Therapeutic compositions and methods

Abstract

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

Description

[0001] Therapeutic compositions and methods

[0002] Field of the Disclosure

[0003] The present disclosure concerns delivery vehicles for active pharmaceutical ingredients (“APIs”). More particularly, but not exclusively, this disclosure concerns a composition, comprising: particles comprising hydrolysable doped silicon; one or more lipids; and an API. The disclosure also concerns related products, medical uses, and methods.

[0004] Background

[0005] A need exists for improvements in vehicles for API delivery. This is required for advances in biomedical research to be fully translated into efficient, safe and cost- effective treatments.

[0006] Recent biological advances have provided insight into new classes of API for the prophylaxis and treatment of many diseases. One example of such a class is nucleic acids. By way of example, a variety of mRNA-based infectious disease vaccines have recently come to light. Messenger RNA (mRNA) -based cancer therapies have also now entered clinical development. Meanwhile, other types of RNA including siRNA, tRNA and oRNA (circular RNA) shows promise for the treatment of various genetic diseases and disorders.

[0007] However, with the promise of new classes of API comes the challenge of how to control API stability during storage. It must also be ensured that APIs actually reach and are taken up by cells once they have been administered to a patient; it may be challenging to stabilise APIs while they are circulating in the body. Moreover there exists a need for tissue or cell targeting, so that an API can be delivered to the correct cells. Once a target cell is reached, then if the API is intended for delivery into the cell (rather than, for example, an API intended to act outside a cell, such as on a cell surface protein) there is also the challenge of how to ensure efficient API uptake by cells. Following such uptake, there is a need to ensure API stability in the cytoplasm.

[0008] In one approach, lipid nanoparticles have been used for the in vivo delivery of fragile APIs such as nucleic acids and especially mRNA. (As used herein, the terms “fragile APIs” and “reactive APIs” may be interchangeable and may refer to APIs which (i) have a half-life upon storage in aqueous solution at about 25 °C of at most one week, measurable by NMR or by GC-MS; and / or (ii) have a half-life in vivo of under about an hour, measurable by assay of a biological sample.) Cold-chain storage is required for API-containing lipid nanoparticles, which limits their distribution and is not energyefficient or cost-effective. There remains a need for delivery vehicles with an increased capability for stabilising fragile APIs, both during storage (so that they can be stored at higher temperatures) and while they are circulating in the body (so that more API reaches target cells more quickly).

[0009] Meanwhile, some lipid nanoparticle-based delivery vehicles may display cytotoxicity (see Example 3 hereinbelow). Commonly used lipid nanoparticles rely on exogeneous cationic lipid(s) (i.e., lipid(s) which has or have a net positive charge at a pH of about 7.4) containing multiple amine groups. Although well-suited to the electrostatic loading of polyanionic nucleic acids (including various form of RNA) these amine-rich species may lead to cytotoxicity, immunogenicity, and non-specific tissue accumulation. Similar problems occur with other, polymer-based, amine-rich delivery systems. There exists a need for improved delivery vehicles which also have an acceptably low toxicity.

[0010] Preferably, new delivery vehicles would also be fully dispersible in an aqueous environment, to ensure ease of delivery, such as by injection in aqueous solution.

[0011] The present disclosure seeks to mitigate the above-mentioned problems. Alternatively or additionally, the present disclosure seeks to provide an improved API delivery vehicle.

[0012] Summary of the Disclosure

[0013] In a first aspect of the present disclosure, there is provided a composition comprising particles comprising hydrolysable doped silicon (especially, hydrolysable silicon which is doped at a level of at least 1 xlO16dopant atoms per cm3), one or more lipids and an API. In a second aspect of the present disclosure, there is provided a composition defined in accordance with the first aspect of the disclosure, for use in a method of preventing or treating a disease or disorder in a human subject.

[0014] In a third aspect, disclosed herein is a method for slowing the degradation of an API, comprising: doping particles comprising hydrolysable silicon to change their zeta potential compared to otherwise identical undoped particles; and contacting the doped particles with one or more lipids and the API.

[0015] In a fourth aspect of the present disclosure, there is provided is a method of preventing or treating a disease or disorder, comprising administering a prophylactically or therapeutically effective amount of a composition defined in accordance with one or both of the first and second aspects of the present disclosure to a human subject in need thereof.

[0016] In a fifth aspect, disclosed herein is the use of a composition defined in accordance with one or both of the first and second aspects of the present disclosure in the manufacture of a medicament for use in a method defined in accordance with one or both of the second and third aspects of the present disclosure.

[0017] In a sixth aspect, disclosed herein is human cell transfection composition, having one or more of the features of a composition defined in accordance with one or both of the first and second aspects of the present disclosure.

[0018] Description of the Drawings

[0019] Embodiments of the present disclosure are now described by way of example only with reference to the accompanying drawings in which:

[0020] Figures 1 and 2 display size and poly dispersity index (PDI) data for undoped

[0021] (SIS0012) and doped (SIS0013) Si-containing delivery vehicles complexed with mRNA. Figure 3 displays gel electrophoresis data exploring the complexation of undoped (SIS0012) and doped (SISOO13) Si-containing delivery vehicles with mRNA.

[0022] Figure 4 displays data on the accessibility to an external reagent of mRNA in undoped (SIS0012) and doped (SISOO13) Si-containing delivery vehicles.

[0023] Figure 5 displays the luminescence observed at a time point of 6 hours after administration of mRNA, using undoped (SIS0012) or doped (SISOO13) Si-containing delivery vehicles, to HEK293 cells.

[0024] Figure 6 displays the luminescence observed at a time point of 24 hours after administration of mRNA, using undoped (SIS0012) or doped (SIS0013) Si-containing delivery vehicles, to HEK293 cells.

[0025] Figure 7 displays the luminescence observed at a time point of 48 hours after administration of mRNA, using undoped (SIS0012) or doped (SIS0013) Si-containing delivery vehicles, to HEK293 cells.

[0026] Figure 8 displays the luminescence observed at a time point of 72 hours after administration of mRNA, using undoped (SIS0012) or doped (SIS0013) Si-containing delivery vehicles, to HEK293 cells.

[0027] Figure 9 displays gel electrophoresis data exploring the complexation of various doped Si-containing delivery vehicles (SIS0013-N, -Q and -T) with mRNA.

[0028] Figure 10 displays data on the accessibility to an external reagent of mRNA in various doped Si-containing delivery vehicles (SIS0013-N, -Q and -T).

[0029] Figure 11 displays the luminescence observed at a time point of 6 hours after administration of mRNA, using various doped Si-containing delivery vehicles (SIS0013-N, -Q and -T), to HEK293 cells. Figure 12 displays the luminescence observed at a time point of 24 hours after administration of mRNA, using various doped Si-containing delivery vehicles (SIS0013-N, -Q and -T), to HEK293 cells.

[0030] Figure 13 displays the luminescence observed at a time point of 48 hours after administration of mRNA, using various doped Si-containing delivery vehicles (SIS0013-N, -Q and -T), to HEK293 cells.

[0031] Figures 14 to 16 display statistical analysis of the luminescence data of Figures 11 to 13.

[0032] Figure 17 displays ClCn7G213R expression in PMBCs of mice treated with undoped (SIS0012) or doped (SIS0013) Si-containing delivery vehicles loaded with siRNA.

[0033] Figure 18 displays expression of ClCn7G213R in bone (femur) cells of mice treated with undoped (SIS0012) or doped (SIS0013) Si-containing delivery vehicles loaded with siRNA.

[0034] Figure 19 displays CTX blood test results of mice treated with undoped (SIS0012) or doped (SIS0013) Si-containing delivery vehicles loaded with siRNA.

[0035] Figure 20 displays UV-vis absorbance data for alkaline phosphatase under the conditions described in Example 5, which investigates the stabilisation of this pH- and temperature-sensitive protein with a doped Si-containing delivery vehicle.

[0036] Figure 21 displays further UV-vis absorbance data for alkaline phosphatase, complementing Figure 20.

[0037] Figure 22 displays gel electrophoresis data exploring the complexation of various doped Si-containing delivery vehicles (SIS0013-N, -Q and -T) with siRNA. Figure 23 displays gel electrophoresis data exploring the complexation of a DPPC, DOPE and PAL-KTTKS-containing doped Si-containing delivery vehicle with siRNA.

[0038] Figure 24 displays gel electrophoresis data exploring the complexation of a DPPC, DOPE and PAL-KTTKS-containing doped Si-containing delivery vehicle with mRNA.

[0039] Figure 25 displays further gel electrophoresis data exploring the complexation of a DPPC, DOPE and PAL-KTTKS-containing doped Si-containing delivery vehicle with siRNA, when the delivery vehicle / siRNA complex is prepared according to a different method, compared to Figure 23.

[0040] Figure 26 shows luciferase assay results for Example 8, wherein luciferase activity was higher when SIS0013 (doped Si) was used, for both mRNAl and mRNA2, than when SIS0012 (undoped Si) was used.

[0041] Figure 27 is a transmission electron microscope (TEM) image that shows the aggregation of silicon particles, as described herein.

[0042] Detailed Description

[0043] While the subject-matter of the present disclosure is described and illustrated below with reference to particular embodiments, it will be appreciated by those skilled in the art that the subject-matter lends itself to many different variations not specifically illustrated herein. By way of example only, certain possible variations of the present disclosure in all its aspects will now be described.

[0044] Particles comprising hydrolysable doped silicon

[0045] The particles comprising hydrolysable doped silicon may be pure or substantially pure doped silicon. The particles may be another hydrolysable doped silicon-containing material. If the particles are not pure doped silicon, they comprise at least about 50% by weight silicon, i.e. they comprise at least about 50% by weight silicon atoms, based on the total mass of atoms in the particles. For example, the silicon particles may contain at least about 60, about 70, about 80, about 90 or about 95 % by weight silicon. The particles may show a rate of hydrolysis, for example in PBS buffer at room temperature, of at least 10% of the rate of hydrolysis of pure silicon particles of the same dimensions. Assays for hydrolysis of silicon-containing material are widely known in the art; see, for example, WO 2011 / 001456, incorporated herein by reference in its entirety.

[0046] Although the particles may contain traces of silica, silica is not hydrolysable silicon. At least about half of the silicon atoms in the particles may be in the form of elemental silicon (or doped elemental silicon).

[0047] The particles may, especially, be nanoparticles. The nanoparticles may have a mean diameter in a range of about 1 nm to about 500 nm, especially about 1 nm to about 250 nm, more especially about 1 nm to about 100 nm, preferably about 1 nm to about 50 nm, especially about 3 nm to about 50 nm, more especially about 3 nm to about 30 nm, (such as about 10 nm).

[0048] A particularly preferred range for the nanoparticles’ mean diameter is about 1 nm to about 30 nm. This may have advantages in terms of the nanoparticles’ aggregation, as described hereinbelow.

[0049] The dimensions of nanoparticulate objects, incliding the particles’ mean diameter, may be measured, for example, by scanning electron microscopy (SEM) or by transmission electron microscopy (TEM).

[0050] The particles may be porous, especially mesoporous. Particles comprising hydrolysable doped silicon can be made porous by standard techniques such as contacting the particles with a hydrofluoric acid (HF) / ethanol mixture and applying a current (“HF etching”). By varying the HF concentration and the current density and time of exposure, the density of pores and their size can be controlled and can be monitored by scanning electron micrography and / or nitrogen adsorption desorption volumetric isothermic measurement. If the particles are porous, their total surface area will be increased by virtue of their porosity. For example, their surface area may be increased by at least about 50 % or at least about 100 %, compared to the surface area of a corresponding non-porous particle. In many circumstances, porous particles will in reality have a much greater increase in total surface area by virtue of their porosity. According to certain embodiments the porosity is at least about 30, about 40, about 50 or about 60 %; meaning that, respectively, at least about 30, about 40, about 50 or about 60 % of the particle volume is pore space.

[0051] Average pore diameter may be in a range of from about 0.1 nm to about 10 nm, such as from about 0.1 nm to about 3 nm, e.g. about 2 nm.

[0052] It will be appreciated that the particles may be produced by various techniques familiar to the skilled person.

[0053] The techniques may include, for example, purely physical (sometimes referred to, in the art, as “non-wef ’) processes having bulk silicon (especially, silicon wafer) as the starting material; such as pulsed laser ablation, thermal degradation and ball milling. Thus, the particles may be obtainable by a method comprising or consisting of one or more of pulsed laser ablation, thermal degradation and ball milling, of bulk silicon (especially, silicon wafer).

[0054] Additionally or alternatively, the particles may be produced by chemical (sometimes referred to, in the art, as “wet”) techniques, including but not limited to electrochemical etching of bulk silicon (especially, silicon wafer). Such techniques optionally include the HF etching described above. Thus, the particles may be obtainable by a method comprising or consisting of electrochemical etching of bulk silicon (especially, silicon wafer).

[0055] Once formed, silicon particles may be sorted by size, such, for example, as by air classification, sieving and / or filtration. Thus, the particles may be obtainable by a method comprising one or more of air classification, sieving and / or filtration. Thus, for example, the particles may be obtainable by a method comprising producing silicon particles from bulk silicon, especially from silicon wafer, such as by one or more of pulsed laser ablation, thermal degradation, ball milling, and electrochemical etching, of bulk silicon (especially, silicon wafer); followed, optionally, by sorting by size, such as by air classification, sieving and / or filtration.

[0056] Optionally, the particles may be washed before use, such as in methanol or ethanol, to remove a thin oxidised layer from their surface. In the art, this may be termed “activation”.

[0057] The particles thus obtained may have a narrow size distribution and homogeneous surface chemistry, leading to batch-to-batch reliability and reproducibility of one or more of the advantages described herein.

[0058] Suitable physical and chemical techniques are set out, for example, in WO 2011 / 012867 Al (in the name of SISAF LTD); in Tokarska K et al., Facile production of ultra-fine silicon nanoparticles, R. Soc. Open Sci., 2020, 7: 200736; and in Kim, T., Lee, J. Silicon nanoparticles: fabrication, characterization, application and perspectives, Micro and Nano Syst. Lett, 2023, 11: 18, each of which is incorporated herein by reference in its entirety.

[0059] Dopant

[0060] The particles of the composition comprise hydrolysable doped silicon.

[0061] As used herein, the term “doped silicon” may refer to silicon which behaves as an extrinsic semiconductor due to the presence of dopant atoms. The dopant atoms may be or comprise dopant atoms which are substitutional (taking the place of Si atoms). Additionally or alternatively, the dopant atoms may be or comprise dopant atoms which are interstitial (amongst, not displacing, Si atoms). Optionally, the dopant atoms may be present on the surface of the particles. Optionally, the dopant atoms may be present only on, or only on or close to, the surface of the particles. In this way, the particle may optionally have an undoped silicon core, while having an outer shell, or a surface, which is doped.

[0062] However, preferably, no metal silicate coating is present on the surface of the particles. A surface coating of metal silicate is not doping. Unlike scattering dopant atoms amongst silicon atoms, a surface coating of metal silicate would cover the surface of the particle with the metal silicate compound. Although silicon particles can be coated with metal silicate, to do so may complicate their synthesis and is not necessary for the beneficial effects described herein, such as but not limited to tissue targeting.

[0063] Advantageously, the silicon particles are doped at a level of at least about 1 xlO15, most especially at least about 1 xlO16dopant atoms per cm3.

[0064] For example, the particles may be doped at a level of at least about 1 xlO17, at least about 1 xlO18, or at least about 1 xlO19dopant atoms per cm3.

[0065] The silicon particles may be doped at a level of up to 1 xlO20dopant atoms per 3 cm .

[0066] The silicon particles may be n-doped or p-doped. The silicon particles may be doped with one or more elements selected from B, P, Mg, Cu, Ga, Al, In, Bi, Ge, Li, Xe, N, Au and Pt. Thus, the dopant may be a p-dopant, especially boron. The dopant may be an n-dopant, especially phosphorus.

[0067] The dopant may be incorporated in a hydrolysable silicon matrix, (i) substitutionally, (ii) interstitially and / or (iii) via surface attachment. In this way, an improved API delivery vehicle may be provided.

[0068] When boron is used as the dopant, as is preferred, doping levels of IxlO15dopant atoms per cm3, and IxlO20dopant atoms per cm3may correspond, respectively, to a resistivity of 13.6 Q-cm, and 1.3 m Q-cm. Embodiments wherein boron is the dopant do not exclude silicon which, while doped (e.g., heavily doped) with boron, is additionally doped with other elements (preferably, in such cases, the majority dopant is boron).

[0069] As used herein, the term “heavy doping” is understood to mean doping of at least about 1 xlO15dopant atoms per cm3. In some preferred embodiments, dopant is present at levels of at least about 1 xlO16dopant atoms per cm3. Thus, in particularly preferred embodiments, the dopant is boron which is present at levels of at least about IxlO16boron atoms per cm3. For example, there may be boron present at levels of at least about 1 xlO16boron atoms per cm3and up to about 1 xlO20boron atoms per cm3.

[0070] Where silicon is referred to herein as “undoped” (such as the particles of composition SIS0012 of Examples 1 and 2), it may mean that no or only small amounts of dopant atoms are present, for example at most about IxlO2dopant atoms per cm3. Additionally or alternatively, “undoped” silicon may mean silicon that does not behave as an extrinsic semiconductor.

[0071] Advantageously, doping the particles comprising hydrolysable silicon may have a beneficial effect on the zeta potential of the particles, which may be a proxy for the surface charge of the particles. Doping may provide a zeta potential more suitable for improved loading of an API. It is thought that this may help stabilise an API while it is in circulation in vivo until it reaches a target cell, where it is subsequently released. Thus, more API may reach a target cell in a given time period after administration, compared to when undoped particles are used, leading to more efficient API delivery.

[0072] Doping the particles may increase the half-life in vivo (at about pH 7.4 and about 37 °C) of the API, such as by a factor of about 1.5, especially a factor of about 2. Especially, in the presence of the particles comprising hydrolysable doped silicon and the one or more lipids, the API may have a half-life in vivo of more than about 1 hour, especially more than about 2 hours, more especially more than about 6 hours. It will be appreciated that the term “half-life in vivo of the API”, as used herein, may refer to the elimination half-life in vivo of the API, i.e. the time period taken for the amount of the API, once administered, to reduce by about half. It will also be appreciated that the term “amount of the API” may refer to the amount of the API or a derivative thereof having the same or substantially the same intended pharmaceutical effect. It will also be appreciated that the term “half-life in vivo", as used herein, may refer to a systemic half-life of the API (inside the body, especially the human body); as opposed to the half-life of the API on an external body surface, such as when present on the skin surface (outside the body, even if in contact with it).

[0073] The doping of the particles may optionally change their zeta potential by a magnitude of at least about 5 mV, especially at least about 10 mV, compared to undoped particles.

[0074] Doping with a p-dopant may lead to a more positive zeta potential, leading to improved binding with an API having a net negative charge, especially an API having a net negative charge at a pH of about 7.4 (because this is a typical physiological pH), especially a nucleic acid, more especially mRNA, siRNA or tRNA. Thus, in some embodiments, the hydrolysable 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.

[0075] P-doping may still provide improved binding with an API having a net positive charge, especially an API having a net positive charge at a pH of about 7.4 (because this is a typical physiological pH), especially where the particles’ zeta potential is further modulated by other components such as the one or more lipids (e.g., phospholipid(s)) being present. Without wishing to be bound by theory, it is thought that electron transfer may occur from p-doped silicon (e.g., boron-doped silicon) to other constituents, thereby modulating particle size and charge, and hence nucleic acid binding capability.

[0076] The p-doping of the particles may optionally increase their zeta potential by at least about 5 mV, especially at least about 10 mV, compared to undoped particles.

[0077] Meanwhile, doping with an n-dopant may lead to a more negative zeta potential, leading to improved binding with a positively charged API. Thus, in some embodiments, the particles comprising hydrolysable doped silicon are doped with an n- dopant and the API is an API having a net positive charge, especially an API having a net positive charge at a pH of about 7.4.

[0078] N-doping may still provide improved binding with an API having a net negative charge, especially an API having a net negative charge at a pH of about 7.4 (because this is a typical physiological pH), especially where the particles’ zeta potential is further modulated by other components such as the one or more lipids (e.g., cationic lipid(s)) being present. Additionally or alternatively, n-doped silicon may be able to protect the one or more lipids, especially zwitterionic or positively charged lipids, from degradation. This may indirectly help to bind and stabilise an API, for example a nucleic acid, even where the API is negatively charged (especially, mRNA).

[0079] The n-doping of the particles may optionally decrease their zeta potential by at least about 5 mV, especially at least about 10 mV, compared to undoped particles.

[0080] P-doping or n-doping of particles comprising hydrolysable silicon may lead to improved binding with zwitterionic or neutral APIs. Thus, in some embodiments, the particles comprise hydrolysable p-doped silicon and the API is zwitterionic or neutral. In other embodiments, the particles comprise hydrolysable n-doped silicon and the API is zwitterionic or neutral.

[0081] Conventional liposomal transfection compositions (which do not comprise silicon) tend to comprise a significant quantity (such as a majority % by weight) of cationic lipid, especially amine-rich cationic lipid; wherein the positive charge of the cationic lipid is intended to stabilise negatively charged APIs, particularly nucleic acid. However, such cationic lipid may not be cost-effective and may potentially not have an adequate safety profile for all clinical applications, such as administration to certain patient groups, e.g. infants, the elderly, or pregnant women. As described herein, the particles provide the potential to use less or no cationic lipid, especially less or no “exotic” or amine-rich cationic lipid, thus may provide improved cost-effectiveness and safety. Relatedly, the particles’ zeta potential may also be modulated by other components of the composition, as evidenced by the compositions SIS0013-T, SIS0013-N and SIS0013-Q of Examples 2 and 6 herein; and the lipidated oligopeptide containing composition of Example 7. Doping of the particles may help maintain a more positive or more negative (as the case may be) zeta potential than otherwise, meaning that components having certain functionalities can be added that would otherwise increase / decrease (as the case may be) the particles’ zeta potential too much, to maintain satisfactory API binding and delivery.

[0082] Moreover, a change in zeta potential which may result from doping hydrolysable silicon, may not be the only beneficial effect of the doping. A change in zeta potential may not be the only reason for increased success of API binding and delivery. Also, as described herein, other components besides silicon may influence API binding and delivery. By way of illustration, SIS0013-N, SIS0013-Q and SIS0013- T of Examples 2 and 6 display advantages for negatively charged mRNA binding and delivery despite a less positive zeta potential compared to SIS0013 of Example 1.

[0083] Meanwhile, as set out in Example 1 hereinbelow ^Materials and Methods: Method for preparing SIS0012 or SIS0013 Method for preparation of SIS0012-mRNA or SIS0013-mRNA complexes’"') a delivery vehicle in accordance with the present disclosure (e.g. SIS0013) can be prepared, stored, and delivered to a clinic, then complexed with an API such as mRNA before it is administered to a patient. The delivery vehicle is able to be stored separately from the mRNA, until close to the time when it is administered to a patient. Since the delivery vehicle does not contain the reactive API such as mRNA while it is being stored, there is no need for storage at especially cold temperatures such as below 4 °C in order specifically to stabilise the API. This is a different approach to conventional lipid nanoparticle delivery vehicles, which are usually stored already encapsulating an API.

[0084] Moreover, the doped Si-containing delivery vehicle disclosed herein has a stabilising effect on a reactive API, such as a nucleic acid (especially, mRNA) when complexed with it. As a result, the delivery vehicle of the disclosure may optionally be stored already complexed to the API. This may be possible at not especially cold temperatures. For example, the composition comprising the API could be stored at temperatures of about 0 °C or above, especially of about 3-5 °C, i.e. the temperature of a typical commercially available refrigerator; or at about 15-30 °C, i.e. typical room temperature.

[0085] The doped Si-containing delivery vehicle can also stabilise APIs while they are circulating in the body. Additionally or alternatively, it can ensure efficient API uptake by cells. Additionally or alternatively, it can stabilise the API in the cytoplasm of a cell. For example, this may be advantageous where the API is mRNA which is preferably delivered safely to ribosomes in the cell cytoplasm fortranslation. Boron doping (which is p doping) of the silicon may stabilise mRNA (which is negatively charged due to its phosphate backbone). This may especially provide improved protection from degradation and thus more effective cell transfection with the mRNA.

[0086] The manufacture of doped silicon is well-understood in the semiconductor industry and includes ion implantation and diffusion methods, making doped silicon per se readily available. As an example of a diffusion method, silicon powder and a doping reagent (for example B2O3 for boron doping) are mixed under N2 atmosphere at a temperature of 1050 °C - 1175 °C for a few minutes, to allow the dopant (such, for example, as boron) to diffuse into the silicon.

[0087] Lipids

[0088] The one or more lipids may be bound to the surface of the particles. The one or more lipids may be complexed with the API, such as a nucleic acid. The one or more lipids may comprise an ionisable lipid. The one or more lipids may comprise a lipid which has a net positive charge at a pH of about 7.4, also referred to herein as a “cationic lipid.”

[0089] It has been found that surface treating the particle with a lipid may help to control the rate of release of the API (e.g. nucleic acid, especially mRNA or siRNA). The type of lipid used to treat the surface of the silicon containing particle may help modulate the rate of API release. Treating a hydrolysable doped silicon particle with a lipid may have a beneficial effect on the surface charge of the particles. It may provide a zeta potential more suitable for improved loading of an API, especially a nucleic acid (such, for example, as siRNA, short activating RNA, short hairpin RNA or mRNA). It may help to control the rate of API release at a target site.

[0090] By way of example, treatment of particles comprising hydrolysable pure silicon with phosphatidylcholine (PC), phosphatidylethanolamine (PE) and / or lecithin may promote a negative zeta potential (with a zeta potential ranging from about -60 to about -20 mV). Meanwhile, treatment with stearylamine and / or DOTAP may promote a positive zeta potential (with a zeta potential ranging from about 0 to about +40 mV). As described herein, doping of silicon particles modulates their zeta potential. Thus, a p-dopant (preferably, boron) can make the particles’ zeta potential more positive (e.g., a typical value of about -40 mV for pure silicon may become about -25 mV upon doping). Consequently, p-doped (e.g. boron-doped) silicon treated with a cationic lipid can more easily achieve a more positive zeta potential. For example, treatment of 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. In consequence, a positive zeta potential can be achieved with a lower amount of cationic lipid or with a wider range of cationic lipids (including those which are non-toxic; such as those which are not amine-rich), when doped silicon is used. Even if the cationic lipid degrades during storage, resulting in a partial loss of positive charge of the lipid, the zeta potential of the particle can remain more comfortably positive for longer.

[0091] The one or more lipids preferably exclude toxic lipids. The one or more lipids may exclude toxic exogenous lipids having a positive charge at physiological pH (known in the art as exotic cationic lipids); such lipids may have an unfavourable toxicity profile, for example by triggering an innate immune response upon administration to a human patient, especially when they are amine-rich. Although well- suited to the electrostatic loading of polyanionic nucleic acids (including various form of RNA) these (amine-rich) exotic lipid species may lead to cytotoxicity, immunogenicity, and may even lead, contrary to what is desired, to non-specific tissue accumulation, thus counteracting the aim of targeted delivery. Furthermore, they may be complex to synthesise and may therefore be expensive.

[0092] Thus, the one or more lipids may exclude amine-rich lipid. Amine-rich lipids may be defined as having more than 2, 3 or 4 nitrogen atoms per molecule of lipid. In sharp contrast, the one or more lipids preferably contain up to 1 nitrogen atom per molecule of lipid.

[0093] Moreover, it has been found that the beneficial effects disclosed herein may be achieved without necessarily being tied to one or more specific lipid compounds. The one or more lipids may play a role in charge-charge interactions, such as in modulating the zeta potential at the surface of silicon particles, so as to enable better binding of the API. This effect may be seen across a wide range of lipids. Lipids as a class show trends in properties, especially in terms of inter-molecular interactions, enabling the compositions of the present disclosure to be implemented with different lipids and in differing amounts, compared to the specific lipids disclosed in the Examples below.

[0094] The one or more lipids may help to modulate the rate of hydrolysis of the doped silicon, such that the doped silicon hydrolyses to bioavailable orthosilicic acid (OSA) degradation product; rather than insoluble polymeric hydrolysis products. Controlling the rate of hydrolysis of the doped silicon may influence the rate of release of API associated with the doped silicon. Controlling the rate of API release may modulate the length of the time period during which protection of the API is sustained, especially concerning protection in vivo in the presence of various bodily fluids. Thus, more API may be delivered to a target cell in a given time period, than for an otherwise identical composition.

[0095] Lipids are generally understood to include fatty acids and fatty acid derivatives, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids and polyketides. As used in the present application, the term “lipid” may encompass lipidated oligopeptide (a term used interchangeably herein with the term lipopeptide) wherein a short peptide sequence (such as a peptide sequence having 3 to 20 amino acid residues, such as 5 to 15 amino acid residues, especially 3, 4, or 5 amino acid residues, and most especially 5 amino acid residues) is conjugated to one or more fatty acid chains (especially a fatty acid chain having a 10 to 24 carbon chain length, preferably, a 12 to 18 carbon chain length; for example a 14, 15 or 16 carbon chain length; for instance, the peptide moiety may optionally be lipidated with a palmitoyl, cetyl or myristoyl moiety).

[0096] Without wishing to be bound by theory, while some lipids are investigated in the Examples hereinbelow, the mechanism by which the one or more lipids act may be due to properties of the class of lipids, such as their behaviour in response to chargecharge interactions; and may thus be generalisable beyond the exemplified lipids.

[0097] The one or more lipids may thus 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.

[0098] Preferably, the one or more lipidated oligopeptides each comprise 3 to 20 amino acid residues. Thus, the lipidated oligopeptide may be a lipidated tetrapeptide, lipidated pentapeptide or lipidated hexapeptide.

[0099] Preferably, the amino acid residues include at least one amino acid residue (for example, about 2 or about 3 amino acid residues) that is cationic at a pH of about 7.4 (physiological pH), such, for example, as lysine or arginine. For example, the lipidated oligopeptide may include one or more (for example, about 2) lysine resides.

[0100] An especial example of a lipidated oligopeptide (“lipopeptide”) is palmitoyl- pentapeptide-4 (CAS number 214047-00-4; abbreviated as PAL-KTTKS).

[0101] 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 is or are positively charged at a pH of about 7.4 (i.e., about physiological pH) such, for example, as one or both of lysine and arginine.

[0102] The lipidated oligopeptide may especially be used in combination with one or more phospholipids, such as DOPE or DPPC. The alkyl chain of a lipidated oligopeptide molecule may be assimilated in a phospholipid bilayer, while the surface of the bilayer is decorated with the peptide moiety. Without wishing to be bound by theory, it is thought that a peptide moiety of the lipidated oligopeptide can enable the targeting of one or more specific tissues and / or cells. Meanwhile, where the peptide moiety bears a positive charge at a pH of about 7.4 (i.e., about physiological pH), it may stabilise negatively charged APIs (e.g. nucleic acids, especially mRNA or siRNA).

[0103] The one or more lipids may be or comprise one or more of: 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-PEG2000).

[0104] The one or more lipids may be or comprise one or more structural lipids (e.g. a cholesterol-based lipid). However, the one or more lipids may optionally exclude structural lipid. Thus, the one or more lipids may exclude sterols; especially, they may exclude cholesterol. It has been found that the presently disclosed compositions need not rely on these types of lipid, which traditional API delivery systems typically rely on. Thus, the compositions disclosed herein have the potential to provide alternatives to API delivery systems reliant on these types of lipids, especially cholesterol. Where cholesterol is not available or where its use is otherwise not possible (e.g., due to its effect in the body) this may be advantageous.

[0105] The one or more lipids may optionally include one or more of: phosphatidylcholine (PC); hydrogenated PC; stearylamine (SA); di oleoylphosphatidylethanolamine (DOPE); cholesteryl 3 -N- (dimethylaminoethyl)carbamate hydrochloride (DC-chol); l,2-dioleoyl-3- trimethylammonium-propane (DOTAP); PEGylated l,2-Distearoyl-sn-glycero-3- phosphoethanolamine (DSPE), such as DSPE-PEG2000; and derivatives thereof.

[0106] In certain embodiments, the lipid is selected from the group consisting of phosphatidylethanolamine (PE), phosphatidylcholine (PC), stearylamine (SA), or any combination thereof. The lipid or lipids component may, in some embodiments, be or comprise a cationic lipid. The term “cationic lipid” refers to molecules having a net positive charge at pH 7.4 (physiological pH), having a cationic head group attached via some spacer to a hydrophobic tail. Examples include DTDTMA (ditetradecyl trimethyl ammonium), DOTMA (2,3-dioleyloxypropyl-l-trimentyl ammonium), DHDTMA (dihexadecyl trimethyl ammonium); di oleoyl-3 -trimethylammonium propane (DOTAP); and stearylamine (SA). The positive charge may typically be stabilised by a negative counterion.

[0107] Thus, the one or more lipids may optionally be or comprise DOTAP. DOTAP exists in an S and an R enantiomeric form, and may be present as the S-, R- form or as a racemate. Optionally, of the total DOTAP present by weight, the R and S forms may be in approximately equal amounts (i.e. no more than about 60 % of the total DOTAP present by weight, of either form). In other embodiments at least about 80, 90, 95, 98, or 99 % of total DOTAP is in the R-form. In other embodiments at least about 80, 90, 95, 98, or 99 % of total DOTAP is in the S-form.

[0108] Nonetheless, as described herein, doping of the silicon may enable less cationic lipid, especially less toxic or “exotic” cationic lipid, to be used, compared to conventional compositions for API delivery (such as lipid nanoparticles which comprise cationic lipid). While exotic cationic lipids may be suitable for electrostatic loading of polyanionic nucleic acids (including various form of RNA) their amine-rich nature may cause cytotoxicity, im unogenicity, and non-specific tissue accumulation.

[0109] Thus, the one or more lipids may optionally exclude cationic lipid, especially toxic cationic lipid. As described herein, cationic lipid may not be necessary when doped silicon, especially p-doped silicon, is used.

[0110] Thus, the one or more lipids may be or comprise one or more of: one or more phospholipids (e.g. DOPE); and one or more polyethylene glycol (PEG) lipids (e.g. DSPE-PEG2000). Overall, the hydrolysable doped silicon-containing particles disclosed herein can provide the potential to use less lipid (especially less cationic lipid, most especially less of a toxic cationic lipid) in API delivery vehicles, compared to conventional API delivery vehicles which do not contain hydrolysable doped silicon particles (e.g. conventional liposomal nucleic acid delivery vehicles, such as those typically used for mRNA delivery in vivo). Additionally or alternatively, the hydrolysable doped silicon particles can provide the potential for API delivery vehicles to be formulated with a wider range of lipids while still providing transfection efficiency, storage stability, and / or targeted delivery to a particular type of tissue, or to a particular type of cell. In turn, this may lead to reduced reliance in the field on specific lipids, particularly cationic lipids, especially toxic cationic lipids and / or cationic lipids which are formulated specifically for the purpose of API delivery and which may therefore not be cost- effective or easily accessible.

[0111] The one or more lipids may have an average molecular weight in the range of about 500 to about 1000.

[0112] The ratio of the one or more lipids (by which is meant all lipid components in the composition) to silicon may be in a range of from about 40: 1 to about 1 :1, especially a range of about 20: 1 to about 1 : 1; such, for example, as a ratio of about 16: 1, when the components are assembled for manufacture of a delivery system, i.e. before any further processing is carried out (such further processing may be, for example, the step “Extrusion” of “Materials and Methods” of Example 1 hereinbelow).

[0113] As described herein, the one or more lipids may especially comprise or be a phospholipid. The term “phospholipid”, as used herein, may refer to a lipid comprising a fatty acid chain and a phosphate group. Phospholipids may carry a negative charge, unlike a cationic lipid which is positively charged. However, phospholipids are typically zwitterionic compounds comprising both positive and negatively charged components, resulting in no overall charge. As such, phospholipids are typically classified as neutral lipids. Suitable phospholipids may be or include glycerophospholipids. Especially suitable phospholipids may be or include those in which the polar head group is linked to quaternary ammonium moieties, such as phosphatidylcholine (PC) or hydrogenated phosphatidylcholine. The phospholipid may be, or be derived from, lecithin. A preferred phospholipid is DOPE (phosphatidyl ethanolamine or 1,2-dioleoyl-sn- gly cero-3 -phosphoethanol amine) .

[0114] Preferably, the side chain(s) of the phospholipid may be aliphatic side chain(s) with about 15 or more carbon atoms, or an ether side chain with about 6 or more repeating ether units, such as a polyethylene glycol or polypropylene glycol chain.

[0115] Lipids with ether side chains may be referred to as “PEG-lipids” or “PEG- ylated” lipids. Thus, as used in the present application, the term “lipid” may cover PEG lipids. Thus, according to preferred embodiments, the one or more lipids may comprise or be one or more polyethylene glycol (PEG) lipids, especially PEGylated DSPE, such as DSPE-PEG2000.

[0116] The one or more lipids may optionally comprise or consist substantially of phosphatidylcholine (PC), hydrogenated phosphatidylcholine, stearylamine (SA), or combinations thereof.

[0117] The one or more lipids may optionally comprise at least about 5 % (e.g., at least about 30 % or at least about 50 %) by weight of PC, based on the total weight of the one or more lipids.

[0118] The one or more lipids may optionally comprise at least about 5 % (e.g., at least about 30 % or at least about 50 %) by weight of hydrogenated PC, based on the total weight of the one or more lipids.

[0119] The one or more lipids may optionally comprise at least about 5 % (e.g., at least about 30 % or at least about 50 %) by weight of SA, based on the total weight of the one or more lipids. The one or more lipids may optionally comprise or consist essentially of PC and SA, optionally in a ratio by weight of PC to SA in a range of from about 1 : 1 to about 20: 1.

[0120] The one or more lipids may optionally comprise or consist essentially of the combination of DOPE, SA, and DC-cholesterol (DC-chol). The ratio by weight of DOPE : SA may be in a range of from about 1 : 1 to about 10: 1. The ratio by weight of DOPE : DC-cholesterol may be in a range of from about 1 : 1 to about 5: 1. The ratio by weight of SA : DC-cholesterol may be in a range of from about 1 : 1 to about 1 :5.

[0121] In certain preferred embodiments, the one or more lipids may optionally comprise or consist essentially of a combination of DOTAP, DOPE and a PEG-lipid (especially DSPE-PEG2000). The ratio by weight of DOTAP : DOPE may be in a range of from about 1 :2 to about 2: 1; such, for example, as about 1 : 1. The ratio by weight of DOTAP: PEG-lipid may be in a range of from about 10: 1 to about 5: 1; such, for example, as about 7:1. The ratio by weight of DOPE:PEG-lipid may be in a range of from about 10: 1 to about 5: 1; such, for example, as about 7: 1.

[0122] Amino acid s)

[0123] The composition disclosed herein may comprise one or more amino acids. In its broadest sense, the term “amino acid” encompasses any artificial or naturally occurring organic compound containing an amine (-NH2) and carboxyl (-COOH) functional group. It includes a, P, y and 6 amino acids. It includes an amino acid in any chiral configuration. The amino acid may, especially, be a naturally occurring a amino acid. It may be a proteinogenic amino acid or a non-proteinogenic amino acid (such as carnitine, levothyroxine, hydroxyproline, ornithine or citrulline).

[0124] The one or more amino acids may help stabilise the doped silicon particles themselves. In vivo, the one or more amino acids may help to modulate the rate of hydrolysis of the doped silicon, such that the doped silicon hydrolyses to bioavailable orthosilicic acid (OSA) degradation product; rather than insoluble polymeric hydrolysis products. In this way, the one or more amino acids may complement the function of the one or more lipids of the present disclosure. Controlling the rate of hydrolysis of the doped silicon may influence the rate of release of API associated with the doped silicon. Controlling the rate of API release may modulate the length of the time period during which protection of the API is sustained, especially concerning protection in vivo in the presence of various bodily fluids. Thus, more API may be delivered to a target cell in a given time period, than for an otherwise identical composition.

[0125] Without wishing to be bound by theory, while some amino acids are investigated in the Examples hereinbelow, the mechanism by which the one or more amino acids act may be due to properties of the class of amino acids, such as their behaviour in response to charge-charge interactions; and may thus be generalisable beyond the exemplified amino acids.

[0126] In preferred embodiments, the amino acid(s) may comprise or consist essentially of glycine, arginine and / or tyrosine; most especially glycine.

[0127] Additionally or alternatively, amino acids which are neutral or positively charged at physiological pH (about pH 7.4), such as tyrosine or arginine, may stabilise negatively charged APIs (e.g. nucleic acids such as mRNA). Meanwhile, amino acids which are neutral or negatively charged at physiological pH (about pH 7.4) may stabilise positively charged APIs. Nonetheless, the interplay of charge-based and / or other interactions (for example, steric interactions) resulting from the combination of doped Si, lipid(s) and amino acid(s) may be such that amino acid(s) which are positively charged at physiological pH may help to stabilise positively charged APIs, or amino acid(s) which are negatively charged at physiological pH may help to stabilise negatively charged APIs.

[0128] The ratio by weight of the one or more lipids (i.e. total lipid components) to the amino acid(s) may be in a range of from about 40: 1 to about 1 : 1; such, for example, as about 32: 1.

[0129] Optionally, the composition may specifically comprise the amino acid tyrosine in addition to the amino acid(s) described hereinabove. Optionally, the composition may specifically comprise tyrosine instead of the amino acid(s) described hereinabove. Thus, it will be understood that while tyrosine is an amino acid, it may optionally be present as a separate, further component, for the purposes of the present disclosure, distinct from the amino acid(s) described hereinabove. Thus, when tyrosine is present as a further component distinct from and in addition to the amino acid(s) described hereinabove (as exemplified by composition SIS0013-T of Example 3 hereinbelow) it will be understood that the calculation of a ratio by weight of the one or more lipids to amino acid(s) in a range of from about 40: 1 to about 1 : 1; such, for example, as about 32: 1, disclosed above, does not include the amount of additional, distinct tyrosine.

[0130] Non-reducing disaccharide

[0131] Additionally or alternatively, the composition may comprise one or more nonreducing disaccharides, especially trehalose, as exemplified, for instance, by the compositions of Example 1 hereinbelow. The ratio by weight of the one or more lipids (i.e. total lipid components) to non-reducing disaccharide may be in a range of from about 20: 1 to about 1 : 1; such, for example, as about 16: 1.

[0132] APIs

[0133] It will be understood that the composition of the present disclosure may be described as a pharmaceutical composition, being for the delivery of an active pharmaceutical ingredient (API).

[0134] The API may, for example, be a fragile API. As used herein, the terms “fragile APIs” and “reactive APIs” may be interchangeable and may refer to APIs which (i) have a half-life upon storage in aqueous solution at about 25 °C of at most one week, measurable by NMR or by GC-MS; and / or (ii) have a half-life in vivo of under about an hour, measurable by assay of a biological sample.

[0135] The API may be any pharmaceutically active compound; thus, for example, it will be understood that the term “API” encompasses pro-drugs. Especially, the API may be a nucleic acid, more especially siRNA or mRNA. Meanwhile, in other preferred embodiments, the API may be a protein. The API may be for administration by injection, orally, intranasally or topically; especially, by injection or orally.

[0136] Nucleic acid APIs

[0137] The API may preferably be or comprise a nucleic acid, especially linear or circular RNA. The RNA may be small interfering RNA (siRNA), small activating RNA (saRNA), small hairpin RNA (shRNA), transfer RNA (tRNA) or messenger RNA (mRNA), especially mRNA (e.g., mRNA that encodes a protein of a pathogenic organism).

[0138] Other nucleic acids for use in accordance with the present disclosure include: double- and single-stranded DNA; DNA:RNA hybrids; peptide:DNA hybrids; and peptide:RNA hybrids.

[0139] RNA and DNA may be naturally occurring or chemically modified to enhance their therapeutic properties, such as enhanced activity, increased serum stability, reduced off-targeting and lower immunological activation. Chemical modifications to RNA and DNA may include any modifications commonly known in the art. As used herein, the term “naturally occurring” means of natural human or animal origin. It will be understood that a molecular structure that is the same as a naturally occurring molecular structure may nevertheless be synthesized in vitro, such as when mRNA is synthesized by in vitro transcription (IVT).

[0140] Thus, as used herein, the terms nucleic acid, DNA and RNA also include known types of modifications, for example, labels which are known in the art, methylation, “caps”, substitution of one or more of the naturally occurring nucleotides with an analogue, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), with negatively charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), and with positively charged linkages (e.g., aminoalklyphosphoramidates, aminoalkylphosphotriesters), those containing pendant moieties, such as, for example, proteins (including nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide or oligonucleotide.

[0141] Similarly, as used herein, the terms “nucleoside” and “nucleotide” will include those moieties which contain not only the known purine and pyrimidine bases, but also other heterocyclic bases which have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, or other heterocycles. Modified nucleosides or nucleotides will also include modifications on the sugar moiety, e.g., wherein one or more of the hydroxyl groups are replaced with a halogen, an aliphatic group, or are functionalized as ethers, amines, or the like. Other modifications to nucleotides or polynucleotides involve rearranging, appending, substituting for, or otherwise altering functional groups on the purine or pyrimidine base which form hydrogen bonds to a respective complementary pyrimidine or purine, e.g., isoguanine, isocysteine, and the like. In some embodiments, the oligonucleotides and / or probes include at least one, two, three or four modified nucleotides.

[0142] In some embodiments, the nucleic acids such as the RNAs disclosed herein include one or more universal bases. As used herein, the term “universal base” refers to a nucleotide analogue that can hybridize to more than one nucleotide selected from A, U / T, C, and G. In some embodiments, the universal base can be selected from the group consisting of deoxyinosine, 3-ntiropyrrole, 4-nitroindole, 6-nitroindole, 5-nitroindole.

[0143] In its broadest sense, the term “saRNA” encompasses small activating RNA, comprising RNA molecules which operate within the RNA activation (RNAa) pathway. The saRNA may be double-stranded. The saRNA may have a length in a range of about 5 to about 50 base pairs, especially about 10 to about 40 base pairs, more especially about 10 to about 30 base pairs.

[0144] In its broadest sense, the term “shRNA” encompasses small hairpin RNA, comprising RNA molecules which operate within the RNA interference (RNAi) pathway. The shRNA may be single stranded while also having base pairing thereby forming a hairpin loop. The single strand of the shRNA may have a length in a range of about 10 to about 100 bases, especially about 25 to about 75 base pairs, more especially about 40 to about 70 base pairs; which may then form a hairpin loop.

[0145] In its broadest sense, the term “siRNA” encompasses small interfering RNA, comprising RNA molecules which operate within the RNA interference (RNAi) pathway. siRNA is sometimes known as short interfering RNA or silencing RNA. The siRNA may be double stranded. The siRNA may have a length in a range of from about 5 to about 50 base pairs, especially about 10 to about 40 base pairs, more especially about 15 to about 30 base pairs. Examples 4, 6 and 7 hereinbelow investigate siRNA APIs in the compositions of the present disclosure.

[0146] In its broadest sense, the term “tRNA” encompasses transfer RNA, comprising RNA molecules which serve, during protein synthesis, as links (or adaptors) between mRNA molecules and growing chains of amino acids. The primary structure of the tRNA may have a length in a range of from about 20 to about 200 nucleotides, especially about 50 to about 100 nucleotides. The tRNA may have a cloverleaf secondary structure. The tRNA may have an L-shaped tertiary structure.

[0147] In its broadest sense, the term “mRNA” encompasses messenger RNA for the synthesis of protein(s). It may encompass mRNA comprising a 5-prime cap and / or a poly-adenylated terminus. Alternatively, one or both of those features may be absent. Typically, the mRNA may be single stranded. The coding region of the mRNA may be at least about 100, especially at least about 500, more especially at least about 1000 bases in length. Examples 1, 2 and 7 hereinbelow investigate mRNA APIs in the compositions of the present disclosure.

[0148] The mRNA may encode an antigen, thereby providing a composition which is a vaccine. The antigen may be a bacterial, parasitic or fungal antigen. The antigen may be a viral antigen, especially a viral antigen of one of the viral diseases described hereinbelow; more especially an antigen of a respiratory virus, for example an antigen of SARS-CoV-2, for example an antigen deriving from the spike protein of SARS- CoV-2.

[0149] 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 vicilins, legumins, albumins; one or more plant defence related proteins; and one or more profilins).

[0150] The mRNA may encode a protein that modulates an immune, autoimmune, or inflammatory disease (including, but not limited to, lupus, atherosclerosis, chronic obstructive pulmonary disease, inflammatory bowel disease, multiple sclerosis, psoriasis, a rheumatic disease, uveitis, atopic dermatitis, and pulmonary fibrosis).

[0151] The mRNA may encode a tumour-specific antigen. As used herein, the term tumour-specific antigen may refer to an antigen that arises, in one or more malignant cancer cells, from non-synonymous somatic mutation (leading to a neoantigen) or viral- integrated mutation (leading to an onco-viral antigen). Tumour-specific antigens may thus refer to antigens that are completely absent from (not expressed by) non-cancerous (healthy, normal) cells.

[0152] The mRNA may encode a tumour-associated antigen. As used herein, the term tumour-associated antigen may refer to an antigen that is over-expressed in a malignant cancer cell, compared to a non-cancerous (healthy, normal) cell, for example due to genetic amplification or post-translational modifications. The term tumour-associated antigen may encompass overexpressed antigens (which term may refer to proteins that are moderately expressed in non-cancerous (healthy, normal) cells, but expressed abundantly in malignant cancer cells); differentiation antigens (which term may refer to proteins that are selectively expressed by the cell lineage from which the malignant cells evolved, an example being prostate-specific antigen); and cancer-germline antigens (which term may refer to antigens that are normally limited to reproductive tissues, but which are aberrantly expressed in a malignant cancer cell; for example, melanoma antigen family A3 (MAGE-A3); New York Esophageal Squamous Cell Carcinoma- 1 Antigen (NY-ESO-1); and Preferentially Expressed Antigen in Melanoma (PRAME)).

[0153] The mRNA may encode multiple proteins, thereby providing more effective pharmacological activity. The mRNA may encode multiple antigens, especially multiple viral antigens.

[0154] The mRNA may additionally encode an adjuvanting protein. An adjuvant may additionally or alternatively be provided as a further component of the composition in addition to the API.

[0155] As evidenced by Examples 1, 2, 4, 6 and 7 hereinbelow, it has been found that doping of silicon particles can help the disclosed compositions deliver an API more effectively to a cell, especially where the API is a nucleic acid, more especially siRNA or mRNA. In particular, the doped silicon-containing compositions disclosed herein may mitigate or address the problem of how to ensure APIs reach cells once they have been administered to a patient, including how to stabilise APIs while they are circulating in the body. They may mitigate or meet the need for tissue or cell targeting, so that an API can be delivered to the correct cells. Once a target cell is reached, they may help ensure efficient API uptake by cells. Following uptake by a cell, they may help to ensure API stability in the cytoplasm; for example, stabilisation and release of an mRNA API in the cytoplasm over an appropriate time period so that more mRNA can be translated successfully.

[0156] Both of the types of mRNA used in Examples 1 and 2 hereinbelow (LUC mRNAl and LUC mRNA2) are chemically modified compared to naturally occurring mRNA. Such chemical modification may potentially make transfection more difficult. The increased ability of doped Si-containing SIS0013, compared to undoped Si- containing SIS0012, to provide for transfection of chemically modified mRNA, is particularly advantageous and surprising. Thus, in some embodiments, the API may be mRNA which is chemically modified compared to naturally occurring mRNA. Accordingly, the method disclosed herein may comprise an in vivo step of transfecting a human cell with a nucleic acid.

[0157] The doped silicon-containing compositions disclosed herein for delivery of an API may be non-toxic. For example, they may be substantially fully biodegradable as described herein, in that the silicon may degrade to non-toxic orthosilicic acid in vivo.

[0158] The doped silicon-containing compositions disclosed herein for delivery of an API may be fully dispersible in an aqueous environment, to ensure ease of delivery, such as by injection in aqueous solution.

[0159] Complexation of components, especially particle / lipid / API complexation

[0160] Preferably, the particles comprising hydrolysable doped silicon are complexed with the one or more lipids thus forming a delivery vehicle for transport of the API. Thus, when the API is added, it also becomes complexed with the particles and / or the lipid. Put another way, the particles and lipid are organised into a delivery vehicle that is loaded with the API. Advantageously, this may make the API less liable to react with one or more external reactive species. The API may be less at risk of degradation catalysed by enzymes external to the complex, especially in vivo, such as during circulation in the body and / or in a cell cytoplasm; this may especially be the case where the API is a nucleic acid, more especially mRNA.

[0161] In its broadest sense, as used here, the term “complexed with” may encompass ionic and / or covalent and / or physical interactions, and especially may encompass charge-charge interactions, such as those resulting from the particles’ zeta potential.

[0162] Thus, preferably, the zeta potential of the particles, especially when modulated by the one or more lipids and any other components present, is such as to attract and facilitate binding of the API.

[0163] In preferred embodiments wherein amino acid(s) is or are present, the amino acid(s) may also complex with the particles, lipid(s) and / or API. The amino acid(s), especially when charged, may modulate the particles’ zeta potential thus modulating API and / or lipid complexation with the particles.

[0164] When other advantageous components are present, such as those described hereinbelow, they may also complex with the particles, lipid(s), amino acid(s) and / or API.

[0165] The one or more lipids may be formed of, or may comprise, one or more lipid structures. Said structures may be or comprise one or more of: micelles, incomplete micelles, liposomes, incomplete liposomes and (e.g., solid or semi-solid) lipid globules, as described herein.

[0166] Meanwhile, preferably, the API (espeically, RNA, most especially mRNA) is bound to (especially, electrostatically co-ordinated with) the particles comprising hydrolysable doped silicon. Preferably, at least about 50, 60 or 70 % of API in the composition is bound to the particles in this way.

[0167] In turn, preferably, the particles comprising hydrolysable doped silicon are bound to the surface of, and / or are present in the interior of, the one or more lipid structures.

[0168] Particle-API binding may be especially effective at reducing or preventing API degradation if the particles comprising hydrolysable doped silicon aggregate into chains of the particles. Thus, preferably, the particles are aggregated. Doping of the silicon may facilitate such aggregation, for example by reducing particle-particle repulsion.

[0169] The chains may extent into the interior of lipidic structures (such as liposomes, incomplete liposomes, micelles, incomplete micelles and / or lipid globules) formed by the one or more lipids. API bound to particles in chains embedded in the interior of lipidic structures may be especially shielded from water molecules, thus shielded from degradation by hydrolysis. The lipidic structures (such as liposomes, incomplete liposomes, micelles, incomplete micelles and / or lipid globules) may typically have a mean diameter of about 50 nm to about 500 nm; especially about 100 nm to about 500 nm. Meanwhile, the particles comprising hydrolysable doped silicon may typically have a mean diameter in a range of from about 1 nm to about 50 nm, such as about 1 nm to about 30 nm. This relative difference in size may optimise the formation of aggregates (especially, chains) of the particles being embedded in, and / or decorating the surface of, the lipidic structures.

[0170] Thus, preferably, particles comprising hydrolysable doped silicon are bound to the surface of, and / or are present in the interior of, one or more of: micelles, incomplete micelles, liposomes, incomplete liposomes, and (e.g., solid or semi-solid) lipid globules; and API is bound to the particles comprising hydrolysable doped silicon. Preferably, the particles comprising hydrolysable doped silicon are present in one or more aggregates of particles comprising hydrolysable doped silicon, especially one or more aggregates comprising or consisting of chains of the particles, most especially chains that extend into the interior of the one or more lipid structures, such as into the interior of one or more of: micelles, incomplete micelles, liposomes, incomplete liposomes, and (e.g., solid or semi-solid) lipid globules (especially, liposomes and / or lipid globules). An amino acid (especially, glycine, arginine and / or tyrosine, such as glycine) may also be associated with (e.g., bound non-covalently to) the particles comprising hydrolysable doped silicon; and / or associated with (e.g., on the surface of) the one or more lipid structures.

[0171] As used herein, the term liposomal lipid particle, or the term liposome, may have its normal 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 visualised as a lipid “bubble” surrounding an interior space. The interior space may be a hydrophilic environment.

[0172] The composition may in some embodiments comprise one or more liposomes.

[0173] I.e., the one or more lipids may be formed of, or may comprise, one or more liposomes. The composition may in some embodiments comprise incomplete liposomes. In that sense, their interior space may be accessible from the exterior. An incomplete liposome may be visualised as an incomplete lipid “bubble” wherein there are one or more gaps in the (approximately spherical) lipid bilayer surface. Thus, suitably, the one or more lipids may be formed of, or may comprise, one or more incomplete liposomes.

[0174] Suitably, the one or more lipids may be formed of, or may comprise, one or more incomplete liposomes and / or one or more (complete) liposomes.

[0175] Thus, the present composition may comprise the particles comprising hydrolysable doped silicon associated with one or more liposomes and / or one or more incomplete liposomes, wherein the API is associated with (especially, bound to) the particles comprising hydrolysable doped silicon. An amino acid (especially, glycine, arginine and / or tyrosine, such as glycine) may also be associated with the particles comprising hydrolysable doped silicon.

[0176] Optionally, the API may be encapsulated by the one or more lipids.

[0177] Liposomes are especially suitable for encapsulating APIs. Thus, the API may optionally be encapsulated in a liposome, or, preferably, the API may be partially encapsulated in an incomplete liposome. Where the liposome is an incomplete liposome, there exists a route for the API to cross between the liposome interior and exterior.

[0178] The optionally present liposomes or incomplete liposomes, may have a mean diameter in a range of from about 50 nm to about 500 nm, especially about 50 nm to about 300 nm, more especially about 100 nm to about 300 nm.

[0179] The API may be associated non-covalently (especially, by virtue of ionic interactions) with the exterior surface of a liposome or of an incomplete liposome. 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 located in non-covalent association with the exterior surface.

[0180] Thus, at least about 10 %, about 30 %, or about 50 % of the API may be fully encapsulated within the interior space of the (optionally, incomplete) liposome, with the remainder being located in non-covalent association with the exterior surface of the (optionally, incomplete) liposome.

[0181] API may be bound non-covalently to particles comprising hydrolysable doped silicon that 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 bound non-covalently to particles comprising hydrolysable doped silicon that are, in turn, bound to the surface of one or more liposomes and / or one or more incomplete liposomes.

[0182] API may be bound non-covalently to particles comprising hydrolysable doped silicon that are, in turn, in the interior of one or more liposomes and / or one or more incomplete liposomes. At least about 50, 60 or 70 % of the API may preferably be bound non-covalently to particles comprising hydrolysable doped silicon that are in the interior of one or more liposomes and / or one or more incomplete liposomes.

[0183] Preferably, API (espeically, RNA, most especially mRNA) is bound non- covalently to particles comprising hydrolysable doped silicon that are in the interior of one or more liposomes and / or one or more incomplete liposomes; and API (espeically, RNA, most especially mRNA) is bound non-covalently to particles comprising hydrolysable doped silicon that are bound to the surface of one or more liposomes and / or one or more incomplete liposomes.

[0184] In some embodiments, the composition may be free or substantially free of liposomes; and / or may be free or substantially free of incomplete liposomes.

[0185] Optionally, the one or more lipids may be formed of, or may comprise, one or more lipid monolayers. Optionally, the one or more lipids may be or comprise one or more micelles or incomplete micelles. It will be understood that micelles have similar characteristics to liposomes, except that micelles’ walls are formed of lipid monolayer; whereas liposomes’ walls are formed of lipid bilayer. Thus, a micelle may refer to a vesicle having at least one lipid monolayer, which may be approximately spherical in shape. A micelle, similarly to a liposome, may be visualised as a lipid “bubble” surrounding an interior space. The interior space may be a hydrophilic environment.

[0186] Thus, the present composition may comprise the particles comprising hydrolysable doped silicon associated with one or more micelles and / or one or more incomplete micelles, wherein the API is associated with (especially, bound to) the particles comprising hydrolysable doped silicon.

[0187] The optionally present micelles may have a mean diameter in a range of from about 50 nm to about 500 nm, especially about 50 nm to about 300 nm, more especially about 100 nm to about 300 nm.

[0188] API may be bound non-covalently to particles comprising hydrolysable doped silicon that 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 bound non-covalently to particles comprising hydrolysable doped silicon that are, in turn, bound to the surface of one or more micelles and / or one or more incomplete micelles.

[0189] API may be bound non-covalently to particles comprising hydrolysable doped silicon that are, in turn, in the interior of one or more micelles and / or one or more incomplete micelles. At least about 50, 60 or 70 % of the API may preferably be bound non-covalently to particles comprising hydrolysable doped silicon that are in the interior of one or more micelles and / or one or more incomplete micelles.

[0190] Preferably, API (espeically, RNA, most especially mRNA) is bound non- covalently to particles comprising hydrolysable doped silicon that are in the interior of one or more micelles and / or one or more incomplete micelles; and API (espeically, RNA, most especially mRNA) is bound non-covalently to particles comprising hydrolysable doped silicon that are bound to the surface of one or more micelles and / or one or more incomplete micelles. In some embodiments, the composition may be free or substantially free of micelles; and / or may be free or substantially free of incomplete micelles.

[0191] The one or more lipids may be formed of, or may comprise, one or more lipid globules, each globule optionally being surrounded by a layer of surfactants. Lipid globules do not enclose an interior space or cavity. Instead, they are solidly formed, or substantially solidly formed, of lipid, in which other components, such as the particles comprising hydrolysable doped silicon to which are bound API molecules, may be dispersed. Thus, the globules’ interior may be studded with the particles comprising hydrolysable doped silicon; with API molecules, in turn, being bound (non-covalently) to the particles comprising hydrolysable doped silicon. Additionally or alternatively (preferably, additionally), the particles comprising hydrolysable doped silicon, to which are (non-covalently) bound API molecules, may be bound to the surface of one or more lipid globules.

[0192] Thus, preferably, the present composition comprises the particles comprising hydrolysable doped silicon associated with (especially, dispersed within and / or bound onto 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 hydrolysable doped silicon.

[0193] The optionally present lipid globules may have a mean diameter in a range of from about 50 nm to about 500 nm, especially about 50 nm to about 300 nm, more especially about 100 nm to about 300 nm.

[0194] API may be bound non-covalently to particles comprising hydrolysable doped silicon that are bound to the surface of one or more lipid globules. Up to about 10 or 20 % of the API may be bound non-covalently to particles comprising hydrolysable doped silicon that are, in turn, bound to the surface of one or more lipid globules.

[0195] API may be bound non-covalently to particles comprising hydrolysable doped silicon that are, in turn, in the interior of one or more lipid globules. At least about 50, 60 or 70 % of the API may preferably be bound non-covalently to particles comprising hydrolysable doped silicon that are in the interior of one or more lipid globules.

[0196] Preferably, API (espeically, RNA, most especially mRNA) is bound non- covalently to particles comprising hydrolysable doped silicon that are in the interior of one or more lipid globules; and API (espeically, RNA, most especially mRNA) is bound non-covalently to particles comprising hydrolysable doped silicon that are bound to the surface of one or more lipid globules.

[0197] Silicon particle aggregates

[0198] The particles comprising hydrolysable doped silicon may coalesce into aggregates of particles comprising hydrolysable doped silicon, for example as shown in the transmission electron microscope (TEM) image of Figure 27.

[0199] Thus, the described composition may comprise aggregates (especially, chains) of the particles comprising hydrolysable doped silicon.

[0200] As used herein, the term “aggregate of particles comprising hydrolysable doped silicon” may refer to a cluster of particles wherein nearest-neighbour particles are in contact with each other. Such clusters may have varying configurations, such as substantially spherical clusters of particles and / or chains of particles. Particularly preferred are configurations comprising or consisting of chains of particles.

[0201] Thus, the composition may comprise one or more aggregates of particles comprising hydrolysable doped silicon. Preferably, the aggregates comprise one or more chains of the particles.

[0202] There may, for example, be present at least about 2, 3 or 4 chains on average per aggregate.

[0203] The one or more aggregates may comprise one or more branched chains of the particles. Thus, the one or more aggregates may be formed of, or may comprise, branches formed from chains of the particles. There may, for example, be present at least about 2, 3 or 4 branches per aggregate.

[0204] The one or more aggregates of particles comprising hydrolysable doped silicon may be associated with the one or more lipids, for example embedded in lipid and / or attached to the surface of lipid.

[0205] The one or more aggregates of particles comprising hydrolysable doped silicon may be associated with one or more lipid structures described herein, for example embedded within and / or attached to one or more lipid structures.

[0206] As described herein, the one or more lipid structures may be formed of or may comprise one or more of: micelles, incomplete micelles, liposomes, incomplete liposomes and lipid globules. Thus, the one or more aggregates of particles comprising hydrolysable doped silicon may be associated with (for example, embedded in and / or attached to the surface of) one or more of lipid micelles, incomplete lipid micelles, liposomes, incomplete liposomes and lipid globules.

[0207] In particular, the one or more aggregates of particles comprising hydrolysable doped silicon may be embedded in or attached to the surface of one or more of: liposomes; incomplete liposomes; and lipid globules.

[0208] The ratio of the longest dimension of an aggregate to the longest dimension of a lipid structure may on average be about 1 :5 to 5 :1, especially about 1 :3 to 3 : 1; especially when the lipid structure is or comprises liposomes and / or lipid globules and the one or more aggregates are embedded therein or attached to the surface thereof. This may be measured, for example, by TEM as shown in Figure 27.

[0209] The average longest dimension of an aggregate may be about 50 nm to about 500 nm, especially about 50 nm to about 200 nm, such as about 50 to about 150 nm, such as when measured by TEM. In particular, the one or more aggregates may be or comprise one or more chains of particles, with the average length of a chain being about 50 nm to about 500 nm, especially about 50 nm to about 200 nm, such as about 50 to about 150 nm. The average cross-sectional diameter of a chain may be about 5 nm to about 50 nm, such as about 5 nm to about 30 nm.

[0210] The ratio of the longest dimension of an individual particle comprising hydrolysable doped silicon to the longest dimension of a lipid structure may on average be in a range of from about 1 : 100 to about 1 :2, especially about 1 : 100 to about 1 :5, more especially about 1 : 100 to about 1 :9; especially when the lipid structure is or comprises liposomes and / or lipid globules and the one or more aggregates are embedded therein or attached to the surface thereof. This may be measured, for example, by TEM as shown in Figure 27.

[0211] When the one or more aggregates are present, the average (e.g., mean) diameter of a particle comprising hydrolysable doped silicon may preferably be about 1 nm to about 50 nm, especially about 5 nm to about 50 nm, more especially about 1 nm to about 30 nm, yet more especially about 5 nm to about 20 nm, such as about 10 nm. Additionally or alternatively, the particles may be porous and may have an average (e.g., mean) pore diameter of about 0.1 to about 5 nm, such as about 2 nm.

[0212] In turn, API may be bound (non-covalently) to one or more of the particles in the aggregates.

[0213] Thus, the particles of the one or more aggregates may bind the API (especially, when the API is or comprises mRNA). When the one or more aggregates are or comprise chains of the particles, such chains may extend into the interior of the lipid structure(s) (especially, into liposomes, incomplete liposomes, and / or lipid globules). In this way, the chains may provide a route for the API to be better encapsulated in the lipid. Without wishing to be bound by theory, it is thought that this may shield the API from degradation, especially by shielding it from enzymes (espeically, in vivo) and preventing or reducing API molecules being available to react with water molecules. It is thought that as the silicon particles degrade over time, API may be released, thus enabling protection of the API until it reaches a target site for release. The presence of charged API may itself facilitate aggregation of the particles. For example, when the API is nucleic acid (especially, mRNA) its negative charge (due to a phosphate backbone) may induce aggregation of the particles. Additionally or alternatively, the presence of Si-0 species on the particles’ surface may induce particleparticle interactions to facilitate aggregation.

[0214] Thus, preferably, the one or more aggregates are or comprise one or more chains of the particles comprising hydrolysable doped silicon, wherein API is bound to the particles, and the one or more chains extend into the interior of the lipid structure(s), especially, into liposomes, incomplete liposomes, or lipid globules. This may provide a stabler environment for the API. Additionally or alternatively, it may enable increased API uptake by the lipid structures, compared to no such aggregate(s) being present. This is in contrast to conventional liposomal delivery vehicles, for which inefficient API uptake may be a problem; for example, it is thought that up to about 80 % of conventional liposomal delivery vehicles formulated into commercially available therapeutic compositions may be “empty” of API.

[0215] Optionally, substantially no particles comprising hydrolysable doped silicon are present as isolated particles; instead, substantially all the particles are present in aggregates.

[0216] Aggregation may be promoted when the particles comprising hydrolysable doped silicon have an average diameter of about 1 nm to about 50 nm, especially about 5 nm to about 50 nm, more especially about 1 nm to about 30 nm, yet more especially about 5 nm to about 20 nm, such as about 10 nm. Such particle diameters may be below the typical diameter of lipidic structures (as described herein) that may form spontaneously (optionally, promoted by extrusion) from the one or more lipids.

[0217] Thus, preferably, the described composition comprises aggregates (especially, chains) of the particles comprising hydrolysable doped silicon, wherein the particles comprising hydrolysable doped silicon have an average diameter of about 1 nm to about 50 nm, especially about 5 nm to about 50 nm, more especially about 1 nm to about 30 nm, yet more especially about 5 nm to about 20 nm, such as about 10 nm. Aggregation may be promoted when the composition is extruded, for instance in the manner set out in the step “ Extrusion” of “Materials and Methods” of Example 1 hereinbelow. Extrusion may be or comprise extrusion through a porous membrane having average pore diameters of about 0.01 pm to about 1 pm, such as about 0.05 pm to about 0.6 pm.

[0218] Accordingly, the composition may be (or be formed from) an extruded composition.

[0219] Preferably, extrusion occurs before addition of the API. Thus, the composition may be formed from an extruded composition comprising the particles and the one or more lipids, to which API has been added.

[0220] Therefore, the composition may suitably be an extruded composition that comprises aggregates (especially, chains) of the particles comprising hydrolysable doped silicon, wherein the particles comprising hydrolysable silicon have an average diameter of about 1 nm to about 50 nm, especially about 5 nm to about 50 nm, more especially about 5 nm to about 30 nm, yet more especially about 5 nm to about 20 nm, such as about 10 nm.

[0221] Other advantageous components

[0222] Optionally, the composition further comprises one or more other advantageous components.

[0223] The one or more other advantageous components may be or comprise tyrosine or a derivative thereof. Thus, optionally, in addition to the above-described amino acid, the specific amino acid tyrosine may be present as a further component, as exemplified by the composition “SIS0013-T” of Examples 3 and 6 hereinbelow. Optionally, the ratio by weight of the one or more lipids (i.e., total lipid components) to the tyrosine may be in a range of from about 130: 1 to about 30: 1, such as about 80: 1. Additionally or alternatively, the composition may comprise nicotinamide adenine dinucleotide (NAD) or a derivative thereof, as exemplified by the composition “SIS0013-N” of Examples 3 and 6 hereinbelow. Optionally, the ratio by weight of the one or more lipids (i.e., total lipid components) to the NAD may be in a range of from about 130: 1 to about 30: 1, such as about 80: 1.

[0224] Additionally or alternatively, the composition may comprise quercetin or a derivative thereof, as exemplified by the composition “SIS0013-Q” of Examples 3 and 6 hereinbelow. Optionally, the ratio by weight of the one or more lipids (i.e., total lipid components) to the as quercetin may be in a range of from about 130: 1 to about 30: 1, such as about 80: 1.

[0225] The composition may optionally further comprise a peptide containing a cell surface receptor- (for example, integrin-) recognition sequence that confers a degree of cell specificity. The peptide may have a "head group" containing a cell surface receptor recognition sequence and additionally a "tail" that can bind non-covalently to the API (e.g., nucleic acid, such as mRNA) and / or the doped silicon.

[0226] Where the API is a nucleic acid, the composition may optionally further comprise a polycationic nucleic acid-binding component. The term “polycationic nucleic acid-binding component” is well known in the art and may refer to polymers having at least 3 repeats of cationic amino acid residues or other cationic unit bearing positively charged groups, such polymers being capable of complexion with a nucleic acid under physiological conditions. An example of a nucleic acid-binding polycationic molecule is an oligopeptide comprising one or more cationic amino acids. A polycationic nucleic acid-binding component may, for example, be an oligo-lysine molecule, an oligo-histidine molecule, an oligo-arginine molecule, an oligo-ornithine molecule, an oligo-diaminopropionic acid molecule, an oligo-diaminobutyric acid molecule, or a combined oligomer comprising or consisting of any combination of histidine, arginine, lysine, ornithine diaminopropionic acid, and diaminobutyric acid residues. Further examples of polycationic components include dendrimers and polyethylenimine. Subjects and their diseases and disorders

[0227] According to preferred embodiments, the subject to whom the composition disclosed herein is administered, is a human subject. The age of the human subject may be in a range of from 1 month or above, optionally 1 year or above. The age of the human subject may be in a range of less than 100 years.

[0228] The disease or disorder in accordance with the present disclosure may be an infectious disease. As used herein, the term “infectious” may be used to refer to a disease which is liable to be transmitted from one organism to another, especially from one human to another.

[0229] The infectious disease may be a viral, bacterial, fungal, or parasitic disease; especially a viral disease.

[0230] Where the disease is a viral disease, it may be that of a respiratory virus, such, for example, as respiratory syncytial virus (RSV), parainfluenza virus (HPIV), 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 an influenza virus.

[0231] The viral disease may be that of a 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.

[0232] The disease or disorder may be a genetic disease or disorder.

[0233] In some embodiments, the genetic disorder may be characterised by a deficiency in the expression of one or more proteins, especially one or more enzymes. The genetic disorder may be a multifactorial disorder, i.e. not confined to any specific pattern of single gene inheritance and likely to be associated with multiple genes effects together with the effects of environmental factors; such, for example, as schizophrenia, diabetes, asthma, depression, epilepsy, heart disease or hypothyroidism.

[0234] The genetic disorder may involve one or more mutations in one or more genes.

[0235] Thus, the genetic disorder may be a monogenic disorder, liable to occur if at least one mutation occurs in a single gene. Where the genetic disorder is a monogenic disorder, it may involve one mutation in the single gene or more than one mutation in the single gene. Examples of monogenic disorders include sickle cell anaemia, cystic fibrosis, Huntington’s disease or Duchene muscular dystrophy.

[0236] The genetic disorder may involve one or more mutations in more than one gene. By way of non-limiting example, the genetic disorder may involve more than one mutation in a first gene and one mutation in a second gene.

[0237] The genetic disorder may be a disorder liable to occur if at least one mutation occurs in at least one gene amongst a set of genes; especially, such a genetic disorder may be osteopetrosis.

[0238] The genetic disorder may be Angelman syndrome; Canavan disease; Charcot- Marie-Tooth disease; colour blindness; cri du chat syndrome; cystic fibrosis; DiGeorge syndrome; Down syndrome; Duchene muscular dystrophy; familial hypercholesterolemia; haemochromatosis type 1; haemophilia; Klinefelter syndrome; neurofibromatosis; phenylketonuria; polycystic kidney disease; Prader-Willi syndrome; Scheuermann's disease; sickle cell disease; spinal muscular atrophy; Tay- Sachs disease; or Turner syndrome.

[0239] In its broadest sense, as used herein, the term genetic disorder may encompass cancer. The cancer may be or involve a blood cancer (such, for example, as a leukaemia, a lymphoma, or a myeloma) or a solid tumour (such, for example, as a sarcoma; a carcinoma; a carcinosarcoma; or a lymphoma). Thus, especially, the cancer may be a cancer of the blood, skin, brain, prostate, breast, lung, oesophagus, stomach, small intestine, pancreas, colon and / or rectum, central nervous system, urinary bladder, thyroid, kidney, uterine corpus, oral cavity, or ovary.

[0240] In more detail, the cancer may be or involve a cancer of the pulmonary system, a brain cancer, a cancer of the gastrointestinal tract, a skin cancer, a genitourinary cancer, a pancreatic cancer, a lung cancer, a medulloblastoma, a basal cell carcinoma, a glioma, a breast cancer, a prostate cancer, a testicular cancer, an oesophageal cancer, a hepatocellular cancer, a gastric cancer, a gastrointestinal stromal tumour (GIST), a colon cancer, a colorectal cancer, an ovarian cancer, a melanoma, a neuroectodermal tumour, head and neck cancer, a sarcoma, a soft-tissue sarcoma, fibrosarcoma, myxosarcoma, liposarcoma, a chondrosarcoma, an osteogenic sarcoma, a chordoma, an angiosarcoma, an endotheliosarcoma, a lymphangiosarcoma, a lymphangioendotheliosarcoma, a synovioma, a mesothelioma, a leiomyosarcoma, a cervical cancer, a uterine cancer, an endometrial cancer, a carcinoma, a bladder carcinoma, an epithelial carcinoma, a squamous cell carcinoma, an adenocarcinoma, a bronchogenic carcinoma, a renal cell carcinoma, a hepatoma, a bile duct carcinoma, a neuroendocrine cancer, a carcinoid tumour, diffuse type giant cell tumour, or glioblastoma.

[0241] Preparation, storage, stability and administration of the disclosed compositions

[0242] Preventing a disease or disorder in a human subject may comprise administering a prophylactically effective amount of a composition disclosed herein to a human subject, wherein the subject is in need thereof; for example, identified by a physician or other healthcare practitioner as being in need thereof. Meanwhile, treating a disease or disorder in a human subject may comprise administering a therapeutically effective amount of a composition disclosed herein to a human subject in need thereof.

[0243] Dosage amounts of the composition disclosed herein may be varied so as to obtain an amount of the API which is effective to achieve the desired prophylactic and / or therapeutic response for a given subject, without being toxic to the subject. A suitable dosage amount of the composition may be the amount of the composition which is the lowest dosage amount effective for the API to produce a therapeutic and / or prophylactic effect.

[0244] The selected dosage amount, form and regime will each depend upon a variety of factors. Such 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 the treatment, the presence of other drugs, compounds and / or materials used in combination with the API, the age, sex, weight, condition, general health and prior medical history of the subject being treated, and other such factors well known in the medical arts.

[0245] The composition may be administered by intramuscular or intravenous injection (encompassing transdermal delivery via a patch), orally (encompassing sublingual administration), intranasally, dermally, or by any other suitable route; especially by injection or orally.

[0246] Preferably, the composition may be administered by injection, such, for example, as intravenous or intramuscular injection. Optionally when the composition is administered by injection, the subject is monitored for symptoms or signs of a hypersensitivity response, such, for example, as a vaccine-associated hypersensitivity response.

[0247] Also preferably, the composition may be administered orally or intranasally. Compositions suitable for oral administration may be presented as discrete dosage forms, especially liquids or aerosol sprays each containing a predetermined amount of the composition. Such dosage forms may be prepared by any of the well-known methods of pharmacy.

[0248] Thus, it is especially preferred that the composition is a composition comprising hydrolysable silicon particles doped at a level of at least about 1 xlO16dopant atoms per cm3, one or more lipids and an active pharmaceutical ingredient (API), which is for use in a method of preventing or treating a disease or disorder in a human subject, wherein the method comprises administering the composition to the human subject by injection, orally or intranasally (especially, by injection or orally). Suitably, the composition may further comprise one or more amino acids for the reasons described hereinabove. A murine model of administration by injection is set out, for instance, in Example 4 hereinbelow.

[0249] The composition may be combined in an intimate admixture with a pharmaceutical carrier, according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration. Any of the usual pharmaceutical media may be employed as carriers, such as, for example, one or more of water, oils, and alcohols (encompassing glycols). The forms in which the disclosed compositions may be incorporated for administration, especially when formulated for administration by injection, orally or intranasally, may include aqueous solutions in saline. The composition may further include one or more pharmaceutically acceptable additives and excipients, such as one or more of the following: detackifiers, anti-foaming agents, buffering agents, polymers, antioxidants, chelating agents, viscomodulators, tonicifiers, odorants, opacifiers, suspending agents, fillers, plasticizers, flavouring agents, preservatives, colouring agents, diluents, binders, disintegrating agents and mixtures thereof.

[0250] The prevention or attenuation of the action of microorganisms may be brought about by the inclusion of various antibacterial and antifungal agents; such as one or more of the following: parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

[0251] The composition disclosed herein may be provided in a sterile solution, by incorporating the composition in the required amount in an appropriate solvent (with various other ingredients, where appropriate) by any of the well-known methods of pharmacy. The composition disclosed herein may be provided in a sterile dispersion, by incorporating the composition in the required amount in an appropriate sterile vehicle (with various other ingredients, where appropriate). The composition disclosed herein may be provided as a sterile powder (e.g., for the subsequent preparation of sterile injectable solutions), such as by vacuum-drying and freeze-drying (lyophilisation) techniques which yield a powder of the composition.

[0252] Optionally, the composition may be stored before being administered to the subject. The composition may be stored at a temperature in a range of 0 °C or above, especially 4 °C or above, for a period of at least 1 week (optionally, up to 6 months, especially up to 1 year) prior to administering the composition to the subject.

[0253] In certain preferred embodiments, the particles comprising hydrolysable doped silicon and the one or more lipids are stored together but without the API. Shortly before administration to the subject, such as not more than about 3 weeks, about 2 weeks or about 1 week, especially not more than about 2 days, more especially not more than about 1 day before administration to the subject, they will be combined with the API (for example, via steps such as those described in Example 1 hereinbelow). Advantageously, the particles and lipid may be storage stable for an extended period of time, especially a period of several months, such as about 6 months or about 12 months; they may be stored in this way without appreciable degradation. When the API is a reactive API, especially mRNA, it may advantageously be synthesised in situ or synthesised off-site and delivered to a clinical setting at the last possible moment, then combined with the particles and lipid(s).

[0254] Nonetheless, it has also been found that an API can be stabilised by the particles comprising hydrolysable doped silicon, including that the API can be stabilised during storage. Thus, in some embodiments, the particles can increase the storage stability of the API, especially where the API is or comprises nucleic acid such as mRNA. In some embodiments, the particles can increase the stability at 25 °C of the API, especially where the API is or comprises nucleic acid such as mRNA.

[0255] Thus, optionally, the composition being stored comprises all of the following components: particles comprising hydrolysable doped silicon; one or more lipids; and an active pharmaceutical ingredient (API). If so, the composition, once formulated, may optionally be stored for only a short period, by which may be meant a period of up to about 3 weeks, about 2 weeks or about 1 week prior to administration to the subject.

[0256] In some embodiments, the particles can increase the stability of the API during circulation in vivo, especially where the API is or comprises nucleic acid such as mRNA. In some embodiments, the particles can protect the API from degradation, especially enzymatic degradation, especially where the API is or comprises nucleic acid such as mRNA. Thus, the doped silicon-containing compositions disclosed herein may mitigate or address the problem of how to ensure APIs reach cells once they have been administered to a patient, including how to stabilise APIs while they are circulating in the body.

[0257] Meanwhile, the disclosed composition comprising hydrolysable doped silicon may mitigate or meet the need for tissue or cell targeting, so that an API can be delivered to the correct cells.

[0258] Additionally or alternatively, once a target cell is reached, the disclosed composition may mitigate or solve the challenge of how to ensure efficient API uptake by the cell. For example, the disclosed composition may assist trafficking of the API from the exterior of the cell into the cytoplasm.

[0259] Following uptake of the API by a cell, the disclosed composition may mitigate or solve the problem of how to prevent the API degrading too quickly in the cytoplasm. It is thought that the particles can increase the stability of the API in the cytoplasm of a cell, especially stability against enzymatic degradation, especially where the API is or comprises nucleic acid such as mRNA. Examples

[0260] Examples 1 and 2 disclose the investigation of mRNA complexation with delivery vehicles containing undoped vs. doped silicon.

[0261] Particle size and zeta potential measurements were obtained; gel retardation assays and Quant-iT RiboGreen RNA assays were performed. mRNA transfection by delivery vehicles containing doped silicon was also investigated through in vitro transfection of human embryonic kidney (HEK293) cells with luciferase-coding mRNA, observed by bioluminescence imaging of cell cultures.

[0262] Example 1

[0263] Silicon particle-containing delivery vehicles with (“SIS0013”) or without (“SIS0012”) dopant were compared for their ability to entrap and stabilise an API and deliver it to cells.

[0264] As the test APIs, two different luciferase mRNAs were selected. The successful transfection of a cell using luciferase mRNA can be measured by a luciferase assay, enabling reliable and accurate determination of the success rate of API delivery to cells in an in vitro culture.

[0265] Moreover, mRNA is a relatively reactive molecule and it can be expected that a delivery vehicle capable of stabilising and successfully delivering mRNA to cells, will be capable of stabilising and successfully delivering other APIs. By studying a reactive API, the confidence that the results of the present studies can be extrapolated to other (e.g., less reactive) APIs is increased.

[0266] Materials and Methods

[0267] Composition of SIS0012 and SIS0013

[0268] SIS0012 (undoped Si) formulation:

[0269] - 7.25 mg DOTAP (1.45 mL)

[0270] - 7.3 mg DOPE (1.46 mL)

[0271] - 1.45 mg DSPE-PEG2000

[0272] 1 mg Si nanoparticles (avg. diameter sub 30 nm, ascertainable by SEM) 1 mg trehalose

[0273] 0.5 mg glycine

[0274] 9 mL nuclease-free water

[0275] SIS0013 (doped Si) formulation:

[0276] - 7.25 mg DOTAP (1.45 mL)

[0277] - 7.3 mg DOPE (1.46 mL)

[0278] - 1.45 mg DSPE-PEG2000

[0279] 1 mg boron-doped Si nanoparticles, circa 5x1018boron atoms / cm3(avg. diameter sub 30 nm, ascertainable by SEM)

[0280] 1 mg trehalose

[0281] 0.5 mg glycine

[0282] 9 mL nuclease-free water

[0283] Additional details of the above-disclosed components of SIS0012 and SIS0013 are as follows. a) Si (“SiNPs”): as porous silicon particles of sub 30 nm average diameter. Doped for SIS0013 (5xl018boron atoms / cm3). Activated by exposure to methanol (to remove surface oxidation and ensure particle uniformity) followed by slow evaporation, producing (dry, solid) powder, which is activated SiNPs. b) Trehalose (“THR”): as solid powder. c) Glycine (“GLY”) : as solid powder. d) SiNPs + GLY + THR solution: as a brownish suspension, obtained via sonication for 60 minutes of a suspension in 50 ml nuclease-free water of: 50 mg activated SiNPs; 50 mg THR; and 25 mg GLY. e) DOTAP-C1 Solution: as a solution obtained by mixing 50 mg DOTAP in 10 ml of methanol, followed by sonication at 40 °C for 30 minutes, until fully dissolved. f) DOPE Solution: as a solution, obtained by mixing 50 mg DOPE in 10 ml of methanol, followed by sonication at 40 °C for 30 minutes, until fully dissolved. g) mPEG2000-DSPE Solution: as a solution, obtained by mixing 40 mg of mPEG2000-DSPE in 8 ml of methanol, followed by sonication at 40 °C for 30 minutes, until fully dissolved.

[0284] Method for preparing SIS0012 or SIS0013

[0285] Lipid film preparation a) Mix all lipids (DOPE, DOTAP and mPEG2000-DSPE) in a glass round bottomed flask. b) Evaporate solvent with rotary evaporator in water bath at 40 °C.

[0286] Rehydration of the film a) Add 1ml of the brownish suspension of SiNPs (SIS0013: doped; SIS0012: undoped) + GLY + THR (Img / ml SiNPs; 0.5 mg / ml GLY; Img / ml THR) to the lipid film, along with 9 ml nuclease free water (total volume of the brownish suspension and nuclease free water together is thus 10 ml). b) Cover the flask with parafilm, then agitate the flask in a water bath at 60 °C for 10 minutes, thus rehydrating the lipid film by means of the 10ml of liquid. c) Leave resultant suspension to rest at room temperature for a few hours before storing at 4 °C.

[0287] Extrusion

[0288] Pass the suspension obtained in step (c) of "Re hydration of the film" through a polycarbonate membrane filter having 0.4 pm and 0.1 pm pore diameters. Pass the suspension 10 times, at 60 °C, through each pore diameter.

[0289] The resultant products are SIS0012 where undoped SiNPs are used and SIS0013 where doped SiNPs are used. Luciferase-coding mRNAs

[0290] The details of the two luciferase-coding mRNAs used as test APIs are as follows.

[0291] (1) Firefly luciferase-coding mRNA having 2315 bases (mod-LUC RNA; 2mg / mL) was obtained (referred to herein as “LUC mRNAl”, “Luc mRNAl” or simply “mRNAl”).

[0292] (2) Meanwhile, EZ Cap™ Firefly Luciferase mRNA having 1921 bases (modified by 5-moUTP), also luciferase-coding, was obtained (referred to herein as “LUC mRNA2”, “Luc mRNA2” or simply “mRNA2”)

[0293] Materials for gel electrophoresis and RiboGreen assays

[0294] The materials used for the gel electrophoresis assays and RiboGreen assays of the present Example (and of subsequent Examples where analogous assays were used) are set out in Table 1.

[0295] Table 1 - Materials for gel electrophoresis and RiboGreen assays Materials for luciferase assays and HEK293 cultures

[0296] The materials used for luciferase assays and HEK293 cultures of the present Example (and of subsequent Examples where analogous cultures were used) are set out in Table 2.

[0297] Table 2 - Materials for luciferase assays and HEK293 cultures

[0298] Method for preparation of SIS0012-mRNA or SIS0013-mRNA complexes

[0299] For preparation of SIS0012-mRNA or SIS0013-mRNA complexes with different mRNA loading w / w ratios, the required volumes of SIS0012 or SIS0013 suspension (with a nominal total lipid concentration of 1.6 mg / mL) were mixed with the required volumes of mRNA stock solution (with concentration of 2 mg / mL) and the final concentration of LUC mRNA was adjusted using nuclease-free water before the complexation incubation step according to the subsequent assay protocol. The samples were mixed thoroughly by gently pipetting and were incubated at room temperature for 60 min to allow for complexation to complete. Following incubation, samples were stored at 4 °C prior to their use in subsequent assays. For the Examples hereinbelow wherein another API is used instead of mRNA, an analogous procedure for API loading was followed.

[0300] Table 3 - mRNA: SIS0012-mRNA or SIS0013-mRNA complexes at different w / w ratios

[0301] Method for particle size and zeta potential measurements

[0302] Particle sizes, poly dispersity and zeta potentials were determined using dynamic and electrophoretic light scattering (Malvern Zetasizer Pro (Red Advance), Malvern, UK). Size measurements were performed using DTS0012 disposable cuvettes and surface charge measurements were obtained using a DTS 1070 folded capillary zeta cell. Analysis was performed for empty SIS0012, empty SIS0013, SIS0012-mRNA and SIS0013-mRNA. For size determination, a backscatter detection at an angle of 173 was used. Particle sizes were obtained as the Z-average [nm] by fitting a correlation function using a cumulate method. A 20 pL sample of empty SIS0012, empty SIS0013, SIS0012-mRNA or SIS0013-mRNA was mixed with 980 pL nuclease-free water to make a total of 1000 pL volume before adding into the disposable cuvette. A total of 4 scans were performed to reduce signal to noise ratio, selecting the best 3 scans as an average size and PDI (n = 3). A sample volume of 150 pL was mixed with 850 pL nuclease-free water before inserting into the folded capillary cell (via a ImL syringe) and measured at 25 °C. A total of 5 scans were performed to eliminate signal to noise ratio and the best 3 scans were selected to record an average zeta potential (n = 3). Method for gel retardation assays

[0303] To assess the formation of SIS0012-mRNA or SIS0013-mRNA complexes, nanoparticles dispersed in nuclease-free water were combined with mRNA at the various loading ratios set out in Table 3 (i.e. 1 :4, 1 :7.2, 1 : 12 or 1 :24 w / w of mRNA to total lipids in SIS0012 or SIS0013) and analysed by electrophoresis on an E-Gel™ 1% agarose gel (ThermoFisher, UK) in E-Gel™ Power Snap Electrophoresis Device (ThermoFisher, UK) for 8 mins. An equal amount of 150 ng mRNA was loaded into each well, naked mRNA was used as a control. The gel was visualized using the E- Gel™ Power Snap Electrophoresis Camera (ThermoFisher, UK).

[0304] Method for accessible mRNA assays

[0305] For evaluating the amount of accessible mRNA within the complexes, Quant- iT RiboGreen RNA reagent from Invitrogen (ThermoFisher, UK) was used. The experimental procedure was performed according to the manufacturer’s guidelines. For analysis, Varioskan LUX multimode plate reader (ThermoFisher, UK) was used at an excitation wavelength of 480 nm and an emission wavelength of 520 nm. The test was performed in lx Tris-EDTA (TE)-buffer (10 mM Tris-HCl and 1 mM EDTA). All samples were measured in triplicate, and the results reported as mean ± SD.

[0306] Method for in vitro cell transfection assessments

[0307] A human embryonic kidney (HEK293) cell line was used to evaluate transfection. Cells were cultivated in Dulbecco's Modified Eagle's Medium (Euroclone, Italy) supplemented with 10% inactivated foetal bovine serum (FBS, Gibco, UK), 1% L-Glutamine (Euroclone, Italy) and 1% Pen / Strep (Euroclone, Italy), incubated under 5% CO2 at 37 °C and passaged following standard laboratory procedures. Twenty -four hours prior to transfection, HEK293 cells were seeded at 1 x 104 cells per well on 96- well black plates with transparent bottoms (or 2 * 104 cells per well for assay at the 72h time point) in 100 pl of complete medium without antibiotic. Cells were incubated at standard culture conditions (37 °C, 5% CO2) for 24h and the next day the medium was replaced with lOOpl of transfection medium containing: 10 pl of transfection complex with Ipg mRNA prepared in nuclease free water, 10 pl of serum free DMEM with 1% L-glutamine and diluted with 80uL of complete growth medium (DMEM with 10% FBS and 1% L-glutamine). 10 pg / ml naked mRNA diluted in complete medium or no mRNA (medium only) were used as negative controls.

[0308] Following completion of the transfection procedure, cells were transferred to an incubator and grown at standard conditions (37°C with 5% CO2). The translation product of LUC-mRNA was detected by luciferase assay (Bright-Glo Luciferase kit, Promega, USA) performed at 6h, 24h, 48h or 72h post-treatment following the manufacturer’s protocol. Briefly, the plates were equilibrated at room temperature prior to addition of 100 pl / well assay reagent and were incubated for 15 minutes at room temperature. Luciferase activity was assessed through a luminometer apparatus (Hamamatsu Photonics bioluminescence imager, Hamamatsu Photonics Italy S.R.L, Arese, Italy - integrated with Digital Camera and camera controller, C4742-98 - Software Wasabi, v. 1.5, Hamamatsu Photonics).

[0309] Results

[0310] Dynamic and electrophoretic light scattering results

[0311] The results of the investigation of surface charge of SIS0012 and SIS0013, complexed with LUC mRNAl, at 24: 1, 12: 1, 7.2: 1 and 4: 1 w / w ratios of mRNA to total lipids, are shown in Tables 4a-4d.

[0312] Table 4a - Characterisation of empty SIS0012 and empty SIS0013

[0313] As Table 4a shows, SIS0012 (without mRNA bound) has a more negative zeta potential than SIS0013.

[0314] Table 4b — Characterisation of SIS0012 complexed with LUC mRNAl at different w / w ratios (mRNA : total lipids)

[0315] Table 4c - Characterisation of SIS0013 complexed with LUC mRNAl at different w / w ratios (mRNA : total lipids)

[0316] Figures 1 and 2 display the size and poly dispersity index (PDI) data for SIS0012 and SIS0013 in accordance with Tables 4a to 4c.

[0317] SIS0012 results. As Tables 4a-4b and Figure 1 show, PDI remained quite stable at all binding ratios. However, an increase in nanoparticle hydrodynamic diameter was observed when SIS0012 complexed with mRNA, which plateaued when more mRNA was added, particularly at ratios of 7.2: 1 and 4:1. The plateau suggests mRNA binding saturation was reached. Zeta potential became less positive with increasing mRNA loading and was negative at a 4: 1 ratio (i.e., with the largest amount of mRNA); it is thought that this may be due to the presence of weakly bound / unbound mRNA.

[0318] SIS0013 results. As Tables 4a, 4c and Figure 2 show, PDI increased with increasing mRNA, thus advantageously providing a delivery vehicle which is especially dispersible in an aqueous environment. Over-accumulation of large pieces of lipid appears to have been prevented with SISOO13; whereas this is a known problem with lipid nanoparticles. Meanwhile, a steady increase in hydrodynamic diameter was observed for SIS0013 when complexed with mRNA, without the plateau observed for SIS0012, suggesting mRNA binding saturation was not reached as early for SIS0013 as for SIS0012. Zeta potential decreased with increasing mRNA, but did not fall as far as for SIS0012. Overall, slow and steady particle growth with increasing mRNA was observed, without undesirable lipid accumulation.

[0319] It may be concluded that SIS0013 can be loaded with a greater amount w / w negatively charged mRNA than SIS0012, i.e., displays increased binding efficiency; which may be due to the more positive initial zeta potential of SIS0013, which in turn may be attributed to the modulation of the SIS0013 zeta potential by the dopant boron.

[0320] Gel retardation assay results

[0321] Figure 3 shows the results of gel electrophoresis to study complexation of SIS0012 and SIS0013 with LUC mRNAl.

[0322] Wells were loaded with SIS0012 / LUC mRNAl or SIS0013 / LUC mRNAl complexes (an equal amount in each well) at increasing ratios from 1 :4 to 1 :24 (w / w mRNA to total lipids). By way of explanation: unbound mRNA migrated through the gel, while mRNA bound by SIS0012 or SIS0013 remained immobilized within wells.

[0323] Full binding of nucleic acid was observed for mRNA : total lipid ratios of 1 :7.2, 1 : 12 and 1 :24 for both SIS0012 and SIS0013. No mRNA migrated through the gel at these ratios.

[0324] At a ratio of 1 :4, with the greatest amount of mRNA relative to SIS0012 or SIS0013, some mRNA was observed to migrate through the gel for both SIS0012 and SIS0013.

[0325] Thus, the ratio of 1 :4 provides particular ground for a comparison between SIS0012 and SIS0013. A weaker band was observed for SIS0013 at a ratio of 1 :4. Thus, confirming the results of the above size and zeta potential analysis, SIS0013 displays increased binding efficiency compared to SIS0012. In other words, it appears SIS0013 is able to bind more mRNA, w / w, than SIS0012. When an excess of mRNA is present, SIS0013 binds a greater % w / w of the total mRNA present, than does SIS0012.

[0326] Similar results were obtained for SIS0012 and SIS0013 complexed with LUC mRNA2.

[0327] Thus, SIS0013 may represent the most efficient delivery vehicle, able to bind more mRNA w / w than SIS0012. From this, it may also be derived that SIS0013 may additionally offer improved binding, thus enhanced nucleic acid stability, when mRNA amounts are added which are below the mRNA saturation threshold for SIS0012. At these amounts, the charge interaction between the SIS0013 and mRNA may be more attractive than between otherwise identical SIS0012 and mRNA.

[0328] As described hereinbelow, SIS0013 also shows efficient uptake of mRNA by cells and faster resultant luciferase expression than does SIS0012.

[0329] Accessible mRNA Quant-iT RiboGreen assay results

[0330] To evaluate how much mRNA is effectively bound to SIS0012 and SIS0013 and not accessible to external reactive species, the amount of mRNA accessible to a fluorescent dye was taken as an indirect parameter showing how much unbound or superficially bound mRNA there was following the complexation.

[0331] The dye in the Quant-iT™ RiboGreen™ RNA Reagent has very high binding affinity and high specificity for RNA which allows for quantification of very low concentrations of RNA in samples. The dye is only able to bind to free RNA or RNA which is weakly bound on the outermost surface of a complex, whereas RNA which is more tightly bound in a complex is not accessible to the dye.

[0332] Both SIS0012 and SIS0013 showed low mRNA accessibility at the very lowest ratio of 1 :24, as shown in Figure 4. Table 5 shows higher mRNA accessibility at other ratios, indicating that more mRNA was unbound or weakly bound on the outermost surface of a complex, for SIS0012, than for SISOO13.

[0333] The % of mRNA available to react with the dye, i.e. with an external reactive species, was consistently greater for SIS0012 than SISOO13, particularly with increasing amounts of mRNA and especially at a w / w ratio of mRNA to total lipids of 1 :4, i.e. when the greatest amount of mRNA is present. This suggests that SISOO13 consistently binds more mRNA in a manner that makes the mRNA unavailable to react with external reactive species, than SIS0012.

[0334] It may be deduced that SISOO13 is capable of binding more mRNA less superficially than SIS0012, meaning that SISOO13 may be able to better stabilise mRNA (and other APIs) in the presence of reactive species, such as the Quant-iT™ RiboGreen™ dye. As a result, stabilisation both in storage and in vivo may be improved. One advantage may be the stabilisation of a reactive API such as mRNA after administration and during circulation in vivo, so that enough API reaches target cells for the necessary effect, such as luciferase expression in the present studies. Another advantage may be the stable delivery of an API into a target cell. Meanwhile, there may also occur stabilisation of an API in the cell cytoplasm.

[0335] Table 5 — mRNA accessibility (% superficially bound) for SIS0012 and SIS0013 at different w / w ratios of mRNA to total lipids Cell transfection results

[0336] 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 mRNAl), SIS0012 / luciferase (Luc mRNA2, SIS0013 / luciferase (Luc mRNAl), or SIS0013 / luciferase (Luc mRNA2). Translation product was detected via Bright-Glo luciferase assay, performed at 6h, 24h, 48h or 72h posttransfection, using a luminometer apparatus, as described herein.

[0337] As a guide, Table 6 sets out the contents of each well triplicate:

[0338] Table 6 — Guide to the contents of wells in Figures 5 to 8 Summary: at each time point, SIS0013 was observed to be more effective in mRNA transfection, across a range of mRNA to total lipid ratios, than SIS0012 and especially at earlier time points, suggesting the rate of transfection with SIS0013 is higher than with SIS0012. Thus, doping of hydrolysable silicon appears to cause more effective and faster API uptake, such that the effect of the API on a target cell is more quickly and reliably obtained.

[0339] Both the LUC mRNAl and LUC mRNA2 are chemically modified compared to naturally occurring mRNA. Such chemical modification may have the potential to make transfection more difficult. The increased ability of doped Si-containing SIS0013, compared to undoped Si-containing SIS0012, to provide for transfection of chemically modified mRNA, is particularly advantageous and surprising.

[0340] 6h post-transfection: the data 6h post-transfection is summarised in Table 7 and Figure 5. A luciferase signal was observed in wells treated with SIS0012 or SIS0013 at a 1 :24 w / w ratio (mRNA: total lipids), regardless of the mRNA used (LUC mRNAl or LUC mRNA2), with a more pronounced signal deriving from the doped Si- containing SIS0013 complexes than the undoped Si-containing SIS0012 complexes. SISOO13 also showed a signal at a 1 : 12 w / w ratio (mRNA: total lipids), for mRNA2. The reduced chemical modification of mRNA2 compared to mRNAl provides a rationale why transfection of cells using mRNA2 appears to be marginally more effective than with mRNAl for the same complex SISOO13.

[0341] Table 7 — summary of results shown in Figure 5 at 6 hours post-transfection

[0342] 24h post-transfection: Figure 6 and Table 8 show that at 24h posttransfection, a luminescence signal was observed for SIS0013 complexed to mRNAl or mRNA2, at ratios of 1 :24 and 1 : 12. A signal was also visible at a ratio of 1 :7.2 for SISOO 13 complexed with mRNA2.

[0343] In comparison, SIS0012 showed a signal with mRNAl or mRNA2 at a 1 :24 ratio and only with mRNA2 at a ratio of 1 : 12. Table 8 — summary of results shown in Figure 6 at 24 hours post-transfection

[0344] 48h post-transfection: Figure 7 and Table 9 show the results of the assay at 48h post-transfection. Table 9 - summary of results shown in Figure 7 at 48 hours post-transfection

[0345] 72h post-transfection: Figure 8 and Table 10 show the results of the assay at 72h post-transfection. Table 10 — summary of results shown in Figure 8 at 72 hours post-transfection

[0346] Example 2 - SIS0013 variants with mRNA

[0347] Example 2 discloses investigations carried out on the effect of functionalising the doped Si-containing SIS0013 of Example 1 with different components.

[0348] Materials and methods

[0349] Except where otherwise specified, the same materials and methods were used as in Example 1. SIS0013 variants

[0350] Modified SISOO13 compositions SIS0013-N, SIS0013-T and SISOO13-Q were respectively prepared by adding, as a further component, 0.2mg of NAD, tyrosine (“TYR”) or quercetin (“QUE”) (NAD, TYR and QUE were obtained from Sigma Aldrich). The NAD, TYR or QUE was added together with the Si nanoparticles in step (a) of the “Rehydration of the film" protocol set out in “Materials and Methods" of Example 1 above.

[0351] Results

[0352] Dynamic and electrophoretic light scattering results

[0353] The surface charge of SIS0013-N, SIS0013-T and SIS0013-Q complexed with LUC mRNAl, when empty (i.e., unloaded with mRNA) and at 24: 1, 12: 1, 7.2: 1 and 4: 1 w / w ratios of mRNA to total lipids, was investigated. The results are shown in Tables l la-l lc.

[0354] Table Ila - Characterisation of SIS0013-N with LUC mRNAl at different ratios of total lipids : mRNA

[0355] * Although this is less positive than SIS0012 of Example 1, it is thought to be more positive than if the silicon particles were not doped. Table 11b - Characterisation of SIS0013-T with LUC mRNAl at different ratios of total lipids : mRNA

[0356] * This is less positive than for SIS0013 of Example 1 but more positive than for SIS0012; it is thought to be more positive than if the silicon particles were not doped.

[0357] Table 11c - Characterisation of SIS0013-Q with LUC mRNAl at different ratios of total lipids : mRNA * This is similar to SIS0012 of Example 1, but it is thought to be more positive than if the silicon particles were not doped.

[0358] Overall, sizes were in the ranges of 200-400 nm; however at a 7.2: 1 ratio of total lipids : mRNA, precipitation was observed upon complexation, which led to an increase in size. Without wishing to be bound by theory, it is thought that at a 7.2: 1 ratio, there may have been some undesirable lipid accumulation due to charge interactions between the negatively charged RNA and the positively charged DOTAP. Once a tipping point is reached in the formation of large lipid pieces, precipitation may occur. It is thought that this effect could be modulated by small changes in doping of the Si, and / or by providing counterions, such as from NaCl or KC1. In any event, this data point does not undermine the overall trend.

[0359] Zeta potential measurements followed the trend ofSIS0013 in Example 1 above.

[0360] Gel retardation assay results

[0361] Figure 9 shows the results of gel electrophoresis to study complexation of SIS0013-N, SIS0013-T and SIS0013-Q with LUC mRNAl. Naked mRNA was used as reference control and DNA ladder was used as size guide.

[0362] Accessible mRNA Quant-iT RiboGreen assay results

[0363] Similarly to Example 1, to evaluate how much mRNA is effectively bound to SIS0013-N, SIS0013-T and SIS0013-Q and not accessible to external reactive species, the amount of mRNA accessible to Quant-iT™ RiboGreen™ RNA fluorescent dye was taken as an indirect parameter showing how much unbound or superficially bound mRNA there was following the complexation.

[0364] Similar behaviour was observed to SIS0013 in Example 1 above. In more detail: all formulations showed a low mRNA accessibility at a w / w ratio of mRNA to total lipid of 1 :24. At mRNA to total lipid w / w ratios of 1 :12, 1 :7.2 and 1 :4, SIS0013-N, SIS0013-T and SIS0013-Q showed lower mRNA accessibility, thus better shielding against external reactive species, than SIS0013; with a more pronounced effect observed for SIS0013-T. These results are shown in Figure 10.

[0365] Cell transfection results

[0366] Similarly 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 using SISOO 13 / luciferase (Luc mRNAl), SISOO 13 / luciferase (Luc mRNA2), SIS0013-N / luciferase (Luc mRNAl), SIS0013-N / luciferase (Luc mRNA2), SIS0013-T / luciferase (Luc mRNAl), SIS0013-T / luciferase (Luc mRNA2), SIS0013-Q / luciferase (Luc mRNAl) and SIS0013-Q / luciferase (Luc mRNA2). A w / w mRNA : total lipid ratio of 1 :24 was selected for investigation. The 1 :24 ratio worked for SIS0013 at 6h, 24h and 48h post-transfection in Example 1 and can therefore be expected to provide a good basis for comparison between SIS0013 and its variants at 6h, 24h or 48h post-transfection.

[0367] Translation product was detected via Bright-Glo luciferase assay, performed at 6h, 24h or 48h post-transfection, using a luminometer apparatus, as described in Example 1.

[0368] At 6 h post-transfection, all SIS0013 variants showed transfection for both LUC-mRNA models (1 and 2), with the most pronounced signal for SIS0013 complexed with mRNA2. The results are shown in Figure 11.

[0369] Similarly to the results observed in Example 1, higher luciferase activity was observed at 24h. The results are shown in Figure 12.

[0370] At 48 h, all SIS0013 variants showed transfection for both LUC-mRNA models (1&2), with more pronounced signals for SIS0013 and SIS013-T. The results are shown in Figure 13.

[0371] These results demonstrate that even when doped Si is combined with a variety of quite different components, its advantageous effects are maintained; NAD, QUE and TYR differ from each other significantly in their properties and behaviour.

[0372] In order to assess the statistical significance of the differences in luminescence between SIS0013 and its variants, a statistical analysis was performed for the time point with the greatest luminescence intensity, i.e. 24 h.

[0373] The analysis was done by means of ROI via software elaboration and by calculating the Mean ± SD value for each triplicate variant series of SIS0013, SIS0013- N, SIS0013-Q and SIS0013-T complexed with mRNA. The maximum value of the ROI was set for the SIS0013 complexed with mRNA2 followed by RM one way ANOVA analysis. The statistical analysis, shown in Figures 14 to 16, confirmed SIS0013 and SIS0013-T are the transfection formulations resulting in the greatest luminescence, which may mean they provide the most effective delivery vehicles. It is notable that a modulation of the intensity signal appears to be obtainable through SIS0013 functionalisation with different components, such as SIS0013-T, SIS0013-Q and SIS0013-N. Nonetheless, all the tested formulations were satisfactorily effective.

[0374] More specifically, Figure 14 is a graphical plot of luminescence intensity at 24h post transfection for SIS0013 / luciferase (Luc mRNAl), SIS0013 / luciferase (Luc mRNA2), SIS0013-N / luciferase (Luc mRNAl), SIS0013-N / luciferase (Luc mRNA2), SIS0013-T / luciferase (Luc mRNAl), SISOO 13 -T / luciferase (Luc mRNA2), SIS0013- Q / luciferase (Luc mRNAl) and SIS0013-Q / luciferase (Luc mRNA2). Values are reported as mean values ± SD (analysis performed in triplicate). Adjacent histogram columns were plotted for head-to-head comparison of transfection efficiency achieved with LUC mRNAl complexes vs. the corresponding LUC mRNA2 complexes.

[0375] Figure 15 shows statistical analysis of the luminescence signal intensity for SISOO 13 and its variants, complexed with mRNAl (RM One way ANOVA, with Geisser Greenhouse correction, followed by Dunnett’s multiple comparison test, with individual variance computed for each comparison; analysis of luminescence signal at 24 hrs, *p=0.05).

[0376] Meanwhile, Figure 16 shows statistical analysis of the luminescence signal intensity for SISOO 13 and its variants, complexed with LUC mRNA2 (RM One way ANOVA, with Geisser Greenhouse correction, followed by Dunnett’s multiple comparison test, with individual variance computed for each comparison; analysis of luminescence signal at 24 hrs, *p=0.05).

[0377] Example 3 - cell viability studies

[0378] In this example, cell viability was investigated for cells transfected with mRNAl or LUC mRNA2 using SIS0013 and its variants SIS0013-N, SIS0013-T and SIS0013-Q. Cells were monitored for cytotoxicity. The results were compared to the viability of cells transfected with mRNAl or LUC mRNA2 using commercial transfection reagents, DharmaFect 1 and Lipofectamine 3000.

[0379] Materials and Methods

[0380] Except where otherwise specified, the same materials and methods were used as in Example 1.

[0381] The same time points post-transfection (6h, 24h, 48h, 72h) and mRNA : lipid w / w ratios (1 :4, 1 :7.2, 1 : 12, 1 :24) were investigated, as in Example 1.

[0382] Dharmafect 1 and Lipofectamine 3000

[0383] Commercial transfection reagents DharmaFect 1 and Lipofectamine 3000 were obtained as set out in Table 12.

[0384] Table 12 - Dharma Feet 1 and Lipofectamine 3000

[0385] Results

[0386] Cell transfection results

[0387] Under otherwise identical conditions (including mRNA : total lipid ratio w / w), DharmaFect 1 and Lipofectamine 3000 displayed cytotoxicity by 24h following transfection (as evidenced by increased LDH signal and Caspase 3 / 7 signal, in addition to altered morphology when cells are analysed under the microscope), while the SIS0013, SIS0013-T, SIS0013-Q and SIS0013-N formulations showed no alteration of cell morphology and cell viability at any analysed time point.

[0388] This indicates that SIS0013, SIS0013-T, SIS0013-Q and SIS0013-N have an improved cell safety profile, compared to conventional transfection reagents such as DharmaFect 1 and Lipofectamine 3000. Example 4 - An in vivo study on osteopetrosis type II (AD02)

[0389] AD02 is a heritable osteosclerotic bone disorder that results from dysfunctional osteoclast activity. AD02 is caused by missense mutations in the chloride channel 7 (CLCn7) gene and is characterized by osteosclerotic with multiple fractures. AD02 can result in osteomyelitis, sight impairment (as osteopetrosis may lead to an occlusion of the macul r hole, thereby compressing the optic nerve ) and bone marrow failure (Alam et al., 2017, Bone, 94:34-41). Currently, there is no cure for AD02.

[0390] In this example, a mouse model was used to investigate silencing of ClCn7G213R in AD02 mice, using SIS0012 or SIS0013 as an siRNA delivery platform. Results were compared between SIS0012 and SIS0013.

[0391] Materials and Methods

[0392] The same protocols were used as in Example 1. Instead of the mRNA of Example 1, siRNA was used, loaded in an analogous protocol to that set out for mRNA in Example 1 above. siRNA specific for ClCn7G213R siRNA specific for ClCn7G213R was obtained as set out in Capulli et al., 2015, Clin. Molec. Therap. 4, e248 (incorporated herein by reference in its entirety).

[0393] Naming conventions for the constructs used are set out in Table 13.

[0394] Table 13 — Constructs used in Example 4

[0395] AD02 mutant mice (age: 10 days) were obtained as set out in Alam et al., 2017, Bone, 94:34-41 (incorporated herein by reference in its entirety). Mice were intraperitoneally injected with one of the four constructs set out in Table 13 (n=5 mice per group). Injection was repeated 3 times per week for a total duration of 2 weeks.

[0396] Results

[0397] ClCn7G213R expression in mouse PMBCs was assayed for each treatment group; the results are shown in Figure 17. Meanwhile, Figure 18 shows bone expression of ClCn7G213R in mouse femur. Figure 19 shows CTX blood test results (CTX is a marker for bone turnover). The results are summarised in Table 14.

[0398] Table 14 — AD02 mice study results

[0399] Both AD02+SIS0012-siRNA (undoped Si) and AD02+SIS0013-siRNA (doped Si) led to a statistically significant downregulation of ClCn7G213R (p<0.02).

[0400] However, surprisingly, administration of ADO2+SIS0013 -siRNA (doped Si) additionally resulted in faster bone turnover, when compared to AD02+SIS0012- siRNA (undoped Si) (Figure 19, CTX evaluation). Thus, a doped Si-containing delivery vehicle appears to enable enhanced symptom reduction of AD02 treated with siRNA, compared to the undoped Si-containing delivery vehicle.

[0401] Example 5 - Stabilisation of Alkaline Phosphatase by SIS0012 vs. SISOO13

[0402] Example 5 investigates the stabilisation of a pH and temperature sensitive protein API (i.e., a relatively fragile API, but of a different nature to mRNA and siRNA investigated in the preceding Examples) by SIS0012 (containing undoped Si) and SIS0013 (containing doped Si).

[0403] The investigated API was alkaline phosphatase, an enzyme that exists in various forms, catalyses the degradation of various proteins, and may be found in all tissues in the human body. Alkaline phosphatase exhibits a significant loss of activity at low pH and at high temperature.

[0404] Materials and methods

[0405] The same protocols were used as in Example 1, except that instead of the mRNA of Example 1, alkaline phosphatase was used as the API, loaded in the protocol set out below.

[0406] Alkaline phosphatase, isolated from bovine intestine and supplied as a recombinant enzyme of 56 kD, expressed in the yeast, Pichia Pastoris. was obtained from Sigma Aldrich / Merck (The Old Brickyard, New Rd, Gillingham, Dorset, SP8 4XT). An aqueous stock solution of alkaline phosphatase (ALP) was prepared at a concentration of 1 U / ml. 1 U (pmol / min) is defined as the amount of ALP that catalyses the conversion of one micromole of PNPP per minute at 37 °C and pH 7.4.

[0407] A 20 mM solution of 4-nitrophenyl phosphatase (PNPP) was prepared using Tris Buffer at pH 7.4 (lOOmM / L).

[0408] ALP solutions were prepared at the following concentrations: 0.1, 0.5, 1, 5, 10, 50, and 100 mU / ml from the lU / ml stock solution, in 15ml test tubes. These were then mixed, in Eppendorf tubes, with the prepared 20 mM solution of PNPP Tris buffer. The tubes were incubated in a water bath at 37 °C for 30 minutes, following which, UV-Vis absorbance measurements were made at 405nm as shown in Figure 20.

[0409] ALP was loaded onto SIS0012 and SIS0013 as follows.

[0410] 1. 3 sets of 8 Eppendorf tubes were prepared:

[0411] A 8 Eppendorf tubes were prepared, each containing 50pL of ALP (50mU / ml) and 500pL of SIS0012 (undoped Si). After adding these components to the tubes, they were mixed, vortexed and refrigerated overnight.

[0412] B 8 Eppendorf tubes were prepared, each containing 50pL of ALP (50mU / ml) and 500pL of SIS0013 (boron doped Si). After adding these components to the tubes, they were mixed, vortexed and refrigerated overnight.

[0413] C 8 Eppendorf tubes were prepared, each containing 50pL of ALP (50mU / ml) and 500pL of Tris buffer. After adding these components to the tubes, they were mixed, vortexed and refrigerated overnight.

[0414] 2. Following their preparation, all Eppendorf tubes were placed in a water bath at 50 °C. Each of the eight tubes, in each of the three sets of tubes A to C, was removed from the water bath after 1, 2, 5, 10, 20, 40 or 60 minutes. 3. 300pL of PNPP was then added to all Eppendorf tubes in all sets A to C. The tubes were mixed and vortexed. They were then placed in a water bath at 37 °C for 30 minutes, during which time dephosphorylation of the PNPP occurred.

[0415] 4. Following this, UV- Vis analysis was carried out on all samples in all sets A to C (at 405nm). The results are shown in Figure 21. In more detail: alkaline phosphatase activity was monitored by measuring changes in the concentration, using UV-Vis absorbance as a proxy, of its substrate 4-nitrophenyl phosphatase (PNPP) the structure of which is set out below. The greater the PNPP concentration, the lower the alkaline phosphatase activity, thus the more this protein API had degraded, thus the less it was stabilised. -

[0416] Results

[0417] Free alkaline phosphatase showed significant degradation compared to both SIS0012 and SIS0013. The activity of free alkaline phosphatase decreased with increasing incubation time at 50 °C.

[0418] Surprisingly, the activity of alkaline phosphatase loaded on SISOO13 (doped Si) was approximately 20-30 % higher than the activity of alkaline phosphatase loaded on SIS0012. It is thought that this is due to improved stabilisation against degradation of alkaline phosphatase, by doped Si compared to undoped Si.

[0419] Example 6 - SIS0013 variants with siRNA

[0420] Example 6 investigates the effect of varying functionalisation of SIS0013 on its function as a delivery vehicle for siRNA. Materials and methods

[0421] Except where otherwise specified, the same materials and methods were used as in Example 2. Instead of the mRNA of Example 2, siRNA was used, loaded in an analogous protocol to that set out for mRNA in Example 1 above.

[0422] The complexation of SIS0013-N, SIS0013-Q and SIS0013-T with siRNA specific for ClCn7G213R and having dTdT overhangs (obtained as set out in Capulli et al., 2015, Clin. Molec. Therap. 4, e248) was investigated.

[0423] Results

[0424] Figure 22 shows gel electrophoresis results that indicate siRNA was successfully and fully bound in these formulations.

[0425] Dynamic light scattering measurements were also performed, using a Zetasizer (obtainable from Malvern Instruments) to assess size and charge, both prior to and following the formation of siRNA complexes, as shown in the table below. As the table indicates, size increases were observed after siRNA complexation, along with a 10- 15mV drop in surface charge.

[0426] Table 15 — size, PDI and zeta potential of SIS0013-N, SIS0013-Q and SIS0013-T before and after complexation with siRNA Materials and methods - continued

[0427] The omission of DOTAP from the NAD, TYR or QUE-containing compositions SIS0013-N, SIS0013-T and SISOO13-Q was investigated in order to determine the behaviour of doped Si-containing compositions upon further variance of other components in the composition besides the doped Si.

[0428] Thus, DPPC / DOPE formulations were prepared following analogous protocols to those of Examples 1 and 2 above except omitting DOTAP from the total lipids. Tests were performed with (i) 0.2 mg or (ii) 1 mg of NAD, TYR and QUE. The formulations are set out in Tables 16 to 18 below.

[0429] Table 16 - Composition of DPPC / DOPE SIS0013 functionalised with beta nicotinamide adenine dinucleotide (NAD). Table 17 - Composition of DPPC / DOPE SIS0013 functionalised with quercetin (QUE).

[0430] Table 18 - Composition of DPPC / DOPE SIS0013 functionalised with tyrosine (TYR)

[0431] | Formulation | Lipid phase | Hydrating solution

[0432] Results - continued

[0433] ZetaSizer measurements were obtained, showing negative zeta potentials across all formulations, irrespective of the amount (0.2 mg or 1 mg) of NAD, TYR or QUE, as shown in Table 19 below.

[0434] Table 19 - DPPC / DOPE SIS0013 functionalised with 0.2mg or 1 mg of NAD, TYR and QUE. It is thought that the differentially functionalised compositions of Example 6 are likely to display API stabilisation and delivery behaviour similar to SIS0013.

[0435] Example 7 - Lipopeptide components

[0436] Lipopeptides, also known as peptide amphiphiles (PA), were explored as yet another alternative component of compositions containing doped silicon particles. It is thought that lipopeptides may offer a solution to the problem of replacing or reducing the amount of cationic lipids in transfection compositions. Lipopeptides consist of an alkyl chain conjugated to a peptide sequence. It is thought that their alkyl chain may be assimilated in lipid bilayers, while the surface of the bilayer is decorated with the peptide moiety.

[0437] An exemplary PA is the molecule palmitoyl pentapeptide-4 (abbreviated as PAL-KTTKS). The two cationic lysine residues may perform a similar function to cationic lipid, such as DOTAP, exhibiting an electrostatic interaction with negatively charged APIs such as RNA.

[0438] Materials and methods

[0439] DPPC and DOPE were selected as neutral lipids to formulate with PAL- KTTKS.

[0440] Full details of the DPPC, DOPE and PAL-KTTKS-containing formulation are provided in Table 20 below.

[0441] Table 20 - DPPC, DOPE and Pal-KTTKS with boron-doped silicon nanoparticles.

[0442] The formulation had a positive zeta potential (measured with a Zetasizer, obtainable from Malvern Instruments), of 54.38 ± 2.11. Although this is lower than SIS0012 of Example 1, it is thought to be more positive than if the silicon particles were not doped.

[0443] It is thought that during assembly of the lipid film, PAL-KTTKS is arranges itself in the lipid bilayer with the peptide portion exposed on the nanoparticle surface. Furthermore, the lysine residues on that surface may contribute positive charge to the formulation.

[0444] The formulation was assessed for its ability to electrostatically bind to siRNA specific for ClCn7G213R and having dTdT overhangs (obtained as set out in Capulli et al., 2015, Clin. Molec. Therap. 4, e248); and to ALDEVRON DASHER GFP mRNA.

[0445] Results

[0446] Gel electrophoresis analysis was performed. Full complexation was not observed for siRNA. Well 3 of Figure 23 shows the gel electrophoresis results for siRNA.

[0447] Full complexation was observed for mRNA. Well 3 of Figure 24 shows the gel electrophoresis results for mRNA.

[0448] To address the partial complexation of siRNA with DPPC / DOPE / PAL-KTTKS, an alternative loading method was adopted. In the alternative loading method, compared to the protocol described previously, the following steps were adopted:

[0449] 1. A thin lipid film was prepared by dissolving DPPC, DOPE and Pal-KTTKS in methanol and evaporating using a rotary evaporator.

[0450] 2. The lipid film was rehydrated with a suspension containing boron-doped silicon with trehalose and glycine as well as either siRNA or mRNA. Rehydration was performed at 40 °C for 10 mins to ensure no lipid-silicon film remained on the walls of the round bottomed rotary evaporation flask.

[0451] Well 4 of Figure 25 shows gel electrophoresis of the complex after the alternative loading method was used, indicating successful complete complexation of siRNA.

[0452] Lipopeptides are highly versatile molecules; they may be fine-tuned, by altering their alkyl chain and / or their peptide sequence. It is thought that customisation of the peptide sequence may enhance cell and / or tissue targeting. In the field of gene therapy, customisation of the peptide may enhance electrostatic interactions with nucleic acids, such as RNA, particularly mRNA. As an example, PAL-KTTKS, when formulated with DPPC and DOPE, resulted in a positively charged surface, as confirmed by zeta potential.

[0453] Meanwhile, lipopeptides, being amphiphilic molecules, have very similar properties to surfactants, which can self-assemble to form micelles; it is thought that this is due at least in part to the alkyl chain being amenable to hydrophobic interactions, whilst the peptide sequence can form inter-molecular hydrogen bonding. Phospholipids, such as DPPC and DOPE, are also able to self-assemble into liposomes. Thus, when incorporating PAs, of which PAL-KTTKS is a representative example (although other lipopeptides may be used) the alkyl chain is able to form hydrophobic interactions with DPPC and DOPE leading to liposomal structures.

[0454] At the same time, the silicon nanoparticles provide structural stability for the complex as a whole. As evidenced by the preceding examples, doped Si particles are able to interact with lipid(s), including lipopeptide(s); and with other species such as NAD, QUE or TYR, via non-covalent (electrostatic) interactions, thus promoting longterm stability and effective delivery of an API.

[0455] Example 8

[0456] Example 8 compares transfection by SIS0012 and SIS0013 in a further cell line, L6C5 (a commercially available musculoskeletal mouse cell line).

[0457] Materials and methods

[0458] Similarly to Example 1, cells were transfected with SIS0012 / luciferase (Luc mRNAl), SIS0012 / luciferase (Luc mRNA2), SIS0013 / luciferase (Luc mRNAl), or SIS0013 / luciferase (Luc mRNA2). Translation product was detected via Bright-Glo luciferase assay, performed at 24h post-transfection, using a luminometer apparatus, as described herein. Results

[0459] Results are shown in Figure 26. Luciferase activity was higher when SIS0013 was used, for both mRNAl and mRNA2, than when SIS0012 was used. This suggests improved transfection efficiency for SIS0013 than SIS0012.

[0460] ***

[0461] Where in the foregoing description, features or limitations are mentioned which have equivalents that are known, evident or foreseeable to those skilled in the art in the light of the present disclosure, then such equivalents are incorporated herein as if particularly set forth. Reference should be made primarily to the claims for determining the scope of the subject-matter of the present disclosure. The scope of protection sought by the present application further encompasses any such equivalents. It will also be appreciated by those skilled in the art that features or limitations of the disclosed subject-matter that are described as preferable, suitable, advantageous, convenient or the like may be optional and may not limit the scope of the independent claim(s) or the protection sought unless explicitly stated otherwise. Moreover, it is to be understood that such optional features or limitations, while of potential benefit in some implementations of the disclosed subject-matter, may be undesirable, and may therefore be absent or omitted in other implementations.

Claims

Claims1. A composition comprising:(i) particles comprising hydrolysable doped silicon;(ii) one or more lipids; and(iii) an active pharmaceutical ingredient (API).

2. A composition according to claim 1, wherein the particles comprising hydrolysable doped silicon differ in zeta potential compared to otherwise identical particles comprising hydrolysable undoped silicon, thereby attracting, binding and / or stabilising the API.

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

4. A composition according to claim 3, wherein the particles comprising hydrolysable p-doped silicon have a more positive zeta potential compared to particles comprising hydrolysable undoped silicon, thereby attracting, binding and / or stabilising 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, especially phosphorus.

7. A composition according to claim 6, wherein the particles comprising hydrolysable n-doped silicon have a more negative zeta potential compared to particles comprising hydrolysable undoped silicon, thereby attracting, binding and / or stabilising 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. A composition according to any preceding claim, wherein the API is neutral or zwitterionic at a pH of about 7.4.

10. A composition according to any preceding claim, wherein the hydrolysable doped silicon particles are doped at a level of at least about 1 xlO16dopant atoms3 per cm .

11. A composition according to any preceding claim, wherein the hydrolysable doped silicon particles are doped at a level of up to about 1 xlO20dopant atoms3 per cm .

12. A composition according to any preceding claim, wherein the API is or comprises a nucleic acid.

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

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

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

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

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

18. A composition according to any preceding claim, wherein the composition further comprises an amino acid.

19. A composition according to any preceding claim, wherein the composition comprises tyrosine.

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

21. A composition according to any preceding claim, wherein the composition further comprises a non-reducing disaccharide, such as trehalose.

22. A composition according to any preceding claim, wherein the composition further comprises NAD.

23. A composition according to any preceding claim, wherein the composition further comprises quercetin.

24. A composition according to any preceding claim, wherein the one or more lipids comprise an ionisable lipid.

25. A composition according to claim 24, wherein the one or more lipids comprise a lipid which has a net positive charge at a pH of 7.4.

26. A composition according to any preceding claim, wherein the one or more lipids comprise one or more lipidated oligopeptides.

27. A composition according to claim 26, wherein the one or more lipidated oligopeptides each contain an oligopeptide moiety having about 3 to about 20 amino acid residues and a fatty acid chain having 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 of the API, especially enzymatic degradation.

29. A composition according to any preceding claim, wherein the API, in the absence of the particles comprising hydrolysable doped silicon and the one or more lipids, has a half-life in vivo of less than about 1 hour.

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

31. A composition according to any preceding claim, wherein the particles increase the intracellular stability of the API.

32. A composition according to any preceding claim, wherein the particles increase the stability at about 25 °C of the API.

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 on the surface of the particles.

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

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

38. A composition according to claim 37, wherein within the one or more aggregates the particles comprising hydrolysable doped silicon have an average diameter in a range of from about 1 nm to about 50 nm.

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

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

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

42. A composition according to any one of claims 37 to 41, wherein the one or more lipidic structures are or comprise one or more of: liposomes, incomplete liposomes, micelles, incomplete micelles, and lipid globules.

43. A composition according to any preceding claim for use in a method of preventing or treating a disease or disorder 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 a human cell with the nucleic acid.

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

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

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

48. A 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, orally, dermally or intranasally, especially by injection or orally.

49. A 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 response.

50. A composition for use according to claim 49, wherein the hydrolysable doped silicon particles are doped at a level of at least about 1 xlO16dopant atoms per cm3and the method comprises administering the composition to the human subject by injection, orally or intranasally.

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

52. A method for slowing the degradation of an API, comprising:(i) doping particles comprising hydrolysable silicon to change their zeta potential compared to otherwise identical undoped particles; and(ii) contacting the doped particles with one or more lipids and the API.

53. A method according to claim 52, wherein step (i) comprises doping the particles with a p-dopant to render their zeta potential more positive compared to otherwise identical undoped particles.

54. A method according to claim 53, wherein the API has a net negative charge at a pH of about 7.4, especially where the API is a nucleic acid.

55. A method according to claim 52, wherein step (i) comprises doping the particles with an n-dopant to render their zeta potential more negative compared to otherwise identical undoped particles.

56. A method according to 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 disorder, comprising administering a prophylactically or therapeutically effective amount of a composition as defined in any one of claims 1 to 51 to a human subject in need thereof.

58. A method according to claim 57, having one or more of the features of a 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 of the features of the composition as defined in any one of claims 1 to 51.

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