumulation of aggregates, despite the use of a large membrane. [Figure 74C] Figure 74 shows the initial large-scale (1 L) operation to generate hybrid lipid particles. (C) The accumulation of aggregates on the 0.8 μm and 0.4 μm membranes resulted in substantially higher operating pressures than at the 50 mL scale (Method 4). [Figure 74D] Figure 74 shows the first large-scale (1L) operation to produce hybrid lipid particles. (D) The DLS properties of the hybrid lipid particles are not adversely affected and are within the reference range.

[0050] [Figure 75A] Figure 75 shows the production of a 2L batch of hybrid lipid particles. (A) Schematic diagram of the workflow used. Note that the TFF process included two steps: ultrafiltration to concentrate the sample from 4L to 2L, followed by diafiltration to remove MeOH and free lipids. [Figure 75B] Figure 75 shows the production of a 2L batch of hybrid lipid particles. (B) The extrusion process was impaired by significant accumulation of aggregates on the membrane, resulting in a loss of membrane performance. [Figure 75C] Figure 75 shows the production of a 2L batch of hybrid lipid particles. (C) For the completed batch, the average size and PDI were outside the reference range. [Figure 75D] Figure 75 shows the production of a 2L batch of hybrid lipid particles. (A) Schematic diagram of the workflow used. (D) This is due to the degradation of the extrusion membrane with excessive accumulation of aggregates, indicating that a pre-filtration step is necessary after activation of hybrid lipid particles when producing them on a large scale. [Figure 76A] Figure 76 illustrates the effect of pre-filtration of the activated silicon nanoparticle suspension on improving the process. (A) Schematic diagram of Method 7 and the specified modified workflow. [Figure 76B] Figure 76 illustrates the effect of pre-filtration of the activated silicon nanoparticle suspension on improving the process. (B) In this case, there was no significant accumulation of aggregates on the extruded membrane. [Figure 76C] Figure 76 illustrates the effect of pre-filtration of the activated silicon nanoparticle suspension on process improvement (C-E). This is reflected in significantly lower operating pressure during extrusion compared to the protocol without pre-filtration (C), resulting in sshLNP with well within-reference-range DLS characteristics at both small (D) and 1L batch sizes (E). [Figure 76D] Figure 76 illustrates the effect of pre-filtration of the activated silicon nanoparticle suspension on process improvement (C-E). This is reflected in significantly lower operating pressure during extrusion compared to the protocol without pre-filtration (C), resulting in sshLNP with well within-reference-range DLS characteristics at both small (D) and 1L batch sizes (E). [Figure 76E] Figure 76 illustrates the effect of pre-filtration of the activated silicon nanoparticle suspension on process improvement (C-E). This is reflected in significantly lower operating pressure during extrusion compared to the protocol without pre-filtration (C), resulting in sshLNP with well within-reference-range DLS characteristics at both small (D) and 1L batch sizes (E). [Figure 77A] Figure 77 shows that hybrid lipid particles produced on a small scale (using lipid thin film hydration, Method 2) and on a large scale (using Method 7) exhibit similar physical and functional properties. (A) The large-scale process produced slightly smaller final particles with comparable PDI. Panels (A-C) show the mean ± SD for samples analyzed in triplicate, and panel D shows the mean ± SD for three independent biological replicas. [Figure 77B]Figure 77 shows that hybrid lipid particles generated on a small scale (using lipid thin film hydration, Method 2) and on a large scale (using Method 7) exhibit similar physical and functional properties. (B) Zeta potential was not affected by scaling. Panels (A-C) show the mean ± SD for samples analyzed in triplicate, and panel D shows the mean ± SD for three independent biological replicas. [Figure 77C] Figure 77 shows that hybrid lipid particles produced on a small scale (using lipid thin film hydration, Method 2) and on a large scale (using Method 7) exhibit similar physical and functional properties. (C) Both methods yielded hybrid lipid particles with comparable RNA encapsulation efficiency. Panels (A-C) show the mean ± SD for samples analyzed in triplicate, and panel D shows the mean ± SD for three independent biological replicas. [Figure 77D] Figure 77 shows that hybrid lipid particles produced on a small scale (using lipid thin film hydration, Method 2) and on a large scale (using Method 7) exhibit similar physical and functional properties. (D) Large-scale production also maintained the transfection efficiency of HEK293 cells with mRNA encoding firefly luciferase (fLuc). Fluctuation intensity was measured to determine fLuc expression levels 24 hours after transfection. Lipofectamine 2000 was used as a positive control and untreated cells as a negative control. [Figure 78A] Figure 78 shows that reverse mixing after silicon nanoparticle activation does not impair the properties of the hybrid lipid particles. (A) Workflows of methods 4 and 5, showing only the difference in mixing order. [Figure 78B] Figure 78 shows that back mixing after silicon nanoparticle activation does not impair the properties of the hybrid lipid particles. (B) Comparison of DLS-derived parameters of samples prepared via methods 4 and 5. In this experiment, the final zeta potential was lower (graph on the right), but subsequent experiments showed that method 5 can reliably produce hybrid lipid particles with zeta potentials within the reference range, including on a large scale. [Figure 79]Figure 79 shows that when a pre-filtration step is introduced before extrusion (Method 6), the operating pressure on the 0.8 μm extruded membrane and the 0.4 μm extruded membrane is significantly reduced compared to the method without pre-filtration (Method 4).

[0051] [Figure 80A] Figure 80 shows the development of an HPLC-based assay for lipid recovery. (A) Chromatogram of a mixed lipid standard solution containing DOTAP, DOPE, and mPEG2000-DSPE components in the same molar ratios used to prepare the hybrid lipid particles. (B) Calibration curve of the lipid reference solution used in the assay of Method 5. (C) Chromatogram of the post-extrusion sample produced using Method 5, showing the presence of two unidentified impurity peaks. In this sample, observed lipid recovery rates were 86%, 81%, and 76% for DOTAP, DOPE, and mPEG2000-DSPE, respectively. (D) An alternative UPLC assay is currently under development to achieve improved peak shapes, particularly for pegylated lipids. The chromatograms show representative results for the mixed lipid standard solution. [Figure 80B-1] Figure 80 shows the development of an HPLC-based assay for lipid recovery. (B) Calibration curve of the lipid reference solution used in the assay of Method 5. [Figure 80B-2] As described in Figure 80B-1. [Figure 80C] Figure 80 shows the development of an HPLC-based assay for lipid recovery. (C) Chromatogram of the extruded sample produced using Method 5, showing the presence of two unidentified impurity peaks. In this sample, the observed lipid recovery rates were 86%, 81%, and 76% for DOTAP, DOPE, and mPEG2000-DSPE, respectively. [Figure 80D-1] Figure 80 shows the development of an HPLC-based assay for lipid recovery. (D) In ​​particular, an alternative UPLC assay is currently under development to achieve improved peak shapes for pegylated lipids. The chromatogram shows typical results for a mixed lipid standard solution. [Figure 80D-2] As described in Figure 80D-1. [Figure 81A] Figure 81(A) shows that using two sets of extrusion films in parallel increases the surface area of ​​the film available for extrusion. [Figure 81B] Figure 81(B) shows that, in combination with pre-filtration of activated silicon nanoparticles, this prevented the accumulation of residues on the 0.8 μm and 0.4 μm films. Yellow discoloration was observed on the 0.1 μm film, suggesting the removal of small aggregates and / or oversized particles from the sample. [Figure 82] Figure 82 is a transmission electron microscope (TEM) image of hybrid lipid particles according to one embodiment of the present invention. This shows the aggregation of silicon particles as described herein. The particles labeled "lipid" are hybrid lipid particles according to an embodiment of the present invention. This image also shows relatively small silicon particles that form part of the aggregates extending from the outside of the hybrid lipid particles to the inside of the hybrid lipid particles. [Modes for carrying out the invention]

[0052] According to a first aspect of the present invention, A. A step of mixing one or more lipids in a solvent or solvent mixture with a suspension of inorganic material particles in a solvent or solvent mixture in an aqueous medium, then, B. The process of passing the mixture obtained from step A through the pores of the extruded film. A method for producing a suspension of hybrid lipid particles, comprising: A method is provided in which hybrid lipid particles have an average diameter at least twice that of the average diameter of inorganic material particles.

[0053] According to a second aspect of the present invention, an aqueous suspension of hybrid lipid particles having an average diameter of 50 to 150 nm is provided. The hybrid lipid particles comprise one or more types of lipids and particles of an inorganic material, wherein the average diameter of the hybrid lipid particles is at least twice the average diameter of the inorganic material particles.

[0054] According to a third aspect of the present invention, an aqueous suspension of liposomal lipid particles having an average diameter of 50 to 150 nm is provided. The liposomal lipid particles comprise an active compound (particularly an API), one or more lipids, and particles of an inorganic material, wherein the average diameter of the hybrid lipid particles is at least twice the average diameter of the inorganic material particles.

[0055] A fourth aspect of the present invention provides a method for producing an aqueous suspension of liposome lipid particles according to the third aspect of the present invention from an aqueous suspension of hybrid lipid particles according to the second aspect of the present invention, the method comprising the step of contacting the aqueous suspension of hybrid lipid particles with an active compound (particularly an API).

[0056] According to a fifth aspect of the present invention, a freeze-dried powder of liposomal lipid particles having an average diameter of 50 to 150 nm is provided, wherein the liposomal lipid particles comprise a mixture of one or more cationic or ionizable lipids, one or more further lipids selected from neutral and polar lipids, and optionally one or more additional lipid components, wherein the liposomal lipid particles comprise inorganic material particles (preferably hydrolyzable silicon particles) having an average diameter of up to 1 / 2 the average diameter of the liposomal lipid particles, the weight ratio of inorganic material particles to lipids is 1:2 to 1:100 (preferably 1:10 to 1:100, more preferably 1:20 to 1:10), and the liposomal lipid particles further comprise one or more active compounds, for example, one or more active pharmaceutical ingredients (APIs) in which at least a portion is encapsulated inside the liposomal particles.

[0057] According to a sixth aspect of the present invention, a freeze-dried powder of hybrid lipid particles having an average diameter of 50 to 150 nm is provided, wherein the hybrid lipid particles comprise a mixture of one or more cationic lipids or ionizable lipids, one or more further lipids selected from neutral lipids and polar lipids, and optionally one or more additional lipid components, wherein the hybrid lipid particles comprise inorganic material particles (preferably hydrolyzable silicon particles) having an average diameter of up to half the average diameter of the hybrid lipid particles, and the weight ratio of inorganic material particles to lipids is 1:2 to 1:100 (preferably 1:10 to 1:100, more preferably 1:20 to 1:10).

[0058] According to a seventh aspect of the present invention, a pharmaceutical composition comprising an aqueous suspension of the hybrid lipid particles of the present invention, or a freeze-dried powder of the hybrid lipid particles of the present invention, A pharmaceutical composition is provided which comprises an aqueous suspension of the liposomal lipid particles of the present invention, or a lyophilized powder of the liposomal lipid particles of the present invention.

[0059] Further embodiments of the present invention provide the use of the pharmaceutical composition of the present invention as a pharmaceutical, and related methods for medical treatment.

[0060] The present invention also, (b) (i) Mixing one or more lipids in a solvent or solvent mixture with a suspension of inorganic material particles in a solvent or solvent mixture in an aqueous medium. (ii) Passing the mixture obtained from step (i) through the pores of the extruded film, (iii) Optionally, purify and / or sterilize the suspension by tangential flow filtration. To obtain an aqueous suspension of hybrid lipid particles manufactured by, and (b) Provide an aqueous suspension of lipid particles containing a pharmaceutically active ingredient (API), which is produced by a method comprising contacting the hybrid lipid particles with an active compound, in particular a pharmaceutically active ingredient (API).

[0061] The present invention also, (c) (i) Mixing one or more lipids in a solvent or solvent mixture with a suspension of inorganic material particles in a solvent or solvent mixture in an aqueous medium. (ii) Passing the mixture obtained from step (i) through the pores of the extruded film. To obtain an aqueous suspension of hybrid lipid particles manufactured by, and (d) Provide an aqueous suspension of hybrid lipid particles containing a pharmaceutically active ingredient (API), which is produced by a method comprising purifying and / or sterilizing the suspension by tangential flow filtration.

[0062] Inorganic material particles According to all aspects of the present invention, the inorganic material particles may be any inorganic material comprising a mixture of one or more inorganic materials. In preferred embodiments of all aspects of the present invention, the inorganic material particles are hydrolyzable silicon particles or particles containing hydrolyzable silicon. Preferably, such particles containing hydrolyzable silicon consist of at least 70%, at least 80%, at least 90%, and most preferably at least 98% hydrolyzable silicon (collectively referred to as "hydrolyzable silicon particles" or interchangeably "hydrolyzable silicon particles").

[0063] Solvents and activating solvents, and mixtures thereof Inorganic material particles can be optionally activated by exposing them to a solvent or solvent mixture according to the method of the present invention. Preferably, the solvent or solvent mixture is an activating solvent or an activating solvent mixture. An activating solvent or activating solvent mixture can be understood as a solvent or solvent mixture that “activates” the inorganic material particles (preferably hydrolyzable silicon particles). Activation should be understood as including washing contaminants from the particles so that the particles can adequately interact with other components present in the method or product of the present invention. “Activation” may also include the removal of contaminants from any pores present in the particles. Contaminants include soluble contaminants such as surface oxides and hydroxides, as well as solid contaminants such as surface “dust.” Preferably, the solvent is an organic compound, such as a volatile organic compound such as an alcohol. Preferably, the activating solvent mixture contains methanol. More preferably, the activating solvent is methanol. Optionally, the activating solvent may be evaporated from the inorganic material particles before the method of the present invention proceeds to subsequent steps. Alternatively, the activating solvent is not pre-evaporated. According to certain embodiments, the activating solvent is toxic and / or not recognized as an approved pharmaceutical ingredient. It should be understood that the solvent or solvent mixture to which one or more lipids are supplied may be the same solvent or solvent mixture to which the inorganic material particles are suspended, or it may be a different solvent or solvent mixture.

[0064] According to certain preferred embodiments of the entirety of the present invention, “activation” with a solvent or solvent mixture comprises surface treatment of particles of an inorganic material, which are hydrolyzable silicon particles or particles containing hydrolyzable silicon. Surface treatment with a solvent or solvent mixture should be understood to include surface treatment with one or more alcohols. Particularly preferred alcohols include methanol, benzyl alcohol, and methanol (or mixtures thereof). The “surface treatment” of hydrolyzable silicon optionally includes Si-O(CH2) on the surface of the particles. xCH3 portion (where x is 0 for methanol, 1 for ethanol, etc.), Si-(CH2) x This may include the formation of a CH3 portion and / or a Si-H portion, -(CH2) x CH3 may be replaced by other carbon-containing groups, such as an aromatic ring, if the alcohol is or contains benzyl alcohol.

[0065] Hydrolyzable silicon As used herein, “hydrolyzable silicon” includes pure elemental silicon; however, perfect purity is not required. Conversely, the present invention is not intended to include pure silica (including sand, quartz, and silica gel). A key requirement is that the material is hydrolyzable, that is, it tends to decompose into soluble products such as orthosilicic acid (OSA) under physiological conditions. According to certain embodiments, the definition of “hydrolyzable silicon” is satisfied if at least half of the mass of the material is hydrolyzed into soluble products within one month of injection into the subject (e.g., after intramuscular or subcutaneous injection).

[0066] Hydrolyzable silicon according to a particular embodiment of the present invention is preferably mesoporous. That is, hydrolyzable silicon according to a particular embodiment of the present invention has pores with a diameter of 2 to 50 nm.

[0067] Hydrolyzable silicon particles may be purchased commercially or produced by some preferred method.

[0068] In certain embodiments, the particles containing hydrolyzable silicon may be pure silicon or substantially pure silicon.

[0069] Alternatively, the particles may be another hydrolyzable silicon-containing material. If the particles are not pure silicon, they contain at least about 50 wt% silicon, i.e., at least about 50 wt% silicon atoms, based on the total mass of atoms in the particles. For example, silicon particles may contain at least about 60 wt%, about 70 wt%, about 80 wt%, about 90 wt%, or about 95 wt% silicon. The particles may exhibit a hydrolysis rate of at least 10% of that of pure silicon particles of the same size, for example, in PBS buffer at room temperature. Assays for the hydrolysis of silicon-containing materials are widely known in the art. See, for example, International Publication 2011 / 001456, which is incorporated in its entirety herein by reference.

[0070] The particles may contain trace amounts of silica, but this silica is not hydrolyzable silicon. At least about half of the silicon atoms in the particles may be in the form of elemental silicon (or doped elemental silicon).

[0071] The particles may be nanoparticles in particular. Nanoparticles according to a particular embodiment may have a nominal diameter in the range of about 1 to about 500 nm, particularly about 1 to about 250 nm, and even more particularly about 1 to about 100 nm (e.g., about 30 nm). As used herein, the term “nominal diameter” may mean the average diameter, and at least about 90% of the total mass of particles in the sample falls within the specified size range.

[0072] The particles may be porous, particularly mesoporous. Using standard techniques, for example, particles containing hydrolyzable doped silicon can be made porous by contacting them with a hydrofluoric acid (HF) / ethanol mixture and applying an electric current. The density and size of the pores can be controlled by varying the HF concentration, current density, and exposure time, and can be monitored by scanning electron microscopy and / or nitrogen adsorption / desorption volumetric isothermic measurement. If the particles are porous, their total surface area increases due to their porosity. For example, their surface area may increase by at least about 50% or at least about 100% compared to the surface area of ​​the corresponding non-porous particles. In many situations, the total surface area of ​​porous particles actually increases much more due to their porosity. According to a particular embodiment, the porosity is at least about 30, about 40, about 50, or about 60%, meaning that at least about 30, about 40, about 50, or about 60% of the particle volume is in pore space, respectively. The pore diameter may be in the range of about 1 nm to about 50 nm, for example, about 1 nm to about 5 nm.

[0073] Doped silicon For use in all aspects of the present invention, hydrolyzable silicon preferably contains (or consists of) hydrolyzable doped silicon.

[0074] As used herein, the term “dope silicon” may refer to silicon that behaves as an exogenous semiconductor due to the presence of a dopant atom, whether the dopant atom is substitutional (in place of a Si atom) or interstitial (between Si atoms (without substitution)).

[0075] Advantageously, silicon particles are 1 cm 3 Each unit is at least approximately 1 x 10 15 pieces, and more specifically, at least about 1 × 10⁻⁶ 16 It is doped at the level of individual dopant atoms.

[0076] For example, the particles may be doped at a level of at least about 1×10 3 per cm 17 atoms, at least about 1×10 18 atoms, or at least about 1×10 19 atoms of dopant atoms.

[0077] The silicon particles may be doped at a level of up to 1×10 3 per cm 20 atoms of dopant atoms.

[0078] The silicon particles may be n-doped or p-doped. The silicon particles may be doped with one or more elements selected from B, P, Mg, Cu, Ga, Al, In, Bi, Ge, Li, Xe, N, Au, and Pt. Thus, the dopant may be a p-dopant, particularly boron. The dopant may be an n-dopant, particularly phosphorus.

[0079] When boron is used as the dopant, preferably, the doping levels of 1×10 3 per cm 15 atoms of dopant atoms and 1×10 3 per cm 20 atoms of dopant atoms correspond to resistivities of 13.6 Ω-cm and 1.3 mΩ-cm (milliohm centimeter), respectively. Embodiments where boron is the dopant do not exclude silicon that is doped with boron (e.g., highly doped) but is further doped with other elements (preferably, in such cases, most of the dopant is boron).

[0080] As used herein, the term "heavy doping" is understood to mean doping with at least about 1×10 3 per cm 15 atoms of dopant atoms. In some preferred embodiments, the dopant is at least about 1×10 3 per cm 16It exists at the level of individual dopant atoms. Therefore, in a particularly preferred embodiment, the dopant is present at 1 cm 3 Each unit is at least approximately 1 x 10 16 This is boron that exists at the level of individual boron atoms. For example, 1 cm 3 Each unit is at least approximately 1 x 10 16 A number of boron atoms, and 1 cm 3 The maximum winning value is approximately 1 x 10 20 Boron can exist at the level of individual boron atoms.

[0081] In this specification, when silicon is referred to as "undoped" (such as the particles of composition SIS0012 in Examples 1 and 2), it means that there are no dopant atoms present, or only small amounts present, e.g., 1 cm 3 The maximum winning value is approximately 1 x 10 2 This could mean the presence of a certain number of dopant atoms. Additionally or alternatively, "undoped" silicon could mean silicon that does not behave as an exogenous semiconductor.

[0082] Doping with silicon particles can improve various functions described herein, particularly the ability to make the interior of the hybrid lipid particles according to the present invention accessible. Additionally or alternatively, doping with silicon particles can improve the stability of active compounds (particularly APIs) inside the liposome particles according to the present invention.

[0083] The semiconductor industry provides a ready supply of properly doped silicon and extensive expertise in silicon doping techniques. The production of doped silicon is well understood in the semiconductor industry and includes ion implantation and diffusion methods, making the doped silicon readily available. As an example of the diffusion method, silicon powder and a doping reagent (e.g., B2O3 for boron doping) are mixed in an N2 atmosphere at a temperature of 1050°C to 1175°C for several minutes to diffuse the dopant (e.g., boron) into the silicon.

[0084] Manufacturing of inorganic material particles It is understood that particles can be produced by various techniques well known to those skilled in the art.

[0085] This technology may include purely physical (sometimes referred to as "non-wetting" in the art) processes, such as pulsed laser ablation, pyrolysis, and ball milling, using bulk inorganic solid materials (particularly silicon wafers) as starting materials. Thus, particles may be obtained by a method comprising or consisting of one or more of pulsed laser ablation, pyrolysis, and ball milling of the bulk inorganic material (particularly silicon wafers).

[0086] Additionally or alternatively, particles may be produced by chemical (sometimes referred to in the art as "wetting") techniques, including, but not limited to, electrochemical etching of bulk inorganic materials (particularly silicon wafers). Such techniques optionally include the HF etching described above. Thus, particles may be obtained by methods comprising, or consisting of, electrochemical etching of bulk inorganic materials (particularly silicon wafers).

[0087] Inorganic material particles (e.g., hydrolyzable silicon particles) can be sorted by size after formation by methods such as air classification, sieving, and / or filtration. Therefore, particles may be obtained by methods including one or more of air classification, sieving, and / or filtration.

[0088] Therefore, for example, particles may be obtained from a solid material, particularly from a silicon wafer, by a method that includes generating inorganic solid material particles by, for example, pulsed laser ablation, thermal decomposition, ball mill grinding, and electrochemical etching of a bulk inorganic material (particularly a silicon wafer), followed optionally by sorting by size by air classification, sieving, and / or filtration.

[0089] If desired, the particles may be washed before use, for example, in methanol or ethanol. In the art, this is sometimes referred to as "activation," which is described elsewhere in this specification.

[0090] The inorganic solid material particles obtained in this manner have a narrow size distribution and uniform surface chemical properties, which can result in batch-by-batch reliability and the reproducibility of one or more of the advantages described herein.

[0091] Suitable physical and chemical techniques are described, for example, in International Publication No. 2011 / 012867 (in the name of SISAF LTD), each of which is incorporated in whole by reference herein; Tokarska K et al., Facile production of ultra-fine silicon nanoparticles, R.Soc. Open Sci., 2020, 7:200736; and Kim, T., Lee, J. Silicon nanoparticles: fabrication, characterization, application and perspectives, Micro and Nano Syst. Lett, 2023, 11:18.

[0092] Dimensions of hybrid lipid particles and liposome particles According to all aspects of the present invention, the hybrid lipid particles and liposomal lipid particles of the present invention have an average diameter of 50 nm to 400 nm, for example, 50 nm to 200 nm, for example, 60 nm to 150 nm, for example, 60 nm to 120 nm, for example, 60 nm to 100 nm. Particles having an average diameter of 1 nm to 100 nm may be called nanoparticles (NPs) or lipid nanoparticles (LNPs). The hybrid lipid particles of the present invention that are silicon-stabilized hybrid lipid nanoparticles may be called sshLNPs. According to a particular preferred embodiment using hydrolyzable silicon particles, the hybrid lipid particles have an average diameter of 50 nm to 200 nm (for example, 70 nm to 160 nm), and the hydrolyzable silicon particles have an average diameter of 4 nm to 20 nm (for example, 6 nm to 16 nm).

[0093] Hybrid lipid particles The hybrid lipid particles of the present invention include micelles, incomplete micelles, liposomes, incomplete liposomes, and (e.g., solid or semi-solid) lipid spheres.

[0094] In some embodiments, the hybrid lipid particles according to the present invention may include a lipid bilayer surrounding an aqueous internal space (or, in the freeze-dried form, an empty internal space). However, the lipid bilayer is not one that completely seals the internal space from the environment outside the particle, and the internal space is at least partially accessible. The internal space can be accessed by active compounds, particularly hydrophilic compounds, which would normally be difficult to pass through the lipid bilayer, especially negatively charged nucleic acids, a typical example being mRNA. This accessibility can be achieved by discontinuities in the lipid bilayer or by pathways through the lipid bilayer provided by the presence of particles of inorganic material (e.g., hydrolyzable silicon particles) that penetrate the lipid bilayer.

[0095] Liposome lipid particles The liposome lipid particles according to the present invention include a lipid bilayer surrounding an aqueous internal space. In contrast to the hybrid lipid particles of the present invention, the lipid bilayer completely seals the internal space from the external environment of the particle, thereby protecting any material held within the internal space, such as APIs held within the internal space.

[0096] Transfer between hybrid lipid particles and liposomal lipid particles It is surprising that the hybrid lipid particles of the present invention are stable over long periods. It could be predicted that the lipid bilayer, imperfectly sealed for storage, would effectively convert the hybrid lipid particles of the present invention into liposome particles. This is not the case. In the absence of active compounds or APIs, the hybrid lipid particles of the present invention have been found to be stable over long periods. For example, according to some embodiments, such stability can be defined as the internal space of the particle remaining accessible rather than sealed from the external environment. According to certain embodiments, such stability may manifest as at least 90% of the internal space of the hybrid lipid particle remaining accessible from the external environment for at least 1 week, at least 2 weeks, or at least 4 weeks in an aqueous suspension at room temperature (understood as 25°C throughout this specification). According to other embodiments, such stability may manifest as at least 90% of the internal space of the hybrid lipid particle remaining accessible from the external environment for at least 4 weeks, at least 8 weeks, at least 16 weeks, or at least 32 weeks in an aqueous suspension at 4°C. According to other embodiments, such stability may manifest in at least 90% of the internal space of the hybrid lipid particles, remaining accessible from the external environment for at least 4 weeks, at least 8 weeks, at least 16 weeks, at least 32 weeks, or at least 64 weeks in an aqueous suspension at -20°C. According to certain embodiments, such stability may manifest in at least 90% of the internal space of the hybrid lipid particles, remaining accessible from the external environment for at least 8 weeks, at least 16 weeks, or at least 32 weeks in a lyophilized powder obtained by lyophilizing an aqueous suspension of the particles. Any suitable method may be conveniently used to assess the accessibility of the internal space. For example, accessibility may be assessed using a reporter mRNA or labeled (e.g., fluorescently tagged, immunolabeled, or radiolabeled) mRNA. According to certain embodiments, such stability may manifest in the ability of the hybrid lipid particles of the present invention to react with active compounds, particularly APIs (e.g., mRNA), to produce liposome particles of the present invention.The liposome particles of the present invention preferably exhibit good stability for active compounds (e.g., APIs). For example, when maintained at 4°C for 3 or 6 months, less than 50% of the active compound, particularly APIs, such as mRNA, is degraded.

[0097] Location of inorganic materials The hybrid lipid particles and liposome lipid particles of the present invention comprise particles of an inorganic material (e.g., hydrolyzable silicon particles). Preferably, some of these particles are arranged exposed on the surface of the particle, some are arranged within the lipid bilayer, and some are arranged inside the lipid particle. According to a particular embodiment, at least 10% of the total inorganic material particles are arranged exposed on the surface of the particle, at least 10% of the total inorganic material particles are arranged within the lipid bilayer, and at least 10% of the total inorganic material particles are arranged inside the lipid particle.

[0098] Arrangement of aggregated inorganic material particles According to certain embodiments, particles of an inorganic material (e.g., hydrolyzable silicon particles) are present in one or more aggregates of the hybrid lipid particles of the present invention. Such aggregates may optionally consist of 10 to 200 particles. They may optionally contain, or consist of, chains of particles, most particularly within one or more lipid structures, such as micelles, incomplete micelles, liposomes, incomplete liposomes, and (e.g., solid or semi-solid) lipid spheres (especially liposomes and / or lipid spheres).

[0099] In embodiments using hydrolyzable silicon, the hydrolyzable silicon particles may spontaneously aggregate into particle aggregates, for example, as shown in the transmission electron microscope (TEM) image in Figure 82. Spontaneous aggregation is observed to be particularly pronounced when the hydrolyzable-doped silicon particles have an average diameter of about 1 nm to about 50 nm, particularly about 1 nm to about 30 nm, and even more particularly about 5 nm to about 20 nm, for example, about 10 nm. Such particle diameters may, if desired, be significantly lower than the typical diameter of the hybrid lipid particles of the present invention.

[0100] Therefore, preferably, the hybrid lipid particles of the present invention may include aggregates (particularly chains) of hydrolyzable doped silicon particles, the hydrolyzable doped silicon particles having an average diameter of about 1 nm to about 50 nm, particularly about 1 nm to about 30 nm, and even more particularly about 5 nm to about 20 nm, for example, about 10 nm.

[0101] Aggregation can occur particularly readily in manufacturing methods including extrusion, such as those of the present invention. Extrusion may optionally be or include extrusion through a porous membrane having an average pore diameter of about 0.01 μm to about 1 μm, for example, about 0.05 μm to about 0.6 μm. Preferably, extrusion is performed before the addition of the API. Thus, hybrid lipid particles may be formed before the optional addition of the API.

[0102] As used herein, the term “aggregate of hydrolyzable silicon particles” may refer to a collection of silicon particles in which the nearest silicon particles are in contact with each other. Such collections may have various configurations, such as substantially spherical collections of particles and / or chains of particles. Configurations containing or consisting of chains of particles are particularly preferred.

[0103] Therefore, the hybrid lipid particles of the present invention may contain one or more aggregates of hydrolyzable silicon particles. Preferably, the aggregates contain one or more chains of particles.

[0104] One or more aggregates of hydrolyzable silicon particles may be associated with one or more types of lipids, for example, they may be embedded in lipids and / or attached to the surface of lipids.

[0105] One or more aggregates of hydrolyzable silicon particles may associate with one or more lipid structures described herein, for example, they may be embedded in and / or attached to one or more lipid structures including micelles, incomplete micelles, liposomes, incomplete liposomes, and lipid spheres. Therefore, one or more aggregates of hydrolyzable silicon particles may associate with one or more of lipid micelles, incomplete lipid micelles, liposomes, incomplete liposomes, and lipid spheres (for example, they may be embedded in and / or attached to their surfaces). In particular, one or more aggregates of hydrolyzable silicon particles may be embedded in or attached to one or more of liposomes, incomplete liposomes, and lipid spheres.

[0106] The ratio of the longest dimension of the aggregate to the longest dimension of the lipid structure can be, on average, about 1:5 to 5:1, and especially about 1:3 to 3:1, particularly when the lipid structure is liposomes and / or lipid spheres, or contains them, with one or more aggregates embedded therein or attached to their surface. This can be measured, for example, by TEM as shown in Figure 82.

[0107] The average longest dimension of the aggregates, as measured by TEM, for example, may be approximately 50 nm to approximately 500 nm, particularly approximately 50 nm to approximately 200 nm, for example, approximately 50 nm to approximately 150 nm. In particular, one or more aggregates may be or contain one or more chains of particles, and the average length of the chains is approximately 50 nm to approximately 500 nm, particularly approximately 50 nm to approximately 200 nm, for example, approximately 50 nm to approximately 150 nm. The average cross-sectional diameter of the chains may be approximately 5 nm to approximately 50 nm, for example, approximately 5 nm to approximately 30 nm.

[0108] The ratio of the longest dimension of individual hydrolyzable silicon particles to the longest dimension of the lipid structure can be in the range of approximately 1:100 to approximately 1:2 on average, particularly approximately 1:100 to approximately 1:5, and even more particularly approximately 1:100 to approximately 1:9, especially when the lipid structure is liposomes and / or lipid spheres, or contains them, with one or more aggregates embedded therein or attached to their surface. This can be measured, for example, by TEM as shown in Figure 82.

[0109] If one or more aggregates are present, the average (e.g., mean) diameter of the particles within the aggregates is preferably about 1 nm to about 50 nm, more particularly about 1 nm to about 30 nm, and even more particularly about 5 nm to about 20 nm, for example, about 10 nm. Additionally or alternatively, the particles may be porous and have an average (e.g., mean) pore diameter of about 0.1 to about 5 nm, for example, about 2 nm.

[0110] Activation of inorganic materials According to certain preferred embodiments of the entirety of the present invention, the inorganic material particles are hydrolyzable silicon material particles. Preferably, such particles are activated by the use of a solvent. "Activation" with a solvent or solvent mixture includes surface treatment of the particles containing hydrolyzable silicon. Surface treatment with a solvent or solvent mixture should be understood to include surface treatment with one or more alcohols. Particularly preferred alcohols include methanol, benzyl alcohol, and methanol (or mixtures thereof). "Surface treatment" of hydrolyzable silicon includes Si-O(CH2) on the surface of the particles. x CH3 portion (where x is 0 for methanol, 1 for ethanol, etc.), Si-(CH2) x This may include the formation of a CH3 portion and / or a Si-H portion, -(CH2) xCH3 may be replaced by other carbon-containing groups, such as aromatic rings, if the alcohol is or contains benzyl alcohol. According to certain embodiments of the method of the present invention, silicon nanoparticles are filtered immediately after activation to reduce their aggregation.

[0111] Configuration of API in liposome particles of the present invention The liposome particles of the present invention contain an active compound, particularly an API. The API may be located within the internal space of the particle (i.e., encapsulated) and / or may be located in association with the outer surface of the particle noncovalently. According to a particular embodiment, at least 10% of the API is located within the internal space of the particle (i.e., encapsulated), and / or at least 10% of the API is located in association with the outer surface of the liposome particle noncovalently. According to a particular embodiment, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% of the API is completely encapsulated within the internal space of the particle, and the remainder is optionally located in association with the outer surface of the liposome particle noncovalently.

[0112] Therefore, the hybrid lipid particles of the present invention may include hydrolyzable silicon particles associated with one or more liposomes and / or one or more incomplete liposomes, and the API is associated with (in particular bound to) the hydrolyzable silicon particles. Amino acids (in particular glycine, arginine, and / or tyrosine, e.g., glycine) may also be associated with the hydrolyzable silicon particles.

[0113] The liposomes or incomplete liposomes present, if desired, may have an average diameter in the range of approximately 50 nm to approximately 400 nm, particularly approximately 50 nm to approximately 200 nm, and even more particularly approximately 60 nm to approximately 100 nm.

[0114] The API may be non-covalently bonded to hydrolyzable silicon particles bound to the surface of one or more liposomes and / or one or more incomplete liposomes. Up to about 10% to 20% of the API may be non-covalently bonded to hydrolyzable silicon particles bound to the surface of one or more liposomes and / or one or more incomplete liposomes.

[0115] The API may be non-covalently bonded to hydrolyzable silicon particles located inside one or more liposomes and / or one or more incomplete liposomes. Preferably, at least about 50%, at least about 60%, or at least about 70% of the API may be non-covalently bonded to hydrolyzable silicon particles located inside one or more liposomes and / or one or more incomplete liposomes.

[0116] Preferably, the API (especially RNA, most especially mRNA) is non-covalently bound to hydrolyzable silicon particles located inside one or more liposomes and / or one or more incomplete liposomes, and the API (especially RNA, most especially mRNA) is non-covalently bound to hydrolyzable silicon particles bound to the surface of one or more liposomes and / or one or more incomplete liposomes.

[0117] In some embodiments, the hybrid lipid particles of the present invention may not contain liposomes, or may not contain substantially liposomes, and / or may not contain incomplete liposomes, or may not contain substantially incomplete liposomes.

[0118] If desired, one or more lipids may be formed from or comprise one or more lipid monolayers. If desired, one or more lipids may be one or more micelles or incomplete micelles, or comprise them. It is understood that micelles have similar properties to liposomes, except that their walls are formed from lipid monolayers, whereas liposome walls are formed from lipid bilayers. Therefore, a micelle may refer to a vesicle having at least one lipid monolayer, which may be substantially spherical in shape. Similar to liposomes, micelles can be visualized as lipid "bubbles" surrounding an internal space. The internal space may be a hydrophilic environment.

[0119] Therefore, the hybrid lipid particles of the present invention may include hydrolyzable silicon particles associated with one or more micelles and / or one or more incomplete micelles, and the API is associated with (in particular bound to) the hydrolyzable silicon particles.

[0120] The micelles present, as desired, may have an average diameter in the range of approximately 50 nm to 400 nm, particularly 50 nm to 200 nm, and even more particularly 60 nm to 100 nm.

[0121] The API may be non-covalently bonded to hydrolyzable silicon particles attached to the surface of one or more micelles and / or one or more incomplete micelles. Up to about 10% to 20% of the API may be non-covalently bonded to hydrolyzable silicon particles attached to the surface of one or more micelles and / or one or more incomplete micelles.

[0122] The API may be non-covalently bonded to hydrolyzable silicon particles located within one or more micelles and / or one or more incomplete micelles. Preferably, at least about 50%, at least about 60%, or at least about 70% of the API may be non-covalently bonded to hydrolyzable silicon particles located within one or more micelles and / or one or more incomplete micelles.

[0123] Preferably, the API (especially RNA, most especially mRNA) is non-covalently bonded to hydrolyzable silicon particles located inside one or more micelles and / or one or more incomplete micelles, and the API (especially RNA, most especially mRNA) is non-covalently bonded to hydrolyzable silicon particles bound to the surface of one or more micelles and / or one or more incomplete micelles.

[0124] In some embodiments, the hybrid lipid particles of the present invention may be omitted from micelles or substantially omitted from micelles, and / or may be omitted from incomplete micelles or substantially omitted from incomplete micelles.

[0125] One or more lipids may be formed from or contain one or more lipid spheres, each sphere optionally surrounded by a layer of surfactant. The lipid spheres do not enclose internal spaces or cavities. Instead, they are formed robustly or substantially robustly from lipids, and other components, such as hydrolyzable silicon particles to which API molecules are bound, may be dispersed therein. Thus, hydrolyzable silicon particles may be scattered inside the spheres, and the API molecules are (non-covalently) bound to the hydrolyzable silicon particles. Additionally or alternatively (preferably additionally), hydrolyzable silicon particles to which API molecules are (non-covalently) bound may be bound to the surface of one or more lipid spheres.

[0126] Accordingly, the hybrid lipid particles of the present invention may comprise hydrolyzable silicon particles associated with (particularly dispersed in and / or bound to the surface of) one or more (solid or substantially solid, i.e., non-hollow) lipid spheres, wherein the API is associated with (particularly bound to) the hydrolyzable silicon particles.

[0127] The lipid spheres present as desired may have an average diameter in the range of approximately 50 nm to 400 nm, particularly 50 nm to 200 nm, and even more particularly 60 nm to 100 nm.

[0128] The API may be non-covalently bonded to hydrolyzable silicon particles attached to the surface of one or more lipid spheres. Up to about 10% to 20% of the API may be non-covalently bonded to hydrolyzable silicon particles attached to the surface of one or more lipid spheres.

[0129] The API may be non-covalently bonded to hydrolyzable silicon particles located inside one or more lipid spheres. Preferably, at least about 50%, at least about 60%, or at least about 70% of the API may be non-covalently bonded to hydrolyzable silicon particles located inside one or more lipid spheres.

[0130] Preferably, the API (especially RNA, most particularly mRNA) is non-covalently bound to hydrolyzable silicon particles located inside one or more lipid microspheres, and the API (especially RNA, most particularly mRNA) is non-covalently bound to hydrolyzable silicon particles bound to the surface of one or more lipid microspheres.

[0131] Relative dimensions According to all aspects of the present invention, the hybrid lipid particles or liposomal lipid particles have an average diameter at least twice the average diameter of the inorganic material particles. In some embodiments, the relative sizes may vary more significantly, for example, the liposomal lipid particles may have an average diameter at least 3, 4, 5, 6, 7, 8, 10, 12, 15, or 20 times the average diameter of the inorganic material particles. In some embodiments, the liposomal lipid particles may have an average diameter at least 3 to 10 times the average diameter of the inorganic material particles, or at least 5 to 20 times the average diameter of the inorganic material particles. In some embodiments, the hybrid lipid particles or liposomal lipid particles have an average diameter of 50 nm to 400 nm, while the inorganic material particles (e.g., hydrolyzable silicon particles) have an average diameter of 10 nm to 60 nm (while the relative diameter remains within the listed range).

[0132] Notes regarding average diameter In this specification, particle size is described as average diameter. Particle diameter can be measured by any preferred method, including dynamic light scattering, electron microscopy, and size exclusion. Preferably, the particles have a diameter distribution around the average diameter such that 80% of the particles have a diameter within ±25% of the average diameter. This is especially true after filtration and extrusion, which are known to enhance monodispersity.

[0133] Extruded membrane In accordance with the present invention, any suitable extruded membrane may be used. Preferably, the extruded membrane is used as part of an in-flow extrusion system comprising the extruded membrane and a flow pump (e.g., an HPLC pump). Preferred extruded membranes include those containing polycarbonate. Preferred pore sizes include pore sizes of 0.05 μm to 1.2 μm, for example, 0.08 μm to 1.0 μm, for example, 0.8 μm, 0.4 μm, or 0.1 μm.

[0134] Extrusion method The flow rate may depend on the total volume to be extruded and the area of ​​the extrusion membrane used. For example, a flow rate of 10 to 100 ml / min may be used. The extrusion pressure may be selected to achieve a sufficient flow rate. Extrusion is usually carried out at a high temperature. This is necessary to increase the fluidity of the lipid used. The exact optimal temperature may depend on the exact lipid formulation used, but extrusion is usually preferred to be carried out at 50°C to 70°C, for example, 55°C to 65°C, for example, about 60°C.

[0135] According to certain preferred embodiments of the present invention, multiple extrusions are typically performed. For example, the same material may be extruded at least six times, or at least eight times. According to certain embodiments, it is preferable to perform multiple extrusions using a membrane with a decreasing pore size. For example, multiple extrusions may be performed using a membrane with a relatively large pore size, or first, multiple extrusions may be performed using a membrane with a medium pore size, and then multiple extrusions may be performed using a membrane with a relatively small pore size. In certain preferred embodiments, multiple extrusions (e.g., at least two or at least three) may be performed using a membrane with a pore size of 0.6 μm to 1.0 μm (e.g., 0.8 μm), first, multiple extrusions (e.g., at least two or at least three) may be performed using a membrane with a pore size of 0.3 μm to 0.5 μm, and then multiple extrusions (e.g., at least two or at least three) may be performed using a membrane with a pore size of 0.08 μm to 0.2 μm (e.g., 0.1 μm).

[0136] Reduction of inorganic materials by extrusion As described above, the present invention is based in part on the discovery that inorganic material particles (such as silicon-containing material particles) tend to be removed from the hybrid lipid particles of the present invention during extrusion, but this does not pose a problem for the sustained stability and favorable properties of the hybrid lipid particles. If inorganic material particles are present at a relatively high initial concentration, it appears that their concentration can be reduced without disadvantage during extrusion. Therefore, in certain embodiments of a preferred first aspect of the present invention, the extrusion step (step B) results in the retention of at least 50%, at least 60%, at least 70%, or 80% (by weight) of the inorganic material particles present in step A.

[0137] Lipid components According to all aspects of the present invention, a lipid mixture used to form the hybrid lipid particles of the present invention, or comprising the liposome lipid particles or hybrid lipid particles of the present invention, preferably comprises one or more cationic lipids or ionizable lipids and one or more further lipids selected from neutral lipids and polar lipids, and preferably optionally comprising one or more additional lipid components.

[0138] The charge on the lipids (and therefore its classification, for example, as cationic, anionic, or zwitterionic) is preferably evaluated at pH 7.4, and can be evaluated, for example, in a physiologically suitable pH 7.4 phosphate buffer.

[0139] According to all aspects of the present invention, a mixture of one or more cationic lipids or ionizable lipids and one or more neutral lipids or polar lipids contains at least one cationic lipid or ionizable lipid. Preferably, the proportion (as a molar ratio) of the total cationic lipids or total ionizable lipids is 20-70% of the total lipids, for example, 30-60% or 40-60%.

[0140] According to certain embodiments, the cationic lipid or ionizable lipid is a cationic lipid. The cationic lipid can 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 mixtures thereof. Mixtures containing DOTAP are particularly preferred. According to certain embodiments, at least half or all of the cationic lipid is DOTAP.

[0141] According to other embodiments, the cationic lipid or ionizable lipid is an ionizable lipid. The ionizable lipid may be selected from the group consisting of [(4-hydroxybutyl)azandiyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate, heptadecan-9-yl8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate, 7-[(2-hydroxyethyl)[8-(nonyloxy)-8-oxooctyl]amino]heptyl2-octyldecanoate, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate, and DODMA, and mixtures thereof.

[0142] According to a particular embodiment, the cationic lipid component or the 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).

[0143] The lipid blend may optionally further contain one or more neutral or polar lipids. The neutral phospholipid DOPE (dioleoylphosphorylethanolamine), PC (phosphatidylcholine), and lecithin (a PC-dominant mixture) are all examples of noncationic phospholipids that can be used as neutral or polar lipids according to the present invention.

[0144] According to a particular preferred embodiment, the lipid blend consists entirely of cationic lipids and phospholipids. For example, the lipid blend may consist of approximately equal amounts of DOTAP and DOPE.

[0145] The lipid blend may optionally further contain additional lipid components, such as conjugate lipids (e.g., pegylated lipids) and / or steroid / sterol components (e.g., cholesterol). In certain preferred embodiments, the lipid blend contains neither substantial amounts of conjugate lipids (e.g., pegylated lipids) nor substantial amounts of steroid / sterols. In any embodiment, it is preferable that the particles of the present invention are cholesterol-free. In other embodiments, small amounts of cholesterol may be present, for example, constituting less than 10%, less than 8%, less than 5%, less than 2%, less than 1%, or less than 0.5% (by weight) of the total lipids present.

[0146] Lipids are generally understood to include fatty acids and fatty acid derivatives, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, and polyketides. As used in this application, the term “lipid” may encompass lipid-conjugated oligopeptides (a term used herein interchangeably with the term “lipopeptide”) in which a short peptide sequence (3 to 20 amino acid residues, e.g., 5 to 15 amino acid residues, particularly 3, 4, or 5 amino acid residues, most particularly a peptide sequence having 5 amino acid residues, etc.) is conjugated to one or more fatty acid chains (particularly 10 to 24 carbon chain lengths, preferably 12 to 18 carbon chain lengths; e.g., fatty acid chains having carbon chain lengths of 14, 15, or 16 carbon chain lengths; e.g., the peptide portion may optionally be lipid-conjugated by a palmitoyl, cetyl, or myristoyl portion).

[0147] Therefore, one or more types of lipids may contain one or more types of lipid-modified oligopeptides. Preferably, one or more types of lipid-modified oligopeptides each contain a fatty acid chain having about 12 to about 18 carbon atoms.

[0148] Preferably, one or more lipid-modified oligopeptides each contain 3 to 20 amino acid residues. Therefore, the lipid-modified oligopeptides may be lipid-modified tetrapeptides, lipid-modified pentapeptides, or lipid-modified hexapeptides.

[0149] Preferably, the amino acid residues include at least one amino acid residue (e.g., about two or three amino acid residues) that is cationic at a pH of about 7.4 (physiological pH), such as lysine or arginine. For example, the lipid-containing oligopeptide may contain one or more (e.g., about two) lysine residues.

[0150] A special example of a lipid-containing oligopeptide ("lipopeptide") is palmitoyl-pentapeptide-4 (CAS number 214047-00-4; abbreviated as PAL-KTTKS).

[0151] Therefore, preferably, one or more lipids may include, or are composed of, one or more lipid-containing oligopeptides, particularly those having one or more amino acid residues that are positively charged at a pH of about 7.4 (i.e., approximately physiological pH), such as lysine and arginine, or one or both.

[0152] Lipidized oligopeptides can be used in particular in combination with one or more phospholipids such as DOPE or DPPC. The alkyl chain of the lipidized oligopeptide molecule can be assimilated within the phospholipid bilayer, but the surface of the bilayer is decorated with the peptide moiety. While not wishing to be bound by theory, it is thought that the peptide moiety of the lipidized oligopeptide may enable the targeting of one or more specific tissues and / or cells. On the other hand, if the peptide moiety has a positive charge at approximately 7.4 pH (i.e., near physiological pH), negatively charged APIs (e.g., nucleic acids, particularly mRNA or siRNA) can be stabilized.

[0153] One or more types of lipids include one or more cationic lipids (e.g., DOTAP); one or more phospholipids (e.g., DOPE); and one or more polyethylene glycol (PEG) lipids (e.g., DSPE-PEG). 2000 ) may be one or more of the above, or may include them.

[0154] One or more lipids may be, or may contain, one or more structural lipids (e.g., cholesterol lipids). However, one or more lipids may optionally exclude structural lipids. Thus, one or more lipids may exclude sterols, and in particular cholesterol. It has been found that the compositions currently disclosed do not need to rely on these types of lipids on which conventional API delivery systems typically depend. Therefore, the compositions disclosed herein have the potential to provide an alternative to API delivery systems that depend on these types of lipids, in particular cholesterol. This may be advantageous when cholesterol is unavailable or otherwise unusable (e.g., due to its action in the body).

[0155] One or more lipids include phosphatidylcholine (PC); hydrogenated PC; stearylamine (SA); dioleoylphosphatidylethanolamine (DOPE); cholesteryl 3β-N-(dimethylaminoethyl)carbamate hydrochloride (DC-chol); 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP); and DSPE-PEG. 2000 The product may optionally contain one or more of the following: pegylated 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) and its derivatives.

[0156] In certain embodiments, the lipid is selected from the group consisting of phosphatidylethanolamine (PE), phosphatidylcholine (PC), stearylamine (SA), or any combination thereof.

[0157] Lipids or lipid components may be or may include cationic lipids in some embodiments. The term “cationic lipid” refers to a molecule having a cationic head group bonded to a hydrophobic tail via several spacers, and having a net positive charge at pH 7.4 (physiological pH). Examples include DTDTMA (ditetradecyltrimethylammonium), DOTMA (2,3-dioleyloxypropyl-1-trimethylammonium), DHDTMA (dihexadecyltrimethylammonium); dioleoyl-3-trimethylammoniumpropane (DOTAP); and stearylamine (SA). The positive charge can usually be stabilized by a negative counterion.

[0158] Therefore, one or more lipids may be or may contain DOTAP as desired. DOTAP exists in S-enantiomer and R-enantiomer forms and may exist in S-form, R-form, or racemic form. Optionally, the R-form and S-form may be present in approximately equal amounts by weight of the total DOTAP (i.e., the total DOTAP of any one form may be present at about 60% by weight or less). In other embodiments, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% of the total DOTAP is in R-form. In other embodiments, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% of the total DOTAP is in S-form.

[0159] Nevertheless, as described herein, doping with silicon may allow for a reduction in the use of cationic lipids such as DOTAP compared to conventional compositions for API delivery (such as lipid nanoparticles containing cationic lipids).

[0160] Therefore, one or more types of lipids may optionally exclude cationic lipids. When doped silicon, particularly p-doped silicon, is used as described herein, cationic lipids may not be necessary.

[0161] Therefore, one or more types of lipids include one or more types of phospholipids (e.g., DOPE); and one or more types of polyethylene glycol (PEG) lipids (e.g., DSPE-PEG). 2000 ) may be one or more of the above, or may include them.

[0162] Overall, the inorganic material particles disclosed herein may offer the possibility of reducing the lipids used in API delivery vehicles (particularly cationic lipids, e.g., DOTAP) compared to conventional API delivery vehicles that do not contain such particles (e.g., conventional liposomal nucleic acid delivery vehicles, e.g., those commonly used for in vivo mRNA delivery). Additionally or alternatively, inorganic material particles may offer the possibility of formulating API delivery vehicles with a wider range of lipids while still providing transfection efficiency, storage stability, and / or targeted delivery to specific types of tissues or cells. This, in turn, may reduce the reliance in the art on specific lipids, particularly cationic lipids, especially cationic lipids that are specifically formulated for API delivery and may therefore be cost-ineffective or not readily accessible.

[0163] One or more types of lipids may have an average molecular weight in the range of approximately 500 to 1000.

[0164] The ratio of one or more lipids (meaning the total lipid components in the composition) to silicon may be in the range of about 40:1 to about 1:1, particularly in the range of about 20:1 to about 1:1, for example, a ratio of about 16:1, when the components are assembled for the manufacture of the delivery system, i.e., before any further processing is performed.

[0165] As described herein, one or more types of lipids may include, or may be, phospholipids in particular. The term "phospholipid" as used herein may refer to lipids containing fatty acid chains and phosphate groups. Unlike positively charged cationic lipids, phospholipids may be negatively charged. However, phospholipids are usually zwitterionic compounds containing both positively and negatively charged components, and are therefore neutral overall. Thus, phospholipids are typically classified as neutral lipids.

[0166] Preferred phospholipids may be or may contain glycerophospholipids. Particularly preferred phospholipids may be those in which a polar head group is linked to a quaternary ammonium moiety, such as phosphatidylcholine (PC) or hydrogenated phosphatidylcholine, or may contain such phospholipids. The phospholipid may be or may be derived from lecithin. A preferred phospholipid is DOPE (phosphatidylethanolamine, or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine).

[0167] Preferably, the phospholipid side chain may be an aliphatic side chain with about 15 or more carbon atoms, or an ether side chain with about 6 or more repeating ether units, such as a polyethylene glycol chain or a polypropylene glycol chain.

[0168] Lipids having either a side chain may be called "PEG-lipids" or "pegylated" lipids. Therefore, as used in this application, the term "lipid" may encompass PEG lipids. Accordingly, according to a preferred embodiment, one or more lipids may be one or more polyethylene glycol (PEG) lipids, particularly DSPE-PEG. 2000 This may include, or could include, pegged DSPEs such as the following.

[0169] One or more lipids may optionally contain phosphatidylcholine (PC), hydrogenated phosphatidylcholine, stearylamine (SA), or a combination thereof, or may substantially consist of them.

[0170] One or more types of lipids may optionally contain at least about 5% (by weight) (e.g., at least about 30% or at least about 50%) of polypropylene (PC) based on the total weight of one or more types of lipids.

[0171] One or more types of lipids may optionally contain at least about 5% (by weight) (e.g., at least about 30% or at least about 50%) of hydrogenated polycarbonate (PC) based on the total weight of one or more types of lipids.

[0172] One or more types of lipids may optionally contain at least about 5% (by weight) (e.g., at least about 30% or at least about 50%) of SA, based on the total weight of one or more types of lipids.

[0173] One or more types of lipids may, if desired, contain PC and SA in a PC to SA weight ratio ranging from approximately 1:1 to approximately 20:1, or may be substantially composed of them.

[0174] One or more lipids may optionally contain a combination of DOPE, SA, and DC-, or may be substantially composed of them.

[0175] In certain preferred embodiments, one or more lipids are DOTAP, DOPE, and PEG-lipids (particularly DSPE-PEG). 2000 The combination of ) may be included as desired, or may essentially consist of them. The weight ratio of DOTAP:DOPE is in the range of approximately 1:2 to approximately 2:1, for example, approximately 1:1. The weight ratio of DOTAP:PEG-lipid is in the range of approximately 10:1 to approximately 5:1, for example, approximately 7:1. The weight ratio of DOPE:PEG-lipid is in the range of approximately 10:1 to approximately 5:1, for example, approximately 7:1.

[0176] Additional ingredients It has been found that the stability of the hybrid lipid particles and liposomal lipid particles of the present invention can be further enhanced in the presence of one or more amino acids and / or one or more non-reducing disaccharides. Therefore, the stability of active compounds (particularly APIs) in the liposomal lipid particles of the present invention can be further enhanced. Accordingly, the method of the present invention may be carried out as desired in the presence of one or more amino acids and / or one or more non-reducing disaccharides. Lipid particles of all embodiments of the present invention may further contain non-reducing disaccharides and / or amino acids. A preferred non-reducing disaccharide is trehalose. A preferred amino acid is glycine. In certain preferred embodiments, the use of both glycine and trehalose is preferred. These components can electrostatically coordinate with inorganic materials such as silicon, thereby enhancing the overall stability of the system. Glycine is particularly preferred due to its hydrophilic, moderate to mild nonpolar nature, and because glycine exists as a zwitterion in aqueous solutions at physiological pH (approximately pH 7.4). Glycine binds to the surface of inorganic materials (e.g., silicon) and, due to its zwitterionic nature, can electrostatically coordinate with both positive and negative charges as well as partial charges, thereby increasing particle stability.

[0177] In its broadest sense, the term "amino acid" encompasses any artificial or naturally occurring organic compound containing an amine (-NH2) functional group and a carboxyl (-COOH) functional group. This includes α-amino acids, β-amino acids, γ-amino acids, and δ-amino acids. This includes amino acids in any chiral configuration. Amino acids can be, in particular, naturally occurring α-amino acids. These can be proteinaceous or non-proteinaceous amino acids (carnitine, levothyroxine, hydroxyproline, ornithine, or citrulline).

[0178] One or more amino acids can help to stabilize the inorganic material itself, particularly silicon particles. In vivo, one or more amino acids can help to regulate the hydrolysis rate of silicon particles, such that silicon is hydrolyzed into biologically available orthosilicic acid (OSA) degradation products rather than insoluble polymer hydrolysis products. Controlling the hydrolysis rate of silicon in vivo can affect the release rate of the active compound (particularly the API).

[0179] By controlling the release rate of the active compound (particularly the API), the length of the period during which the protection of the active compound (particularly the API) is sustained can be adjusted, particularly with respect to in vivo protection in the presence of various body fluids. Thus, more active compound (particularly the API) can be delivered to the target cells over a given period than in an otherwise identical composition.

[0180] In a preferred embodiment, the amino acid can comprise or consist essentially of glycine.

[0181] Additionally or alternatively, amino acids that are neutral or positively charged at physiological pH (about pH 7.4), such as tyrosine or arginine, can help to stabilize negatively charged APIs (such as nucleic acids like mRNA). On the other hand, amino acids that are neutral or negatively charged at physiological pH (about pH 7.4) can help to stabilize positively charged APIs. Nevertheless, the interactions, charge-based and / or other (such as steric, etc.) interactions resulting from the combination of the inorganic material, lipid, and amino acid can be such that positively charged amino acids at physiological pH can help to stabilize positively charged active compounds (particularly the API), or negatively charged amino acids at physiological pH can help to stabilize negatively charged active compounds, particularly negatively charged APIs.

[0182] The weight ratio of one or more lipids (i.e., the total lipid component) to amino acid can be within the range of about 40:1 to about 1:1, such as about 32:1, etc.

[0183] If desired, the composition may specifically contain the amino acid tyrosine in addition to the amino acids described herein. If desired, the composition may specifically contain tyrosine instead of the amino acids described herein. Therefore, it is understood that tyrosine is an amino acid, but for the purposes of this disclosure, tyrosine may optionally be present as a separate and further component distinct from the amino acids described herein. Therefore, if tyrosine is present as a separate and further component distinct from the amino acids described herein, in addition to the amino acids described herein, it is understood that the calculation of the range of one or more lipids to amino acids, such as about 40:1 to about 1:1, for example, about 32:1 disclosed above, does not include the amount of additional different tyrosine.

[0184] Additionally or alternatively, one or more non-reducing disaccharides, particularly trehalose, may be included. The weight ratio of one or more lipids (i.e., total lipid components) to non-reducing disaccharides may be in the range of about 20:1 to about 1:1, for example, about 16:1.

[0185] Particle stability The hybrid lipid particles and liposome particles of the present invention exhibit enhanced dimensional stability compared to corresponding particles that do not contain inorganic material particles (e.g., hydrolyzable silicon material) according to the present invention. This enhanced dimensional stability manifests as resistance to the particles coalescing into larger particles. According to certain embodiments, the coalescence rate at 5°C is up to half that of equivalent corresponding inorganic material particles (except for the absence of hydrolyzable silicon particles according to the present invention, which have the same composition). According to certain embodiments, at least 90% of the particles did not coalesce and maintained their original size after being stored for 3 months in an aqueous solution at physiological pH (approximately 7.4) at 5°C.

[0186] Charge stability The surface charge of lipid particles, including the hybrid lipid particles and liposomal lipid particles of the present invention, can be estimated using the zeta potential (interfacial dynamic potential) parameter. As an empirical rule, suspensions of particles in nuclease-free water with low zeta potentials (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.

[0187] According to certain embodiments, the hybrid lipid particles and liposomal lipid particles of the present invention have a value of >±40mV, more preferably >±45mV, >±50mV, or >±60mV. Preferably, the zeta potential is increased by at least ±10mV in the presence of hydrolyzable silicon (i.e., the zeta potential is the same as that of the present invention, but at least ±10mV greater than the zeta potential of equivalent lipid particles in the absence of inorganic material particles (e.g., hydrolyzable silicon particles according to the present invention)).

[0188] The presence of inorganic materials such as hydrolyzable silicon also suppresses the loss of positive charge in cationic lipids. This is known as lipid aging, and is preferably slowed by at least 2 times, at least 4 times, at least 8 times, or at least 16 times in the lipid particles of the present invention (at 5°C).

[0189] Nucleic acid stability The liposomal lipid particles of the present invention act to protect active compounds and APIs. The liposomal lipid particles of the present invention particularly act to protect active compounds and APIs, which are nucleic acids (particularly RNA, and even more particularly mRNA) electrostatically complexed on the interior and surface of the liposomal lipid particles. The present invention makes it easier to store therapeutic formulations, such as vaccines, for example, allowing them to be stored at 5°C or room temperature instead of below zero. They also increase stability and reduce nucleic acid degradation during lyophilization, rehydration, transport, and storage. According to certain embodiments of the present invention, in particular, the half-life of an active compound or API, where the active compound or API is mRNA, is extended by at least 100 times, at least 1000 times, or at least 10000 times compared to the corresponding mRNA not complexed with the liposomal lipid particles of the present invention. According to certain embodiments, the half-life of mRNA is extended by at least 10 times, at least 100 times, or at least 1000 times compared to the corresponding mRNA complexed with equivalent liposomal lipid particles lacking the inorganic material (e.g., hydrolyzable silicon) component particles according to the present invention. The half-life can be measured in a physiologically compatible aqueous solution at pH 7.4 and 5°C. According to certain embodiments, particularly when the active compound or API is a nucleic acid such as RNA, the half-life can be measured in natural physiological lipids. For example, the half-life can be measured in blood or in blood components such as plasma, in vivo, ex vivo, or in vitro. For example, the half-life can be measured in vitro in human plasma. Such assays can be particularly demanding for active components or APIs that are RNA, because human plasma is known to contain substances known to degrade RNA, such as enzymes.

[0190] Method of the present invention A method for producing an aqueous suspension of hybrid lipid particles comprises step A, mixing one or more lipids in a solvent or solvent mixture with a suspension of inorganic material particles in a solvent or solvent mixture in an aqueous medium. This step may be carried out as desired using one or more different apparatuses and methods, including bulk mixing methods or bulk mixing apparatuses, and microfluidic mixing methods or microfluidic mixing apparatuses.

[0191] Processes that exist upon request The method of the present invention may optionally include an additional step D, after step B and, if present, step C, in which the hybrid lipid particles of the present invention are contacted with an active compound, in particular a pharmaceutically active ingredient (API). Such a method optionally produces liposomal lipid particles of the present invention. According to certain embodiments of the method of the present invention, there is an interval of at least one week, at least one month, or at least six months between step B and step D, and between step C and step D, if present. Optionally, the hybrid lipid particles are maintained at 4°C or 20°C during that interval.

[0192] Filtration available upon request The method of the present invention may optionally include an additional step C after step B, in which the suspension is purified, concentrated, and / or sterilized by tangential flow filtration.

[0193] Therefore, according to a first aspect of the present invention, an additional step C may be inserted between steps B and D. Preferably, step C is carried out using tangential flow filtration, but any suitable filtration process can be used. One of the purposes of filtration is to increase the size uniformity of hybrid lipid particles by filtration through a membrane having a size cutoff of a desired particle size (e.g., 100 nm). Surprisingly, it has also been found that filtration removes from the solution many inorganic material particles (e.g., hydrolyzable silicon particles) that do not strongly associate with the hybrid lipid particles, i.e., particles that are not bound to the surface of or within the lipid particles. This is despite the fact that the inorganic material particles (e.g., hydrolyzable silicon particles) are smaller than the hybrid lipid particles and the filtration membrane exclusion size. Therefore, in its first aspect, a filtration step may be optionally inserted between steps B and C of the present invention.

[0194] After filtration, the total weight percentage of hybrid lipid particles composed of inorganic material particles (e.g., hydrolyzable silicon particles) may be less than 20%, for example, less than 10%, less than 5%, or 1%. Keeping the amount of inorganic material such as silicon low reduces concerns about side effects of inorganic material such as silicon.

[0195] In certain embodiments, the filtration, if desired, is diafiltration. In certain embodiments, this diafiltration can maintain the concentrations of these materials in the product by using a diafiltration solution containing a non-reducing disaccharide such as trehalose and an amino acid such as glycine.

[0196] Absence of solvent evaporation step The method of the present invention does not necessarily require a solvent evaporation step. According to a preferred embodiment of the method of the present invention, such a method does not include a solvent evaporation step. In particular, such a method does not include a step of evaporating the solvent used to activate inorganic material particles (e.g., hydrolyzable silicon particles) nor a step of evaporating the solvent used to mix one or more lipids. Preferably, substantially all solvents (especially all alcohols such as methanol) are removed without substantial evaporation by the filtration method described herein.

[0197] In-process controlled processes According to a preferred embodiment of the method of the present invention, such a method further comprises one or more of the in-process quality control steps that may be present. The method may optionally include a visual inspection of complete dissolution of lipids prior to mixing in step A. Alternatively or additionally, the method may optionally include, after step B, measurement of dynamic light scattering (DLS) parameters, measurement of mean hydrodynamic size parameters, measurement of polydispersity (PDI), and / or measurement of zeta potential. Optionally, this measurement is compared to product specifications, and if the measurement fails to meet product specifications, extrusion step B is optionally repeated, followed optionally by repeated measurements and repeated comparisons with product specifications.

[0198] Pharmaceutical compositions and their use The present invention further intends to utilize the hybrid lipid particles and liposomal lipid particles of the present invention for formulating pharmaceuticals that also fall within the scope of the present invention. Such pharmaceuticals include injectable formulations (such as injectable vaccines), topical creams, capsules, tablets, and ointments. They also include pharmaceutical precursors or pharmaceuticals, e.g., dehydrated (lyophilized) and concentrated products, which must be diluted and / or rehydrated before use.

[0199] temperature The temperature employed during extrusion is generally about 60°C, for example, 50°C to 70°C. Such temperatures, as may be preferred in certain embodiments, threaten the integrity of certain APIs, for example, when the API is a nucleic acid such as RNA (siRNA, saRNA, mRNA). As an example of the method of the present invention, there is first a step of generating the hybrid lipid particles of the present invention in the absence of the API. There is optionally a step of transporting the hybrid lipid particles for further use, a step optionally present of lyophilizing it, freezing it, or storing it prior to further use, and then a step of contacting the hybrid lipid particles with the API to generate a suspension of the liposomal lipid particles of the present invention. This final step is preferably carried out at a relatively low and thus milder temperature. For example, this final step can be carried out at room temperature (25°C), or at a temperature slightly higher than freezing (0°C). According to certain embodiments, this final step is carried out at 0°C to 30°C, for example, 0°C to 25°C, or 0°C to 10°C.

[0200] Other method features In a particular preferred embodiment, step A of the method of the present invention involves mixing one or more lipids and inorganic material particles in an aqueous medium, wherein the lipids are provided in a solvent (e.g., an alcohol such as methanol). The use of such a solvent advantageously ensures proper mixing between the lipids. It may be considered necessary to evaporate the solvent, especially if it is a toxic solvent such as methanol. The presence of the solvent may be considered detrimental to the formation of lipid particles. Surprisingly, the inventors have found that good formation of hybrid lipid particles occurs even when the solvent does not evaporate and the lipids and particles are mixed in an aqueous medium according to step A of the method of the present invention, and the lipids are provided in a solvent, e.g., an alcohol such as methanol. Such a method preferably includes a downstream step of purifying the suspension by tangential flow filtration (e.g., the method as described in step C of the method of the present invention). Such a step has been found to be suitable for removing the solvent from the suspension. If an activating solvent or solvent mixture is used to activate particles of inorganic material (such a solvent may be the same solvent or solvent mixture to which lipids are provided), TFF may be used in a subsequent step to remove the activating solvent. TFF may be used optionally in a diafiltration method. In certain embodiments, this diafiltration method may maintain the concentrations of these materials in the product by using a diafiltration solution containing a non-reducing disaccharide such as trehalose and an amino acid such as glycine.

[0201] mixed method Lipid particles can be produced by various processes, and conventional methods often involve hydrating lipid thin films. While such methods may be effective, they can be fragile for the production of lipid particles and involve inconsistent sizing and low encapsulation efficiency. The present invention, for example, involves mixing one or more lipids in a solvent or solvent mixture with a suspension of inorganic material particles in a solvent or solvent mixture, and then mixing the solvent or solvent mixture having one or more lipids and the suspension of inorganic material particles into an aqueous medium.

[0202] According to a particular embodiment, this final mixing may be carried out by injecting a solvent or solvent mixture into an aqueous medium, or by any other method including a bulk apparatus or microfluidics apparatus that provides effective and efficient mixing of lipids and inorganic particles of inorganic material.

[0203] According to certain preferred embodiments, it has been found that further improvements in production efficiency and product quality can be achieved by mixing a solvent or solvent mixture with an aqueous medium using a rapid mixing method.

[0204] The rapid mixing methods according to embodiments of the present invention include cross-flow injection or T-junction mixing. Such methods are adaptations of solvent injection techniques and result in rapid mixing of organic solutions / suspensions and aqueous solutions / suspensions. They have been found to enable the reliable generation of small hybrid lipid particles, and particle size can be conveniently controlled by varying the injection flow rate and / or pressure. Figure 68 shows examples of various rapid mixing methods according to certain embodiments of the present invention, particularly in its first aspect. These include cross-flow injection, T-junction mixing, microhydrodynamic focusing, staggered herringbone mixing (SHM), baffle mixing, segment flow micromixing, and toroidal / biferrated micromixing.

[0205] Active pharmaceutical ingredients and other active compounds In certain embodiments of the present invention, the active pharmaceutical component may be some pharmaceutically active compound. It is preferably a hydrophilic compound, such as a negatively charged compound, such as a nucleic acid. In other embodiments, the methods and products of the present invention include other types of “active compounds.” Such other active compounds are not necessarily pharmaceutically active compounds. For example, the active compound may be a compound useful as a cosmetic, a research tool, or a plant protection compound. It is preferably a hydrophilic compound, such as a negatively charged compound, such as a nucleic acid.

[0206] The API or other active compounds according to the present invention may, for example, be fragile compounds. As used herein, the terms “reactive compound” and “fragile compound” may be interchangeable and may both refer to compounds that (i) tend to decompose completely when stored at about 25°C for more than about one week, and / or (ii) have an in vivo half-life of less than about one hour.

[0207] An API can be any pharmaceutically active compound, and therefore, for example, the term "API" is understood to encompass prodrugs. In particular, an API can be a nucleic acid, and more specifically, siRNA or mRNA. On the other hand, in other preferred embodiments, an API can be a protein.

[0208] Nucleic acids for use in the present invention Since RNA is particularly susceptible to degradation in the absence of the protection provided by the present invention, the present invention is particularly suitable for use with some nucleic acid, and more particularly with RNA. Accordingly, according to certain preferred embodiments of the entirety of the present invention, the nucleic acid is RNA. RNA may optionally be siRNA. RNA may optionally be mRNA. For example, RNA may be mRNA encoding a vaccine antigen. RNA may optionally be chemically modified or sequence-modified to enhance its stability and prevent its degradation. According to certain embodiments of the present invention, RNA is chemically modified to enhance its stability or to prevent its degradation. However, in certain preferred embodiments, RNA is not chemically modified, because it has been found that the liposomal lipid particles of the present invention can provide sufficient protection against RNA degradation, and therefore, for example, RNA modification is not necessary and such treatment is unnecessary.

[0209] According to certain embodiments of the entirety of the present invention, the nucleic acid is DNA. According to other preferred embodiments, the nucleic acid is RNA. The nucleic acid may be siRNA, mRNA, saRNA, or shRNA. The nucleic acid may be of some preferred length, but is typically 10 to 30 nucleotides long for siRNA, saRNA, or shRNA, or 200 to 2000 nucleotides long for mRNA. The nucleic acid may be double-stranded or single-stranded, or, in particular, chemically single-stranded in the case of siRNA, saRNA, or shRNA, but having one or more regions of base pairing (and optionally having unpaired overhangs). The nucleic acid may be chemically modified as desired (e.g., by the use of N1-methylpsoiduridine substitution), or its sequence may be modified (e.g., by UTR shortening). Preferably, the nucleic acid (i.e., RNA) may not be modified (especially chemically modified) as it may be unnecessary to provide stability. According to a particular embodiment, the nucleic acid may optionally contain a 5-prime cap and / or a poly-A tail, but may be RNA without other modifications.

[0210] The RNA according to the present invention may be small interfering RNA (siRNA), small activated RNA (saRNA), small hairpin RNA (shRNA), or messenger RNA (mRNA), and more particularly mRNA (e.g., mRNA encoding a protein of a pathogenic organism).

[0211] Other nucleic acids for use in accordance with this disclosure include double-stranded DNA and single-stranded DNA, DNA:RNA hybrids, peptide:DNA hybrids, and peptide:RNA hybrids.

[0212] RNA and DNA may exist naturally or may be chemically modified to enhance their therapeutic properties, such as increased activity, increased serum stability, reduced off-targeting, and decreased immunological activation. Chemical modifications to RNA and DNA may include any modifications commonly known in the art.

[0213] Accordingly, as used herein, the terms nucleic acids, DNA, and RNA also include known types of modifications, such as labels, methylation, "caps," substitution of one or more naturally occurring nucleotides by analogues, internucleotide modifications, such as those having uncharged bonds (e.g., methylphosphonates, phosphotriesters, phosphoramides, carbamates, etc.), negatively charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.), and positively charged bonds (e.g., aminoalkylphosphoramides, aminoalkylphosphotriesters), those containing pendant portions, such as proteins (including nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those having intercalators (e.g., acridine, psoralens, etc.), those containing chelating agents (e.g., metals, radioactive metals, boron, metal oxides, etc.), those containing alkylating agents, those having modified bonds (e.g., alpha-anomeric nucleic acids, etc.), and unmodified forms of polynucleotides or oligonucleotides.

[0214] Similarly, as used herein, the terms “nucleoside” and “nucleotide” include moieties containing not only known purine and pyrimidine bases but also other modified heterocyclic bases. Such modifications include methylated purines or methylated pyrimidines, acylated purines or acylated pyrimidines, or other heterocyclic bases. Modified nucleosides or nucleotides also include modifications to the sugar moiety, for example, here in which one or more hydroxyl groups are substituted with halogens, aliphatic groups, or functionalized with ethers, amines, etc. Other modifications to nucleotides or polynucleotides involve rearranging, adding, substituting, or otherwise altering functional groups on the purine or pyrimidine base, such as isoguanine, isocysteine, etc., which form hydrogen bonds with their respective complementary pyrimidines or purines. In some embodiments, oligonucleotides and / or probes contain at least one, at least two, at least three, or at least four modified nucleotides.

[0215] In some embodiments, nucleic acids such as RNA disclosed herein include one or more universal bases. As used herein, the term “universal base” refers to a nucleotide analog that can hybridize to multiple nucleotides selected from A, U / T, C, and G. In some embodiments, the universal base may be selected from the group consisting of deoxyinosine, 3-nitropyrrole, 4-nitroindole, 6-nitroindole, and 5-nitroindole.

[0216] In its broadest sense, the term "saRNA" encompasses small activating RNA molecules, including RNA molecules that operate within the RNA activation (RNAa) pathway. SaRNAs can be double-stranded. They can have lengths ranging from approximately 5 to approximately 50 base pairs, particularly from approximately 10 to approximately 40 base pairs, and even more particularly from approximately 10 to approximately 30 base pairs.

[0217] In its broadest sense, the term "shRNA" encompasses small hairpin RNAs, including RNA molecules that operate within the RNA interference (RNAi) pathway. shRNAs can be single-stranded but also possess base pairings, thereby forming hairpin loops. A single strand of shRNA can have a length ranging from approximately 10 to 100 bases, particularly 25 to 75 base pairs, and even more specifically 40 to 70 base pairs, which allows for the formation of hairpin loops.

[0218] In its broadest sense, the term "siRNA" encompasses small interfering RNAs, including RNA molecules that operate within the RNA interference (RNAi) pathway. siRNA is sometimes known as short interfering RNA or silencing RNA. siRNA can be double-stranded. siRNA can have lengths ranging from approximately 5 to approximately 50 base pairs, particularly approximately 10 to approximately 40 base pairs, and even more particularly approximately 15 to approximately 30 base pairs.

[0219] In its broadest sense, the term "mRNA" encompasses messenger RNA for protein synthesis. This may include mRNA containing a 5-prime cap and / or polyadenylated ends, or it may lack one or both of these features. Typically, mRNA can be single-stranded. The coding region of mRNA can be at least about 100 nucleotides long, particularly at least about 500 nucleotides long, and even more particularly at least about 1000 nucleotides long.

[0220] mRNA encodes an antigen, thereby providing a composition that is a vaccine. The antigen may be a viral antigen, in particular a viral antigen of one of the viral diseases described below herein, and more particularly an antigen of a respiratory virus, for example, an antigen of SARS-CoV-2, for example, an antigen derived from the spike protein of SARS-CoV-2.

[0221] mRNA can encode multiple proteins, thereby potentially providing more effective pharmacological activity. mRNA can encode multiple antigens, particularly multiple viral antigens.

[0222] mRNA may further encode an adjuvanting protein. The adjuvant may be provided additionally or alternatively as a further component of the composition in addition to the API.

[0223] Complexation of components, particularly particle / lipid / API complexation. Preferably, inorganic material particles are complexed with one or more lipids to form a delivery vehicle for transporting the active compound or API. Thus, upon addition, the active compound or API complexes with the particles and / or lipids. In other words, the particles and lipids are organized into a delivery vehicle supporting the active compound or API. Advantageously, this can make the active compound or API less reactive with one or more external reactants. The active compound or API may have a low risk of degradation catalyzed by enzymes outside the complex, particularly in vivo, for example, during circulation in the body and / or cytoplasm. This may be particularly true if the active compound or API is a nucleic acid, and more particularly mRNA.

[0224] In its broadest sense, the term “complexed with” as used herein may encompass ionic and / or covalent and / or physical interactions, and may particularly encompass charge-charge interactions such as those arising from the zeta potential of particles.

[0225] Therefore, preferably, the zeta potential of the particles is such that it attracts and promotes the binding of the active compound or API, particularly when regulated by one or more lipids and some other components present.

[0226] In preferred embodiments where amino acids are present, the amino acids may also complex with particles, lipids, and / or active compounds / APIs. In particular, if charged, amino acids may modulate the zeta potential of particles and thus modulate the complexation of active compounds / APIs and / or lipids with particles.

[0227] Tangential flow filtration The method of the present invention optionally comprises one or more tangential flow filtration (TFF) steps. The TFF may be configured to concentrate the aqueous suspension according to the present invention. Additionally or alternatively, the TFF may be used to remove a solvent used in the initial steps of the method, such as methanol.

[0228] The use of TFF is particularly preferred as part of a particular preferred embodiment of the present invention, which lacks the step of solvent evaporation.

[0229] Favorable properties and product parameters Both the particles of the present invention, hybrid lipid particles, and liposomal lipid particles are preferably 60 to 120 nm in size, have a polydispersity index (PDI) of 0.100 to 0.200, and a zeta potential of 50 to 70 mV.

[0230] Composition of hybrid lipid particles and liposomal lipid particles In all embodiments, the hybrid lipid particles and liposome lipid particles of the present invention preferably have the following configuration in addition to the lipid structure. The lipid particles may have particles of inorganic material (e.g., hydrolyzable silicon particles) incorporated into the lipid bilayer and partially or completely exposed on the surface of the hybrid particle or liposome particle, so as to be available for interacting with active compounds, particularly APIs, and even more particularly nucleic acids. Preferably, at least 10%, at least 20%, at least 30%, or at least 50% of the total inorganic material particles (e.g., hydrolyzable silicon particles) are accessible on the surface of the lipid particles and are not completely encapsulated within the lipid structure. The active compounds, particularly nucleic acids or other APIs, if present, are mainly arranged electrostatically bound to the surface of the lipid particles. For example, according to certain embodiments that exist, more than 90% of the total active compounds (particularly nucleic acids or other APIs) are bound to the surface of the lipid particles, and less than 10% are encapsulated within the lipid structure. In some embodiments, zero or substantially zero (e.g., less than 0.5%) of the total active compounds present (particularly nucleic acids or other APIs) are encapsulated within the lipid structure. In other embodiments, there is an even equal division between the encapsulated active compounds or APIs and the active compounds or APIs associated with the surface of the lipid particles. For example, according to some embodiments, at least 10% of the active compounds or APIs are associated with the surface of the particles, and at least 10% of the active compounds or APIs are encapsulated.

[0231] Treatment methods and products related to treatment The product of the present invention may be used in a therapeutic method, or may be a product for use in a therapeutic method. The method of the present invention may further include subsequent steps constituting a therapeutic method.

[0232] Treatment methods include the treatment or prevention of a disease or disorder. In some embodiments, treatment methods may include downregulation of gene expression by siRNA. In other embodiments, treatment methods may include vaccination against cancer or against infectious diseases by delivery of mRNA encoding an antigen (or fragment thereof) or a causative agent of an infectious disease (e.g., the spike protein of SARS-CoV-2).

[0233] A disease or disorder may be an infectious disease. As used herein, the term “infectious” may be used to mean a disease that is easily transmitted from one organism to another, in particular from one person to another.

[0234] Infectious diseases can be viral diseases, bacterial diseases, fungal diseases, or parasitic diseases, and especially viral diseases.

[0235] If the disease is a viral disease, it may be caused by a respiratory virus, such as respiratory syncytial virus (RSV), parainfluenza virus (HPIV), metapneumovirus (HMPV), rhinovirus (HRV), coronavirus, such as SARS-CoV (especially SARS-CoV-1, and even more specifically SARS-CoV-2), adenovirus (HAdV), enterovirus (EV), bocavirus (HBoV), parechovirus (HPeV), or influenza virus.

[0236] Viral diseases may be caused by dengue virus, Ebola virus, encephalomyocarditis virus, hepatitis virus, herpesvirus, human immunodeficiency virus, human papillomavirus, human T-lympotropic virus, measles virus, monkeypox virus, mumps virus, poliovirus, rabies virus, rotavirus, rubella virus, varicella-zoster virus, West Nile virus, yellow fever virus, or Zika virus.

[0237] The disease or disorder may be a hereditary disease or hereditary disorder.

[0238] In some embodiments, the hereditary disorder may be characterized by a deficiency in the expression of one or more proteins, particularly one or more enzymes.

[0239] Hereditary disorders, such as schizophrenia, diabetes, asthma, depression, epilepsy, heart disease, or hypothyroidism, can be multifactorial disorders, meaning they are not limited to any specific pattern of single-gene genetic makeup, but are likely to be associated with the influence of multiple genes along with environmental factors.

[0240] Hereditary disorders may involve one or more mutations in one or more genes.

[0241] Therefore, hereditary disorders can be monogenic disorders, which are more likely to occur when at least one mutation occurs in a single gene. If a hereditary disorder is monogenic, it may involve one mutation in a single gene or multiple mutations in a single gene. Examples of monogenic disorders include sickle cell anemia, cystic fibrosis, Huntington's disease, or Duchenne muscular dystrophy.

[0242] A genetic disorder may involve multiple mutations in one of several genes. As a non-limiting example, a genetic disorder may involve multiple mutations in a first gene and one mutation in a second gene.

[0243] A genetic disorder may be one that is likely to occur when at least one mutation occurs in at least one gene within a set of genes. In particular, such a genetic disorder may be osteopetrosis.

[0244] Hereditary disorders may include Angelman syndrome, Canavan disease, Charcot-Marie-Tooth disease, color blindness, cat-cry syndrome, cystic fibrosis, DiGeorge syndrome, Down syndrome, Duchenne muscular dystrophy, familial hypercholesterolemia, hemochromatosis type 1, hemophilia, Klinefelter syndrome, neurofibromatosis, phenylketonuria, polycystic kidney disease, Prader-Willi syndrome, Scheuermann disease, sickle cell anemia, spinal muscular atrophy, Tay-Sachs disease, or Turner syndrome.

[0245] In its broadest sense, as used herein, the term hereditary disorder may encompass cancer. Cancer may be or may be a blood cancer (e.g., leukemia, lymphoma, or myeloma) or a solid tumor (e.g., sarcoma, carcinoma, carcinosarcoma, or lymphoma).

[0246] Therefore, cancer can be, in particular, cancer of the blood, skin, brain, prostate, breast, lung, esophagus, stomach, small intestine, pancreas, colon, and / or rectum, central nervous system, bladder, thyroid, kidney, uterine body, oral cavity, or ovaries.

[0247] More specifically, cancer includes lung cancer, brain cancer, gastrointestinal cancer, skin cancer, genitourinary cancer, pancreatic cancer, lung cancer, medulloblastoma, basal cell carcinoma, glioma, breast cancer, prostate cancer, testicular cancer, esophageal cancer, hepatocellular carcinoma, gastric cancer, gastrointestinal stromal tumor (GIST), colon cancer, colorectal cancer, ovarian cancer, melanoma, neuroectodermal tumor, head and neck cancer, sarcoma, soft tissue sarcoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, It may be or may be associated with osteogenic sarcoma, chordoma, angiosarcoma, endosarcoma, lymphangiosarcoma, lymphangiosarcoma, synoviomas, mesothelioma, leiomyosarcoma, cervical cancer, uterine cancer, endometrial cancer, carcinoma, bladder cancer, epithelial carcinoma, squamous cell carcinoma, adenocarcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, neuroendocrine carcinoma, carcinoid tumor, diffuse giant cell tumor, or glioblastoma.

[0248] Preparation, storage, stability, and administration of the disclosed pharmaceutical composition (also simply referred to as the “Composition”). The treatment or prevention of a disease or disorder according to the present invention may include administering a preventive effective amount of the pharmaceutical composition disclosed herein to a subject (particularly a human subject) that is in need and identified as such by, for example, a physician or other healthcare professional. On the other hand, the treatment of a disease or disorder in a human subject may include administering a therapeutic effective amount of the pharmaceutical composition disclosed herein to a subject that is in need.

[0249] The dosage of the pharmaceutical compositions disclosed herein may be varied to obtain an amount of API that is not toxic to the subject and is effective in achieving a desired prophylactic and / or therapeutic response to a given subject. A preferred dosage of the composition may be the amount of the composition that is the minimum effective dose for the API to produce a therapeutic and / or prophylactic effect.

[0250] The selected dosage, form, and regimen depend on various factors, each of which may include, for example, the activity of the API, the route of administration, the timing of administration, the rate of excretion or metabolism of the API, the rate and degree of absorption, the duration of treatment, the presence of other drugs, compounds, and / or materials used in combination with the API, the age, sex, weight, condition, overall health status, and prior medical history of the person being treated, as well as other such factors well known in the medical field.

[0251] The composition may be administered by intramuscular or intravenous injection (including transdermal delivery via patch), orally (including sublingual administration), intranasally, or by any other suitable route.

[0252] Preferably, the composition may be administered by injection, for example, intravenous or intramuscular injection. If the composition is administered by injection, the subject may be monitored for symptoms or signs of hypersensitivity reactions, for example, vaccine-related hypersensitivity reactions.

[0253] Furthermore, the composition may preferably be administered orally or intranasally. Compositions suitable for oral administration may be provided in individual dosage forms, particularly as liquids or aerosol sprays containing predetermined amounts of the composition. Such dosage forms may be prepared by any well-known pharmaceutical method.

[0254] The composition may be closely mixed with a pharmaceutical carrier according to conventional pharmaceutical formulation techniques. The carrier may take a wide variety of forms depending on the desired form of the preparation for administration. Any of the common pharmaceutical media may be used as the carrier, for example, one or more of water, oil, and alcohol (including glycols). In particular, when formulated for administration by injection, oral administration, or nasal administration, the forms in which the disclosed pharmaceutical composition may be incorporated for administration may include aqueous solutions in physiological saline. The composition may further contain one or more pharmaceutically acceptable additives and excipients, such as antifungals, defoamers, buffers, polymers, antioxidants, chelating agents, viscosity modifiers, tonicity modifiers, odorants, opacifiers, suspending agents, fillers, plasticizers, flavoring agents, preservatives, colorants, diluents, binders, disintegrants, and mixtures thereof.

[0255] Prevention or reduction of microbial activity can be achieved by including various antibacterial and antifungal agents, such as one or more of parabens, chlorobutanol, phenol, sorbic acid, and thimerosal.

[0256] The compositions disclosed herein may be provided in sterile solutions by incorporating the required amount of the composition into a suitable solvent (with various other components, as appropriate) by any known pharmaceutical method. The compositions disclosed herein may be provided in sterile dispersions by incorporating the required amount of the composition into a sterile vehicle (with various other components, as appropriate). The compositions disclosed herein may be provided as sterile powders (for example, for the subsequent preparation of sterile injection solutions) by techniques such as vacuum drying and freeze-drying (lyophilization) which result in a powder of the composition.

[0257] If desired, the composition may be stored before administration to the subject. The composition may be stored at a temperature of 0°C or higher, particularly 4°C or higher, for a period of at least one week (optionally up to six months, and especially up to one year) before administration to the subject.

[0258] In some embodiments, liposome particles can enhance the stability of an active compound, such as an API, during in vivo circulation, particularly when the API is or contains nucleic acids such as mRNA. In some embodiments, particles can protect the API from degradation, particularly enzymatic degradation, particularly when the API is or contains nucleic acids such as mRNA. Thus, the compositions disclosed herein can mitigate or address the problem of how to ensure access to cells after the API has been administered to a patient, including how to stabilize the API while it is circulating in the body.

[0259] On the other hand, the disclosed compositions may reduce or satisfy the need for tissue or cell targeting so that the API can be delivered to the correct cells.

[0260] Additionally or alternatively, the disclosed compositions may mitigate or solve the challenge of ensuring efficient API uptake by target cells when accessed. For example, the disclosed compositions may assist in the transport of APIs from outside the cell into the cytoplasm.

[0261] Following the uptake of the API by cells, the disclosed compositions may mitigate or solve the problem of how to prevent the API from degrading too rapidly in the cytoplasm. Liposome particles are thought to enhance the stability of the API in the cytoplasm of cells, particularly its resistance to enzymatic degradation, especially when the API is or contains nucleic acids such as mRNA.

[0262] Relationship between aspects of the invention The method according to the first aspect of the present invention may optionally be used to produce an aqueous suspension of hybrid lipid particles according to the second aspect of the present invention, and such a suspension may optionally be used to produce a lyophilized powder of hybrid lipid particles according to the present invention. Such a product may optionally be used to produce an aqueous suspension of liposomal lipid particles according to the present invention. Such an aqueous suspension may optionally be used to produce a lyophilized powder liposomal lipid particles according to the present invention. The advantage of the method and products of the present invention compared to methods for lipid particles that attempt to encapsulate APIs or other active ingredients during initial lipid particle formation as described above is that the encapsulation method requires encapsulation to be carried out at the high temperature (usually 50-70°C) that is typically required to form lipid particles. Such high temperatures can degrade heat-sensitive active ingredients such as RNA molecules. The present invention makes it possible to prepare hybrid lipid particles before the active ingredients delivered by the lipid particles are introduced. The present invention also makes it possible for hybrid lipid particles to exist for extended periods (e.g., in aqueous suspension or in lyophilized powder form) as an intermediate storage or commodity for the subsequent production of the final product, which may be liposomal lipid particles.

[0263] An aqueous suspension of the lipid particles of the present invention may be prepared using the freeze-dried powder of the lipid particles of the present invention.

[0264] The pharmaceutical composition of the present invention may be prepared using the freeze-dried powder of the lipid particles of the present invention and / or the aqueous suspension of the lipid particles of the present invention.

[0265] The hybrid lipid particles and liposomal lipid particles of the present invention can be prepared as desired using the method of the present invention.

[0266] Features that are optionally present as part of one aspect of the present invention as described herein or claimed herein are understood to be, where appropriate, also optionally present in other aspects of the present invention. [Examples]

[0267] Various aspects and embodiments of the present invention are described below in the following non-limiting examples.

[0268] The examples may also illustrate subject matter that is not necessarily within the scope of the present invention but is included to aid in understanding the present invention or to provide a comparison with the present invention.

[0269] method We developed a process for producing silicon-stabilized hybrid lipid nanoparticles (sshLNPs) using a combination of flow extrusion to form sshLNPs with desired particle size and surface charge characteristics, followed by tangential flow filtration (TFF) for concentration and purification of the solution. To target various tissues, we developed two formulations of sshLNPs, one containing pegylated lipids and one without.

[0270] This process generates intermediate hybrid lipid particles on which nucleic acids are supported immediately before the packing and finishing operation. Adding nucleic acids at this stage minimizes degradation. This also provides an opportunity for post-stage customization of sshLNP.

[0271] The combination of flow extrusion and TFF provides a highly scalable manufacturing process that can reliably produce sshLNPs on a scale ranging from less than 1L to over several hundred L, making sshLNPs suitable for use in a variety of products, from personalized pharmaceuticals to mass-produced goods.

[0272] Figure 1A shows one embodiment of the method of the present invention. Silicon nanoparticles (SiNPs) are activated by dispersion in methanol as an activating solvent. A filtration step may be used, optionally present, to reduce aggregation of silicon nanoparticles. Lipids DOTAP-cl, DOPE, and optionally mPEG2000-DSPE are mixed by dissolving them in methanol. An aqueous solution supplemented with trehalose and glycine is first mixed with the SiNP suspension. The dissolved lipid / methanol stream is then injected. The resulting mixture is extruded through a membrane with a reduced pore size and then subjected to TFF to remove excess trehalose, glycine, methanol, and lipids. IPC1 and 2 are quality controls in which the product is inspected against specifications.

[0273] Manufacturing of sshLNP 1.1.Biocourier sshLNP composition [Table 1]

[0274] 1. DOTAP-Cl 1,2-Dioleoyloxy-3-trimethylammonium propane chloride, (UNII:3R78UC794Z, CAS number 132172-61-3) [ka]

[0275] 2. DOPE 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine, (UNII:JNP6V6AI0U, CAS number 4004-05-1) [ka]

[0276] 3. mPEG2000-DSPE N-(carbonyl-methoxypolyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, sodium salt, (UNII:3L6NN8ZZKU, CAS number 147867-65-0) [ka]

[0277] Description of the manufacturing process of methanol injection combined with TF Silicon nanoparticles (500 mg) are dispersed in methanol (25 mL) and allowed to stand for 30 minutes with stirring to activate. The dispersion is filtered through a 0.8 μm hydrophilic polyethersulfone filter. Trehalose (400 mg) and glycine (200 mg) are dissolved in nuclease-free water (380 ml) and mixed with the activated silicon nanoparticle dispersion (20 mL) by stirring at 50°C for 60 minutes.

[0278] In the case of pegged sshLNP Dissolve DOTAP-Cl (3500 mg) in methanol (350 mL) by stirring at 40°C for 30 minutes. Dissolve DOPE (3500 mg) in methanol (350 mL) by stirring at 40°C for 30 minutes. Dissolve mPEG2000-DSPE (650 mg) in methanol (65 mL) by stirring at 40°C for 30 minutes (IPC 1).

[0279] Mix DOTAP-Cl solution (297 mL), DOPE solution (299 mL), and mPEG2000-DSPE solution (59 mL). Slowly inject the mixed lipid solution (640 mL) into the mixture of silicon nanoparticles, trehalose, and glycine at a flow rate of 10 mL / min. After the addition is complete, add nuclease-free water to bring the total volume to 4 L.

[0280] Or in the case of non-pegged sshLNP Dissolve DOTAP-Cl (3500 mg) in methanol (350 mL) by stirring at 40°C for 30 minutes. Dissolve DOPE (3500 mg) in methanol (350 mL) by stirring at 40°C for 30 minutes (IPC 1).

[0281] Mix DOTAP-Cl solution (320 mL) and DOPE solution (340 mL). Slowly inject the mixed lipid solution (640 mL) into the mixture of silicon nanoparticles, trehalose, and glycine at a flow rate of 10 mL / min. After the addition is complete, add nuclease-free water to bring the total volume to 4 L.

[0282] The resulting mixture is extruded through two sets of 47 mm polycarbonate extrusion films (arranged in parallel to increase the effective surface area) with decreasing pore sizes (3 × 0.8 μm, 3 × 0.4 μm, and 3 × 0.1 μm in each extrusion set). A second extrusion through the 3 × 0.1 μm film may be required to obtain the desired physical properties (IPC 2).

[0283] Extruded mixture, 0.5m 2 Ultrafiltration is performed using tangential flow filtration with a polyethersulfone membrane (100 kDa molecular weight cutoff). After concentrating to a volume of 2 L, the mixture is diafiltration using 10 diafiltration volumes (20 L) of a solution containing trehalose (0.1 mg / mL) and glycine (0.05 mg / mL).

[0284] After diafiltration, the solution is filtered through a 0.2 μm polyethersulfone membrane.

[0285] Unlike conventional processes where the composition of the product is directly proportional to the amount of material introduced, flow extrusion / TFF is a dynamic process that modifies the material content. As developed, this process produces sshLNPs with important chemical and physical attributes that result in effective delivery of nucleic acids.

[0286] The process involves four key unit operations.

[0287] a. Preparation and mixing of solutions Trehalose and glycine are dissolved in nuclease-free water, and lipids are dissolved in methanol.

[0288] Silicon nanoparticles are dispersed in methanol and activated. Activation modifies the contact angle of the silicon nanoparticle surface to increase the dispersion of the silicon nanoparticles. Activation also promotes the expression of hydroxyl groups on the surface of the silicon nanoparticles, thereby facilitating electrostatic interactions and physical absorption of other excipients on the surface. The initial silicon nanoparticle dispersion contains 20 mg / mL of silicon; using lower concentrations of silicon may affect the physical properties of the resulting LNPs, such as average particle size, polydispersity index, and zeta potential.

[0289] This dispersion is filtered through a 0.8 μm hydrophilic polyethersulfone (PES) membrane filter to remove silicon aggregates and excess particles. Without this filtration, the extrusion membrane may become clogged, which could potentially affect the physical properties of the resulting LNPs, such as average particle size, polydispersity index, and zeta potential.

[0290] A solution of trehalose and glycine is mixed with a silicon dispersion, and then a lipid solution is slowly added to form lipid nanoparticles. However, at this stage, the LNPs have an average particle size and polydispersity index that are larger than the desired values.

[0291] Formation of lipid particles by flow injection is preferable to the formation of thin films by evaporation and subsequent rehydration. The thin-film / rehydration process has several limitations, including the difficulty in obtaining uniform dispersion. The thin-film / rehydration method may also present challenges regarding its manufacturability at the specific production scales mentioned above.

[0292] b. Flow extrusion To reduce the average particle size and polydispersity to a desired range, the mixture is then extruded through a series of polycarbonate films with progressively smaller pore sizes (3 × 0.8 μm, 3 × 0.4 μm, and 3 × 0.1 μm).

[0293] Gradually reducing the pore size of the membrane decreases the extrusion pressure and associated mechanical stress during the process. Using three membranes at each stage yields multiple extrusion cycles. This technique of reducing pore size, along with multiple extrusion cycles, produces LNPs with controlled and reproducible physical properties (average particle size, polydispersity index, and zeta potential).

[0294] During this process, further silicon is removed, and some lipids are retained on the membrane.

[0295] c. Tangential Flow Filtration (TFF) TFF using a polyethersulfone membrane (100 kDa molecular weight cutoff) performs two functions: ultrafiltration (UF) and diafiltration (DF). First, the LNP solution is concentrated by UF to reduce its volume by 50%, and then purified by DF using a 10-fold increase in diafiltration volume to remove methanol and free (unbound) lipids. Since trehalose and glycine have low molecular weights, these molecules also permeate the membrane. To compensate for the loss of these molecules, a diafiltration solution containing both trehalose and glycine is used to maintain the concentrations of these materials in the product. TFF does not significantly affect the physical properties of LNP and does not remove silicon.

[0296] d. Final filtration Upon completion of TFF, the resulting solution is filtered through a 0.2 μm polyethersulfone filter to remove microbial contamination and generate a low bioburden sshLNP Biocourier.

[0297] Next, this product can be complexed with mRNA at room temperature.

[0298] In summary, the following process operations control the main product attributes.

[0299] [Table 2]

[0300] method Analysis method Inflow extrusion Inflow extrusion was performed using a Knauer K-501 HPLC pump with a 50 ml / min stainless steel pump head. The flow rate was set according to the extrusion volume in the range of 10 ml / min to 50 ml / min. 0.8 μm, 0.4 μm, and 0.1 μm extruded membranes were used at a high temperature of 60°C using a heat bath. The holder containing the membranes was immersed in water to achieve the required temperature. The extrusion process was investigated by DLS to measure size and size distribution, as well as by zeta potential analysis. The extrusion pressure on the membrane as a function of time and extrusion volume was also monitored. Other parameters investigated for inflow extrusion were the extrusion flow rate, the number of extruded membranes, and the use of a pre-extrusion pre-filtration step using a syringe filter.

[0301] Tangential flow filtration Tangential flow filtration (TFF) was used to remove methanol and other impurities such as unassociated lipids and any potential degradation products.

[0302] TFF used TangenX SIUS PD cassettes with standard notch offsets for 50ml and 100ml volumes. Specifications of the cassettes used: [Table 3]

[0303] The micelle lipid particles were concentrated twice and washed with 10 diafiltration volumes (DV) of THR (trehalose) and GLY (glycine) solutions to remove organic solvents. Note that a diafiltration solution containing 0.1 mg / ml of THR and 0.05 mg / ml of GLY was used to prevent the loss of these components during TFF. The in-process samples were then analyzed after TFF.

[0304] For a 2kg batch, the cassette used has the following specifications (standard notch offset): [Table 4]

[0305] The only difference between the two cassettes is the membrane area. A larger membrane allows for the filtration of a larger volume.

[0306] DLS and zeta potential measurements were used to evaluate the surface charge, size, and size distribution of the samples throughout the entire extrusion process and after TFF. Zeta potential, mean diameter (Ζ), and PDI are shown for the experiments described. The feasibility of currently available HPLC methods for lipid quantification after extrusion and after TFF was investigated using the starting material as the standard for calibration curves. 1 Methanol removal was investigated by 1H-NMR analysis and headspace gas chromatography.

[0307] Manufacturing and feasibility testing for inflow extrusion and TFF was conducted under non-GMP conditions, without the use of additional measures for low bioburden treatment. The TFF cassettes were reused for manufacturing various batches after washing.

[0308] When using nuclease-free water, the manufacturing process employed additional measures, such as wearing gloves disinfected with 70% v / v ethanol, routine cleaning of workstations and equipment with 70% v / v ethanol, and the use of sterile laboratory equipment and sampling in a laminar flow cabinet.

[0309] During extrusion, the solution passed through Whatman 25mm polycarbonate membrane filters with specified pore sizes of 0.4 μm and 0.8 μm. Three membranes were used in series for each extrusion step. To achieve extrusion and push the fluid through the membranes, a Knauer K501 HPLC pump with a 50 ml / min stainless steel pump head was used with a water bath to maintain an operating temperature of 60°C. Before each extrusion step, the membranes were washed with MeOH (20 ml) and rinsed with Milli-Q water (20 ml). The flow rate used for extrusion was 10 ml / min. Finally, DLS and zeta potential analysis were performed throughout the entire process to evaluate the feasibility of direct addition of SiNPs to aqueous solutions and the feasibility of the extrusion process, and to monitor the characterization of SiNPs throughout the entire experimental process.

[0310] Example 1 - Description of a conventional "evaporation" manufacturing process used to generate a baseline value for comparison. The "evaporation" manufacturing process for micelle lipid particle production shown in Figure 1 begins with dissolving selected lipids DOTAP, DOPE, and mPEG2000-DSPE in methanol. Simultaneously, exposure to methanol activates mesoporous hydrolyzable silicon nanoparticles (SiNPs). Subsequently, the methanol solvent is evaporated using 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). An appropriate volume of lipids is continuously transferred to a round-bottom flask, and the methanol is evaporated by rotational evaporation. As a result of this evaporation process, a lipid film is formed on the flask wall. Next, a suspension containing dispersed silicon, THR, and GLY is added to the flask containing the lipid film for lipid rehydration.

[0311] Example 2 - Use of film extrusion Proof of concept that membrane extrusion works Micelle lipid particles were prepared using a general protocol. Activated SiNPs were mixed with THR and GLY in water, and the resulting solution was sonicated at 50°C for 60 minutes. For lipid membrane hydration, the lipid membrane was continuously exposed to an appropriate volume of aqueous solution containing NP-THR-GLY. Lipid membranes were formed by mixing various methanol lipid solutions at concentrations indicated in the general protocol and evaporating the solvent by rotational evaporation. Pre-activated SiNPs were used without further exposure to MeOH. The general procedure for sample MVI0001 is shown in Figure 3.

[0312] [Table 5]

[0313] DOTAP-Cl, DOPE, and mPEG2000-DSPE lipid solutions were prepared at concentrations of 5 mg / ml by dissolving 25 mg of each lipid in 5 ml of MeOH. The lipid solutions were then thoroughly sonicated at 40°C for 30 minutes. After sonication, appropriate volumes of each lipid solution were transferred to 10 ml glass round-bottom flasks, as shown in Table 2.

[0314] [Table 6]

[0315] The solvent was evaporated by rotational evaporation under the conditions P=300 mbar, T=40°C, and t=30 minutes to generate a lipid film. Next, for lipid film hydration, the lipid film was exposed to 1 ml of aqueous solution containing SiNP-THR-GLY at 60°C for 5 minutes. MilliQ water was added last up to 10 ml. After the preparation of the crude sample, inflow extrusion was performed as described in the first part of this section and as shown in Figure 1. In summary, the sample was extruded at room temperature at 10 ml / min using a Knauer K501 HPLC pump through three consecutively arranged 25 mm extruded membranes with a pore size of 0.4 μm and three consecutively arranged 25 mm extruded membranes with a pore size of 0.1 μm. Before each extrusion step, the membrane was washed with 20 ml of MeOH and rinsed with 20 ml of MilliQ water.

[0316] A visual inspection of the solutions obtained in each step is shown in Figure 4.

[0317] The formation of a fine precipitate was observed in the SiNP dispersion containing THR and GLY. The successful formation of a lipid film was confirmed by the presence of an opaque film on the wall of the round-bottom flask. When the SiNP solution was added to the lipid film, the formation of a larger precipitate was observed. The precipitate disappeared upon extrusion. After extrusion, the solution appeared clear. DLS and zeta potential analysis were performed on the generated MVI0001 samples before and after each extrusion step. The obtained values ​​were compared with reference values ​​and are shown in Table 3.

[0318] [Table 7]

[0319] Figure 5 shows the analysis of the zeta mean, PDI, and zeta potential for MVI0001 throughout the extrusion process.

[0320] Experimental observations confirmed that extrusion improved the monodispersity of the sample. Large precipitates were captured by the 0.4 μm film, and the solution after 0.1 μm extrusion appeared completely clear. The zeta potential was largely maintained throughout the entire extrusion process. The mean zeta, PDI, and zeta potential values ​​were similar to the reference values ​​indicated by SiSaf, as shown in Table 3.

[0321] Example 3 This experiment investigated the direct addition of unpre-activated SiNPs to a lipid membrane. For activation, the unpre-activated SiNPs were initially exposed to MeOH for 30 minutes. After activation, the SiNPs were directly added to an aqueous THR-GLY solution and sonicated at 50°C for 60 minutes. Finally, the lipid membrane formed by the evaporation of MeOH from the methanol lipid solution was exposed to an aqueous solution containing SiNP-THR-GLY. The general procedure for sample MVI0002 is shown in Figure 6.

[0322] To prepare this sample, 20 mg of unpre-activated SiNP was initially exposed to 1 ml of MeOH (Table 3).

[0323] [Table 8]

[0324] Simultaneously, 20 mg of THR and 10 mg of GLY were weighed in the same tube, and 19 ml of MilliQ water was added to the tube. After 30 minutes of MeOH exposure, the SiNPs were considered activated and were therefore added to the THR-GLY solution, which was then sonicated at 50°C for 60 minutes. The final concentration of the SiNP-THR-GLY stock solution was the same as that shown in Experiment MVI0001 in Table 1.

[0325] As described in MVI0001, lipid solutions of DOTAP-Cl, DOPE, and mPEG2000-DSPE were prepared at a concentration of 5 mg / ml, and appropriate volumes of each lipid solution were transferred to 10 ml glass round-bottom flasks as shown in Table 2. Next, the solvent was evaporated by rotational evaporation under the conditions P=300 mbar, T=40°C, and t=30 minutes to form a lipid membrane. Subsequently, for lipid membrane hydration, the lipid membrane was exposed to 1 ml aqueous solution containing NP-THR-GLY at 60°C for 5 minutes. MilliQ water was finally added up to 10 ml.

[0326] After preparing the crude samples, inflow extrusion was performed at a high temperature of 60°C using a Knauer K501 HPLC pump and a flow rate of 10 ml / min, as shown in Figure 6, through 0.4 μm and 0.1 μm extruded membranes. Before each extrusion step, the membranes were washed with 20 ml of MeOH and rinsed with 20 ml of MilliQ water.

[0327] A visual inspection of the solutions obtained in each step is shown in Figure 7.

[0328] In the experimental sample MVI0002, SiNPs were initially exposed to MeOH for activation. As shown in Figure 7, a large deposit of SiNPs was observed at the bottom of the cuvette. Precipitation in the SiNP dispersion containing THR and GLY was also observed. A lipid film was successfully formed, as indicated by the presence of an opaque film on the wall of the round-bottom flask. When the SiNP solution was added to the lipid film, the formation of a very large precipitate and deposit was observed at the bottom of the round-bottom flask. The precipitate disappeared after extrusion. After extrusion, the solution appeared clear. DLS and zeta potential analysis were performed on the generated MVI0002 sample before extrusion and after each extrusion step. The obtained values ​​were compared with the SiSaf reference values ​​and are shown in Table 5.

[0329] [Table 9]

[0330] Figure 8 shows the investigation of the zeta mean, PDI, and zeta potential for sample MVI0002 throughout the extrusion process.

[0331] Consistent with experimental observations for sample MVI0001, extrusion improved the monodispersity of the analyzed sample. Large precipitates were captured by the 0.4 μm film, and the solution after 0.1 μm extrusion appeared completely clear. Zeta potential was largely maintained throughout the entire extrusion process. PDI and zeta potential values ​​were similar to the baseline (Table 5), but the mean zeta potential was higher than the baseline.

[0332] Example 4 - Elimination of the evaporation step in sample MVI0003 The direct injection of lipid solutions into aqueous solutions containing SiNP-THR-GLY was investigated. For activation, inactivated SiNPs were first exposed to MeOH for 30 minutes. After activation, SiNPs were directly added to the THR-GLY aqueous solution and sonicated at 50°C for 60 minutes. Finally, the lipid solution was prepared and slowly injected into the aqueous solution containing SiNP-THR-GLY. The general procedure for MVI0003 is shown in Figure 9.

[0333] To prepare this sample, a SiNP-THR-GLY solution was prepared as described for sample MVI0002. In summary, for activation, 20 mg of SiNP was exposed to 1 ml of MeOH for 30 minutes (see Table 4). 20 mg of THR and 10 mg of GLY were weighed in the same tube, and 19 ml of MilliQ water was added to the tube. After activation, SiNP was added to the THR-GLY solution, and the solution was sonicated at 50°C for 60 minutes. The final concentration of the SiNP-THR-GLY stock solution was the same as that of samples MVI0001 and MVI0002, and as shown in Table 1.

[0334] As described for sample MVI0001, lipid solutions of DOTAP-Cl, DOPE, and mPEG2000-DSPE were prepared at a concentration of 5 mg / ml, and appropriate volumes of each lipid solution were transferred to 10 ml glass vials, as shown in Table 2. Next, 1 ml of aqueous solution containing SiNP-THR-GLY was transferred to a stirred glass vial. Then, the lipids were collected using a 10 ml sterile syringe and slowly injected into the SiNP solution using a ProSense NE1000 syringe pump. After setting the correct diameter of the syringe used, the syringe pump flow rate was set to 3.2 ml / min. Subsequently, MilliQ water was added to a final volume of 10 ml. After dilution, the solution was allowed to stand and stir for 30 minutes to homogenize it.

[0335] After preparing the crude sample, in flow extrusion was performed at 60°C using a Knauer K501 HPLC pump at 10 ml / min, as shown in Figure 9, through 0.4 μm and 0.1 μm extruded membranes, similar to the experiment with sample MVI0002. Here again, before each extrusion step, the membranes were washed with 20 ml of MeOH and rinsed with 20 ml of MilliQ water.

[0336] A visual inspection of the solutions obtained in each step is shown in Figure 10.

[0337] In experiment MVI0003, as in experiment MVI0002, SiNP was initially exposed to MeOH for activation. As shown in Figure 10, SiNP deposits were observed at the bottom of the cuvette. The formation of large precipitates in the SiNP dispersion in the presence of THR and GLY was also observed. When lipids were added to the SiNP solution, the formation of precipitates was similarly observed. However, when lipids were directly injected slowly into the SiNP solution using a syringe, the SiNP solubility seemed to improve, and less precipitate was observed compared to MVI0002 (Figure 7). The precipitate disappeared after extrusion. After extrusion, the solution appeared clear. DLS and zeta potential analysis were performed on the MVI0003 sample before extrusion and after each extrusion step. The obtained values ​​were compared with the SiSaf reference values ​​and are shown in Table 6.

[0338] [Table 10]

[0339] Figure 11 shows the analysis of the zeta mean, PDI, and zeta potential for MVI0003 throughout the extrusion process.

[0340] Here again, extrusion improved the monodispersity of the MVI0003 sample. Large precipitates were captured by the 0.4 μm film, and the solution after 0.1 μm extrusion appeared completely clear. The zeta potential was largely maintained throughout the entire extrusion process. As shown in Table 6, the mean zeta, PDI, and zeta potential values ​​were similar to the baseline values.

[0341] Table 7 shows the DLS characteristics of all three samples analyzed after 0.1 μm extrusion.

[0342] [Table 11]

[0343] Observations of samples MVI0001 and MVI0003 indicate that the DLS characteristics of the extruded samples are consistent with the baseline. Furthermore, sample MVI0001 visually matches the predicted results, while sample MVI0003 is characterized by the presence of large aggregates in the SiNP solution. These observations already suggest that the evaporation procedure was well reproduced in test MVI0001 and that direct injection of lipids into the unpre-activated SiNP solution is feasible. It is important to note that this final experimental method is best suited for scaling up the micelle lipid particle manufacturing process.

[0344] Example 5 - Optimization of Inflow Extrusion The inflow extrusion process was further investigated by evaluating the following: 1. Use of an additional 0.8 μm membrane to capture large silicon aggregates (MVI0007) 2. Reproducibility of experimental results when using nuclease-free water (MVI0008) 3. Use of a 0.8 μm syringe filter for a pre-filtration step before inflow extrusion (MVI0011) 4. Extrusion of large (1L) volume micellar lipid particle solution - (MVI0012) 5. Extrusion pressure generated under various experimental conditions

[0345] Representative results for each experiment are shown below.

[0346] Investigating the use of an additional 0.8 μm extruded membrane to capture large aggregates (MVI0007) To remove large SiNP aggregates, the use of an additional extruded membrane with a pore size of 0.8 μm was investigated. These aggregates can indeed cause the experimental variability observed in reported experiments. The experimental conditions for sample MVI0007 using the additional 0.8 μm membrane are shown in Figure 17.

[0347] Micelle lipid particles were prepared in a final volume of 50 ml, as shown in Figure 17. Inflow extrusion was performed at 60°C through a 3-row 25 mm extruded membrane with a pore size of 0.8 μm, a 3-row 25 mm extruded membrane with a pore size of 0.4 μm, and a 3-row 25 mm extruded membrane with a pore size of 0.1 μm. A visual inspection of the solutions obtained in each step of Experiment MVI0007 is shown in Figure 18.

[0348] In experiment MVI0007, the formation of precipitates was observed in a SiNP dispersion containing THR and GLY. When lipids were added to the SiNP solution, precipitate formation was also observed. The precipitates disappeared upon extrusion. After complete extrusion, the solution appeared clear.

[0349] For comparison, Figure 19A shows photographs of the 0.4 μm and 0.1 μm films after extrusion without using the 0.8 μm film. Figure 19B shows photographs of the 0.8 μm, 0.4 μm, and 0.1 μm films after extrusion.

[0350] In Figure 19A, the 0.4 μm extruded film appears to be completely covered by a brown layer of SiNPs, already indicating that in the absence of the 0.8 μm film, it captured most of the aggregates. Conversely, as shown in Figure 19B, in the presence of the 0.8 μm extruded film, it appears to be covered by a brown layer of SiNPs, indicating that it captured most of the aggregates in the solution.

[0351] DLS and zeta potential analysis were performed on the MVI0007 sample before extrusion and after each extrusion step. The results of the DLS analysis of the sample are shown in Table 8 and Figure 20.

[0352] [Table 12]

[0353] DLS analysis confirmed that the 0.8 μm extruded film removed most of the SiNP aggregates, as indicated by the decrease in the average Z value after 0.8 μm extrusion.

[0354] Zeta potential measurements after 0.1 μm extrusion showed relatively low counts. Therefore, further concentrated samples were analyzed to achieve better zeta potential values. The dilution factors investigated were 20x (value indicated by SiSaf) and 5x. The results of this investigation are shown in Figure 21.

[0355] Figure 21 shows that the use of a 5x dilution factor improved the characterization of the zeta potential signal. Note that the reference dilution value is 20x. Interestingly, the average zeta diameter obtained using a 20x dilution factor is 72 nm, while the average zeta diameter obtained using a 5x dilution factor is slightly larger (80 nm). The reference value is 108 nm.

[0356] Example 6 - Process step reduction In this example, the use of nuclease-free water for the generation of 50 ml batches of micelle lipid particles was investigated. The experimental conditions used in this experiment were the same as those used for sample MVI0007, as shown in Figure 17. Note that in this case, the experimental procedure was slightly modified to reduce the exposure time of SiNPs to a non-sterile environment and to avoid unnecessary steps that could lead to microbial contamination. Therefore, instead of transferring the SiNPs to a tube containing THR-GLY aqueous solution after activation, the SiNPs were diluted by directly adding the THR-GLY aqueous solution into the same tube in which the SiNPs were activated. Inflow extrusion through 0.8 μm, 0.4 μm, and 0.1 μm membranes was performed at 60°C.

[0357] Figure 22 shows a photograph of the solution obtained for the labeled sample MVI0008.

[0358] As shown in Figure 22, substantial precipitate formation was still observed in the MVI0008 sample before extrusion. DLS analysis was performed on the MVI0008 sample before extrusion and after each extrusion step. The results of the DLS analysis for the three samples are shown in Table 9 and Figure 23.

[0359] [Table 13]

[0360] In this experiment, micelle lipid particles were prepared using nuclease-free water, referencing the baseline sample MVI0007. Experimental observations showed that experiment MVI0008, using nuclease-free water, successfully reproduced MVI0007 and therefore the predicted characteristics. The precipitate was largely captured by the 0.8 μm membrane. Extrusion improved the monodispersity of all samples analyzed. The solution after 0.1 μm extrusion appeared completely clear. Importantly, alteration of the addition order did not affect the experimental results.

[0361] Example 7 - Use of optional pre-filtration step In this example, the use of a pre-inflow extrusion pre-filtration step to filter out large aggregates from the SiNP solution was investigated. The final volume of this experiment was increased to 100 ml. Furthermore, to reduce the volume of lipid-injected MeOH in the final solution, a MeOH lipid stock solution was prepared at an increased concentration of 10 mg / ml (instead of 5 mg / ml). A 0.8 μm hydrophilic polyethersulfone syringe filter was used for pre-filtration. Figure 24 shows the strategy used in this experiment, which was named MVI0011.

[0362] After filtering the micelle lipid particles using a 0.8 μm hydrophilic polyethersulfone syringe filter, inflow extrusion was performed. The photograph of the syringe filter shown in Figure 24 already indicates that the pre-filtration step captured most of the large aggregates on the SiNP solution, as shown by the brownish layer on the syringe filter and the absence of the same layer on the 0.8 μm extruded membrane. Conversely, in experiments where this pre-filtration step was not performed, the 0.8 μm membrane appeared to be completely covered by the brownish layer of SiNP (see Figure 19).

[0363] DLS analysis was performed on the MVI0011 sample after each pre-filtration and extrusion process. The results of the DLS and zeta potential investigations for the sample are shown in Table 10 and Figure 26.

[0364] [Table 14]

[0365] The Ζ mean value improved after filtration, consistent with the visual observations shown in Figure 25, indicating that large aggregates were removed from the SiNP solution. The DLS measurement for sample MVI0011 was in agreement with the predicted value after extrusion.

[0366] Finally, the use of a more concentrated stock lipid solution was also investigated. Increasing the MeOH concentration did not affect the DLS and zeta potential characteristics of the micelle lipid particles.

[0367] Example 8 - Demonstration of Large Batch In this embodiment, to demonstrate the feasibility of a large-volume batch extrusion process, we investigate the generation of micelle lipid particles in a final volume of 1 L. This is considered an intermediate step preceding generation. In this experiment, named MVI0012, we investigated the use of an extrusion membrane with a diameter of 47 mm.

[0368] For the preparation of micelle lipid particles, the experimental conditions shown in Figure 17 were used, with the volume adjusted to a final batch size of 1 L. In summary, SiNP-THR-GLY aqueous solutions were prepared using a standard procedure of SiNP activation in MeOH followed by SiNP dilution in THR-GLY aqueous solution. To do this, 100 mg of unpre-activated SiNP was weighed and transferred to a 200 ml sterile Falcon tube, and 5 ml of MeOH was added for activation. THR-GLY aqueous solution was prepared by weighing 100 mg of THR and 50 mg of GLY and adding 95 ml of MilliQ water. After activating the SiNPs at room temperature for 30 minutes, the THR-GLY solution was added to the SiNPs and sonicated at 50°C for 60 minutes.

[0369] Next, 750 mg of DOTAP-Cl, 750 mg of DOPE, and 200 mg of mPEG2000-DSPE were weighed out, and 10 mg / ml stock solutions of DOTAP-Cl, DOPE, and mPEG2000-DSPE were prepared by adding 75 ml of MeOH to solubilize DOTAP-Cl and DOPE, and 20 ml of MeOH to solubilize mPEG2000-DSPE. The lipid solutions were then sonicated at 40°C for 30 minutes. 100 ml of SiNP-THR-GLY solution was transferred to a 1 L sterile bottle. The volumes of each lipid solution to be added to the SiNP solution at this scale are shown in Table 11. Due to the large volume of the mixed lipid solution, it was slowly added using an HPLC pump. The flow rate used for addition was 6 ml / min. Finally, MilliQ water was added up to 1 L.

[0370] [Table 15]

[0371] Inflow extrusion was performed at 60°C through 0.8 μm, 0.4 μm, and 0.1 μm films. Figure 27 shows photographs of the obtained solutions.

[0372] Interestingly, unlike other experiments, the solution after 0.8 μm extrusion appeared yellow, suggesting that some of the silicon had passed through the membrane. This is supported by a visual examination of the extruded membrane shown in Figure 28, which reveals the presence of a brown layer on the 0.4 μm membrane, which was not observed in other experiments.

[0373] The results of the DLS survey on the samples are shown in Table 12 and Figure 29.

[0374] [Table 16]

[0375] Experimental observations for experiment MVI0012 showed that DLS and zeta potential measurements were consistent with the baseline values ​​after extrusion. Furthermore, while the use of a 47 mm membrane reduced the extrusion pressure, increasing the lipid concentration in the initial MeOH lipid solution reduced the amount of MeOH in the final solution.

[0376] Example 9 - Analysis by HPLC This section investigates the development of a method for HPLC-CAD quantification of lipid content in solution. To do this, the use of Waters Xbridge Phenyl column (SKU: 186003352) was investigated: [Table 17]

[0377] A 40 mM ammonium acetate solution was used as the elution buffer. The gradient profile is shown in Table 13.

[0378] [Table 18]

[0379] Next, 1 mg / mL solutions of each lipid were prepared and injected separately into phenyl columns.

[0380] A 1:1:1 mixture of all three lipid solutions was also prepared and investigated using the same method. The chromatogram of the mixed solution is shown in Figure 31.

[0381] In the case of a phenyl column, the lipid DOPE showed a peak eluting at 15.95 minutes. DSPE-mPEG2k showed a broad peak at 16-23 minutes, mainly related to the polydispersity of the PEG chain. Finally, DOTAP-Cl eluted at approximately 24 minutes. In the case of DOTAP-Cl, a slight overlap with the peak was observed at the end of the method, caused by the abrupt solvent switch. Therefore, as shown in Figure 33, the method initially used was optimized for earlier lipid elution.

[0382] [Table 19]

[0383] Finally, the lipid mixture was prepared and analyzed using the modified method at the following final product concentrations.

[0384] [Table 20]

[0385] Figure 34 shows the chromatogram of the lipid mixture.

[0386] Experimental observations showed good separation of lipid peaks using the developed HPLC method. Therefore, this method was used to evaluate the lipid content in the extrusion solution for all samples.

[0387] The determination of the lipid content in the extruded sample is based on the creation of calibration curves for three lipids, followed by HPLC sample analysis using the method described above. A detailed procedure and the results obtained for a representative experiment, Experiment MVI0010, are presented here. A table including the lipid recovery rates for all analyzed samples is also shown at the end of the examples.

[0388] To quantify the lipids in the extruded sample, a standard lipid solution was prepared using the prepared lipid stock solution as a starting material. The standards used to create the calibration curve are shown in Table 14.

[0389] [Table 21]

[0390] Calibration curves were created by determining and analyzing the chromatograms of each standard solution and reporting the peak area versus lipid concentration for each lipid. Figure 37 shows the calibration curves for each lipid obtained for sample MVI0010 using the standards shown in Table 14.

[0391] The lipid recovery rate after extrusion was determined using calibration curves for three lipids. Sample 10 was prepared following the general experimental procedure for MeOH exposure of SiNPs for activation of the SiNP solution and injection of lipids into the SiNP solution (see Figure 17). The solution was then extruded through 0.8 μm, 0.4 μm, and 0.1 μm membranes at 60°C, as described above. The sample was then analyzed by HPLC. The chromatogram is shown in Figure 38.

[0392] The area of ​​each peak was used to determine the lipid concentration in the extruded sample. Table 15 shows the lipid recovery rate after extrusion for experiment MVI0010.

[0393] [Table 22]

[0394] Finally, Table 16 summarizes the HPLC content of all samples analyzed after extrusion using the described procedure.

[0395] [Table 23]

[0396] The experimental results showed the following average recovery rates after extrusion: [Table 24]

[0397] Example 10 - Stability over time In the chromatograms of the extruded samples, the inventors noted the presence of unknown peaks at 7 min, 8 min, and 9.5 min (see Figure 36 for a representative example). To investigate potential sources of contamination, the chromatograms of nuclease-free water and THR-GLY solution samples were first analyzed, both of which are shown in Figure 41.

[0398] As shown in Figure 41, GLY and THR eluted directly upon injection. Consequently, the peaks at 7, 8, and 9.5 minutes do not appear to be related to GLY and THR.

[0399] To evaluate the emergence of unknown peaks, the HPLC and DLS characteristics of the samples were analyzed over time. Samples were stored at room temperature (RT) and 4°C, and HPLC content and DLS characteristics were measured at time 0 (at time of generation), 1 week later, and 2 weeks later.

[0400] (i) Investigation of samples stored at room temperature The changes in lipid content in samples stored at room temperature were determined at different time points, specifically at time 0, 1 week later, and 2 weeks later. To do this, calibration curves were created for each time point. Then, for each lipid, the change in lipid concentration was determined by comparing the lipid concentration measured at time 0 with the lipid concentration measured at the different time points. Figure 44 shows the chromatograms of the samples stored at room temperature for up to 2 weeks.

[0401] To further investigate the stability of the samples over time, DLS characteristics were measured. The results are shown in Table 17.

[0402] [Table 25]

[0403] Table 17 shows that the DLS characteristics of samples stored at room temperature are largely maintained over time.

[0404] (ii) Investigation of samples stored at 4°C The same procedure used for samples stored at room temperature was employed to determine the change in lipid content at different time points for samples stored at 4°C. Calibration curves were then constructed for each time point, and the lipid concentration was determined by comparing the concentration measured at time 0 with the concentration over time. Figure 44 shows chromatograms of the samples analyzed at 4°C at different time points.

[0405] The characteristics of DLS and zeta potential were measured over time. The results are shown in Table 18.

[0406] [Table 26]

[0407] As shown in Table 18, the DLS characteristics are largely maintained over time even in samples stored at 4°C.

[0408] Experimental observations of the investigated samples suggested a decrease in lipid concentration over time in samples stored at 4°C and room temperature. This decrease may also be attributable to instrumental variability due to the low concentrations measured for each lipid, and low peak areas can lead to large experimental variability. Interestingly, increasing the injection volume increased the recovery rate of DSPE-mPEG lipids (114%). This observation also indicates instrumental variability in the obtained results.

[0409] Example 11 - Evaluation of tangential flow rate Tangential flow filtration (TFF) is a rapid and efficient method for the separation and purification of biomolecules. In the context of this study, TFF is used to purify a sample by diafiltration and ultimately concentrate the sample to meet the desired concentration range of components. Diafiltration washes small molecules through a membrane with a specified pore size, while larger molecules are retained in the residual solution. For this reason, diafiltration was used to remove MeOH. In this study, diafiltration was performed by adding the diafiltration solution to the sample supply reservoir via a second pump at the same rate at which the filtrate was produced. In this way, the volume in the sample reservoir remains constant, but MeOH is freely permeated through the membrane and washed away. During TFF, THR and GLY can be removed due to their small molecular weight. For this reason, a THR-GLY solution was used at the same final concentrations as the diafiltration solution (0.1 mg / ml THR and 0.05 mg / ml GLY).

[0410] One diafiltration volume is constructed by adding a volume of diafiltration solution equal to the volume of the product in the system to the supply reservoir, and then concentrating it back to the starting volume. Therefore, for a 100 mL starting sample, 1 DV = 100 mL. It has been previously observed that using 10 DV, continuous diafiltration dramatically reduces the MeOH content to within the 100 ppm range (European standard for acceptable MeOH content <3000 ppm).

[0411] For the feasibility test of TFF, a film with the following characteristics was used: • Surface 0.02m 2 MWCO 100kDa • HyStream membrane. The HyStream membrane is inherently highly hydrophilic and resistant to contamination from hydrophobic species such as lipids. This membrane has good chemical resistance. • Architecture LP screen. This type of channel is best suited for clear feed flows across a wide range of viscosities.

[0412] The general TFF conditions used in this study are shown in Figure 46.

[0413] Samples were prepared by exposing NP to MeOH for activation, followed by dilution in an aqueous THR-GLY solution. A mixed lipid solution was then appropriately prepared and injected into an NP-THR-GLY dispersion. The solution was then first extruded through 0.8 μm, 0.4 μm, and 0.1 μm membranes at 60°C, followed by purification by TFF using the experimental conditions described in Figure 46. To test the effect of TFF on the system, DLS characteristics, HPLC lipid content, and MeOH content must be evaluated before and after TFF. DLS characteristics and HPLC content analysis were performed as described in the previous section. 1 The MeOH content was investigated using 1H-NMR.

[0414] A common method for determining the MeOH content in a sample is based on the creation of a MeOH calibration curve. A detailed procedure and the results obtained for a representative experiment, Experiment MVI0010, are presented here. At the end of the examples, a table containing the determined MeOH content for all analyzed samples is reported.

[0415] MeOH standard solutions with a final volume of 1 ml were prepared at various MeOH concentrations (Table 21).

[0416] [Table 27]

[0417] 10% D2O by volume was added to the standard (100 μL of D2O). 600 μL of each standard was placed in an NMR cuvette. 1 All standards were analyzed using 1H-NMR. The calibration curve shown in Figure 47 was prepared by reporting the area of ​​the peak associated with the MeOH signal at each MeOH concentration.

[0418] After creating a standard curve, the samples were analyzed to determine the MeOH content. Pre-TFF samples were prepared by appropriately diluting the original samples to match the range of the calibration curve. For sample 11, a 1000-fold dilution was used because the predicted methanol content in the sample was approximately 30% of the sample volume (see Table 2 for example). Post-TFF samples were concentrated and analyzed. The final volume of each sample was 1000 μL. Therefore, 10% by volume of D2O was added to the sample (100 μL of D2O). 600 μL of each sample was placed in an NMR cuvette. 1 Analysis was performed using 1H-NMR. Subsequently, the concentration of MeOH in the sample before and after TFF was determined using the area of ​​the MeOH peak in the NMR spectrum.

[0419] Figure 48 shows representative NMR signals of sample 10 before and after TFF.

[0420] Table 20 shows the quantification of MeOH in sample 10 before and after TFF.

[0421] [Table 28]

[0422] The MeOH content was estimated considering the density of the MeOH-H2O mixture at 0.988 kg / L [2].

[0423] The MeOH content of the other analyzed samples, namely samples 08 and 09, was determined using the same procedure as described for determining the MeOH content of sample 10. Table 21 shows the MeOH content of all analyzed samples before and after TFF (Total Fat Filling).

[0424] [Table 29]

[0425] The experimental observation results for all samples analyzed were 105 from 10 2 This shows that the MeOH concentration decreases by three orders of magnitude as a result of TFF.

[0426] As already mentioned, in order to determine the feasibility of this process, the HPLC and DLS characteristics of the samples need to be evaluated after TFF. These data for all samples analyzed before and after TFF are shown in Tables 35 and 36.

[0427] [Table 30]

[0428] [Table 31]

[0429] Experimental observations indicate that DLS characteristics are largely maintained after TFF. Finally, lipid recovery rates are also maintained after TFF.

[0430] Note that sample MVI0010 was concentrated 2-fold by TFF. This was necessary to increase the silicon content to match the desired silicon concentration in the final product.

[0431] Example 12 - Further optimization and reduction of silicon This embodiment demonstrates further optimization of the process for developing micelle lipid particles. Three different experimental conditions were investigated, as listed below:

[0432] i. Preparation of a micelle lipid particle batch by filtering the SiNP solution after activation. ii. Preparation of a micelle lipid particle batch using 1 / 5 of the normally used amount of Si (4 mg). iii. Preparation of micelle lipid particle batches without the presence of DSPE-mPEG2000.

[0433] Preparation of a micelle lipid particle batch by filtering the SiNP solution after activation (MVI0013)

[0434] Figure 51 shows the experimental procedure for preparing a micelle lipid particle batch, in which the SiNP solution is filtered after activation. This experiment was named MVI0013.

[0435] As shown in Figure 52, micelle lipid particles were prepared in a final volume of 50 ml. The SiNP solution was manually filtered after MeOH activation using a 0.8 μm hydrophilic polyethersulfone syringe filter. Inflow extrusion was performed at 60°C through 25 mm extrusion membranes with pore sizes of 0.8 μm, 0.4 μm, and 0.1 μm. Visual inspection of the solutions obtained in each step of Experiment MVI0007 is shown in Figure 52. Photographs of the 0.8 μm hydrophilic polyethersulfone syringe filter and extruded membrane after the filtration and extrusion steps are shown in Figure 53.

[0436] In Figure 52, the filtered solution appears yellowish without the presence of large aggregates, already suggesting the removal of most of the aggregated SiNPs in the solution. This observation is further confirmed in Figure 53, as indicated by the presence of a brown layer of NPs on the 0.8 μm syringe filter shown in Figure 53A. Furthermore, the 0.8 μm and 0.4 μm extruded membranes were completely clean, as indicated by the absence of brownish regions, while the 0.1 μm membrane still captured some small aggregates of SiNPs.

[0437] DLS and zeta potential analysis were performed on the MVI0013 sample before extrusion and after each extrusion step. The obtained values ​​were compared with the SiSaf reference values. The results of the DLS analysis for the sample are shown in Table 37 and Figure 54.

[0438] [Table 32]

[0439] DLS analysis shows that the 0.8 μm syringe filter removes most of the SiNP aggregates, as indicated by the initial Z-mean value (pre-extrusion value). The Z-mean value is largely maintained up to the 0.4 μm extrusion step, confirming aggregate removal by the syringe filter. Extrusion pressure is also shown in Figure 55 and shows similar indications: the pressure on the 0.8 μm and 0.4 μm films is almost zero, confirming the absence of large aggregates in the extrusion solution, although some pressure is shown at the 0.1 μm pore size of the film. Finally, PDI and zeta potential values ​​are largely maintained throughout the entire process.

[0440] [Table 33]

[0441] Experimental observations in experiment MVI0013 show that the DLS characteristics are consistent with the predicted values. However, the inventors noted the absence of a clear peak in zeta potential analysis (Figure 56). Finally, HPLC quantification shows a lipid recovery rate of 90%.

[0442] (ii) Preparation of a micelle lipid particle batch using 1 / 5 of the Si content (MVI0014). Figure 57 shows the experimental procedure for preparing a micelle lipid particle batch, in which the SiNP solution is filtered after activation. This experiment was named MVI0014.

[0443] As shown in Figure 57, micelle lipid particles were prepared in a final volume of 50 ml. As shown in Table 39, SiNP solutions were generated by reducing the amount of silicon typically used in all other experiments.

[0444] [Table 34]

[0445] To do this, 4 mg of inactivated SiNPs were weighed out, and the concentration of the SiNP solution was reduced to 4 mg / ml by exposing them to 1 ml of MeOH. A visual inspection of the resulting solution is shown in Figure 58.

[0446] DLS and zeta potential analysis were performed on the MVI0014 sample before extrusion and after each extrusion step. The obtained values ​​were compared with SiSaf reference values. The results of the DLS analysis for the sample are shown in Table 40 and Figure 59.

[0447] [Table 35]

[0448] Figure 60 shows the investigation of extrusion pressure for MVI0013. As shown in Figure 60, the extrusion pressure values ​​are relatively low, which may be related to the small amount of silicon used in this experiment.

[0449] Finally, the HPLC content was investigated. The results are shown in Table 41.

[0450] [Table 36]

[0451] Experimental observations in experiment MVI0014 showed that the DLS characteristics were in agreement with the predicted values. HPLC quantification showed an 80% lipid recovery rate.

[0452] (iii) Preparation of a micelle lipid particle batch without the presence of DSPE-mPEG2000 (MVI0015) Figure 61 shows the experimental procedure for preparing a micelle lipid particle batch, in which the SiNP solution is filtered after activation. This experiment was named MVI0015. Note that after extrusion, the sample was concentrated twice by performing TFF to remove MeOH.

[0453] As shown in Figure 61, micelle lipid particles were prepared in a final volume of 200 ml (before TFF). The lipid concentrations in the final solution were modified as shown in Table 42.

[0454] [Table 37]

[0455] A visual inspection of the obtained solution is shown in Figure 62.

[0456] After extrusion, TFF was performed as described in Section 2.6 Feasibility of Tangential Flow Filtration, and the MVI0015 sample was purified and concentrated 2-fold. DLS and zeta potential characteristics were investigated. The results are shown in Table 43 and Figure 63.

[0457] [Table 38]

[0458] We also investigated the HPLC content, and the results are shown in Table 44.

[0459] [Table 39]

[0460] lastly, 1 The MeOH content was investigated by 1H-NMR. The results are shown in Table 45.

[0461] [Table 40]

[0462] The MeOH content measured in MVI0015 after TFF was below 50 ppm, the lowest concentration point on the calibration curve. This value may not be accurate. However, it indicates almost complete removal of MeOH from the sample.

[0463] Experimental observations in experiment MVI0015 (absence of DSPE-mPEG2000) showed that the DLS characteristics were consistent with the baseline, indicating that the DLS characteristics were largely maintained after TFF. HPLC quantification showed a lipid recovery rate of 90% before TFF, but the inventors observed a decrease in lipid recovery rate after TFF (from 90% to 60%).

[0464] Example 13 - Determination of silicon content The silicon content of SiSaf was determined by ICP-OES by examining samples prepared using the various experimental techniques described in the previous section. Specifically, the silicon content in samples prepared using the original SiSaf experimental procedure by exposure of a lipid membrane with pre-activated SiNPs (experiment MVI0004) was determined and compared with the silicon content measured using a modified experimental procedure of lipid injection into an unpre-activated SiNP solution (unpre-hydrated SiNPs + lipid injection). Table 46 provides a brief description of the analyzed samples for convenience.

[0465] [Table 41]

[0466] Table 47 summarizes the results of the ICP-OES survey for all the samples analyzed.

[0467] [Table 42]

[0468] The baseline silicon content in the final product is 2.1 mg / L. This value was also observed in experiment MVI0004, which replicated the original SiSaf procedure. On the other hand, using the experimental procedure shown in MVI0005C and MVI0009 (unpre-hydrated SNiP + lipid injection), the silicon content was found to be 1.2 mg / L. By the 2x enrichment method via TFF applied to MVI0010, as shown in Section 2.6 Feasibility of Tangential Flow Filtration, the inventors restored the silicon content, finding it to be 2.18 mg / L in the sample after TFF. Interestingly, a certain variation in results was observed in samples MVI0011-MVI0015, where the experimental procedure was modified, as shown in Table 46.

[0469] Example 14 - Production of a 2kg batch This example shows the results of producing a 2 kg micelle lipid particle batch. The experimental procedure for producing the 2 kg batch is shown in Figure 64. This experiment was named MVI0022.

[0470] DLS and zeta potential, HPLC for determining lipid content, and MeOH quantification 1 The obtained batches were characterized by 1H-NMR.

[0471] For calculation purposes, the density of the SiNP-lipid dispersion is set to 1 g / cm³. 3 This assumption was made. As a result, 4 L of SNiP-lipid solution was concentrated twice by TFF. The final volume was 2 L, which was equivalent to 2 kg. Table 48 shows the theoretical concentrations of all components before and after TFF. Note that the concentrations of THR and GLY were maintained after TFF because the THR-GLY solution was used at the same final concentrations as the diafiltration solution (0.1 mg / ml THR and 0.05 mg / ml GLY).

[0472] [Table 43]

[0473] To prepare a 2 kg micelle lipid particle batch, a SiNP-THR-GLY aqueous solution was prepared using a standard procedure involving SiNP activation in MeOH (MeOH dispersion + sonication), followed by SiNP dilution in a THR-GLY aqueous solution.

[0474] To do this, 400 mg of unpre-activated SiNP was weighed and transferred to a 50 ml sterile Falcon tube, and 20 ml of MeOH was added for activation. A THR-GLY aqueous solution was prepared by dissolving 400 mg of THR and 200 mg of GLY in 380 ml of nuclease-free water (in a Nalgene sterile bottle). After activating the SiNP at room temperature for 30 minutes, the SiNP was added to the THR-GLY solution, and the resulting dispersion was sonicated at 50°C for 60 minutes. 10 mg / ml stock solutions of DOTAP-Cl, DOPE, and mPEG2000-DSPE were prepared by dissolving 3000 mg of DOTAP-Cl in 300 ml of MeOH, 3000 mg of DOPE in 300 ml of MeOH, and 600 mg of mPEG2000-DSPE in 60 ml of MeOH. The lipid solutions were then sonicated at 40°C for 30 minutes. 400 ml of SiNP-THR-GLY solution was transferred to a 5 L sterile bottle. Lipids were slowly added using a Knauer K501 HPLC pump at a flow rate of 6 ml / min. After lipid addition, nuclease-free water was added up to 4 L, and the batch was stirred for homogenization. Table 49 shows the volume of stock solution prepared and the volume used to produce the 4 L solution.

[0475] [Table 44]

[0476] 4 L of the crude product was prepared without any particular problems and stored overnight at 4°C. As usual, the presence of large SiNP aggregates was observed.

[0477] Next, inflow extrusion was performed at 60°C through three consecutively arranged 47 mm extruded membranes with pore sizes of 0.8 μm, 0.4 μm, and 0.1 μm. A Knauer K501 pump equipped with a pressure sensor and a stainless steel pump head with a flow rate of 50 ml / min was used for extrusion. The flow rate was set to 50 ml / min.

[0478] Table 50 shows the results of the visual inspection of MVI0022.

[0479] [Table 45]

[0480] In particular, as shown in Figure 65, the SiNP aggregates saturated the film. The extruded film was replaced during extrusion due to pressure fluctuations.

[0481] TFF was performed according to the MVI0010 conditions. In MVI0022, the surface area of ​​the TFF cassette was set to 0.5 m². 2 The TFF film was increased while maintaining other characteristics of the cassette used for small batch extrusion (HyStream, MWCO 100kD, LP screen channel). Importantly, prior to TFF, the system was deheated by washing the entire system with a 0.2M NaOH solution for 1 hour. This technique was introduced at this stage for the production of 2kg batches. Deheating is widely used to remove exothermic substances from the contact material.

[0482] The results of the DLS survey for MVI0022 are shown in Table 51 and Figure 66.

[0483] [Table 46]

[0484] The results of the HPLC investigation of MVI0022 are shown in Table 52. Note that the sample was diluted with MeOH, as shown in Section 2.4.3 on page 32. The sample before TFF was diluted 2-fold, while the sample after TFF was diluted 4-fold to match the range of the calibration curve.

[0485] [Table 47]

[0486] Table 53 shows the results of the 1H-NMR investigation for MeOH quantification in MVI0022.

[0487] [Table 48]

[0488] Finally, the silicon recovery rate of the TFF-treated sample, as determined by ICP-OES, was 2.9 mg / L.

[0489] The experimental observations regarding experiment MVI0022 are as follows: DLS measurement values • Zeta mean is slightly higher than predicted • Small batch 132nm MVI0022 vs 108nm baseline vs 78nm average • The zeta mean was largely maintained after TFF. • PDI was consistent with the predicted value before TFF (0.175) and slightly increased after TFF (0.248). • The zeta potential matches the predicted values ​​(64.5mV) before and after TFF.

[0490] HPLC measurement values • Pre-TFF analysis showed a high lipid recovery rate (95%) exceeding the predicted concentration. • Post-TFF analysis showed a high lipid recovery rate (100%) exceeding the predicted concentration.

[0491] 1H-NMR measurement values • MeOH is not detected in samples after TFF.

[0492] ICP-OES measurement values • Silicon recovery rate matched the predicted value.

[0493] In particular, the inventors observed a general trend where size increases with increasing extrusion volume, as shown in Figure 67.

[0494] The presence of SiNPs can cause clogging of the extruded membrane during inflow extrusion, which can lead to experimental variations in the Ζ mean. To avoid membrane solidification, a pre-filtration step (for membranes of 0.8 μm or thicker) can be used immediately after SiNP activation.

[0495] The extruded film was replaced during extrusion due to pressure fluctuations. For the next experiment to increase the extruded surface area, inflow extrusion through two parallel extrusion holders containing extruded films of the same pore size can be considered.

[0496] Time is a critical parameter for the extrusion process. Increasing the extrusion flow rate by changing the pump / pump head has a positive effect on the extrusion time (the one currently used by the inventors has an inherent limit of 30 ml / min, which decreases over time due to film solidification).

[0497] Finally, based on these experimental observations, 0.5m 2 The TFF film can be used in 5kg batch sizes.

[0498] Summary of Examples These examples investigate the feasibility of an expandable process for the production of micelle lipid particles according to the present invention. The feasibility of direct addition of activated SiNPs from methanol to aqueous solution, and the feasibility of direct addition of lipids to SiNPs, followed by inflow extrusion and tangential flow filtration posttreatment, was investigated to generate an expandable process for the production of micelle lipid particles. Experimental results demonstrated the feasibility of these steps. DLS and zeta potential characteristics were consistent with the predicted characteristics. Lipid recovery was 70%–80% of the initial lipid concentration after extrusion (DOPE: 79.2%, DSPE-mPEg: 76.6%, DOTAP-Cl: 70.4%). All of these characteristics were largely maintained after TFF. Furthermore, TFF demonstrated successful removal of MeOH from all samples investigated, and the feasibility of doubling the solution concentration to match the optimal concentration of components in the final product was also demonstrated.

[0499] HPLC analysis of lipid content revealed the presence of an unknown peak. HPLC analysis of the sample over time showed a decrease in lipid concentration. However, this decrease in concentration, as indicated by the HPLC measurements, may be attributable to instrument variability due to the low measured concentrations. Supporting this hypothesis, increasing the injection volume increased the recovery rate of DSPE-mPEG lipids. Further investigation of this characteristic could be performed, for example, by LC-MS. Silicon recovery showed significant variability. Currently, increased concentration by TFF post-treatment is necessary to bring the silicon recovery rate closer to a baseline.

[0500] Tables 54 and 55, which summarize the characteristics of all the samples analyzed, are shown on the following page.

[0501] [Table 49]

[0502] [Table 50]

[0503] Example 15 - Storage Stability The storage stability of the hybrid lipid particles of the present invention, formulated using silicon, was tested to compare them with lipid nanoparticles formulated without silicon or other inorganic particles. Biocourier MVI0012 and MVI0012 particles were prepared according to the method described above. Prior art comparison particles (LNPs) were also prepared using the same method as MVI0012, but with silicon particles or other inorganic particles. The particles were then stored at room temperature at 4°C for several weeks. The particle size and their surface charge were determined at intervals. The results are shown in Figure 69. It can be seen that the positively charged surface of the particles of the present invention withstood at room temperature for at least 24 weeks and at 4°C for at least 80 weeks. It is assumed that this positive surface charge (zeta potential >30mV) inhibited particle coalescence, and from Figure 69, it can be seen that the particle size remained constant throughout the test period, in contrast to the LNP control.

[0504] Example 16 - Size stability after RNA loading To demonstrate the stability of the “empty” hybrid lipid particles of the present invention, the particles of Example 15 were prepared without the use of an active API. This example demonstrates that the addition of mRNA to the particles of the present invention does not disrupt their stability. Empty biocourier MVI0012 and LNP particles were mixed with mRNA in various ratios, and their size and size monodispersity (PDI) were measured. The results are shown in Figure 70, and the results show that, unlike the situation with conventional LNP particles, the particles of the present invention retain their small size when complexed with mRNA in all ratios tested, enabling even higher mRNA loading without disrupting the particles.

[0505] Materials and methods of Examples 17 and 18 Silicon nanoparticles were commercially supplied as electrochemically etched powder (purity ≥ 98%; manufactured by American Elements, Inc., Los Angeles, CA; or The Porous Silicon Company, Salzburg, Austria). Trehalose and glycine (USP / PhEur specifications) were purchased from Merck. The following lipids were supplied by Lipoid GmbH, Ludwigshafen, Germany: 1,2-Dioleoyl-3-trimethylammonium propane chloride (DOTAP chloride; CAS number 132172-61-3); 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE; CAS number 4004-05-1); and N-(carbonyl-methoxypolyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, sodium salt (mPEG2000 DSPE; CAS number 147867-65-0). Nuclease-free water produced in-house was used throughout the procedure. The mean hydrodynamic particle size, PDI, and zeta potential measurements were obtained using a Zetasizer Nano (Malvern Instruments, UK), as reported in previous studies (Baran-Rachwalska et al., 2020; Maurizi et al., 2023).

[0506] Formation of sshLNPs by lipid film hydration (Figure 71). In Method 1, as previously described (Baran-Rachwalska et al., 2020), silicon nanoparticles were activated by suspending them in MeOH (20 mg / mL), stirring for 0.5 hours, and then slowly evaporating them. These were then added to an aqueous solution of trehalose (1 mg / mL) and glycine (0.5 mg / mL) at 1 mg / mL, and the mixture was sonicated at 50°C for 1 hour. Separately, lipid solutions of DOTAP chloride, DOPE, and optionally mPEG2000-DSPE were prepared in MeOH at 5 mg / mL and sonicated at 40°C for 0.5 hours. Aliquots of these solutions (1.44 mL, 1.46 mL, and 0.29 mL, respectively) were transferred to 10 mL round-bottom flasks and mixed. A lipid film was formed by removing the solvent by rotational evaporation at 40°C for 0.5 hours, followed by hydration of the lipid film by adding 1 mL of the above aqueous solution and incubating at 60°C for 5 minutes. Water was added to a final volume of 10 mL (dehydration-rehydration method), and the mixture was then extruded through series-arranged 5 × 0.4 μm and 5 × 0.1 μm polycarbonate hydrophilic membranes (Whatman Nucleopore) using an Avanti Polar Lipid Extruder combined with manual injection via a gastight Hamilton syringe. The membranes were pre-washed with MeOH and water (20 mL each) before use. In Method 2, MeOH was not evaporated from the activated silicon nanoparticles before further use. Instead, aliquots of the activated suspension were added directly to a trehalose / glycine aqueous solution to achieve the same final concentrations as above: 1 mg / mL of SiNP, 1 mg / mL of trehalose, and 0.5 mg / mL of glycine. Hybrid lipid particle formation and extrusion were then carried out as described above.

[0507] Formation of hybrid lipid particles by direct, low-speed injection of lipids (Figure 72). In Method 3, SiNP (20 mg) was activated in MeOH (1 mL) as described above, and then combined with an aqueous solution (19 mL) of trehalose (20 mg) and glycine (10 mg). The mixture was sonicated at 50°C for 1 hour. Separately, a lipid stock solution was prepared and combined in the same manner as in Method 1. Using a PSNE100 syringe pump (ProSense, Munich, Germany), the combined lipid solution was added to 1 mL of the above aqueous solution at a rate of 3.2 mL / min. The volume was increased to 10 mL with water, the solution was stirred for 0.5 hours, and then extruded as described above.

[0508] A modified method with an added 0.8 μm extrusion step (Figure 72D). Method 4 was performed on a five-fold larger scale (i.e., a final volume of 50 mL) and followed the same procedure as Method 3, except that a 3 × 0.8 μm polycarbonate hydrophilic membrane (Whatman Nucleopore) was also incorporated in series before the 3 × 0.4 μm and 3 × 0.1 μm membranes during the extrusion process.

[0509] Other improvements to the small-scale protocol (Figures 72E, 78). In Method 5, the order of addition used to prepare the aqueous solution was changed. The pre-prepared aqueous solution of trehalose / glycine was added directly to the activated methanol suspension of SiNP, instead of in reverse as in Method 4. The final concentrations of silicon nanoparticles (1 mg / mL), trehalose (1 mg / mL), and glycine (0.5 mg / mL) in the intermediate aqueous solution were not changed, and the remaining procedure was carried out as before. Method 6 was carried out on twice the scale of Method 4 (i.e., a final volume of 100 mL) and introduced two new modifications. First, the lipid stock solution was prepared at 10 mg / mL instead of 5 mg / mL, and then aliquots of DOTAP chloride (7.20 mL), DOPE (7.30 mL), and mPEG2000-DSPE (1.45 mL) were combined and added to the pre-prepared aqueous solution as described above. After adjusting the volume to 100 mL with water, the mixture was pre-filtered through a 0.8 μm hydrophilic polyethersulfone (PES) syringe filter and extruded as in Method 4.

[0510] Diafiltration using TFF conditions (Figure 73). SIUS PD 0.02m 2 (LP)HyS 100kD cassette (XP100LP2L; Repligen, Waltham, MA) is used in volumes up to 100mL, and SIUS 0.5m 2 A 100kD TFF cassette (NC1095082; Fisher Scientific, Waltham, MA) was used for a larger volume. After the initial ultrafiltration step (usually up to 0.5 times the initial input volume) as shown, the sample was diafiltration with 10 volumes of aqueous solution containing 0.1 mg / mL trehalose and 0.05 mg / mL glycine to remove MeOH and unbound lipids while maintaining the concentrations of these two excipients. This was achieved by using a second pump to introduce the trehalose / glycine solution into the flow path at the same flow rate as the main system pump (Figure 73A). Figures 73C and 73D show the results of repeated runs of TFF by Method 5 and subsequent diafiltration only, or TFF by ultrafiltration (up to half the initial volume) and subsequent diafiltration.

[0511] Methanol content analysis. Standard aqueous solutions containing methanol in water containing 10% D2O, with known MeOH concentrations (50, 100, 300, and 500 ppm (v / v; Figure 73B, inset). 1 Calibration curves were created by measuring the 1H NMR peak area (δ = 3.34 ppm; Gottlieb et al., 1997). The pre- and post-TFF samples were analyzed in H2O / D2O (90:10), and the MeOH content was estimated by referring to the calibration curves. Due to the high MeOH content, the pre-TFF sample was diluted by a factor of 1:1,000 before obtaining the NMR spectrum.

[0512] Lipid recovery analysis. Lipid quantification relied on HPLC analysis using a Waters XBridge BEH Phenyl Column (130 Å, 5 μm, 4.6 × 150 mm) in combination with a charged aerosol detector (CAD). Buffer A was 40 mM NH4OAc and buffer B was 100% MeOH, and the following elution gradient was used: 25% A for 1 minute, increasing to 5% A over 6 minutes, holding at 5% A for 18 minutes, increasing to 25% A over 0.1 minutes, and then holding at 25% A for 4.9 minutes (flow rate 1 mL / min). Calibration curves were created for DOTAP chloride, DOPE, and mPEG2000-DSPE (with retention times of 12.8 min, 15.1 min, and 20.8 min, respectively; see Figure 80) to enable quantification. Standard solutions for DOTAP chloride and DOPE were prepared at concentrations of 0.1, 0.2, 0.5, 0.7, and 0.9 mg / mL. For mPEG2000-DSPE, reference solutions were prepared at concentrations of 0.01, 0.05, 0.15, 0.2, and 0.3 mg / mL. Peak area was plotted against concentration to determine the lipid concentration in the test sample. Lipid recovery was calculated as the ratio of the observed concentration to the theoretical concentration if all lipids were incorporated into sshLNP, and expressed as a percentage.

[0513] The first large-scale test run (1L scale, Figure 74) for generating sshLNPs was performed. Silicon nanoparticles (100 mg) were activated by suspending them in MeOH (5 mL) in a sterile 50 mL Falcon tube and incubating them at room temperature for 0.5 hours. Then, an aqueous solution (95 mL) of trehalose (100 mg) and glycine (50 mg) was added directly to the silicon nanoparticle suspension, and the mixture was sonicated at 50°C for 1 hour. Lipid stock solutions were prepared by dissolving DOTAP chloride (750 mg), DOPE (750 mg), and mPEG2000-DSPE (200 mg) in 10 mg / mL MeOH, and sonicating the resulting mixture at 40°C for 0.5 hours. Next, aliquots of DOTAP chloride (72.5 mL), DOPE (73.0 mL), and mPEG2000-DSPE (14.5 mL) solutions were combined (i.e., to achieve final concentrations of 0.725, 0.730, and 0.145 mg / mL, respectively). The pre-prepared aqueous solution (100 mL) was transferred to a sterile 1 L bottle, and methanol-mixed lipid solution (160 mL) was injected at 6 mL / min using a Knauer K501 HPLC pump. After the addition was complete, the volume was increased to 1 L with water, and inflow extrusion was performed at 60°C at a flow rate of 85 mL / min using 3 × 0.8 μm polycarbonate hydrophilic membranes, 3 × 0.4 μm polycarbonate hydrophilic membranes, and 3 × 0.1 μm polycarbonate hydrophilic membranes (47 mm diameter, Whatman Nucleopore) arranged in series as described above.

[0514] A second large-scale test run (2L scale, Figure 6) was conducted for the generation of hybrid lipid particles. As described above, SiNP (400 mg) was activated in MeOH (20 mL). An aqueous solution (380 mL) of trehalose (400 mg) and glycine (200 mg) was prepared in a sterile bottle, and then the suspension of activated SiNP was directly added to it. The resulting dispersion was sonicated at 50°C for 1 hour. As described above, stock solutions of DOTAP chloride (3.00 g), DOPE (3.00 g), and mPEG2000-DSPE (600 mg) were prepared at 10 mg / mL in MeOH. A 400 mL aqueous solution was transferred to a 5 L sterile bottle, and then, using an HPLC pump, the combined lipid mixture (290 mL of DOTAP chloride stock solution, 292 mL of DOPE stock solution, and 58 mL of mPEG2000-DSPE stock solution; total volume 640 mL) was injected into the bottle at a rate of 6 mL / min. Water was added until the final volume reached 4 L, and stirring was continued until the mixture was homogenized. The crude mixture was stored overnight at 4°C, and then inflow extrusion was performed as described in the previous example. Under TFF conditions (using a SIUS 0.5 m² 100 kD cassette as shown in Figure 73A), the mixture was first deheated with 0.2 M NaOH for 1 hour, and then the 4 L sample was concentrated to 2 L by ultrafiltration and diafiltration against 10 volumes (i.e., 20 L) of aqueous solutions of 0.1 mg / mL trehalose and 0.05 mg / mL glycine. The final hybrid lipid particle samples and intermediate hybrid lipid particle samples were analyzed using the procedure described above, except that the pre-TFF and post-TFF samples were diluted with MeOH at a ratio of 1:2 and 1:4, respectively, to match the range of the calibration curve for lipid content analysis.

[0515] Modified procedure for hybrid lipid particles, including pre-filtration of aggregates (Figure 76D). Method 7 was identical to Method 5 (50 mL scale), except for an additional pre-filtration step of activated silicon nanoparticles. Specifically, silicon nanoparticles (20 mg) were suspended in MeOH (1 mL) and activated by stirring at room temperature for 0.5 hours, and the mixture was then manually filtered through a 0.8 μm hydrophilic PES syringe filter. A pre-prepared aqueous solution (19 mL) of trehalose (20 mg) and glycine (10 mg) was added directly to the silicon nanoparticle suspension, and the dispersion was sonicated at 50°C for 1 hour. Separately, stock solutions of DOTAP chloride, DOPE, and mPEG2000-DSPE were prepared at 10 mg / mL in MeOH while being sonicated at 40°C for 0.5 hours. Aliquotes of these solutions (3.63 mL, 3.65 mL, and 0.73 mL, respectively) were combined, and the mixed lipid solution was injected into a 5 mL aliquot of aqueous solution at a rate of 3.2 mL / min using a syringe pump. The volume was increased to 50 mL with water, stirred at room temperature for 0.5 hours, and then extruded as in Method 4.

[0516] Demonstration of large batches using aggregate prefiltration (1 L scale, Figure 76E). Silicon nanoparticles (100 mg) were activated by stirring in MeOH (5 mL) at room temperature for 0.5 hours. To remove large aggregates, the suspension was manually filtered through three consecutive 0.8 μm hydrophilic PES syringe filters. The resulting sample was combined with a pre-prepared aqueous solution (95 mL) of trehalose (100 mg) and glycine (50 mg) while vigorously magnetically stirring (500 rpm) at 50°C for 1 hour. Separately, stock solutions of DOTAP chloride (750 mg), DOPE (750 mg), and mPEG2000-DSPE (160 mg) were prepared at 10 mg / mL in MeOH while sonicating at 40°C for 0.5 hours. Aliquots of these solutions (72.5 mL, 73.0 mL, and 14.5 mL, respectively) were combined, and the mixed lipid solution was then injected into an aqueous solution (in a 1 L polycarbonate container) at a rate of 6 mL / min using a Knauer BlueShadow 80P HPLC pump. The volume was increased to 1 L using water (added at 100 mL / min with stirring using the same pump), and stirring was continued at room temperature for 0.5 hours for homogenization. Inflow extrusion was performed at 60°C and a flow rate of 85 mL / min using two sets of extrusion membranes arranged in parallel (three each of pore sizes 0.8 μm, 0.4 μm, and 0.1 μm). The TFF conditions were deheated using 0.2 M NaOH for 1 hour, rinsed with water, and methanol was removed by diafiltration as outlined above.

[0517] Evaluation of RNA encapsulation efficiency (Figure 77C). As previously reported (Maurizi et al., 2023), the Quant-iT RiboGreen Assay Kit (Fisher Scientific, UK) was used according to the manufacturer's instructions. A Varioskan LUX microplate reader (Thermo Fisher Scientific) was used for analysis (excitation 480 nm, emission 520 nm).

[0518] Example 17 - Elimination of the Evaporation Step Conventional methods for producing lipid nanoparticles may include at least two solvent evaporation steps. Firstly, inorganic particles may be activated using a solvent and then evaporated in the first evaporation step. Secondly, the inorganic particles and lipids may be mixed in a solvent and the solvent may be evaporated to form a lipid film. The first evaporation step was previously considered necessary to ensure proper activation of the inorganic particles. The second evaporation step, resulting in the formation of a lipid film, and the subsequent hydration of the film, were previously considered necessary for the accurate hydration and formation of lipid nanoparticles. This example demonstrates that one or both of the evaporation steps can be eliminated.

[0519] As described above, small-scale production of lipid nanoparticles can be carried out using a workflow based on lipid thin-film hydration followed by flow extrusion. Such a method is illustrated in Figure 71A and labeled “Method 1”. Two consecutive inflow extrusion steps were used through a film having continuously small pore sizes (0.4 μm and 0.1 μm) to reduce the operating pressure on the film and ensure that the final particles had the desired dimensions. Trehalose (1 mg / mL) and glycine (0.5 mg / mL) were also included in the aqueous phase as excipients, as it has been found that the hybrid lipid particles are further stabilized, possibly by coordination with silicon nanoparticles (SiNPs).

[0520] The presence of trehalose and glycine, when in appropriate ratios, promotes the dispersibility of silicon particles in a mixed water-alcohol environment. Amino acid excipients are also included to regulate the hydrolysis rate of silicon and promote the formation of orthosilicic acid rather than insoluble polymeric silicon species (see International Publication 2011 / 012867 for further details).

[0521] To develop this method for large-scale applications, it was necessary to eliminate two solvent evaporation steps: the removal of MeOH from the activated silicon nanoparticle suspension and the removal of MeOH from the dissolved lipids used to produce the thin film. The first of these steps was eliminated by directly mixing the activated silicon nanoparticle suspension with a trehalose / glycine aqueous solution, followed by lipid thin film hydration. Such a method is indicated as "Method 2" and illustrated in Figure 71A. This method resulted in an initial hybrid lipid nanoparticle suspension with noticeably more precipitate than before (see Figure 71B, lower image; upper image shows Method 1), but this difference was eliminated after extrusion by producing particles with a low polydispersity index (PDI) and a slightly higher zeta potential (see Figure 71C). Preferably, the finished hybrid lipid nanoparticles should have a zeta potential of +50 to +70 mV, which indicates good to excellent colloidal stability and a minimal tendency toward aggregation (due to charge repulsion). It can be seen that eliminating the first evaporation step does not hinder the formation of hybrid lipid particles with acceptable zeta potential and polydispersity.

[0522] To demonstrate that lipid thin-film evaporation and subsequent hydration steps could be eliminated without disadvantage, a direct, slow injection of a methanol-lipid solution into an aqueous silicon nanoparticle suspension was attempted (labeled "Method 3" and illustrated in Figure 72A). This method resulted in reduced precipitation (Figure 72B), and the resulting hybrid lipid particle sample exhibited comparable average size (102 ± 0.17 nm), PDI (0.135 ± 0.01), and zeta potential (56.0 ± 0.81 mV) to the previous batch (Figure 72B). This established the feasibility of a further expandable direct injection mixing technique. To ensure reproducibility, the inventors introduced two in-process inspections (IPCs) into the method (Figure 72A): a visual inspection of complete lipid dissolution before mixing (IPC 1), and specific reference ranges for dynamic light scattering (DLS) parameters after extrusion: namely, mean hydrodynamic size of 75–140 nm, PDI of 0.1–0.2, and zeta potential of +50–+70 mV (IPC 2). If necessary, the 0.1 μm extrusion process is repeated according to IPC 2.

[0523] Other method adjustments were investigated before scaling up (Figure 72C, labeled "Methods 4-6"). Most notably, an additional inflow extrusion step (0.8 μm pore size) was attempted to remove large silicon nanoparticle aggregates, resulting in a good reduction in the average size of the final hybrid lipid particles (Figure 72D, "Method 4"). Conversely (Figure 72C, "Method 5", Figure 78), adding a trehalose / glycine solution to the activated methanol suspension of silicon nanoparticles did not affect the results. Including a 0.8 μm pre-filtration step after initial hybrid lipid particle formation reduced aggregate accumulation on the extruded membrane (Figure 72E, "Method 6"), significantly reduced the operating pressure on the 0.8 μm membrane (Figure 79), and essentially yielded sshLNPs with altered properties (Figure 72F).

[0524] For the commercial manufacture of pharmaceuticals, organic solvents (i.e., MeOH) must preferably be removed from the final product so that any residual levels are below regulatory limits (e.g., <3,000 ppm for European medicines; European Medicines Agency, 2022). For this purpose, tangential flow filtration (TFF) may be used, as illustrated in Figure 73A. For initial feasibility testing (up to 100 mL scale), 100 kDa MWCO and 0.02 m 2 A TFF cassette with a membrane surface area was used. Under these conditions, trehalose and glycine are also removed from the solution, so a second pump was used to inject a diafiltration aqueous solution of 0.1 mg / mL trehalose and 0.05 mg / mL glycine at a constant flow rate matching the flow rate of filtrate production. Diafiltration of 10 volumes of this solution reduced the MeOH content from approximately 20% v / v after extrusion to well within acceptable regulatory limits. 1The concentration was reduced to approximately 200 ppm based on 1H NMR while simultaneously maintaining the desired level of trehalose / glycine. The final process included an initial out-of-time filtration step to generate a completed hybrid lipid nanoparticle suspension with the desired silicon concentration (see below) and low residual MeOH level (approximately 450 ppm; Figure 73B) by reducing the input volume by 50% before diafiltration.

[0525] After TFF, the DLS properties of the extruded sshLNPs were largely maintained. The degree of lipid recovery was measured using HPLC (Figure 80). The results showed a significant decrease in lipid content, with recovery rates for each lipid dropping to approximately 10% when diafiltration alone was applied. However, this effect was significantly mitigated by employing a modified TFF procedure combining both ultrafiltration and diafiltration. The silicon content of the final hybrid lipid nanoparticle samples was also measured using inductively coupled plasma emission spectroscopy (ICP-OES). In batches subjected to TFF using diafiltration alone, the silicon content was 1.19 mg / L compared to the target value of 2.1 mg / L based on products prepared using the original lipid thin-film hydration method (Figure 71A). The analytical methods described above guided further optimization efforts toward scaling up. These can also be used for appropriate quality control at different stages during commercial production. To determine residual MeOH content... 1 As an alternative to the 1H NMR method, a headspace GC assay may be used (Maurizi et al., 2023).

[0526] Example 18 - Large-scale test operation The first large-scale test run (illustrated in Figure 74A) incorporated three inflow extrusion steps (i.e., a scaled-up version of Method 4 in Example 17 using the modifications introduced in Method 5 of Example 17), but the TFF protocol was omitted at this stage. Here, to limit the volume of MeOH in the system, the lipid stock solution was prepared at 10 mg / mL instead of 5 mg / mL as before. At the 1 L scale, the main problem encountered was the accumulation of even larger insoluble aggregates on the extruded membrane, despite the use of a larger membrane (47 mm in diameter compared to the previous 25 mm). This resulted in higher operating pressure during extrusion (Figure 74C), but the DLS properties of the sample were not adversely affected (Figure 74D). Production of 2 L batches was demonstrated by using a two-step TFF protocol (Figure 75A), in which case an even larger membrane surface area (0.5 m²) was required. 2 A cassette with the following characteristics was used. As previously noted, the main problem encountered was during extrusion, when the membrane became saturated with insoluble aggregates (Figure 75B), and the membrane had to be changed midway through the procedure due to pressure fluctuations. The extruded sample showed suboptimal visual and DLS characteristics (Figure 75C), but further close process monitoring revealed that the average particle size gradually increased with the extrusion volume (Figure 75D), exceeding the reference range only after approximately 1.5 L of the total 4 L volume had been processed. After TFF, residual MeOH was below the detection limit. When a 100 mL aliquot of the pre-TFF sample was subjected to an additional extrusion step through a 3 × 0.1 μm membrane, the average particle size decreased from 129 ± 0.44 nm to 105 ± 0.38 nm, and the PDI decreased from 0.17 ± 0.02 to 0.11 ± 0.01 (both well within the reference range), confirming that extrusion membrane overload is a problem during processing of large batches, and demonstrating that such a problem can be easily mitigated by using additional extrusion.

[0527] Considering these results, the inventors modified the procedure to reduce precipitation during sshLNP formation. In small-scale test runs without TFF, when the activated suspension of SiNPs was filtered before mixing with the trehalose / glycine solution (Figure 7A, Method 7), aggregates were well removed and no significant deposition of insoluble material was observed on the extruded membrane (Figure 7B). The operating pressure during extrusion was even lower (Figure 7C), and the DLS properties of the resulting sshLNPs were well within the reference range (Figure 7D). To implement this method on a larger scale, two sets of larger 47 mm extruded membranes were used in parallel (Figures 76A and 81), which resulted in successful production of 1 L batches of sshLNPs using an optimized protocol (Figure 76E). In the current embodiment, a final 0.2 μm filtration step is further used to reduce bioburden.

[0528] Following the development of kilogram-scale production protocols, it was also essential to establish equivalent properties between sshLNPs produced by the original lipid thin-film hydration technique (Method 2) and the optimized large-scale process (Method 7). The inventors found that while scaling up resulted in slightly smaller particles on average (Figure 77A), the PDI and zeta potentials were essentially identical (Figures 77A and 77B). Both batches exhibited superior mRNA encapsulation efficiency as determined by the RiboGreen assay (Figure 77C). In cell transfection experiments, sshLNPs produced by either pathway efficiently introduced mRNA encoding firefly luciferase (fLuc) into HEK293 cells, resulting in significantly higher transgene expression after 24 hours compared to lipofectamine 3000 as a reference (Figure 77D). Therefore, sshLNPs produced by the large-scale methods developed herein are physically and functionally equivalent to those produced by the original dehydration-rehydration process.

[0529] Particular consideration of Examples 17 and 18 To realize the full potential of lipid nanoparticles, clinical transitions require reliable manufacturing processes capable of producing particles with relevant properties on a multi-kilogram scale. Examples 17 and 18 demonstrate such processes.

[0530] The convergent manufacturing method generates platform intermediate (i.e., "empty") hybrid silicon nanoparticles suitable for subsequent nucleic acid loading before filling / finishing operations, which can be considerably separated in time and distance from the original manufacturing process. This ability is conferred by the presence of silicon nanoparticles, some of which remain accessible on the surface and stabilize the lipid membrane. Their presence mitigates the well-known tendency for aggregation or fusion of LNPs over time (i.e., Ostwald maturation), which is a limiting factor in the shelf life of current formulations (Gindy et al., 2014; Nag et al., 2022). The incorporation of SiNPs also allows for the desired omission of the cholesterol component of conventional LNPs, which accounts for approximately 40 mol% of the lipid content in commercially available RNA-LNP products (Sun et al., 2023). In the hybrid lipid particles of the present invention, the interaction between phospholipids and silicon results in enhanced structural integrity and reduces the risk of rupture during extrusion. Interestingly, this also appears to produce final particles with an imperfectly sealed lipid bilayer that remains accessible internally for nucleic acid loading.

[0531] Another advantage of silicon components is that they allow for greater flexibility in formulations, meaning that the lipid composition can be easily modified without significantly altering the manufacturing process. For example, hybrid lipid particles can be formulated without using pegylated lipids, which are essential components of conventional LNPs but are sometimes associated with safety and efficacy concerns. It is well known that pegylation can induce anti-PEG antibodies and potentially trigger premature release of RNA payloads via antibody binding (Shi et al., 2022; Senti et al., 2022). Therefore, non-pegylated hybrid lipid particles may be a clinically useful option for individuals with a history of PEG hypersensitivity reactions (Chen et al., 2023; Ibrahim et al., 2022).

[0532] Furthermore, the compatibility of the hybrid lipid particles of the present invention with point-of-care RNA loading minimizes the degradation of nucleic acid drugs while providing opportunities for late-stage customization and individualization of therapeutic formulations. In fact, this “post-loading” concept has recently been highlighted as a potential strategy to overcome remaining challenges in the field of RNA medicine (Li et al., 2023). Low delivery efficiency, short shelf life, and high market barriers to entry (i.e., the cost of developing and manufacturing optimized LNP formulations) are particularly recognized as current limitations hindering the growth of this field (Verma et al., 2023), and sshLNP can potentially address all of these challenges.

[0533] Since 2020, addressing the cold distribution requirements and limited shelf life faced by COVID-19 vaccines has been a priority. Notable recent innovations have resulted in improvements to lyophilized mRNA-LNP formulations that maintain stability and transfection efficiency for several weeks at 4°C (or even room temperature) (Ai et al., 2023; Meulewaeter et al., 2023; Shirane et al., 2023). However, the commercial scalability of these workflows has not yet been established, and they do not solve the problem of needing to incorporate the RNA component early in the production process. Therefore, in some cases, extensive optimization of LNP formulations may still be necessary. As an example, identifying the optimal formulation for patisiran (the first FDA-approved RNA therapeutic delivered by LNP) required screening of more than 300 ionizable lipids alone (Kulkarni et al., 2019). In contrast, our own studies using ADO2 mice required only an initial screening of seven sshLNP formulations to identify promising lead candidates for siRNA delivery to bone (Maurizi et al., 2022). Therefore, the Bio-Courier platform shows promise in accelerating the clinical transition of RNA therapeutics.

[0534] Based on this, the stabilizing effect of the hybrid lipid particles of the present invention is also potent enough to eliminate the need for chemical modification of RNA, as demonstrated in the aforementioned in vivo studies using unmodified siRNA (Baran-Rachwalska et al., 2020; Maurizi et al., 2023). In contrast, all current FDA-approved oligonucleotide therapeutics include chemical modifications (Bost et al., 2021) involving five siRNA-based products (Friedrich & Aigner, 2022). The use of unmodified (or minimally modified) RNA and sshLNP is another factor that could further accelerate the transition by reducing the significant time and cost required to develop current constructs, which often necessitate a complex series of chemical modifications.

[0535] In short, sshLNPs show great potential as a platform technology for improving RNA delivery, but the methods previously reported for their generation are not readily applicable to scaling up.

[0536] References Mikhail (1961), "Densities and Viscosities of Methanol-Water Mixtures," (Journal of Chemical & Engineering Data, vol. 6, no. 4, p. 533-537, 1961. Wang, C., Zhang, Y., and Dong, Y. (2021).Lipid nanoparticle-mRNA formulations for therapeutic applications.Acc.Chem. Res. 54, 4283-4293. Curreri, A., Sankholkar, D., Mitragotri, S., and Zhao, Z. (2023).RNA therapeutics in the clinic.Bioeng.Transl.Med. 8, e10374. Moss, KH, Popova, P., Hadrup SR, Astakhova, K., and Taskova, M. (2019).Lipid nanoparticles for delivery of therapeutic RNA oligonucleotides.Mol. Pharm. 16, 2265-2277. Verma, M., Ozer, I., Xie, W., Gallagher, R., Teixeira, A., and Choy, M. (2023).The landscape for lipid-nanoparticle-based genomic medicines.Nat. Rev. Drug Discov.22, 349-350.

[0537] Schoenmaker, L., Witzigmann, D., Kulkarni, J.A., Verbeke, R., Kersten, G., Jiskoot, W., and Crommelin, D.J.A.(2021). mRNA-lipid nanoparticle COVID-19 vaccines:Structure and stability.Int. J.Pharm. 601, 120586. Bege, M., and Borbas, A.(2022).The medicinal chemistry of artificial nucleic acids and therapeutic oligonucleotides.Pharmaceuticals (Basel) 15, 909. Egli, M., and Manoharan, M.(2019).Re-engineering RNA molecules into therapeutic agents.Acc.Chem. Res. 52, 1036-1047. Nag, K., Sarker, M.E.H., Kumar, S., Khan, H., Chakraborty, S., Islam, M.J., Baray, J.C., Khan, M.K., Mahmud, A., Barman, U., et al.(2022).DoE-derived continuous and robust process for manufacturing of pharmaceutical-grade wide-range LNPs for RNA-vaccine / drug delivery.Sci. Rep. 12, 9394. Cameau, E., Zhang, P., Ip, S., Mathiasson, L., and Stenklo, K.(2022).Process & analytical insights for GMP manufacturing of mRNA lipid nanoparticles.Cell Gene Ther. Insights 8, 621-635. Catignol, P., and Lim, H.(2022).Overcoming bottlenecks in mRNA manufacturing (Pharma’s Almanac). https: / / www.pharmasalmanac.com / articles / overcoming-bottlenecks-in-mrna-manufacturing.

[0538] Daniel, S., Kis, Z., Kontoravdi, C., and Shah, N.(2022).Quality by Design for enabling RNA platform production processes.Trends Biotechnol.40, 1213-1228. Andoh, E.A., and Yu, H.(2023).A two-stage decision-support approach for improving sustainable last-mile cold chain logistics operations of COVID-19 vaccines.Ann. Oper.Res. 328, 75-105. Khairi, L.N.H.M., Fahrni, M.L., and Lazzarino, A.I.(2022).The race for global equitable access to COVID-19 vaccines.Vaccines (Basel) 10, 1306. Saffie-Siebert, S., Gibson, A., and Dehsorkhi, A.(2023).Bio-Courier:A new delivery technology to increase the global accessibility of RNA therapeutics (Manufacturing Chemist). https: / / www.manufacturingchemist.com / news / article_page / Bio-Courier_a_new_delivery_technology_to_increase_the_global_accessibility_of_RNA_therapeutics / 212116. Baran-Rachwalska, P., Torabi-Pour, N., Sutera, F.M., Ahmed, M., Thomas, K., Nesbit, M.A., Welsh, M., Moore, C.B.T., and Saffie-Siebert, S.R.(2020).Topical siRNA delivery to the cornea and anterior eye by hybrid silicon-lipid nanoparticles.J.Control.Release 326, 192-202. Maurizi, A., Patrizii, P., Teti, A., Sutera, F.M., Baran-Rachwalska, P., Burns, C., Nandi, U., Welsh, M., Torabi-Pour, N., et al.(2023).Novel hybrid silicon-lipid nanoparticles deliver a siRNA to cure autosomal dominant osteopetrosis in mice.Implications for gene therapy in humans.Mol. Ther. Nucleic Acids 33, 925-937.

[0539] Saffie-Siebert, R.S., Ahmed, M., and Sutera, F.(2023).Compositions comprising doped silicon particles, and related methods (WIPO Patent No. WO 2023 / 002222 A1). Saffie-Siebert, R.S., and Sutera, F.(2023).Protection of biological species from degradation (WIPO Patent No. WO 2023 / 002224 A1). Hou, X., Zaks, T., Langer, R., and Dong, Y.(2021).Lipid nanoparticles for mRNA delivery.Nat. Rev. Mater.6, 1078-1094. Kulkarni, J.A., Witzigmann, D., Chen, S., Cullis, P.R., and van der Meel, R.(2019).Lipid nanoparticle technology for clinical translation of siRNA therapeutics.Acc.Chem. Res. 9, 2435-2444. Maurizi, A., Patrizii, P., Teti, A., Sutera, F.M., Hadainamrei, R., Dehsorkhi, A., and Saffie-Siebert, S.(2022).Development of a potential clinical product for treating CLCN7-dependent autosomal dominant osteopetrosis type 2 (ADO2).Bone Rep. 16, 101199. Li, D., Liu, C., Li, Y., Tenchov, R., Sasso, J.M., Zhang, D., Li, D., Zou, L., Wang, X., and Zhou, Q.(2023).Messenger RNA-based therapeutics and vaccines:What’s beyond COVID-19?ACS Pharmacol.Transl.Sci. 6, 943-969.

[0540] Ai, L., Li, Y., Zhou, L., Yao, W., Zhang, H., Hu, Z., Han, J., Wang, W., Wu, J., Xu, P., et al.(2023).Lyophilized mRNA-lipid nanoparticle vaccines with long-term stability and high antigenicity against SARS-CoV-2.Cell Discov.9, 9. Meulewaeter, S., Nuytten, G., Cheng, M.H.Y., De Smedt, S.C., Cullis, P.R., De Beer, T., Lentacker, I., and Verbeke, R.(2023).Continuous freeze-drying of messenger RNA lipid nanoparticles enables storage at higher temperatures.J.Control.Release 357, 149-160. Shirane, D., Tanaka, H., Sakurai, Y., Taneichi, S., Nakai, Y., Tange, K., Ishii, I., and Akita, H.(2023).Development of an alcohol dilution-lyophilization method for the preparation of mRNA-LNPs with improved storage stability.Pharmaceutics 15, 1819. Gindy, M.E., Feuston, B., Glass, A., Arrington, L., Haas, R.M., Schariter, J., and Stirdivant, S.M.(2014).Stabilization of Ostwald ripening in low molecular weight amino lipid nanoparticles for systemic delivery of siRNA therapeutics.Mol. Pharm. 11, 4143-4153. Sun, H., Zhang, Y., Wang, G., Yang, W., and Xu, Y.(2023). mRNA-based therapeutics in cancer treatment.Pharmaceutics 15, 622. Bost, J.P., Barriga, H., Holme, M.N., Gallud, A., Maugeri, M., Gupta, D., Lehto, T., Valadi, H., Esbjorner, E.K., et al.(2021).Delivery of oligonucleotide therapeutics:Chemical modifications, lipid nanoparticles, and extracellular vesicles.ACS Nano 15, 13993-14021.

[0541] Friedrich, M., and Aigner, A.(2022).Therapeutic siRNA:State-of-the-art and future perspectives.BioDrugs 36, 549-571. Gottlieb, H.E., Kotlyar, V., and Nudelman, A.(1997).NMR chemical shifts of common laboratory solvents as trace impurities.J.Org. Chem. 62, 7512-7515. Shi, D., Beasock, D., Fessler, A., Szebeni, J., Ljubimova, J.Y., Afonin, K.A., and Dobrovolskaia, M.A.(2022).To PEGylate or not to PEGylate:Immunological properties of nanomedicine’s most popular component, polyethylene glycol and its alternatives.Adv. Drug.Deliv.Rev. 180, 114079. Senti, M.E., de Jongh, C.A., Dijkxhoorn, K., Verhoef, J.J.F., Szebeni, J., Storm, G., Hack, C.E., Schiffelers, R.M., Fens, M.H., and Boross, P.(2022).Anti-PEG antibodies compromise the integrity of PEGylated lipid-based nanoparticles via complement.J.Control.Release 341, 475-486. Chen, W.-A., Chang, D.-Y., Chen, B.-M., Lin, Y.-C., Barenholz, Y., and Roffler, S.R.(2023).Antibodies against poly(ethylene glycol) activate innate immune cells and induce hypersensitivity reactions to PEGylated nanomedicines.ACS Nano 17, 5757-5772. Ibrahim, M., Ramadan, E., Elsadek, M.E., Emam, S.E., Shimizu, T., Ando, H., Ishima, Y., Elgarhy, O.H., Sarhan, H.A., Hussein, A.K., et al.(2022).Polyethylene glycol (PEG):The nature, immunogenicity, and role in the hypersensitivity of PEGylated products.J.Control.Release 351, 215-230.

Claims

1. A. A step of mixing one or more lipids in a solvent or solvent mixture with a suspension of inorganic material particles in a solvent or solvent mixture in an aqueous medium, then, B. The process of passing the mixture obtained from step A through the pores of an extrusion membrane. A method for producing an aqueous suspension of hybrid lipid particles, comprising: The hybrid lipid particles have an average diameter at least twice the average diameter of the inorganic material particles. The aforementioned method.

2. The method according to claim 1, wherein the particles of the inorganic material include hydrolyzable silicon, or particles made of hydrolyzable silicon, or particles made of said silicon.

3. The method according to claim 1 or claim 2, wherein step B and optionally step A are performed at a temperature of 50°C to 70°C.

4. The method according to claim 1, claim 2, or claim 3, wherein the inorganic material particles are configured to impart tensile strength to one or more lipid membranes in the hybrid lipid particles.

5. The method according to any one of claims 1 to 4, wherein the mixture is passed through the pores of an extruded film at least three times, the pore size cutoff of the film is 0.05 μm to 1 μm in diameter, and optionally, the passage does not substantially change the size, PDI, and charge of the particles.

6. The method according to any one of claims 1 to 5, wherein after step B, there is an additional step C of purifying and / or sterilizing the suspension by tangential flow filtration, wherein step C optionally removes the activating solvent and / or the solvent of step A, and the tangential flow filtration optionally uses a diafiltration solution containing an amino acid such as glycine and a disaccharide such as trehalose.

7. The method according to any one of claims 1 to 6, wherein an additional step D is performed after step B (and step C, if present), in which the micellar lipid particles are brought into contact with an active compound, in particular a pharmaceutically active ingredient (API).

8. The method according to claim 7, wherein step D is performed at a temperature below 20°C (for example, below 10°C, below 5°C, or below 3°C), and the API is an RNA molecule, particularly mRNA.

9. The method according to any one of claims 1 to 8, further comprising freeze-drying the suspension after all the steps described in the claim, or between steps C and D, to produce a freeze-dried powder containing micellar lipid particles.

10. The method according to any one of claims 1 to 9, wherein the one or more lipids include at least one cationic lipid and at least one polar lipid.

11. The method according to claim 10, wherein the one or more lipids consist of at least DOTAP, DOPE, and mPEG2000-DSPE, and these are optionally in a molecular ratio of 2-7:2-7:1-2.

12. The method according to any one of claims 1 to 11, wherein the particles of the inorganic material are hydrolyzable silicon particles activated by exposure to an alcohol such as methanol, ethanol, or benzyl alcohol.

13. The method according to any one of claims 1 to 12, which does not include the step of solvent evaporation.

14. The method according to any one of claims 1 to 13, comprising one or more quality control steps selected from measuring dynamic light scattering (DLS) parameters, measuring mean hydrodynamic size parameters, measuring polydispersity (PDI), and / or measuring zeta potential, optionally comparing the measurement to product specifications, and optionally repeating extrusion step B if the measurement does not meet the product specifications.

15. An aqueous suspension of hybrid lipid particles having an average diameter of 50 to 150 nm, wherein the hybrid lipid particles comprise a mixture of one or more cationic lipids or ionizable lipids, one or more further lipids selected from neutral lipids and polar lipids, and optionally one or more additional lipid components, wherein the hybrid lipid particles comprise inorganic material particles (e.g., hydrolyzable silicon particles) having an average diameter of 1 / 2 or more of the average diameter of the hybrid lipid particles, and the weight ratio of the inorganic material particles to the lipids is 1:2 to 1:100 (preferably 1:10 to 1:100, more preferably 1:20 to 1:10).

16. The aqueous suspension of hybrid lipid particles according to claim 15, wherein the aqueous suspension of the hybrid lipid particles has an average diameter of 80 to 200 nm, and the inorganic material particles have an average diameter of 2 to 20 nm and are hydrolyzable silicon particles present in one or more aggregated chains of hydrolyzable lipid particles extending from the outside of the hybrid lipid particles to the inside of the hybrid lipid particles.

17. An aqueous suspension of hybrid lipid particles according to claim 15 or claim 16, further comprising one or more active compounds, for example, one or more active pharmaceutical ingredients (APIs).

18. An aqueous suspension of liposome lipid particles having an average diameter of 50 to 150 nm, wherein the liposome lipid particles comprise a mixture of one or more cationic lipids or ionizable lipids, one or more further lipids selected from neutral lipids and polar lipids, and optionally one or more additional lipid components, wherein the liposome lipid particles comprise inorganic material particles (e.g., hydrolyzable silicon particles) having an average diameter of 1 / 2 or more of the average diameter of the liposome lipid particles, the weight ratio of the inorganic material particles to lipids is 1:2 to 1:100 (preferably 1:10 to 1:100, more preferably 1:20 to 1:10), and the aqueous suspension further comprises one or more active compounds, such as one or more active pharmaceutical ingredients (APIs), in which at least a portion is encapsulated inside the liposome particles.

19. A method for producing liposome lipid particles containing an active compound (e.g., API), comprising performing the method according to any one of claims 1 to 14, and then contacting the active compound with the hybrid lipid particles in an aqueous suspension under conditions such that the hybrid lipid particles are converted into liposome lipid particles.

20. A freeze-dried powder of hybrid lipid particles or liposomal lipid particles having an average diameter of 50 to 150 nm, wherein the hybrid lipid particles or liposomal lipid particles comprise a mixture of one or more cationic lipids or ionizable lipids, one or more further lipids selected from neutral lipids and polar lipids, and optionally one or more additional lipid components, wherein the hybrid lipid particles or liposomal lipid particles comprise inorganic material particles (e.g., hydrolyzable silicon particles) having an average diameter of 1 / 2 or more of the average diameter of the liposomal lipid particles, the weight ratio of the inorganic material particles to lipids is 1:2 to 1:100 (preferably 1:10 to 1:100, more preferably 1:20 to 1:10), and the freeze-dried powder further comprises one or more active compounds, such as one or more active pharmaceutical ingredients (APIs), in which at least a portion is encapsulated inside the hybrid lipid particles or liposomal lipid particles.

21. A freeze-dried powder of hybrid lipid particles having an average diameter of 50 to 150 nm, wherein the hybrid lipid particles comprise a mixture of one or more cationic lipids or ionizable lipids, one or more further lipids selected from neutral lipids and polar lipids, and optionally one or more additional lipid components, wherein the hybrid lipid particles also comprise inorganic material particles (e.g., hydrolyzable silicon particles) having an average diameter of 1 / 2 or more of the average diameter of the hybrid lipid particles, and the weight ratio of the inorganic material particles to the lipids is 1:2 to 1:100 (preferably 1:10 to 1:100, more preferably 1:20 to 1:10).

22. An aqueous suspension of hybrid lipid particles according to claim 15, claim 16, or claim 17, wherein the active compound (e.g., API) is a nucleic acid, such as mRNA, or a freeze-dried powder according to claim 20, claim 21, or claim 22.

23. A pharmaceutical composition comprising an aqueous suspension of hybrid lipid particles according to claim 15, claim 16, or claim 17, an aqueous suspension of liposome lipid particles according to claim 18, or a lyophilized powder according to claim 20 or claim 21.

24. The pharmaceutical composition according to claim 23, which is a vaccine composition optionally filled into a vaccine delivery device such as a syringe.

25. A pharmaceutical composition according to claim 23 or claim 24 for use as a pharmaceutical.

26. A pharmaceutical composition for use as a pharmaceutical according to claim 25 for treating or preventing a target disease or disorder.

27. A pharmaceutical composition for use as a vaccine against infectious diseases, according to claim 25.

28. A method for treating or preventing a disease or disorder of a subject requiring such treatment, comprising administering a pharmaceutical composition according to any one of claims 23 to 27 to the subject.

29. A method for providing a preventive vaccine to a subject, comprising administering a pharmaceutical composition according to any one of claims 23 to 27 to the subject.

30. (a) (i) Mixing one or more lipids in a solvent or solvent mixture with a suspension of inorganic material particles in a solvent or solvent mixture in an aqueous medium. (ii) Passing the mixture obtained from step (i) through the pores of the extruded film, and (iii) Optionally, purify and / or sterilize the suspension by tangential flow filtration. To obtain an aqueous suspension of hybrid lipid particles manufactured by, and (b) Contacting the hybrid lipid particles with an active compound, particularly a pharmaceutically active ingredient (API). A method for producing an aqueous suspension of lipid particles containing a pharmaceutically active ingredient (API).

31. (a) (i) Mixing one or more lipids in a solvent or solvent mixture with a suspension of inorganic material particles in a solvent or solvent mixture in an aqueous medium. (ii) Passing the mixture obtained from step (i) through the pores of the extruded film. To obtain an aqueous suspension of hybrid lipid particles manufactured by, and (b) Purify and / or sterilize the suspension by tangential flow filtration. A method for producing an aqueous suspension of hybrid lipid particles containing a pharmaceutically active ingredient (API).

32. (a) (i) Mixing one or more lipids in a solvent or solvent mixture with a suspension of inorganic material particles in a solvent or solvent mixture in an aqueous medium. (ii) Passing the mixture obtained from step (i) through the pores of the extruded film, and (iii) Optionally, purify and / or sterilize the suspension by tangential flow filtration. To obtain an aqueous suspension of hybrid lipid particles manufactured by, and (b) Contacting the hybrid lipid particles with an active compound, particularly a pharmaceutically active ingredient (API). An aqueous suspension of lipid particles containing a pharmaceutically active ingredient (API), produced by a method comprising [the specified method].

33. (a) (i) Mixing one or more lipids in a solvent or solvent mixture with a suspension of inorganic material particles in a solvent or solvent mixture in an aqueous medium. (ii) Passing the mixture obtained from step (i) through the pores of the extruded film. To obtain an aqueous suspension of hybrid lipid particles manufactured by, and (b) Purify and / or sterilize the suspension by tangential flow filtration. An aqueous suspension of hybrid lipid particles containing a pharmaceutically active ingredient (API), produced by a method comprising [the specified method].

Images