Therapeutic compositions and methods

Abstract

Therapeutic compositions and methods. Disclosed is a composition comprising: a particle comprising hydrolysable doped silicon; one or more lipids; and an active pharmaceutical ingredient (API). Also disclosed are related products, methods and uses thereof.

Description

Technical Field

[0001] The present disclosure relates to delivery vehicles for active pharmaceutical ingredients ("APIs"). More specifically, but not limited to, the present disclosure relates to a composition comprising: particles comprising hydrolyzable doped silicon; one or more lipids; and the API. The present disclosure also relates to related products, medical uses, and methods. Background Art

[0002] There is a need for improved API delivery vehicles, which are necessary to fully translate advances in biomedical research into effective, safe, and cost-effective therapies.

[0003] Recent biological advances have provided new insights into a new class of APIs for preventing and treating many diseases. One example of this class is nucleic acids. For example, a variety of mRNA-based vaccines for infectious diseases have recently emerged. Messenger RNA (mRNA)-based cancer therapies are now entering clinical development. Meanwhile, other types of RNA, including siRNA, tRNA, and oRNA (circular RNA), are showing promise in treating a variety of genetic diseases and conditions.

[0004] However, the prospect of a new class of APIs also brings the challenge of how to control the stability of the API during storage. It is also necessary to ensure that once the API is administered to the patient, it can actually reach and be taken up by the cells; when the API circulates in the body, it may be challenging to maintain its stability. In addition, there is a need for tissue or cell targeting so that the API can be delivered to the correct cells. Once it reaches the target cell, if the API is intended to be delivered into the cell (rather than, for example, an API intended to act outside the cell, such as on a cell surface protein), how to ensure that the API is effectively taken up by the cell is also a challenge. After cellular uptake, there is a need to ensure the stability of the API in the cytoplasm.

[0005] In one approach, lipid nanoparticles have been used to deliver fragile APIs, such as nucleic acids, particularly mRNA, in vivo. (The terms "fragile API" and "reactive API" as used herein are interchangeable and may refer to an API that: (i) has a half-life of up to one week after storage in an aqueous solution at about 25°C, as measured by NMR or GC-MS; and / or (ii) has an in vivo half-life of less than about 1 hour as measured by an assay in a biological sample.) Lipid nanoparticles containing APIs require cold chain storage, which limits their distribution and is not energy-efficient or cost-effective. There remains a need for a delivery vehicle that can enhance the stability of fragile APIs, both during storage (so that they can be stored at higher temperatures) and during circulation in the body (so that more API reaches target cells faster).

[0006] At the same time, some delivery vehicles based on lipid nanoparticles may show cytotoxicity (see Example 3 below). Commonly used lipid nanoparticles rely on exogenous cationic lipids containing multiple amine groups (i.e., lipids with a net positive charge when pH is about 7.4). Although these amine-rich materials are very suitable for the electrostatic load of polyanionic nucleic acids (including various forms of RNA), they may cause cytotoxicity, immunogenicity and nonspecific tissue accumulation. Other amine-based delivery systems based on polymers also have similar problems. Therefore, there is a demand for improved delivery vehicles also with acceptable low toxicity.

[0007] Preferably, the new delivery vehicle should also be able to fully disperse in an aqueous environment to ensure ease of delivery, for example by injection into an aqueous solution.

[0008] The present disclosure aims to alleviate the above problems.Alternatively or additionally, the present disclosure aims to provide an improved API delivery vehicle. Summary of the Invention

[0009] In a first aspect of the present disclosure, there is provided a composition comprising: particles comprising hydrolyzable doped silicon (particularly, having a doping level of at least 1×10 16 dopant atoms / cm 3 hydrolyzable silicon), one or more lipids, and an API.

[0010] In a second aspect of the present disclosure, there is provided a composition as defined in the first aspect of the present disclosure for use in a method of preventing or treating a disease or disorder in a human subject.

[0011] In a third aspect, disclosed herein is a method for slowing degradation of an API, comprising: doping particles comprising hydrolyzable silicon such that their zeta potential is altered compared to otherwise identical undoped particles; and contacting the doped particles with one or more lipids and the API.

[0012] In a fourth aspect of the present disclosure, a method for preventing or treating a disease or disorder is provided, comprising: administering a preventively or therapeutically effective amount of the composition defined according to one or both of the first and second aspects of the present disclosure to a human subject in need thereof.

[0013] In a fifth aspect, disclosed herein is the use of a composition as defined in one or both of the first and second aspects of the disclosure in the manufacture of a medicament for use in a method as defined in one or both of the second and third aspects of the disclosure.

[0014] In a sixth aspect, disclosed herein is a human cell transfection composition having one or more features of the composition defined according to one or both of the first and second aspects of the disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Embodiments of the present disclosure will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0016] Figure 1 and Figure 2 Particle size and polydispersity index (PDI) data are shown for undoped (SIS0012) and doped (SIS0013) Si-containing delivery vehicles complexed with mRNA.

[0017] Figure 3 Shown are gel electrophoresis data investigating the complexation of undoped (SIS0012) and doped (SIS0013) Si-containing delivery vectors with mRNA.

[0018] Figure 4 Data showing accessibility of mRNA to external agents in undoped (SIS0012) and doped (SIS0013) Si-containing delivery vehicles.

[0019] Figure 5 Shown are luminescence observed at a 6-hour time point following administration of mRNA to HEK293 cells using undoped (SIS0012) or doped (SIS0013) Si-containing delivery vehicles.

[0020] Figure 6 Shown are luminescence observed at the 24 hour time point following administration of mRNA to HEK293 cells using undoped (SIS0012) or doped (SIS0013) Si-containing delivery vehicles.

[0021] Figure 7 Shown are luminescence observed at the 48 hour time point following administration of mRNA to HEK293 cells using either undoped (SIS0012) or doped (SIS0013) Si-containing delivery vehicles.

[0022] Figure 8 Shown are luminescence observed at the 72 hour time point following administration of mRNA to HEK293 cells using undoped (SIS0012) or doped (SIS0013) Si-containing delivery vehicles.

[0023] Figure 9 Shown are gel electrophoresis data exploring the complexation of various doped Si-containing delivery vehicles (SIS0013-N, -Q, and -T) with mRNA.

[0024] Figure 10 Data showing the accessibility of mRNA to external agents in various doped Si-containing delivery vehicles (SIS0013-N, -Q, and -T).

[0025] Figure 11Shown are the luminescence observed at a 6-hour time point after mRNA administration to HEK293 cells using various doped Si-containing delivery vehicles (SIS0013-N, -Q, and -T).

[0026] Figure 12 Shown are the luminescence observed at the 24-hour time point after mRNA administration to HEK293 cells using various doped Si-containing delivery vehicles (SIS0013-N, -Q, and -T).

[0027] Figure 13 Shown are the luminescence observed at the 48-hour time point after mRNA administration to HEK293 cells using various doped Si-containing delivery vehicles (SIS0013-N, -Q, and -T).

[0028] Figures 14 to 16 exhibit Figures 11 to 13 Statistical analysis of luminescence data.

[0029] Figure 17 Shown is the expression of ClCn7G213R in mouse PMBCs treated with undoped (SIS0012) or doped (SIS0013) siRNA-loaded Si-containing delivery vehicles.

[0030] Figure 18 Shown is the expression of ClCn7G213R in mouse bone (femur) cells treated with undoped (SIS0012) or doped (SIS0013) siRNA-loaded Si-containing delivery vehicles.

[0031] Figure 19 Shown are the results of CTX blood tests in mice treated with undoped (SIS0012) or doped (SIS0013) siRNA-loaded Si-containing delivery vehicles.

[0032] Figure 20 Presented are ultraviolet-visible (UV-vis) absorbance data for alkaline phosphatase under the conditions described in Example 5, which investigated the stability of this pH- and temperature-sensitive protein with doped Si-containing delivery vehicles.

[0033] Figure 21 Shows further UV-vis absorbance data for alkaline phosphatase, supplementing Figure 20 .

[0034] Figure 22 Shown are gel electrophoresis data exploring the complexation of various doped Si-containing delivery vehicles (SIS0013-N, -Q, and -T) with siRNA.

[0035] Figure 23Shown are gel electrophoresis data investigating the complexation of doped Si-containing delivery vehicles containing DPPC, DOPE, and PAL-KTTKS with siRNA.

[0036] Figure 24 Presented are gel electrophoresis data investigating the complexation of doped Si-containing delivery vehicles containing DPPC, DOPE, and PAL-KTTKS with mRNA.

[0037] Figure 25 Further gel electrophoresis data are presented to investigate the complexation of siRNA with doped Si-containing delivery vehicles containing DPPC, DOPE, and PAL-KTTKS, wherein the delivery vehicle / siRNA complexes are prepared according to the Figure 23 Compared with different preparation methods.

[0038] Figure 26 The luciferase assay results of Example 8 are shown, wherein when SIS0013 (doped with Si) was used, the luciferase activities of both mRNA1 and mRNA2 were higher than when SIS0012 (not doped with Si) was used.

[0039] Figure 27 is a transmission electron microscope (TEM) image showing the aggregation of silicon particles described herein. DETAILED DESCRIPTION

[0040] Although the subject matter of the present disclosure will be described and illustrated below in conjunction with specific embodiments, it will be understood by those skilled in the art that the subject matter itself can be subjected to many different variations, which are not specifically described herein. Some possible variations of all aspects of the present disclosure will now be described by way of example only.

[0041] Particles comprising hydrolyzable doped silicon

[0042] The particles comprising hydrolyzable doped silicon may be pure or substantially pure doped silicon.

[0043] The particles can be another hydrolyzable doped silicon-containing material. If the particles are not pure doped silicon, they include at least about 50% (by weight) silicon, that is, based on the total mass of atoms in the particles, they include at least about 50% (by weight) silicon atoms. For example, the silicon particles can contain at least about 60%, about 70%, about 80%, about 90% or about 95% (by weight) silicon. The hydrolysis rate of these particles (e.g., at room temperature in PBS buffer) can show at least 10% of the hydrolysis rate of pure silicon particles of the same size. Determination of hydrolysis of silicon-containing materials is well known in the art; see, for example, WO2011 / 001456, which is incorporated herein by reference in its entirety.

[0044] Although the particles may contain trace amounts of silicon dioxide, the silicon dioxide is not hydrolyzable silicon.At least about half of the silicon atoms in the particles may be in the form of elemental silicon (or doped elemental silicon).

[0045] The particles can be nanoparticles in particular. The average diameter of the nanoparticles can be in the range of about 1 nm to about 500 nm, in particular in the range of about 1 nm to about 250 nm, more particularly in the range of about 1 nm to about 100 nm, preferably in the range of about 1 nm to about 50 nm, in particular in the range of about 3 nm to about 50 nm, more particularly in the range of about 3 nm to about 30 nm (e.g., about 10 nm).

[0046] The particularly preferred range of the average diameter of the nanoparticles is from about 1 nm to about 30 nm. As described below, this facilitates the aggregation of the nanoparticles.

[0047] The size of nanoparticle objects, including the average diameter of the particles, can be measured, for example, by scanning electron microscopy (SEM) or transmission electron microscopy (TEM).

[0048] The particles can be porous, in particular mesoporous. Particles comprising hydrolyzable doped silicon can be made porous by standard techniques, such as contacting the particles with a hydrofluoric acid (HF) / ethanol mixture and applying an electric current ("HF etching"). By varying the HF concentration, current density, and exposure time, the density of pores and their size can be controlled and monitored by scanning electron microscopy and / or nitrogen adsorption-desorption volume isothermal measurements. If the particles are porous, their total surface area will be increased due to their porosity. For example, their surface area can be increased by at least about 50% or at least about 100% compared to the surface area of ​​the corresponding non-porous particles. In many cases, porous particles actually increase their total surface area more due to their porosity. According to certain embodiments, the porosity is at least about 30%, about 40%, about 50%, or about 60%; this means that at least about 30%, about 40%, about 50%, or about 60% of the volume of the particle is pore space, respectively.

[0049] The average pore size may be in the range of about 0.1 nm to about 10 nm, such as about 0.1 nm to about 3 nm, for example about 2 nm.

[0050] It will be appreciated that particles may be prepared by a variety of techniques with which the skilled artisan will be familiar.

[0051] These techniques may include, for example, purely physical (sometimes referred to in the art as "non-wet") processes starting from bulk silicon (especially silicon wafers); such as pulsed laser ablation, thermal degradation, and ball milling. Thus, particles may be obtained by a process comprising one or more of, or a combination of, pulsed laser ablation, thermal degradation, and ball milling of bulk silicon (especially silicon wafers).

[0052] Additionally or alternatively, the particles can be prepared by chemical (sometimes referred to in the art as "wet") techniques, including but not limited to electrochemical etching of bulk silicon (especially silicon wafers). Such techniques optionally include the HF etching described above. Thus, the particles can be obtained by a process comprising or consisting of electrochemical etching of bulk silicon (especially silicon wafers).

[0053] Once the silicon particles are formed, they can be screened according to particle size, for example by air screening, sieving and / or filtering. Thus, the particles can be obtained by a method comprising one or more of air screening, sieving and / or filtering.

[0054] Thus, for example, the particles may be obtained by a method comprising preparing silicon particles from bulk silicon (especially silicon wafers), for example by subjecting bulk silicon (especially silicon wafers) to one or more of pulsed laser ablation, thermal degradation, ball milling and electrochemical etching; and then, optionally, screening according to particle size, for example by air screening, sieving and / or filtering.

[0055] Optionally, the particles can be washed before use, for example in methanol or ethanol, to remove a thin oxide layer on their surface. This is known in the art as "activation."

[0056] The particles thus obtained can have a narrow particle size distribution and uniform surface chemistry, thereby achieving batch-to-batch reliability and reproducibility of one or more of the advantages described herein.

[0057] Suitable physical and chemical techniques are described, for example, in WO 2011 / 012867 A1 (in 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 NanoSyst.Lett, 2023, 11:18, the entire contents of each of which are incorporated herein by reference.

[0058] dopant

[0059] The particles of the composition include hydrolyzable doped silicon.

[0060] As used herein, the term "doped silicon" may refer to silicon that behaves as an impure semiconductor due to the presence of dopant atoms. The dopant atoms may be or include substitutional dopant atoms (replacing Si atoms). Additionally or alternatively, the dopant atoms may be or include interstitial dopant atoms (located between Si atoms and not substituted). Optionally, the dopant atoms may be present on the surface of the particles. Optionally, the dopant atoms may be present only on the surface of the particles, or only on or near the surface of the particles. In this way, the particles may optionally have an undoped silicon core while having a doped shell or surface.

[0061] However, preferably, no metal silicate coating is present on the particle surface. The metal silicate surface coating is undoped. Unlike scattered dopant atoms between silicon atoms, the metal silicate surface coating covers the particle surface with a metal silicate compound. While silicon particles can be coated with metal silicates, doing so can complicate their synthesis and is not necessary for the beneficial effects described herein (such as, but not limited to, tissue targeting).

[0062] Advantageously, the doping level of the silicon particles is at least about 1×10 15 dopant atoms / cm 3 , especially at least about 1×10 16 dopant atoms / cm 3 .

[0063] For example, the doping level of the particles may be at least about 1×10 17 dopant atoms / cm 3 , at least about 1x10 18 dopant atoms / cm 3 or at least about 1x10 19 dopant atoms / cm 3 .

[0064] The doping level of silicon particles can be up to 1x10 20 dopant atoms / cm 3 .

[0065] The silicon particles may be n-doped or p-doped. The silicon particles may be doped with one or more elements selected from the group consisting of B, P, Mg, Cu, Ga, Al, In, Bi, Ge, Li, Xe, N, Au, and Pt. Thus, the dopant may be a p-dopant, particularly boron. The dopant may be an n-dopant, particularly phosphorus.

[0066] Dopants can be incorporated into the hydrolyzable silicon matrix by (i) substitution, (ii) interstitial, and / or (iii) surface attachment. In this way, improved API delivery vehicles can be provided.

[0067] When boron is preferably used as a dopant, 1x1015 dopant atoms / cm 3 and 1x10 20 dopant atoms / cm 3 The doping levels of 13.6 Ω-cm and 1.3 mΩ-cm may correspond to resistivities of 13.6 Ω-cm and 1.3 mΩ-cm, respectively. Embodiments in which boron is the dopant do not exclude silicon, while doped (e.g., heavily doped) with boron, but also doped with other elements (in which case the primary dopant is preferably boron).

[0068] The term "heavily doped" as used herein is understood to mean a doping concentration of at least about 1x10 15 dopant atoms / cm 3 In some preferred embodiments, the dopant content is at a level of at least about 1×10 16 dopant atoms / cm 3 Thus, in a particularly preferred embodiment, the dopant is boron at a level of at least about 1 x 10 16 boron atoms / cm 3 For example, the boron content level is at least about 1x10 16 boron atoms / cm 3 , at most about 1x10 20 boron atoms / cm 3 .

[0069] Silicon is referred to herein as "undoped" (e.g., particles of composition SIS0012 in Examples 1 and 2), which may mean that no or only a small amount of dopant atoms are present, e.g., at most about 1 x 10 2 dopant atoms / cm 3 Additionally or alternatively, "undoped" silicon may mean silicon that does not behave as an impurity-containing semiconductor.

[0070] Advantageously, doping particles containing hydrolyzable silicon can have a beneficial effect on the particle's zeta potential, which can serve as an indicator of the particle's surface charge. Doping can provide a zeta potential more suitable for improved API loading. It is believed that this may help stabilize the API circulating in the body until it reaches target cells and is subsequently released. As a result, compared to using undoped particles, more API can reach target cells within a given period of time after administration, resulting in more efficient API delivery.

[0071] Doping the particles can extend the in vivo half-life of the API (at about pH 7.4 and about 37° C.), for example by about 1.5 times, in particular by about 2 times. In particular, in the presence of particles comprising hydrolyzable doped silicon and one or more lipids, the in vivo half-life of the API can be greater than about 1 hour, in particular greater than about 2 hours, and more in particular greater than about 6 hours. It should be understood that the term “in vivo half-life of the API” as used herein may refer to the in vivo elimination half-life of the API, i.e., the time period required for the amount of the API to be reduced by about half once administered. It should also be understood that the term “amount of the API” may refer to the amount of the API or its derivative having the same or substantially the same intended pharmacological effect. It should also be understood that the term “in vivo half-life” as used herein may refer to the systemic half-life of the API (in vivo, in particular in humans); this is in contrast to the half-life of the API on the surface in vitro, for example, when present on the surface of the skin (even if in contact with the skin, it is in vitro).

[0072] Doping of the particles may optionally change their zeta potential by an order of at least about 5 mV, in particular at least about 10 mV, compared to undoped particles.

[0073] Doping with a p-dopant can result in a higher positive zeta potential, thereby improving binding to net negatively charged APIs, especially those with a net negative charge at a pH of about 7.4 (as this is a typical physiological pH), especially nucleic acids, more especially mRNA, siRNA, or tRNA. Therefore, in some embodiments, the hydrolyzable doped silicon particles are doped with a p-dopant and the API is a negatively charged API, especially a nucleic acid, more especially mRNA or siRNA.

[0074] P-doping can also provide improved binding to APIs with a net positive charge, especially those with a net positive charge at a pH of about 7.4 (as this is a typical physiological pH), especially when the zeta potential of the particle is further modulated by other components, such as the presence of one or more lipids (e.g., phospholipids). Without wishing to be bound by theory, it is believed that electron transfer from p-doped silicon (e.g., boron-doped silicon) to other components can occur, thereby modulating particle size and charge, and thereby nucleic acid binding capacity.

[0075] P-doping of the particles may optionally increase their zeta potential by at least about 5 mV, in particular at least about 10 mV, compared to undoped particles.

[0076] At the same time, doping with an n-dopant can result in a more negative zeta potential, thereby improving binding to positively charged APIs. Thus, in some embodiments, the particles comprising hydrolyzable doped silicon are doped with an n-dopant and the API is a net positively charged API, particularly an API with a net positive charge at a pH of about 7.4.

[0077] N-doping can also provide improved binding to APIs with a net negative charge, especially those with a net negative charge at a pH of about 7.4 (as this is a typical physiological pH), especially when the zeta potential of the particle is further modulated by other components, such as the presence of one or more lipids (e.g., cationic lipids). Additionally or alternatively, n-doped silicon may be able to protect one or more lipids (especially zwitterionic or positively charged lipids) from degradation. This can indirectly help bind and stabilize APIs, such as nucleic acids, even if the API is negatively charged (especially mRNA).

[0078] N-doping of the particles may optionally reduce their zeta potential by at least about 5 mV, in particular at least about 10 mV, compared to undoped particles.

[0079] P-doping or n-doping of particles comprising hydrolyzable silicon can result in improved binding to zwitterionic or neutral APIs. Thus, in some embodiments, the particles comprise hydrolyzable p-doped silicon and the API is zwitterionic or neutral. In other embodiments, the particles comprise hydrolyzable n-doped silicon and the API is zwitterionic or neutral.

[0080] Conventional liposome transfection compositions (which do not include silicon) often include large amounts (e.g., majority % by weight) of cationic lipids, particularly amine-rich cationic lipids, wherein the positive charge of the cationic lipids is intended to stabilize negatively charged APIs, particularly nucleic acids. However, such cationic lipids may not be cost-effective and may not be sufficiently safe in all clinical applications, such as administration to certain patient populations (e.g., infants, the elderly, or pregnant women). As described herein, particles offer the potential to use less or no cationic lipids, particularly less or no "foreign" or amine-rich cationic lipids, thereby providing improved cost-effectiveness and safety.

[0081] Relatedly, the zeta potential of the particles can also be modulated by other components in the composition, as demonstrated herein for compositions SIS0013-T, SIS0013-N, and SIS0013-Q in Examples 2 and 6, and for the lipidated oligopeptide-containing composition in Example 7. Doping of the particles may help maintain a more positive or negative (as the case may be) zeta potential than would otherwise be the case, meaning that components with functionalities that would otherwise excessively increase / decrease (as the case may be) the zeta potential of the particles can be added to maintain satisfactory API binding and delivery.

[0082] Furthermore, the change in zeta potential caused by doping with hydrolyzable silicon may not be the only beneficial effect of doping. The change in zeta potential may not be the sole reason for the improved success rate of API binding and delivery. Furthermore, as described herein, other components besides silicon may also affect API binding and delivery. For example, despite having lower positive zeta potentials compared to SIS0013 in Example 1, SIS0013-N, SIS0013-Q, and SIS0013-T in Examples 2 and 6 exhibit advantages in binding and delivery of negatively charged mRNA.

[0083] At the same time, as described in Example 1 below ("Materials and Methods: Methods of Preparing SIS0012 or SIS0013; Methods of Preparing SIS0012-mRNA or SIS0013-mRNA Complexes"), a delivery vehicle according to the present disclosure (e.g., SIS0013) can be prepared, stored, and transported to a clinic, and then complexed with an API (e.g., mRNA) before administration to a patient. The delivery vehicle can be stored separately from the mRNA until close to the time of administration to the patient. Because the delivery vehicle does not contain a reactive API (e.g., mRNA) during storage, it does not need to be stored at particularly low temperatures (e.g., below 4°C) specifically to stabilize the API. This is a different approach from conventional lipid nanoparticle delivery vehicles, which are typically stored in a form that is already coated with the API.

[0084] In addition, the doped Si-containing delivery vector disclosed herein has a stabilizing effect on reactive APIs (e.g., nucleic acids, especially mRNA) when complexed with them. Therefore, the delivery vector disclosed herein can optionally be stored in a form that has been complexed with the API. This is feasible at temperatures that are not particularly cold. For example, a composition comprising an API can be stored at a temperature of about 0°C or higher, in particular about 3-5°C (i.e., the temperature of a typical commercial refrigerator); or about 15-30°C (i.e., typical room temperature).

[0085] Doping with Si-containing delivery vehicles can also stabilize the API in the body's circulation. In addition or alternatively, it can ensure that the API is effectively taken up by the cells. In addition or alternatively, it can stabilize the API in the cytoplasm of the cell. For example, when the API is mRNA, this may be advantageous because it is preferably safely delivered to the ribosomes in the cytoplasm of the cell for translation. Boron-doped (i.e., p-doped) silicon can stabilize mRNA (negatively charged due to its phosphate backbone). This can, in particular, provide improved protection to prevent its degradation, thereby more efficiently utilizing mRNA for cell transfection.

[0086] The production of doped silicon is well known in the semiconductor industry and includes ion implantation and diffusion methods, which make doped silicon itself readily available. An example of a diffusion method is mixing silicon powder and a dopant (e.g., B2O3 for boron doping) at a temperature of 1050°C to 1175°C for several minutes under an N2 atmosphere to diffuse the dopant (e.g., boron) into the silicon.

[0087] lipids

[0088] One or more lipids can be bound to the particle surface. One or more lipids can be complexed with an API (e.g., a nucleic acid). One or more lipids can include ionizable lipids. One or more lipids can include lipids with a net positive charge at a pH of about 7.4, also referred to herein as "cationic lipids."

[0089] It has been found that surface treatment of particles with lipids may help control the release rate of APIs (eg nucleic acids, particularly mRNA or siRNA).The type of lipid used to treat the surface of the silicon-containing particles may help to modulate the release rate of the API.

[0090] Treating hydrolyzable silicon-doped particles with lipids can have a beneficial effect on the surface charge of the particles. This can provide a zeta potential more suitable for improved API loading (especially nucleic acids such as siRNA, short activating RNA, short hairpin RNA, or mRNA). This can help control the release rate of the API at the target site.

[0091] For example, treating particles comprising hydrolyzable pure silicon with phosphatidylcholine (PC), phosphatidylethanolamine (PE) and / or lecithin can promote the generation of a negative zeta potential (zeta potential range of about -60 to -20 mV). Simultaneously, treating with stearylamine and / or DOTAP can promote the generation of a positive zeta potential (zeta potential range of about 0 to about +40 mV). As described herein, doping of silicon particles adjusts their zeta potential. Thus, a p-dopant (preferably boron) can impart a higher positive zeta potential to the particles (e.g., a typical value of pure silicon is about -40 mV, which can be reduced to about -25 mV after doping). Thus, p-doped (e.g., boron-doped) silicon treated with cationic lipids can more easily achieve a higher positive zeta potential. For example, treating p-doped (e.g., boron-doped) silicon with stearylamine and / or DOTAP can achieve a zeta potential of about +20 mV to about +60 mV. Therefore, when using doped silicon, a positive zeta potential can be achieved even with small amounts of cationic lipids or with a wider range of cationic lipids (including non-toxic, e.g., lipids that are not rich in amines). Even if the cationic lipids degrade during storage, resulting in a partial loss of the positive charge of the lipids, the zeta potential of the particles can remain positive for a longer period of time.

[0092] One or more lipids preferably do not include toxic lipids. One or more lipids may not include toxic exogenous lipids (known in the art as external cationic lipids) with a positive charge at physiological pH; Such lipids may have adverse toxicity, for example, when administered to human patients, triggering an innate immune response, especially when they are rich in amines. Although these (amine-rich) exogenous lipid species are very suitable for the electrostatic loading of polyanionic nucleic acids (including various forms of RNA), they may cause cytotoxicity, immunogenicity, and may even cause nonspecific tissue accumulation contrary to expectation, thereby resisting the purpose of targeted delivery. In addition, their synthesis may be complicated, so the cost may be higher.

[0093] Thus, the one or more lipids may not include amine-rich lipids. Amine-rich lipids can be defined as lipids containing more than 2, 3, or 4 nitrogen atoms per lipid molecule. In stark contrast, the one or more lipids preferably contain at most 1 nitrogen atom per lipid molecule.

[0094] In addition, it has been found that the beneficial effects disclosed herein can be achieved without relying on one or more specific lipid compounds. One or more lipids may play a role in charge-charge interactions, such as regulating the zeta potential of the silicon particle surface, thereby enabling better binding of the API. This effect can be observed in a variety of lipids. Lipids, as a category, show convergence in properties (especially in terms of intermolecular interactions), which enables the compositions of the present disclosure to be implemented with different lipids and in different amounts (compared to the specific lipids disclosed in the examples below).

[0095] One or more lipids can help regulate the hydrolysis rate of the doped silicon so that the doped silicon is hydrolyzed 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 regulate the length of time over which API protection is maintained, particularly with respect to in vivo protection in the presence of various body fluids. Thus, more API can be delivered to target cells in a given period of time compared to a composition with the same other ingredients.

[0096] Lipid is generally understood to include fatty acids and fatty acid derivatives, glycerolipids, glycerophospholipids, sphingolipids, glycolipids and polyketides. The term "lipid" used in this application can include lipidated oligopeptides (a term used interchangeably with the term "lipopeptide" in this article), wherein a short peptide sequence (e.g., a peptide sequence with 3 to 20 amino acid residues, e.g., 5 to 15 amino acid residues, especially 3, 4 or 5 amino acid residues, most especially 5 amino acid residues) is coupled to one or more fatty acid chains (especially fatty acid chains with 10 to 24 carbon chain lengths, preferably 12 to 18 carbon chain lengths; e.g., 14, 15 or 16 carbon chain lengths; e.g., the peptide moiety can optionally be lipidated with a palmitoyl, hexadecyl or myristoyl moiety).

[0097] Without wishing to be bound by theory, although a number of lipids are studied in the examples below, the mechanism of action of one or more of the lipids may be due to properties of the lipid class, such as their responsiveness to charge-charge interactions; therefore, it may be generalizable to lipids other than those exemplified.

[0098] Thus, the one or more lipids may comprise one or more lipidated oligopeptides.Preferably, the one or more lipidated oligopeptides each comprise a fatty acid chain having in the range of about 12 to about 18 carbon atoms.

[0099] Preferably, the one or more lipidated oligopeptides each comprise 3 to 20 amino acid residues.Thus, the lipidated oligopeptide may be a lipidated tetrapeptide, a lipidated pentapeptide or a lipidated hexapeptide.

[0100] Preferably, the amino acid residues include at least one amino acid residue (e.g., about 2 or about 3 amino acid residues) that is cationic at a pH of about 7.4 (physiological pH), such as lysine or arginine. For example, the lipidated oligopeptide may include one or more (e.g., about 2) lysine residues.

[0101] A particular example of a lipidated oligopeptide ("lipopeptide") is palmitoyl pentapeptide-4 (CAS No. 214047-00-4; abbreviated PAL-KTTKS).

[0102] Thus, preferably, the one or more lipids may comprise or be one or more lipidated oligopeptides, particularly those having one or more amino acid residues that are positively charged at a pH of about 7.4 (i.e., about physiological pH), such as one or both of lysine and arginine.

[0103] Lipidated oligopeptides can be used in combination with one or more phospholipids (e.g., DOPE or DPPC). The alkyl chains of the lipidated oligopeptide molecules can be incorporated into the phospholipid bilayer, and the surface of the bilayer is modified by the peptide portion. Without wishing to be bound by theory, it is believed that the peptide portion of the lipidated oligopeptide can target one or more specific tissues and / or cells. At the same time, when the peptide portion is positively charged at a pH of about 7.4 (i.e., about physiological pH), it can stabilize negatively charged APIs (e.g., nucleic acids, especially mRNA or siRNA).

[0104] The one or more lipids can be or include one or more of the following: 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 ).

[0105] The one or more lipids can be or include one or more structural lipids (e.g., cholesterol lipids). However, the one or more lipids may optionally not include structural lipids. Therefore, the one or more lipids may not include sterols; in particular, they may not include cholesterol. It has been found that the compositions disclosed herein do not need to rely on these types of lipids, while traditional API delivery systems generally rely on these types of lipids. Therefore, the compositions disclosed herein may provide alternatives for API delivery systems that rely on these types of lipids (especially cholesterol). This may be advantageous when cholesterol is not available or cannot be used (e.g., due to its effect in the body).

[0106] The one or more lipids may optionally include one or more of the following: phosphatidylcholine (PC); hydrogenated PC; stearylamine (SA); dioleoylphosphatidylethanolamine (DOPE); cholesteryl 3β-N-(dimethylaminoethyl)carbamate hydrochloride (DC-chol); 1,2-dioleoyl-3-trimethylammonium propane (DOTAP); PEGylated 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), such as DSPE-PEG 2000 ; and its derivatives.

[0107] In certain embodiments, the lipid is selected from phosphatidylethanolamine (PE), phosphatidylcholine (PC), stearylamine (SA), or any combination thereof.

[0108] In some embodiments, the lipid or lipid component can be or include a cationic lipid. The term "cationic lipid" refers to a molecule with a net positive charge at pH 7.4 (physiological pH), with a cationic head group connected to a hydrophobic tail via some spacers. Examples include DTDTMA (ditetradecyltrimethylammonium), DOTMA (2,3-dioleoyloxypropyl-1-trimethylammonium), DHDTMA (dicetyltrimethylammonium); dioleoyl-3-trimethylammonium propane (DOTAP); and stearylamine (SA). Positive charges can typically be stabilized by negative counterions.

[0109] Thus, one or more lipids may optionally be or include DOTAP. DOTAP exists in the form of S and R enantiomers and may exist in the form of S, R, or racemates. Optionally, the content of the R and S forms in the total DOTAP present by weight may be approximately equal (i.e., neither form exceeds about 60% of the total DOTAP present by weight). In other embodiments, at least about 80%, 90%, 95%, 98%, or 99% of the total DOTAP is the R form. In other embodiments, at least about 80%, 90%, 95%, 98%, or 99% of the total DOTAP is the S form.

[0110] Nevertheless, as described herein, compared to conventional compositions for API delivery (e.g., lipid nanoparticles comprising cationic lipids), silicon doping can reduce the use of cationic lipids, especially the use of less toxic or "exotic" cationic lipids. Although exogenous cationic lipids may be suitable for electrostatic loading of polyanionic nucleic acids (including various forms of RNA), their amine-rich nature may lead to cytotoxicity, immunogenicity, and nonspecific tissue accumulation.

[0111] Thus, the one or more lipids may optionally exclude cationic lipids, particularly toxic cationic lipids.As described herein, cationic lipids may not be necessary when doped silicon is used, particularly when p-doped silicon is used.

[0112] Thus, the one or more lipids may be or include one or more of the following: one or more phospholipids (e.g., DOPE); and one or more polyethylene glycol (PEG) lipids (e.g., DSPE-PEG 2000 ).

[0113] In general, the hydrolyzable, doped silicon-containing particles disclosed herein can provide the potential for using fewer lipids (particularly fewer cationic lipids, most particularly fewer toxic cationic lipids) in API delivery vehicles compared to conventional API delivery vehicles that do not include hydrolyzable silicon-doped particles (e.g., conventional liposomal nucleic acid delivery vehicles, e.g., those commonly used for in vivo mRNA delivery). Additionally or alternatively, the hydrolyzable silicon-doped particles can provide the potential for API delivery vehicles to be formulated with a wider range of lipids while also providing transfection efficiency, storage stability, and / or targeted delivery to specific types of tissues or specific types of cells. This, in turn, can reduce the field's reliance on specific lipids, particularly cationic lipids, particularly toxic cationic lipids and / or cationic lipids specifically formulated for API delivery purposes, which may not be cost-effective or readily available.

[0114] The average molecular weight of the one or more lipids may range from about 500 to about 1000.

[0115] The ratio of one or more lipids (which refers to all lipid components in the composition) to silicon can be in the range of about 40:1 to about 1:1, in particular in the range of about 20:1 to about 1:1; for example, when the components are assembled to make the delivery system, i.e., before any further processing (such further processing can be, for example, the "extrusion" step in the "Materials and Methods" section of Example 1 below), the ratio is about 16:1.

[0116] As described herein, one or more lipids may particularly include or be phospholipids. The term "phospholipid" as used herein may refer to a lipid comprising a fatty acid chain and a phosphate group. Phospholipids can be negatively charged, unlike cationic lipids which are positively charged. However, phospholipids are typically zwitterionic compounds comprising positively and negatively charged components and are therefore generally uncharged. Therefore, phospholipids are typically classified as neutral lipids.

[0117] Suitable phospholipids can be or include glycerophospholipids. Particularly suitable phospholipids can be or include phospholipids having a polar head group attached to a quaternary ammonium salt moiety, such as phosphatidylcholine (PC) or hydrogenated phosphatidylcholine. The phospholipid can be lecithin or can be derived from lecithin. A preferred phospholipid is DOPE (phosphatidylethanolamine or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine).

[0118] Preferably, the side chain of the phospholipid 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.

[0119] Lipids with ether side chains may be referred to as "PEG-lipids" or "PEGylated" lipids. Thus, as used in this application, the term "lipid" may encompass PEG lipids. Thus, according to a preferred embodiment, the one or more lipids may include or be one or more polyethylene glycol (PEG) lipids, in particular PEGylated DSPE, such as DSPE-PEG 2000 .

[0120] The one or more lipids may optionally include or consist essentially of phosphatidylcholine (PC), hydrogenated phosphatidylcholine, stearylamine (SA), or a combination thereof.

[0121] The one or more lipids can optionally include at least about 5% (eg, at least about 30% or at least about 50%) by weight PC, based on the total weight of the one or more lipids.

[0122] The one or more lipids can optionally include at least about 5% (eg, at least about 30% or at least about 50%) by weight hydrogenated PC, based on the total weight of the one or more lipids.

[0123] The one or more lipids can optionally comprise at least about 5% (eg, at least about 30% or at least about 50%) by weight SA, based on the total weight of the one or more lipids.

[0124] The one or more lipids may optionally comprise, or consist essentially of, PC and SA, optionally in a weight ratio of PC to SA ranging from about 1:1 to about 20:1.

[0125] The one or more lipids may optionally include or consist essentially of a combination of DOPE, SA, and DC-cholesterol (DC-chol). The weight ratio of DOPE:SA may be in the range of about 1:1 to about 10:1. The weight ratio of DOPE:DC-cholesterol may be in the range of about 1:1 to about 5:1. The weight ratio of SA:DC-cholesterol may be in the range of about 1:1 to about 1:5.

[0126] In certain preferred embodiments, the one or more lipids may optionally include DOTAP, DOPE, and PEG-lipids (particularly DSPE-PEG 2000 ). The weight ratio of DOTAP:DOPE can be in the range of about 1:2 to about 2:1; for example, about 1:1. The weight ratio of DOTAP:PEG-lipid can be in the range of about 10:1 to about 5:1; for example, about 7:1. The weight ratio of DOPE:PEG-lipid can be in the range of about 10:1 to about 5:1; for example, about 7:1.

[0127] amino acids

[0128] The compositions disclosed herein may include one or more amino acids. In a broad sense, the term "amino acid" includes any artificial or naturally occurring organic compound containing an amine (-NH2) and a carboxyl (-COOH) functional group. It includes α, β, γ, and δ amino acids. It includes amino acids of any chiral configuration. The amino acid may particularly be a naturally occurring α amino acid. It may be a proteinogenic amino acid or a non-proteinogenic amino acid (e.g., carnitine, levothyroxine, hydroxyproline, ornithine, or citrulline).

[0129] One or more amino acids can help stabilize the doped silicon particles themselves. In vivo, one or more amino acids can help regulate the hydrolysis rate of the doped silicon so that the doped silicon is hydrolyzed into bioavailable orthosilicic acid (OSA) degradation products; rather than insoluble polymer hydrolysis products. In this way, one or more amino acids can complement the function of one or more lipids of the present disclosure. Controlling the hydrolysis rate of the doped silicon can affect the release rate of the API associated with the doped silicon. Controlling the API release rate can regulate the length of the time period over which API protection is maintained, especially with respect to in vivo protection in the presence of various body fluids. Therefore, more API can be delivered to target cells in a given time period compared to a composition with the same other ingredients.

[0130] Without wishing to be bound by theory, although a number of amino acids are studied in the examples below, the mechanism of action of one or more amino acids may be due to properties of the amino acid class, such as their reactivity to charge-charge interactions; therefore, it may be generalized to amino acids other than those exemplified.

[0131] In preferred embodiments, the amino acids may comprise or consist essentially of glycine, arginine and / or tyrosine; most particularly glycine.

[0132] Additionally or alternatively, amino acids that are neutral or positively charged at physiological pH (about pH 7.4), such as tyrosine or arginine, can stabilize negatively charged APIs (e.g., nucleic acids, such as mRNA). At the same time, amino acids that are neutral or negatively charged at physiological pH (about pH 7.4) can stabilize positively charged APIs. Nevertheless, charge-based interactions and / or other interactions (e.g., steric interactions) generated by the combination of doped silicon, lipids, and amino acids can be such that positively charged amino acids at physiological pH can help stabilize positively charged APIs, or negatively charged amino acids at physiological pH can help stabilize negatively charged APIs.

[0133] The weight ratio of the one or more lipids (ie, the total lipid component) to the amino acids can range from about 40:1 to about 1:1; for example, about 32:1.

[0134] Optionally, in addition to the amino acid described above, the composition can particularly include the amino acid tyrosine. Optionally, the composition can particularly include tyrosine to replace the amino acid described above. Therefore, it should be understood that although tyrosine is an amino acid, for the purposes of this disclosure, it can optionally exist as a separate, additional component, and is different from the amino acid described above. Therefore, when tyrosine exists as an additional component different from the amino acid described above and in addition to the amino acid described above (as shown in the composition SIS0013-T of Example 3 below), it should be understood that the weight ratio of one or more lipids to amino acid is calculated in the range of about 40: 1 to about 1: 1; for example, as described above, about 32: 1, which does not include the amount of additional, different tyrosine.

[0135] Non-reducing disaccharides

[0136] Additionally or alternatively, the composition can include one or more non-reducing disaccharides, particularly trehalose, for example, as shown below in the composition of Example 1. The weight ratio of the one or more lipids (i.e., the total lipid component) to the non-reducing disaccharide can be in the range of about 20:1 to about 1:1; for example, about 16:1.

[0137] API

[0138] It will be understood that the compositions of the present disclosure may be described as pharmaceutical compositions for the delivery of an active pharmaceutical ingredient (API).

[0139] For example, the API can be a friable API. The terms "friable API" and "reactive API" are used interchangeably herein and may refer to an API that (i) has a half-life of up to one week after storage in aqueous solution at about 25° C. as measured by NMR or GC-MS; and / or (ii) has an in vivo half-life of less than about 1 hour as measured by an assay in a biological sample.

[0140] The API can be any pharmaceutically active compound; thus, for example, it is understood that the term "API" encompasses prodrugs. In particular, the API can be a nucleic acid, more particularly an siRNA or an mRNA. Meanwhile, in other preferred embodiments, the API can be a protein.

[0141] The API can be administered by injection, orally, nasally or topically; in particular by injection or orally.

[0142] Nucleic Acid API

[0143] The API may preferably be or include a nucleic acid, in particular a linear or circular RNA. The RNA may be a small interfering RNA (siRNA), a small activating RNA (saRNA), a short hairpin RNA (shRNA), a transfer RNA (tRNA) or a messenger RNA (mRNA), in particular mRNA (e.g., an mRNA encoding a protein of a pathogenic organism).

[0144] Other nucleic acids for use in accordance with the present disclosure include: double-stranded and single-stranded DNA; DNA:RNA hybrids; peptide:DNA hybrids; and peptide:RNA hybrids.

[0145] RNA and DNA can be naturally occurring or can be chemically modified to enhance their therapeutic properties, such as enhancing activity, improving serum stability, reducing off-target effects, and reducing immune activation. Chemical modifications of RNA and DNA can include any modification known in the art. As used herein, the term "naturally occurring" refers to natural human or animal origin. It should be understood that molecular structures identical to naturally occurring molecular structures can also be synthesized in vitro, such as by synthesizing mRNA through in vitro transcription (IVT).

[0146] Thus, the terms nucleic acid, DNA, and RNA as used herein also include known types of modifications, such as labels, methylation, "capping," substitution of one or more naturally occurring nucleotides with an analog, internucleotide modifications, such as those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), those with negatively charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), and those with positively charged linkages (e.g., aminoalkylphosphamides, aminoalkylphosphotriesters), those containing side chain moieties, such as proteins (including nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalating agents (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidized metals, etc.), those containing alkylating agents, those with modified linkages (e.g., alpha isomeric nucleic acids, etc.), as well as unmodified forms of polynucleotides or oligonucleotides.

[0147] Likewise, the terms "nucleoside" and "nucleotide" as used herein will include those parts that contain not only known purine and pyrimidine bases, but also other modified heterocyclic bases. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, or other heterocycles. Modified nucleosides or nucleotides also include modifications to the sugar moiety, for example, wherein one or more hydroxyl groups are substituted with halogens, aliphatic groups, or are functionalized to ethers, amines, and the like. Other modifications to nucleotides or polynucleotides include rearranging, adding, replacing, or otherwise changing the functional groups on the purine or pyrimidine bases that form hydrogen bonds with the corresponding complementary pyrimidines or purines (e.g., isoguanine, isocysteine, etc.). In some embodiments, the oligonucleotides and / or probes include at least one, two, three, or four modified nucleotides.

[0148] In some embodiments, nucleic acids (e.g., RNA) disclosed herein include one or more universal bases. As used herein, the term "universal base" refers to a nucleotide analog that can hybridize with more than one nucleotide selected from A, U / T, C, and G. In some embodiments, the universal base can be selected from deoxyinosine, 3-nitropyrrole, 4-nitroindole, 6-nitroindole, and 5-nitroindole.

[0149] In a broad sense, the term "saRNA" encompasses small activating RNAs, including RNA molecules that operate in the RNA activation (RNAa) pathway. The saRNA can be double-stranded. The length of the saRNA can range from about 5 to about 50 base pairs, particularly about 10 to about 40 base pairs, and more particularly about 10 to about 30 base pairs.

[0150] In a broad sense, the term "shRNA" encompasses short hairpin RNAs, including RNA molecules that operate in the RNA interference (RNAi) pathway. shRNAs can be single-stranded and can also undergo base pairing, thereby forming a hairpin loop. The single-stranded length of the shRNA can be in the range of about 10 to about 100 base pairs, particularly about 25 to about 75 base pairs, and more particularly about 40 to about 70 base pairs; a hairpin loop can then be formed.

[0151] In a broad sense, the term "siRNA" encompasses small interfering RNAs, including RNA molecules that operate in the RNA interference (RNAi) pathway. siRNA is sometimes also referred to as short interfering RNA or silencing RNA. siRNA can be double-stranded. The length of the siRNA can be in the range of about 5 to about 50 base pairs, particularly about 10 to about 40 base pairs, more particularly about 15 to about 30 base pairs. Examples 4, 6, and 7 below investigate the siRNA API in the compositions of the present disclosure.

[0152] In a broad sense, the term "tRNA" encompasses transfer RNAs, including RNA molecules that serve as a connection (or adapter) between an mRNA molecule and a growing chain of amino acids during protein synthesis. The length of the primary structure of a tRNA can be in the range of about 20 to about 200 nucleotides, in particular about 50 to about 100 nucleotides. The tRNA can have a clover-shaped secondary structure. The tRNA can have an L-shaped tertiary structure.

[0153] In a broad sense, the term "mRNA" encompasses messenger RNA for synthesizing proteins. It may include mRNA containing a 5' end cap and / or a polyadenylated end. Alternatively, one or both of these features may not be present. Typically, the mRNA may be single-stranded. The coding region of the mRNA may be at least about 100 bases in length, particularly at least about 500 bases, and more particularly at least about 1000 bases in length. Examples 1, 2, and 7 below investigate the mRNA API in the compositions of the present disclosure.

[0154] The mRNA can encode an antigen, thereby providing a composition for use as a vaccine. The antigen can be a bacterial, parasitic, or fungal antigen. The antigen can be a viral antigen, particularly an antigen of one of the viral diseases described below; more specifically, an antigen of a respiratory virus, such as an antigen of SARS-CoV-2, for example, an antigen derived from the SARS-CoV-2 spike protein.

[0155] The mRNA may encode an allergen (including but not limited to one or more nut allergens; which in turn include but are not limited to: one or more seed storage proteins, such as vicilin, legumin, albumin; one or more plant defense-related proteins; and one or more profilins).

[0156] The mRNA may encode a protein that modulates immune, autoimmune, or inflammatory diseases including, but not limited to, lupus, atherosclerosis, chronic obstructive pulmonary disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatism, uveitis, atopic dermatitis, and pulmonary fibrosis.

[0157] mRNA can encode tumor-specific antigens. The term "tumor-specific antigen" as used herein may refer to an antigen produced by a non-synonymous somatic mutation (resulting in a new antigen) or a viral integration mutation (resulting in a tumor virus antigen) in one or more malignant tumor cells. Thus, a tumor-specific antigen may refer to an antigen that is completely absent (not expressed) in non-tumor (healthy, normal) cells.

[0158] mRNA can encode tumor-associated antigens. The term tumor-associated antigen used herein may refer to an antigen that is overexpressed in malignant cells compared to non-tumor (healthy, normal) cells, for example due to gene amplification or post-translational modification. The term tumor-associated antigen may encompass overexpressed antigens (the term may refer to proteins that are moderately expressed in non-tumor (healthy, normal) cells but are expressed in large quantities in malignant cells); differentiation antigens (the term may refer to proteins selectively expressed by cell lineages evolved from malignant cells, such as prostate-specific antigens); and tumor germline antigens (the term may refer to antigens that are normally limited to reproductive tissues but abnormally expressed in malignant cells; for example, melanoma antigen family A3 (MAGE-A3); New York esophageal squamous cell carcinoma-1 antigen (NY-ESO-1); and melanoma preferentially expressed antigens (PRAME)).

[0159] mRNA can encode multiple proteins, thereby providing more effective pharmacological activity. mRNA can encode multiple antigens, especially multiple viral antigens.

[0160] The mRNA may also encode an adjuvant protein.The adjuvant may additionally or alternatively be provided as an additional component of the composition in addition to the API.

[0161] As shown in Examples 1, 2, 4, 6 and 7 below, it has been found that doping with silicon particles can help the disclosed compositions deliver APIs to cells more effectively, especially when the API is a nucleic acid, more specifically siRNA or mRNA. Specifically, the doped silicon-containing compositions disclosed herein can alleviate or solve the problem of how to ensure that the API reaches cells after administration to a patient, including how to stabilize the API circulating in the body. They can alleviate or meet the needs of tissue or cell targeting so that the API can be delivered to the correct cells. Once at the target cells, they can help ensure that the API is effectively taken up by the cells. After being taken up by the cells, they can help ensure the stability of the API in the cytoplasm; for example, stabilizing and releasing the mRNA API in the cytoplasm within an appropriate period of time, thereby successfully translating more mRNA.

[0162] Compared to naturally occurring mRNA, both types of mRNA (LUC mRNA1 and LUC mRNA2) used in Examples 1 and 2 below were chemically modified. Such chemical modifications may make transfection more difficult. The enhanced ability of Si-doped SIS0013 to transfect chemically modified mRNA compared to undoped Si-doped SIS0012 is particularly advantageous and surprising. Therefore, in some embodiments, the API can be a chemically modified mRNA compared to naturally occurring mRNA.

[0163] Thus, the methods disclosed herein can include an in vivo step of transfecting human cells with the nucleic acid.

[0164] The doped silicon-containing compositions disclosed herein for delivering APIs can be non-toxic. For example, they can be substantially completely biodegradable as described herein because silicon can be degraded into non-toxic orthosilicic acid in the body.

[0165] The doped silicon-containing compositions disclosed herein for delivery of APIs can be fully dispersed in aqueous environments to ensure ease of delivery, such as by injection into aqueous solutions.

[0166] Component complexation, especially particle / lipid / API complexation

[0167] Preferably, the particles comprising hydrolyzable doped silicon are complexed with one or more lipids to form a delivery vehicle for transporting the API. Thus, when the API is added, it also becomes complexed with the particles and / or lipids. In other words, the particles and lipids are combined into a delivery vehicle that carries the API. Advantageously, this can make the API less susceptible to reaction with one or more external reactive substances. The API may be less likely to be at risk of being degraded by enzymes outside the complex, especially in vivo, for example in the circulation and / or in the cytoplasm of cells; this is especially true when the API is a nucleic acid, especially mRNA.

[0168] In a broad sense, the term "complexed with" as used herein may encompass ionic and / or covalent and / or physical interactions, and in particular may encompass charge-charge interactions, such as interactions caused by the zeta potential of the particles.

[0169] Thus, preferably, the zeta potential of the particles, especially when modulated by the lipid(s) and any other components present, is such as to attract and promote the binding of the API.

[0170] In preferred embodiments where amino acids are present, the amino acids may also complex with the particles, lipids and / or API. Amino acids, particularly when charged, may modulate the zeta potential of the particles, thereby modulating the complexation of the API and / or lipid with the particles.

[0171] When other advantageous components are present (such as those described below), they may also be complexed with the particles, lipids, amino acids, and / or API.

[0172] One or more lipids can be formed by one or more lipid structures, or can include one or more lipid structures.Described structure can be or include following one or more: micelle, incomplete micelle, liposome, incomplete liposome and (for example, solid or semisolid) lipid sphere, as described herein.

[0173] At the same time, preferably, the API (especially RNA, most especially mRNA) is bound to the particles comprising hydrolyzable doped silicon (especially by electrostatic coordination). Preferably, at least about 50%, 60% or 70% of the API in the composition is bound to the particles in this manner.

[0174] In turn, preferably, the particles comprising hydrolyzable doped silicon are bound to the surface of one or more lipid structures and / or are present within the interior of one or more lipid structures.

[0175] Particle-API binding can be particularly effective in reducing or preventing API degradation if the particles comprising hydrolyzable doped silicon aggregate into chains of particles. Therefore, preferably, the particles aggregate. Silicon doping can promote such aggregation, for example by reducing repulsive forces between particles.

[0176] The chains can extend into the interior of a lipid structure (e.g., liposomes, incomplete liposomes, micelles, incomplete micelles, and / or lipid globules) formed by one or more lipids. The API is bound to the particles embedded in the chains within the lipid structure, which can, among other things, shield the particles from water molecules and thus from hydrolytic degradation.

[0177] The average diameter of the lipid structures (e.g., liposomes, incomplete liposomes, micelles, incomplete micelles, and / or lipid globules) is typically from about 50 nm to about 500 nm, particularly from about 100 nm to about 500 nm. Meanwhile, the average diameter of the particles comprising hydrolyzable doped silicon is typically in the range of from about 1 nm to about 50 nm, for example, from about 1 nm to about 30 nm. This relative size difference can optimize the formation of particle aggregates (particularly chains) that are embedded in the lipid structures and / or modify the surface of the lipid structures.

[0178] Thus, preferably, the particles comprising hydrolyzable doped silicon are bound to the surface of and / or present in the interior of one or more of the following: micelles, incomplete micelles, liposomes, incomplete liposomes and (e.g., solid or semi-solid) lipid globules; and the API is bound to the particles comprising hydrolyzable doped silicon. Preferably, the particles comprising hydrolyzable doped silicon are present in one or more aggregates of particles comprising hydrolyzable doped silicon, in particular one or more aggregates comprising or consisting of chains of particles, most particularly chains extending into the interior of one or more lipid structures, for example extending into the interior of one or more of the following: micelles, incomplete micelles, liposomes, incomplete liposomes and (e.g., solid or semi-solid) lipid globules (in particular liposomes and / or lipid globules). Amino acids (in particular glycine, arginine and / or tyrosine, such as glycine) may also be associated (e.g., non-covalently) with the particles comprising hydrolyzable doped silicon; and / or associated (e.g., on the surface thereof) with one or more lipid structures.

[0179] As used herein, the term "liposomal lipid particle" or the term "liposome" may have its conventional meaning in the art. Thus, it may refer to a vesicle having at least one lipid bilayer, which may be approximately spherical in shape. A liposome may be viewed as a lipid "bubble" enclosing an interior space. This interior space may be a hydrophilic environment.

[0180] In some embodiments, the composition may include one or more liposomes. That is, the one or more lipids may be formed from one or more liposomes, or may include one or more liposomes.

[0181] In some embodiments, said composition can include incomplete liposomes. In this sense, their inner space can be entered from the outside. Incomplete liposomes can be regarded as incomplete lipid " bubbles", in which (approximately spherical) lipid bilayer surfaces have one or more gaps. Therefore, suitably, one or more lipids can be formed by one or more incomplete liposomes, or can include one or more incomplete liposomes.

[0182] Suitably, the one or more lipids may be formed from or may comprise one or more incomplete liposomes and / or one or more (complete) liposomes.

[0183] Thus, the present composition may comprise particles comprising hydrolyzable doped silicon associated with one or more liposomes and / or one or more incomplete liposomes, wherein the API is associated with (particularly bound to) the particles comprising hydrolyzable doped silicon. Amino acids (particularly glycine, arginine and / or tyrosine, e.g. glycine) may also be associated with the particles comprising hydrolyzable doped silicon.

[0184] Optionally, the API may be encapsulated by one or more lipids.

[0185] Liposomes are particularly suitable for encapsulating APIs. Thus, the API can optionally be encapsulated in liposomes, or preferably, the API can be partially encapsulated in incomplete liposomes. When the liposomes are incomplete liposomes, there are pathways for the API to pass through the interior and exterior of the liposomes.

[0186] The average diameter of the optional liposomes or incomplete liposomes may be in the range of about 50 nm to about 500 nm, particularly in the range of about 50 nm to about 300 nm, and more particularly in the range of about 100 nm to about 300 nm.

[0187] The API may be non-covalently associated with the exterior surface of the liposome or incomplete liposome (particularly via ionic interactions). The API may be located within the interior space of the (optionally incomplete) liposome (i.e., at least partially encapsulated). Preferably, at least about 10% of the API is located within the interior space. Additionally or alternatively, at least 10% of the API may preferably be non-covalently associated with the exterior surface.

[0188] Thus, at least about 10%, about 30%, or about 50% of the API may be completely encapsulated within the (optionally, incomplete) interior space of the liposomes, with the remainder located in non-covalent association on the (optionally, incomplete) exterior surface of the liposomes.

[0189] The API may be non-covalently bound to particles comprising hydrolyzable doped silicon, which are 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 bound to particles comprising hydrolyzable doped silicon, which are in turn bound to the surface of one or more liposomes and / or one or more incomplete liposomes.

[0190] The API may be non-covalently bound to particles comprising hydrolyzable doped silicon, which in turn are located within one or more liposomes and / or one or more incomplete liposomes. Preferably, at least about 50%, 60%, or 70% of the API may be non-covalently bound to particles comprising hydrolyzable doped silicon, which are located within one or more liposomes and / or one or more incomplete liposomes.

[0191] Preferably, the API (especially RNA, most especially mRNA) is non-covalently bound to particles comprising hydrolyzable doped silicon, which are located inside one or more liposomes and / or one or more incomplete liposomes; and the API (especially RNA, most especially mRNA) is non-covalently bound to particles comprising hydrolyzable doped silicon, which are bound to the surface of one or more liposomes and / or one or more incomplete liposomes.

[0192] In some embodiments, the composition may be free or substantially free of liposomes; and / or may be free or substantially free of incomplete liposomes.

[0193] Optionally, the one or more lipids may be formed from one or more lipid monolayers, or may include one or more lipid monolayers. Optionally, the one or more lipids may be or include one or more micelles or incomplete micelles. It will be understood that micelles have similar properties to liposomes, except that the wall of a micelle is formed from a lipid monolayer, whereas the wall of a liposome is formed from a lipid bilayer. Thus, a micelle may refer to a vesicle having at least one lipid monolayer, which may be approximately spherical in shape. Similar to liposomes, micelles may be viewed as a lipid "bubble" enclosing an interior space. This interior space may be a hydrophilic environment.

[0194] Thus, the present composition may comprise particles comprising hydrolyzable doped silicon associated with one or more micelles and / or one or more incomplete micelles, wherein the API is associated with (particularly bound to) the particles comprising hydrolyzable doped silicon.

[0195] The average diameter of the optional micelles may be in the range of about 50 nm to about 500 nm, specifically about 50 nm to about 300 nm, and more specifically about 100 nm to about 300 nm.

[0196] The API may be non-covalently bound to particles comprising hydrolyzable doped silica, which are bound 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 bound to particles comprising hydrolyzable doped silica, which are in turn bound to the surface of one or more micelles and / or one or more incomplete micelles.

[0197] The API may be non-covalently bound to particles comprising hydrolyzable doped silicon, which in turn are located within one or more micelles and / or one or more incomplete micelles. Preferably, at least about 50%, 60%, or 70% of the API may be non-covalently bound to particles comprising hydrolyzable doped silicon, which are located within one or more micelles and / or one or more incomplete micelles.

[0198] Preferably, the API (especially RNA, most especially mRNA) is non-covalently bound to particles comprising hydrolyzable doped silicon, which are located in the interior of one or more micelles and / or one or more incomplete micelles; and the API (especially RNA, most especially mRNA) is non-covalently bound to particles comprising hydrolyzable doped silicon, which are bound to the surface of one or more micelles and / or one or more incomplete micelles.

[0199] In some embodiments, the composition may be free or substantially free of micelles; and / or may be free or substantially free of incomplete micelles.

[0200] The one or more lipids may be formed from one or more lipid globules, or may include one or more lipid globules, each of which is optionally coated with a layer of surfactant. The lipid globules do not enclose an internal space or cavity. Instead, they are formed from the lipids in a solid state or substantially solid state, in which other components may be dispersed, such as particles comprising hydrolyzable doped silicon and associated with the API molecule. Thus, the interior of the lipid globules may be filled with particles comprising hydrolyzable doped silicon; in turn, the API molecule is bound (non-covalently) to the particles comprising hydrolyzable doped silicon. Additionally or alternatively (preferably additionally), particles comprising hydrolyzable doped silicon and associated (non-covalently) with the API molecule may be bound to the surface of one or more lipid globules.

[0201] Thus, preferably, the present composition comprises particles comprising hydrolyzable doped silicon associated with (especially dispersed within and / or bound to the surface of) one or more (solid or substantially solid; i.e. non-hollow) lipid globules, wherein the API is associated with (especially bound to) the particles comprising hydrolyzable doped silicon.

[0202] The average diameter of the optional lipid globules may be in the range of about 50 nm to about 500 nm, particularly about 50 nm to about 300 nm, more particularly about 100 nm to about 300 nm.

[0203] The API may be non-covalently bound to particles comprising hydrolyzable doped silicon, which are bound to the surface of one or more lipid globules. Up to about 10% or 20% of the API may be non-covalently bound to particles comprising hydrolyzable doped silicon, which are in turn bound to the surface of one or more lipid globules.

[0204] The API may be non-covalently bound to particles comprising hydrolyzable doped silicon, which in turn are located within the interior of one or more lipid globules. Preferably, at least about 50%, 60%, or 70% of the API may be non-covalently bound to particles comprising hydrolyzable doped silicon, which are located within the interior of one or more lipid globules.

[0205] Preferably, the API (especially RNA, most especially mRNA) is non-covalently bound to particles comprising hydrolyzable doped silicon, which are located in the interior of one or more lipid globules; and the API (especially RNA, most especially mRNA) is non-covalently bound to particles comprising hydrolyzable doped silicon, which are bound to the surface of one or more lipid globules.

[0206] Silicon particle aggregates

[0207] The particles comprising hydrolyzable doped silicon may be agglomerated into aggregates of particles comprising hydrolyzable doped silicon, e.g. Figure 27 Transmission electron microscopy (TEM) images are shown.

[0208] Thus, the composition may contain aggregates (especially chains) of particles comprising hydrolyzable doped silicon.

[0209] As used herein, the term "aggregates of particles comprising hydrolyzable doped silicon" may refer to clusters of particles in which the nearest neighboring particles are in contact with one another. Such clusters may have various configurations, such as substantially spherical particle clusters and / or particle chains. Configurations comprising or consisting of particle chains are particularly preferred.

[0210] Thus, the composition may comprise one or more aggregates of particles comprising hydrolysable doped silicon. Preferably, these aggregates comprise one or more chains of particles.

[0211] For example, there may be an average of at least about 2, 3, or 4 chains present per aggregate.

[0212] One or more aggregates may include one or more particle branches. Thus, one or more aggregates may be formed by branches formed by particle chains, or include branches formed by particle chains. For example, each aggregate may have at least about 2, 3, or 4 branches.

[0213] One or more aggregates of particles comprising hydrolyzable doped silicon may be associated with one or more lipids, such as embedded in the lipids and / or attached to the surface of the lipids.

[0214] One or more aggregates of particles comprising hydrolyzable doped silicon may be associated with, eg, embedded in and / or attached to, one or more lipid structures described herein.

[0215] As described herein, the one or more lipid structures can be formed from or include one or more of the following: micelles, incomplete micelles, liposomes, incomplete liposomes, and lipid spheres. Thus, one or more particle aggregates comprising hydrolyzable doped silicon can be associated with (e.g., embedded in and / or attached to the surface of) one or more of the lipid micelles, incomplete lipid micelles, liposomes, incomplete liposomes, and lipid spheres.

[0216] Specifically, one or more aggregates of particles comprising hydrolyzable doped silicon may be embedded in or attached to the surface of one or more of: liposomes; incomplete liposomes; and lipid spheres.

[0217] The ratio of the longest dimension of the aggregate to the longest dimension of the lipid structure may be, on average, about 1:5 to 5:1, in particular about 1:3 to 3:1; in particular when the lipid structure is or comprises a liposome and / or lipid globule and one or more aggregates are embedded therein or attached to its surface. This can be measured, for example, by transmission electron microscopy (TEM), e.g. Figure 27 shown.

[0218] The average value of the longest dimension of the aggregate can be from about 50 nm to about 500 nm, especially from about 50 nm to about 200 nm, for example from about 50 nm to about 150 nm, for example when measured by transmission electron microscopy (TEM). Specifically, one or more aggregates can be or include one or more particle chains, wherein the average length of the chain is from about 50 nm to about 500 nm, especially from about 50 nm to about 200 nm, for example from about 50 nm to about 150 nm. The average cross-sectional diameter of the chain can be from about 5 nm to about 50 nm, for example from about 5 nm to about 30 nm.

[0219] The average value of the ratio of the longest dimension of a single particle comprising hydrolyzable doped silicon to the longest dimension of the lipid structure may be from about 1:100 to about 1:2, in particular from about 1:100 to about 1:5, more in particular from about 1:100 to about 1:9; in particular when the lipid structure is or comprises a liposome and / or lipid globule, and one or more aggregates are embedded therein or attached to its surface. This can be measured, for example, by transmission electron microscopy (TEM), such as Figure 27 shown.

[0220] When one or more aggregates are present, the average diameter of the particles comprising hydrolyzable doped silicon is preferably from about 1 nm to about 50 nm, particularly from about 5 nm to about 50 nm, more particularly from about 1 nm to about 30 nm, even more particularly from about 5 nm to about 20 nm, for example about 10 nm. Additionally or alternatively, the particles may be porous, and the average (e.g., mean) pore size may be from about 0.1 nm to about 5 nm, for example, about 2 nm.

[0221] In turn, the API may be bound (non-covalently) to one or more particles in the aggregate.

[0222] Thus, one or more aggregate particles can be bound to the API (especially when the API is or includes mRNA). When one or more aggregates are or include particle chains, such chains can extend into the interior of the lipid structure (especially liposomes, incomplete liposomes and / or lipid spheres). In this way, these chains can provide a path for the API to be better encapsulated by lipids. Without wishing to be bound by theory, it is believed that this can shield the API from degradation, especially shielding it from enzymatic degradation (especially in vivo), and preventing or reducing the reaction of the API molecules with water molecules. It is believed that as the silicon particles degrade over time, the API can be released, thereby being able to protect the API until it reaches the target release site.

[0223] The presence of charged APIs can itself promote particle aggregation. For example, when the API is a nucleic acid (particularly mRNA), its negative charge (due to the phosphate backbone) can induce particle aggregation. Additionally or alternatively, the presence of Si-O species on the particle surface can induce particle-particle interactions, thereby promoting aggregation.

[0224] Thus, preferably, the one or more aggregates are or include one or more chains comprising particles of hydrolyzable doped silicon, wherein the API is bound to the particles and one or more chains extend into the interior of the lipid structure, particularly a liposome, incomplete liposome, or lipid globule. This can provide a more stable environment for the API. Additionally or alternatively, it can increase the uptake of the API into the lipid structure compared to the absence of such aggregates. This is in contrast to conventional liposomal delivery vehicles, which can suffer from low API uptake efficiency; for example, it is believed that up to about 80% of conventional liposomal delivery vehicles formulated into commercially available therapeutic compositions may be "empty" for the API.

[0225] Optionally, substantially no particles comprising hydrolyzable doped silicon are present as isolated particles; rather, substantially all particles are present as aggregates.

[0226] Aggregation can be promoted when the average diameter of the particles comprising hydrolyzable doped silicon is from about 1 nm to about 50 nm, particularly from about 5 nm to about 50 nm, more particularly from about 1 nm to about 30 nm, still more particularly from about 5 nm to about 20 nm, for example, about 10 nm. Such particle diameters can be smaller than the typical diameter of lipid structures (as described herein) that can be formed spontaneously (optionally, promoted by filtration) from one or more lipids.

[0227] Therefore, preferably, the composition contains aggregates (especially chains) of particles comprising hydrolyzable doped silicon, wherein the average diameter of the particles comprising hydrolyzable doped silicon is from about 1 nm to about 50 nm, especially from about 5 nm to about 50 nm, more especially from about 1 nm to about 30 nm, still more especially from about 5 nm to about 20 nm, for example about 10 nm.

[0228] Aggregation can be promoted when the composition is filtered, for example, as described below in the "Filtration" step of "Materials and Methods" in Example 1. Filtration can be or include filtration through a porous membrane having an average pore size of about 0.01 μm to about 1 μm (e.g., about 0.05 μm to about 0.6 μm).

[0229] Thus, the composition may be (or formed from) a leached composition.

[0230] Preferably, filtration occurs before the addition of the API.Thus, the composition may be formed from a filtrate composition comprising the particles and the one or more lipids, to which the API has been added.

[0231] Thus, the composition may suitably be a leachable composition comprising aggregates (especially chains) of particles comprising hydrolyzable doped silicon, wherein the average diameter of the particles comprising hydrolyzable silicon is from about 1 nm to about 50 nm, especially from about 5 nm to about 50 nm, more especially from about 5 nm to about 30 nm, still more especially from about 5 nm to about 20 nm, for example about 10 nm.

[0232] Other beneficial components

[0233] Optionally, the composition further comprises one or more other advantageous components.

[0234] One or more other advantageous components can be or include tyrosine or a derivative thereof. Thus, optionally, in addition to the above amino acids, the specific amino acid tyrosine can be present as an additional component, as shown in the composition "SIS0013-T" in Examples 3 and 6 below. Optionally, the weight ratio of one or more lipids (i.e., the total lipid component) to tyrosine can be in the range of about 130:1 to about 30:1, for example, about 80:1.

[0235] Additionally or alternatively, the composition can include nicotinamide adenine dinucleotide (NAD) or a derivative thereof, as exemplified below in the composition "SIS0013-N" in Examples 3 and 6. Optionally, the weight ratio of the one or more lipids (i.e., the total lipid component) to NAD can be in the range of about 130:1 to about 30:1, for example about 80:1.

[0236] Additionally or alternatively, the composition can include quercetin or a derivative thereof, as exemplified by the composition "SIS0013-Q" in Examples 3 and 6 below. Optionally, the weight ratio of the one or more lipids (i.e., the total lipid component) to the quercetin can be in the range of about 130:1 to about 30:1, for example, about 80:1.

[0237] The composition may also optionally include a peptide containing a cell surface receptor (e.g., integrin) recognition sequence that confers a degree of cell specificity. The peptide may have a "head group" containing the cell surface receptor recognition sequence and a "tail" that can non-covalently bind to the API (e.g., nucleic acid, such as mRNA) and / or doped silicon.

[0238] When the API is a nucleic acid, the composition can also optionally include a polycationic nucleic acid binding component. The term "polycationic nucleic acid binding component" is well known in the art and can refer to a polymer having at least 3 repetitions of a cationic unit with a cationic amino acid residue or other positively charged groups, which polymer can be complexed with nucleic acids under physiological conditions. Examples of polycationic molecules that bind nucleic acids are oligopeptides that include one or more cationic amino acids. The polycationic nucleic acid binding component can 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 combination of oligomers comprising any combination of histidine, arginine, lysine, ornithine, diaminopropionic acid, and diaminobutyric acid residues, or consisting of any combination of these residues. Other examples of polycationic components include dendrimers and polyethyleneimine.

[0239] Subjects and their diseases and conditions

[0240] According to a preferred embodiment, the subject to which the compositions disclosed herein are administered is a human subject. The age of the human subject can be in the range of 1 month or more, optionally 1 year or more. The age of the human subject can be in the range of less than 100 years old.

[0241] A disease or condition according to the present disclosure may be a contagious disease.As used herein, the term "contagious" may be used to refer to a disease that is easily transmitted from one organism to another, especially from one person to another.

[0242] Infectious diseases can be viral, bacterial, fungal or parasitic; particularly viral diseases.

[0243] When the disease is a viral disease, it may be a respiratory virus, such as respiratory syncytial virus (RSV), parainfluenza virus (HPIV), human metapneumovirus (HMPV), rhinovirus (HRV), coronavirus, such as SARS-CoV (especially SARS-CoV-1, more especially SARS-CoV-2), adenovirus (HAdV), enterovirus (EV), bocavirus (HBoV), parechovirus (HPeV) or influenza virus.

[0244] The viral disease can be dengue virus, Ebola virus, encephalomyocarditis virus, hepatitis virus, herpes virus, human immunodeficiency virus, human papillomavirus, human T-lymphotropic virus, measles virus, monkeypox virus, mumps virus, polio virus, rabies virus, rotavirus, rubella virus, varicella-zoster virus, West Nile virus, yellow fever virus or Zika virus.

[0245] The disease or condition may be a genetic disease or condition.

[0246] In some embodiments, the genetic disorder may be characterized by defective expression of one or more proteins, particularly one or more enzymes.

[0247] Genetic disorders can be multifactorial, meaning they are not confined to any particular single-gene inheritance pattern but may involve the effects of multiple genes as well as environmental factors; for example, schizophrenia, diabetes, asthma, depression, epilepsy, heart disease, or hypothyroidism.

[0248] A genetic disorder may involve one or more mutations in one or more genes.

[0249] Thus, a genetic disorder can be a single gene disorder, which can occur if at least one mutation occurs in a single gene. When a genetic disorder is a single gene disorder, it can involve one mutation or more than one mutation in a single gene. Examples of single gene disorders include sickle cell anemia, cystic fibrosis, Huntington's disease, or Duchenne muscular dystrophy.

[0250] A genetic disorder may involve one or more mutations in more than one gene. By way of non-limiting example, a genetic disorder may involve more than one mutation in a first gene and one mutation in a second gene.

[0251] The genetic disorder may be a disorder that may occur if at least one mutation occurs in at least one gene of a group of genes; in particular, the genetic disorder may be osteopetrosis.

[0252] The genetic disorder can be Angelman syndrome; Canavan disease; Charcot-Marie-Tooth disease; color blindness; Cry-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; osteochondritis; sickle cell disease; spinal muscular atrophy; Tay-Sachs disease; or Turner syndrome.

[0253] In a broad sense, "genetic disorder" as used herein can encompass cancer. The cancer can be or include a blood cancer (e.g., leukemia, lymphoma, or myeloma) or a solid tumor (e.g., sarcoma; carcinoma; carcinosarcoma, or lymphoma).

[0254] Thus, 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, uterus, oral cavity or ovary.

[0255] In more detail, the cancer can be or include pulmonary system cancer, brain cancer, gastrointestinal cancer, skin cancer, genitourinary cancer, pancreatic cancer, lung cancer, medulloblastoma, basal cell carcinoma, glioma, breast cancer, prostate cancer, testicular cancer, esophageal cancer, hepatocellular carcinoma, gastric cancer, gastrointestinal stromal tumor (GIST), colon cancer, colorectal cancer, ovarian cancer, melanoma, neuroectodermal tumor, head and neck cancer, sarcoma, soft tissue sarcoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, leiomyosarcoma, cervical cancer, uterine cancer, endometrial cancer, carcinoma, bladder cancer, epithelial cancer, squamous cell carcinoma, adenocarcinoma, bronchogenic carcinoma, renal cell carcinoma, liver cancer, bile duct cancer, neuroendocrine carcinoma, carcinoid tumor, diffuse giant cell tumor or glioblastoma.

[0256] Preparation, storage, stability and administration of the disclosed compositions

[0257] Prevention of a disease or condition in a human subject can include administering a prophylactically effective amount of a composition disclosed herein to a human subject, wherein the subject is in need thereof; for example, a subject in need thereof determined by a physician or other healthcare practitioner. Simultaneously, treatment of a disease or condition in a human subject can include administering a therapeutically effective amount of a composition disclosed herein to a human subject in need thereof.

[0258] The dosage of the compositions disclosed herein can be varied to obtain an amount of the API that is effective to achieve the desired prophylactic and / or therapeutic response for a given subject without causing toxicity to the subject. The appropriate dosage of the composition can be the lowest effective dose that allows the API to produce a therapeutic and / or prophylactic effect.

[0259] The selected dose, dosage form, and regimen will each depend on a variety of factors. These factors may include, for example, the activity of the API, the route of administration, the time of administration, the rate of excretion or metabolism of the API, the rate and extent 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, general health, and past medical history of the subject being treated, and other such factors well known in the medical arts.

[0260] The composition may be administered intramuscularly or intravenously (including transdermal delivery via patches), orally (including sublingual administration), intranasally, dermally or by any other suitable route; in particular by injection or oral administration.

[0261] Preferably, the composition can be administered by injection, such as intravenous or intramuscular injection. Optionally, when the composition is administered by injection, the subject is monitored for symptoms or signs of hypersensitivity reactions, such as vaccine-related hypersensitivity reactions.

[0262] Also preferably, the composition can be administered orally or intranasally. Compositions suitable for oral administration can be in the form of discrete dosage forms, particularly liquids or aerosols, each containing a predetermined amount of the composition. Such dosage forms can be prepared by any known pharmaceutical method.

[0263] Therefore, it is particularly preferred that the composition comprises a doping level of at least about 1x10 16 dopant atoms / cm 3 A composition comprising hydrolyzable silicon particles, one or more lipids, and an active pharmaceutical ingredient (API) for use in a method for preventing or treating a disease or condition in a human subject, wherein the method comprises administering the composition to the human subject by injection, oral administration, or intranasal administration (particularly by injection or oral administration). Suitably, for the reasons described above, the composition may also include one or more amino acids. For example, a mouse model for administration by injection is provided in Example 4 below.

[0264] According to conventional pharmaceutical preparation technology, said composition can be combined with pharmaceutical carrier in close mixing.Carrier can adopt various forms, specifically depending on the desired formulation form of administration.Any conventional pharmaceutical medium can be used as carrier, such as one or more in water, oil and alcohol (including glycol).The form of disclosed composition can depend on administration, especially when being formulated for injection, oral or intranasal administration, can include physiological saline solution.Said composition can also include one or more pharmaceutically acceptable additives and excipients, such as one or more of the following: antitack agent, defoamer, buffer, polymer, antioxidant, chelating agent, viscosity modifier, tension agent (tonicifier), flavor enhancer, opacifier, suspending agent, filler, plasticizer, flavoring, preservative, coloring agent, diluent, adhesive, disintegrant and mixture thereof.

[0265] The action of microorganisms can be prevented or reduced by the addition of various antibacterial and antifungal agents, for example, one or more of the following: parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

[0266] Compositions disclosed herein can be provided as sterile solutions, i.e., by adding the desired amount of the composition to an appropriate solvent (suitably adding various other ingredients) by any known pharmaceutical method. Compositions disclosed herein can be provided as sterile dispersions, i.e., by adding the desired amount of the composition to an appropriate sterile carrier (suitably adding various other ingredients). Compositions disclosed herein can be provided as sterile powders (e.g., for subsequent preparation of sterile injection solutions), such as powders obtained by vacuum drying and freeze drying (lyophilization) techniques.

[0267] Optionally, the composition can be stored before administration to a subject. The composition can be stored at a temperature in the range of 0°C or above, in particular 4°C or above, for at least 1 week (optionally, up to 6 months, in particular up to 1 year) before administration to a subject.

[0268] In certain preferred embodiments, particles comprising hydrolyzable doped silicon are stored together with one or more lipids, but without the API. They are combined with the API (e.g., by the steps described in Example 1 below) shortly before administration to a subject, for example, no more than about 3 weeks, about 2 weeks, or about 1 week, especially no more than about 2 days, more especially no more than about 1 day, before administration to a subject. Advantageously, the particles and lipids can be stably stored for a long time, especially several months, such as about 6 months or about 12 months; they can be stored in this way without significant degradation. When the API is a reactive API (especially mRNA), it can advantageously be synthesized in situ or synthesized externally and delivered to the clinical environment at the last possible moment and then combined with the particles and lipids.

[0269] Nevertheless, it has also been found that particles comprising hydrolyzable doped silicon can stabilize the API, including stabilizing the API during storage. Thus, in some embodiments, the particles can improve the storage stability of the API, particularly when the API is or comprises a nucleic acid (e.g., mRNA). In some embodiments, the particles can improve the stability of the API at 25°C, particularly when the API is or comprises a nucleic acid (e.g., mRNA).

[0270] Thus, optionally, the stored composition comprises all of the following components: particles comprising hydrolyzable doped silicon; one or more lipids; and an active pharmaceutical ingredient (API). If so, the composition, once formulated, can optionally be stored for only a short period of time, which can mean a period of up to about 3 weeks, about 2 weeks, or about 1 week prior to administration to a subject.

[0271] In some embodiments, the particles can improve the stability of the API during circulation in vivo, especially when the API is or includes a nucleic acid (e.g., mRNA). In some embodiments, the particles can protect the API from degradation, especially enzymatic degradation, especially when the API is or includes a nucleic acid (e.g., mRNA). Thus, the doped silicon-containing compositions disclosed herein can alleviate or solve the problem of how to ensure that the API reaches cells after administration to a patient, including how to stabilize the API while circulating in vivo.

[0272] At the same time, the disclosed compositions comprising hydrolyzable doped silicon can alleviate or meet the requirements of tissue or cell targeting, thereby enabling the delivery of APIs to the correct cells.

[0273] Additionally or alternatively, once the target cell is reached, the disclosed compositions can alleviate or resolve the difficulty of ensuring efficient uptake of the API by the cell. For example, the disclosed compositions can facilitate transport of the API from the outside of the cell into the cytoplasm.

[0274] After the API is taken up by the cell, the disclosed compositions can alleviate or solve the problem of how to prevent the API from being degraded too quickly in the cytoplasm. It is believed that the particles can increase the stability of the API in the cytoplasm, especially the stability against enzymatic degradation, especially when the API is or includes a nucleic acid (e.g., mRNA).

[0275] Example

[0276] Examples 1 and 2 disclose studies of mRNA complexed with delivery vehicles containing undoped and doped silicon.

[0277] Particle size and zeta potential were measured; gel retardation assays and Quant-iT RiboGreen RNA assays were performed. mRNA transfection via the silicon-doped delivery vehicle was also investigated by in vitro transfection of human embryonic kidney (HEK293) cells with mRNA encoding luciferase and observation by bioluminescence imaging of the cell cultures.

[0278] Example 1

[0279] Delivery vehicles containing silicon particles with a dopant ("SIS0013") or without a dopant ("SIS0012") were compared for their ability to encapsulate and stabilize the API and deliver it to cells.

[0280] Two different luciferase mRNAs were selected as test APIs. Successful transfection of cells with luciferase mRNA can be measured using a luciferase assay, enabling reliable and accurate determination of the success rate of API delivery to cells in in vitro culture.

[0281] Furthermore, mRNA is a relatively reactive molecule, and it can be expected that delivery vehicles that can stably and successfully deliver mRNA to cells will also be able to stably and successfully deliver other APIs. By studying reactive APIs, the confidence that the results of this study can be generalized to other (e.g., less reactive) APIs is increased.

[0282] Materials and methods

[0283] Composition of SIS0012 and SIS0013

[0284] SIS0012 (undoped Si) formulation:

[0285] -7.25mg DOTAP (1.45mL)

[0286] -7.3mg DOPE (1.46mL)

[0287] -1.45mg DSPE-PEG 2000

[0288] -1 mg Si nanoparticles (average diameter less than 30 nm, as determined by SEM)

[0289] -1mg trehalose

[0290] -0.5mg glycine

[0291] -9mL nuclease-free water

[0292] SIS0013 (doped Si) formulation:

[0293] -7.25mg DOTAP (1.45mL)

[0294] -7.3mg DOPE (1.46mL)

[0295] -1.45mg DSPE-PEG 2000

[0296] -1mg boron-doped Si nanoparticles, approximately 5x10 18 boron atoms / cm 3 (Average diameter is less than 30 nm, confirmed by SEM)

[0297] -1mg trehalose

[0298] -0.5mg glycine

[0299] -9mL nuclease-free water

[0300] Further details of the above components of SIS0012 and SIS0013 are as follows.

[0301] a) Si ("SiNP"): porous silicon particles with an average diameter of less than 30 nm. For SIS0013, the doped (5x10 18 boron atoms / cm 3 Activation was performed by exposure to methanol (to remove surface oxidation and ensure particle homogeneity), followed by slow evaporation, yielding a (dry, solid) powder, the activated SiNPs.

[0302] b) Trehalose ("THR"): solid powder.

[0303] c) Glycine ("GLY"): solid powder.

[0304] d) SiNP+GLY+THR solution: a brown suspension obtained by sonicating a suspension in 50 ml of nuclease-free water for 60 minutes, comprising: 50 mg activated SiNPs; 50 mg THR; and 25 mg GLY.

[0305] e) DOTAP-Cl solution: 50 mg of DOTAP was mixed with 10 ml of methanol and then sonicated at 40° C. for 30 minutes until the solution was completely dissolved.

[0306] f) DOPE solution: 50 mg of DOPE was mixed with 10 ml of methanol and then sonicated at 40° C. for 30 minutes until DOPE was completely dissolved.

[0307] g) mPEG2000-DSPE solution: 40 mg of mPEG2000-DSPE was mixed with 8 ml of methanol, and then sonicated at 40° C. for 30 minutes until completely dissolved to obtain a solution.

[0308] Preparation method of SIS0012 or SIS0013

[0309] Lipid film preparation

[0310] a) All lipids (DOPE, DOTAP and mPEG2000-DSPE) were mixed in a glass round-bottom flask.

[0311] b) Evaporate the solvent using a rotary evaporator in a 40°C water bath.

[0312] Film rehydration

[0313] a) 1 ml of brown suspension of SiNPs (SIS0013: doped; SIS0012: undoped) + GLY + THR (1 mg / ml SiNPs; 0.5 mg / ml GLY; 1 mg / ml THR) and 9 ml of nuclease-free water were added to the lipid film (therefore, the total volume of the brown suspension and nuclease-free water was 10 ml).

[0314] b) The flask was sealed with parafilm and then shaken in a 60°C water bath for 10 minutes to allow 10 ml of liquid to rehydrate the lipid film.

[0315] c) The resulting suspension was allowed to stand at room temperature for several hours and then stored at 4°C.

[0316] Filter out

[0317] The suspension obtained in step (c) "rehydration of the membrane" was passed through polycarbonate membrane filters having pore sizes of 0.4 μm and 0.1 μm, respectively. The suspension was passed through each pore size 10 times at 60°C.

[0318] The resulting products are SIS0012 (using undoped SiNPs) and SIS0013 (using doped SiNPs).

[0319] mRNA encoding luciferase

[0320] Details of the two luciferase encoding mRNAs used as test APIs are as follows.

[0321] (1) A 2315-base mRNA encoding firefly luciferase (mod-LUC RNA; 2 mg / mL) (referred to herein as "LUC mRNA1," "Luc mRNA1," or simply "mRNA1") was obtained.

[0322] (2) At the same time, the 1921-base EZ Cap sequence encoding luciferase was obtained. TM Firefly luciferase mRNA (5-moUTP modified) (referred to herein as "LUC mRNA2," "Luc mRNA2," or simply "mRNA2").

[0323] Materials used for gel electrophoresis and RiboGreen detection

[0324] The materials used for gel electrophoresis and RiboGreen detection in this example (and subsequent examples using similar detection methods) are listed in Table 1.

[0325] Table 1 - Materials used for gel electrophoresis and RiboGreen detection

[0326] Reagents CAS number <![CDATA[E-Gel TM 1Kb plus Express DNA Ladder]]> N / A <![CDATA[E-Gel TM EX Agarose (1%) N / A <![CDATA[Quant-iT TM RiboGreen TM RNA Reagents]]> N / A Tris buffer (100 mM, pH 7.4) N / A EDTA 6381-92-6 Nuclease-free water ("NFW") 7732-18-5

[0327] Materials used for luciferase assay and HEK293 culture

[0328] The materials used for the luciferase assays and HEK293 cultures in this example (and subsequent examples using similar cultures) are listed in Table 2.

[0329] Table 2 - Materials used for luciferase assays and HEK293 cultures

[0330] Reagents CAS number Dulbecco's modified medium N / A <![CDATA[Fetal bovine serum, heat-inactivated, qualified, One Shot TM > N / A L-Glutamine N / A Penicillin-Streptomycin Solution 100x N / A PBS, sterile pH 7.4 N / A Trypsin / EDTA, 0.5% 9002-07-7 <![CDATA[Bright-Glo TM Luciferase Assay System]]> N / A

[0331] Preparation method of SIS0012-mRNA or SIS0013-mRNA complex

[0332] To prepare SIS0012-mRNA or SIS0013-mRNA complexes with different mRNA loading weight ratios (w / w), the required volume of SIS0012 or SIS0013 suspension (nominal total lipid concentration of 1.6 mg / mL) was mixed with the required volume of mRNA stock solution (concentration of 2 mg / mL) and the final concentration of LUC mRNA was adjusted using nuclease-free water according to the subsequent detection protocol before the complex incubation step. The sample was thoroughly mixed by gentle pipetting and incubated at room temperature for 60 minutes to allow complexation to complete. After incubation, the sample was stored at 4°C until subsequent detection.

[0333] For the examples below where another API was used instead of mRNA, similar API loading procedures were followed.

[0334] Table 3 - mRNA:SIS0012-mRNA or SIS0013-mRNA complexes at different weight ratios

[0335]

[0336] Particle size and zeta potential measurement methods

[0337] Particle size, polydispersity, and zeta potential were determined using dynamic and electrophoretic light scattering (Malvern Zetasizer Pro (Red Advance), Malvern, UK). Particle size determination was performed using a DTS0012 disposable cuvette, and surface charge determination was obtained using a DTS1070 folded capillary zeta cell. Unloaded SIS0012, unloaded SIS0013, SIS0012-mRNA, and SIS0013-mRNA were analyzed. To determine particle size, backscatter detection at 173 degrees was used. The Z-average [nm] was obtained as the particle size by fitting the correlation function using the accumulation method. 20 μL of unloaded SIS0012, unloaded SIS0013, SIS0012-mRNA, or SIS0013-mRNA sample was mixed with 980 μL of nuclease-free water to a total volume of 1000 μL and then added to the disposable cuvette. A total of 4 scans were performed to reduce the signal-to-noise ratio, and the best 3 scans were selected as the average particle size and PDI (n = 3). A volume of 150 μL of sample was mixed with 850 μL of nuclease-free water, then injected into a folded capillary cell (via a 1 mL syringe) and measured at 25°C. A total of 5 scans were performed to eliminate the signal-to-noise ratio, and the best 3 scans were selected to record the average zeta potential (n = 3).

[0338] Gel retardation test method

[0339] To evaluate the formation of SIS0012-mRNA or SIS0013-mRNA complexes, nanoparticles dispersed in nuclease-free water were combined with mRNA at various loading ratios listed in Table 3 (i.e., the weight ratio of mRNA to total lipids in SIS0012 or SIS0013 was 1:4, 1:7.2, 1:12, or 1:24) and plated on E-Gel. TM Power Snap electrophoresis apparatus (ThermoFisher, UK) was used to electrophoresed the E-Gel TM Electrophoresis analysis was performed on 1% agarose gel (ThermoFisher, UK) for 8 minutes. Each well was loaded with an equal amount of 150 ng mRNA, and naked mRNA was used as a control. TM The gel was visualized using a Power Snap electrophoresis imager (ThermoFisher, UK).

[0340] Available mRNA detection methods

[0341] To assess the amount of mRNA available in the complex, Quant-iT RiboGreen RNA reagent from Invitrogen (ThermoFisher, UK) was used. The experimental procedures were performed according to the manufacturer's instructions. For analysis, a Varioskan LUX multimode microplate reader (ThermoFisher, UK) with an excitation wavelength of 480 nm and an emission wavelength of 520 nm was used. The test was performed in 1x Tris-EDTA (TE) buffer (10 mM Tris-HCl and 1 mM EDTA). All samples were measured three times, and the results are expressed as mean ± standard deviation (SD).

[0342] In vitro cell transfection assessment method

[0343] Human embryonic kidney (HEK293) cell line was used to evaluate transfection. Cells were cultured in Dulbecco's Modified Eagle's Medium (Euroclone, Italy) supplemented with 10% inactivated fetal bovine serum (FBS, Gibco, UK), 1% L-glutamine (Euroclone, Italy) and 1% penicillin / streptomycin (Euroclone, Italy) at 37°C and 5% CO2 and passaged according to standard laboratory procedures. 24 hours before transfection, HEK293 cells were plated at 1 × 10 cells / mL in 100 μl of complete medium without antibiotics. 4 Cells / well were seeded in a 96-well black clear-bottom plate (or 2×10 4 Cells were incubated at 4% 4% CO2 for 24 hours under standard culture conditions (37°C, 5% CO2). The next day, the medium was replaced with 100 μl of transfection medium containing: 10 μl of transfection complex (containing 1 μg of mRNA prepared in nuclease-free water), 10 μl of serum-free DMEM (containing 1% L-glutamine), and diluted with 80 μl of complete growth medium (DMEM containing 10% FBS and 1% L-glutamine). 10 μg / ml naked mRNA diluted in complete medium or no mRNA (medium alone) served as negative controls.

[0344] After the transfection step is completed, cells are transferred to an incubator and grown under standard conditions (37°C, 5% CO 2 ). After treatment, 6 hours, 24 hours, 48 ​​hours or 72 hours, according to the manufacturer's protocol, the translation product of LUC-mRNA is detected by luciferase assay (Bright-Glo luciferase test kit, Promega, USA). In brief, before adding the detection reagent of 100 μl / well, culture plate is balanced at room temperature and then incubated at room temperature for 15 minutes. By photometer device (Hamamatsu Photonics bioluminescence imager, Hamamatsu Photonics Italy SRL, Arese, Italy-its integrated digital camera and camera controller, C4742-98-Wasabi software, v.1.5, Hamamatsu Photonics) luciferase activity is assessed.

[0345] result

[0346] Dynamic and electrophoretic light scattering results

[0347] The results of surface charge studies of SIS0012 and SIS0013 complexed with LUC mRNA1 (mRNA to total lipid weight ratios of 24:1, 12:1, 7.2:1, and 4:1) are shown in Tables 4a-4d.

[0348] Table 4a - Characteristics of unloaded SIS0012 and unloaded SIS0013

[0349]

[0350] As shown in Table 4a, SIS0012 (not bound to mRNA) has a more negative zeta potential than SIS0013.

[0351] Table 4b - Characteristics of SIS0012 complexed with LUC mRNA1 at different weight ratios (mRNA:total lipid)

[0352]

[0353] Table 4c - Characteristics of SIS0013 complexed with LUC mRNA1 at different weight ratios (mRNA:total lipid)

[0354]

[0355] Figure 1 and Figure 2 Particle size and polydispersity index (PDI) data for SIS0012 and SIS0013 according to Tables 4a to 4c are presented.

[0356] SIS0012 results. As shown in Tables 4a-4b and Figure 1 As shown, the PDI remained fairly stable at all binding ratios. However, when SIS0012 was complexed with mRNA, an increase in the hydrodynamic diameter of the nanoparticles was observed, which plateaued as more mRNA was added, particularly at ratios of 7.2:1 and 4:1. This plateau indicated that mRNA binding had reached saturation. The positive zeta potential gradually decreased with increasing mRNA loading, reaching a negative value at a ratio of 4:1 (i.e., when the amount of mRNA was maximum); this is thought to be due to the presence of weakly bound / unbound mRNA.

[0357] SIS0013 results. As shown in Tables 4a, 4c and Figure 2 As shown, the PDI increases with increasing mRNA, thus favoring a delivery vehicle that is particularly dispersible in aqueous environments. SIS0013 appears to prevent excessive accumulation of bulky lipids, a known issue with lipid nanoparticles. Furthermore, when SIS0013 complexed with mRNA, its hydrodynamic diameter was observed to steadily increase, without the plateau observed for SIS0012, suggesting that mRNA binding for SIS0013 does not reach saturation as early as for SIS0012. The zeta potential decreased with increasing mRNA, but not as rapidly as for SIS0012. Overall, slow and steady particle growth was observed with increasing mRNA, without undesirable lipid accumulation.

[0358] It can be concluded that SIS0013 can load a larger weight ratio of negatively charged mRNA than SIS0012, i.e., it exhibits higher binding efficiency; this may be due to the higher initial positive zeta potential of SIS0013, which in turn may be attributed to the modulation of the zeta potential of SIS0013 by the dopant boron.

[0359] Gel retardation test results

[0360] Figure 3 Shown are gel electrophoresis results for studying the complexation of SIS0012 and SIS0013 with LUC mRNA1.

[0361] Wells were loaded with either SIS0012 / LUC mRNA1 or SIS0013 / LUC mRNA1 complexes (equal amounts in each well) at ratios increasing from 1:4 to 1:24 (mRNA to total lipid weight ratio). This is explained by the fact that unbound mRNA migrated through the gel, while mRNA bound to SIS0012 or SIS0013 remained immobilized within the wells.

[0362] Complete binding of nucleic acids for both SIS0012 and SIS0013 was observed when the ratio of mRNA to total lipid was 1:7.2, 1:12, and 1:24. At these ratios, no mRNA migrated through the gel.

[0363] At a ratio of 1:4, the amount of mRNA relative to either SIS0012 or SIS0013 was greatest, with some mRNA of both SIS0012 and SIS0013 observed to migrate through the gel.

[0364] Therefore, the 1:4 ratio provides a unique basis for comparison between SIS0012 and SIS0013. At a 1:4 ratio, a weaker band was observed for SIS0013. Therefore, confirming the results of the particle size and zeta potential analyses above, SIS0013 exhibits higher binding efficiency than SIS0012. In other words, SIS0013 appears to be able to bind more mRNA (weight ratio) than SIS0012. When an excess of mRNA is present, SIS0013 binds a greater percentage of total mRNA weight (% w / w) than SIS0012.

[0365] Similar results were obtained when SIS0012 and SIS0013 were complexed with LUC mRNA2.

[0366] Therefore, SIS0013 may represent the most efficient delivery vehicle, capable of binding more mRNA weight-to-weight (w / w) than SIS0012. It can also be inferred that SIS0013 may also provide improved binding, thereby enhancing nucleic acid stability, when the amount of mRNA added is below the mRNA saturation threshold of SIS0012. At these amounts, the charge interactions between SIS0013 and mRNA may be more attractive than those between otherwise identical SIS0012 and mRNA.

[0367] As described below, SIS0013 also exhibited efficient cellular uptake of mRNA and resulted in faster expression of luciferase than SIS0012.

[0368] Quant-iTRiboGreen assay results for available mRNA

[0369] To assess how much mRNA is efficiently bound to SIS0012 and SIS0013 and cannot be accessed by external reactive species, the amount of mRNA accessible to the fluorescent dye was used as an indirect parameter to indicate how much unbound or superficially bound mRNA remains after complexation.

[0370] Quant-iT TM RiboGreen TMThe dye in the RNA reagent has a high binding affinity and specificity for RNA, allowing quantification of very low concentrations of RNA in the sample. The dye binds only to free RNA or RNA weakly bound to the outermost surface of the complex; more tightly bound RNA within the complex is not accessible to the dye.

[0371] like Figure 4 As shown, both SIS0012 and SIS0013 exhibited low mRNA accessibility at the lowest ratio of 1:24.

[0372] Table 5 shows that for SIS0012, mRNA accessibility at other ratios is higher, indicating that there is more unbound mRNA or RNA weakly bound to the outermost surface of the complex than for SIS0013.

[0373] For SIS0012, the percentage (%) of mRNA available for reaction with the dye (i.e., with the external reactive substance) was consistently higher than for SIS0013, especially as the amount of mRNA increased, and particularly when the weight ratio (w / w) of mRNA to total lipid was 1:4 (i.e., when the maximum amount of mRNA was present). This suggests that SIS0013 consistently binds to more mRNA than SIS0012, in a manner that renders the mRNA unavailable for reaction with the external reactive substance.

[0374] It can be inferred that SIS0013 is able to bind more deeply to more mRNA than SIS0012, which means that in reactive substances (such as Quant-iT TM RiboGreen TM In the presence of dyes, SIS0013 may be able to better stabilize mRNA (and other APIs). Therefore, the stability of both storage and in vivo can be improved. One advantage is that after administration and during circulation in the body, the reactive API (such as mRNA) can remain stable so that enough API reaches the target cells to play the necessary role, such as luciferase expression in this study. Another advantage is that the API is stably delivered to the target cells. At the same time, the API can also remain stable in the cytoplasm of the cell.

[0375] Table 5 - mRNA accessibility (percentage of superficial binding (%)) of SIS0012 and SIS0013 at different mRNA to total lipid weight ratios (w / w)

[0376]

[0377] Cell transfection results

[0378] A series of in vitro transfection studies were performed in HEK293 cells to compare the performance of SIS0012 and SIS0013. 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 using the Bright-Glo luciferase assay, as described herein, using a luminometer setup, 6, 24, 48, or 72 hours after transfection.

[0379] As a guide, Table 6 lists the contents of three replicate wells:

[0380] Table 6 - Pair Figures 5 to 8 Guidelines for Mesopore Content

[0381]

[0382] Summary: At various time points, SIS0013 was observed to transfect mRNA more efficiently than SIS0012 across a range of mRNA to total lipid ratios, especially at earlier time points, suggesting that SIS0013 has a higher transfection rate than SIS0012. Therefore, the doping of hydrolyzable silicon appears to result in more efficient and rapid uptake of the API, enabling more rapid and reliable effect of the API on target cells.

[0383] Compared to naturally occurring mRNA, both LUC mRNA1 and LUC mRNA2 are chemically modified. Such chemical modifications may have the potential to make transfection more difficult. The increased ability of Si-doped SIS0013 to transfect chemically modified mRNAs compared to undoped Si-doped SIS0012 is particularly advantageous and surprising.

[0384] 6 hours after transfection: The data 6 hours after transfection are summarized in Tables 7 and Figure 5 Regardless of which mRNA was used (LUC mRNA1 or LUC mRNA2), luciferase signal was observed in wells treated with SIS0012 or SIS0013 at a 1:24 weight ratio (w / w) (mRNA:total lipids), and the signal from the Si-doped SIS0013 complex was more pronounced than the signal from the undoped Si-doped SIS0012 complex. At a 1:12 weight ratio (w / w) (mRNA:total lipids), SIS0013 also showed a signal for mRNA2. Compared to mRNA1, the chemical modification of mRNA2 is reduced, which explains why, for the same complex SIS0013, cell transfection with mRNA2 appears to be slightly more efficient than with mRNA1.

[0385] Table 7- Figure 5 Summary of results shown 6 hours after transfection

[0386]

[0387] 24 hours after transfection: Figure 6 As shown in Table 8, 24 hours after transfection, luminescent signals of SIS0013 complexed with mRNA1 or mRNA2 were observed at a ratio of 1:24 and 1:12. A signal of SIS0013 complexed with mRNA2 was also seen at a ratio of 1:7.2.

[0388] In contrast, at a ratio of 1:24, SIS0012 showed signals with either mRNA1 or mRNA2, and at a ratio of 1:12, only with mRNA2.

[0389] Table 8- Figure 6 Summary of results shown 24 hours after transfection

[0390]

[0391] 48 hours after transfection: Figure 7 Table 9 shows the detection results 48 hours after transfection.

[0392] Table 9- Figure 7 Summary of results shown 48 hours after transfection

[0393]

[0394] 72 hours after transfection: Figure 8 Table 10 shows the detection results 72 hours after transfection.

[0395] Table 10- Figure 8 Summary of results shown 72 hours after transfection

[0396]

[0397] Example 2 - SIS0013 variants and mRNA

[0398] Example 2 discloses a study on the effect of functionalizing the Si-doped SIS0013 of Example 1 with different components.

[0399] Materials and methods

[0400] Unless otherwise specified, the materials and methods used were the same as in Example 1.

[0401] SIS0013 variants

[0402] 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 additional components (NAD, TYR, and QUE were obtained from Sigma Aldrich). NAD, TYR, or QUE was added along with the Si nanoparticles in step (a) of the "Film Rehydration" protocol described in the "Materials and Methods" section of Example 1 above.

[0403] result

[0404] Dynamic and electrophoretic light scattering results

[0405] The surface charge of SIS0013-N, SIS0013-T, and SIS0013-Q complexed with LUC mRNA1 was studied in the absence of mRNA and at weight ratios of mRNA to total lipids (w / w) of 24:1, 12:1, 7.2:1, and 4:1. The results are shown in Tables 11a-11c.

[0406] Table 11a - Properties of SIS0013-N complexed with LUC mRNA1 at different total lipid:mRNA ratios

[0407]

[0408] *While this is less positive than the SIS0012 of Example 1, it is believed to be more positive than that of undoped silicon particles.

[0409] Table 11b - Properties of SIS0013-T complexed with LUC mRNA1 at different total lipid:mRNA ratios

[0410]

[0411] *This is less positive than the value for SIS0013 of Example 1, but more positive than that for SIS0012; it is believed to be more positive than that for undoped silicon particles.

[0412] Table 11c - Properties of SIS0013-Q complexed with LUC mRNA1 at different total lipid:mRNA ratios

[0413]

[0414]

[0415] *This is similar to SIS0012 of Example 1, but is believed to be more positive than that of undoped silicon particles.

[0416] In general, particle diameter is within the range of 200-400nm; However, when the ratio of total lipid: mRNA is 7.2: 1, precipitation is observed during compounding, which causes particle diameter to increase. Without wishing to be bound by theory, it is believed that at a ratio of 7.2: 1, due to the charge interaction between negatively charged RNA and positively charged DOTAP, some undesirable lipid accumulation may exist. Once the formation of large lipid fragments reaches a critical point, precipitation may occur. It is believed that this effect can be regulated by subtle changes in Si doping and / or by providing counterions (e.g., from NaCl or KCl). In any case, this data point does not destroy the overall trend.

[0417] The zeta potential measurement results were consistent with the trend of SIS0013 in Example 1 above.

[0418] Gel retardation test results

[0419] Figure 9 Shown are the results of gel electrophoresis investigating the complexes of SIS0013-N, SIS0013-T, and SIS0013-Q with LUC mRNA 1. Naked mRNA was used as a reference control and a DNA ladder was used as a size guide.

[0420] Available mRNAQuant-iTRiboGreen assay results

[0421] Similar to Example 1, to assess how much mRNA was effectively bound to SIS0013-N, SIS0013-T, and SIS0013-Q and could not be accessed by external reactive substances, Quant-iT TM RiboGreen TM The amount of mRNA that can be captured by RNA fluorescent dyes serves as an indirect parameter to represent the amount of unbound or superficially bound mRNA after complexation.

[0422] Similar performance was observed as for SIS0013 in Example 1 above. In more detail: When the weight ratio of mRNA to total lipids (w / w) was 1:24, all formulations showed lower mRNA accessibility. When the weight ratio of mRNA to total lipids (w / w) was 1:12, 1:7.2, and 1:4, SIS0013-N, SIS0013-T, and SIS0013-Q showed lower mRNA accessibility, thus shielding external reactive substances better than SIS0013; the effect of SIS0013-T was observed to be more significant. These results are shown in Figure 2. Figure 10 shown.

[0423] Cell transfection results

[0424] Similar to Example 1, a series of in vitro transfection studies were performed in HEK293 cells to compare the performance of SIS0013, SIS0013-N, SIS0013-T, and SIS0013-Q. Cells were transfected with 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 / Luciferin (Luc mRNA1), and SIS0013-Q / Luciferase (Luc mRNA2).

[0425] A weight ratio (w / w) of mRNA to total lipids of 1:24 was selected for study. In Example 1, a 1:24 ratio was effective for SIS0013 at 6, 24, and 48 hours post-transfection, and thus is expected to provide a good basis for comparison between SIS0013 and its variants at 6, 24, or 48 hours post-transfection.

[0426] 6 hours, 24 hours or 48 hours after transfection, the translation products were detected by Bright-Glo luciferase assay using a luminometer device as described in Example 1.

[0427] At 6 hours post-transfection, all SIS0013 variants showed transfection in both LUC-mRNA models (1 and 2), with the signal of SIS0013 complexed with mRNA2 being the most obvious. Figure 11 shown.

[0428] Similar to the results observed in Example 1, higher luciferase activity was observed at 24 hours. Figure 12 shown.

[0429] At 48 hours, all SIS0013 variants showed transfection in both LUC-mRNA models (1 and 2), with SIS0013 and SIS013-T showing more pronounced signals. Figure 13 shown.

[0430] These results demonstrate that the advantageous effects of Si doping persist even when combined with diverse and disparate components; the properties and performance of NAD, QUE, and TYR differ significantly from one another.

[0431] To evaluate the statistical significance of the luminescence differences between SIS0013 and its variants, statistical analysis was performed on the time point of maximum luminescence intensity (i.e., 24 h).

[0432] The analysis was performed using the ROI method optimized by the software and by calculating the mean ± SD values ​​for each of the three variant series SIS0013, SIS0013-N, SIS0013-Q, and SIS0013-T complexed with mRNA. SIS0013 complexed with mRNA2 was set as the maximum value of the ROI, and then a one-way analysis of variance (ANOVA) was performed on the RM.

[0433] like Figures 14 to 16 As shown, statistical analysis confirmed that SIS0013 and SIS0013-T were the transfection formulations that resulted in the strongest luminescence, which may mean that they provide the most efficient delivery vectors. Notably, by functionalizing SIS0013 with different components (e.g., SIS0013-T, SIS0013-Q, and SIS0013-N), it appears that modulation of signal intensity can be achieved. Nevertheless, all formulations tested had satisfactory effects.

[0434] More specifically, Figure 14 Figure 2 shows the luminescence intensity of 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) 24 hours after transfection. Values ​​are reported as mean ± standard deviation (SD) (analysis was repeated three times). Adjacent histograms were plotted to allow for a head-to-head comparison of the transfection efficiency achieved with the LUC mRNA1 complex and the corresponding LUC mRNA2 complex.

[0435] Figure 15 Statistical analysis of the intensity of luminescence signals of SIS0013 and its variants complexed with mRNA1 is shown (RM one-way ANOVA with Geisser Greenhouse correction followed by Dunnett's multiple comparison test to calculate individual differences for each comparison; luminescence signal analysis at 24 hours, *p=0.05).

[0436] at the same time, Figure 16Statistical analysis of the intensity of luminescence signals of SIS0013 and its variants complexed with LUC mRNA2 is shown (RM one-way ANOVA with Geisser Greenhouse correction followed by Dunnett's multiple comparison test to calculate individual differences for each comparison; luminescence signal analysis at 24 hours, *p=0.05).

[0437] Example 3 - Cell viability studies

[0438] In this example, the cell viability of cells transfected with mRNA1 or LUC mRNA2 using SIS0013 and its variants SIS0013-N, SIS0013-T, and SIS0013-Q was studied. Cell cytotoxicity was monitored and 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.

[0439] Materials and methods

[0440] Unless otherwise specified, the materials and methods used were the same as in Example 1.

[0441] The time points after transfection studied (6 h, 24 h, 48 h, 72 h) and the weight ratios (w / w) of mRNA:lipid (1:4, 1:7.2, 1:12, 1:24) were the same as in Example 1.

[0442] Dharmafect 1 and Lipofectamine 3000

[0443] The availability of commercially available transfection reagents DharmaFect 1 and Lipofectamine 3000 is shown in Table 12.

[0444] Table 12 - DharmaFect 1 and Lipofectamine 3000

[0445] Reagents supplier Supply Number DharmaFect 1 Horizon Dharmacon T-2001-03 Lipofectamine 3000 Invitrogen L3000-008

[0446] result

[0447] Cell transfection results

[0448] Under otherwise identical conditions, including the weight (w / w) ratio of mRNA to total lipids, DharmaFect 1 and Lipofectamine 3000 exhibited cytotoxicity 24 hours after transfection (as evidenced by increased LDH and Caspase 3 / 7 signals, and altered morphology when cells were analyzed under a microscope), whereas SIS0013, SIS0013-T, SIS0013-Q, and SIS0013-N formulations showed no alterations in cell morphology or viability at any time point analyzed.

[0449] This suggests that SIS0013, SIS0013-T, SIS0013-Q, and SIS0013-N have improved cell safety compared to conventional transfection reagents such as DharmaFect 1 and Lipofectamine 3000.

[0450] Example 4 - In vivo studies of osteosclerosis type II (ADO2)

[0451] ADO2 is an inherited osteosclerotic bone disease caused by dysfunctional osteoclast activity. ADO2 is caused by missense mutations in the chloride channel 7 (CLCn7) gene and is characterized by osteosclerosis with multiple fractures. ADO2 can lead to osteomyelitis, visual impairment (because osteosclerosis can lead to obstruction of the macular hole, thereby compressing the optic nerve), and bone marrow failure (Alam et al., 2017, Bone, 94:34-41). Currently, there is no cure for ADO2.

[0452] In this example, SIS0012 or SIS0013 was used as an siRNA delivery platform to investigate the silencing of ClCn7G213R in ADO2 mice using a mouse model. The results between SIS0012 and SIS0013 were compared.

[0453] Materials and methods

[0454] The same protocol was used as in Example 1. siRNA was used instead of the mRNA of Example 1 and loading was performed following a similar protocol as shown for the mRNA in Example 1 above.

[0455] ClCn7G213R-specific siRNA

[0456] ClCn7G213R-specific siRNA was obtained as described in Capulli et al., 2015, Clin. Molec. Therap. 4, e248 (incorporated herein by reference in its entirety).

[0457] The naming convention of the constructs used is shown in Table 13.

[0458] Table 13 - Constructs used in Example 4

[0459]

[0460] ADO2 mutant mice (10 days old) were obtained as described in Alam et al., 2017, Bone, 94:34-41 (incorporated herein by reference in its entirety). Mice were injected intraperitoneally with one of the four constructs shown in Table 13 (n = 5 mice per group). Injections were repeated three times per week for a total of 2 weeks.

[0461] result

[0462] For each treatment group, the expression of ClCn7G213R in mouse PMBCs was detected; the results are shown in Figure 2. Figure 17 As shown. At the same time, Figure 18 Shown is bone expression of ClCn7G213R in mouse femur. Figure 19 The results of the CTX blood test are shown (CTX is a marker of bone turnover). The results are summarized in Table 14.

[0463] Table 14 - ADO2 mouse study results

[0464]

[0465]

[0466] Both ADO2+SIS0012-siRNA (undoped with Si) and ADO2+SIS0013-siRNA (doped with Si) resulted in statistically significant downregulation of ClCn7G213R (p<0.02).

[0467] However, surprisingly, administration of ADO2+SIS0013-siRNA (doped with Si) additionally resulted in accelerated bone turnover compared to ADO2+SIS0012-siRNA (undoped with Si) ( Figure 19 , CTX assessment). Therefore, compared with the undoped Si-containing delivery vehicle, the doped Si-containing delivery vehicle seemed to be able to more strongly alleviate the symptoms of ADO2 treated with siRNA.

[0468] Example 5 - Effects of SIS0012 and SIS0013 on the stability of alkaline phosphatase

[0469] Example 5 investigated the stabilizing effects of SIS0012 (containing undoped Si) and SIS0013 (containing doped Si) on pH- and temperature-sensitive protein APIs (i.e., relatively fragile APIs, but with different properties from the mRNA and siRNA studied in the previous examples).

[0470] The API under investigation is alkaline phosphatase, an enzyme that exists in multiple forms and catalyzes the degradation of various proteins and can be found in all tissues of the human body. Alkaline phosphatase significantly loses its activity at low pH and high temperature.

[0471] Materials and methods

[0472] The same protocol as in Example 1 was used, except that alkaline phosphatase was used as the API instead of the mRNA in Example 1, and loading was performed according to the following protocol.

[0473] Alkaline phosphatase, isolated from calf small intestine, was provided as a 56 kD recombinant enzyme expressed in Pichia pastoris and obtained from Sigma Aldrich / Merck (The Old Brickyard, New Rd, Gillingham, Dorset, SP8 4XT). A stock solution of alkaline phosphatase (ALP) was prepared in water at a concentration of 1 U / ml. 1 U (μmol / min) is defined as the amount of ALP required to catalyze the conversion of one micromole of PNPP per minute at 37°C and pH 7.4.

[0474] A 20 mM solution of 4-nitrophenyl phosphatase (PNPP) was prepared using Tris buffer (100 mM / L) at pH 7.4.

[0475] ALP solutions were prepared from a 1 U / ml stock solution at the following concentrations: 0.1, 0.5, 1, 5, 10, 50, and 100 mU / ml, and placed in 15 ml test tubes. These were then mixed with the prepared 20 mM PNPP Tris buffer solution in an Eppendorf tube. The tubes were incubated in a 37°C water bath for 30 minutes, and then UV-Vis absorbance was measured at 405 nm, as shown in Figure 2. Figure 20 shown.

[0476] Load ALP to SIS0012 and SIS0013 as follows:

[0477] 1. Prepare 3 sets of 8 microcentrifuge tubes:

[0478] A. Prepare 8 microcentrifuge tubes, each containing 50 μL ALP (50 mU / ml) and 500 μL SIS0012 (undoped with Si). After adding these components to the microcentrifuge tubes, mix them, vortex them, and refrigerate them overnight.

[0479] B. Prepare 8 microcentrifuge tubes, each containing 50 μL ALP (50 mU / ml) and 500 μL SIS0013 (boron-doped Si). After adding these components to the microcentrifuge tubes, mix them, vortex them, and refrigerate them overnight.

[0480] C. Prepare 8 microcentrifuge tubes, each containing 50 μL of ALP (50 mU / ml) and 500 μL of Tris buffer. Add these components to the microcentrifuge tubes, mix well, vortex, and refrigerate overnight.

[0481] 2. After preparation, place all microcentrifuge tubes in a 50°C water bath. After 1, 2, 5, 10, 20, 40, or 60 minutes, remove eight microcentrifuge tubes from each of the three sets of microcentrifuge tubes (A to C) from the water bath.

[0482] 3. Next, add 300 μL of PNPP to all microcentrifuge tubes in groups A through C. Mix and vortex the tubes. Then, place them in a 37°C water bath for 30 minutes to allow PNPP to dephosphorylate.

[0483] 4. Afterwards, all samples from groups A to C were subjected to UV-Vis analysis (at 405 nm). The results are as follows: Figure 21 More specifically, alkaline phosphatase activity is monitored by measuring changes in the concentration of its substrate, 4-nitrophenyl phosphatase (PNPP), using ultraviolet-visible (UV-Vis) absorbance as an indicator. The structure of the substrate is shown below. Higher PNPP concentrations indicate lower alkaline phosphatase activity, indicating greater degradation of the protein API and, therefore, poorer stabilization.

[0484]

[0485] result

[0486] Free alkaline phosphatase showed significant degradation compared to both SIS0012 and SIS0013. At 50°C, the activity of free alkaline phosphatase decreased with increasing incubation time.

[0487] Surprisingly, the activity of alkaline phosphatase loaded on SIS0013 (doped with Si) was about 20-30% higher than that loaded on SIS0012. This is believed to be because doping with Si improves the stability of alkaline phosphatase against degradation compared to undoped Si.

[0488] Example 6 - SIS0013 variants and siRNA

[0489] Example 6 studied the effects of different functionalizations of SIS0013 on its function as a siRNA delivery vector.

[0490] Materials and methods

[0491] Unless otherwise stated, the materials and methods used were the same as in Example 2. siRNA was used instead of the mRNA of Example 2 and loading was performed following a similar protocol as shown for the mRNA in Example 1 above.

[0492] Complexation of SIS0013-N, SIS0013-Q, and SIS0013-T with siRNA specific for ClCn7G213R and with dTdT overhangs (obtained as described in Capulli et al., 2015, Clin. Molec. Therap. 4, e248) was studied.

[0493] result

[0494] Figure 22 The gel electrophoresis results shown indicate that siRNA was successfully and completely incorporated into these formulations.

[0495] Dynamic light scattering measurements were also performed to assess particle size and charge using a Zetasizer (available from Malvern Instruments) before and after siRNA complex formation, as shown in the table below. As shown in the table, an increase in particle size was observed after siRNA complexation, accompanied by a 10-15 mV decrease in surface charge.

[0496] Table 15 - Particle size, PDI, and zeta potential of SIS0013-N, SIS0013-Q, and SIS0013-T before and after complexing with siRNA

[0497]

[0498] Materials and Methods—Continued

[0499] The removal of DOTAP from compositions SIS0013-N, SIS0013-T, and SIS0013-Q containing NAD, TYR, or QUE was investigated to determine how the doped Si-containing compositions would perform after further changes to the composition components other than the dopant Si.

[0500] Therefore, DPPC / DOPE formulations were prepared according to a similar protocol as in Examples 1 and 2, except that DOTAP was removed from the total lipids. Experiments were performed using either (i) 0.2 mg or (ii) 1 mg of NAD, TYR, and QUE. The formulations are shown in Tables 16 to 18 below.

[0501] Table 16 - Composition of DPPC / DOPE SIS0013 functionalized with beta nicotinamide adenine dinucleotide (NAD).

[0502]

[0503] Table 17 - Composition of DPPC / DOPE SIS0013 functionalized with quercetin (QUE).

[0504]

[0505] Table 18 - Composition of DPPC / DOPE SIS0013 functionalized with tyrosine (TYR)

[0506]

[0507]

[0508] Results - continued

[0509] Zetasizer measurements were obtained showing negative zeta potential for all formulations regardless of the amount of NAD, TYR, or QUE (0.2 mg or 1 mg), as shown in Table 19 below.

[0510] Table 19 - DPPC / DOPE SIS0013 functionalized with 0.2 mg or 1 mg NAD, TYR, and QUE.

[0511] sample Particle size PDI ζ-no load DPPC / DOPE-NAD-0.2mg 78.46±2.65 0.18±0.04 -20.76±0.68 DPPC / DOPE-NAD-1mg 88.46±1.22 0.16±0.05 -25.75±0.57 DPPC / DOPE-QUE-0.2mg 86.0±0.63 0.17±0.02 -25.81±0.82 DPPC / DOPE-QUE-1mg 78.35±2.19 0.18±0.02 -20.2±1.43 DPPC / DOPE-TYR-0.2mg 84.16±2.33 0.19±0.03 -24.99±0.44 DPPC / DOPE-TYR-1mg 83.21±2.57 0.16±0.03 -18.81±0.46

[0512] It is believed that the differently functionalized compositions in Example 6 may exhibit similar API stability and delivery performance as SIS0013.

[0513] Example 7 - Lipopeptide Components

[0514] Lipopeptides, also known as amphiphilic peptides (PAs), have been developed as another alternative component of compositions containing doped silica particles.

[0515] It is believed that lipopeptides may provide a solution to the problem of replacing or reducing the amount of cationic lipids in transfection compositions. Lipopeptides consist of an alkyl chain coupled to a peptide sequence. It is believed that their alkyl chains can be incorporated into the lipid bilayer, which then becomes surface-modified with the peptide moiety.

[0516] An exemplary PA is the palmitoyl pentapeptide-4 molecule (abbreviated as PAL-KTTKS). The two cationic lysine residues can perform a similar function to cationic lipids (eg, DOTAP), exhibiting electrostatic interactions with negatively charged APIs (eg, RNA).

[0517] Materials and methods

[0518] DPPC and DOPE were selected as neutral lipids to formulate with PAL-KTTKS.

[0519] Table 20 below provides details of formulations containing DPPC, DOPE, and PAL-KTTKS.

[0520] Table 20 - DPPC, DOPE, and PAL-KTTKS with Boron-Doped Silicon Nanoparticles

[0521]

[0522] This formulation had a positive zeta potential of 54.38 ± 2.11 (measured using a Zetasizer from Malvern Instruments). Although this potential was lower than that of SIS0012 from Example 1, it was believed to be more positive than that of undoped silicon particles.

[0523] It is believed that during lipid membrane assembly, PAL-KTTKS arranges itself within the lipid bilayer, exposing the peptide to the nanoparticle surface. Furthermore, lysine residues on this surface can contribute positive charges to the formulation.

[0524] The electrostatic binding ability of the formulations to ClCn7G213R-specific siRNA with dTdT overhangs (obtained as described in Capulli et al., 2015, Clin. Molec. Therap. 4, e248) was evaluated.

[0525] result

[0526] Gel electrophoresis analysis was performed. Complete complexation of the siRNA was not observed. Figure 23 Well 3 shows the gel electrophoresis results of siRNA.

[0527] Complete complexation of the mRNA was observed. Figure 24 Well 3 shows the gel electrophoresis results of mRNA.

[0528] To address the issue of partial complexation of siRNA with DPPC / DOPE / PAL-KTTKS, an alternative loading method was employed. Compared to the previously described protocol, the following steps were employed in this alternative loading method:

[0529] 1. A thin lipid film was prepared by dissolving DPPC, DOPE and Pal-KTTKS in methanol and evaporating using a rotary evaporator.

[0530] 2. Rehydrate the lipid film with a suspension containing boron-doped silica, trehalose, glycine, and siRNA or mRNA. Rehydrate at 40°C for 10 minutes to ensure that no residual lipid-silica film remains on the walls of the round-bottom rotary evaporator.

[0531] Figure 25 Well 4 shows gel electrophoresis of the complex after using the alternative loading method, indicating successful and complete complexation of the siRNA.

[0532] Lipopeptides are versatile molecules that can be fine-tuned by varying their alkyl chains and / or peptide sequences. It is believed that tailoring the peptide sequence can enhance cell and / or tissue targeting. In the field of gene therapy, peptide customization can enhance electrostatic interactions with nucleic acids (e.g., RNA, particularly mRNA). For example, when PAL-KTTKS is formulated with DPPC and DOPE, the PAL-KTTKS surface exhibits a positive charge, as confirmed by zeta potential.

[0533] At the same time, lipopeptides, as amphiphilic molecules, have properties very similar to those of surfactants and can self-assemble to form micelles; this is believed to be at least in part due to the alkyl chains' tendency to undergo hydrophobic interactions, while the peptide sequences can form intermolecular hydrogen bonds. Phospholipids, such as DPPC and DOPE, are also capable of self-assembling into liposomes. Therefore, when PA is introduced (of which PAL-KTTKS is a representative example, although other lipopeptides can also be used), the alkyl chains are able to form hydrophobic interactions with DPPC and DOPE, thereby forming a liposomal structure.

[0534] At the same time, silicon nanoparticles provide structural stability to the entire complex. As shown in the previous examples, Si-doped particles can interact with lipids (including lipopeptides) and other substances (such as NAD, QUE or TYR) through non-covalent (electrostatic) interactions, thereby promoting long-term stability and effective delivery of APIs.

[0535] Example 8

[0536] Example 8 compares the transfection of SIS0012 and SIS0013 in another cell line, L6C5, a commercially available musculoskeletal mouse cell line.

[0537] Materials and methods

[0538] Cells were transfected with SIS0012 / luciferase (Luc mRNA1), SIS0012 / luciferase (Luc mRNA2), SIS0013 / luciferase (Luc mRNA1), or SIS0013 / luciferase (Luc mRNA2) similar to Example 1. 24 hours after transfection, translation products were detected by the Bright-Glo luciferase assay using a luminometer device as described herein.

[0539] result

[0540] The results are as follows Figure 26 When SIS0013 was used, the luciferase activity of both mRNA1 and mRNA2 was higher than that when SIS0012 was used. This indicates that the transfection efficiency of SIS0013 was improved compared to SIS0012.

[0541] ***

[0542] If features or limitations mentioned in the previous description have equivalents that are known, clear or foreseeable to those skilled in the art in light of this disclosure, such equivalents are incorporated herein as if specifically set forth. The scope of the subject matter of the present disclosure should be determined primarily with reference to the claims. The scope of protection sought by this application also includes any such equivalents. It will also be understood by those skilled in the art that, unless expressly stated otherwise, features or limitations of the disclosed subject matter that are described as preferred, suitable, advantageous, convenient, etc. may be optional and should not limit the scope of the independent claims or the scope of protection sought. Furthermore, it will be understood that such optional features or limitations, while potentially beneficial in some implementations of the disclosed subject matter, may be undesirable in other implementations and therefore may not be present or omitted.

Claims

1. A composition comprising: (i) particles comprising hydrolyzable doped silicon; (ii) one or more lipids; as well as (iii) Active pharmaceutical ingredient (API).

2. The composition of claim 1 , wherein the particles comprising hydrolyzable doped silicon differ in zeta potential compared to otherwise identical particles comprising hydrolyzable undoped silicon, thereby attracting, binding and / or stabilizing the API.

3. A composition according to claim 1 or claim 2, wherein the dopant of the particles is or comprises a p-dopant, in particular boron.

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

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

4.

6. A composition according to claim 1 or claim 2, wherein the dopant of the particles is or comprises an n-dopant, in particular phosphorus.

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

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

4.

9. The composition of any one of the preceding claims, wherein the API is neutral or zwitterionic at a pH of about 7.

4.

10. The composition of any one of the preceding claims, wherein the hydrolyzable doped silicon particles have a doping level of at least about 1 x 10 16 dopant atoms / cm 3 .

11. The composition of any preceding claim, wherein the hydrolyzable doped silicon particles have a doping level of at most about 1 x 10 20 dopant atoms / cm 3 .

12. The composition of any preceding claim, wherein the API is or comprises a nucleic acid.

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

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

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

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

17. A composition according to any one of the preceding claims, wherein the API is or comprises a protein.

18. The composition of any preceding claim, wherein the composition further comprises an amino acid.

19. The composition of any preceding claim, wherein the composition comprises tyrosine.

20. The composition of claim 19, wherein the tyrosine is present together with or in place of the amino acid of claim 18.

21. The composition of any preceding claim, wherein the composition further comprises a non-reducing disaccharide, such as trehalose.

22. The composition of any preceding claim, wherein the composition further comprises NAD.

23. The composition of any preceding claim, wherein the composition further comprises quercetin.

24. The composition of any one of the preceding claims, wherein the one or more lipids comprise an ionizable lipid.

25. The composition of claim 24, wherein the one or more lipids comprise lipids with a net positive charge at pH 7.

4.

26. The composition of any one of the preceding claims, wherein the one or more lipids comprise one or more lipidated oligopeptides.

27. The composition of claim 26, wherein the one or more lipidated oligopeptides each comprise an oligopeptide portion having from about 3 to about 20 amino acid residues and a fatty acid chain having from about 12 to about 18 carbon atoms, preferably wherein at least one of the amino acid residues is positively charged at a pH of about 7.

4.

28. A composition according to any preceding claim, wherein the particles prevent or reduce degradation, particularly enzymatic degradation, of the API.

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

30. The composition of any preceding claim, wherein the particles increase the in vivo half-life of the API.

31. The composition of any preceding claim, wherein the particles provide increased intracellular stability of the API.

32. The composition of any preceding claim, wherein the particles provide increased stability of the API at about 25°C.

33. A composition according to any preceding claim, wherein the dopant of the particles is or comprises an interstitial dopant.

34. A composition according to any preceding claim, wherein the dopant of the particles is or comprises a substitutional dopant.

35. A composition according to any preceding claim, wherein the dopant of the particles is or comprises a dopant located on the surface of the particles.

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

37. A composition according to any preceding claim comprising one or more aggregates of said particles comprising hydrolysable doped silicon.

38. The composition of claim 37, wherein in the one or more aggregates, the particles comprising hydrolyzable doped silicon have an average diameter in the range of about 1 nm to about 50 nm.

39. A composition according to claim 37 or 38, wherein the one or more aggregates comprise chains of particles comprising hydrolyzable doped silicon.

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

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

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

43. A composition according to any preceding claim for use in a method of preventing or treating a disease or condition in a human subject.

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

45. A composition for use according to claim 43 or claim 44, wherein the disease or condition is an infectious disease.

46. ​​A composition for use according to claim 43 or claim 44, wherein the disease or condition is a genetic disease or condition.

47. A composition for use according to any one of claims 43 to 46, wherein the human subject is in the range of about 1 month or older, optionally about 1 year or older.

48. The composition for use according to any one of claims 43 to 47, wherein the method comprises administering the composition to the human subject by injection, oral administration, transdermal administration or intranasal administration, in particular by injection or oral administration.

49. The composition for use 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 composition for use according to claim 49, wherein the hydrolyzable doped silicon particles have a doping level of at least about 1 x 10 16 dopant atoms / cm 3 , and the method comprises administering the composition to the human subject by injection, orally or intranasally.

51. The composition for use according to any one of claims 43 to 50, wherein the method comprises the step of storing the composition comprising the API at a temperature range of about 0°C or above for 1 week or longer prior to administering the composition to the subject.

52. A method for slowing down API degradation, comprising: (i) doping particles comprising hydrolyzable silicon such that their zeta potential is altered compared to otherwise identical undoped particles; as well as (ii) contacting the doped particles with one or more lipids and the API.

53. The method of claim 52, wherein step (i) comprises doping the particles with a p-dopant to impart a more positive zeta potential than otherwise identical undoped particles.

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

55. The method of claim 52, wherein step (i) comprises doping the particles with an n-dopant to impart a more negative zeta potential than an otherwise identical undoped particle.

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

4.

57. A method of preventing or treating a disease or condition comprising administering to a human subject in need thereof a prophylactically or therapeutically effective amount of a composition as defined in any one of claims 1 to 51.

58. A method according to claim 57 having one or more features of the method as defined in 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 medicament for use in a method as defined in any one of claims 52 to 58.

60. A human cell transfection composition having one or more features of a composition as defined in any one of claims 1 to 51.

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