Protection of biological species from degradation

Abstract

A method of preparing an injectable or oral storage-stable formulation comprising contacting a biological species with a delivery system comprising biocompatible solid particles to form a complex; then, optionally, lyophilizing the complex to form a powder; and then dispersing the complex in a biodegradable gel material to form the formulation comprising the complex embedded in the biodegradable gel material. Also relates to products, methods, and medical uses.

Description

[Technical field]

[0001] The present invention relates to methods and compositions, particularly injectable or oral compositions, for protecting biological species, such as nucleic acids, antigens, and vaccines, from degradation. Such methods and compositions are particularly useful, but not limited to, in the field of pharmacology. [Background technology]

[0002] There is a need for improved non-medicinal excipients for bioactive agents, particularly in the field of injectable or oral formulations. The provision of suitable non-medicinal excipients is required for the full translation of advances in biomedical research into effective, safe and cost-effective treatments. In particular, there is a need to stabilize biological species to reduce degradation during storage. There is also a need for biocompatible non-medicinal excipients for oral or injectable compositions that allow for oral or injection administration without the intervening steps of (i) extraction of the active biological species from the composition and (ii) reconstitution.

[0003] Biological species, such as nucleic acids, antigens, and vaccines, can be effective agents for use in the treatment or prevention of various medical conditions. However, unlike small chemicals, biological species can be difficult to store and often require storage at low temperatures (e.g., −20° C.) or cryogenic temperatures (e.g., −70° C.). This makes it difficult to store and transport many pharmaceutical products, including biological species, to patients, especially in developing countries.

[0004] One reason for the instability of pharmaceutical products containing biological species is that the manufacturing process of biological species, especially biological species containing nucleic acids such as mRNA, pDNA, saRNA, shRNA, and siRNA, results in enzyme residues present in the product. For example, the use of microbial fermentation expression systems to produce biological species results in crude products containing enzyme residues that are difficult to completely remove. Even with significant downstream processing, some low-level contaminants remain that can degrade the biological species. In particular, mRNA is susceptible to enzymatic and chemical degradation. Enzyme activity can be slowed down by low temperature and / or lyophilization of the biological species, but each of these solutions has its drawbacks. Furthermore, even if the biological species can be made storage stable by low temperature or removal of water from the storage medium, instability of the resulting product upon conversion of the stored preparation into a formulation suitable for administration remains a problem. Upon reconstitution of a lyophilized preparation or thawing of a frozen preparation, enzyme residue contaminants that were dormant during storage are reactivated, resulting in a reduced shelf life of the final formulation. Additionally, the presence of water and free radicals in biological systems, such as mRNA-lipid nanoparticle vaccines, can lead to hydrolysis of the phosphate backbone or clipping of the phosphates, rendering the nucleic acid unreadable.

[0005] Several mRNA vaccines against SARS-CoV-2, including the Pfizer BioNTech vaccine BNT162b2 ("Comirnaty") and the Moderna CX-024414 vaccine, require cold chain storage and transportation. This limits access to the vaccine in low-income countries and adds cost and logistical complexity in all markets. Cold chain requirements also limit the amount of vaccine that can be manufactured, stored, and transported, thus limiting vaccine supply and often preventing effective deployment of the vaccine in the short time frame that may be critical to curbing the spread of infection. It may be advantageous if the vaccine can be stored and transported at standard refrigerator temperatures (approximately -5°C) or room temperature (approximately 20°C). It may also be beneficial if the vaccine can withstand higher temperatures (e.g., 30°C, 40°C, or 50°C) for storage, or at least in the short term for transportation and distribution purposes.

[0006] Maintaining RNA stability in injectable compositions (e.g., in vaccine compositions) by low temperatures, and also posing logistical challenges, has the technical limitation that the RNA must be thawed before injection and must remain stable in the body for a sufficient time after injection to show sufficient biological activity. This may require stability to be maintained during transit in the body and / or escape from endosomal compartments. In vivo stability must also be maintained for a sufficient time for sufficient translation into protein to occur and / or for transport to the location in the body where translation into protein occurs.

[0007] There is a need for a preparation capable of stabilizing biological species, such as mRNA vaccines, that allows for long-term storage at standard refrigerator temperatures, room temperature, or above. Similarly, there is a need for stabilizing preparations for delivery of biological species to cells in vivo, such that the biological species payload is released only upon internalization into the desired cells. Preparations for stabilizing biological species ideally contain only pharma-ceutically acceptable non-active ingredients, so that the preparation can be administered, particularly orally or by injection, without the need to extract the active biological species from the preparation and then reconstitute it. For example, preparations used to stabilize biological species can advantageously be administered directly, particularly orally or by injection, or can be administered, particularly orally or by injection, after the addition of additional drugs or non-active ingredients, for example diluted with a solvent, or after a simple reconstitution step.

[0008] Gel materials, especially hydrogels, have been used previously as adhesives in various biomedical applications. For example, hydrogels are used to seal wounds in tissues and organs, to adhere bioelectronics to the body, or to adhere drug delivery devices, such as patches, to the skin. Numerous biocompatible, biodegradable hydrogels have been developed for such purposes, as described by Bovone et al. in ACS Biomater. Sci. Eng. April 2021, “Engineering Hydrogel Adhesion for Biomedical Applications via Chemical Design of the Junction”. However, the use of biocompatible gels to stabilize biological species complexed with biocompatible solid particles, especially in injectable or oral preparations, has not been suggested so far. Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention is based on the realization that when a biological species is contacted with a delivery system comprising biocompatible solid particles to form a complex, and then the complex is embedded in a biodegradable gel material, the degradation of the delivery system, and therefore the degradation of the biological species, can be stopped or at least delayed, especially when the particles comprise hydrolyzable silicon. Embedding the complex in a biodegradable gel material can therefore be used to stabilize the complex. A stabilized complex can better protect the biological species contained therein from degradation during storage or in vivo.

[0010] Additionally, the delivery system may also function as a biocompatible non-active ingredient for oral or injectable administration of a biological species, allowing for oral or injectable administration without the intervening steps of (i) extraction of the active biological species from the composition and (ii) reconstitution. [Means for solving the problem]

[0011] Thus, according to a first aspect of the present invention there is provided a method for preparing a preparation, advantageously an oral or injectable preparation, and advantageously a storage stable preparation, comprising: (i) contacting a biological species with a delivery system comprising biocompatible solid particles to form a complex; then (ii) optionally lyophilizing said complex to form a powder; and then (iii) dispersing said complex in a biodegradable gel material to form a preparation comprising said complex embedded in said biodegradable gel material.

[0012] According to a second aspect of the present invention, there is provided a method of protecting a biological species from degradation comprising: (i) contacting the biological species with a delivery system comprising biocompatible solid particles to form a complex; then (ii) optionally lyophilizing the complex to form a powder; and then (iii) dispersing the complex in a biodegradable gel material to form a preparation, advantageously an oral or injectable preparation, comprising the complex embedded in the biodegradable gel material.

[0013] The method of the second aspect of the invention may further comprise (iv) optionally a step of storing said preparation, and then (v) a step of preparing a medicament from said preparation for administration, preferably for administration orally or by injection, preferably without any intervening step of extraction and reformulation of the active biological species from the composition. Thus, in a preferred embodiment, the step (v) of preparing said medicament does not include any extraction step, such that the medicament contains all the components of the preparation. Thus, the preparation step may, for example, simply be or include a dilution and / or reconstitution step.

[0014] According to a third aspect of the present invention, there is provided a preparation, preferably an oral or injectable preparation, comprising a biodegradable gel matrix embedded with a biological species complexed with a delivery system comprising solid biocompatible particles. The preparation of the third aspect of the present invention is advantageously a storage-stable preparation. The preparation of the third aspect of the present invention advantageously comprises a lipid component comprising a cationic lipid and / or an ionizable lipid, and optionally a non-reducing disaccharide.

[0015] The preparation of the third aspect of the invention (preferably an oral preparation or an injectable preparation) may be prepared according to the first aspect of the invention. The preparation of the third aspect of the invention is typically a gel or solid. The preparation of the third aspect of the invention may be a pharmaceutical composition suitable for direct administration. For example, the preparation may be administered topically, e.g. a gel for administration to the skin or a membrane or surface of the body, or the preparation may be a solid oral formulation or an injectable formulation (e.g. an injectable solution). Alternatively, the preparation of the third aspect of the invention may be suitable for conversion into a pharmaceutical composition for administration, in particular for oral administration or administration by injection, during the preparation process. For example, the preparation may be reconstituted and / or diluted to prepare an injectable solution, reformulated as a lotion or ointment, reformulated into an oral formulation, or included as an active component of a transdermal delivery patch with multiple microneedles for injecting the composition through one or more layers of the skin. Advantageously, the entire preparation of the third aspect of the invention may be suitable for conversion into a pharmaceutical composition for administration without the need to extract the components of the preparation other than the optional removal of a solvent such as water (e.g. in an evaporation or freeze-drying step). During conversion into a pharmaceutical composition, additional components (e.g. , pharma- ceutically acceptable non-medicinal ingredients, diluents, and adjuvants) may be added to the preparation. Thus, the preparation of the third aspect of the invention and medicaments prepared therefrom meet the need for biocompatible non-medicinal ingredients, especially for oral or injectable compositions, that allow administration, especially oral administration or administration by injection, without the intervening step of extracting the active biological species from the composition and without the intervening step of reconstitution. This may be particularly true when the biocompatible solid particles comprise hydrolyzable silicon.

[0016] According to a fourth aspect of the invention there is provided a method of preparing a medicament comprising a biological species for administration, advantageously oral administration or administration by injection, comprising either preparing a preparation according to the method of the first aspect of the invention or providing a preparation according to the third aspect of the invention, then (ii) optionally storing the preparation; and then (iii) preparing the medicament comprising the biological species for administration (such as a dilution and / or reconstitution step), preferably for administration oral administration or administration by injection.

[0017] Thus, the method of the fourth aspect of the invention, when it comprises preparing a preparation by the method of the first aspect of the invention, may comprise contacting a biological species with a delivery system comprising solid biocompatible particles to form a complex; then, optionally, freeze-drying the complex to form a powder; then dispersing the complex in a biodegradable gel material to form a preparation; then, optionally storing the preparation; and then preparing a medicament comprising the biological species and the delivery system for administration, preferably for administration orally or by injection.

[0018] Alternatively, the method of the fourth aspect of the invention when it involves preparing a preparation according to the third aspect of the invention may comprise preparing a preparation, preferably an oral or injectable preparation, comprising a biological species complexed with a delivery system comprising biocompatible solid particles embedded in a biodegradable gel matrix; and then preparing a medicament comprising the biological species and the delivery system for administration, preferably oral or by injection.

[0019] The step of preparing the medicament for administration may be, for example, a step of preparing the medicament for administration by injection (such as, for example, diluting and / or reconstituting a biodegradable gel material containing dispersed biological species and a delivery system), or in another example, it may be a step of preparing the medicament for oral administration (e.g., by tableting with pharma- ceutically acceptable non-medicinal ingredients such as fillers, disintegrants, etc.).

[0020] According to a fifth aspect of the invention there is provided a method of treating or preventing a disease or disorder comprising administering, preferably orally or by injection, a medicament according to the fourth aspect of the invention (e.g. by subcutaneous or intramuscular injection) to a subject in need thereof.

[0021] Thus, the method of the fifth aspect of the invention may comprise contacting a biological species with a delivery system comprising biocompatible solid particles to form a complex; then optionally lyophilizing the complex to form a powder; then dispersing the complex in a biodegradable gel material to form a preparation, preferably an oral or injectable preparation; then optionally storing the preparation; then preparing a medicament comprising the biological species and the delivery system for administration, preferably for oral administration or administration by injection; and then administering the medicament, preferably orally or by injection, to a subject in need of administration.

[0022] Alternatively, the method of the fifth aspect of the invention may comprise preparing a preparation, preferably an oral or injectable preparation, comprising a biodegradable gel material having embedded therein a biological species complexed to a delivery system comprising biocompatible solid particles; then preparing a medicament comprising the biological species and the delivery system for administration, preferably for oral administration or administration by injection (such as diluting and / or reconstituting the biodegradable gel material comprising the embedded biological species and the delivery system); and then administering the medicament, preferably orally or by injection, to a subject in need thereof. The step of preparing a medicament for administration, preferably for oral administration or administration by injection, may for example be or include a step of diluting and / or reconstituting the biodegradable gel material comprising the embedded biological species and the delivery system; or in another example, may be or include a step of preparing the medicament for oral administration by tableting with pharma- ceutically acceptable non-active ingredients (fillers, disintegrants, etc.). Administering the medicament can be, for example, by subcutaneous or intramuscular injection, or by oral administration of a solid formulation.

[0023] Preferably, the method for preparing a medicament of the fourth aspect of the invention and / or the method for treating or preventing a disease or disorder of the fifth aspect of the invention does not include any extraction step, and thus the medicament for administration, preferably for oral administration or administration by injection, comprises all the components of the (preferably oral or injectable) preparation produced by the method of the first aspect of the invention, or the (preferably oral or injectable) preparation of the third aspect of the invention. A method (of the fourth or fifth aspect of the invention) that does not include an extraction step may comprise a step of removing a solvent, such as water, from the (preferably oral or injectable) preparation produced by the method of the first aspect of the invention, or the (preferably oral or injectable) preparation of the third aspect of the invention (e.g. an evaporation, freeze-drying or filtration step).

[0024] In a sixth aspect, the present invention provides a medicament (preferably oral or injectable). The medicament may comprise a biological species complexed with a delivery system embedded in a biodegradable gel material. Alternatively, the medicament may comprise a biological species complexed with a delivery system together with a compound capable of forming a biodegradable gel matrix dispersed in a carrier system, which may be a liquid carrier system, such as an aqueous solution, or a solid carrier system. The medicament may be, for example, an injectable formulation comprising a biological species complexed with a delivery system together with a compound capable of forming a biodegradable gel matrix dispersed in a liquid carrier system. Alternatively, the medicament may be, for example, a solid or gel formulation (for example for oral administration) comprising a biological species complexed with a delivery system and co-embedded in a biodegradable gel.

[0025] In the methods of the first, second, fourth and fifth aspects of the invention, after the step of contacting the biological species with the delivery system, the biological species in contact with the delivery system is optionally lyophilized in a lyophilization step to form a powder. It has been found that lyophilization can be advantageous to enhance the stability of the preparation (e.g. during optional storage of the preparation).

[0026] The biological species is optionally one or more of a nucleic acid, an antigen, and a vaccine, or optionally comprises one or more of a nucleic acid, an antigen, and a vaccine. The biological species typically comprises a nucleic acid, in particular an siRNA or an mRNA, or a plasmid DNA. When the biological species is a vaccine, the method of preventing a disease or disorder of the fifth aspect of the invention can be a method of vaccination.

[0027] The delivery systems of the first to sixth aspects of the present invention are particulate delivery systems comprising biocompatible (and optionally biodegradable) solid particles. Delivery systems comprising biocompatible particles advantageously comprise particles of hydrolyzable silicon. The delivery system may, for example, comprise biocompatible particles of hydrolyzable silicon in the presence of a lipid component comprising at least one lipid (such as a cationic lipid or an ionizable lipid) and, optionally, a non-reducing disaccharide (such as trehalose).

[0028] Thus, the method of the first aspect of the invention or the method of the second aspect of the invention (and consequently the method of the fourth aspect of the invention or the method of the fifth aspect of the invention) may comprise contacting a biological species comprising a nucleic acid with a delivery system comprising biocompatible particles of hydrolysable silicon in the presence of at least one lipid (such as a cationic or ionizable lipid) and optionally a non-reducing disaccharide (such as trehalose); optionally then freeze-drying the biological species in contact with the delivery system to form a powder; and then embedding the combined biological species and delivery system in a biodegradable gel material. The preparation of the third aspect of the invention and / or the medicament of the sixth aspect of the invention may comprise a biological species comprising a nucleic acid complexed to particles comprising hydrolysable silicon, at least one lipid, such as a cationic or ionizable lipid, and optionally a non-reducing disaccharide (such as trehalose), embedded in a biodegradable gel material.

[0029] The biological species or one or more components thereof contacted with the delivery system may function as a vector for delivery of the biological species or one or more other components thereof to a cell (e.g., as a transfection or other vector that can be internalized and allows endosomal escape to deliver the biological species to the cytoplasm). A transfection vector may be used to target the biological species to a cell. Thus, the biological species may include a nucleic acid for transcription within the cell, together with a transfection vector, particularly a non-viral transfection vector, or other components that provide a vector that allows uptake by immune cells resulting in endosomal escape of the nucleic acid for release into the cytoplasm.

[0030] A (preferably oral or injectable) preparation produced by the method of the first or second aspect of the invention or provided by the third aspect of the invention may advantageously be stored at relatively high temperatures (e.g. -10°C or higher, in particular 4°C or higher) for a substantial period of time (e.g. 6 weeks or more) without substantial degradation, e.g. without degradation of 10% or more (such as 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% or more) of the biological species. Furthermore, when a (preferably oral or injectable) preparation of the third aspect of the invention is prepared as an administrable medicament, e.g. by the method of the fourth aspect of the invention, the biological species present in the medicament advantageously remain protected from degradation, as compared to, e.g. a conventional reconstituted or thawed pharmaceutical product comprising a biological species.

[0031] In the case of solid structures, the movement of the molecules is restricted and their normal resonance amplitude is reduced. When a composition containing biological species and trace contaminants such as enzymes (e.g., residual enzyme species from the manufacturing process of the biological species) is bound to a matrix structure, it is hypothesized that the contaminants (e.g., enzymes) in the system do not have sufficient movement to act on the biological species and cause its degradation. For example, the enzymes (e.g., residual enzyme species) do not have sufficient freedom of movement to bind to the correct portion of the nucleic acid or other biological species and do not have the ability to perform enzymatic cleavage. Similarly, without wishing to be bound by theory, it is believed that the number of water molecules available for enzyme-catalyzed reactions with the biological species may be reduced, for example, water molecules may be removed by the reaction of water with hydrolyzable silicon.

[0032] Thus, embedding a complex of a biological species with a delivery system comprising particles of a biocompatible solid material within a biocompatible gel has been found to stabilize the complex and preserve the components of the complex both during storage and in vivo after administration to the human body.

[0033] Contact of biological species with a delivery system comprising particles of a biocompatible solid material, particularly hydrolyzable silicon, has been found to be particularly effective in preventing degradation of the biological species. It is hypothesized that the biological species, such as one or more nucleic acids, bind to the surface of the porous silicon, protecting cleavage sites of the nucleic acid, such as sites on the phosphate backbone, from enzymatic degradation.

[0034] It is believed that there are three main factors that contribute to the overall product stability during storage: prevention of degradation of biological species (e.g., RNA); retardation of oxidation of lipids (e.g., lipids involved in transfection); and colloidal stability of the particulate suspension, which prevents particle aggregation. The preparations and medicaments of the present invention advantageously provide some or all of these three factors. Furthermore, it is believed that after administration, preferably oral or by injection, the complex of biological species and delivery system can remain stabilized in vivo by the presence of the gel until it is completely degraded. This enhanced biological stability can help to increase the likelihood that the biological species will be delivered to the desired location (e.g., internalized by the target cells). [Brief description of the drawings]

[0035] [Figure 1] 13 is a gel electrophoresis image of a liquid mRNA-Biocourier preparation embedded in sodium hyaluronate biodegradable gel upon preparation and after 6 hours of storage at room temperature (RT) or 40° C. [Diagram 2] Gel electrophoresis images of liquid mRNA-Biocourier preparations embedded in sodium hyaluronate biodegradable gel after 24 and 48 hours of storage at room temperature (RT) or 40° C. [Diagram 3] Gel electrophoresis images of lyophilized and reconstituted mRNA-Biocourier preparations embedded in sodium hyaluronate biodegradable gels upon preparation and after 6 hours of storage at room temperature (RT) or 40° C. [Figure 4] Gel electrophoresis images of lyophilized and reconstituted mRNA-Biocourier preparations embedded in sodium hyaluronate biodegradable gel after 24 and 48 hours of storage at room temperature (RT) or 40° C. [Diagram 5] 13 shows gel retardation images of siRNA-Biocourier biodegradable gel preparations immediately after preparation and after 2 hours of storage at room temperature (RT) or 40° C. [Figure 6]Gel retardation images of siRNA-Biocourier biodegradable gel preparations after 24-48 hours of storage at room temperature (RT) or 40° C. [Figure 7] Gel electrophoresis images of siRNA-SIS0012 biodegradable gel preparations immediately after preparation (0 hours) and after 2 hours of storage at room temperature. [Figure 8] Gel electrophoresis images of siRNA-SIS0012 biodegradable gel preparations after 4-6 hours of storage at room temperature. [Figure 9] Gel electrophoresis images of siRNA-SIS0012 biodegradable gel preparations after 24-48 hours of storage at room temperature. [Figure 10] Gel electrophoresis images of siRNA-SIS0012 biodegradable gel preparations immediately after preparation (0 hours) and after 2 hours of storage at 40°C. [Figure 11] Gel electrophoresis images of siRNA-SIS0012 biodegradable gel preparations after 4-6 hours of storage at 40°C. [Figure 12] 13 is a gel electrophoresis image of siRNA-SIS0012 biodegradable gel preparation after 24-48 hours of storage at 40° C. [Figure 13] Gel electrophoresis images of siRNA-SIS0013 biodegradable gel preparations immediately after preparation (0 hours) and after 2 hours of storage at room temperature. [Figure 14] Gel electrophoresis images of siRNA-SIS0013 biodegradable gel preparations after 4-6 hours of storage at room temperature. [Figure 15] Gel electrophoresis images of siRNA-SIS0013 biodegradable gel preparations after 24-48 hours of storage at room temperature. [Figure 16] Gel electrophoresis images of siRNA-SIS0013 biodegradable gel preparations immediately after preparation (0 hours) and after 2 hours of storage at 40°C. [Figure 17] Gel electrophoresis images of siRNA-SIS0013 biodegradable gel preparations after 4-6 hours of storage at 40°C. [Figure 18]13 is a gel electrophoresis image of siRNA-SIS0013 biodegradable gel preparation after 24-48 hours of storage at 40° C. [Figure 19] 13 is a gel electrophoresis image of freshly prepared siGLO delivery system complexes in liquid form (Liq) or resuspended lyophilized powder (FD). [Figure 20] Gel electrophoresis images of siGLO delivery system complexes in liquid form (Liq) or resuspended lyophilized powder (FD) after 24-48 hours of storage at room temperature (RT) or 4°C. [Figure 21] Gel electrophoresis images of siGLO delivery system complexes in liquid form (Liq) or resuspended lyophilized powder (FD) after 5 and 7 days of storage at room temperature (RT) or 4°C. [Figure 22] 13 is a gel electrophoresis image of siGLO delivery system complexes in liquid form (Liq) or resuspended lyophilized powder (FD) after 14 and 28 days of storage at room temperature (RT) or 4° C. [Diagram 23] 13 is a gel electrophoresis image of the siGLO delivery system complexes freshly prepared, as resuspended lyophilized powder (FD), or loaded into sodium hyaluronate (SH) hydrogel. [Figure 24] Gel electrophoresis images of siGLO delivery system complexes as resuspended lyophilized powder (FD) or loaded into sodium hyaluronate (SH) hydrogels after 24-48 hours of storage at 40°C. [Diagram 25] Gel electrophoresis images of siGLO delivery system complexes as resuspended lyophilized powder (FD) or loaded into sodium hyaluronate (SH) hydrogels after 5 and 7 days of storage at 40°C. [Figure 26] 13 shows gel electrophoresis images of siGLO delivery system complexes as resuspended lyophilized powder (FD) or loaded into sodium hyaluronate (SH) hydrogels after 14 and 28 days of storage at 40° C. [Figure 27]1 is a chart showing MRSA bacterial populations on the skin of human volunteers after inoculation and treatment with a peptide API in a conventional chassis; loaded with a preparation of the invention; or a control (no peptide). [Figure 28] 1 is a chart showing results of the MRSA-Biofilm mouse nasal carriage model testing silicon-containing preparations. [Figure 29] FIG. 1 shows gel electrophoresis images of SIS0012 and SIS0013 with NAD, TYR, and QUE when loaded with siRNA. [Diagram 30] FIG. 13 shows a gel electrophoresis image of the DPPC / PAL-KTTKS-DOPE preparation of Example 6 when loaded with siRNA. [Diagram 31] FIG. 13 shows further gel electrophoresis images of the DPPC / PAL-KTTKS-DOPE preparation of Example 6 when loaded with siRNA. [Diagram 32] FIG. 13 shows further gel electrophoresis images of the DPPC / PAL-KTTKS-DOPE preparation of Example 6 when loaded with mRNA. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] Without wishing to be bound by any theory, the inventors have come to realise that complexing nucleic acids or other biological species with solid biocompatible particles such as hydrolysable silicon limits the mobility (molecular movement) of not only the biological species but also the positively charged components that may be present in the composition such as lipids, polymers and peptides used to form the lipid membrane. This allows them to stabilize their positive charge and therefore retain their function of stabilising the biological species. It is believed that modified and derivatised silicon particles retain their positive charge and prevent dissociation of the biological species and the positively charged components, thereby stabilising the integrity of the biological species as well as the positively charged components. Unlike liposomes or lipid nanoparticles, which are the result of oil / water / surfactant interactions, which are believed to still contain some water pockets within the lipid nanoparticles and thus expose the biological species to an aqueous environment, the complexes of the present invention advantageously have no or very little water present. Thus, there is no solvent / aqueous environment for enzymes or free radicals to operate. This dual effect of silicon binding and reduced local water availability may also account for the increased stability of biological species when stored at room temperature.

[0037] Based on that realization, the inventors have investigated the effect of entrapment of the entire complex in a matrix system (of a biodegradable gel material) to further protect and immobilize the components of the complex. Physical protection of biological species, such as nucleic acids, from the environment is provided by a combination of encapsulation by a lipid layer and adsorption to a solid support (e.g., particles containing hydrolyzable silicon) in the complex of the invention, and by entrapment of the entire complex in a matrix system (of a biodegradable gel material). This provides a high level of protection from degradation by avoiding interactions with external species. While adsorption or encapsulation alone can limit some of these interactions, such as the access of enzymes and microorganisms, contact with environmental moisture, and mobility of biological species, inclusion in a matrix system (of a biodegradable gel material) has been found to be highly advantageous in preventing interactions during long-term storage under mild conditions. The effectiveness of gels, materials that necessarily contain a significant amount of liquid (usually water) to swell the polymer matrix in order to stabilize the complex, was surprising, as the presence of liquid was previously thought to destabilize the complex and / or result in degradation of the biological species or other components. Thus, gels have been widely used in the past not for protection of active ingredients, particularly in oral or injectable preparations, or to promote storage stability, but for applications such as tissue bonding or topical application.

[0038] A further advantage of the (preferably oral or injectable) preparations of the present invention (e.g. the preparations of the third aspect of the present invention) is that they are ready to be converted into a medicament (e.g. the medicament of the sixth aspect of the present invention) for administration, in particular for oral administration or administration by injection, without any extraction step. Thus, the preparations may be, for example, ready to be diluted and injected without an extraction step, or ready to be made into a solid oral formulation without an extraction step. Thus, the preparations meet the need for biocompatible non-medicinal ingredients, in particular oral or injectable compositions, that allow administration, in particular oral administration or administration by injection, without an intervening step of extraction and reformulation of the active biological species from the composition. Without wishing to be bound by any theory, it is believed that this may be the case since the solid particles of the delivery system of the present invention are biocompatible. Thus, it may be especially the case that the particles comprise hydrolyzable silicon. In contrast, for example, silica particles administered orally or by injection are not biocompatible and may cause irritation.

[0039] Oral or injectable preparations and medicines The methods, preparations and medicaments of the first to sixth aspects of the invention are, or preferably result in, oral or injectable products. Gels such as those present in the preparations and medicaments of the third or sixth aspects of the invention have not previously been widely used to protect (or promote storage stability of) biological species in oral or injectable preparations, but have been used more for applications such as tissue bonding, or for topical applications such as cosmetics, or for treating burns.

[0040] For example, advantageously, the entire preparation of the third aspect of the invention may be suitable for conversion into a pharmaceutical composition for administration without the need to extract the components of the preparation other than the optional removal of solvents such as water (e.g. in an evaporation or lyophilization step). It will also be appreciated that additional components (such as additional pharma- ceutically acceptable non-medicinal ingredients, diluents, and adjuvants) may be added to the preparation upon conversion into a pharmaceutical composition, still without an extraction and / or reconstitution step.

[0041] Thus, the preparation of the third aspect of the invention and medicaments prepared therefrom fulfil the need for a biocompatible non-medicinal ingredient for oral or injectable compositions which allows for oral or injection administration without the intervening steps of extracting the active biological species from the composition and without the intervening steps of reconstitution. This may be particularly the case where the biocompatible solid particles comprise hydrolysable silicon.

[0042] Thus, in a preferred embodiment, the preparation is an injectable preparation and the medicament is an injectable medicament. The mode of injection can be subcutaneous or intradermal (e.g. by a transdermal patch containing microneedles); intramuscular; or intravenous. Thus, provided herein is an injection device comprising the preparation of the third aspect of the invention or a medicament prepared from the preparation of the third aspect of the invention. The injection device may be or may comprise a syringe comprising a plunger, a cartridge (also known as a barrel) and a needle. The cartridge of the syringe may comprise the preparation of the third aspect of the invention or a medicament prepared from the preparation of the third aspect of the invention. The cartridge may comprise a single dose or multiple doses, for example 1 dose, 2 doses, 3 doses, 4 doses or 5 doses of said preparation or medicament. The syringe may be or may comprise a safety syringe. The syringe may be or may comprise a disposable syringe. The injector may be or include a hypodermic injector. The injection device may be or include an automatic injection system (such as an injection pen or a system including an injection pen). The injection device may be or include a needle-free injector. The injection device may be or include a transdermal delivery device, such as a delivery patch with multiple microneedles for injection. The injection device may be or include an automatic injector. The injection device may be or include an infusion pump device. In all its embodiments, the injection device comprises the preparation of the third aspect of the invention or a medicament prepared from the preparation of the third aspect of the invention.

[0043] Also provided herein is an oral formulation comprising the preparation of the third aspect of the invention or a medicament prepared from the preparation of the third aspect of the invention. The oral formulation may contain one or more physiologically compatible carriers and / or non-medicinal ingredients and may be in solid (e.g., tablet, capsule, or powder) or liquid (e.g., solution) form. Tablets and capsules may be prepared with binders (e.g., syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone); fillers (such as lactose, sucrose, corn starch, calcium phosphate, sorbitol, or glycine); lubricants (such as magnesium stearate, talc, polyethylene glycol, or silica); and surfactants (such as sodium lauryl sulfate). Liquid compositions may contain conventional additives such as suspending agents such as sorbitol syrup, methylcellulose, sugar syrup, gelatin, carboxymethylcellulose, or edible fats; emulsifiers such as lecithin or acacia; vegetable oils such as almond oil, coconut oil, cod liver oil, or peanut oil; preservatives such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT). Oral formulations may be or contain liquid compositions that can be encapsulated (e.g., in gelatin) to prepare unit dosage forms. Solid oral formulations may include tablets, two-piece hard shell capsules, and soft elastic gelatin (SEG) capsules. The solid oral formulation may be or may include a dry shell preparation, typically comprising gelatin at a concentration of about 40% to about 60%, a plasticizer (such as glycerin, sorbitol, or propylene glycol) at a concentration of about 20% to about 30%, and water at a concentration of about 30% to about 40%. Other materials such as preservatives, dyes, opacifiers, and flavorings may also be present. The liquid fill material may include a solid form of the preparation or medicament, or a liquid form of the preparation or medicament, dissolved, solubilized, or dispersed (with a suspending agent such as beeswax, hydrogenated castor oil, or polyethylene glycol 4000) in a vehicle such as mineral oil, vegetable oil, triglyceride, glycol, polyol, and surfactant, or a combination of such vehicles.

[0044] Extraction process Advantageously, in the method of the fourth aspect of the invention, the preparation of the medicament for administration does not include any extraction step, so that the medicament includes all the components of the preparation. As used herein, the term "extraction step" may refer to the removal of a biological species from a composition containing said species. The removal step may include one or more of physical separation (such as centrifugation and / or filtration) and chemical separation (e.g. dissolving said species in a solvent in which the other components of the composition are poorly soluble and / or dissolving said other components in one or more solvents in which said species are poorly soluble; this may be followed by physical separation, such as filtration as described above, and / or evaporation, such as rotary evaporation). If the biological species is unstable under the extraction conditions, especially if the biological species is or may contain a nucleic acid, such as mRNA, extraction may risk degradation of the biological species. Furthermore, as used herein, the term "reconstitution" or "reconstituting" may refer to the addition of one or more non-medicinal ingredients to the biological species after it has been extracted from a previous composition.

[0045] Advantageously, preparations and medicaments are provided in all aspects of the invention which, as described herein, eliminate the extraction and reconstitution steps, or alternatively, eliminate only the extraction step.

[0046] Delivery System The delivery systems of the first to sixth aspects of the present invention typically comprise particles of a biocompatible material (e.g. particles of hydrolysable silicon) together with a lipid component which typically comprises at least one cationic or ionisable lipid, and optionally a non-reducing disaccharide (such as trehalose).

[0047] Biocompatible particles The delivery system comprises particles of a solid material that is biocompatible. Thus, the particles are not harmful to living tissue. Advantageously, the particles are also biodegradable and can be decomposed in vivo into degradation products that are not harmful to living tissue. The particles are solid particles that do not melt or pyrolyze at temperatures below 60°C, preferably below 80°C.

[0048] Suitable known biocompatible solid materials that may be used in the present invention are metal particles, metalloid particles, especially silicon particles, and graphene particles. Metal nanoparticles, especially metal nanoparticles including gold and iron oxide, are widely used in medicine for diagnostic and therapeutic purposes and are used as delivery systems for drugs and vaccines.

[0049] The biocompatible solid particles may contain silicon (e.g., mesoporous silicon particles). Advantageously, the biocompatible particles may be pure silicon or another hydrolyzable silicon-containing material. If the biocompatible solid particles are not pure silicon, they contain 50% or more silicon by weight, i.e., 50% or more silicon atoms by weight based on the total mass of atoms in the particle. For example, the silicon particles may contain 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more silicon. The silicon particles preferably exhibit a hydrolysis rate (e.g., in PBS buffer at room temperature) that is 10% or more of the hydrolysis rate of pure silicon particles of the same size. Assays for hydrolysis of silicon-containing materials are widely known in the art, see, for example, WO 2011 / 001456. It will be understood that silica (SiO2) nanoparticles that do not contain 50% elemental silicon by weight do not fall within the definition of silicon nanoparticles. Silica nanoparticles are also not hydrolyzable, since hydrolysis of silica is thermodynamically unfavorable under conditions present in vivo. Hydrolyzable silicon particles have the advantage that they can degrade in vivo into the beneficial orthosilicic acid (e.g. when prepared as described in WO 2011 / 012867). Additional components in the delivery system, such as lipid components and / or amino acids, advantageously act as a stabiliser or stabilisers, as discussed in WO 2011 / 012867, which modify the rate of hydrolysis of the silicon in the delivery system in vivo and / or inhibit the rate of orthosilicic acid polymerisation upon degradation of the delivery system in vivo.

[0050] According to all aspects of the invention, the particles, including those containing hydrolyzable silicon, may be nanoparticles. The nanoparticles have a nominal diameter of 5 nm to 300 nm, such as 8 nm to 200 nm, such as 10 nm to 150 nm, such as 15 nm to 100 nm (such as 18 nm to 80 nm). The nominal diameter may refer to the average diameter, and at least 90% of the total mass of particles in a sample of particles may fall within the specified size range. The particle size may be determined or confirmed by transmission electron microscopy (TEM), for example, using the NIST-NCL Joint Assay Protocol, PCC-X, Version 1.1, Revised February 2010, "Measuring the size of nanoparticles using TEM" (https: / / tsapps.nist.gov / publication / get_pdf.cfm?pub_id=854083).

[0051] The biocompatible particles are preferably etched. When the particles are etched, their total surface area increases because their porosity increases. For example, the surface area may increase by 50% or more or 100% or more with respect to the surface area of ​​the corresponding non-porous particle. In many circumstances, the porous particles of all aspects of the present invention actually have a much larger increase in total surface area due to their porosity. Particles containing hydrolyzable silicon can be made porous by standard techniques such as contacting the particles with a hydrofluoric acid (HF) / ethanol mixture and applying an electric current. By varying the HF concentration and the current density and exposure time, the density of the pores and their size can be controlled and monitored by scanning electron microscopy and / or nitrogen adsorption-desorption volumetric isotherm measurements. According to certain embodiments, the porosity is 30% or more, 40% or more, 50% or more, or 60% or more. This means that 30%, 40%, 50%, or 60% of the particle volume, respectively, is pore space. The pore size is preferably in the range of 1 nm to 50 nm (for example, 5 nm to 25 nm).The particles are preferably porous, and more preferably mesoporous.

[0052] The particles of all aspects of the invention can be conveniently prepared by techniques conventional in the art (e.g., by grinding processes) or other known techniques for particle size reduction. Silicon-containing particles can be made from sodium silicate particles, colloidal silica, or silicon wafer material. Macro- or micro-scale particles can be ground in a ball mill, a planetary ball mill, or other size reduction mechanisms. The resulting particles can be air classified or sieved to recover the particles. Plasma methods and laser ablation can also be used for particle generation.

[0053] Lipids The delivery systems of the first to fifth aspects of the present invention typically contain a lipid component.

[0054] Lipids in the art are generally understood to include fatty acids and fatty acid derivatives, glycerolipids, glycerophospholipids, sphingolipids, glycolipids, and polyketides.

[0055] As used in this application, the term "lipid" may also encompass lipidated oligopeptides (a term used interchangeably herein with the term lipopeptide) in which a short peptide sequence (such as a peptide sequence having 3-20 amino acid residues, e.g., having 5-15 amino acid residues, particularly 3, 4 or 5 amino acid residues, and especially 5 amino acid residues) is conjugated to one or more fatty acid chains (particularly fatty acid chains having a carbon chain length of 10-24, preferably 12-18 carbon chain length; e.g., 14, 15 or 16 carbon chain lengths; e.g., the peptide moiety may be optionally lipidated with palmitoyl, cetyl or myristoyl moieties).

[0056] The lipidated oligopeptide may optionally be a lipidated tetrapeptide, a lipidated pentapeptide, or a lipidated hexapeptide. Preferably, the amino acid residue comprises at least one amino acid residue (e.g., 2 or 3 amino acid residues) that is cationic at a pH of 7.4 (physiological pH), such as lysine or arginine. For example, the lipidated oligopeptide may comprise one or more (e.g., 2) lysine residues. Thus, a specific example is palmitoyl-pentapeptide-4 (CAS number 214047-00-4; abbreviated as PAL-KTTKS).

[0057] [ka]

[0058] Thus, in certain preferred embodiments according to all aspects of the invention, the one or more lipids are or comprise one or more lipidated oligopeptides, particularly having one or more amino acid residues that are cationic at pH 7.4 (physiological pH; examples include lysine and arginine).

[0059] The lipidated oligopeptide may be used in combination with one or more phospholipids, such as DOPE or DPPC. The alkyl chains of the lipopeptide may be advantageously assimilated into the phospholipid bilayer, while the surface of the bilayer may be decorated with peptide moieties. In this way, the peptide may provide tissue and / or cell targeting, for example, when the peptide carries a cationic charge at physiological pH, it may stabilize negatively charged APIs, such as nucleic acids, such as mRNA.

[0060] According to all aspects of the present invention, the lipid component advantageously comprises a cationic lipid and / or an ionizable lipid. The lipid component may additionally comprise a helper lipid (e.g., a phospholipid); a structured lipid (e.g., a cholesterol-based lipid); and / or a PEG lipid. A preferred lipid component comprises at least a cationic lipid or an ionizable lipid and a phospholipid, and optionally a PEG lipid and / or a structured lipid, preferably a PEG lipid and / or a structured lipid.

[0061] The type of lipid used to treat the surface of the biodegradable biocompatible solid particles of the delivery system can affect its degradation rate in vivo. For example, it has been found that the presence of at least one lipid in a delivery system containing silicon particles allows the hydrolysis rate of silicon to be controlled so that silicon hydrolyzes into bioavailable orthosilicic acid decomposition products rather than into insoluble polymer hydrolysis products. It has also been found that surface treating particles with lipids can help the uptake of biological species such as nucleic acids by the delivery system and / or help control the release rate of biological species from the delivery system. In particular, it has been found that surface treating silicon particles with lipids has a beneficial effect on the surface charge of silicon particles, providing the silicon particles with the necessary zeta potential to allow improved loading of siRNA (small interfering RNA) or mRNA (messenger RNA) and controlling the release rate of loaded molecules at target sites.

[0062] According to all aspects of the invention, the lipid or lipids of the lipid component may have an average molecular weight in the range of 300 to 1200 Daltons, such as 500 to 1000 Daltons.

[0063] The lipid component may be or may include a cationic lipid. The term "cationic lipid" refers to a positively charged molecule with a cationic head group attached to a hydrophobic tail via some spacer. Examples include DTDTMA (ditetradecyltrimethylammonium), DOTMA (2,3-dioleyloxypropyl-1-trimentylammonium), DHDTMA (dihexadecyltrimethylammonium), DOTAP (1,2-dioleoyl-3-trimethylammonium propane), and SA (stearylamine). The positive charge is usually stabilized by a negative counterion (e.g., chloride ion). The negative charge of the lipid may also or additionally be stabilized by association with the material of the solid biocompatible particle (e.g., by association with hydrolyzable silicone). The presence of cationic lipids has been found to promote cellular internalization by cells in the tissue or organ of interest. The positively charged cationic lipids facilitate the uptake of the delivery system and the biological species, and allow for the transfer of the nucleic acid component of the biological species in the cells of the tissue or organ of interest that produces a therapeutic effect.

[0064] The lipid component may be or may include an ionizable lipid. The term "ionizable lipid" refers to a lipid having a group capable of being positively charged, usually having a Lewis base (hydrogen acceptor) head group attached (e.g., via some spacer) to a hydrophobic tail. An ionizable lipid usually includes a head group that includes a tertiary amine moiety. An ionizable lipid may be neutral at physiological pH, but becomes cationic at lower pH (e.g., below pH 6.5), such as the pH found within the vacuole as part of endosomal escape. Ionizable lipids are described, for example, in Nano Lett. 2020 Mar 11; 20(3): 1578-1589. Many vaccine platforms use lipid systems that are neutral at physiological pH, then enter the endosome and become cationic only when the pH is reduced to about pH 6.2, and the lipids that become cationic then allow for endosomal escape. A delivery system containing neutral lipids allows more particles to travel from muscle to lymph nodes and be phagocytosed by dendritic cells. The ionizable lipids can be stabilized by association with the material of the solid biocompatible particles (e.g., by association with hydrolyzable silicones).

[0065] The lipid component may include a phospholipid as a helper lipid. The term "phospholipid" refers to a lipid that includes a fatty acid chain and a phosphate group. Phospholipids are typically neutral molecules in that they may have no overall charge or may have a negative charge, unlike positively charged cationic lipids. Phospholipids are typically zwitterionic compounds that include both positively and negatively charged components, but do not include an overall charge. Thus, phospholipids are typically classified as neutral lipids. Particularly suitable phospholipids are glycerophospholipids. Particularly suitable phospholipids are those in which the polar head group is attached to a quaternary ammonium moiety, such as phosphatidylcholine (PC) or hydrogenated phosphatidylcholine. Other examples of phospholipids are fusogenic lipids, such as DOPE (phosphatidylethanolamine or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine). The type of lipid may be selected depending on the nature of the preparation, with neutral or negatively charged phospholipids being preferred for aprotic preparations, and positively charged cationic lipids and small CH3 chain lipids being preferred for protic preparations. The phospholipid may be lecithin or may be derived from lecithin. Preferably, the side chain (or chains) of the phospholipid is an aliphatic side chain (or chains) having 15 or more carbon atoms, or an ether side chain having 6 or more repeating ether units (such as a polyethylene glycol or polypropylene glycol chain).

[0066] The lipid component may additionally or alternatively include a PEG lipid. A lipid having a polyether side chain may be referred to as a "PEG lipid" or a "PEGylated" lipid. The PEG lipid may be a phospholipid such as 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) having a PEG side chain (e.g., DSPE-mPEG2000).

[0067] The lipid component may include one or more of phosphatidylcholine (PC), hydrogenated PC, stearylamine (SA), dioleoylphosphatidylethanolamine (DOPE), cholesteryl 3β-N-(dimethylaminoethyl)carbamate hydrochloride (DC)-cholesterol, and derivatives thereof. In certain embodiments, the lipid component may consist essentially of phosphatidylcholine, hydrogenated phosphatidylcholine, stearylamine, or combinations thereof.

[0068] Preferably, according to all aspects of the invention, the ratio of lipid(s) (i.e. total lipid component) to silicon, before any further processing of the material (i.e. by filtration or sterilization processes), is between 0.5:1 and 45:1, such as between 0.8:1 and 20:1, 1:1 and 16:1, 1:1 and 12:1, 1:1 and 11:1, 1:1 and 10:1, 1:1 and 9:1, 1:1 and 8:1, 1:1 and 13:1, 2:1 and 12:1, 2:1 and 11:1, 2:1 and 10:1, 2:1 and 9:1, 2:1 and 8:1, such as between 1:1 and 7:1, 2:1 and 7:1, 3:1 and 6:1, 4:1 and 5:1. A lipid molar ratio to silicon of 0.8:1 to 20:1 has been found to be particularly advantageous, for example 16:1, 12:1, 8:1 or 2.5:1. Advantageously, this lipid to silicon ratio provides a multilamellar vesicle system capable of controlling and stabilizing the release of nucleic acids in contact with the hydrolyzable silicon particles and promoting the controlled release of the bioavailable degradation product of silicon, i.e. orthosilicic acid.

[0069] Advantageously, the lipid component can have a significant effect on the surface charge of the silicon particles. Particles containing hydrolyzable silicon treated with phosphatidylcholine (PC), phosphatidylethanolamine (PE), and lecithin demonstrate a negative surface charge when subjected to zeta potential analysis (range -60 to -20 mV, silicon:lipid ratios ranging from 1:1 to 1:3). Particles surface treated with stearylamine demonstrate a positive zeta potential (range 0 to 40 mV, silicon:lipid ratios ranging from 1:1 to 1:3). The lipid component can be stabilized by association with the material of the solid biocompatible particles (e.g., by association with hydrolyzable silicon). Conversely, the solid biocompatible particles, hydrolyzable silicon particles, can be stabilized by association with the lipid component.

[0070] Non-reducing disaccharides According to all aspects of the invention, the delivery system optionally includes at least one non-reducing disaccharide, which may be optionally selected from sucrose, trehalose, raffinose, stachyose, and verbascose, or any mixture thereof, and most preferably the non-reducing disaccharide is trehalose or a mixture comprising trehalose.

[0071] When the delivery system comprises silicone particles, the non-reducing disaccharide (e.g. trehalose, or a mixture comprising trehalose) is optionally present in a weight ratio to the silicone of 1000:1 or more, 100:1 or more, 50:1 or more, 10:1 or more, 1:1 or more, or 0.5:1 or more. Preferably, the non-reducing disaccharide is trehalose, optionally present in a weight ratio to the silicone of 1000:1 or more, 100:1 or more, 50:1 or more, 10:1 or more, 1:1 or more, or 0.5:1 or more.

[0072] It is hypothesized that non-reducing disaccharides, especially trehalose, may act as dehydration protectants. Non-reducing disaccharides may act as cocoons that trap biomolecules within a glassy sugar matrix such that protein movement is restricted by the sugar matrix. Trehalose may be particularly effective due to its ability to transition between one crystalline form and another without relaxing its structural integrity, and / or due to its particularly high glass transition temperature compared to other disaccharides such as sucrose. Additionally, in amorphous trehalose, there are localized pockets of crystalline dihydrate that capture residual water molecules and immobilize them when water is scarce. An additional benefit of including a non-reducing disaccharide such as trehalose is that the presence of the non-reducing disaccharide facilitates resuspension of the powdered material.

[0073] amino acid The delivery systems according to all aspects of the present invention may include the optional presence of one or more additional amino acids.

[0074] In its broadest sense, the term "amino acid" encompasses any artificial or naturally occurring organic compound that contains an amine (-NH2) functional group and a carboxyl (-COOH) functional group. The term "amino acid" includes alpha amino acids, beta amino acids, gamma amino acids, and delta amino acids. The term "amino acid" includes amino acids of any chiral configuration. According to some embodiments (e.g., when the nanoparticles of the invention are prepared with one or more of PC, hydrogenated PC, SA, DOPE, DC-cholesterol, and derivatives thereof), the amino acid is preferably a naturally occurring alpha amino acid. It may be a proteinogenic or non-proteinogenic amino acid (such as carnitine, levothyroxine, hydroxyproline, ornithine, or citrulline).

[0075] In a preferred embodiment, the amino acid comprises arginine, histidine, lysine, proline, or glycine, or a mixture thereof, in particular a mixture of arginine and glycine or a mixture of glycine and lysine. Preferably, the amino acid is arginine, glycine, a combination of glycine and arginine, or a combination of glycine and lysine. In a particularly preferred embodiment, the amino acid comprises glycine or a combination of glycine and lysine. Such amino acids may function to stabilize solid biocompatible particles, in particular biodegradable biocompatible particles such as silicon particles. Amino acids advantageously control (e.g., reduce the rate of) hydrolysis of silicon both during storage and in vivo.

[0076] When the delivery system comprises silicon particles, the amino acid (e.g., glycine or a mixture of glycine and lysine) is optionally present in a weight ratio to the silicon of 500:1 or more, 50:1 or more, 5:1 or more, 2.5:1 or more, 1:1 or more, or 0.5:1 or more, or 0.05:1 or more. Preferably, the amino acid is glycine, which is optionally present in a weight ratio to the silicon of 500:1 or more, 50:1 or more, 5:1 or more, 2.5:1 or more, 1:1 or more, or 0.5:1 or more, or 0.05:1 or more. Advantageously, this ratio of amino acid to silicon further influences and stabilizes the release rate of biological species, such as RNA molecules, associated with the particles.

[0077] According to all aspects of the invention, the delivery system advantageously comprises a lipid component (e.g. comprising a cationic lipid) and a biocompatible particle treated with an amino acid. The delivery system may additionally comprise a non-reducing disaccharide such as trehalose.

[0078] biological species According to all aspects of the present invention, the biological species may include nucleic acid, antigen, or vaccine. Nucleic acid may be DNA or RNA, in particular RNA, or plasmid DNA. RNA may be, for example, mRNA or saRNA, shRNA or siRNA, in particular mRNA vaccine. mRNA may be self-amplifying mRNA that can be amplified in vivo. The method and preparation of the present invention have been found to be particularly effective in stabilizing RNA, which is particularly susceptible to degradation, both ex vivo during storage and in vivo after administration to a patient.

[0079] The nucleic acid may be derived from a biological source (e.g., in vitro transcribed or plasmid nucleic acid). Advantageously, the preparation of the invention protects the nucleic acid (e.g., RNA) derived from a biological source from degradation by enzymes from the biological source. Thus, the preparation of the third aspect of the invention may protect biologically derived nucleic acid from degradation by enzymes from the biological source during storage. Similarly, the method of the second aspect of the invention may be a method of protecting nucleic acid derived from a biological source from degradation by enzymes from the biological source. Certain embodiments of the biological material species according to the invention comprise nucleic acid (such as mRNA) derived from a biological source and low but measurable levels of degradative enzymes derived from that biological source.

[0080] In its broadest sense, the term "siRNA" encompasses small interfering RNA (siRNA), sometimes known as small interfering RNA or silencing RNA, and includes double-stranded RNA molecules of 5-50 base pairs in length, e.g., 10-45 base pairs, 15-40 base pairs, 20-30 base pairs, and particularly 20-25 base pairs in length, that operate within the RNA interference (RNAi) pathway.

[0081] The term "mRNA" encompasses messenger RNA and may optionally include mRNAs that contain a 5 prime cap and / or polyadenylated end. Alternatively, either of these features may be absent. mRNAs may be, in certain embodiments, 100 base pairs or more, 200 base pairs or more, 300 base pairs or more, 500 base pairs or more, 1000 base pairs or more in length.

[0082] The RNA according to the embodiments of all aspects of the present invention may be naturally occurring or chemically modified to enhance their therapeutic properties, such as enhanced activity, increased serum stability, reduced off-target, and reduced immunological activation. Chemical modifications to RNA may include any modifications generally known in the art. The mRNA of the present invention may code for multiple proteins. For example, a viral antigen and an adjuvant protein, or multiple viral antigens.

[0083] The biological species may be an antigen, or an mRNA encoding an antigen, thereby providing a preparation that is a vaccine. The antigen may be an antigen of a pathogen or a viral antigen, such as an antigen of SARS-CoV-2 (e.g., an antigen derived from the spike protein of SARS-CoV-2).

[0084] Nucleic acids such as RNA for use in accordance with the present invention include double- and single-stranded DNA, RNA, DNA:RNA hybrids, and hybrids between PNA (peptide nucleic acid), RNA, or DNA. The term also includes known types of modifications, such as, for example, labels known in the art, methylation, "capping," substitution of one or more naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, modifications with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), modifications with negatively charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), and modifications with positively charged linkages (e.g., aminoalkyl phosphoramidates, aminoalkyl phosphotriesters), modifications that include pendant moieties such as proteins (including nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), modifications with interfering agents (e.g., acridine, psoralen, etc.), modifications that include chelators (e.g., metals, radioactive metals, boron, metal oxides, etc.), modifications that include alkylating agents, modifications with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of polynucleotides or oligonucleotides.

[0085] As used herein, the terms "nucleoside" and "nucleotide" are understood to include moieties that contain not only known purine and pyrimidine bases, but also other heterocyclic bases that have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, or other heterocycles. Modified nucleosides or nucleotides also include modifications to the sugar moiety, such as where one or more of the hydroxyl groups are replaced with halogens, aliphatic groups, or functionalized as ethers, amines, or the like. Other modifications to nucleotides or polynucleotides include rearranging, adding, substituting, or otherwise changing functional groups on the purine or pyrimidine bases that form hydrogen bonds to the respective complementary pyrimidines or purines (e.g., isoguanine and isocysteine, etc.). In some embodiments, the oligonucleotides and / or probes include one or more, two or more, three or more, or four or more modified nucleotides.

[0086] In some embodiments, a nucleic acid, such as an RNA, disclosed herein comprises one or more universal bases. As used herein, the term "universal base" refers to a nucleotide analog that can hybridize to two or more 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, 5-nitroindole.

[0087] Biological species containing siRNA may optionally include siRNA synthetically produced by chemical synthesis outside of a biological system. Such siRNA may be produced without nuclease degradation. However, a preferred method of producing longer nucleic acids such as mRNA (e.g., for use in vaccine preparations) involves the use of biological systems. mRNA is usually purified from the biological system to reduce the level of degradative enzymes (e.g., RNA-ase). It may be difficult to completely eliminate all degradative enzymes. This may usually require storage at low temperatures to minimize enzyme activity. However, according to the present invention, it has been found that by preparing nucleic acids (such as mRNA) to form complexes with the delivery system of the present invention, particularly a delivery system comprising particles comprising hydrolyzable silicon, lipid components, and non-reducing disaccharides, and then dispersing the complexes in a biodegradable gel matrix, the shelf life of the mRNA in the preparation can be extended, and perhaps the need for low temperature storage can be abolished, reduced, or reduced.

[0088] Thus, the method of the second aspect of the invention includes a method of protecting biological species (e.g., mRNA, saRNA, shRNA, or siRNA) from degradation due to enzymes present in the nucleic acid preparation. Preferably, enzymatic degradation at room temperature (20°C) is reduced by at least half, more preferably at least 5-fold, at least 10-fold, at least 35-fold, at least 50-fold, at least 100-fold, at least 500-fold, or at least 1000-fold compared to an equivalent composition not containing particles of hydrolyzable silicon-containing material. Preferably, the biological species in the preparation of the invention has a half-life at 4°C of at least 3 months, at least 6 months, or at least 12 months. It has been found that the preparation prepared by the method of the invention does not degrade when stored at 40°C for at least 48 hours, or when stored at room temperature (20°C) for at least 8 days.

[0089] Association of biological species with particles The biological species and the delivery system that contacts it are advantageously associated together in a complex. Usually, the biological species is loaded into a delivery system that includes biocompatible solid particles (e.g., silicon particles), on which lipid components (e.g., including cationic lipids and / or ionizable lipids) are preloaded, preferably with phospholipids and PEG lipids and / or structured lipids, and optionally with other components such as non-reducing disaccharides (especially trehalose) and amino acids. Thus, the contact between the biological species and the delivery system usually forms a complex that includes biocompatible solid particles (especially silicon particles) loaded with lipid components, non-reducing disaccharides, and the biological species, along with any additional components such as amino acids.

[0090] Advantageously, 80% or more (e.g., 90% or more) of the weight of the nucleic acid (e.g., siRNA or mRNA) or other biological species present in the preparation of all aspects of the present invention is associated with the particles of the delivery system. By this is meant that the biological species is non-covalently associated with the biocompatible biodegradable particles (e.g., nucleic acid is non-covalently associated with silicon particles). It is hypothesized that if this occurs, the random movement of the biological species is reduced (e.g., due to the presence of degradative enzymes in the preparation), reducing the chance of the biological species being degraded.

[0091] The rate of degradation of the silicon particles and the end of the association with the biological species is governed by the hydrolysis of the silicon in the particles. Because this rate can be controlled, the rate at which the nucleic acid or other biological species (such as siRNA, saRNA, shRNA, or mRNA) becomes bioavailable can also be controlled to avoid dose dumping and / or ensure gradual release over a suitable long period of time.

[0092] It has been found that treating lipid-treated biodegradable biocompatible particles with amino acids (e.g., one or more of glycine, arginine, lysine, and histidine, preferably glycine, or a combination of glycine and lysine) provides beneficial stabilizing effects on biological species such as nucleic acids (e.g., mRNA, saRNA, shRNA, or siRNA). In particular, treating lipid-treated particles with amino acids has been shown to stabilize biological species such as RNA in biological fluids (e.g., in ocular tissue). Lipid-treated particles prepared in this manner with amino acids are particularly suitable for delivery to the body (e.g., by transdermal injection, intravitreal injection, and intraocular implantation).

[0093] To aid in the complexation of biological species with the particles of the delivery system, the delivery system may optionally further comprise a polycationic nucleic acid binding component. The term "polycationic nucleic acid binding component" is well known in the art and refers to a polymer with three or more repeating cationic amino acid residues or other cationic units carrying positively charged groups, which can form a complex with nucleic acid under physiological conditions. An example of a nucleic acid-binding polycationic molecule is an oligopeptide that contains one or more cationic amino acids. Such an oligopeptide may be, for example, an oligolysine molecule, an oligohistidine molecule, an oligoarginine molecule, an oligoornithine molecule, an oligodiaminopropionic acid molecule, or an oligodiaminobutyric acid molecule, or a complex oligomer that contains or consists of any combination of histidine, arginine, lysine, ornithine diaminopropionic acid, and diaminobutyric acid residues. Further examples of polycationic components include dendrimers and polyethyleneimines.

[0094] Favourable combinations According to all aspects of the present invention, a particularly preferred embodiment relates to an active ingredient that is a nucleic acid (especially an mRNA, and especially an mRNA that codes for an antigen of an mRNA vaccine). In some embodiments, a cationic lipid (e.g., a lipid that is or contains DOTAP) is present. However, as shown in the examples herein below, it has been found that DOTAP is not necessary for the preparation. Thus, according to the examples, in a particularly preferred embodiment, the lipid(s) is or comprises one or more of a phospholipid (such as DPPC and / or DOPE) and a lipidated oligopeptide with one or more amino acid residues that are cationic at pH 7.4 (physiological pH; examples include lysine and arginine). Optionally, one or more sugars (especially trehalose) and / or one or more amino acids (especially glycine) are also present.

[0095] Alternatively, in other preferred embodiments, the lipid or lipids are or comprise one or more phospholipids (such as DPPC and / or DOPE) and are formulated with one or more coenzymes (e.g., NAD); one or more flavanols (e.g., quercetin), and / or one or more amino acids (e.g., tyrosine). Optionally, one or more sugars (especially trehalose) and / or one or more amino acids (especially glycine) are also present.

[0096] Biodegradable Gel Materials A "gel" is defined by IUPAC as a non-fluid colloidal or polymeric network that is extended throughout its entire volume by a liquid. A gel is usually a dilute cross-linked system that does not flow in the steady state and is composed of two or more components, one of which is a liquid present in a significant amount and the other of which is a cross-linked polymer matrix swollen by the liquid. Cross-links at the network junctions can be provided by chemical bonds between the polymer chains or by physical cohesion of the polymer chains at the network junctions. The biodegradable gel material of the present invention is a gel that does not have harmful effects on the human body and degrades in vivo into non-harmful degradation products.

[0097] In step (iii) of the method of the invention (i.e. the method of the first, second, fourth and fifth aspects of the invention), the biological species and the delivery system are dispersed in a biodegradable gel material. Similarly, the preparation of the third aspect of the invention comprises a biodegradable gel material. The medicament of the sixth aspect of the invention may comprise a biodegradable gel material or may comprise a residue of a biodegradable gel material. The material forming the biodegradable gel matrix of the preparation of the third aspect of the invention is advantageously present in the medicament of the sixth aspect of the invention, but not necessarily in gel form. For example, the material forming the biodegradable gel may be dissolved in the liquid medium of the medicament of the sixth aspect of the invention. Advantageously, the material forming the biodegradable gel matrix is ​​not removed during preparation of the medicament.

[0098] Gel materials are typically polymer networks formed by physical aggregation of polymer chains. The aggregation can be the result of, for example, hydrogen bonding and / or crystallization, resulting in locally ordered regions that act as network junctions. The swollen network can be a thermoreversible gel, where the locally ordered regions are thermoreversible. Alternatively, the gel material can be a covalent polymer network (e.g., a network formed by cross-linking polymer chains).

[0099] The gels may be in the form of xerogels, in which the liquid swelling agent (e.g., water) is removed, leaving a network of crosslinked polymers open. Because such xerogels lack water, the implanted delivery system and biological species are protected from degradation mechanisms that involve water.

[0100] The gel may be a hydrogel, where the swelling agent is water. Hydrogels are essentially hydrated materials, where the polymer network often comprises a small portion of the total volume. Thus, compared to dry materials, adhesion of hydrogels relies primarily on sparse and loosely packed adhesive junctions surrounded by water. Hydrogel materials are usually formed from hydrophilic polymers that can absorb water without dissolving in water. Hydrogel materials can absorb 5% water by weight, usually 10% water by weight or more. Hydrogels may be formed from natural, synthetic, or semi-synthetic polymers that are physically or covalently crosslinked. Hydrogels of natural origin include chitosan-based hydrogels, alginate-based hydrogels, fibrin-based hydrogels, gelatin-based hydrogels, cellulose-based hydrogels, and hyaluronic acid-based hydrogels. Examples include some naturally occurring starches, such as potato starch. Synthetic hydrogels include poly(ethylene glycol) or poly(vinyl alcohol) polymer matrices. Semi-synthetic hydrogels are usually gelatin methacryloyl hydrogels, which are gelatin-based polymers functionalized with synthetic methacryloyl groups, and functionalized natural hydrogels, such as hydroxypropylmethylcellulose (HPMC) "hypromellose". The hydrogels used in the present invention are biocompatible and biodegradable. Preferred hydrogels include hyaluronic acid-based hydrogels, such as sodium hyaluronate, and cellulose-based hydrogels, such as hypromellose.

[0101] It is believed that the gel protects the embedded material from degradation, at least in part, due to the reduced ability of two functional groups of the embedded material to react with one another. Thus, water-containing gels, i.e., hydrogels, have been found to be effective in stabilizing the composites of the invention due to their ability to immobilize species in the gel matrix, which prevents degradation reactions from occurring.

[0102] It is believed that non-covalent interactions between the solid particles (e.g., silicon particles) of the delivery system stabilize the gel structure. In particular, it has been found that the non-covalently crosslinked gel structure is stabilized by the embedding of the solid particles of the delivery system. As a result, the gel structure is then stabilized while protecting the embedded delivery system and biological species from degradation. Thus, it has been found that the preparation based on the biodegradable gel of the present invention is advantageous for the storage and protection of biological materials from degradation without the need for permeable binding within the gel matrix. Furthermore, the storage-stable preparation comprising the biodegradable gel can be used as a medicament or can be used to prepare a medicament without the need for extraction of biological species.

[0103] Preparation of preparations Preparation of particles of biocompatible biodegradable materials The delivery system includes particles of a biocompatible solid material, which may be metallic materials, metalloid materials, typically gold, carbon tubes, graphene, silica, porous elemental materials including porous diamond, gold, silver, and hydrolyzable silicon.

[0104] In a preferred embodiment, the solid biocompatible particles are hydrolyzable silicon particles. Hydrolyzable silicon can be conveniently prepared by conventional techniques in the art (e.g., grinding process) or other known techniques for particle size reduction. Silicon-containing particles can be made from sodium silicate particles, colloidal silica, or silicon wafer material in certain embodiments. Macro- or micro-scale particles are ground in a ball mill, planetary ball mill, plasma or laser ablation, or other size reduction equipment. The resulting particles can be air classified to recover nanoparticles. It is also possible to use plasma and laser ablation for nanoparticle generation.

[0105] Porous silicon particles can be produced, for example, by anodically etching silicon wafers with aqueous HF acid, followed by crushing and sieving or air classifying the etched wafers to obtain a uniform particle size selection. Alternatively, silicon powder can be prepared or purchased and then etched to increase the porosity of the powder. Etching can increase the surface area of ​​the silicon material by 1.5 times or more, 2 times or more, 2.5 times or more, 3 times or more, 3.5 times or more, or 4 times or more relative to the surface area of ​​a non-porous material of comparable size. By varying the HF concentration and the current density and exposure time, the density of the pores and their size can be controlled and monitored by scanning electron microscopy and / or nitrogen adsorption / desorption volumetric isotherm measurements.

[0106] According to certain embodiments, the porosity is 30% or more, 40% or more, 50% or more, or 60% or more. This means that 30%, 40%, 50%, or 60% of the particle volume is in the pore space, respectively. Preferred pore sizes are in the range of 1 nm to 50 nm (e.g., 5 nm to 25 nm).

[0107] Formation of a delivery system Additional components of the delivery system may be loaded onto the particles of biocompatible biodegradable material prior to contact with the biological species, simultaneously with contact of the particles with the biological species, or after contact of the particles with the biological species. Typically, the non-reducing disaccharide component and the amino acid component are combined with the particles prior to contact with the biological species. The lipid component of the delivery system may be combined with the particles prior to contact with the biological species, simultaneously with contact of the particles with the biological species, or after contact of the particles with the biological species.

[0108] The delivery system may be prepared by dispersing the particles (e.g., porous silicon nanoparticles) in a volatile alcohol solvent (such as methanol, ethanol, or propanol, e.g., methanol) to activate the particles, followed by mixing with an aqueous solution containing a non-reducing disaccharide (e.g., trehalose) and an amino acid (e.g., glycine). Alternatively, the solvent is removed from the activated particles, and the activated particles are dispersed in nuclease-free water with the non-reducing disaccharide (e.g., trehalose) and an amino acid (e.g., glycine). The lipid is then added as a solution or dispersion (e.g., in methanol). Sonication may optionally be used to facilitate dispersion. The resulting dispersion is extruded in a flowing state at elevated temperature (e.g., 60°C) onto an extrusion membrane. Multiple passes may be employed. After extrusion, methanol and potential other impurities are removed, e.g., using tangential flow filtration.

[0109] Contact of the biological species with the delivery system In step (i) of the method of the first and second aspects of the invention, as well as certain embodiments of the method of the fourth and fifth aspects of the invention, the biological species is contacted with the delivery system. The contacting step may, for example, only involve mixing a solution of the biological species with the delivery system under conditions that result in complexation of the biological species with the delivery system. The contacting step may involve contacting the biological species with particles of a biocompatible biodegradable material before adding the lipid component. Addition of the lipid component after contacting the biological species with the particles of a biocompatible biodegradable material may promote the formation of a lipid encapsulation layer on the surface of the particles that encapsulates both the biocompatible biodegradable material and the biological species. Typically, particles of a biocompatible biodegradable material such as porous hydrolyzable silicon are mixed with a non-reducing disaccharide, a biological species, and optionally an amino acid (or amino acids), and then contacted with the lipid component to form lipid encapsulated particles, in which the biological species is associated with silicon or other biocompatible biodegradable material within the lipid encapsulation.

[0110] It is hypothesized that delivery systems containing particles, particularly particles of hydrolyzable silicon, increase the stability of biological species, particularly nucleic acids, by restricting the mobility (molecular movement) of the biological species. In addition, when additional components of the delivery system, such as lipids and peptides, are bound to the silicon particles, a stabilized complex can be formed in which both the hydrolyzable silicon and the additional components of the delivery system, such as lipids and peptides, are stabilized. For example, the lipid components can form a lipid membrane around the silicon particles, which protects the silicon from hydrolysis, stabilizes the lipid molecules from degradation, and stabilizes the biological species, such as RNA, complexed to the silicon. It is believed that positively charged species, such as cationic lipids and other lipid components with positive charges, including phospholipids, bind to the silicon, thereby stabilizing the integrity of the positively charged components. Unlike liposomes, which contain oil / water / surfactant interactions, there is no water in the systems of the present invention, which contain lipid-silicon and other organic molecules in a non-vesicular structure, thus eliminating an aqueous environment for enzymes or free radicals to operate. It is therefore believed that by including the biological species with a non-vesicular structure formed by silicon particles and lipid components, the biological species is protected from degradation.

[0111] The solid material may optionally be conveniently passed through a filter (a process sometimes referred to as "extrusion") or subjected to a tangential flow filtration / sterilization process, depending on the nature of the desired product profile.

[0112] Freeze drying In step (ii) of the methods of the first and second aspects of the invention, as well as certain embodiments of the methods of the fourth and fifth aspects of the invention, the biological species and the delivery system are preferably freeze-dried (e.g. to form a powder). Freeze-drying removes water from the biological species and the delivery system, allowing the dry powder to be dispersed in a high concentration biodegradable gel in the subsequent dispersion step (iii), thereby minimizing the presence of water. It is also envisaged that the freeze-drying step may facilitate complexation of the biological species and the delivery system (e.g. facilitating binding of nucleic acid and lipid components to the silicon particles).

[0113] Dispersion of biological species and delivery systems in biodegradable gels. In step (iii) of the method of the first and second aspects of the invention, as well as of certain embodiments of the method of the fourth and fifth aspects of the invention, the biological species and the delivery system are dispersed in a biodegradable gel. Dispersion in a biodegradable gel typically involves preparation of an aqueous dispersion of a biodegradable gel material and introduction of the biological species and the delivery system to the aqueous dispersion, either as an aqueous dispersion or as a solid powder. Preferably, the biological species and the delivery system are freeze-dried after contact and introduced into the biodegradable gel as a powder. After dispersing the biological species and the delivery system in the aqueous dispersion of the biodegradable gel, the resulting dispersion may be stored as a liquid dispersion or may be converted into a solid or semi-solid gel (e.g., in a gelation step, optionally including removal of water). The biological species and the delivery system are advantageously embedded in the matrix of the biodegradable gel. Embedding in a biodegradable gel may facilitate immobilization of the biological species, lipid components, and any contaminants that may promote degradation (such as enzymes), thus halting degradation.

[0114] Pharmaceuticals The (optionally oral or injectable) medicament of the present invention (e.g., the medicament of the sixth aspect of the present invention) may be, for example, a liquid formulation or a solid formulation. The medicament may be in the form of an injectable formulation that includes a biological species complexed with a delivery system together with a compound capable of forming a hydrogel matrix dispersed in a liquid carrier system. Solid formulations include formulations for oral administration (such as capsules and tablets, suppositories, dissolving films for sublingual administration, etc.), and transdermal delivery patches (such as a delivery patch using multiple microneedles for injection).

[0115] The preparation or medicament of the invention may be provided in a delivery device (eg, an injection device such as a syringe or multiple microneedles). EXAMPLES

[0116] Various aspects and embodiments of the present invention are illustrated with reference to the following non-limiting examples. To the extent that the examples are outside the scope of some aspects of the present invention, they are included as comparative examples.

[0117] Summary of Experimental Results Binding efficacy studies were performed with the delivery systems dispersed in an adhesive-like biodegradable gel matrix and continuously exposed to 40°C for over 48 hours. In particular, gel retardation assays were performed on siRNA loaded into delivery system preparations SIS0012&MVI0010 (silicone) and SIS0013 (boron-doped silicon) embedded in sodium hyaluronate (1 w / w%) biodegradable gel or hypromellose (2 w / w%) biodegradable gel after storage at room temperature or 40°C, and analyzed by gel electrophoresis at the indicated time points (0 h, 2 h, 4 h, 6 h, 24 h, and 48 h). The gel electrophoresis system used for these experiments consisted of a gel electrophoresis device, a precast agarose gel (1%, with electrophoresis buffer, and electrodes mounted within the gel), and a camera. Samples, controls (naked siRNA), and DNA ladders were mixed with the required amount of loading buffer to a total volume of 20 μL. The amount of RNA used per well cell was 200 ng / well, which is in accordance with standard protocols for agarose gel assays and is subsaturating.

[0118] The results revealed that the nucleic acid binding ability of delivery systems SIS0012&MVI0010 (with silicon) and delivery system SIS0013 (with boron-doped silicon) was maintained even when the samples were continuously exposed to 40°C for 48 hours. It was observed that all of the tested delivery system preparations were able to form complexes with siRNA and completely stop its migration through the gel. Furthermore, both SIS0012 and SIS0013 preparations were found to be stable at room temperature and at 40°C for up to 48 hours.

[0119] Preparation of lipid-functionalized silicon nanoparticle (SiNP) delivery systems The lipid components were dissolved in methanol. At the same time, 30 nm diameter porous silicon dioxide nanoparticles (SiNPs) of either undoped or boron-doped silicon from American Elements (CAS number 7440-21-3, supplier code SI-E-0181M-NP100N) were dispersed in methanol, followed by evaporation of the solvent in a slow evaporation process to obtain activated SiNPs. This activation step is aimed at making the SiNPs suitable for dispersion in water. The activated SiNPs are dispersed in nuclease-free water with glycine and, optionally, trehalose.

[0120] For the preparation of conjugate SIS0012 and conjugate SIS0013, a dispersion of activated SiNPs with glycine and trehalose in nuclease-free water is used to hydrate the lipids. The dispersion is extruded onto an extrusion filter. After solvent evaporation, the lipid-functionalized SiNP delivery system is collected.

[0121] For the preparation of MVI0010, lipids are solubilized in methanol and then slowly injected into a dispersion of SiNPs with glycine and trehalose in nuclease-free water, followed by application of tangential flow filtration using 3 cycles at 0.8 μm, 3 cycles at 0.4 μm, and 3 cycles at 0.1 μm.

[0122] The compositions of the delivery systems SIS0012 / MVI0010 and SIS0013 are shown in Table 1.

[0123] [Table 1]

[0124] Characterization of boron-doped silicon present in SIS0013 Single-sided polished wafer, CZ Diameter: 150±0.2mm Orientation: (100)±1° Type: p / boron Resistivity: 0.014±25%Ohmcm. 5×10 18 atoms / cm3 Close to. Primary flat: 57.50±2.5mm Primary flat 1 position: D <100> ~{110} Thickness: 675±15μm TTV:≦18μm TIR:≦5μm The thin film is approximately 40% porous and up to 50 μm thick before being crushed into particles.

[0125] Preparation of stock solutions (a) Si-NP+GLY+THR: 50 mg of SiNPs, 50 mg of trehalose and 25 mg of glycine were suspended in 50 ml of nuclease-free water and sonicated for 60 min. (b) DOTAP-Cl: 50 mg of DOTAP was solubilized in 10 ml of methanol and sonicated until completely solubilized. (c) DOPE: 60 mg of DOPE was solubilized in 12 ml of methanol and sonicated until completely solubilized. (d) mPEG2000-DSPE: 40 mg of mPEG2000-DSPE was solubilized in 8 ml of methanol and sonicated until completely solubilized.

[0126] Thin film preparation (a) The amounts of lipids shown in Table 1 were transferred from stock solutions into clean glass round-bottom flasks and mixed. (b) The solvent was evaporated using a rotary evaporator with a 23° C. water bath and vacuum.

[0127] Rehydration of thin films (a) Add Si-NP+GLY(+THR) solution to obtain a lipid:SiNP ratio of 16:1 to the lipid thin film and adjust the final volume to 10 mL with nuclease-free water. (b) Cover the flask with Parafilm, rehydrate the thin film, and agitate the flask in a water bath (60° C.) for 5 minutes. (c) Divide 1 mL of the sample in the RNA-free Eppendorf into a total of 10 Eppendorfs. Store the suspension in the refrigerator.

[0128] Extrusion Process (a) The lipid suspension is passed 20 times through membrane filters with pore sizes of 0.4 μm and 0.1 μm at 60° C.

[0129] Example 1 Assessment of the stability of commercially available mRNA-loaded preparations embedded in sodium hyaluronate biodegradable gels at different temperatures using agarose gel electrophoresis Preparation of mRNA delivery system complex The Dasher GFP mRNA stock solution has a concentration of 1 mg / mL in nuclease-free water. This stock solution was diluted with an equal volume of nuclease-free water to give a working solution with a concentration of 0.5 mg / mL.

[0130] The mRNA delivery system complexes were prepared by mixing 50 μL (25 μg) of mRNA solution with 200 μL of delivery system dispersion (delivery system / RNA weight ratio of 12:1). The mixture was incubated at room temperature for 40 min to allow complete complexation and then used in liquid form or lyophilized and redispersed in 250 μL of nuclease-free water. The mRNA delivery system complexes were then embedded by mixing with an equal volume (250 μL) of sodium hyaluronate hydrogel. To prepare controls, naked mRNA, either in liquid form or in lyophilized and reconstituted solution, was mixed with an equal volume of sodium hyaluronate hydrogel (1 w / w%). Naked mRNA solution of the same concentration was also used as an additional control. The final concentration of mRNA in all samples was 0.05 μg / μL. To minimize the risk of cross-contamination during storage and analysis, the final preparation was divided into 30 μL aliquots (for each analysis time point). All samples and controls were stored at two different temperatures, room temperature and in a water bath set at 40°C. The amount of RNA used per well cell was 200ng / well, which follows the standard protocol for agarose gel assays, which is less than saturating. The performance of a gel electrophoresis assay, like any other biological assay, can depend to some extent on the correct parameters used. The molecular weight of the nucleic acid, the choice of the correct buffer, and the gel loading amount can all be important. 200ng / well was chosen to ensure that the gel was neither underloaded nor overloaded.

[0131] Agarose gel electrophoresis At the indicated time points (0, 6, 24, and 48 hours), the mRNA delivery system-biodegradable gel preparations and controls were subjected to gel electrophoresis. A DNA ladder (E-Gel™ 1Kb plus Express DNA ladder) was also used as a size guide. Samples were loaded onto E-Gel™ agarose gels (1%) in an E-Gel™ Power Snap electrophoresis device. Gels were transilluminated and imaged at 3 and 7 minutes using an E-Gel™ Power Snap electrophoresis camera. The total amount of mRNA loaded onto the gel was 0.2 μg / well for all samples and controls.

[0132] Test 1: Stability of liquid mRNA delivery system complexes Agarose gel electrophoresis images of liquid mRNA delivery system preparations embedded in sodium hyaluronate hydrogels immediately after preparation and up to 48 hours after storage at either room temperature or 40° C. are shown in Figures 1 and 2. Figure 1 shows gel electrophoresis images of liquid mRNA delivery system preparations embedded in sodium hyaluronate (SH) hydrogels (3, 4, 6, 7) as prepared and after 6 hours of storage at room temperature (RT) or 40° C. Figure 2 shows gel electrophoresis images of liquid mRNA delivery system preparations embedded in sodium hyaluronate hydrogels (3, 4, 6, 7) after 24 hours and 48 hours of storage at room temperature (RT) or 40° C. Naked mRNA (1, 8) and naked mRNA embedded in hydrogels (2, 5) were used as controls.

[0133] As observed, both naked mRNA (1, 8) and mRNA embedded in hydrogel (2, 5) migrated through the agarose gel. Also, some of the hyaluronic acid co-migrated with the mRNA (2, 5), which resulted in a smear band rather than the sharp band observed with naked mRNA (1, 8). Naked mRNA (8) kept at 40°C started to degrade after 48 hours, as indicated by a fuzzy and ambiguous band. Similarly, mRNA embedded in hydrogel and stored at 40°C (5) showed a weak signal after 48 hours, suggesting some degradation of the mRNA. In the mRNA delivery system biodegradable gel preparations (3, 4, 6, 7), there was some leakage of hyaluronic acid from the well into the agarose gel in the area near the well. However, the short migration distance in the gel suggests that there was no mRNA release from the biodegradable gel.

[0134] Study 2: Stability of lyophilized and reconstituted mRNA delivery system complexes Agarose gel electrophoresis images of freeze-dried and reconstituted mRNA delivery system preparations embedded in sodium hyaluronate hydrogels immediately after preparation and up to 48 hours after storage at either room temperature or 40° C. are shown in Figures 3 and 4. Figure 3 shows gel electrophoresis images of freeze-dried and reconstituted mRNA delivery system preparations embedded in sodium hyaluronate (SH) hydrogels (3, 4, 6, 7) upon preparation and after 6 hours of storage at room temperature (RT) or 40° C. Figure 4 shows gel electrophoresis images of freeze-dried and reconstituted mRNA delivery system preparations embedded in sodium hyaluronate (SH) hydrogels (3, 4, 6, 7) after 24 hours and 48 hours of storage at room temperature (RT) or 40° C. Naked mRNA (1, 8) and freeze-dried and reconstituted mRNA embedded in hydrogels (2, 5) were used as controls.

[0135] Similar to what was observed for the liquid mRNA delivery system preparations of Figures 1 and 2, lyophilized and reconstituted mRNA delivery system preparations embedded in hydrogels (3, 4, 6, 7) stopped the migration of the mRNA through the agarose gel and were stable for up to 48 hours at both room temperature and 40°C, whereas lyophilized and reconstituted mRNA simply loaded onto the hydrogel (2, 5) passed through the gel, indicating no complexation between the hydrogel and the mRNA. From these data it can be inferred that the mRNA delivery system preparations (3, 4, 6, 7) are stable during and after the lyophilization process and do not impair their association or stability.

[0136] Example 2 Assessment of the stability of siRNA loaded into a delivery system and embedded in sodium hyaluronate (2 w / w%) hydrogels after storage at room temperature or 40°C using agarose gel electrophoresis Preparation of siRNA delivery system complexes siGLO™ Green stock solution was prepared by dissolving 50 nMol (0.66 mg) of siRNA powder in 1 mL of nuclease-free water.

[0137] Sodium hyaluronate biodegradable gel was prepared by dissolving the powder in nuclease-free water to a concentration of 2 w / w%.

[0138] The siRNA delivery system complexes were prepared by mixing 4.5 μL of siGLO™ Green stock solution (equivalent to 3 μg of siRNA) with 23 μL of SIS0012 delivery system solution, SIS0013 delivery system solution, and MVI0010 delivery system solution (i.e., lipid to siRNA weight ratio of 12) and incubated at room temperature to ensure complete complexation. The siGLO delivery system complexes were then lyophilized overnight. The lyophilized powder was then dissolved in 3 μL of nuclease-free water and embedded in 23 μL of sodium hyaluronate (SH) biodegradable gel. A siGLO-MVI0010 preparation that was not lyophilized and loaded into the biodegradable gel was also used.

[0139] All preparations were prepared as 25 μL aliquots (per analysis time point), stored either at room temperature or in a water bath set at 40° C. for up to 48 hours, and analyzed by gel electrophoresis. The amounts of siGLO delivery system biodegradable gel preparation, naked siGLO, and DNA ladder used in the gel retardation assay, as well as the total amount of siRNA / DNA per well, are shown in Table 2:

[0140] [Table 2]

[0141] Agarose gel electrophoresis At the indicated time points (0, 6, 24, and 48 hours), samples were analyzed by agarose gel electrophoresis using an E-Gel™ Power Snap electrophoresis device and E-Gel™ agarose (1%) gels. Samples, naked siGLOs, and DNA ladders were loaded onto the gels in a volume of 20 μL / well. Naked siRNAs and DNA ladders were diluted in nuclease-free water (NFW) and then loaded onto the agarose gels according to Table 1, but the preparations were used without any dilution. Gels were imaged after 3 and 7 minutes using an E-Gel™ Power Snap electrophoresis camera.

[0142] Study 3: Stability of lyophilized and reconstituted siRNA delivery system complexes Gel electrophoresis images of the loaded siRNA delivery system biodegradable gel preparations stored at room temperature or at 40° C. are shown in FIG. 5 and FIG.

[0143] As can be seen, all preparations efficiently complexed the siRNA and stopped it from migrating through the gel. The complexes were stable for 6 hours at both room temperature and 40°C, as indicated by no siRNA passing through the gel. However, after 24 hours, a significant amount of siRNA was released into the gel by the MVI0010 preparation in liquid stored at 40°C (9), indicating the lack of stability of this preparation at high temperatures. On the other hand, in the siRNA-SIS0012 and SIS0013 preparations embedded in biodegradable gels (6, 7), only a trace amount of siRNA was observed passing through the gel after 48 hours of storage at 40°C, which was slightly higher in the SIS0013 biodegradable gel preparation (7) compared to the SIS0012 biodegradable gel preparation (6) and the MVI0010 biodegradable gel preparation (8). As can be inferred from these data, the siRNA delivery system preparations embedded in sodium hyaluronate biodegradable gels were stable for 48 h at both room temperature (2, 3, 4) and 40 °C (6, 7, 8), whereas the siRNA-MVI0010 preparations in liquid form (5, 9) were stable only at room temperature (5) and started to release siRNA from 24 h at 40 °C (9). These findings suggest the role of the biodegradable gel matrix in stabilizing the siRNA delivery system complexes, especially at elevated temperatures.

[0144] conclusion Mixing the siRNA delivery system complex with high concentration sodium hyaluronate hydrogel resulted in stability for 48 h at room temperature and at 40° C. siRNA-MVI0010 complexes that were not mixed with hydrogel (5, 9) were stable for 48 h at room temperature (5) but less than 24 h at 40° C. (9), indicating the role of sodium hyaluronate hydrogel in stabilizing the siRNA-MVI0010 complex.

[0145] Example 3 After storage at room temperature or at 40° C., the stability of siRNA loaded into delivery system preparations SIS0012 and SIS0013 embedded in sodium hyaluronate (2 w / w%) or hypromellose (4 w / w%) hydrogels is evaluated using agarose gel electrophoresis.

[0146] Preparation of siRNA delivery system biodegradable gel formulation ADO2-siRNA (dTdT overhang) and siGLO™ Green stock solutions were prepared by dissolving siRNA powder in nuclease-free DI water according to Table 1. 250 nMol (33.33 mg) ADO2 siRNA was dissolved in 0.5 mL water to obtain a 6.66 mg / mL solution, and further diluted with water to a 1 μg / μL or 66.5 ng / μL solution. 50 nMol (0.66 mg) siGLO™ Green siRNA was dissolved in 1 mL water to obtain a 0.66 mg / mL solution, and then further diluted with water to a 66.5 ng / μL solution.

[0147] Sodium hyaluronate and hypromellose hydrogels were prepared by dissolving the powders in nuclease-free water to concentrations of 2 w / w% and 4 w / w%, respectively.

[0148] siRNA delivery system-hydrogel formulations were prepared keeping the lipid / siRNA weight ratio constant at 12. The amounts of siRNA, delivery system, and hydrogel used in the preparation of the different formulations are shown in Table 3. The formulations were prepared in small aliquots (32-33 μL for the formulations containing ADO2 siRNA and siGLO™ Green, respectively) and stored either at room temperature or in a 40° C. water bath for up to 48 h.

[0149] [Table 3]

[0150] Agarose gel electrophoresis At the indicated time points (0, 2, 4, 6, 24, and 48 hours), the preparations were analyzed by agarose gel electrophoresis. The gel electrophoresis system used for these experiments consisted of a gel electrophoresis device, a precast agarose gel (with 1%, buffer, and electrophoresis electrodes mounted within the gel), and a camera. Samples, controls (naked siRNA), and DNA ladder were mixed with the required amount of gel loading buffer to a total volume of 20 μL according to Table 8 and then loaded onto the gel. The gel was then inserted into the device chamber, and samples, controls, and DNA ladder were loaded and run for 7 minutes. The gel was transilluminated and imaged with a camera at 3 and 7 minutes.

[0151] [Table 4]

[0152] Study 4: Stability of siRNA-loaded SIS0012 preparations Gel electrophoresis images of SIS0012 biodegradable gel preparations loaded with ADO2-siRNA or siGLO™ Green and stored at room temperature are shown in Figures 7-9. As can be seen, all preparations complexed the loaded siRNA and stopped it migrating through the gel, regardless of the order of combination of the components (i.e., delivery system, biodegradable gel, and siRNA). In contrast, siRNA loaded into the biodegradable gel without a delivery system, used as a negative control, passed through the gel, indicating no binding between the siRNA and the biodegradable gel.

[0153] Nevertheless, a closer look at the images reveals that in the preparation where siRNA was first mixed with sodium hyaluronate and then combined with SIS0012, complexation was not complete and a small amount of siRNA passed through the gel. This is likely due to the high viscosity of sodium hyaluronate biodegradable gel, which caused the siRNA to be unevenly dispersed in the biodegradable gel. As a result, when the delivery system was added, the siRNA could not be completely mixed with the delivery system, and the remaining uncomplexed siRNA passed through the gel. This observation suggests that for efficient complexation of siRNA, either the delivery system should be mixed with the biodegradable gel before the addition of siRNA, or the delivery system should be mixed with the siRNA and then embedded within the biodegradable gel. The intensity of the siRNA band in this particular preparation was significantly lower after 48 hours of storage at room temperature, which suggested the degradation of free / unbound siRNA.

[0154] As can be inferred from the images, there was no difference between ADO-2 siRNA and siGLO™ Green in terms of complexation and stability. It is also noteworthy that the two types of biodegradable gels behaved differently in terms of releasing unbound siRNA into the agarose gel. The siRNA released from hypromellose appeared to have fewer fragments, while the siRNA released from sodium hyaluronate appeared as a mixture of fragments of different sizes. Perhaps a certain percentage of hyaluronic acid may also migrate with the siRNA through the gel due to its negative charge.

[0155] Gel retardation images of SIS0012 biodegradable gel-siRNA preparations stored at 40°C are shown in Figures 10-12. As can be seen, the SIS0012 biodegradable gel-siRNA preparations stored at 40°C behaved in a similar manner to the preparations stored at room temperature. Similarly, in the preparation where siRNA was first mixed with sodium hyaluronate and then combined with SIS0012, some of the siRNA passed through the gel, indicating a lack of efficient complexation due to non-uniform distribution of the siRNA within the biodegradable gel. However, the lack of siRNA bands in this preparation after 4 hours suggests faster degradation of unbound siRNA at 40°C.

[0156] Study 5: Stability of siRNA-loaded SIS0013 preparations Gel retardation images of SIS0013 biodegradable gel preparations loaded with either ADO-2 siRNA or siGLO™ Green and stored at room temperature are shown in Figures 13-15. As is evident from these figures, embedding siRNA in the biodegradable gel before mixing with the delivery system leads to inefficient complexation for the siRNA-SIS0013 hyaluronic acid preparation, similar to that observed for the siRNA-SIS0012 hyaluronic acid preparation. The higher intensity of the siRNA band compared to that observed for SIS0012 indicates a larger proportion of unbound siRNA due to poor dispersion of siRNA in the biodegradable gel. This observation further confirms the previous presumption that for efficient complexation of siRNA, either the siRNA should be mixed with the delivery system first and then embedded in the biodegradable gel, or the siRNA should be added to a homogenous mixture of the delivery system and the biodegradable gel. Again, the decrease in intensity of the siRNA band observed for this preparation after 4-6 hours and disappearance of the band after 24 hours indicates degradation of unbound siRNA over time.

[0157] Gel retardation images of SIS0013 biodegradable gel preparations loaded with either ADO2 siRNA or siGLO™ Green and stored at 40° C. are shown in Figures 16-18. Except for the preparation in which the siRNA was first embedded into the hyaluronic acid biodegradable gel and then combined with the delivery system, all preparations appeared to be stable for 48 hours, as indicated by the lack of siRNA transfer, in a manner similar to the samples stored at room temperature. The fading of the signal for the siRNA associated with this preparation after 4 hours indicates rapid degradation of unbound siRNA at 40° C.

[0158] observation Loading siRNA into SIS0012 and SIS0013 delivery system preparations with lipid / siRNA weight ratio of 12 mixed with sodium hyaluronate biodegradable gels ranging from 1-2% or hypromellose biodegradable gels 2-4% resulted in complete complexation of siRNA, suggesting this ratio as the optimal ratio for complexation. The preparations were stable for 48 hours at both room temperature and 40°C. For efficient mixing and complexation of siRNA with the delivery system, it is recommended to either mix the siRNA with the delivery system before embedding the siRNA in the biodegradable gel or mix the delivery system with the biodegradable gel before adding the siRNA. This approach will ensure uniform dispersion of siRNA in the mixture, which may allow complete complexation of siRNA with the delivery system. There was no difference between ADO-2 siRNA and siGLO™ Green with respect to complexation with the delivery system, suggesting that siGLO™ Green can be used as an alternative to ADO-2 siRNA for complexation and stability assays by gel electrophoresis.

[0159] Example 4 This experiment was performed to provide validation of stabilization data for siRNA by itself or siRNA complexed with the gel-embedded Bio-Courier delivery system at various temperatures (4°C, RT, and 40°C) using agarose gel electrophoresis with deliberate overloading.

[0160] To reaffirm that the lack of detectable siRNA bands in previous studies (Examples 1, 2, and 3) was not due to underloading of RNA into the agarose gel wells or degradation of RNA at the edges of the wells, the agarose gel well cells were deliberately overloaded with an siRNA amount of 2.6 ug / well.

[0161] siGLO was loaded into different Biocourier preparations in liquid and lyophilized form. The siGLO-Biocourier complexes embedded in hydrogels were examined at different storage temperatures using agarose gel electrophoresis.

[0162] Each well cell was overloaded with siRNA either by itself or complexed with the Bio-Courier delivery system. The purpose of this experiment was to determine whether the lack of siRNA shear or smear bands for the Bio-Courier delivery system complexed with the biological species by itself and / or embedded in the gel (experiments related to Examples 1, 2, and 3) was due not to low level detection of siRNA or eventual degradation of the biological species in the well, but to the binding effectiveness provided by the presence of the solid particle system complexed with the biological species and embedded in the biodegradable gel.

[0163] Preparation of siRNA delivery system complexes The siRNA delivery system preparation was prepared by mixing the delivery system solution with the siRNA solution (0.66 mg / mL) at a weight ratio of 12:1. The mixture was incubated at room temperature for 40 minutes to allow complete complexation and then used in liquid form or lyophilized overnight before resuspending in nuclease-free water. For samples loaded onto hydrogels, the lyophilized powder was added directly to the hydrogel. The samples were divided into 30 μL aliquots and stored at different temperatures (room temperature, 4° C., and 40° C.). At the designated time points shown in Table 5, the samples were analyzed by gel electrophoresis.

[0164] [Table 5]

[0165] Agarose gel electrophoresis The siGLO delivery system complexes were analyzed by agarose gel electrophoresis using E-Gel™ agarose gels (1%) in an E-Gel™ Power Snap electrophoresis device. Naked siGLO stored under similar conditions was used as a control in all cases. 20 μL of each sample (containing a total of 2.6 μg of siGLO) was loaded onto the agarose gel. For the control (naked siGLO) and DNA ladder, 2 μL of concentrated solution was diluted with 18 μL of nuclease-free water before loading onto the agarose gel. The gels were transilluminated and imaged at 3 and 7 min using an E-Gel™ Power Snap electrophoresis camera.

[0166] Test 5. Validation of the siRNA delivery system complexes at various temperatures when agarose gels are fully overloaded with siRNA. Gel electrophoresis images of the siGLO delivery system complex immediately after preparation are shown in Figures 19-26. In the lyophilized preparation, complete complexation was observed and no free siGLO passed through the gel, whereas in the liquid preparation, some traces of siGLO passed through the gel, indicating the presence of some unbound siGLO. Nevertheless, the amount of unbound siGLO was not significant compared to the large amount of siGLO loaded into the preparation.

[0167] The presence of strong signals at the edge of all wells suggests the lack of siGLO degradation in any of the siGLO delivery system complexes and the stability of the preparations under refrigerated and room temperature conditions for up to 28 days. The only band that did not remain at the edge of the agarose gel ladder was our control siRNA free form / siGLO, which migrated in the agarose gel as previously observed. The band intensity of naked siGLO used as a control was very strong, sharp and fully detectable, confirming that the system was functional and thus able to indicate whether there was unbinding of siRNA complex drug delivery. It is important to keep in mind that due to the very large amount of siGLO loaded into the agarose gel that produced a very strong signal, it was not possible to detect the slight decrease in signal caused by partial degradation of naked siGLO, as observed in Examples 1, 2 and 3, where the gel assay was performed under non-saturating conditions.

[0168] When preparations were exposed to 40°C, they also proved stable for up to 7 days, with the presence of strong signal at the edge of the well indicating no siGLO degradation. However, after 28 days, the signal from siGLO-GEN AVE1001 was no longer observed at the edge of the well, suggesting degradation of siGLO, whereas siGLO-MVI0015 still showed a fairly strong signal up to 28 days.

[0169] Observations and Findings Stabilization of nucleic acids, mRNA, and siRNA by incorporation into a delivery system containing particles of biocompatible silicone ("Biocourier") has been demonstrated using agarose gel electrophoresis assays. The binding of RNA to the complexes remains stable at various temperatures, indicating that the complexes are stable over time without loss of binding. Embedding the RNA-Biocourier complexes into hydrogels further improves the stability of the RNA, which offers the possibility of using RNA without the need for cryogenic storage. Sufficient overloading of the agarose gel with siRNA by itself or complexed with Biocourier confirmed the validity of the stabilization data obtained for the RNA-Biocourier complexes in hydrogels. When the agarose gel retardation assay was used under saturating conditions, i.e., with overloaded gel wells, no significant shear or smear bands were observed again as previously reported in Experiment 1, Experiment 2, and Experiment 3. The lack of significant shear or banding under saturating conditions was not due to degradation of the siRNA or lack of siRNA detectability due to an insufficient amount of biological species complexed into the gel, but was due to the efficiency of binding provided by the Biocourier delivery system and its embedding in the biodegradable gel. The efficiency of binding was indicated by the intensity of the bands at the edge of the wells, which were similar to that of the saturated naked siRNA controls that migrated into the agarose gel wells.

[0170] Example 5: Investigation into Methicillin-Resistant Staphylococcus aureus (MRSA) The formulations shown in the table below were prepared having peptides as API: [Table 6]

[0171] To prepare the preparation, the lipid components were dissolved in methanol. At the same time, porous silicon nanoparticles (SiNPs) with a diameter of 30 nm (obtained from American Elements, CAS number 7440-21-3, supplier code SI-E-0181M-NP100N) were dispersed in methanol, followed by evaporation of the solvent in a slow evaporation process to obtain activated SiNPs. This activation step aims to make the SiNPs suitable for dispersion in water. The activated SiNPs were dispersed in nuclease-free water together with glycine and arginine and peptide APIs.

[0172] After solvent evaporation, the lipid film was hydrated with an aqueous dispersion of functionalized SiNPs. The delivery system was then dispersed in the hydrogel, as detailed below. The prepared samples were refrigerated until further analysis.

[0173] 0.11%, 0.33%, or 1% w / w peptide-loaded preparations 1% Hypromellose Hydrogel Gel C: Embedded in Hypromellose 0.5% + Pluronic 0.5%

[0174] In vivo analysis: application to human skin 10 μL of 1 × 10 7 The mice were inoculated with a suspension of MRSA / ml. In a volume of 50 μL, Peptides in hypromellose gel alone (at various concentrations, see FIG. 27); Peptides bound to silicon-containing preparations of the invention using hypromellose gel (at various concentrations, see FIG. 27); No peptide (control) The samples were incubated with one of the following for 1 hour at 37° C. and 5% CO2 and then applied to the inoculated skin. After a further hour of incubation, the number of viable bacteria was determined microbiologically. The results are expressed as colony forming units (CFU). These results are shown in FIG. 27.

[0175] Additional in vivo analysis: Application to mouse nasal transport Using an in vivo MRSA-Biofilm mouse nasal transport model, selected preparations, namely B1 PS-peptide and C1-PS peptide, aimed at reducing MRSA bacterial load in the nasal cavity were tested. The proposed preparations aimed to stabilize the peptides at nasal pH and temperature, which improves their activity against MRSA. As shown in Figure 28, the preparations demonstrated enhanced performance in reducing MRSA bacterial counts by more than 90%. Details of the preparations used are given below.

[0176] Gel B1-Peptide: 1% peptide-loaded preparation bound to the silicon-containing preparation described above embedded in 1% hypromellose hydrogel Gel C1-Peptide: 1% peptide-loaded preparation bound to the silicon-containing preparation described above, embedded in hypromellose 0.5% + pluronic 0.5% GEL-Peptide: Peptide at 1% concentration embedded in 1% hypromellose hydrogel Peptide-PBS: Peptide at 1% concentration embedded in 1% hypromellose hydrogel

[0177] Example 6: Complexation of additional silicon-containing preparations with mRNA or siRNA Alternatives were investigated to replace the cationic lipids such as DOTAP used in SIS0012 and SIS0013.

[0178] One such alternative is lipopeptides. These are amphiphiles consisting of a lipid chain (generally 12-18 carbon atoms long) conjugated to a peptide sequence (generally 3-20 amino acid residues). A specific example of such a lipopeptide is palmitoyl pentapeptide-4 (abbreviated as PAL-KTTKS).

[0179] [ka]

[0180] PAL-KTTKS is believed to be a good candidate for an alternative to cationic lipids due to its cationic lysine residues that aid in binding to negatively charged RNA.

[0181] Other candidates investigated in SIS0012 and SIS0013 include NAD, tyrosine (TYR), and quercetin (QUE). It is believed that these ligands can enhance the organ-specific uptake of RNA and improve cellular internalization and targeting. In addition, these ligands can help provide a positively charged environment for the binding of negatively charged RNA.

[0182] Nicotinamide adenine dinucleotide (NAD) is a coenzyme. In its oxidized form, NAD+, it has the following structure: [ka]

[0183] Tyrosine is a naturally occurring amino acid that has the following structure at physiological pH (pH 7.4): [ka]

[0184] Quercetin is a flavanol having the following structure: [ka]

[0185] NAD, TYR, and QUE: Investigation of additions or substitutions to DOTAP in SIS0012 and SIS0013 Modified SIS0012 and modified SIS0013 compositions were prepared by adding 0.2 mg of NAD, TYR, and QUE to the normal composition described above (i.e., in addition to DOTAP). Then, complexation with siRNA was evaluated. Figure 29 shows the gel electrophoresis results, which show that siRNA was successfully and completely bound in these preparations.

[0186] Dynamic light scattering measurements were also performed using a Zetasizer (available from Malvern Instruments) to assess size and charge both before and after siRNA complexation, as shown in the table below. As the table shows, an increase in size was observed after siRNA complexation, along with a 10-15 mV decrease in surface charge.

[0187] [Table 7]

[0188] Next, replacement of DOTAP with NAD, TYR, or QUE (rather than simply adding NAD, TYR, or QUE to a DOTAP-containing composition) was investigated.

[0189] In this study, DPPC and DOPE were chosen for use with NAD, TYR, or QUE because they are zwitterionic lipids that do not play a significant role in surface charge at neutral pH.

[0190] Therefore, DPPC / DOPE preparations were prepared using (i) 0.2 mg and (ii) 1 mg of NAD, TYR, and QUE, as shown in the table below.

[0191] [Table 8]

[0192] [Table 9]

[0193] [Table 10]

[0194] As shown in the table below, Zetasizer measurements were obtained that revealed negative zeta potentials in all preparations, regardless of the amount of NAD, TYR, or QUE.

[0195] [Table 11]

[0196] Lipopeptides: Exploring the substitution of DOTAP in SIS0012 and SIS0013 Lipopeptides, also known as peptide amphiphiles (PAs), were investigated as another alternative to DOTAP.

[0197] It is believed that lipopeptides may provide a solution to the problem of replacing or reducing the amount of cationic lipids such as DOTAP in transfection compositions. Lipopeptides consist of alkyl chains conjugated to peptide sequences. These alkyl chains can be assimilated into lipid bilayers, while the surface of the bilayer is decorated with peptide moieties.

[0198] An exemplary PA is the molecule palmitoyl pentapeptide-4 (abbreviated as PAL-KTTKS, see above). The two cationic lysine residues may function similarly to cationic lipids such as DOTAP, which exhibit electrostatic interactions with negatively charged RNA.

[0199] DPPC and DOPE were selected as neutral lipids to prepare PAL-KTTKS. Various preparations were prepared with different silicon nanoparticles or without silicon at all; and with pH 4 buffer (to investigate the effect of pH).

[0200] Complete details of the DPPC, DOPE, and PAL-KTTKS containing preparations are shown in the table below.

[0201] [Table 12]

[0202] All eight preparations had positively charged surfaces with zeta potentials (measured with a Zetasizer available from Malvern Instruments) as shown in the table below.

[0203] Based on these results, it is believed that during lipid film assembly, PAL-KTTKS positions itself in the lipid bilayer with the peptide moiety exposed to the nanoparticle surface, and furthermore, the lysine residues on its surface contribute to the positively charged preparation.

[0204] [Table 13]

[0205] All preparations were evaluated for their ability to electrostatically bind to siRNA and mRNA.

[0206] Gel electrophoresis analysis was performed. No complete complexation was observed for siRNA. However, complete complexation was observed for mRNA. Figure 30 shows the gel electrophoresis results for siRNA, and Figure 31 shows the gel electrophoresis results for mRNA.

[0207] To address the partial complexation of siRNA with DPPC / DOPE / PAL-KTTKS, an alternative loading method was employed. Figure 32 shows gel electrophoresis of the complex after using the alternative loading method, which indicates successful complete complexation of siRNA. (The bright spot in lane 3 of Figure 32 is an artifact of the imaging device.)

[0208] In an alternative loading method, the following steps were adopted compared to the above protocol: 1. A lipid film was prepared by dissolving DPPC, DOPE, and Pal-KTTKS in methanol and evaporating using a rotary evaporator. 2. The lipid film was rehydrated with a suspension containing activated silicon (SIS0012) or activated boron-doped silicon (SIS0013) along with trehalose and glycine, and either siRNA or mRNA. Rehydration was carried out at 40° C. for 10 min to ensure that no lipid-silicon film remained on the walls of the round-bottom rotary evaporator flask.

[0209] Lipopeptides are very versatile molecules, and they can be fine-tuned by modifying their alkyl chains and / or their peptide sequences. It is believed that peptide customization can enhance cell and / or tissue targeting. In the field of gene therapy, peptide customization can result in electrostatic interactions with nucleic acids (e.g., RNA, especially mRNA). As an example, PAL-KTTKS, when prepared with DPPC and DOPE, resulted in a positively charged surface, as confirmed by the zeta potential.

[0210] Lipopeptides, being amphiphilic molecules, have properties very similar to surfactants that can self-assemble to form micelles. This is at least partially due to the fact that the peptide sequence can form intermolecular hydrogen bonds, while the alkyl chains favor hydrophobic interactions. Phospholipids such as DPPC and DOPE can also self-assemble into liposomes. Thus, when incorporating PA, of which PAL-KTTKS is a representative example (although other lipopeptides may be used), the alkyl chains can form hydrophobic interactions with DPPC and DOPE to obtain liposomal structures.

[0211] On the one hand, silicon nanoparticles provide structural stability to the complex as a whole and can interact with lipids and other ligands (such as lipopeptides, NAD, QUE, or TYR) via non-covalent (electrostatic) interactions, thus promoting long-term stability and binding of nucleic acids.

Claims

**Claim 1** A method for preparing an injectable or orally storage-stable preparation, comprising: (i) contacting a delivery system containing biocompatible solid particles with a biological substance species to form a complex; then, (ii) optionally, lyophilizing the complex to form a powder; and then, (iii) dispersing the complex in a biodegradable gel material to form the preparation containing the complex embedded in the biodegradable gel material The method as described above. **Claim 2** The method according to claim 1, wherein the delivery system further comprises a lipid component containing a cationic lipid and / or an ionizable lipid, and optionally a non-reducing disaccharide. **Claim 3** The method according to claim 1, wherein the biocompatible solid particles contain hydrolyzable silicon. **Claim 4** A biodegradable gel material-containing, The biodegradable gel material contains a delivery system containing biocompatible solid particles, a lipid component containing a cationic lipid and / or an ionizable lipid, and optionally a non-reducing disaccharide, and a biological substance species complexed with the delivery system is embedded therein. An injectable or orally storage-stable preparation. **Claim 5** The preparation according to claim 4, wherein the biocompatible solid particles contain hydrolyzable silicon. **Claim 6** (i) preparing a preparation by the method according to any one of claims 1 to 3, or preparing the preparation according to claim 4 or claim 5; then, (ii) optionally, storing the preparation; then, (iii) preparing a medicament containing a biological substance species and a delivery system for oral administration or injection A method for preparing a medicament containing a biological substance species for oral administration or injection, comprising the above steps. **Claim 7** The method according to claim 6, wherein the step (iii) of preparing the medicament does not include any extraction step such that the medicament contains all the components of the preparation. **Claim 8** The method according to claim 6, wherein in the step (ii) of storing the preparation, the preparation is stored at a temperature exceeding 4°C for 3 months or more. **Claim 9** A biological substance species complexed with a delivery system, wherein the complex is embedded in a biodegradable gel material, or A complex of the biological substance species and the delivery system, together with a compound capable of forming a matrix of a biodegradable gel dispersed in a carrier system, wherein the delivery system comprises particles of hydrolyzable silicon, a lipid component containing a cationic lipid, and optionally a non-reducing disaccharide, an oral or injectable pharmaceutical.

10. The pharmaceutical according to claim 9, wherein the pharmaceutical is in the form of an injectable solution.

11. (i) contacting a delivery system comprising biocompatible solid particles with a biological substance species to form a complex; then, (ii) optionally, lyophilizing the complex to form a powder; and then, (iii) dispersing the complex in a biodegradable gel material to form an oral preparation or an injectable preparation containing the complex embedded in the biodegradable gel material A method for protecting a biological substance species from degradation.

12. The method according to claim 11, wherein the delivery system further comprises a lipid component containing a cationic lipid and / or an ionizable lipid, and optionally a non-reducing disaccharide.

13. The method according to claim 11, wherein the biocompatible solid particles comprise hydrolyzable silicon.

14. An injection device comprising the preparation according to claim 4 or claim 5, or the pharmaceutical according to claim 9 or claim 10.

15. An oral formulation comprising the preparation according to claim 4 or claim 5, or the pharmaceutical according to claim 9 or claim 10.

16. The method according to any one of claims 1 to 3 and claims 11 to 13, wherein the delivery system comprises the non-reducing disaccharide trehalose.

17. The method according to any one of claims 1 to 3 and claims 11 to 13, wherein the biological substance species comprises one or more of nucleic acids, antigens, and vaccines.

18. The method according to claim 17, wherein the biological substance species comprises nucleic acids and optionally one or more enzymes.

19. The method according to any one of claims 1 to 2 or claim 12, wherein the lipid component contains a cationic lipid and / or an ionizable lipid, and the lipid component further comprises a phospholipid, and optionally a PEG lipid and / or a structural lipid.

20. The method according to any one of claims 1 to 3 and claims 11 to 13, wherein the delivery system comprises an amino acid.

21. The method according to claim 20, wherein the amino acid is selected from one or more of arginine, histidine, lysine, proline, and glycine.

22. The method according to claim 21, wherein the amino acid is glycine or a combination of glycine and arginine or lysine.

23. The delivery system according to claim 4 or claim 5 or the pharmaceutical according to claim 9 or claim 10, wherein the delivery system contains the non-reducing disaccharide trehalose.

24. The preparation according to claim 4 or claim 5 or the pharmaceutical according to claim 9 or claim 10, wherein the biological substance species contains one or more of nucleic acid, antigen, and vaccine.

25. The preparation or pharmaceutical according to claim 24, wherein the biological substance species contains nucleic acid and optionally one or more enzymes.

26. The preparation according to claim 4 or claim 5 or the pharmaceutical according to claim 9 or claim 10, wherein the lipid component contains a cationic lipid and / or an ionizable lipid, and the lipid component further contains a phospholipid and optionally a PEG lipid and / or a structural lipid.

27. The preparation according to claim 4 or claim 5 or the pharmaceutical according to claim 9 or claim 10, wherein the delivery system contains an amino acid.

28. The preparation or pharmaceutical according to claim 27, wherein the amino acid is selected from one or more of arginine, histidine, lysine, proline, and glycine.

29. The preparation or pharmaceutical according to claim 28, wherein the amino acid is glycine or a combination of glycine and arginine or lysine.

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