Therapeutic composition and therapeutic method

Abstract

Therapeutic compositions and methods. Compositions comprising hydrolyzable doped silicone particles, one or more lipids, and a pharmacokinetic agent (API) are disclosed. Related products, methods, and uses thereof are also disclosed.

Description

[Technical Field]

[0001] This disclosure relates to a delivery vehicle for active pharmaceutical ingredients ("APIs"). More specifically, but not exclusively, this disclosure relates to compositions comprising hydrolyzable doped silicon particles, one or more lipids, and APIs. This disclosure also relates to related products, medical applications, and methods. [Background technology]

[0002] Improvements to API delivery methods are needed. This is necessary to fully translate advances in biomedical research into efficient, safe, and cost-effective treatments.

[0003] Recent biological advances have provided insights into a new class of APIs for the prevention and treatment of many diseases. Nucleic acids are one example of this class. For instance, various mRNA-based infectious disease vaccines have recently emerged. Messenger RNA (mRNA)-based cancer therapies are also currently in clinical development. Meanwhile, other types of RNA, such as siRNA, tRNA, and oRNA (circular RNA), are showing promise for treating various genetic diseases and disorders.

[0004] However, while a new class of APIs promises, challenges also arise regarding how to control the stability of APIs during storage. Furthermore, it is necessary to ensure that APIs actually reach and are taken up by cells after administration to a patient, and stabilizing APIs while they circulate in the body can be difficult. In addition, it is necessary to target tissues or cells so that APIs can be delivered to the correct cells. If the goal is for the API to be delivered into the cell after reaching the target cell (e.g., rather than APIs intended to act extracellularly, such as cell surface proteins), there are also challenges in ensuring efficient absorption of the API by the cell. After such uptake, it is necessary to ensure the stability of the API within the cytoplasm.

[0005] One approach involves using lipid nanoparticles for in vivo delivery of fragile APIs, particularly mRNA. (As used herein, the terms “unstable API” and “reactive API” are interchangeable and may refer to APIs that (i) have a half-life of up to one week when stored in aqueous solution at approximately 25°C and are measurable by NMR or GC-MS, and / or (ii) have an in vivo half-life of less than approximately one hour and are measurable by assays of biological samples.) API-containing lipid nanoparticles require cold-chain storage, which limits their distribution and makes them energy-inefficient or cost-inefficient. There is still a need for delivery media that can stabilize fragile APIs both during storage (to allow storage at high temperatures) and in circulation in the body (to allow more APIs to reach target cells faster).

[0006] On the other hand, some lipid nanoparticle-based delivery media exhibit cytotoxicity (see Example 3 below). Typically used lipid nanoparticles rely on exogenous cationic lipids containing multiple amine groups (i.e., lipids with a net positive charge at approximately pH 7.4). While these amine-rich species are suitable for electrostatic loading of polyanionic nucleic acids (including various forms of RNA), they can cause cytotoxicity, immunogenicity, and nonspecific tissue accumulation. Similar problems arise with other polymer-based amine-rich delivery systems. There is a need for improved delivery media that also possess acceptablely low toxicity.

[0007] Preferably, the new delivery medium is completely dispersible in an aqueous environment to ensure easy delivery, such as by injection into an aqueous solution.

[0008] This disclosure aims to mitigate the aforementioned problems. Alternatively or additionally, this disclosure aims to provide an improved API delivery medium. [Overview of the Initiative]

[0009] In a first aspect of this disclosure, hydrolyzable doped silicon (particularly 1 cm) 3 1 x 10 16 A composition is provided comprising particles (hydrolyzable silicon doped at the above dopant atom levels), one or more lipids, and an API.

[0010] A second aspect of this disclosure provides compositions, as defined in accordance with the first aspect of this disclosure, for use in methods for preventing or treating diseases or disorders in human subjects.

[0011] In a third aspect, the present specification discloses a method for delaying the degradation of an API, the method including doping particles containing hydrolyzable silicon to change the ζ potential of the particles as compared to undoped particles that are otherwise the same, and contacting the doped particles with one or more lipids and an API.

[0012] In a fourth aspect of the present disclosure, there is provided a method of preventing or treating a disease or disorder, including administering a prophylactically or therapeutically effective amount of a composition to a human subject who requires a prophylactically or therapeutically effective amount of the composition defined according to one or both of the first and second aspects of the present disclosure.

[0013] In a fifth aspect, the present specification discloses the use of a composition defined according to one or both of the first and second aspects of the present disclosure in the manufacture of a medicament for use in a method defined according to one or both of the second and third aspects of the present disclosure.

[0014] In a sixth aspect, the present specification discloses a human cell transfection composition having one or more characteristics of a composition defined according to one or both of the first and second aspects of the present disclosure.

Brief Description of Drawings

[0015] Embodiments of the present disclosure are described by way of example only with reference to the accompanying drawings.

[0016] [Figure 1] FIG. 1 is a diagram showing the size and polydispersity index (PDI) data of undoped (SIS'0012) Si-containing delivery media and doped (SIS'0013) Si-containing delivery media complexed with mRNA. [Figure 2] FIG. 2 is a diagram showing the size and polydispersity index (PDI) data of undoped (SIS'0012) Si-containing delivery media and doped (SIS'0013) Si-containing delivery media complexed with mRNA. [Figure 3]Figure 3 shows gel electrophoresis data examining the formation of complexes between undoped (SIS0012)Si-containing delivery media and doped (SIS0013)Si-containing delivery media and mRNA. [Figure 4] Figure 4 shows data regarding the accessibility of mRNA to external reagents in undoped (SIS0012)Si-containing delivery media and doped (SIS0013)Si-containing delivery media. [Figure 5] Figure 5 shows the luminescence observed 6 hours after mRNA was administered to HEK293 cells using either an undoped (SIS0012)Si-containing delivery medium or a doped (SIS0013)Si-containing delivery medium. [Figure 6] Figure 6 shows the luminescence observed 24 hours after administering mRNA to HEK293 cells using either an undoped (SIS0012)Si-containing delivery medium or a doped (SIS0013)Si-containing delivery medium. [Figure 7] Figure 7 shows the luminescence observed 48 hours after administering mRNA to HEK293 cells using either an undoped (SIS0012)Si-containing delivery medium or a doped (SIS0013)Si-containing delivery medium. [Figure 8] Figure 8 shows the luminescence observed 72 hours after administration of mRNA to HEK293 cells using either an undoped (SIS0012)Si-containing delivery medium or a doped (SIS0013)Si-containing delivery medium. [Figure 9] Figure 9 shows gel electrophoresis data obtained when examining the complex formation between mRNA and various doped Si-containing delivery media (SIS0013-N, -Q, and -T). [Figure 10] Figure 10 shows data regarding the accessibility of mRNA to external reagents in various doped Si-containing delivery media (SIS0013-N, -Q, and -T). [Figure 11] Figure 11 shows the luminescence observed 6 hours after mRNA was administered to HEK293 cells using various doped Si-containing delivery media (SIS0013-N, -Q, and -T). [Figure 12] Figure 12 shows the luminescence observed 24 hours after mRNA was administered to HEK293 cells using various doped Si-containing delivery media (SIS0013-N, -Q, and -T). [Figure 13] Figure 13 shows the luminescence observed 48 hours after mRNA was administered to HEK293 cells using various doped Si-containing delivery media (SIS0013-N, -Q, and -T). [Figure 14] Figure 14 shows a statistical analysis of the luminescence data from Figures 11 to 13. [Figure 15] Figure 15 shows a statistical analysis of the luminescence data from Figures 11 to 13. [Figure 16] Figure 16 shows a statistical analysis of the luminescence data from Figures 11 to 13. [Figure 17] Figure 17 shows the expression of ClCn7G213R in PMBCs of mice administered with either an undoped (SIS0012)Si-containing delivery medium loaded with siRNA or a doped (SIS0013)Si-containing delivery medium. [Figure 18] Figure 18 shows the expression of ClCn7G213R in bone (femoral) cells of mice administered with either an undoped (SIS0012)Si-containing delivery medium or a doped (SIS0013)Si-containing delivery medium loaded with siRNA. [Figure 19] Figure 19 shows the CTX blood test results of mice administered with either an undoped (SIS0012)Si-containing delivery medium loaded with siRNA or a doped (SIS0013)Si-containing delivery medium. [Figure 20] Figure 20 shows the UV-vis absorbance data of alkaline phosphatase under the conditions described in Example 5. The stabilization of this pH and temperature-sensitive protein by a doped Si-containing delivery medium was investigated. [Figure 21] Figure 21 shows further UV-vis absorbance data for alkaline phosphatase, supplementing Figure 20. [Figure 22]Figure 22 shows gel electrophoresis data for investigating the formation of complexes between various doped Si-containing delivery media (SIS0013-N, -Q, and -T) and siRNA. [Figure 23] Figure 23 shows gel electrophoresis data investigating the formation of complexes between siRNA and doped Si-containing delivery media containing DPPC, DOPE, and PAL-KTTKS. [Figure 24] Figure 24 shows gel electrophoresis data examining complex formation between mRNA and doped Si-containing delivery media containing DPPC, DOPE, and PAL-KTTKS. [Figure 25] Figure 25 shows further gel electrophoresis data to investigate the formation of complexes between doped Si-containing delivery media containing DPPC, DOPE, and PAL-KTTKS and siRNA when the delivery medium / siRNA complexes were prepared by a different method than in Figure 23. [Figure 26] Figure 26 shows the results of the luciferase assay in Example 8. For both mRNA1 and mRNA2, the luciferase activity was higher when using SIS0013 (doped Si) than when using SIS0012 (undoped Si). [Figure 27] Figure 27 shows a transmission electron microscope (TEM) image illustrating the aggregation of silicon particles as described herein. [Modes for carrying out the invention]

[0017] While the subject matter of this disclosure is described and illustrated below with reference to specific embodiments, it will be understood by those skilled in the art that the subject matter is capable of many different modifications not specifically shown herein. Specific possible modifications in any aspect of this disclosure are described below for illustrative purposes only.

[0018] Particles containing hydrolyzable doped silicon The particles containing the hydrolyzable doped silicon may be pure or substantially pure doped silicon.

[0019] The particles may be another hydrolyzable doped silicon-containing material. If the particles are not pure doped silicon, they contain about 50% by weight or more of silicon, that is, about 50% by weight or more of silicon atoms based on the total mass of atoms in the particles. For example, the silicon particles may contain about 60% by weight or more, about 70% by weight or more, about 80% by weight or more, about 90% by weight or more, or about 95% by weight or more of silicon. The hydrolysis rate of the particles, for example, in PBS buffer at room temperature, may be 10% or more of the hydrolysis rate of pure silicon particles of the same size. Assays for the hydrolysis of silicon-containing materials are well known in the art; see, for example, International Publication 2011 / 001456, which is incorporated herein by reference in its entirety.

[0020] The particles may contain trace amounts of silica, but the silica is not hydrolyzable silicon. More than half of the silicon atoms in the particles may be in the form of elemental silicon (or doped elemental silicon).

[0021] The particles may be nanoparticles in particular. The average diameter of the nanoparticles may be in the range of about 1 nm to about 500 nm, particularly about 1 nm to about 250 nm, even more particularly about 1 nm to about 100 nm, preferably about 1 nm to about 50 nm, particularly about 3 nm to about 50 nm, and even more particularly about 3 nm to about 30 nm (for example, about 10 nm).

[0022] A particularly preferred range for the average diameter of nanoparticles is approximately 1 nm to 30 nm. This may offer advantages in terms of nanoparticle aggregation, as will be discussed later.

[0023] The dimensions of nanoparticles (including the average diameter of the particles) can be measured, for example, by a scanning electron microscope (SEM) or a transmission electron microscope (TEM).

[0024] The particles may be porous, particularly mesoporous. Particles containing hydrolyzable doped silicon can be made porous by standard techniques such as contacting the particles with a hydrofluoric acid (HF) / ethanol mixture and passing an electric current through them ("HF etching"). By varying the HF concentration, current density, and exposure time, the density and size of the pores can be controlled and monitored by scanning electron microscopy and / or nitrogen adsorption / desorption volume isothermometry. If the particles are porous, their porosity increases the total surface area of ​​the particles. For example, their surface area may increase by about 50% or more, or about 100% or more, compared to the surface area of ​​the corresponding non-porous particles. In many cases, porous particles actually have a significantly increased total surface area due to their porosity. According to certain embodiments, the porosity is about 30% or more, about 40% or more, about 50% or more, or about 60% or more, meaning that about 30% or more, about 40% or more, about 50% or more, or about 60% or more of the particle volume is pore space, respectively.

[0025] The average pore size may be in the range of approximately 0.1 nm to approximately 10 nm, for example, approximately 0.1 nm to approximately 3 nm, or for example, approximately 2 nm.

[0026] It will be understood that the aforementioned particles can be produced by various techniques well known to those skilled in the art.

[0027] These techniques include, for example, purely physical (also referred to in the art as "non-wet") processes starting from bulk silicon (especially silicon wafers), such as pulsed laser ablation, pyrolysis, and ball milling. Thus, the particles can be obtained by a method comprising, or consisting of, one or more of pulsed laser ablation, pyrolysis, and ball milling of bulk silicon (especially silicon wafers).

[0028] Additionally or alternatively, the particles may be produced by chemical (also referred to in the art as "wet") techniques, including but not limited to electrochemical etching of bulk silicon (particularly silicon wafers). Such techniques optionally include the HF etching described above. Thus, the particles can be obtained by methods including, or comprising, electrochemical etching of bulk silicon (particularly silicon wafers).

[0029] After the silicon particles are formed, they can be classified by size, for example, by air classification, sieving, and / or filtration. Therefore, the particles can be obtained by a method that includes one or more of air classification, sieving, and / or filtration.

[0030] Therefore, for example, the particles can be obtained by a method that includes generating silicon particles from bulk silicon (especially silicon wafers) by one or more of the following: pulsed laser ablation, thermal decomposition, ball milling, and electrochemical etching of bulk silicon (especially silicon wafers), and then optionally classifying them by size by air classification, sieving, and / or filtration.

[0031] Optionally, the particles are washed with methanol or ethanol before use to remove a thin oxide layer from the surface. In the art, this may be referred to as "activation."

[0032] The particles thus obtained have a narrow size distribution and uniform surface chemical properties, resulting in batch-to-batch reliability and reproducibility of one or more of the advantages described herein.

[0033] Appropriate physical and chemical methods are described, for example, in International Publication No. 2011 / 012867(A1) (under the name of SISAF LTD), Tokarska K et al., Facile production of ultra-fine silicon nanoparticles, R. Soc. Open Sci., 2020, 7: 200736, and Kim, T., Lee, J. Silicon nanoparticles: fabrication, characterization, application and perspectives, Micro and Nano Syst. Lett, 2023, 11: 18, each of which is incorporated herein by reference in its entirety.

[0034] Dopant The particles of the aforementioned composition contain hydrolyzable doped silicone.

[0035] As used herein, the term “doped silicon” may refer to silicon that behaves as an exogenous semiconductor due to the presence of dopant atoms. The dopant atoms may be substitutional (substituting for Si atoms) dopant atoms, or may contain substitutional dopant atoms. Additionally or alternatively, the dopant atoms may be interstitial dopant atoms (located between Si atoms and not substituting Si atoms), or may contain interstitial dopant atoms. Optionally, the dopant atoms may be located on the surface of the particle. Optionally, the dopant atoms may be located only on the surface of the particle, or only on or near the surface. Thus, the particle may optionally have an undoped silicon core and a doped shell or surface.

[0036] However, preferably, there is no metal silicate coating on the surface of the particles. The surface coating of the metal silicate is not doping. Different from scattering dopant atoms between silicon atoms, the surface coating of the metal silicate will cover the surface of the particles with a metal silicate compound. The silicon particles can be coated with metal silicate, but doing so may complicate the synthesis and is not necessary for the beneficial effects described herein, such as tissue targeting.

[0037] Advantageously, the silicon particles are doped at a level of at least about 1×10 dopant atoms per cm 3 , most specifically about 1×10 15 dopant atoms or more. 16

[0038] For example, the particles can be doped at a level of at least about 1×10 3 , at least about 1×10 17 , or at least about 1×10 18 dopant atoms or more. [[ID=二十一]] 19

[0039] The silicon particles may be doped with up to 1×10 3 dopant atoms per cm. 20

[0040] The silicon particles may be n-type doped or p-type doped. The silicon particles are doped with one or more elements selected from B, P, Mg, Cu, Ga, Al, In, Bi, Ge, Li, Xe, N, Au, and Pt. Thus, the dopant may be a p-type dopant, particularly boron. The dopant may be an n-type dopant, particularly phosphorus.

[0041] The dopant can be incorporated into the hydrolyzable silicon matrix (i) substitutionally, (ii) interstitially, and / or (iii) by surface attachment. In this way, an improved API delivery medium can be provided.

[0042] Preferably, if boron is used as a dopant, 1 cm 3 1 x 10 15 dopant atoms, and 1 cm 3 1 x 10 20 The doping levels of the dopant atoms may correspond to resistivities of 13.6 Ω-cm and 1,300,000 Ω-cm, respectively. Embodiments in which boron is the dopant do not exclude silicon that is doped with boron (e.g., highly doped) but further doped with other elements (preferably, in such cases, the majority of the dopant is boron).

[0043] As used in this specification, the term "high-concentration doping" refers to 1 cm 3 Approximately 1 x 10 15 This is understood to mean doping with one or more dopant atoms. In some preferred embodiments, the dopant is 1 cm 3 Approximately 1 x 10 16 The above dopant atoms are present. Therefore, in a particularly preferred embodiment, the dopant is 1 cm 3 Approximately 1 x 10 16 This is boron that exists at the level of more than one boron atom. For example, 1 cm 3 Approximately 1 x 10 16 From 1 cm² or more boron atoms 3 Approximately 1 x 10 20 Boron can exist at the level of individual boron atoms.

[0044] In this specification, when silicon is referred to as "undoped" (the particles of composition SIS0012 in Examples 1 and 2), it means that it contains no dopant atoms at all, or only a small amount, e.g., 1 cm³. 3 Approximately 1 x 10 2 This could mean that only up to a certain number of dopant atoms are present. Additionally or alternatively, "undoped" silicon may mean silicon that does not act as an extrinsic semiconductor.

[0045] Advantageously, doping particles with hydrolyzable silicon can have a beneficial effect on the particle's zeta potential, which can serve as a surrogate indicator of the particle's surface charge. Doping can result in a zeta potential suitable for improving the API load. This is thought to help stabilize the API while it is circulating in the body until it reaches target cells and is released. Therefore, compared to using undoped particles, more API reaches target cells within a certain period after administration, enabling more efficient API delivery.

[0046] Doping with the aforementioned particles can increase the in vivo half-life of the API (at approximately pH 7.4 and approximately 37°C) by, for example, about 1.5 times, and particularly about 2 times. In particular, when particles containing hydrolyzable doped silicone are present along with one or more lipids, the API may have an in vivo half-life of about 1 hour or more, more specifically about 2 hours or more, and more specifically about 6 hours or more. As used herein, the term "in vivo half-life of API" may refer to the in vivo decay time of the API, i.e., the time it takes for the administered amount of API to decrease by about half. It will also be understood that the term "amount of API" may refer to the amount of API or its derivatives having the same or substantially the same intended pharmaceutical effect. Furthermore, as used herein, the term "in vivo half-life" may refer to the systemic half-life of the API (in the body, especially in the human body), in contrast to the half-life of the API on external surfaces, such as when present on the skin surface (externally, even in contact).

[0047] Doping of particles may optionally change the zeta potential by approximately 5 mV or more, and especially by approximately 10 mV or more, compared to undoped particles.

[0048] Doping with a p-type dopant can make the zeta potential more positive, which can improve binding to APIs with a net negative charge, particularly those with a net negative charge at pH around 7.4 (as this is a typical physiological pH), especially nucleic acids, more specifically mRNA, siRNA, or tRNA. Therefore, in some embodiments, hydrolyzable doped silicon particles are doped with a p-type dopant, and the API is a negatively charged API, particularly nucleic acids, more specifically mRNA or siRNA.

[0049] p-type doping may further improve binding to APIs with a net positive charge, particularly those with a net positive charge at pH approximately 7.4 (as this is a typical physiological pH). This is especially effective when the particle's zeta potential is further modulated by other components, such as one or more lipids (e.g., phospholipids). While we do not wish to be constrained by theory, it is thought that electron transfer occurs from p-type doped silicon (e.g., boron-doped silicon) to other components, thereby adjusting the particle's size and charge, and thus its nucleic acid binding ability.

[0050] Particle p-type doping can optionally increase the zeta potential by approximately 5 mV or more, and especially by approximately 10 mV or more, compared to undoped particles.

[0051] On the other hand, doping with an n-type dopant makes the zeta potential more negative, which can improve binding with positively charged APIs. Therefore, in some embodiments, particles containing hydrolyzable doped silicon are doped with an n-type dopant, and the API is an API with a net positive charge, in particular an API with a net positive charge at pH approximately 7.4.

[0052] n-type doping may further improve binding to APIs with a net negative charge, particularly those with a net negative charge at pH approximately 7.4 (as this is a typical physiological pH). This is especially effective when the particle's zeta potential is further modulated by other components, such as one or more lipids present (e.g., cationic lipids). Additionally or alternatively, n-type doped silicon may protect one or more lipids, particularly zwitterionic or positively charged lipids, from degradation. This may indirectly help bind and stabilize APIs such as nucleic acids, even when the API is negatively charged (especially mRNA).

[0053] n-type doping of particles can optionally reduce the zeta potential by approximately 5 mV or more, and especially by approximately 10 mV or more, compared to undoped particles.

[0054] P-type or n-type doping of particles containing hydrolyzable silicon can improve the binding with amphoteric or neutral APIs. Therefore, in some embodiments, the particles contain hydrolyzable p-doped silicon, and the API is amphoteric or neutral. In other embodiments, the particles contain hydrolyzable n-doped silicon, and the API is amphoteric or neutral.

[0055] Conventional liposome transfection compositions (silicone-free) tend to contain a significant amount (e.g., a large portion by weight) of cationic lipids, particularly amine-rich cationic lipids, where the positive charge of the cationic lipids is intended to stabilize negatively charged APIs, especially nucleic acids. However, these cationic lipids are not cost-effective and may not have an appropriate safety profile for all clinical applications, such as administration to certain patient groups, including infants, the elderly, or pregnant women. As described herein, the particles offer the possibility of using fewer or no cationic lipids, particularly fewer or no "exotic" or amine-rich cationic lipids, thus improving cost-effectiveness and safety.

[0056] In connection therewith, the zeta potential of the particles may also be modulated by other components in the composition, as demonstrated by the compositions SIS0013-T, SIS0013-N, and SIS0013-Q of Examples 2 and 6 of this specification, and the lipid-containing oligopeptide composition of Example 7. Doping the particles may help maintain a more positive or more negative (depending on the situation) zeta potential than if they were not doped, which means that satisfactory API binding and delivery can be maintained by adding components that have certain functions that would otherwise excessively increase / decrease the zeta potential of the particles (depending on the situation).

[0057] Furthermore, the change in zeta potential resulting from hydrolyzable silicon doping may not be the only beneficial effect of doping. The change in zeta potential may not be the sole reason for improved API binding and delivery success. Also, as described herein, non-silicon components may also affect API binding and delivery. For example, SIS0013-N, SIS0013-Q, and SIS0013-T in Examples 2 and 6 show advantages in the binding and delivery of negatively charged mRNA, despite having a non-positive zeta potential compared to SIS0013 in Example 1.

[0058] On the other hand, as described in Example 1 below ("Materials and Methods: Method for Preparing SIS0012 or SIS0013; Method for Preparing SIS0012-mRNA or SIS0013-mRNA Complexes"), the delivery medium according to this disclosure (e.g., SIS0013) can be prepared, stored, and delivered to a clinic, where it can then be complexed with an API such as mRNA before being administered to a patient. The delivery medium can be stored separately from the mRNA until immediately before administration to a patient. Since the delivery medium does not contain reactive APIs such as mRNA during storage, it does not need to be stored at particularly low temperatures, such as below 4°C, in particular to stabilize the API. This is a different approach from conventional lipid nanoparticle delivery mediums, which are usually stored with the API already encapsulated.

[0059] Furthermore, the doped Si-containing delivery media disclosed herein have a stabilizing effect on reactive APIs, such as nucleic acids (particularly mRNA), when complexed with them. As a result, the disclosed delivery media may optionally be stored already complexed with the API, especially at low temperatures. For example, a composition containing the API can be stored at about 0°C or above, particularly at about 3°C ​​to 5°C, i.e., the temperature of a typical commercial refrigerator, or at about 15°C to 30°C, i.e., typical room temperature.

[0060] The doped Si-containing delivery medium can also stabilize the API while circulating in the body. Additionally or alternatively, it can ensure efficient uptake of the API by cells. Additionally or alternatively, it can stabilize the API within the cytoplasm of cells. For example, this may be advantageous if the API is mRNA and it is preferable that it be safely delivered to cytoplasmic ribosomes for translation. Boron doping of silicon (p-type doping) can stabilize negatively charged mRNA with its phosphate backbone. This may improve protection against degradation in particular, potentially making mRNA-mediated cell transfection more effective.

[0061] The production of doped silicon is well understood in the semiconductor industry and includes methods such as ion implantation and diffusion, making doped silicon readily available. As an example of the diffusion method, silicon powder and doping reagents (e.g., B2O3 in the case of boron doping) are mixed in an N2 atmosphere at a temperature of 1050°C to 1175°C for several minutes to diffuse the dopant (e.g., boron) into the silicon.

[0062] Lipids The one or more types of lipids may be bound to the surface of the particles. The one or more types of lipids may form a complex with an API such as nucleic acids. The one or more types of lipids may include ionizable lipids. The one or more types of lipids may include lipids having a net positive charge at a pH of about 7.4 (also referred to herein as "cationic lipids").

[0063] It has been found that treating the surface of the aforementioned particles with lipids can help control the release rate of APIs (e.g., nucleic acids, particularly mRNA or siRNA). The type of lipid used for surface treatment of silicon-containing particles can help adjust the release rate of the APIs.

[0064] Treating hydrolyzable doped silicon particles with lipids can have beneficial effects on the particle surface charge. This may provide a zeta potential more suitable for improving the loading of APIs, particularly nucleic acids (e.g., siRNA, short activated RNA, short hairpin RNA, or mRNA). This may help control the API release rate at target sites.

[0065] For example, treatment of particles containing hydrolyzable pure silicon with phosphatidylcholine (PC), phosphatidylethanolamine (PE), and / or lecithin can promote a negative zeta potential (zeta potential in the range of approximately -60mV to approximately -20mV). On the other hand, treatment with stearylamine and / or DOTAP can promote a positive zeta potential (zeta potential in the range of approximately 0mV to approximately +40mV). As described herein, doping of silicon particles adjusts their zeta potential. Therefore, p-type dopants (preferably boron) can make the zeta potential of the particles more positive (for example, the typical value of pure silicon, approximately -40mV, can become approximately -25mV with doping). As a result, p-type doped (e.g., boron-doped) silicon treated with cationic lipids can more easily achieve a more positive zeta potential. For example, treating p-type doped (e.g., boron-doped) silicon with stearylamine and / or DOTAP can achieve a zeta potential of approximately +20mV to approximately +60mV. As a result, using doped silicon allows for the achievement of a positive zeta potential with smaller amounts of cationic lipids, or with a wider range of cationic lipids (including non-toxic and amine-free ones). Even if cationic lipids decompose during storage and the positive charge of the lipids is partially lost, the particle's zeta potential can be kept positive for a longer period and more safely.

[0066] The aforementioned one or more types of lipids are preferably free from toxic lipids. The aforementioned one or more types of lipids may not contain toxic exogenous lipids (known in the art as exotic cationic lipids) that have a positive charge at physiological pH. Such lipids, especially when rich in amines, may have undesirable toxicity profiles, such as inducing congenital immune responses when administered to human patients. These (amine-rich) exotic lipid species are suitable for electrostatic loading of polyanionic nucleic acids (including various forms of RNA), but may cause cytotoxicity and immunogenicity, and may even lead to nonspecific tissue accumulation, contrary to undesirable results, potentially hindering the objective of targeted delivery. Furthermore, their complex synthesis can make them expensive.

[0067] Therefore, the one or more types of lipids mentioned above do not necessarily include lipids rich in amines. Lipids rich in amines are defined as having two, three, or more than four nitrogen atoms per lipid molecule. In contrast, the one or more types of lipids mentioned above preferably contain a maximum of one nitrogen atom per lipid molecule.

[0068] Furthermore, it has been found that the beneficial effects disclosed herein can be achieved without necessarily being tied to one or more specific lipid compounds. The one or more lipids can play a role in charge-charge interactions, such as regulating the zeta potential on the surface of silicon particles, thereby improving API binding. This effect can be observed with a wide range of lipids. Lipids, as a class, exhibit characteristic tendencies, particularly in terms of intermolecular interactions, allowing the compositions of this disclosure to be implemented with different lipids and in different amounts compared to the specific lipids disclosed in the following examples.

[0069] The aforementioned one or more lipids help regulate the hydrolysis rate of the doped silicon, resulting in its hydrolysis into bioavailable orthosilicic acid (OSA) degradation products rather than insoluble polymer hydrolysis products. Controlling the hydrolysis rate of the doped silicon can affect the release rate of the API associated with the doped silicon. Controlling the release rate of the API can adjust the duration of protection provided by the API, particularly in vivo in the presence of various bodily fluids. Thus, more API may be delivered to target cells within a given period than with otherwise identical components.

[0070] Lipids are generally understood to include fatty acids and fatty acid derivatives, glycerolipids, glycerophospholipids, sphingolipids, glycolipids, and polyketides. As used in this application, the term “lipid” may encompass lipid-modified oligopeptides (as used herein interchangeably with the term “lipopeptide”) to which a short peptide sequence (e.g., peptide sequences having 3 to 20 amino acid residues, e.g., 5 to 15 amino acid residues, particularly 3, 4, or 5 amino acid residues, and most particularly 5 amino acid residues) is linked to one or more fatty acid chains (especially fatty acid chains having a carbon chain length of 10 to 24 carbon atoms, preferably 12 to 18 carbon atoms, e.g., 14, 15, or 16 carbon atoms; for example, the peptide portion may be lipid-modified with a palmitoyl, cetyl, or myristoyl portion).

[0071] While we do not wish to be constrained by theory, the following examples examine several lipids, but the mechanisms by which one or more of these lipids act can be generalized beyond the exemplified lipids, as they may be due to the properties of the lipid class, such as their behavior in relation to charge-to-charge interactions.

[0072] Therefore, the one or more types of lipids may include one or more types of lipid-modified oligopeptides. Preferably, each of the one or more types of lipid-modified oligopeptides contains a fatty acid chain having about 12 to about 18 carbon atoms.

[0073] Preferably, each of the one or more types of lipid-modified oligopeptides contains 3 to 20 amino acid residues. Therefore, the lipid-modified oligopeptides may be lipid-modified tetrapeptides, lipid-modified pentapeptides, or lipid-modified hexapeptides.

[0074] Preferably, the amino acid residues include one or more amino acid residues (e.g., about two or three amino acid residues) that are cationic at a pH of about 7.4 (physiological pH), such as lysine or arginine. For example, the lipidized oligopeptide may contain one or more (e.g., about two) lysine residues.

[0075] A special example of a lipid-containing oligopeptide ("lipopeptide") is palmitoyl pentapeptide-4 (CAS number: 214047-00-4, abbreviation PAL-KTTKS).

[0076] Therefore, preferably, the one or more lipids may include one or more lipid-modified oligopeptides, particularly lipid-modified oligopeptides having one or more amino acid residues that are positively charged at a pH of about 7.4 (i.e., near physiological pH), such as one or both of lysine and arginine, or may be said lipid-modified oligopeptides.

[0077] The aforementioned lipid-modified oligopeptides can be used in combination with one or more phospholipids, particularly DOPE or DPPC. The alkyl chain of the lipid-modified oligopeptide molecule can be absorbed into the phospholipid bilayer, whose surface is modified by the peptide moiety. While not wishing to be constrained by theory, the peptide moiety of the lipid-modified oligopeptide is thought to enable the targeting of one or more specific tissues and / or cells. Furthermore, if the peptide moiety is positively charged at approximately pH 7.4 (i.e., near physiological pH), it can stabilize negatively charged APIs (nucleic acids, particularly mRNA or siRNA).

[0078] The aforementioned one or more types of lipids include one or more cationic lipids (e.g., DOTAP), one or more phospholipids (e.g., DOPE), and one or more polyethylene glycol (PEG) lipids (e.g., DSPE-PEG). 2000 ) may be one or more of these, or may include them.

[0079] The one or more lipids may be one or more structural lipids (e.g., cholesterol-based lipids), or may contain one or more structural lipids. However, the one or more lipids may optionally exclude structural lipids. Thus, the one or more lipids may exclude sterols, and in particular cholesterol. The compositions currently disclosed have been found not to require reliance on these types of lipids on which conventional API delivery systems typically depend. Therefore, the compositions disclosed herein may be an alternative to API delivery systems that rely on these types of lipids, particularly cholesterol. This may be advantageous when cholesterol is unavailable or unusable for any reason (e.g., due to its effects in the body).

[0080] The aforementioned one or more lipids include phosphatidylcholine (PC), hydrogenated PC, stearylamine (SA), dioleoylphosphatidylethanolamine (DOPE), cholesteryl 3β-N-(dimethylaminoethyl)carbamate hydrochloride (DC-chol), 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), PEGylated 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), for example, DSPE-PEG. 2000 , and one or more of their derivatives may optionally be included.

[0081] In certain embodiments, the lipid is selected from the group consisting of phosphatidylethanolamine (PE), phosphatidylcholine (PC), stearylamine (SA), and any combination thereof.

[0082] In some embodiments, the lipid or lipid component may be a cationic lipid or may contain a cationic lipid. The term "cationic lipid" refers to a molecule that has a net positive charge at pH 7.4 (physiological pH) and has a cationic head group attached to a hydrophobic tail via some spacer. Examples include DTDTMA (ditetradecyltrimethylammonium), DOTMA (2,3-dioleyloxypropyl-1-trimentylammonium), DHDTMA (dihexadecyltrimethylammonium), dioleoyl-3-trimethylammoniumpropane (DOTAP), and stearylamine (SA). The positive charge is usually stabilized by a negative counterion.

[0083] Therefore, the one or more lipids may optionally be DOTAP, or may contain DOTAP. DOTAP exists in the form of S-isomers and R-isomers, and may exist as S-isomer, R-isomer, or racemic mixture. If necessary, the R-isomer and S-isomer may be present in approximately equal amounts by weight of the total DOTAP present (i.e., each form may be about 60% or less of the total DOTAP present by weight). In other embodiments, about 80% or more, about 90% or more, about 95% or more, about 98% or more, or about 99% or more of the total DOTAP is the R-isomer. In other embodiments, about 80% or more, about 90% or more, about 95% or more, about 98% or more, or about 99% or more of the total DOTAP is the S-isomer.

[0084] Nevertheless, as described herein, silicon doping may allow the use of fewer cationic lipids, particularly less toxic or “exotic” cationic lipids, compared to conventional API delivery compositions (such as lipid nanoparticles containing cationic lipids). While exotic cationic lipids may be suitable for electrostatic loading of polyanionic nucleic acids (including various forms of RNA), their high amine content may lead to cytotoxicity, immunogenicity, and nonspecific tissue accumulation.

[0085] Therefore, the one or more types of lipids may optionally exclude cationic lipids, particularly toxic cationic lipids. Cationic lipids may not be necessary when doped silicon, particularly p-type doped silicon, is used as described herein.

[0086] Therefore, the one or more types of lipids include one or more types of phospholipids (e.g., DOPE) and one or more types of polyethylene glycol (PEG) lipids (e.g., DSPE-PEG). 2000 ) may be one or more of them, or may include one or more of them.

[0087] Overall, the hydrolyzable-doped silicon-containing particles disclosed herein may offer the possibility of reducing the amount of lipids used in API delivery media (particularly cationic lipids, and most especially toxic cationic lipids) compared to conventional API delivery media that do not contain hydrolyzable-doped silicon particles (e.g., conventional liposomal nucleic acid delivery media typically used for in vivo mRNA delivery). Additionally or alternatively, hydrolyzable-doped silicon particles may offer the possibility of formulating API delivery media with a wider range of lipids while providing transfection efficiency, storage stability, and / or targeted delivery to specific types of tissues or cells. This may lead to a reduction in the reliance in the art on specific lipids, particularly cationic lipids, especially toxic cationic lipids, and / or cationic lipids that are specifically formulated for API delivery and are therefore potentially cost-ineffective and not readily available.

[0088] The aforementioned one or more types of lipids may have an average molecular weight in the range of approximately 500 to approximately 1000.

[0089] The ratio of one or more lipids (meaning all lipid components in the composition) to silicon may be in the range of about 40:1 to about 1:1, particularly in the range of about 20:1 to about 1:1. For example, when the components are assembled for the manufacture of a delivery system, i.e., before further processing is carried out (such further processing may be, for example, the "extrusion" step in the "Materials and Methods" section of Example 1 herein), the ratio may be such as about 16:1.

[0090] As described herein, the one or more lipids may include phospholipids in particular, or may be phospholipids. As used herein, the term "phospholipid" may refer to lipids containing fatty acid chains and phosphate groups. Unlike positively charged cationic lipids, phospholipids may be negatively charged. However, phospholipids are typically amphoteric compounds containing components with both positive and negative charges, and therefore have no overall charge. For this reason, phospholipids are usually classified as neutral lipids.

[0091] Suitable phospholipids may be or may include glycerophospholipids. Particularly suitable phospholipids include those in which a polar head group is attached to a quaternary ammonium moiety, such as phosphatidylcholine (PC) or hydrogenated phosphatidylcholine. The phospholipid may be or may be derived from lecithin. A preferred phospholipid is DOPE (phosphatidylethanolamine or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine).

[0092] Preferably, the phospholipid side chain may be an aliphatic side chain having about 15 or more carbon atoms, or an ether side chain having about 6 or more repeating ether units, such as a polyethylene glycol or polypropylene glycol chain.

[0093] Lipids having ether side chains are sometimes referred to as "PEG lipids" or "PEGylated" lipids. Therefore, the term "lipid" as used in this application may cover PEG lipids. Accordingly, according to a preferred embodiment, the one or more lipids are one or more polyethylene glycol (PEG) lipids (particularly DSPE-PEG). 2000 It may contain PEGylated DSPEs such as, or it may be one or more types of polyethylene glycol (PEG) lipids.

[0094] The aforementioned one or more lipids may optionally include phosphatidylcholine (PC), hydrogenated phosphatidylcholine, stearylamine (SA), or a combination thereof, or may substantially consist of them.

[0095] The aforementioned one or more types of lipids may optionally contain approximately 5% by weight or more (for example, approximately 30% by weight or approximately 50% by weight or more) of polypropylene (PC) based on the total weight of the aforementioned one or more types of lipids.

[0096] The one or more types of lipids may optionally contain hydrogenated polycarbonate (PC) in an amount of approximately 5% by weight or more (for example, approximately 30% by weight or approximately 50% by weight or more) based on the total weight of the one or more types of lipids.

[0097] The aforementioned one or more types of lipids may optionally contain SA in an amount of approximately 5% by weight or more (for example, approximately 30% by weight or approximately 50% by weight or more) based on the total weight of the aforementioned one or more types of lipids.

[0098] The aforementioned one or more lipids may optionally include PC and SA, or consist essentially of them. Optionally, the weight ratio of PC to SA is within the range of approximately 1:1 to approximately 20:1.

[0099] The aforementioned one or more lipids may optionally include, or essentially consist of, a combination of DOPE, SA, and DC cholesterol (DC-chol). The weight ratio of DOPE:SA may be in the range of approximately 1:1 to approximately 10:1. The weight ratio of DOPE:DC cholesterol may be in the range of approximately 1:1 to approximately 5:1. The weight ratio of SA:DC cholesterol may be in the range of approximately 1:1 to approximately 1:5.

[0100] In certain preferred embodiments, the one or more lipids may optionally be DOTAP, DOPE, and PEG lipids (particularly DSPE-PEG).2000 ) may include, or essentially consist of, a combination of ). The weight ratio of DOTAP:DOPE is in the range of approximately 1:2 to approximately 2:1, for example, approximately 1:1. The weight ratio of DOTAP:PEG lipids is in the range of approximately 10:1 to approximately 5:1, for example, approximately 7:1. The weight ratio of DOPE:PEG lipids is in the range of approximately 10:1 to approximately 5:1, for example, approximately 7:1.

[0101] amino acid The compositions disclosed herein may contain one or more amino acids. In its broadest sense, the term “amino acid” encompasses artificial or natural organic compounds containing amine (-NH2) and carboxyl (-COOH) functional groups. These include α-amino acids, β-amino acids, γ-amino acids, and δ-amino acids. They include amino acids in any chiral configuration. The amino acids may, in particular, be naturally occurring α-amino acids. They may be protein-constituting amino acids or non-protein-constituting amino acids (such as carnitine, levothyroxine, hydroxyproline, ornithine, or citrulline).

[0102] The one or more amino acids may help stabilize the doped silicon particles themselves. In vivo, the one or more amino acids may help regulate the hydrolysis rate of the doped silicon, resulting in the doped silicon being hydrolyzed to bioavailable orthosilicic acid (OSA) degradation products rather than insoluble polymer hydrolysis products. In this way, the one or more amino acids may complement the function of one or more lipids of this disclosure. Controlling the hydrolysis rate of the doped silicon may affect the release rate of the API associated with the doped silicon. Controlling the release rate of the API may adjust the duration of protection provided by the API, particularly in vivo in the presence of various bodily fluids. Thus, more API may be delivered to target cells within a given period than with the otherwise identical composition.

[0103] While we do not wish to be constrained by theory, the following examples examine several amino acids, but the mechanisms by which one or more amino acids act may be generalizable beyond the exemplified amino acids, as they may be due to the properties of the amino acid class, such as their behavior in relation to charge-to-charge interactions.

[0104] In a preferred embodiment, the amino acids may include glycine, arginine, and / or tyrosine, most particularly glycine, or may consist essentially of them.

[0105] Additionally or alternatively, amino acids that are neutral or positively charged at physiological pH (approximately pH 7.4), such as tyrosine or arginine, can stabilize negatively charged APIs (e.g., nucleic acids such as mRNA). Conversely, amino acids that are neutral or negatively charged at physiological pH (approximately pH 7.4) can stabilize positively charged APIs. Nevertheless, the charge-based interactions and / or other interactions (e.g., steric interactions) arising from the combination of doped Si, lipids, and amino acids may lead to situations where positively charged amino acids at physiological pH help stabilize positively charged APIs, or where negatively charged amino acids at physiological pH help stabilize negatively charged active pharmaceutical ingredients.

[0106] The weight ratio of one or more types of lipids (i.e., total lipid components) to the amino acids may be in the range of approximately 40:1 to approximately 1:1, for example, approximately 32:1.

[0107] Optionally, the composition may contain, in addition to the above-mentioned amino acids, specifically the amino acid tyrosine. Optionally, the composition may contain, in place of the above-mentioned amino acids, specifically tyrosine. Therefore, it will be understood that tyrosine, although an amino acid, may optionally be present as a further component different from the above-mentioned amino acids for the purposes of the present invention. Therefore, if tyrosine is present as a further component different from the above-mentioned amino acids (as exemplified in composition SIS0013-T of Example 3 below), it will be understood that the amount of additional different tyrosine is not included in the calculation of the weight ratio of one or more lipids to amino acids in the range of about 40:1 to about 1:1, for example, about 32:1 disclosed above.

[0108] Non-reducing disaccharides Additionally or alternatively, the composition may include one or more non-reducing disaccharides, particularly trehalose, as exemplified by the composition of Example 1 below. The weight ratio of the one or more lipids (i.e., total lipid components) to the non-reducing disaccharides may be in the range of about 20:1 to about 1:1, for example, about 16:1.

[0109] API It will be understood that the compositions of this disclosure may be described as pharmaceutical compositions for delivering active pharmaceutical ingredients (APIs).

[0110] For example, an API may be an unstable API. As used herein, the terms “unstable API” and “reactive API” may be interchangeable and may refer to an API that (i) has a half-life of up to one week when stored in aqueous solution at approximately 25°C and is measurable by NMR or GC-MS, and / or (ii) has an in vivo half-life of less than approximately one hour and is measurable by assays of biological samples.

[0111] The API can be any pharmaceutically active compound. Therefore, it will be understood that the term "API" includes prodrugs, for example. In particular, the API may be a nucleic acid, more specifically siRNA or mRNA. On the other hand, in other preferred embodiments, the API may be a protein.

[0112] The API can be administered by injection, orally, intranasally, or topically, and in particular, by injection or orally.

[0113] Nucleic acid API The API is preferably nucleic acid, particularly linear or circular RNA, or may include such RNA. The RNA may be small interfering RNA (siRNA), small activating RNA (saRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), or messenger RNA (mRNA), particularly mRNA (e.g., pathogenic organism encoding a pathogenic protein).

[0114] Other nucleic acids used in accordance with this disclosure include double-stranded and single-stranded DNA, DNA:RNA hybrids, peptide:DNA hybrids, and peptide:RNA hybrids.

[0115] RNA and DNA may be naturally occurring or chemically modified to enhance therapeutic properties, such as by increasing activity, improving serum stability, reducing off-target effects, or decreasing immune activation. Chemical modifications to RNA and DNA may include any modifications well known in the art. As used herein, "naturally occurring" means of natural human or animal origin. It should be understood that even if the molecular structure is the same as that of a naturally occurring molecule, it may be synthesized in vitro, such as when mRNA is synthesized by in vitro transcription (IVT).

[0116] Therefore, as used herein, the terms nucleic acids, DNA, and RNA also include known types of modifications. For example, these include labels, methylation, "caps," substitutions of one or more naturally occurring nucleotide analogs, internucleotide modifications, such as those having uncharged links (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), negatively charged links (e.g., phosphorothioates, phosphorodithioates, etc.), and positively charged links (e.g., aminoalkylphosphoramidates, aminoalkylphosphotriesters, etc.); those containing pendant portions, such as proteins (including nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.); those containing intercalators (e.g., acridine, psoralens, etc.); those containing chelating agents (e.g., metals, radioactive metals, boron, oxidizing metals, etc.); those containing alkylating agents; those having modified links (e.g., α-anomeric nucleic acids, etc.); and unmodified forms of polynucleotides or oligonucleotides.

[0117] Similarly, the terms “nucleoside” and “nucleotide” as used herein include not only known purine and pyrimidine bases, but also parts containing other modified heterocyclic bases. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, or other heterocyclic bases. Modified nucleosides or nucleotides also include modifications of the sugar moiety, for example, by substituting one or more hydroxyl groups with halogens or aliphatic groups, or by functionalizing them as ethers or amines. Other modifications to nucleotides or polynucleotides include rearrangement, addition, substitution, or other alteration of functional groups on purine or pyrimidine bases that form hydrogen bonds with their respective complementary pyrimidines or purines, such as isoguanines, isocysteines, and similar groups. In some embodiments, oligonucleotides and / or probes contain one or more, two or more, three or more, or four or more modified nucleotides.

[0118] In some embodiments, nucleic acids such as RNA disclosed herein contain one or more universal bases. As used herein, the term “universal base” refers to a nucleotide analog that can hybridize to one or more nucleotides selected from A, U / T, C, and G. In some embodiments, the universal bases may be selected from the group consisting of deoxyinosine, 3-nitropyrrole, 4-nitroindole, 6-nitroindole, and 5-nitroindole.

[0119] In its broadest sense, the term "saRNA" encompasses small activating RNA molecules, including RNA molecules that act within the RNA activation (RNAa) pathway. SaRNAs can be double-stranded. The length of a saRNA can range from approximately 5 to 50 base pairs, particularly from approximately 10 to 40 base pairs, and even more specifically from approximately 10 to 30 base pairs.

[0120] In its broadest sense, the term "shRNA" encompasses small hairpin RNAs, including RNA molecules that act in the RNA interference (RNAi) pathway. shRNAs may be single-stranded, form base pairs, and create hairpin loops. A single strand of shRNA may have a length ranging from approximately 10 to 100 bases, particularly 25 to 75 base pairs, and more particularly 40 to 70 base pairs, and may form a hairpin loop.

[0121] In its broadest sense, the term "siRNA" encompasses small interfering RNAs, including RNA molecules that act in the RNA interference (RNAi) pathway. siRNA can be double-stranded. The length of siRNA can range from about 5 to about 50 base pairs, particularly about 10 to about 40 base pairs, and even more particularly about 15 to about 30 base pairs. Examples 4, 6, and 7 of this specification examine the APIs of siRNA in the compositions of this disclosure.

[0122] In its broadest sense, the term "tRNA" includes transfer RNA, which is an RNA molecule that acts as a link (or adapter) between mRNA molecules and the growing amino acid chain during protein synthesis. The primary structure of tRNA can be approximately 20 to 200 nucleotides long, particularly in the range of approximately 50 to 100 nucleotides. tRNA can have a cloverleaf-shaped secondary structure. tRNA can have an L-shaped tertiary structure.

[0123] In its broadest sense, the term "mRNA" encompasses messenger RNA for protein synthesis. It may also include mRNA consisting of a 5-prime cap and / or polyadenylated ends. Alternatively, it may lack one or both of these features. Typically, mRNA can be single-stranded. The coding region of mRNA can be about 100 nucleotides or longer, particularly about 500 nucleotides or longer, and more particularly about 1000 nucleotides or longer. Examples 1, 2, and 7 of this specification examine the APIs of mRNA in the compositions of the present disclosure.

[0124] mRNA can encode an antigen, thereby providing a composition that constitutes a vaccine. The antigen may be a bacterial antigen, a parasitic antigen, or a fungal antigen. The antigen may be a viral antigen, in particular a viral antigen of any of the viral diseases described herein. More specifically, it may be an antigen of a respiratory virus, such as the antigen of SARS-CoV-2, such as an antigen derived from the spike protein of SARS-CoV-2.

[0125] mRNA may encode allergens (including, but not limited to, one or more nut allergens; one or more seed storage proteins such as bicillin, legumin, and albumin; one or more plant defense-related proteins; and one or more profilins).

[0126] The mRNA may encode proteins that regulate immune disorders, autoimmune disorders, and inflammatory disorders (including, but not limited to, lupus, atherosclerosis, chronic obstructive pulmonary disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatic diseases, uveitis, atopic dermatitis, and pulmonary fibrosis).

[0127] mRNA may encode tumor-specific antigens. In this specification, the term tumor-specific antigen may refer to an antigen arising from a non-synonymous cell mutation (leading to a new antigen) or a fusion viral mutation (leading to an oncoviral antigen) in one or more malignant cancer cells. Therefore, tumor-specific antigens refer to antigens that are not present (not expressed) at all in non-cancerous (healthy, normal) cells.

[0128] mRNA may encode tumor-associated antigens. In this specification, the term tumor-associated antigen may refer to an antigen that is overexpressed in malignant cancer cells compared to non-cancerous cells (healthy normal cells) due to, for example, gene amplification or post-translational modification. The term tumor-associated antigen may include overexpressed antigens (a term referring to proteins that are moderately expressed in non-cancerous (healthy, normal) cells but abundantly expressed in malignant cancer cells), differentiation antigens (a term referring to proteins that are selectively expressed depending on the cell lineage from which malignant cells have evolved, e.g., prostate-specific antigens), and oncogenic germline antigens (this term is usually limited to germ tissue, but may refer to antigens that are abnormally expressed in malignant cancer cells, e.g., melanoma antigen family A3 (MAGE-A3), New York esophageal squamous cell carcinoma-1 antigen (NY-ESO-1), and antigens preferentially expressed in melanoma (PRAME)).

[0129] mRNA can encode multiple proteins, thereby enabling more effective pharmacological activity. mRNA can encode multiple antigens, particularly multiple viral antigens.

[0130] mRNA may further encode adjuvant proteins. Additionally or alternatively, adjuvants can be provided as further components of the composition in addition to the API.

[0131] As is evident from Examples 1, 2, 4, 6, and 7 below, doping with silicon particles has been found to help the disclosed compositions deliver APIs to cells more effectively, particularly when the API is nucleic acid, more specifically siRNA or mRNA. In particular, the doped silicon-containing compositions disclosed herein can mitigate or solve the problem of how to ensure that an API administered to a patient reaches cells, including a method for stabilizing the API while it is circulating in the body. The compositions can mitigate or satisfy the need for targeting of tissues or cells, thereby enabling the delivery of the API to the correct cells. Once reached in target cells, the compositions can facilitate the efficient uptake of the API into the cells. For example, stabilizing the mRNA API in the cytoplasm for an appropriate period of time to allow for more successful translation of the mRNA and release.

[0132] Both types of mRNA (LUC mRNA1 and LUC mRNA2) used in Examples 1 and 2 of this specification are chemically modified compared to naturally occurring mRNA. Such chemical modifications can make transfection more difficult. Doped Si-containing SIS0013 has an improved ability to enable transfection of chemically modified mRNA compared to undoped Si-containing SIS0012, which is particularly advantageous and surprising. Therefore, in some embodiments, the API may be chemically modified mRNA compared to naturally occurring mRNA.

[0133] Therefore, the methods disclosed herein may include an in vivo step of transfecting human cells with nucleic acids.

[0134] The doped silicon-containing compositions disclosed herein for the delivery of APIs may be non-toxic. For example, they may be substantially completely biodegradable, in that the silicon decomposes in vivo into non-toxic orthosilicic acid, as described herein.

[0135] For the delivery of APIs, the doped silicon-containing compositions disclosed herein may be completely dispersible in an aqueous environment to ensure ease of delivery, such as by injection in an aqueous solution.

[0136] Complex formation of components, particularly particle / lipid / API complex formation. Preferably, the particles containing the hydrolyzable doped silicon are complexed with one or more lipids to form a delivery medium for transporting the API. Thus, the API also forms a complex with the particles and / or the lipids when added. In other words, the particles and the lipids are organized into a delivery medium filled with the API. Advantageously, this may make the API less reactive with one or more external reactants. The API may be at a lower risk of being enzymatically degraded outside the complex, particularly in vivo, for example, in circulation and / or in the cytoplasm. This is especially true if the API is nucleic acid, more specifically mRNA.

[0137] In the broadest sense as used herein, the term “complex-formed” may encompass ionic interactions and / or covalent interactions and / or physical interactions, and in particular, charge-charge interactions such as those arising from the zeta potential of the particles.

[0138] Therefore, preferably, the ζ potential of the particles will attract and promote the binding of the API, particularly when regulated by the one or more types of lipids and other components present.

[0139] In preferred embodiments where one or more amino acids are present, the amino acids may also form complexes with the particles, lipids, and / or APIs. The amino acids, especially when charged, modulate the zeta potential of the particles and thus regulate the complex formation between the APIs and / or lipids and the particles.

[0140] If other advantageous components, such as those listed below, are present, they may form complexes with the particles, lipids, amino acids, and / or APIs.

[0141] The aforementioned one or more lipids may be formed from one or more lipid structures, or may contain one or more lipid structures. The structures may be one or more of micelles, incomplete micelles, liposomes, incomplete liposomes, and (e.g., solid or semi-solid) lipid globules, as described herein, or may contain these.

[0142] On the other hand, preferably, the API (in particular RNA, in particular mRNA) is bound (in particular electrostatically conjugated) to particles made of hydrolyzable doped silicon. Preferably, about 50% or more, about 60% or more, or about 70% or more of the API in the composition is bound to the particles in this manner.

[0143] Preferably, the particles made of hydrolyzable doped silicon are bound to the surface of and / or present inside the one or more lipid structures.

[0144] Particle-API bonding is particularly effective in reducing or preventing API degradation when particles containing hydrolyzable doped silicon aggregate in a chain-like manner. Therefore, it is preferable that the particles aggregate. Silicon doping can promote such aggregation, for example, by reducing the repulsive forces between particles.

[0145] The chain may extend into the interior of a lipid structure (such as a liposome, incomplete liposome, micelle, incomplete micelle, and / or lipid sphere) formed by one or more of the lipids. APIs bound to chain-like particles embedded within a lipid structure can be protected from hydrolysis, particularly from water molecules.

[0146] The aforementioned lipid structures (liposomes, incomplete liposomes, micelles, incomplete micelles, and / or lipid spheres, etc.) typically have an average diameter of about 50 nm to about 500 nm, particularly about 100 nm to about 500 nm. On the other hand, particles containing hydrolyzable doped silicon typically have an average diameter in the range of about 1 nm to about 50 nm, for example, about 1 nm to about 30 nm. This relative size difference can optimize the formation of aggregates (especially chains) of particles that are embedded in or modify the surface of lipid structures.

[0147] Therefore, preferably, the particles containing hydrolyzable doped silicone are bound to and / or present inside one or more of micelles, incomplete micelles, liposomes, incomplete liposomes, and (e.g., solid or semi-solid) lipid spheres, and the API is bound to the particles containing hydrolyzable doped silicone. Preferably, the particles containing hydrolyzable doped silicone are present in one or more aggregates of the particles containing hydrolyzable doped silicone, particularly one or more aggregates containing or consisting of chains of the particles, most particularly inside one or more lipid structures, for example, micelles, incomplete micelles, liposomes, incomplete liposomes, and (e.g., solid or semi-solid) lipid spheres (particularly liposomes and / or lipid spheres), containing or consisting of chains extending into the interior of one or more of these structures. Amino acids (in particular glycine, arginine, and / or tyrosine, e.g., glycine) may also associate (e.g., non-covalently) with the particles containing the hydrolyzed positively doped silicon, and / or associate (e.g., on the surface thereof) with one or more lipid structures.

[0148] As used herein, the terms “liposomal lipid particle” or “liposome” may have the common meanings in the art. Therefore, the terms refer to vesicles having one or more lipid bilayers, which may be substantially spherical in shape. Liposomes can be visualized as “bubbles” of lipids surrounding an internal space. This internal space may be a hydrophilic environment.

[0149] In some embodiments, the composition may comprise one or more liposomes. That is, the one or more lipids may be formed from one or more liposomes, or may comprise one or more liposomes.

[0150] In some embodiments, the composition may contain incomplete liposomes. In this sense, the internal space may be accessible from the outside. Incomplete liposomes can be visualized as incomplete lipid "bubbles" with one or more gaps on the surface of a (nearly spherical) lipid bilayer. Preferably, the one or more lipids may be formed from or contain one or more incomplete liposomes.

[0151] Preferably, the one or more lipids may be formed from one or more incomplete liposomes and / or one or more (complete) liposomes, or may contain these.

[0152] Therefore, the composition may contain particles containing hydrolyzable doped silicone associated with one or more liposomes and / or one or more incomplete liposomes, wherein the API is associated (particularly bound) to the hydrolyzable doped silicone particles. Amino acids (particularly glycine, arginine, and / or tyrosine, e.g., glycine) may also be associated with the hydrolyzable doped silicone particles.

[0153] Optionally, the API may be encapsulated with one or more of the lipids.

[0154] Liposomes are particularly well-suited for encapsulating APIs. Therefore, the API may optionally be encapsulated in liposomes, or preferably, partially encapsulated in incomplete liposomes. If the liposomes are incomplete, a pathway exists for the API to traverse between the inside and outside of the liposome.

[0155] The average diameter of optional liposomes or incomplete liposomes may be in the range of approximately 50 nm to approximately 500 nm, particularly approximately 50 nm to approximately 300 nm, and even more particularly approximately 100 nm to approximately 300 nm.

[0156] The API may be non-covalently associated (particularly by ionic interactions) with the outer surface of a liposome or an incomplete liposome. The API may be located within the internal space of an (optionally) incomplete liposome (i.e., at least partially encapsulated). Preferably, about 10% or more of the API is located within the internal space. Additionally or alternatively, it is preferable that 10% or more of the API is located in non-covalent association with the outer surface.

[0157] Therefore, approximately 10% or more, approximately 30% or more, or approximately 50% or more of the API may be completely encapsulated within the internal space of the (optionally) incomplete liposome, while the remainder may be arranged in non-covalent association with the outer surface of the (optionally) incomplete liposome.

[0158] The API may be non-covalently bonded to a particle containing hydrolyzable doped silicon bound to the surface of one or more liposomes and / or one or more incomplete liposomes. Up to about 10% or 20% of the API may be non-covalently bonded to the particle containing hydrolyzable doped silicon, whereas the particle is bound to the surface of one or more liposomes and / or one or more incomplete liposomes.

[0159] The API may be non-covalently bonded to particles containing hydrolyzable doped silicon, where the particles are located inside one or more liposomes and / or one or more incomplete liposomes. Approximately 50% or more, approximately 60% or more, or 70% or more of the API may be non-covalently bonded to particles containing hydrolyzable doped silicon located inside one or more liposomes and / or one or more incomplete liposomes.

[0160] Preferably, the API (in particular RNA, most particularly mRNA) is non-covalently bound to one or more liposomes and / or particles containing hydrolyzable doped silicone inside one or more incomplete liposomes, and the API (in particular RNA, most particularly mRNA) is non-covalently bound to one or more liposomes and / or particles containing hydrolyzable doped silicone bound to the surface of one or more incomplete liposomes.

[0161] In some embodiments, the composition may be liposome-free or substantially liposome-free, and / or may be liposome-free or substantially liposome-free.

[0162] Optionally, the one or more lipids may be formed from one or more lipid monolayers, or may contain one or more lipid monolayers. Optionally, the one or more lipids may be one or more micelles or incomplete micelles, or may contain one or more micelles or incomplete micelles. The micelles have similar properties to liposomes, but differ in that the micelle wall is formed from a lipid monolayer, while the liposome wall is formed from a lipid bilayer. Therefore, a micelle refers to a vesicle having one or more lipid monolayers, and its shape may be approximately spherical. Like liposomes, micelles can be visualized as lipid "bubbles" surrounding an internal space. The internal space may be a hydrophilic environment.

[0163] Therefore, the composition may contain particles comprising hydrolyzable doped silicon associated with one or more micelles and / or one or more incomplete micelles, wherein the API is associated (particularly bound) to the hydrolyzable doped silicon particles.

[0164] The average diameter of optionally present micelles may be in the range of approximately 50 nm to 500 nm, particularly 50 nm to 300 nm, and even more specifically 100 nm to 300 nm.

[0165] The API may be non-covalently bonded to particles containing hydrolyzable doped silicon bonded to the surface of one or more micelles and / or one or more incomplete micelles. Up to about 10% or 20% of the API may be non-covalently bonded to the particles containing hydrolyzable doped silicon, whereas the particles are bonded to the surface of one or more micelles and / or one or more incomplete micelles.

[0166] The API may be non-covalently bonded to particles containing hydrolyzable doped silicon, where the particles reside within one or more micelles and / or one or more incomplete micelles. Approximately 50% or more, approximately 60% or more, or approximately 70% or more of the API may be non-covalently bonded to particles containing hydrolyzable doped silicon residing within one or more micelles and / or one or more incomplete micelles.

[0167] Preferably, the API (in particular RNA, most particularly mRNA) is non-covalently bound to one or more micelles and / or particles containing hydrolyzable doped silicon inside one or more incomplete micelles, and the API (in particular RNA, most particularly mRNA) is non-covalently bound to one or more micelles and / or particles containing hydrolyzable doped silicon bound to the surface of one or more incomplete micelles.

[0168] In some embodiments, the composition may be omitted or substantially omitted from micelles, and / or may be omitted or substantially omitted from incomplete micelles.

[0169] The one or more lipids may be formed from one or more lipid spheres, or may contain one or more lipid spheres, each sphere optionally surrounded by a layer of surfactant. The lipid spheres do not enclose an internal space or cavity. Instead, they are formed from lipids as a solid or substantially solid, and other components, such as particles containing hydrolyzable doped silicone to which API molecules are bound, may be dispersed therein. Thus, particles containing hydrolyzable doped silicone are dispersed inside the spheres, with API molecules (non-covalently) bound to the particles containing hydrolyzable doped silicone. Additionally or alternatively (preferably further), particles containing hydrolyzable doped silicone to which API molecules (non-covalently) are bound may be bound to the surface of one or more lipid spheres.

[0170] Therefore, preferably, the composition comprises particles containing hydrolyzable doped silicon that are associated with (particularly dispersed inside and / or bound to the surface of) one or more (solid or substantially solid, i.e., non-hollow) lipid spheres, wherein the API is associated with (particularly bound to) the hydrolyzable doped silicon particles.

[0171] The average diameter of the optionally present lipid globules may be in the range of approximately 50 nm to 500 nm, particularly 50 nm to 300 nm, and even more particularly 100 nm to 300 nm.

[0172] The API may be non-covalently bonded to a particle containing hydrolyzable doped silicon bound to the surface of one or more lipid spheres. Up to about 10% or 20% of the API may be non-covalently bonded to the hydrolyzable doped silicon particle, whereas the particle is bound to the surface of one or more lipid spheres.

[0173] The API may be non-covalently bonded to particles containing hydrolyzable doped silicon, where the particles are located inside one or more lipid spheres. Approximately 50% or more, approximately 60% or more, or approximately 70% or more of the API may be non-covalently bonded to particles containing hydrolyzable doped silicon located inside one or more lipid spheres.

[0174] Preferably, the API (in particular RNA, most particularly mRNA) is non-covalently bound to a hydrolyzable doped silicon-containing particle located inside one or more lipid spheres, and the API (in particular RNA, most particularly mRNA) is non-covalently bound to a hydrolyzable doped silicon-containing particle bound to the surface of one or more lipid spheres.

[0175] Silicon particle aggregates Particles containing hydrolyzable doped silicon can fuse with aggregates of hydrolyzable doped silicon particles, as shown, for example, in the transmission electron microscope (TEM) image in Figure 27.

[0176] Therefore, the above composition may contain aggregates (especially chains) of particles containing hydrolyzable doped silicon.

[0177] As used herein, the term “aggregates of hydrolyzable doped silicon particles” may refer to clusters of particles in which the nearest adjacent particles are in contact with each other. Such clusters can have various configurations, including clusters of substantially spherical particles and / or chains of particles. Particularly preferred are configurations that include or consist of chains of particles.

[0178] Therefore, the composition may contain one or more aggregates of particles containing hydrolyzable doped silicon. Preferably, the aggregates contain one or more chains of particles.

[0179] For example, each aggregate may contain an average of approximately two or more, three or more, or four or more chains.

[0180] The one or more aggregates may contain one or more branched chains of the particles. Therefore, the one or more aggregates may be formed from branches formed from the chains of the particles, or may contain branches formed from the chains of the particles. For example, there may be about two or more, about three or more, or about four or more branches per aggregate.

[0181] One or more aggregates of the hydrolyzable doped silicon particles may be associated with one or more types of lipids, for example, embedded in the lipids and / or attached to the surface of the lipids.

[0182] One or more aggregates of the hydrolyzable doped silicon particles may associate with one or more lipid structures described herein, for example, by being embedded in and / or attached to one or more lipid structures.

[0183] As described herein, the one or more lipid structures may be formed from or comprise one or more micelles, incomplete micelles, liposomes, incomplete liposomes, and lipid spheres. Accordingly, one or more aggregates of the hydrolyzable doped silicon particles may associate with (e.g., embed and / or adhere to) one or more lipid micelles, incomplete lipid micelles, liposomes, incomplete liposomes, and lipid spheres.

[0184] In particular, one or more aggregates of the hydrolyzable doped silicon particles can be embedded in or attached to the surface of one or more liposomes, incomplete liposomes, and lipid spheres.

[0185] The ratio of the longest dimension of the aggregate to the longest dimension of the lipid structure is, on average, about 1:5 to 5:1, and especially about 1:3 to 3:1, particularly when the lipid structure is liposomes and / or lipid spheres, or contains them, and one or more aggregates are embedded therein or attached to their surface. This can be measured, for example, by TEM as shown in Figure 27.

[0186] The average longest dimension of the aggregates, as measured by TEM, may be approximately 50 nm to 500 nm, particularly approximately 50 nm to 200 nm, for example, approximately 50 nm to 150 nm. In particular, one or more aggregates may be chains of one or more particles, or may contain chains of one or more particles, with an average chain length of approximately 50 nm to 500 nm, particularly approximately 50 nm to 200 nm, for example, approximately 50 nm to 150 nm. The average cross-sectional diameter of the chains may be approximately 5 nm to 50 nm, for example, approximately 5 nm to 30 nm.

[0187] The ratio of the longest dimension of individual particles containing hydrolyzable doped silicon to the longest dimension of the lipid structure is, on average, in the range of about 1:100 to about 1:2, particularly about 1:100 to about 1:5, and even more particularly about 1:100 to about 1:9, especially when the lipid structure is liposomes and / or lipid spheres, or contains them, and one or more aggregates are embedded therein or attached to their surface. This can be measured, for example, by TEM as shown in Figure 27.

[0188] If one or more aggregates are present, the average (e.g., mean) diameter of the hydrolyzable doped silicon particles is preferably about 1 nm to about 50 nm, particularly about 5 nm to about 50 nm, more specifically about 1 nm to about 30 nm, and even more specifically about 5 nm to about 20 nm, for example, about 10 nm. Additionally or alternatively, the particles may be porous and have an average (e.g., central) pore diameter of about 0.1 to about 5 nm, for example, about 2 nm.

[0189] Consequently, the API may be bound (non-covalently) to one or more particles within the aggregate.

[0190] Therefore, one or more aggregate particles can bind to the API (especially if the API is mRNA or contains mRNA). If the one or more aggregates are a chain of particles or contain a chain of particles, such chains may extend into the interior of lipid structures (especially liposomes, incomplete liposomes, and / or lipid spheres). In this way, the chains may provide a pathway for more appropriate encapsulation of the API within the lipid. While we do not wish to be bound by theory, it is conceivable that the API can be protected from degradation, particularly by protecting it from enzymes (especially in vivo) and by preventing or reducing the reaction of the API molecule with water molecules. If the silicon particles degrade over time, the API is released, and it is conceivable that the API can be protected until it reaches the target site of release.

[0191] The mere presence of a charged API may promote the aggregation of the particles. For example, if the API is a nucleic acid (particularly mRNA), its negative charge (due to the phosphate backbone) may induce particle aggregation. Additionally or alternatively, the presence of Si-O species on the particle surface may induce interactions between particles and promote aggregation.

[0192] Therefore, preferably, the one or more aggregates are one or more chains of hydrolyzable doped silicon particles, or include them, the API bound to the particles, and one or more chains extending into the interior of the lipid structure, particularly into liposomes, incomplete liposomes, or lipid spheres. This may provide a more stable environment for the API. Additionally or alternatively, it may be possible to increase the uptake of the API by the lipid structure compared to the absence of such aggregates. This is in contrast to conventional liposome delivery media where inefficient API uptake can be a problem. For example, it is thought that up to about 80% of conventional liposome delivery media formulated into commercially available therapeutic compositions may have "empty" APIs.

[0193] Optionally, particles containing hydrolyzable doped silicon do not exist as virtually isolated particles, but rather virtually all particles exist as aggregates.

[0194] Aggregation can be promoted when the average diameter of the hydrolyzable doped silicon particles is about 1 nm to about 50 nm, particularly about 5 nm to about 50 nm, more specifically about 1 nm to about 30 nm, and even more specifically about 5 nm to about 20 nm, for example, about 10 nm. The diameter of such particles may be smaller than the typical diameter of lipid structures (as described herein) that can be spontaneously formed (optionally promoted by extrusion) from one or more lipids.

[0195] Therefore, preferably, the composition comprises aggregates (particularly chains) of hydrolyzable doped silicon particles, the hydrolyzable doped silicon particles having an average diameter of about 1 nm to about 50 nm, particularly about 5 nm to about 50 nm, more particularly about 1 nm to about 30 nm, and even more particularly about 5 nm to about 20 nm, for example, about 10 nm.

[0196] Aggregation can be promoted, for example, when extruding the composition by the method described in the "Extrusion" step of "Materials and Methods" in Example 1 below. The extrusion may be carried out through a porous membrane having an average pore size of about 0.01 μm to about 1 μm, for example, about 0.05 μm to about 0.6 μm, or may include such extrusion.

[0197] Therefore, the composition may be an extruded composition (or may be formed from an extruded composition).

[0198] Preferably, extrusion is performed before the addition of the API. Therefore, the composition may be formed from an extruded composition to which the API has been added, comprising the particles and one or more lipids.

[0199] Therefore, the composition may be an extruded composition comprising aggregates (particularly chains) of hydrolyzable doped silicon particles, the average diameter of the hydrolyzable silicon particles being about 1 nm to about 50 nm, particularly about 5 nm to about 50 nm, more specifically about 5 nm to about 30 nm, and even more specifically about 5 nm to about 20 nm, for example, about 10 nm.

[0200] Other beneficial ingredients Optionally, the composition may further comprise one or more other beneficial components.

[0201] One or more other advantageous components may be tyrosine or a derivative thereof, or may contain tyrosine or a derivative thereof. Therefore, optionally, in addition to the above amino acids, tyrosine, a specific amino acid, may be present as a further component, as exemplified in the compositions "SIS0013-T" of Examples 3 and 6 below. Optionally, the weight ratio of one or more lipids (i.e., total lipid components) to tyrosine may be in the range of about 130:1 to about 30:1, for example, about 80:1.

[0202] Additionally or alternatively, the composition may contain nicotinamide adenine dinucleotide (NAD) or a derivative thereof, as exemplified by the composition "SIS0013-N" of Examples 3 and 6 below. Optionally, the weight ratio of one or more lipids (i.e., total lipid components) to NAD may be in the range of about 130:1 to about 30:1, for example, about 80:1.

[0203] Additionally or alternatively, the composition may contain quercetin or a derivative thereof, as exemplified by the composition "SIS0013-Q" of Examples 3 and 6 below. Optionally, the weight ratio of one or more lipids (i.e., total lipid components) to quercetin may be in the range of about 130:1 to about 30:1, for example, about 80:1.

[0204] The composition may optionally further include a peptide containing a cell surface receptor (e.g., integrin) recognition sequence that confers a certain degree of cell specificity. The peptide may further have a "head group" containing the cell surface receptor recognition sequence and a "tail" that can non-covalently bond to an API (such as nucleic acids like mRNA) and / or doped silicon.

[0205] If the API is a nucleic acid, the composition may optionally further contain a polycationic nucleic acid-binding component. The term "polycationic nucleic acid-binding component" is well known in the art and refers to polymers having three or more repeating cationic amino acid residues or other cationic units with positively charged groups, such polymers can form complexes with nucleic acids under physiological conditions. An example of a nucleic acid-binding polycation molecule is an oligopeptide containing one or more cationic amino acids. A polycationic nucleic acid-binding component may be, for example, an oligolysine molecule, an oligohistidine molecule, an oligoarginine molecule, an oligoornithine molecule, an oligodiaminopropionic acid molecule, an oligodiaminobutyric acid molecule, or a complex oligomer containing or consisting of any combination of histidine, arginine, lysine, ornithine diaminopropionic acid, and diaminobutyric acid residues. Further examples of polycationic components include dendrimers and polyethyleneimines.

[0206] Subjects and their diseases and disabilities In preferred embodiments, the subjects to whom the compositions disclosed herein are administered are human subjects. The age of the human subjects may be between one month and optionally one year or older. The age of the human subjects may be between 100 years and older.

[0207] The diseases or disorders described herein may be infectious diseases. The term “infectious” as used herein may be used to mean diseases that can be transmitted from one organism to another, in particular from one person to another.

[0208] The aforementioned infectious disease may be a viral, bacterial, fungal, or parasitic disease, particularly a viral disease.

[0209] If the aforementioned disease is a viral disease, it may be a disease caused by respiratory viruses such as syncytial virus (RSV), parainfluenza virus (HPIV), metapneumovirus (HMPV), rhinovirus (HRV), coronaviruses such as SARS-CoV (especially SARS-CoV-1, and even more specifically SARS-CoV-2), adenovirus (HAdV), enterovirus (EV), bocavirus (HBoV), parechovirus (HPeV), or influenza virus.

[0210] Examples of viral diseases include those caused by dengue virus, Ebola virus, encephalomyocarditis virus, hepatitis virus, herpesvirus, human immunodeficiency virus, human papillomavirus, human T lymphotropic virus, measles virus, monkeypox virus, mumps virus, poliovirus, rabies virus, rotavirus, rubella virus, varicella-zoster virus, West Nile virus, yellow fever virus, or Zika virus.

[0211] The aforementioned disease or disorder may be a hereditary disease or disorder.

[0212] In some embodiments, the hereditary disorder may be characterized by a deficiency in the expression of one or more proteins, particularly one or more enzymes.

[0213] The aforementioned hereditary disorders may be multifactorial disorders, meaning they are not limited to specific patterns of single-gene inheritance, but may be associated with the influence of multiple genes along with environmental factors, such as schizophrenia, diabetes, asthma, depression, epilepsy, heart disease, or hypothyroidism.

[0214] The aforementioned genetic disorders may involve one or more mutations in one or more genes.

[0215] Therefore, the aforementioned hereditary disorder may be a monogenic disorder, which can occur when at least one mutation occurs in a single gene. When the aforementioned hereditary disorder is a monogenic disorder, a single gene may contain one mutation or multiple mutations. Examples of monogenic disorders include sickle cell anemia, cystic fibrosis, Huntington's disease, or Duchenne muscular dystrophy.

[0216] The aforementioned hereditary disorder may involve one or more mutations in multiple genes. As a non-limiting example, the aforementioned hereditary disorder may involve multiple mutations in a first gene and one mutation in a second gene.

[0217] The aforementioned hereditary disorder may be a disease that can occur when at least one mutation occurs in at least one gene within a set of genes, and in particular, such a hereditary disorder may be osteopetrosis.

[0218] The aforementioned genetic disorders may be Angelman syndrome, Canavan disease, Charcot-Marie-Tooth disease, color blindness, cri du chat syndrome, cystic fibrosis, DiGeorge syndrome, Down syndrome, Duchenne muscular dystrophy, familial hypercholesterolemia, hemochromatosis type 1, hemophilia, Klinefelter syndrome, neurofibromatosis, phenylketonuria, polycystic kidney disease, Prader-Willi syndrome, Scheuermann disease, sickle cell anemia, spinal muscular atrophy, Tay-Sachs disease, or Turner syndrome.

[0219] In the broadest sense as used herein, the term hereditary disorder may also include cancer. Such cancer may be a blood cancer (e.g., leukemia, lymphoma, myeloma, etc.) or a solid tumor (e.g., sarcoma, carcinoma, carcinosarcoma, lymphoma, etc.), or may be associated with both.

[0220] Therefore, in particular, the cancer may be a cancer of the blood, skin, brain, prostate, breast, lung, esophagus, stomach, small intestine, pancreas, colon and / or rectum, central nervous system, bladder, thyroid, kidney, uterine body, oral cavity, or ovary.

[0221] More specifically, the cancers include: respiratory cancers, brain tumors, gastrointestinal cancers, skin cancers, genitourinary cancers, pancreatic cancers, lung cancers, medulloblastomas, basal cell carcinomas, gliomas, breast cancers, prostate cancers, testicular cancers, esophageal cancers, hepatocellular carcinomas, gastric cancers, gastrointestinal stromal tumors (GISTs), colorectal cancers, colon cancers, ovarian cancers, melanomas, neuroectodermal tumors, head and neck cancers, sarcomas, soft tissue sarcomas, fibrosarcomas, myxosarcomas, liposarcomas, and cartilage sarcomas. It may be or may include tumors, osteosarcoma, chordoma, angiosarcoma, endosarcoma, lymphangiosarcoma, lymphangiosarcoma, synoviomas, mesothelioma, leiomyosarcoma, cervical cancer, uterine cancer, endometrial cancer, carcinoma, bladder cancer, epithelial carcinoma, squamous cell carcinoma, adenocarcinoma, bronchial cancer, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, neuroendocrine carcinoma, carcinoid tumors, diffuse giant cell tumors, or glioblastomas.

[0222] Preparation, storage, stability, and administration of the disclosed composition. Preventing a disease or disorder in a human subject may include administering a prophylactic effective amount of the composition disclosed herein to a subject in need, for example, as identified by a physician or other healthcare professional. Conversely, treating a disease or disorder in a human subject may include administering a therapeutic effective amount of the composition disclosed herein to a human subject in need.

[0223] The dosage of the compositions disclosed herein can be modified to obtain an amount of API that is non-toxic to a subject and effective in achieving a desired prophylactic and / or therapeutic response to a given subject. A suitable dosage of the composition may be the amount of the composition that is the minimum dosage in which the API is effective in producing a therapeutic and / or prophylactic effect.

[0224] The selected dosage, form, and regimen will vary depending on various factors. These factors include, for example, the activity of the API, the route of administration, the timing of administration, the rate of excretion or metabolism of the API, the rate and degree of absorption, the duration of treatment, the presence of other drugs, compounds, and / or materials used in combination with the API, the age, sex, weight, condition, health status, and medical history of the patient, as well as other factors well known in the medical field.

[0225] The composition may be administered by intramuscular or intravenous injection (including transdermal administration by patch), orally (including sublingual administration), intranasally, cutaneously, or by other suitable route, particularly by injection or orally.

[0226] Preferably, the composition may be administered by injection, for example, intravenous or intramuscular injection. Optionally, if the composition is administered by injection, the subject will be monitored for symptoms or signs of hypersensitivity reactions, such as vaccine-related hypersensitivity reactions.

[0227] Preferably, the composition may be administered orally or intranasally. Compositions suitable for oral administration may be provided in individual dosage forms, particularly as liquids or aerosol sprays, each containing a predetermined amount of the composition. Such dosage forms can be prepared by any well-known pharmacopoeia method.

[0228] Therefore, the composition is about 1 × 10 16 More than one dopant atom / cm 3A composition comprising hydrolyzable silicon particles doped at a certain level, one or more lipids, and a pharmacokinetic agent (API), which is particularly preferred for use in methods to prevent or treat diseases or disorders in human subjects, the method comprising administering the composition to human subjects by injection, orally or intranasally (particularly by injection or orally). Preferably, the composition may further contain one or more amino acids for the reasons mentioned above. A mouse model of administration by injection is shown, for example, in Example 4 below.

[0229] The composition may be tightly mixed with a pharmaceutical carrier to form a compound according to conventional pharmaceutical formulation techniques. The carrier may take various forms depending on the desired form of preparation for administration. Any of the common pharmaceutical media can be used as a carrier, for example, one or more of water, oil, and alcohol (including glycols). Forms into which the disclosed composition can be incorporated for administration include aqueous solutions in physiological saline, particularly when formulated for injection, oral, or intranasal administration. The composition may further contain one or more pharmaceutically acceptable additives and excipients, such as detangulating agents, defoaming agents, buffers, polymers, antioxidants, chelating agents, viscosity modifiers, isotonic agents, odorants, opacifiers, suspending agents, fillers, plasticizers, flavoring agents, preservatives, colorants, diluents, binders, disintegrants, and mixtures thereof.

[0230] The prevention or reduction of microbial activity can be achieved by adding one or more various antimicrobial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal.

[0231] The compositions disclosed herein may be provided in the form of sterile solutions by incorporating the required amount of the composition into a suitable solvent (with various other components as appropriate) by any well-known pharmaceutical method. The compositions disclosed herein may be provided as sterile dispersions by incorporating the required amount of the composition into a suitable sterile medium (with various other components as appropriate). The compositions disclosed herein may be provided as sterile powders (for example, for the preparation of subsequent sterile injection solutions) by techniques such as vacuum drying and freeze-drying (freeze-drying) to produce powders of the compositions.

[0232] Optionally, the composition may be stored before being administered to the subject. The composition may be stored for at least one week (optionally, up to six months, particularly up to one year) at a temperature of 0°C or higher, especially 4°C or higher, before being administered to the subject.

[0233] In certain preferred embodiments, particles containing hydrolyzable doped silicon and one or more lipids are stored together without the API. Immediately before administration to the subject, for example, within about 3 weeks, about 2 weeks, or about 1 week, particularly within about 2 days, more particularly within about 1 day, they are combined with the API (for example, via a process as described in Example 1 below). Advantageously, the particles and lipids can be stored stably for a long period, particularly several months, for example, about 6 months or about 12 months, thus without significant degradation. If the API is a reactive API, particularly mRNA, it may be advantageously synthesized in situ, or it may be synthesized in advance and delivered to the clinical environment as close to the immediate vicinity as possible before being combined with the particles and lipids.

[0234] Nevertheless, it has also been found that APIs can be stabilized by particles containing hydrolyzable doped silicon, including being stabilized during storage. Therefore, in some embodiments, the particles can enhance the storage stability of the API, particularly when the API is a nucleic acid such as mRNA, or when the API contains a nucleic acid such as mRNA. In some embodiments, particularly when the API is a nucleic acid such as mRNA, or when the API contains a nucleic acid, the particles can enhance the stability of the API at 25°C.

[0235] Therefore, optionally, the stored composition may include all of the hydrolyzable doped silicone particles, one or more lipids, and active pharmaceutical ingredients (APIs). If so, once formulated, the composition may be stored for a short period, which may mean up to about three weeks, two weeks, or one week prior to administration to the subject, as needed.

[0236] In some embodiments, particularly when the API is a nucleic acid such as mRNA or contains nucleic acids, the particles can enhance the stability of the API during circulation in the body. In some embodiments, particularly when the API is a nucleic acid such as mRNA or contains nucleic acids, the particles can protect the API from degradation, especially enzymatic degradation. Thus, the doped silicon-containing compositions disclosed herein, including methods for stabilizing the API while it is circulating in the body, can mitigate or solve the problem of how to ensure that an API administered to a patient reaches the cells.

[0237] On the other hand, the disclosed compositions containing hydrolyzable doped silicon can reduce or satisfy the need for targeting of tissues or cells, thereby enabling the delivery of the API to the correct cells.

[0238] Additionally or alternatively, upon reaching target cells, the disclosed compositions can mitigate or solve the challenge of ensuring efficient uptake of APIs by cells. For example, the disclosed compositions can assist in the transport of APIs from outside the cell to the cytoplasm.

[0239] Following uptake of the API by cells, the disclosed composition can mitigate or solve the problem of how to prevent the rapid degradation of the API in the cytoplasm. The particles are thought to enhance the stability of the API in the cytoplasm of cells, particularly against enzymatic degradation when the API is a nucleic acid such as mRNA or when the API contains a nucleic acid such as mRNA. [Examples]

[0240] Examples 1 and 2 disclose the study of mRNA complex formation using undoped silicon-containing delivery media versus doped silicon-containing delivery media.

[0241] Particle size and zeta potential measurements were obtained, and gel delay assays and Quant-iT RiboGreen RNA assays were performed. mRNA transfection using doped silicon-containing delivery media was also investigated by in vitro transfection of luciferase-encoding mRNA into human embryonic kidney (HEK293) cells, and observed by bioluminescence imaging of cell cultures.

[0242] Example 1 We compared the ability of a dopant-containing silicon particle delivery medium ("SIS0013") and a dopant-free silicon particle delivery medium ("SIS0012") to capture, stabilize, and deliver APIs to cells.

[0243] Two different luciferase mRNAs were selected as test APIs. The success of cell transfection using the luciferase mRNAs can be measured by a luciferase assay, which allows for a reliable and accurate determination of the success rate of API delivery to cells in in vitro culture.

[0244] Furthermore, since mRNA is a relatively reactive molecule, a delivery medium that can stabilize mRNA and deliver it successfully to cells can be expected to stabilize and deliver other APIs as well. Studying highly reactive APIs increases the reliability of extrapolating current research results to other (e.g., less reactive) APIs.

[0245] Materials and methods Composition of SIS0012 and SIS0013 SIS0012 (undoped Si) formulation: -7.25mg of DOTAP (1.45mL) -7.3mg of DOPE (1.46mL) -1.45mg DSPE-PEG 2000 - 1 mg of Si nanoparticles (average diameter less than 30 nm, verifiable by SEM) -1 mg of trehalose -0.5mg of glycine -9 mL of nuclease-free water SIS0013 (Dope Si) preparation: -7.25mg of DOTAP (1.45mL) -7.3mg of DOPE (1.46mL) -1.45mg DSPE-PEG 2000 -1 mg of boron-doped Si nanoparticles, approximately 5 x 10⁻⁶ 18 Boron atoms / cm 3 (Average diameter less than 30 nm, verifiable by SEM) -1 mg of trehalose -0.5mg of glycine -9 mL of nuclease-free water

[0246] Further details of the components of SIS0012 and SIS0013 disclosed above are as follows: a) Si ("SiNP"): Porous silicon particles with an average diameter of less than 30 nm. SIS0013 (5 × 10 18 Boron atoms / cm 3 The material is doped with ) and activated by exposure to methanol (to remove surface oxidation and ensure particle uniformity), then slowly evaporated to produce a powder (a dry solid) of activated SiNP. b) Trehalose ("THR"): Solid powder. c) Glycine ("GLY"): Solid powder. d) SiNP+GLY+THR solution: A brown suspension obtained by sonicating a suspension containing 50 mg of activated SiNP, 50 mg of THR, and 25 mg of GLY in 50 mL of nuclease-free water for 60 minutes. e) DOTAP-Cl solution: A solution obtained by mixing 50 mg of DOTAP with 10 mL of methanol and sonicating it at 40°C for 30 minutes to completely dissolve it. f) DOPE solution: A solution obtained by mixing 50 mg of DOPE with 10 mL of methanol and sonicating it at 40°C for 30 minutes to completely dissolve it. g) mPEG2000-DSPE solution: A solution obtained by mixing 40 mg of mPEG2000-DSPE with 8 mL of methanol and sonicating it at 40°C for 30 minutes to completely dissolve it.

[0247] Method for preparing SIS0012 or SIS0013 Preparation of lipid films a) Mix all lipids (DOPE, DOTAP, and mPEG2000-DSPE) in a glass round-bottom flask. b) Evaporate the solvent using a rotary evaporator in a 40°C water bath.

[0248] Film rehydration a) Add 1 mL of a brown suspension of SiNP (SIS0013: doped, SIS0012: undoped) + GLY + THR (1 mg / mL of SiNP, 0.5 mg / mL of GLY, 1 mg / mL of THR) to the lipid film together with 9 mL of nuclease-free water (the total volume of the brown suspension and nuclease-free water is 10 mL). b) Cover the flask with parafilm and shake it in a 60 °C water bath for 10 minutes to rehydrate the lipid membrane with 10 mL of liquid. c) After leaving the resulting suspension at room temperature for several hours, store it at 4 °C.

[0249] Extrusion Pass the suspension obtained in step (c) of "Rehydration of the film" through polycarbonate membrane filters with pore sizes of 0.4 μm and 0.1 μm. Pass the suspension through each pore size 10 times at 60 °C.

[0250] The resulting product is SIS0012 using undoped SiNP and SIS0013 using doped SiNP.

[0251] mRNA encoding luciferase Details of the two luciferase-encoding mRNAs used as test APIs are as follows. (1) Obtained firefly luciferase-encoding mRNA (mod-LUC RNA; 2 mg / mL) having 2315 bases (hereinafter referred to as "LUC mRNA1", "Luc mRNA1", or simply "mRNA1"). (2) On the other hand, obtained EZ Cap™ firefly luciferase mRNA having 1921 bases (5-moUTP modified) encoding luciferase (hereinafter referred to as "LUC mRNA2", "Luc mRNA2", or simply "mRNA2").

[0252] Materials for gel electrophoresis and RiboGreen assay Table 1 shows the materials used for the gel electrophoresis assay and RiboGreen assay of this example (and subsequent examples where similar assays were used).

[0253] Table 1: Materials for gel electrophoresis and RiboGreen assays [Table 1]

[0254] Materials for luciferase assays and HEK293 culture Table 2 shows the materials used for the luciferase assay and HEK293 culture in this example (and subsequent examples using similar cultures).

[0255] Table 2: Materials for luciferase assay and HEK293 culture [Table 2]

[0256] Method for preparing SIS0012-mRNA complex or SIS0013-mRNA complex To prepare SIS0012-mRNA complexes or SIS0013-mRNA complexes in w / w ratios for different mRNA loading, the required amount of SIS0012 or SIS0013 suspension (nominal total lipid concentration: 1.6 mg / mL) was mixed with the required amount of mRNA stock solution (concentration: 2 mg / mL). The final LUC mRNA concentration was then adjusted using nuclease-free water before the complex formation incubation step according to the subsequent assay protocol. The samples were gently pipetted and thoroughly mixed, and incubated at room temperature for 60 minutes until complex formation was complete. After incubation, the samples were stored at 4°C until use in the next assay.

[0257] In the following examples, a different API is used instead of mRNA, so the same procedure was followed for loading the API.

[0258] Table 3: mRNA at different w / w ratios: SIS0012-mRNA complex or SIS0013-mRNA complex [Table 3]

[0259] Method for measuring particle size and zeta potential Particle size, polydispersity, and zeta potential were measured using dynamic light scattering and electrophoretic light scattering (Malvern Zetasizer Pro (Red Advance), Malvern, UK). Size measurements were performed using DTS0012 disposable cuvettes, and surface charge measurements were obtained using DTS1070 folded capillary zeta cells. Analysis was performed on empty SIS0012, empty SIS0013, SIS0012-mRNA, and SIS0013-mRNA. Backscatter detection at a 173-degree angle was used for size determination. Particle size was obtained as Z-mean [nm] by fitting a correlation function using the cumulative method. 20 μL samples of empty SIS0012, empty SIS0013, SIS0012-mRNA, or SIS0013-mRNA were mixed with 980 μL of nuclease-free water to a total volume of 1000 μL and added to disposable cuvettes. To reduce the signal-to-noise ratio, a total of four scans were performed, and the three best scans were selected for average size and PDI (n=3). 150 μL of sample was mixed with 850 μL of nuclease-free water and inserted into a folded capillary cell (using a 1 mL syringe) and measured at 25°C. To eliminate the influence of the signal-to-noise ratio (S / N ratio), a total of five scans were performed, and the three best scans were selected to record the average zeta potential (n=3).

[0260] Gel delay assay method To evaluate the formation of the IS0012-mRNA complex or the SIS0013-mRNA complex, nanoparticles dispersed in nuclease-free water were combined with mRNA at various loading ratios (w / w ratio of mRNA to total lipids in SIS0012 or SIS0013 being 1:4, 1:7.2, 1:12, or 1:24) as shown in Table 3 and analyzed by electrophoresis on an E-Gel™ 1% agarose gel (ThermoFisher Scientific, UK) for 8 minutes using an E-Gel™ Power Snap electrophoresis apparatus (ThermoFisher Scientific, UK). An equal amount of 150 ng of mRNA was loaded into each well, and naked mRNA was used as a control. The gel was visualized using an E-Gel™ Power Snap electrophoresis camera (ThermoFisher Scientific, UK).

[0261] Accessible mRNA assay To evaluate the amount of accessible mRNA within the complex, the Quant-iT RiboGreen RNA reagent from Invitrogen (ThermoFisher Scientific, UK) was used. The experimental procedure was carried out according to the manufacturer's guidelines. For the analysis, a Varioskan LUX multimode plate reader (ThermoFisher Scientific, UK) was used with an excitation wavelength of 480 nm and an emission wavelength of 520 nm. The tests were performed in 1× Tris-EDTA (TE) buffer (10 mM Tris-HCl and 1 mM EDTA). All samples were measured in triplicate, and the results were reported as mean ± SD.

[0262] In vitro cell transfection evaluation method To evaluate transfection, a human fetal kidney (HEK293) cell line was used. Cells were cultured in Dulbecco's modified Eagle medium (Euroclone, Italy) supplemented with 10% inactivated fetal bovine serum (FBS, Gibco, UK), 1% L-glutamine (Euroclone, Italy), and 1% Pen / Strep (Euroclone, Italy), incubated at 37°C under 5% CO2, and subcultured according to standard laboratory procedures. 24 hours prior to transfection, HEK293 cells were transfected in 1 × 10⁶ cells per well in 100 μL of antibiotic-free complete medium in a 96-well black plate with a clear bottom. 4 Cells (or 2 × 10 per well in the assay at 72 hours) 4 Cells were seeded. The cells were cultured for 24 hours under standard culture conditions (37°C, 5% CO2), and the following day the medium was replaced with 100 μL of transfection medium. This medium contained 10 μL of a transfection complex containing 1 μg of mRNA prepared with nuclease-free water, and 10 μL of serum-free DMEM containing 1% L-glutamine, which was diluted with 80 μL of complete growth medium (DMEM containing 10% FBS and 1% L-glutamine). 10 μg / mL of naked mRNA diluted in complete medium or no mRNA (medium only) was used as a negative control.

[0263] After the transfection procedure was completed, the cells were transferred to an incubator and cultured under standard conditions (37°C, 5% CO2). The LUC-mRNA translation product was detected by luciferase assay (Bright-Glo Luciferase kit, Promega, USA) at 6, 24, 48, or 72 hours post-treatment, according to the manufacturer's protocol. Briefly, after equilibrating the plate at room temperature, 100 μL / well of assay reagent was added and incubated at room temperature for 15 minutes. Luciferase activity was evaluated by a luminometer (Hamamatsu Photonics bioluminescence imaging system, Hamamatsu Photonics Italy SRL, Italy, integrated with Arese-digital camera and camera controller, C4742-98-Software Wasabi version 1.5, Hamamatsu Photonics).

[0264] result Results of dynamic light scattering and electrophoretic light scattering The surface charges of SIS0012 and SIS0013, which formed complexes with LUC mRNA1, were investigated when the w / w ratio of mRNA to total lipids was 24:1, 12:1, 7.2:1, and 4:1. The results are shown in Tables 4a to 4d.

[0265] Table 4a: Characterization of empty SIS0012 and empty SIS0013 [Table 4a]

[0266] As shown in Table 4a, SIS0012 (without bound mRNA) has a more negative zeta potential than SIS0013.

[0267] Table 4b: Characterization of SIS0012 complexed with LUC mRNA1 at different w / w ratios (mRNA:total lipids) [Table 4b]

[0268] Table 4c: Characterization of SIS0013 complexed with LUC mRNA1 at different w / w ratios (mRNA:total lipids) [Table 4c]

[0269] Figures 1 and 2 show the size and polyvariance index (PDI) data for SIS0012 and SIS0013 according to Tables 4a to 4c.

[0270] Results for SIS0012: As shown in Tables 4a-4b and Figure 1, PDI was very stable at all binding ratios. However, when SIS0012 complexed with mRNA, an increase in the hydrodynamic diameter of the nanoparticles was observed, particularly at the 7.2:1 and 4:1 ratios. Further addition of mRNA caused this increase to plateau. This plateau indicates that mRNA binding reached saturation. The zeta potential decreased in positive values ​​as mRNA loading increased, and became negative at the 4:1 ratio (i.e., maximum mRNA amount). This is thought to be due to the presence of weakly bound or unbound mRNA.

[0271] As shown in Tables 4a, 4c, and Figure 2, the PDI for SIS0013 increased with increasing mRNA, thus favoring a particularly dispersible delivery medium in an aqueous environment. In SIS0013, excessive accumulation of large lipid clumps appeared to be prevented, which is a known issue with lipid nanoparticles. On the other hand, while SIS0013 was observed to steadily increase in hydrodynamic diameter upon complex formation with mRNA, the plateau observed in SIS0012 was not seen. This suggests that mRNA binding saturation was not achieved as quickly in SIS0013 as in SIS0012. The zeta potential decreased with increasing mRNA, but not as much as in SIS0012. Overall, slow and steady particle growth was observed with increasing mRNA, without undesirable lipid accumulation.

[0272] SIS0013 can load a larger amount (w / w) of negatively charged mRNA than SIS0012. That is, we can conclude that binding efficiency is improved. This is due to the more positive initial zeta potential of SIS0013, which may be caused by the modulation of the zeta potential of SIS0013 by the dopant boron.

[0273] Gel delay assay results Figure 3 shows the results of gel electrophoresis to study the complex formation between SIS0012 and SIS0013 and LUC mRNA1.

[0274] Each well was loaded with either the SIS0012 / LUC mRNA1 or SIS0013 / LUC mRNA1 complex (equal amounts in each well), increasing in ratio from 1:4 to 1:24 (mRNA to total lipids (w / w)). The explanation is that unbound mRNA passed through the gel, while mRNA bound to SIS0012 or SIS0013 remained immobilized within the wells.

[0275] In both SIS0012 and SIS0013, complete binding of nucleic acids was observed when the mRNA:total lipid ratio was 1:7.2, 1:12, and 1:24. At these ratios, mRNA did not pass through the gel.

[0276] When the ratio was 1:4, and the amount of mRNA relative to SIS0012 or SIS0013 was highest, it was observed that some mRNA from both SIS0012 and SIS0013 passed through the gel.

[0277] Therefore, the 1:4 ratio provides a specific basis for comparing SIS0012 and SIS0013. A weaker band was observed in SIS0013 at a 1:4 ratio. Thus, the results of the size and zeta potential analysis above indicate that SIS0013 has improved binding efficiency compared to SIS0012. In other words, SIS0013 appears to be able to bind to more mRNA (w / w) than SIS0012. In the presence of excess mRNA, SIS0013 binds to a larger wt (w / w)% of the total mRNA present compared to SIS0012.

[0278] Similar results were obtained with SIS0012 and SIS0013, which formed complexes with LUC mRNA2.

[0279] Therefore, SIS0013 may be the most efficient delivery medium, capable of binding more mRNA (w / w) than SIS0012. From this, it can be inferred that SIS0013 may further improve binding and enhance nucleic acid stability when an amount of mRNA below the mRNA saturation threshold of SIS0012 is added. At these amounts, the charge interaction between SIS0013 and mRNA is more attractive than that between the same SIS0012 and mRNA, except for the undoped state.

[0280] As explained below, SIS0013 also demonstrates faster efficient uptake of mRNA by cells and faster resulting luciferase expression compared to SIS0012.

[0281] Accessible mRNA Quant-iT RiboGreen assay results To evaluate the amount of mRNA that effectively binds to SIS0012 and SIS0013 and is inaccessible to external reactants, the amount of mRNA accessible by the fluorescent dye was adopted as an indirect parameter indicating the amount of unbound or surface-bound mRNA after complex formation.

[0282] Quant-iT® RiboGreen® RNA Reagent exhibits extremely high binding affinity and specificity to RNA, enabling the quantification of very low concentrations of RNA in a sample. The dye can only bind to free RNA or RNA weakly bound to the outermost surface of the complex, while it cannot access RNA that is more strongly bound within the complex.

[0283] As shown in Figure 4, both SIS0012 and SIS0013 exhibited low mRNA accessibility, with the lowest ratio being 1:24.

[0284] Table 5 shows that mRNA accessibility is higher with other ratios, indicating that SIS0012 has more mRNA that is not bound to the outermost surface of the complex, or is weakly bound, than SIS0013.

[0285] The proportion of mRNA that could react with dyes, i.e., external reactants, was consistently higher with SIS0012 than with SIS0013, and this was particularly pronounced as the amount of mRNA increased, especially when the w / w ratio of mRNA to total lipids was 1:4, i.e., when the amount of mRNA was at its maximum. This suggests that SIS0013 consistently binds to more mRNA than SIS0012, preventing the mRNA from reacting with external reactants.

[0286] It can be hypothesized that SIS0013 can bind to more mRNA in a less superficial way than SIS0012, meaning that SIS0013 may be able to stabilize mRNA (and other APIs) more effectively in the presence of reactive species such as Quant-iT® RiboGreen® dyes. As a result, stability during storage and in vivo may be improved. One advantage is that reactive APIs such as mRNA are stabilized after administration and during circulation, ensuring that sufficient APIs reach target cells and achieve the desired effects, such as luciferase expression, as in this study. Another advantage is the stable delivery of APIs to target cells. On the other hand, stabilization of APIs may also occur within the cytoplasm.

[0287] Table 5: Accessibility (surface binding rate) of mRNAs of SIS0012 and SIS0013 at different w / w ratios of mRNA to total lipids. [Table 5]

[0288] Cell transfection results To compare the performance of SIS0012 and SIS0013, a series of in vitro transfection studies were conducted in HEK293 cells. Cells were transfected with SIS0012 / luciferase (Luc mRNA1), SIS0012 / luciferase (Luc mRNA2), SIS0013 / luciferase (Luc mRNA1), or SIS0013 / luciferase (Luc mRNA2). Translational products were detected by Bright-Glo luciferase assays performed at 6, 24, 48, or 72 hours post-transfection using a luminometer, as described herein. As a guide, Table 6 shows the contents of each well in a three-part sequence.

[0289] Table 6: Guide to the contents of the wells in Figures 5-8 [Table 6]

[0290] Summary: At each time point, SIS0013 was observed to be more effective than SIS0012 in mRNA transfection across the entire range of mRNA-to-total lipid ratios, and particularly at earlier time points, the transfection rate with SIS0013 was suggested to be higher than that with SIS0012. Therefore, doping with hydrolyzable silicon is thought to induce more effective and rapid API uptake, leading to a more rapid and reliable effect of the API on target cells.

[0291] Both LUC mRNA1 and LUC mRNA2 are chemically modified compared to naturally occurring mRNA. Such chemical modifications can make transfection more difficult. The doped Si-containing SIS0013 exhibits a particularly advantageous and surprising ability to enable transfection of chemically modified mRNA compared to the undoped Si-containing SIS0012.

[0292] Six hours after transfection: Data obtained six hours after transfection are summarized in Table 7 and Figure 5. Regardless of the mRNA used (LUC mRNA1 or LUC mRNA2), luciferase signals were observed in wells treated with SIS0012 or SIS0013 at a 1:24 w / w ratio (mRNA:total lipids), with signals from the doped Si-containing SIS0013 complex being more pronounced than those from the undoped Si-containing SIS0012 complex. SIS0013 also showed a signal at a 1:12 w / w ratio (mRNA:total lipids) relative to mRNA2. The less chemical modification of mRNA2 compared to mRNA1 explains why transfection of cells using the same SIS0013 complex appears slightly more effective than transfection using mRNA1.

[0293] Table 7: Summary of results 6 hours after transfection shown in Figure 5 [Table 7]

[0294] 24 hours after transfection: Figure 6 and Table 8 show that SIS0013 complexed with mRNA1 or mRNA2 in ratios of 1:24 and 1:12, respectively, and luminescence signals were observed 24 hours after transfection. The signal of SIS0013 complexed with mRNA2 was also confirmed in a ratio of 1:7.2.

[0295] In comparison, SIS0012 showed a signal in a 1:24 ratio with either mRNA1 or mRNA2, and a signal in a 1:12 ratio with mRNA2 only.

[0296] Table 8: Summary of results 24 hours after transfection shown in Figure 6 [Table 8]

[0297] 48 hours after transfection: Figure 7 and Table 9 show the assay results 48 hours after transfection.

[0298] Table 9: Summary of results 48 hours after transfection shown in Figure 7 [Table 9]

[0299] 72 hours after transfection: Figure 8 and Table 10 show the assay results 72 hours after transfection.

[0300] Table 10: Summary of results 72 hours after transfection shown in Figure 8 [Table 10]

[0301] Example 2: SIS0013 variant using mRNA Example 2 discloses the results of an investigation into the effects of functionalizing the doped Si-containing SIS0013 of Example 1 with different components.

[0302] Materials and methods Unless otherwise specified, the same materials and methods as in Example 1 were used.

[0303] SIS0013 variant The modified SIS0013 compositions, SIS0013-N, SIS0013-T, and SIS0013-Q, were prepared by adding 0.2 mg of NAD, tyrosine ("TYR"), or quercetin ("QUE") as further components, respectively (NAD, TYR, and QUE were obtained from Sigma Aldrich). NAD, TYR, or QUE was added together with the Si nanoparticles in step (a) of the "Rehydration of Film" protocol described in "Materials and Methods" of Example 1 above.

[0304] result Results of dynamic light scattering and electrophoretic light scattering The surface charges of SIS0013-N, SIS0013-T, and SIS0013-Q, which formed complexes with LUC mRNA1, were investigated in the empty state (i.e., without mRNA loading) and when the w / w ratio of mRNA to total lipids was 24:1, 12:1, 7.2:1, and 4:1. The results are shown in Tables 11a to 11c.

[0305] Table 11a: Characterization of SIS0013-N containing LUC mRNA1 at different ratios of total lipids to mRNA. [Table 11a] * This is not as positive as SIS0012 in Example 1, but it is considered to be more positive than when the silicon particles are not doped.

[0306] Table 11b: Characterization of SIS0013-T containing LUC mRNA1 at different ratios of total lipids to mRNA. [Table 11b] * This is not as positive as SIS0013 in Example 1, but it is more positive than SIS0012. It is considered to be more positive than when the silicon particles are not doped.

[0307] Table 11c: Characterization of SIS0013-Q containing LUC mRNA1 at different ratios of total lipids to mRNA. [Table 11c] * This is similar to SIS0012 in Example 1, but is considered more positive than when the silicon particles are not doped.

[0308] Overall, the size was within the range of 200-400 nm, but when the ratio of total lipids to mRNA was 7.2:1, precipitation was observed due to complex formation, and the size increased. While we do not wish to be constrained by theory, it is possible that at a ratio of 7.2:1, undesirable lipid accumulation occurred due to charge interactions between negatively charged RNA and positively charged DOTAP. Precipitation may occur when a turning point is reached in the formation of large lipid fragments. This effect can be thought to be modulated by small changes in Si doping and / or by providing counterions such as sodium chloride (NaCl) or potassium chloride (KCl). In any case, this data point does not detract from the overall trend.

[0309] The measurement results for the ζ potential were consistent with the trend of SIS0013 in Example 1 described above.

[0310] Gel delay assay results Figure 9 shows the results of gel electrophoresis to study the complex formation of SIS0013-N, SIS0013-T, and SIS0013-Q with LUC mRNA1. Naked mRNA was used as a reference control, and a DNA ladder was used as a size guide.

[0311] Accessible mRNA Quant-iT RiboGreen assay results Similar to Example 1, to evaluate the amount of mRNA that effectively binds to SIS0013-N, SIS0013-T, and SIS0013-Q and is inaccessible to external reactants, the amount of mRNA accessible by Quant-iT® RiboGreen® RNA fluorescent dye was used as an indirect parameter indicating the amount of unbound or surface-bound mRNA after complex formation.

[0312] Similar behavior to SIS0013 in Example 1 was observed. More specifically, in all formulations, low mRNA accessibility was observed when the w / w ratio of mRNA to total lipids was 1:24. When the w / w ratio of mRNA to total lipids was 1:12, 1:7.2, and 1:4, SIS0013-N, SIS0013-T, and SIS0013-Q showed low mRNA accessibility and superior shielding effect against external reactants compared to SIS0013. A more pronounced effect was observed in SIS0013-T. These results are shown in Figure 10.

[0313] Cell transfection results Similar to Example 1, a series of in vitro transfection tests were performed using HEK293 cells to compare the performance of SIS0013, SIS0013-N, SIS0013-T, and SIS0013-Q. Cells were transfected using SIS0013 / luciferase (Luc mRNA1), SIS0013 / luciferase (Luc mRNA2), SIS0013-N / luciferase (Luc mRNA1), SIS0013-N / luciferase (Luc mRNA2), SIS0013-T / luciferase (Luc mRNA1), SIS0013-T / luciferase (Luc mRNA2), SIS0013-Q / luciferase (Luc mRNA1), and SIS0013-Q / luciferase (Luc mRNA2).

[0314] For consideration, a w / w mRNA:total lipid ratio of 1:24 was selected. Since the 1:24 ratio functioned with SIS0013 at 6, 24, and 48 hours post-transfection in Example 1, it is expected to provide an excellent benchmark for comparing SIS0013 and its variants at 6, 24, or 48 hours post-transfection.

[0315] The translation product was detected by a Bright-Glo luciferase assay performed at 6, 24, or 48 hours post-transfection using a luminometer, as described in Example 1.

[0316] Six hours after transfection, all SIS0013 variants showed transfection with both LUC-mRNA models (1 and 2), with the signal from SIS0013 complexed with mRNA2 being the most prominent. The results are shown in Figure 11.

[0317] Similar to the results observed in Example 1, higher luciferase activity was observed after 24 hours. The results are shown in Figure 12.

[0318] After 48 hours, all SIS0013 variants showed transfection in both LUC-mRNA models (1 and 2), with SIS0013 and SIS013-T showing more pronounced signals. The results are shown in Figure 13.

[0319] These results demonstrate that the favorable effects of doped Si are maintained even when combined with a variety of completely different components. NAD, QUE, and TYR differ significantly from each other in their properties and behavior.

[0320] To evaluate the statistical significance of the differences in luminescence between SIS0013 and its variants, a statistical analysis was performed at the point in time when the luminescence intensity was highest, i.e., over 24 hours.

[0321] The analysis was performed using ROIs obtained through software analysis, by calculating the mean ± SD values ​​for each of the three variant series of SIS0013, SIS0013-N, SIS0013-Q, and SIS0013-T that formed complexes with mRNA. The maximum ROI was set to SIS0013 that formed complexes with mRNA2, and one-way ANOVA was performed.

[0322] Statistical analyses shown in Figures 14-16 confirmed that SIS0013 and SIS0013-T were the transfection formulations that produced the greatest luminescence. This may mean that they provide the most effective delivery medium. Notably, it appears that the intensity signal can be modulated through the functionalization of SIS0013 with various components such as SIS0013-T, SIS0013-Q, and SIS0013-N. Nevertheless, all formulations tested showed satisfactory efficacy.

[0323] Specifically, Figure 14 is a graph of the luminescence intensity 24 hours after transfection for SIS0013 / luciferase (Luc mRNA1), SIS0013 / luciferase (Luc mRNA2), SIS0013-N / luciferase (Luc mRNA1), SIS0013-N / luciferase (Luc mRNA2), SIS0013-T / luciferase (Luc mRNA1), SIS0013-T / luciferase (Luc mRNA2), SIS0013-Q / luciferase (Luc mRNA1), and SIS0013-Q / luciferase (Luc mRNA2). Values ​​are reported as mean ± SD (analysis was performed in triplicate). Adjacent histogram columns were plotted to directly compare the transfection efficiencies achieved with the LUC mRNA1 complex and the corresponding LUC mRNA2 complex.

[0324] Figure 15 shows a statistical analysis of the luminescence signal intensity of SIS0013 and its variants that formed a complex with mRNA1 (RM one-way ANOVA, Geisser-Greenhouse correction, Dunnett's multiple comparison test, calculation of individual variances for each comparison, analysis of luminescence signal over 24 hours). * p=0.05).

[0325] On the other hand, Figure 16 shows a statistical analysis of the luminescence signal intensity of SIS0013 and its mutants that formed a complex with LUC mRNA2 (one-way ANOVA, Geisser-Greenhouse correction, Dunnett's multiple comparison test, calculation of individual variances for each comparison, analysis of luminescence signal over 24 hours). * p=0.05).

[0326] Example 3 - Study on cell viability In this example, the cell viability of cells transfected with mRNA1 or LUC mRNA2 was investigated using SIS0013 and its variants SIS0013-N, SIS0013-T, and SIS0013-Q. Cell toxicity was monitored. The results were compared with the viability of cells transfected with mRNA1 or LUC mRNA2 using the commercially available transfection reagents DharmaFECT 1 and Lipofectamine 3000.

[0327] Materials and methods Unless otherwise specified, the same materials and methods as in Example 1 were used.

[0328] Similar to Example 1, the mRNA:lipid w / w ratios (1:4, 1:7.2, 1:12, 1:24) were examined at the same time points after transfection (6 hours, 24 hours, 48 ​​hours, 72 hours) and in the mRNA:lipid w / w ratios (1:4, 1:7.2, 1:12, 1:24).

[0329] DharmaFECT 1 and Lipofectamine 3000 The commercially available transfection reagents, DharmaFECT 1 and Lipofectamine 3000, were obtained as shown in Table 12.

[0330] Table 12: DharmaFECT 1 and Lipofectamine 3000 [Table 12]

[0331] result Cell transfection results Under other identical conditions (including mRNA:total lipid ratio (w / w)), DharmaFECT 1 and Lipofectamine 3000 showed cytotoxicity within 24 hours after transfection (evidence of increased LDH and Caspase 3 / 7 signaling, in addition to morphological changes observed when cells were analyzed under a microscope). On the other hand, the SIS0013, SIS0013-T, SIS0013-Q, and SIS0013-N formulations did not show any changes in cell morphology or cell viability at any time point.

[0332] This indicates that SIS0013, SIS0013-T, SIS0013-Q, and SIS0013-N have improved cytotoxicity profiles compared to conventional transfection reagents such as DharmaFECT 1 and Lipofectamine 3000.

[0333] Example 4 - In vivo study on osteopetrosis type II (ADO2) ADO2 is a hereditary osteosclerotic bone disorder caused by osteoclast dysfunction. ADO2 is caused by missense mutations in the chloride channel 7 (CLCn7) gene and is characterized by osteosclerosis accompanied by multiple fractures. ADO2 can lead to osteomyelitis, visual impairment (due to macular hole occlusion and compression of the optic nerve caused by osteopetrosis), and bone marrow failure (Alam et al., 2017, Bone, 94:34-41). Currently, there is no treatment for ADO2.

[0334] In this example, SIS0012 or SIS0013 was used as an siRNA delivery platform, and the silencing of ClCn7G213R in ADO2 mice was investigated using a mouse model. The results were compared between SIS0012 and SIS0013.

[0335] Materials and methods The same protocol as in Example 1 was used. Instead of mRNA as in Example 1, siRNA was used and loaded using the same protocol as described for mRNA in Example 1.

[0336] ClCn7G213R-specific siRNA ClCn7G213R-specific siRNA was obtained as described in Capulli et al., 2015, Clin. Molec. Therap. 4, e248 (the entire article is incorporated herein by reference).

[0337] Table 13 shows the naming conventions for the constructs used.

[0338] Table 13: Constructs used in Example 4 [Table 13]

[0339] ADO2 mutant mice (age: 10 days) were obtained as described in Alam et al., 2017, Bone, 94:34-41 (the entire work is incorporated herein by reference). The mice were intraperitoneally injected with one of the four constructs shown in Table 13 (n=5 mice per group). Injections were repeated three times a week for a total of two weeks.

[0340] result ClCn7G213R expression in mouse PMBC was assayed for each treatment group. The results are shown in Figure 17. Meanwhile, Figure 18 shows the bone expression of ClCn7G213R in mouse femurs. Figure 19 shows the results of CTX blood tests (CTX is a bone metabolism marker). The results are summarized in Table 14.

[0341] Table 14: Research results for ADO2 mice [Table 14]

[0342] Both ADO2+SIS0012-siRNA (undoped Si) and ADO2+SIS0013-siRNA (doped Si) resulted in statistically significant downregulation of ClCn7G213R (p<0.02).

[0343] However, surprisingly, administration of ADO2 + SIS0013-siRNA (doped Si) resulted in an even faster bone metabolism compared to ADO2 + SIS0012-siRNA (undoped Si) (Figure 19, CTX evaluation). Therefore, doped Si-containing delivery media appear to enhance the reduction of symptoms in ADO2 patients treated with siRNA compared to undoped Si-containing delivery media.

[0344] Example 5 - Stabilization of alkaline phosphatase by SIS0012 vs. SIS0013 In Example 5, we investigate the stabilization of pH and temperature-sensitive protein APIs (i.e., relatively unstable APIs, but with properties different from the mRNA and siRNA examined in the previous examples) using SIS0012 (containing undoped Si) and SIS0013 (containing doped Si).

[0345] The API examined is alkaline phosphatase, an enzyme that exists in various forms, catalyzes the degradation of various proteins, and is potentially present in all tissues of the human body. Alkaline phosphatase activity is significantly reduced at low pH and high temperatures.

[0346] Materials and methods The same protocol as in Example 1 was used, but instead of mRNA as in Example 1, alkaline phosphatase was used as the API and loaded using the protocol shown below.

[0347] Alkaline phosphatase, isolated from bovine intestines and supplied as a 56 kD recombinant enzyme expressed in the yeast Pichia Pastoris, was obtained from Sigma Aldrich / Merck (The Old Brickyard, New Rd, Gillingham, Dorset, SP8 4XT). A aqueous stock solution of alkaline phosphatase (ALP) was prepared at a concentration of 1 U / mL. 1 U (μmol / min) is defined as the amount of ALP that catalyzes the conversion of 1 micromolar of PNPP per minute at 37°C and pH 7.4.

[0348] A 20 mM solution of 4-nitrophenyl phosphatase (PNPP) was prepared using Tris buffer (100 mM / L) at pH 7.4.

[0349] ALP solutions were prepared in 15 mL test tubes at concentrations of 0.1 mU / mL, 0.5 mU / mL, 1 mU / mL, 5 mU / mL, 10 mU / mL, 50 mU / mL, and 100 mU / mL, starting from a stock solution of 1 U / mL. These were then mixed in Eppendorf tubes with a 20 mM solution of the prepared PNPP Tris buffer. The tubes were incubated in a 37°C water bath for 30 minutes, and then the UV-Vis absorbance was measured at 405 nm as shown in Figure 20.

[0350] ALP was loaded into SIS0012 and SIS0013 as follows.

[0351] 1. Prepare three sets of eight Eppendorf tubes. Eight Eppendorf tubes were prepared. Each tube contained 50 μL of ALP (50 mU / mL) and 500 μL of SIS0012 (undoped Si). After adding these components to the tubes, they were mixed, vortexed, and refrigerated overnight. B. Eight Eppendorf tubes were prepared. Each tube contained 50 μL of ALP (50 mU / mL) and 500 μL of SIS0013 (boron-doped Si). After adding these components to the tubes, they were mixed, vortexed, and refrigerated overnight. Eight Eppendorf tubes were prepared. Each tube contained 50 μL of ALP (50 mU / mL) and 500 μL of Tris buffer. After adding these components to the tubes, they were mixed, vortexed, and refrigerated overnight.

[0352] 2. After preparation, all Eppendorf tubes were placed in a 50°C water bath. Eight tubes were placed in each of the three sets of tubes A-C, and they were removed from the water bath after 1 minute, 2 minutes, 5 minutes, 10 minutes, 20 minutes, 40 minutes, or 60 minutes.

[0353] 3. Next, 300 μL of PNPP was added to all Eppendorf tubes in all sets A to C, and the tubes were mixed and vortexed. Then, the mixture was placed in a 37°C water bath for 30 minutes, during which time dephosphorylation of PNPP occurred.

[0354] 4. Next, UV-Vis analysis (405 nm) was performed on all samples from groups A to C. The results are shown in Figure 21. More specifically, the activity of alkaline phosphatase was monitored by measuring the change in the concentration of its substrate, 4-nitrophenyl phosphatase (PNPP), using UV-Vis absorbance as a proxy. The structure of PNPP is described below. As the PNPP concentration increased, the activity of alkaline phosphatase decreased, and therefore this protein API was more degraded and less stable. [ka]

[0355] result Free alkaline phosphatase showed significant degradation compared to both SIS0012 and SIS0013. The activity of free alkaline phosphatase decreased with increasing incubation time at 50°C.

[0356] Surprisingly, the activity of alkaline phosphatase loaded onto SIS0013 (doped Si) was approximately 20% to 30% higher than that of alkaline phosphatase loaded onto SIS0012. This is thought to be because doped Si showed improved stability against alkaline phosphatase degradation compared to undoped Si.

[0357] Example 6 - SIS0013 variant using siRNA In Example 6, we investigate the effect of changes in the functionalization of SIS0013 on its function as a delivery medium for siRNA.

[0358] Materials and methods Unless otherwise specified, the same materials and methods as in Example 2 were used. Instead of mRNA in Example 2, siRNA was used and loaded using the same protocol as described for mRNA in Example 1 above.

[0359] We investigated the complex formation of SIS0013-N, SIS0013-Q, and SIS0013-T with siRNA specific to ClCn7G213R and possessing a dTdT overhang (obtained as described in Capulli et al., 2015, Clin. Molec. Therap. 4, e248).

[0360] result Figure 22 shows the gel electrophoresis results indicating that the siRNA successfully and completely bound to these formulations.

[0361] As shown in the table below, dynamic light scattering measurements were also performed using a Zetasizer (available from Malvern Instruments) to evaluate the size and charge both before and after siRNA complex formation. As shown in the table below, an increase in size was observed after siRNA complex formation, and the surface charge decreased by 10–15 mV.

[0362] Table 15: Size, PDI, and zeta potential of SIS0013-N, SIS0013-Q, and SIS0013-T before and after complex formation with siRNA. [Table 15]

[0363] Materials and Methods - Continued The behavior of doped Si-containing compositions in response to further changes in other components in the compositions other than doped Si was investigated when DOTAP was omitted from compositions SIS0013-N, SIS0013-T, and SIS0013-Q containing NAD, TYR, or QUE.

[0364] Therefore, DPPC / DOPE formulations were prepared according to the same protocol as in Examples 1 and 2 above, except that DOTAP was omitted from the total lipids. Tests were conducted using (i) 0.2 mg or (ii) 1 mg of NAD, TYR, and QUE. The formulations are shown in Tables 16 to 18 below.

[0365] Table 16: Composition of DPPC / DOPESIS0013 functionalized with β-nicotinamide adenine dinucleotide (NAD). [Table 16]

[0366] Table 17: Composition of DPPC / DOPE SIS0013 functionalized with quercetin (QUE). [Table 17]

[0367] Table 18: Composition of DPPC / DOPE SIS0013 functionalized with tyrosine (TYR) [Table 18]

[0368] Results - continued ZetaSizer measurements showed a negative zeta potential across all formulations, regardless of the amount of NAD, TYR, or QUE (0.2 mg or 1 mg), as shown in Table 19 below.

[0369] Table 19: DPPC / DOPE SIS0013 functionalized with 0.2 mg or 1 mg of NAD, TYR, and QUE [Table 19]

[0370] The differentially functionalized composition of Example 6 is likely to exhibit API stabilization and delivery behavior similar to that of SIS0013.

[0371] Example 7 - Lipopeptide component Lipopeptides, also known as peptide amphiphilic substances (PAs), were studied as yet another alternative component to compositions containing doped silicon particles.

[0372] Lipopeptides are considered to offer a solution to the problem of substituting or reducing the amount of cationic lipids in transfection compositions. Lipopeptides consist of alkyl chains conjugated to a peptide sequence. While the surface of the lipid bilayer is decorated with the peptide portion, the alkyl chains are thought to be absorbed into the lipid bilayer.

[0373] An exemplary PA is the molecular palmitoyl pentapeptide-4 (abbreviated as PAL-KTTKS). Its two cationic lysine residues perform functions similar to those of cationic lipids such as DOTAP and can exhibit electrostatic interactions with negatively charged APIs such as RNA.

[0374] Materials and methods DPPC and DOPE were selected as neutral lipids to be formulated together with PAL-KTTKS.

[0375] Details of formulations including DPPC, DOPE, and PAL-KTTKS are shown in Table 20 below.

[0376] Table 20: DPPC, DOPE, and Pal-KTTKS containing boron-doped silicon nanoparticles [Table 20]

[0377] This formulation exhibited a positive zeta potential of 54.38 ± 2.11 (measured with a Zetasizer available from Malvern Instruments). This is lower than that of SIS0012 in Example 1, but is considered to be more positive than when the silicon particles are not doped.

[0378] During the construction of the lipid membrane, PAL-KTTKS is thought to be positioned within the lipid bilayer with its peptide portion exposed on the nanoparticle surface. Furthermore, the lysine residues on its surface may impart a positive charge to the formulation.

[0379] The ability of the aforementioned formulation to electrostatically bind to ClCn7G213R-specific siRNA with dTdT overhangs (obtained as described in Capulli et al., 2015, Clin. Molec. Therap. 4, e248) and ALDEVRON DASHER GFP mRNA was evaluated.

[0380] result Gel electrophoresis analysis was performed. Complete complex formation was not observed with siRNA. Well 3 in Figure 23 shows the gel electrophoresis results for siRNA.

[0381] Complete complex formation was observed with mRNA. Well 3 in Figure 24 shows the results of mRNA gel electrophoresis.

[0382] To address the partial complex formation between siRNA and DPPC / DOPE / PAL-KTTKS, an alternative loading method was employed. This alternative loading method involved the following steps compared to the previously described protocol.

[0383] 1. Thin lipid films were prepared by dissolving DPPC, DOPE, and Pal-KTTKS in methanol and evaporating them using a rotary evaporator.

[0384] 2. The lipid film was rehydrated with a suspension containing boron-doped silicon, trehalose, glycine, and siRNA or mRNA. Rehydration was carried out at 40°C for 10 minutes, ensuring that no lipid silicon film remained on the walls of the round-bottom rotary evaporation flask.

[0385] Well 4 in Figure 25 shows the gel electrophoresis of the complex after using the alternative loading method, indicating successful complete complex formation of the siRNA.

[0386] Lipopeptides are highly versatile molecules that can be fine-tuned by altering the alkyl chain and / or peptide sequence. Customizing the peptide sequence is thought to potentially enhance cell and / or tissue targeting. In the field of gene therapy, peptide customization may enhance electrostatic interactions with nucleic acids, particularly mRNA. For example, when PAL-KTTKS is formulated with DPPC and DOPE, a positively charged surface is generated, as confirmed by zeta potential.

[0387] On the other hand, lipopeptides, which are amphiphilic molecules, have properties very similar to surfactants, which can self-assemble to form micelles. This is thought to be because the alkyl chain is suitable for hydrophobic interactions, at least in part, while the peptide sequence can form intermolecular hydrogen bonds. Phospholipids such as DPPC and DOPE can also self-assemble into liposomes. Therefore, when PA (a typical example being PAL-KTTKS) is incorporated (although other lipopeptides can also be used), the alkyl chain can form hydrophobic interactions with DPPC and DOPE, and a liposome structure is formed.

[0388] Simultaneously, silicon nanoparticles contribute to the structural stability of the entire complex. As is evident from the examples described above, doped Si particles can interact non-covalently (electrostatically) with lipids, including lipopeptides, and other species such as NAD, QUE, or TYR, promoting the long-term stability and effective delivery of the API.

[0389] Example 8 Example 8 compares transfection with SIS0012 and SIS0013 in the further cell line L6C5 (a commercially available musculoskeletal mouse cell line).

[0390] Materials and methods Similar to Example 1, cells were transfected with SIS0012 / luciferase (Luc mRNA1), SIS0012 / luciferase (Luc mRNA2), SIS0013 / luciferase (Luc mRNA1), or SIS0013 / luciferase (Luc mRNA2). Translation products were detected by a Bright-Glo luciferase assay performed 24 hours post-transfection using a luminometer, as described herein.

[0391] result The results are shown in Figure 26. When SIS0013 was used, luciferase activity was higher for both mRNA1 and mRNA2 than when SIS0012 was used. This suggests that the transfection efficiency of SIS0013 is improved compared to SIS0012. ***

[0392] Where the foregoing description refers to features or limitations that have equivalents known, obvious, or foreseeable to those skilled in the art in light of this disclosure, such equivalents are incorporated herein by reference as if specifically stated. To determine the scope of the subject matter of this disclosure, please refer primarily to the claims. The scope of protection sought by this application also includes such equivalents. Furthermore, it will be understood by those skilled in the art that features or limitations of the disclosed subject matter described as preferred, appropriate, advantageous, convenient, etc., are desired and, unless explicitly stated otherwise, do not limit the scope of the independent claims or the protection sought. Moreover, it should be understood that such desired features or limitations may be desirable but not desirable in some implementations of the disclosed subject matter, and therefore may not be present or may be omitted in other implementations.

Claims

1. (i) Particles containing hydrolyzable doped silicon, (ii) One or more types of lipids, and (iii) Active pharmaceutical ingredients (APIs) A composition containing the following:

2. The composition according to claim 1, wherein the particles containing hydrolyzable doped silicon have a different zeta potential compared to the same particles, except that they contain hydrolyzable undoped silicon, thereby attracting, binding to, and / or stabilizing APIs.

3. The composition according to claim 1 or claim 2, wherein the dopant of the particle is a p-type dopant (particularly boron), or comprises a p-type dopant (particularly boron).

4. The composition according to claim 3, wherein the particles containing hydrolyzable p-doped silicon have a more positive zeta potential than particles containing hydrolyzable undoped silicon, thereby attracting, binding to, and / or stabilizing APIs.

5. The composition according to claim 3 or claim 4, wherein the API has a net negative charge at a pH of approximately 7.

4.

6. The composition according to claim 1 or 2, wherein the dopant of the particle is an n-type dopant (particularly phosphorus), or comprises an n-type dopant (particularly phosphorus).

7. The composition according to claim 6, wherein the particles containing hydrolyzable n-doped silicon have a more negative zeta potential than particles containing hydrolyzable undoped silicon, thereby attracting, binding to, and / or stabilizing APIs.

8. The composition according to claim 6 or claim 7, wherein the API has a net positive charge at a pH of approximately 7.

4.

9. The composition according to any one of claims 1 to 8, wherein the API is neutral or zwitterionic at a pH of approximately 7.

4.

10. The hydrolyzable doped silicon particles are 1 cm ３ Approximately 1 x 10 １６ The composition according to any one of claims 1 to 9, which is doped at the above levels of dopant atoms.

11. The hydrolyzable doped silicon particles are 1 cm ３ Approximately 1 x 10 ２０ The composition according to any one of claims 1 to 10, which is doped at the level of dopant atoms.

12. The composition according to any one of claims 1 to 11, wherein the API is a nucleic acid or comprises a nucleic acid.

13. The composition according to claim 12, wherein the nucleic acid is RNA.

14. The composition according to claim 13, wherein the RNA is a small interfering RNA (siRNA).

15. The composition according to claim 13, wherein the RNA is messenger RNA (mRNA).

16. The composition according to claim 15, wherein the mRNA encodes a protein of a pathogenic organism.

17. The composition according to any one of claims 1 to 16, wherein the API is a protein or comprises a protein.

18. The composition according to any one of claims 1 to 17, further comprising an amino acid.

19. The composition according to any one of claims 1 to 18, wherein the composition comprises tyrosine.

20. The composition according to claim 19, wherein the tyrosine is present in addition to, or in place of, the amino acid according to claim 18.

21. The composition according to any one of claims 1 to 20, wherein the composition further comprises a non-reducing disaccharide (such as trehalose).

22. The composition according to any one of claims 1 to 21, further comprising NAD.

23. The composition according to any one of claims 1 to 22, further comprising quercetin.

24. The composition according to any one of claims 1 to 23, wherein the one or more lipids include ionizable lipids.

25. The composition according to claim 24, wherein the one or more lipids include lipids having a net positive charge at pH 7.

4.

26. The composition according to any one of claims 1 to 25, wherein the one or more lipids comprises one or more lipid-modified oligopeptides.

27. The composition according to claim 26, wherein each of the one or more lipid-modified oligopeptides contains an oligopeptide portion having about 3 to about 20 amino acid residues and a fatty acid chain having about 12 to about 18 carbon atoms, and preferably one or more of the amino acid residues are positively charged at a pH of about 7.

4.

28. The composition according to any one of claims 1 to 27, wherein the particles prevent or reduce the degradation (particularly enzymatic degradation) of the API.

29. The composition according to any one of claims 1 to 28, wherein the API has an in vivo half-life of less than about one hour in the absence of the hydrolyzable doped silicon and particles containing one or more types of lipids.

30. The composition according to any one of claims 1 to 29, wherein the particles increase the in vivo half-life of the API.

31. The composition according to any one of claims 1 to 30, wherein the particles enhance the intracellular stability of the API.

32. The composition according to any one of claims 1 to 31, wherein the particles enhance the stability of API at approximately 25°C.

33. The composition according to any one of claims 1 to 32, wherein the dopant of the particle is an interstitial dopant or comprises an interstitial dopant.

34. The composition according to any one of claims 1 to 33, wherein the dopant of the particle is a substitutional dopant or comprises a substitutional dopant.

35. The composition according to any one of claims 1 to 34, wherein the dopant of the particle is a dopant on the surface of the particle, or comprises a dopant on the surface of the particle.

36. The composition according to claim 35, wherein the dopant is present only on or near the surface of the particles.

37. The composition according to any one of claims 1 to 36, comprising one or more aggregates of particles containing the hydrolyzable doped silicon.

38. The composition according to claim 37, wherein in the one or more aggregates, the average diameter of the particles containing the hydrolyzable doped silicon is about 1 nm to about 50 nm.

39. The composition according to claim 37 or claim 38, wherein the one or more aggregates include a chain of particles containing the hydrolyzable doped silicon.

40. The composition according to any one of claims 37 to 39, wherein the one or more aggregates are embedded in one or more lipid structures.

41. The composition according to any one of claims 37 to 40, wherein mRNA is non-covalently bound to particles in one or more aggregates.

42. The composition according to any one of claims 37 to 41, wherein the one or more lipid structures are one or more of liposomes, incomplete liposomes, micelles, incomplete micelles, and lipid spheres, or comprise one or more of these.

43. A composition according to any one of claims 1 to 42, for use in a method for preventing or treating a disease or disorder in humans.

44. The composition according to claim 43, wherein the API is a nucleic acid, and the method includes an in vivo step of transfecting human cells with the nucleic acid.

45. The composition according to claim 43 or claim 44, wherein the disease or disorder is an infectious disease.

46. The composition according to claim 43 or claim 44, wherein the disease or disorder is a hereditary disease or disorder.

47. The composition according to any one of claims 43 to 46, wherein the age of the human subject is approximately one month or longer, and optionally approximately one year or longer.

48. The composition according to any one of claims 43 to 47, wherein the method comprises administering the composition to a human subject by injection, orally, transdermally, or intranasally, particularly by injection or orally.

49. The composition according to claim 48, wherein the composition is administered by injection, and the method further comprises monitoring the subject for symptoms or signs of a hypersensitivity reaction.

50. The hydrolyzable doped silicon particles are 1 cm ３ Approximately 1 x 10 １６ The composition according to claim 49, wherein the composition is doped at the above levels of dopant atoms, and the method comprises administering the composition to a human subject by injection, orally, or intranasally.

51. The composition according to any one of claims 43 to 50, wherein the method includes the step of storing the composition containing the API at a temperature in the range of about 0°C or higher for a period of one week or more before administering the composition to the subject.

52. A method to slow down API decomposition, (i) Doping particles containing hydrolyzable silicon to change the ζ potential of the particles compared to undoped particles which are identical in all respects except that they are undoped, and (ii) Contacting the doping particles with one or more types of lipids and the API, The method, including the method described above.

53. The method according to claim 52, wherein step (i) includes doping the particles with a p-type dopant to make the zeta potential of the particles more positive than that of undoped particles which are otherwise identical.

54. The method according to claim 53, wherein the API has a net negative charge at a pH of approximately 7.4, particularly when the API is a nucleic acid.

55. The method according to claim 52, wherein step (i) includes doping the particles with an n-type dopant to make the zeta potential of the particles more negative than that of undoped particles which are otherwise identical.

56. The method according to claim 55, wherein the API has a net positive charge at a pH of approximately 7.

4.

57. A method for preventing or treating a disease or disorder, comprising administering a prophylactic or therapeutically effective amount of the composition described in any one of claims 1 to 51 to a human subject who requires a prophylactic or therapeutically effective amount of the composition.

58. The method according to claim 57, having one or more features of the method according to any one of claims 43 to 56.

59. Use of a composition as defined in any one of claims 1 to 51 in the manufacture of a pharmaceutical for use in any one of claims 52 to 58.

60. A human cell transfection composition having one or more of the characteristics of the composition described in any one of claims 1 to 51.

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