Method of encapsulating a biomolecule in silica

Abstract

A method is disclosed for the encapsulation of a biomolecule in a silica shell to form a particle, the method comprising: a) Hydrolysing a mixture of silica precursors, said mixture comprising a funct

Description

Background Biomolecules are increasingly being used as pharmaceuticals, including as vaccines. Biomolecules at the macromolecular level have a range of specific shapes and configurations necessary for their activity. Biomolecules can include, but are not limited to, proteins (e.g., antibodies and enzymes), peptides, viruses, virus-like particles, nucleic acids (e.g., including complexed nucleic acids such as nucleic acid lipid nanoparticles (LNP)s ) or cells. Biomolecules are increasingly being used as vaccines or biopharmaceuticals. However, biomolecules are prone to instability over time, especially in high temperatures. In some instances, biomolecules unfold and / or lose their specific shape or configuration, leading to loss of function. Additionally, or alternatively, biomolecules are prone to chemical or biological degradation. The storage and distribution of biomolecules therefore relies on a “cold chain” of continuous refrigeration, however, use of a cold chain is costly. Furthermore, breaks in the cold chain can occur due to various factors such as an inconsistent supply of electricity. Therefore, there is a need to store and transport biomolecules in a way which protects them from denaturation and removes the reliance on cold chain. The standard method of vaccine preservation is thermostabilisation. This process involves freeze-drying or lyophilisation of the biomolecule. However, many biomolecule formulations do not tolerate this process well due to the increase in effective concentration of metals and excipients which create detrimental effects to the biomolecule. Aluminium adjuvanted vaccine formulations, for example, are typically not suitable for lyophilisation. Preservation methods involving sugar glass have also been proposed, but these have not achieved great practical success since these coatings are easily dissolved. Silica coatings have also been proposed to make biomolecules more thermally stable. One prior technique to form a silica coating around a biomolecule is via an “ensilication” method. Using such an “ensilication” method, a non-functionalised silica precursor, such as tetraethylorthosilicate (TEOS), is first hydrolysed before being contacting with the surface of the biomolecule. The resultant silica grows and polymerises about the surface of the biomolecule to form a continuous protective layer about the biomolecule which is understood to closely match the shape of the biomolecule. The silica is attracted to the surface of the biomolecule through electrostatic interactions and the aggregation of silica monomers and oligomers results in a spontaneous nucleation point that then uses entropy to cover and polymerise about the surface of the biomolecule. The resultant silica shell protects the biomolecule from external surroundings and prevents certain molecules from interacting with the biomolecule inside the silica shell. This approach can be used to preserve and protect the biomolecule, e.g., during storage and transportation from external conditions such as high temperature. Nevertheless, the present inventors sought to improve and optimise ensilication methods, such that this process could be used to ensilicate a wider variety of biomolecules at high yields. Many biomolecules (including biomolecules with a negatively charged surface) were challenging to effectively ensilicate at high yield using prior processes. The present inventors also sought to develop new and improved ensilicated biomolecules. Other silica coating methods are described in the prior art. However, these methods require cloaking the biomolecule with an intermediate layer before silica deposition, typically using a polycationic polymer such as protamine, PEI or polylysine. However, due to the presence of an intermediate layer, these silica coating methods do not provide the same advantages of “shape-matching” as is provided by the ensilication methods mentioned above for certain biomolecules, thereby providing a silica shell coat that is less protective. The use of positively charged polymers and / or templating materials is also undesirable in at least because these polymers can interfere with formulation of the biomolecule, the release of the biomolecule and / or may have undesirable biological effects. Further, the silica coating methods of the present invention are less complicated as they do not require an intermediate templating step of the biomolecule. Other silica coating methods in the prior art involve deposition of biomolecules in a previously prepared silica gel and / or deposition of biomolecules on the surface or through the pores of pre-formed silica nanoparticles. However, the biomolecules are not contained within a continuous silica shell and are therefore less stable and more exposed to external and surrounding conditions. Summary of Invention In the present invention, there is provided a method of encapsulating a biomolecule in a silica shell to form a particle, the method comprising: a) hydrolysing a mixture of silica precursors, said mixture comprising a functionalised silica precursor comprising a Si-C bond and a non-functionalised silica alkoxide precursor b) directly contacting the hydrolysed precursors with an aqueous solution comprising the biomolecule and c) encapsulating the biomolecule in a silica shell to form a particle, such that at least a portion of the silica on the interior surface of the silica shell is functionalised and comprises an Si-C bond. The present invention provides a way to capture a biomolecule in one or more layers of covalently bonded amorphous silica, forming a continuous cage or shell which preserves the shape of the biomolecule, protects the biomolecule from surrounding conditions and prevents denaturation caused by heat and / or aging and / or non-physiological conditions. The method of the present invention represents a novel refinement of the previously described ensilication method which used a single alkoxide silica precursor TEOS. By using a mixture of two silica precursors, one which is functionalised and the other nonfunctionalised, means that the precursors have different chemical properties and therefore different affinities for different parts of the biomolecule surface. For example, it is hypothesised that the non-functionalised silica alkoxide can interact with positively charged portions of the biomolecule surface. The functional group of the functionalised silica precursor, wherein the Si is functionalised by way of a Si-C bond, can now additionally form unique and different interactions with the biomolecule surface, both during formation of the particle and once the particle has been formed. The presence of the functional group on the silica precursor is believed to further help facilitate the growth of the silica shell about the biomolecule surface. By way of example, when a basic or positively charged functional group is used (e.g., a positively charged amino group), this is hypothesised to interact favourably with negatively charged areas of a biomolecule surface, including negatively charged proteins, nucleic acids or viral capsids. The present method results in a distinct product comprising functional groups on the interior surface of the shell which enables form non-covalent interactions with the surface of the biomolecule. In some embodiments, these functional groups can further stabilise the biomolecule once the silica shell has been formed. The particles described herein provide a way of thermally protection and stabilising biomolecules within a silica shell. The present process enables direct ensilication of a wide range of biomolecules which were previously challenging to ensilicate effectively with high yield. The present method also has wide applicability to all types of biomolecule, including proteins, viruses, nucleic acid lipid nanoparticles (LNPs) and cells, with good or even improved ensilication efficiency. Different from prior ensilication methods, wherein the silica shell is formed using only a nonfunctionalised silica precursor (e.g., TEOS), particles of the present invention can be formed in higher yields and / or in a more tuneable process which is applicable to a greater variety and type of biomolecule. The method of the invention is quick to implement and can be applied to any biomolecule in aqueous solution. As such, the new ensilication method can be used to ensilicate biomolecules with a wide range of surface properties and isoelectric points without the need for intermediates or prior cloaking or surface modification of the biomolecule (e.g., using polylysine, PEI, chitosan, PLGA or protamine). The present process is tuneable since the precursor blend can be adjusted to suit the properties of the biomolecule target, e.g., biomolecules with different isoelectronic points. The resultant silica shell can stabilise and protect the biomolecule from surrounding and / or extreme conditions, e.g., including from thermal degradation. The resulting particles prepared by the present method may be used to stabilise the biomolecules for storage or transit. The biomolecules within the particles of the present method can then be released from the silica shell before administration to a subject. A biomolecule released from its silica shell, after transport and / or storage can then be used in a method vaccination. The biomolecule once released from its silica shell can be prepared as a pharmaceutically or physiologically acceptable preparation or composition containing a pharmaceutically or physiologically acceptable carrier, excipient, or diluent. While functionalised silica precursors such as APTES are known, these previously have been used in different technical fields, primarily for attaching organic films to metal oxides. This is, to the present inventor’s knowledge, the first time that such a compound has been implemented in a process of encapsulating biomolecules in silica. Further, it is important to note that the functionalised silica precursor APTES alone was not effective for ensilication, and the ensilication only worked when a blend of functionalised and non-functionalised precursors were mixed and hydrolysed before contacting with the biomolecule surface. Without wishing to be bound by theory, it is believed that, when used alone and in the absence of non-functionalised silica precursor (e.g., TEOS), the presence of the functional group sterically prevents growth of the silica shell around the biomolecule. Methods of the present invention differ and are improved compared to prior art sol-gel processes. While the methods disclosed herein start with silica precursors, as in sol-gel processes, the silica precursor monomers instead polymerise rapidly around the biomolecule itself, and precipitate. A gel is not produced as in conventional sol-gel processes, because if the silica is allowed to gel, it grows into glass which instead damages the biomolecule. Preferred embodiments of the present invention now follows: In some embodiments, the functionalised silica precursor is a basic silica precursor or a positively charged silica precursor. In some embodiments, the functionalised silica precursor is a basic silica precursor, for example, an aminoalkyl alkoxide silica precursor such as APTES and the method comprises acidifying the basic silica precursor before step a. In some embodiments, the basic silica precursor is acidified before mixing with the non-functionalised silica precursor. In some embodiments, the acidification of the basic silica precursor occurs at a pH of between 1 and 5, preferably between 1 and 2. In embodiments where a basic silica precursor is used, it was found that acidification of the basic silica precursor, i.e., pre-acidification of the basic silica precursor, is preferred before step a). This is found to reduce aggregation and promotes formation of a homogenous mixture of precursors which can be more effectively used in an ensilication process. In some embodiments, the functionalised silica precursor is a positively charged silica precursor, for example, an ammonium alkyl alkoxide silica precursor such as C18-TMS (structure disclosed elsewhere herein), and the method does not comprise acidifying the positively charged silica precursor before step a. In some embodiments, the functionalised silica precursor, is functionalised with an aminoalkyl group or ammonium alkyl group. In some embodiments, the functionalised silica precursor is an amino-alkyl alkoxide silica precursor or an ammonium alkyl alkoxide silica precursor. In some embodiments the amino group of the amino-alkyl alkoxide silica precursor is a primary amine. In some embodiments, amino-alkyl alkoxide silica precursor has the formula: RO^ ^OR Si ROZ ' ( NH2 wherein n is 0-4, and / or R is selected from methyl, ethyl, propyl, isopropyl, butyl. In some embodiments, n is preferably 1-3, more preferably 2. In some embodiments, R is methyl, ethyl or propyl, preferably ethyl. In some embodiments, the functionalised silica precursor, or basic silica precursor, is aminoalkyl-alkoxysilane is (3-Aminopropyl)triethoxysilane (APTES), i.e., wherein n is 2 and R is ethyl A basic functionalised silica precursor, such as APTES, was shown to be effective in the method of the invention. The presence of a positively charged amino group (i.e., when acidified in the case of APTES), provides different interactions with the biomolecule surface. This can be particularly effective for biomolecules with a negatively charged regions, or biomolecules with an isoelectric point below 6.5. In some embodiments, amino-alkyl alkoxide silica precursor has the formula: wherein n is 0-4, and / or R is selected from methyl, ethyl, propyl, isopropyl, butyl, optionally methyl or ethyl Ri is selected from methyl, ethyl, propyl, isopropyl, butyl, preferably methyl R2 is selected from methyl, ethyl, propyl, isopropyl, butyl, preferably methyl, and Rais a C1-C25 alkyl, optionally a C1-C20 alkyl, further optionally C18 alkyl. In some embodiments, n is preferably 1-3, more preferably 2. In some embodiments, R is methyl, ethyl or propyl, optionally methyl or ethyl. In some embodiments, the functionalised silica precursor, is Dimethyloctadecyl[3-(trimethylsilyl)propyl]ammonium salt (C18-TMS), e.g., Dimethyloctadecyl[3-(trimethylsilyl)propyl]ammonium chloride salt. A positively charged functionalised silica precursor, was shown to be effective in the method of the invention. The presence of an ammonium group provides different interactions with the biomolecule surface. This can also be particularly effective for biomolecules with a negatively charged regions, or biomolecules with an isoelectric point below 6.5. In preferred embodiments, the non-functionalised silica precursor is a silica alkoxide, more preferably tetraethylorthosilicate (TEOS) or tetrapropylorthosilicate (TPOS), more preferably TEOS. Silica alkoxides (Si(OR)4) and TEOS in particular, were demonstrated to work effectively in the method of the disclosure. Silica monomers, deriving from silica alkoxides, are believed to interact particularly effectively with positively charged regions of the biomolecule surface. TEOS is shown in the examples to be an effective non-functionalised silica precursor. It is also considered to be less toxic than other silica alkoxides. In some embodiments, the silica shell is formed from a mixture of silica precursors comprising a first functionalised silica precursor functionalised with a basic or positively charged group and a second non-functionalised silica precursor (e.g., a silica alkoxide), wherein the deposition of silica is nucleated by non-covalent interactions between the first silica precursor and negatively charged surfaces of the biomolecule, and non-covalent interactions between the second non-functionalised silica precursors and positively charged surface of the biomolecule. In some embodiments, the isoelectric point of the biomolecule is between 0.5 and 12, and optionally wherein the isoelectric point of the biomolecule is less than 6.5. The isoelectric point of the biomolecule may be determined using any suitable method, including electrophoretic light scattering (ELS), Phase Analysis Light Scattering (PALS), or Isoelectric focusing (IEF) gels. As stated above, the method of the invention is applicable for ensilicating a wide range of biomolecules, including those with negatively charged surfaces that were previously challenging to ensilicate with high yields. In some embodiments, the isoelectric point of the biomolecule is determined before step a) of the method. In some embodiments, the functionalised silica precursor and non-functionalised silica precursor are mixed in a ratio from 1:1 to 1:2000, preferably from 1:4 to 1:800. The particular ratio of functionalised silica precursor and non-functionalised silica precursor can be tuned depending on the nature of the biomolecule, e.g., the type or isoelectronic point of the biomolecule. In some embodiments, the biomolecule is selected from a protein, polypeptide, virus, viruslike particle, cell, nucleic acid, or a nucleic acid lipid nanoparticle (LNP). In some embodiments, the biomolecule is a vaccine, optionally wherein the vaccine is selected from a live virus vaccine, live attenuated virus vaccine, an inactivated vaccine, a nucleic acid vaccine, a polypeptide vaccine, heat-killed vaccine, subunit vaccine, or recombinant vaccine. The ensilication method has been shown to be applicable to a wide range of biomolecules including those that are suitable for use as a vaccine. The resulting ensilicated biomolecule is thermally stable and can be preserved in transport and / or storage without cold-chain. The present method is quick to implement and can be applied to any biomolecule in aqueous solution. Preferred parameters of the method are as follows, leading to the most effective ensilication. - In some embodiments, the mixing step takes place for at least 10 minutes, and / or until the mixture becomes homogenous, preferably for between 30 and 60 minutes. -In some embodiments, the contacting takes place instantaneously, or for a period of at least 10 seconds, or at least 30 seconds, or at least 5 minutes. In some embodiments, the contacting takes place for 30 seconds to 120 minutes, optionally from 5 to 60 minutes, further optionally between 10 and 30 minutes. -In some embodiments, the hydrolysing takes place at a pH of less than 5, more preferably at a pH of less than 2, for example, between 1 and 2. In preferred embodiments, step a) of the method comprises mixing the functionalised silica precursor with the non-functionalised silica precursor, before hydrolysing one or more of the silica precursors. In preferred embodiments, step a) of the method comprises mixing the functionalised silica precursor with the non-functionalised silica precursor, before hydrolysing the non-functionalised silica precursor (e.g., TEOS). In some embodiments, the mixing step occurs at an acidic pH, preferably wherein the pH is below 5, and preferably between 2 and 5. This reduces aggregation and prevents gel formation. In some embodiments, the contacting takes place at a pH in the range of between 4 and 10, optionally in the range of 5 to 8. In some embodiments, the ratio of hydrolysed silica to the biomolecule in buffer is from about 1:5 to 1:200, for example, or from about 1:7.5 to 1:150, or about 1:10 to 1:100. In some embodiments, the biomolecule is added in aqueous buffer, optionally wherein the buffer comprises one or more salts containing one or more of a magnesium salt, a calcium salt, or a sodium salt, or a potassium salt and further optionally wherein the buffer is a phosphate buffer, an SM buffer, a modified SM buffer, PBS, Tris, an imidazole buffer, a sucrose buffer, or bis-tris buffer. The method of any preceding claim, wherein in the contacting step, the biomolecule is present in the aqueous solution at a concentration of 0.1 mg / ml to 100 mg / ml. In some embodiments, there is provided method of encapsulating a biomolecule in a silica shell to form a particle, wherein step a) comprises ai) mixing a functionalised silica precursor comprising a Si-C bond, with a non-functionalised silica alkoxide precursor to form a mixture of precursors aii) hydrolysing the mixture of precursors. In some embodiments, wherein the functionalised silica precursor is a basic functionalised silica precursor, the basic functionalised silica precursor is acidified (and therefore may be at least partially hydrolysed) before mixing with and hydrolysing the non-functionalised silica alkoxide precursor. In some embodiments, the acidification of the basic functionalised silica precursor occurs at a pH of between 1 and 5, preferably between 1 and 2. In some embodiments, the biomolecule is provided as a formulation comprising one or more adjuvants, carriers or preservatives. In other words, the ensilication process is compatible with any adjuvants, carriers or preservatives which may be used in combination with the biomolecule to be used as a biopharmaceutical. In preferred embodiments, the method is free of a templating step where the biomolecule is first coated with an intermediate layer, for example, intermediate layers comprising polylysine, polyethyleneimine chitosan, polylactic, protamine or spermine or combinations thereof. This is beneficial as the presence of an intermediate layer, more specifically a positively charged polymer intermediate layer, is expected to compromise release of the biomolecule. Further, the presence of an intermediate layer may . Further, the absence of a templating step and direct deposition of the silica on the biomolecule enables the resultant silica coat to closely resemble the shape of the biomolecule. Finally, methods that do not involve a templating step and is therefore simpler and less complex. Also disclosed herein is a method of encapsulating a nucleic acid lipid nanoparticle (LNP) in a silica shell to form a particle, the method comprising: a) hydrolysing a mixture of silica precursors, said mixture comprising a functionalised silica precursor comprising a Si-C bond and a non-functionalised silica alkoxide precursor b) directly contacting the hydrolysed precursors with an aqueous solution comprising the nucleic acid lipid nanoparticle (LNP) and c) encapsulating the nucleic acid lipid nanoparticle (LNP) in a silica shell to form a particle, such that at least a portion of the silica on the interior surface of the silica shell is functionalised and comprises an Si-C bond. Also disclosed herein is a method of encapsulating a cell in a silica shell to form a particle, the method comprising: a) hydrolysing a mixture of silica precursors, said mixture comprising a functionalised silica precursor comprising a Si-C bond and a non-functionalised silica alkoxide precursor b) directly contacting the hydrolysed precursors with an aqueous solution comprising the cell and c) encapsulating the cell in a silica shell to form a particle, such that at least a portion of the silica on the interior surface of the silica shell is functionalised and comprises an Si-C bond. Also disclosed herein is a method of encapsulating a protein in a silica shell to form a particle, the method comprising: a) hydrolysing a mixture of silica precursors, said mixture comprising a functionalised silica precursor comprising a Si-C bond and a non-functionalised silica alkoxide precursor b) directly contacting the hydrolysed precursors with an aqueous solution comprising the protein and c) encapsulating the protein in a silica shell to form a particle, such that at least a portion of the silica on the interior surface of the silica shell is functionalised and comprises an Si-C bond. The protein may be any suitable protein, including but not limited to, antibodies and enzymes. Also disclosed herein is a method of encapsulating a virus or viral vector in a silica shell to form a particle, the method comprising: a) hydrolysing a mixture of silica precursors, said mixture comprising a functionalised silica precursor comprising a Si-C bond and a non-functionalised silica alkoxide precursor b) directly contacting the hydrolysed precursors with an aqueous solution comprising the virus or viral vector and c) encapsulating the virus or viral vector in a silica shell to form a particle, such that at least a portion of the silica on the interior surface of the silica shell is functionalised and comprises an Si-C bond. Brief Description of Figures Examples, embodiments and experiments illustrating the principles of the disclosure will now be discussed with reference to the accompanying figures in which: Figure 1 depicts the method of the invention. A biomolecule (1) is encapsulated in a continuous silica shell (2), wherein at least some of the silica is functionalised via a Si-C bond. The functional group of the functionalised silica, depicted as *, can interact with the biomolecule surface and can facilitate the growth of the silica shell about the biomolecule. Figure 2 shows an example of the continuous silica shell (2) when formed using a nonfunctionalised silica alkoxide precursor (e.g., TEOS) in combination with a functionalised APTES precursor. The functional group deriving from the functionalised silica precursor is able to interact with the biomolecule surface (1). Figure 3 shows the material % from initial ensilication of Newcastle disease Virus (NDV) using TEOS only and APTES:TEOS. Data shows that for a TEOS only ensilication, using a nonfunctionalised silica precursor, poor ensilication yields are obtained, while using a mixture of functionalised and non-functionalised silica precursors, e.g. TEOS:APTES mixture at a 4:1 ratio, effective ensilication of NDV is achieved. Figure 4 shows the isoelectric point determination of Newcastle Disease Virus (NDV) in 50mM Tris-HCI Figure 5 shows the material, load &ensilication efficiency of ensilicated NDV using APTES:TEOS method Figure 6 shows SDS PAGE analysis of Newcastle Disease Virus (NDV) Ensilication, APTES:TEOS 1:25, Silica 1:50. Lane 1 Pre-stained protein ladder, Lanes 2-4 NDV Stock, Lanes 5-7 Ensilication supernatants and Lanes 8-9 De-ensilicated NDV material. Figure 7 shows western blot analysis of Newcastle Disease Virus (NDV) Ensilication, APTES:TEOS 1:25, Silica 1:50. Lane 1 Pre-stained protein ladder, Lanes 2-4 NDV Stock, Lanes 5-7 Ensilication supernatants and Lanes 8-9 De-ensilicated NDV material. Figure 8 shows an ELISA Assay comparing the stock Newcastle Disease Virus (NDV) and the de-ensilicated material Figure 9 shows an ELISA Assay comparing the stock Newcastle Disease Virus (NDV) and the supernatants from the ensilication of NDV Figure 10 shows the isoelectric point determination of Lysozyme in 50mM Tris-HCl. Figure 11 shows the average Material %, Load % and Ensilication Efficiency from the ensilication of 1mg / ml Lysozyme in 50mM Tris-HCl pH 8 using non-functionalised silica precursor alone (e.g., TEOS), or a mixture of silica functionalised and non-functionalised silica precursors (e.g., APTES and TEOS mixture) added at a 1:50 ratio. Figure 12 shows the specific activity of lysozyme protein, determined through the use of Enzchek assay kit, before and after ensilication. Thermal stability of the Stock and ensilicated material was tested by heating at 95°C for 1 hour. Both un-heated and heated ensilicated material was then de-ensilicated and activity determined alongside the heated and un-heated stock material. Figure 13 shows SDS PAGE analysis of Lysozyme Ensilication utilising both methods -TEOS only and APTES / TEOS combination. Lane 1 Pre-stained protein ladder, Lane 2 Lysozyme stock, Lane 3 Supernatant from TEOS ensilication, Lane 4 Supernatant from APTES / TEOS ensilication, Lane 5 De-ensilication from TEOS made material, Lane 6 De-ensilication from APTES / TEOS made material, Lane 7 De-ensilication from TEOS made material that had been heated at 95° C for 1 hour prior to the de-ensilication process, Lane 8 De-ensilication from APTES / TEOS material that had been heated at 95° C for 1 hour prior to the de-ensilication process and Lane 9 Lysozyme stock that has been heated at 95° C. Figure 14 shows the Isoelectric point determination of BSA in 50mM Tris-HCl. Figure 15 shows the material %, load % and ensilication efficiency from ensilication of BSA. Material % is the total percentage of BSA incorporated into the silica matrix determined from the supernatant concentration; load % is the percentage mass that is BSA in the ensilicated material; and ensilication efficiency is the percentage of starting BSA that could be extracted from the ensilicated material. Figure 16 shows the SDS PAGE analysis of BSA ensilication with both methods - TEOS only and APTES / TEOS combination. Lane 1 pre-stained protein ladder, Lane 2 BSA stock, Lane 3-4 supernatants from TEOS only ensilication, Lane 5-6 supernatants from APTES / TEOS ensilication and Lane 7-8 De-ensilication from APTES / TEOS material. Figure 17 shows the material % (percentage of entity incorporated within the silica matrix) from ensilications of both NIBSC-NDV and Lysozyme with varying ratios of APTES and TEOS in the silica pre-cursor. As the percentage of APTES increases the material % of lysozyme (positively charged) decreases and that on NDV (negatively charged) increases. Figure 18 shows micro-plate ensilications. Rows A&B contain 1mg / ml NIBSC NDV Stock and Rows C&D contain 1mg / ml Lysozyme Stock. In each well silica was added at a 1:50 ratio. Column 1 contained TEOS with no APTES present and Columns 2-6 containing increasing amounts of APTES (i.e., with ratios of 1:200, 1:160, 1:80, 1:40 and 1:20 respectively). Figure 19 shows a well plate used for micro-ensilications of horse radish peroxidase (HRP) showing the formation of ensilicated material as the percentage of APTES increases. A1: TEOS only, A2: 1:200 APTES / TEOS, A3: 1:100 APTES / TEOS, A4: 1:50 APTES / TEOS, A5: 1:25 APTES / TEOS and A6: 1:5 APTES / TEOS. Figure 20 shows SDS PAGE analysis of HRP ensilication with APTES / TEOS silica precursor. Lane 1 pre-stained protein ladder, Lane 2 HRP stock, Lane 3 Ensilication supernatant and Lane 4 De-ensilicated HRP. Figure 21 shows the Optical Density (600nm) of Ensilicated E. coli incubated with LB + Ampicillin growth media for 24 hours at 37° C. Figure 22 shows a 24 well plate used for micro-ensilications showing the formation of ensilicated material of the human lung carcinoma cell line A549. A1-A6: 1:10-1:800 APTES:TEOS with silica ratio 1:10. B1-B6: 1:10-1:800 APTES:TEOS with silica ratio 1:40. C1-C6: 1:10-1:320 TEOS Figure 23 shows pictures of ensilicated human lung carcinoma cell line A549 at different ratios. Figure 24 shows the structure of C18-TMS. Figure 25 shows the percentage of protein (BSA or Lysozyme) incorporated into the silica matrix - Material % for the different silica pre-cursors. Figure 26 shows the material %, Load % and Ensilication efficiency for ensilicated BSA and Lysozyme (LYZ) using TEOS, APTES / TEOS and C18-TMS / TEOS silica pre-cursors. Figure 27 shows SDS PAGE analysis of BSA and Lysozyme (LYZ) ensilication using C18-TMS / TEOS silica pre-cursor. Lane 1, Pre-stained protein ladder, Lane 2 BSA stock, Lane 3 LYZ stock, Lane 4-6 De-ensilicated BSA material and Lanes 7-9 De-ensilicated LYZ. Figure 28 shows the average Material %, Load % and Ensilication Efficiency from the ensilication of Lysozyme (LYZ) with TEOS or TPOS silica precursors and BSA with APTES: TEOS and APTES: TPOS silica precursors. Figure 29 shows SDS PAGE analysis of Lysozyme Ensilications utilising TEOS and TPOS silica pre-cursors. Lane 1 &10 Pre-stained protein ladder, Lane 2 Lysozyme stock, Lanes 3-5De-ensilicated material from Lysozyme ensilications using TEOS and Lanes 6-8De-ensilicated material for Lysozyme ensilications using TPOS. Figure 30 shows SDS PAGE analysis of BSA Ensilications utilising APTES:TEOS and APTES:TPOS silica pre-cursors. Lane 1 Pre-stained protein ladder, Lane 2 BSA stock, Lanes 3-5 De-ensilicated material from BSA ensilications using TEOS and Lanes 6-8 De-ensilicated material for BSA ensilications using TPOS. Figure 31 shows the specific activity of lysozyme protein, determined using Enzchek assay kit. De-ensilicated Lysozyme from ensilications using either TEOS or TPOS silica precursor were tested alongside lysozyme positive and negative stocks Figure 32 shows the material %, Load % and Ensilication efficiency % from the ensilication of a 1:1 mixture of BSA and Lysozyme in 50mM Tris-HCI pH 7 using 1:200 APTES:TEOS. Figure 33 shows SDS PAGE analysis of the ensilication of a 1:1 mixture of BSA and Lysozyme in 50mM Tris-HCI pH 7 using 1:200 APTES:TEOS. Lane 1 pre-stained protein ladder, Lane 2 BSA stock, Lane 3 Lysozyme Stock, Lane 4 BSA and Lysozyme 1:1 mixed stock, Lane 5 Supernatant from the ensilication and Lane 6 De-ensilicated material. Figure 34 shows a turbidity graph of small scale ensilications performed in a 96 well plate for Newcastle Disease virus (NDV) (negatively charged) stock solution and a mixture of NDV and protamine (A positive intermediate). APTES and TEOS were hydrolysed separately and mixed together, at different ratios. The ensilication was deemed successful if the well went turbid. Figure 35 shows a photograph of 24-well plate after addition of silica to eGFP mRNA-LNP. A1- ensilicated eGFP mRNA-LNP to be left in solution, A2- ensilicated eGFP mRNA-LNP to be left in solution and de-ensilicated when needed. Figure 36 shows fluorescent images of A549 cells transfected with ensilicated, de-ensilicated or non-ensilicated eGFP mRNA-LNP for 48h at day 1 of stability study. Figure 37 shows fluorescent images of A549 cells transfected with ensilicated, de-ensilicated or non-ensilicated eGFP mRNA-LNP for 48h at day 4 of stability study. Figure 38 shows fluorescent images of A549 cells transfected with ensilicated, de-ensilicated or non-ensilicated eGFP mRNA-LNP for 48h at day 7 of stability study. Figure 39 shows fluorescent images of A549 cells transfected with ensilicated, de-ensilicated or non-ensilicated eGFP mRNA-LNP for 48h at day 14 of stability study. Figure 40 shows the overall fluorescent at 488 nm excitation and 513nm emission after 48h transfection of A549 cells with ensilicated and de-ensilicated mRNA-LNP. Statistical analysis: ANOVA (with Tuckey post hoc test) *p<0.05, **p<0.01, ***p<0.001 Figure 41 shows the viability of A549 cells after 48h transfection period with ensilicated and de-ensilicated mRNA-LNP. Statistical analysis: ANOVA: non-significant differences. Figure 42 shows the specific activity of lysozyme protein, determined through the use of Enzchek assay kit, of ensilicated material exposed to an acidic environment pre-de-ensilication. Detailed Description The present disclosure provides the aspects mentioned above. Optional and preferred features of the various aspects are described below. Unless otherwise stated, any optional or preferred feature may be combined with any other optional or preferred feature, and with any of the aspects of the invention mentioned herein. All dependent claims can be combined unless context clearly dictates otherwise. The terms used in this specification generally have their ordinary meanings in the art, within the context of this disclosure and in the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance in describing the compositions and methods of the disclosure and how to make and use them. As used herein and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the content clearly dictates otherwise. wt.% herein, unless otherwise specified is w / w%. Various standard methods for measuring properties (e.g. ASTM, ISO, DIN, TAPPI) may be mentioned herein. Unless otherwise stated, the standard to be used is the most recent before the filing date of the present application. As defined herein the term “ensilication” refers to a process wherein a continuous silica shell is formed by polymerising silica monomers about a biomolecule surface. Ensilication process can be used interchangeably with “silica encapsulation”. As defined herein the term “silica monomer” refers to any molecule that can polymerise to form amorphous silica. Silica monomers defined herein include silica monomers that are functionalised, characterized by a presence of an Si-C bond (i.e., wherein a functional group is attached to the silica of the silica monomer via a carbon atom), and non-functionalised silica monomers that do not comprise the presence of an Si-C bond. As defined herein the term “silica precursor” refers to any molecule that can be hydrolysed to form a silica monomer. The silica precursor preferably contains a silicon atom coordinated by one or more alkoxide groups or derivatives of alkoxide groups, preferably two or more alkoxide groups or three or more alkoxide groups. The silica precursors defined herein include functionalised silica precursors characterized by the presence of an Si-C bond (i.e., wherein a functional group is attached to the silica of the silica precursor via a carbon atom) and non-functionalised silica precursors that do not comprise an Si-C bond. In the contacting step of the methods disclosed herein, the silica monomers polymerise about the surface of the biomolecule to form an amorphous silica shell about the biomolecule. As defined herein “native biomolecule” refers to a biomolecule that is not silica encapsulated / ensilicated, i.e., the biomolecule is outside of the silica shell. The terms "treatment" and "treating" herein refer to an approach for obtaining beneficial or desired results in a subject, which includes a prophylactic benefit and optionally also a therapeutic benefit. “Prophylactic benefit” refers to delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. “Therapeutic benefit” refers to eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the patient may still be afflicted with the underlying disorder. The term "comprising" (and related terms such as "comprise" or "comprises" or "having" or "including") includes those embodiments, for example, an embodiment of any composition of matter, composition, method, or process, or the like, that "consists of” or "consists essentially of” the described features. The term “comprises” or “comprising” can be used interchangeably with “includes”. When ranges are used herein, all combinations and sub-combinations of ranges and specific embodiments therein are intended to be included. The term "about" when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range may vary. Typical experimental variabilities may stem from, for example, changes and adjustments necessary during scale-up from laboratory experimental and manufacturing settings to large scale. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Abbreviations used herein have their conventional meaning within the chemical and biological arts, unless otherwise indicated. Precursors The present invention comprises functionalised and non-functionalised silica precursors. Functionalised silica precursor The functionalised silica precursor is any silica precursor which is functionalised with a functional group wherein the Si of the silica precursor is attached to the functional group via carbon. A functionalised silica precursor can be characterized by the presence of an Si-C bond. In some embodiments, the functional group is basic group, positively charged group or positively polar group. In some embodiments, the functional group is a basic group. In some embodiments, the basic group is capable of being protonated to form a positively charged group. In some embodiments, the basic group is capable of forming a hydrogen bond. In some embodiments, the basic group has a pKa of from 8-15, preferably from 8.5-12, or in some embodiments, from 9-11.5, or from 10-11. In some embodiments, the functionalised silica precursor has the formula (I): wherein FG represents the functional group, and wherein n is 1-6, preferably wherein n is 0-4, and more preferably wherein n is 1-3, further preferably wherein n is 2, and / or R is selected from methyl, ethyl, propyl, isopropyl, butyl, preferably ethyl In some embodiments, the functional group (FG) is a basic or positively charged group. In some embodiments, the functional group is an amino group. In some embodiments, the amino group is a primary amine. In some embodiments, the functionalised silica precursor is an amino-alkyl alkoxide silica precursor, more preferably with the formula (II): NH2 (IQ wherein n is 0-6, preferably wherein n is 1-5, and more preferably wherein n is 1-3, and further preferably wherein n is 2, and / or R is selected from methyl, ethyl, propyl, isopropyl, butyl, preferably ethyl. In some embodiments, the functionalised silica precursor has the formula (III), wherein R is selected from methyl, ethyl, propyl, isopropyl, butyl, preferably ethyl In some embodiments, the functionalised silica precursor is aminoalkyl-alkoxysilane is (3-Aminopropyl)triethoxysilane (APTES). In some embodiments, the functional group is an ammonium group. In some embodiments, the functionalised silica precursor is an ammonium-alkyl alkoxide silica precursor, more preferably with the formula (IV) wherein n is 0-6, preferably wherein n is 1-5, and more preferably wherein n is 1-3, and further preferably wherein n is 2, and / or R is selected from methyl, ethyl, propyl, isopropyl, butyl, optionally methyl, Ri is from methyl, ethyl, propyl, isopropyl, butyl; preferably methyl R2 is selected from methyl, ethyl, propyl, isopropyl, butyl; preferably methyl, and Rais a C1-C25 alkyl, optionally a C1-C20 alkyl, further optionally Cis alkyl, wherein the alkyl is preferably an unsubstituted unbranched alkyl. In some embodiments, the functionalised silica precursor has the formula (V), wherein R is selected from methyl, ethyl, propyl, isopropyl, butyl; optionally methyl, and R3 is C1-C20. In some embodiments R3 is Cis alkyl, wherein the alkyl is preferably an unsubstituted unbranched alkyl. In some embodiments, the functionalised silica precursor, is Dimethyloctadecyl[3-(trimethylsilyl)propyl]ammonium salt (C18-TMS), e.g., Dimethyloctadecyl[3- (trimethylsilyl)propyl]ammonium chloride salt. Non-functionalized silica precursor The non-functionalised silica precursor is a silica precursor capable of hydrolysing to form a silica monomer that is not functionalised, i.e., the silica is coordinated with 4 oxygen atoms and does not comprise a Si-C bond. In preferred embodiments, the non-functionalised silica precursor is a silica alkoxide. In some embodiments, the silica alkoxide has the formula (Si(OR4) wherein R is an alkyl chain of 1-6 carbon atoms, preferably 1-3 carbon atoms, more preferably 2 carbon atoms. In some embodiments, the non-functionalised silica precursor is selected from tetra-methoxy-orthosilicate (TMOS), tetra-ethoxy-orthosilicate (TEOS), tetra-propoxy-orthosilicate (TPOS), tetra-butoxy-orthosilicate (TBOS), and tetra (ethoxymethoxy) silane. In some embodiments and examples, the non-functionalized silica precursor is tetraethylorthosilicate (TEOS). In some embodiments and examples, the non-functionalized silica precursor is tetrapropylorthosilicate (TPOS). Biomolecule The biomolecule which can be encapsulated with a silica shell according to the present invention may be any biological molecule, biopharmaceutical, biological entity or biologic. In some embodiments, the biomolecule has secondary or higher order structure important for its function. In some embodiments, the biomolecule has secondary structure, tertiary structure or quaternary structure. The method of the present invention is suitable for forming a silica shell around a wide range of biomolecules with a range of isoelectric points. In some embodiments, the isoelectric point of the biomolecule is between 0.5 and 12, or between 2 and 10, or between 2.5 and 10. The isoelectric point may be determined using any suitable method, e.g.., using PALS as described elsewhere herein. In some embodiments, the isoelectric point of the biomolecule is less than 6.5 (e.g., between 0.5 and 6.5) and / or the biomolecule has a negatively charged surface. In some embodiments, the isoelectric point of the biomolecule is less than 6, or less than 5.5, or less than 5, or less than 4.5, or less than 4, or less than 3.5. In some embodiments, the isoelectric point of the biomolecule is between 2 and 6.5, or between 2.5 and 6.5. In some examples, the isoelectric point of the biomolecule is between 2.5 and 4. In some examples, the isoelectric point of the biomolecule is between 4 and 5.5. In some embodiments, the isoelectric point of the biomolecule is between 5.5. and 6.5. Examples included in the present invention are Newcastle disease virus having an isoelectric point between 3.2-3.5 and Bovine serum albumin having an isoelectric point between 4.5-5.5. In some embodiments, the isoelectric point of the biomolecule is greater than 6.5, (e.g., between 6.5 and 12). In some embodiments, the isoelectric point of the biomolecule is greater than 7, or greater than 7.5, or greater than 8, or greater than 9. These biomolecules are those with neutral or positively charged surfaces. Examples included in the present invention are Lysozymes (isoelectric point ~ 8.6 as determined herein). This demonstrates that the modified method is also applicable to biomolecules that could be ensilicated using previous ensilication methods that only used one non-functionalised silica precursor. Nevertheless, biomolecules that are positively charged can still be ensilicated using the modified method, with higher tunability, and no compromise in thermal stability. In some embodiments, the biomolecule is provided as a formulation comprising one or more adjuvants, carriers or preservatives. In other words, the ensilication process is compatible with any pharmaceutically excipients or formulations used in combination with the biomolecule for delivery as a biopharmaceutical. The adjuvants, carriers or preservatives are distinct from templating polymers (e.g., cationic polymers such as polylysine, chitosan, polylactic acid, protamine and PEI) in prior art methods to cloak the biomolecule before and solely for the purpose of silica coating. The adjuvants, carriers or preservatives are typically present in combination with the biomolecule, i.e., they do not cloak the entire surface of the biomolecule before silica coating. In some embodiments, the adjuvants may comprise one or more of Freund’s adjuvant, monophosphoryl lipid (MPLA), Alum, Squalene or AS04 (e.g., a combination of MPLA and alum). Squalene adjuvants described herein include squalene-based adjuvants AS03, AF03, or M5F9. In some embodiments, the adjuvants comprise a CpG DNA adjuvant (e.g., CpG 1018). In some embodiments, the carriers or preservatives includes emulsifiers, osmolytes, co-solvents or any combination thereof. Examples of emulsifiers may be Tween 20 or Tween 80. Examples of osmolytes include sucrose. Examples of co-solvents may be sorbitol and / or glycerol. The biomolecule may have any suitable size. In some embodiments, the biomolecule has a size of from 1 kDa to 1500 kDa, or from 1 kDa to 500 KDa, or from 10 KDa to 300 kDa. In some embodiments, the biomolecule has a molecular weight greater than 1kDa, or greater than 2kDa, or greater than 3kDa, or greater than 5 kDa, or greater than 10 kDa, or greater than 50 kDa, or greater than 100 kDa, or greater than 200 kDa. The molecular weight can be determined by any suitable method, e.g, by gel electrophoresis or SDS-PAGE. In some embodiments, the biomolecule is selected from is selected from a protein, polypeptide, virus, virus-like particle, cell, nucleic acid or a nucleic acid lipid nanoparticle (LNP). In some embodiments, the silica shell comprises one biomolecule type. In some embodiments, the silica shell comprises a plurality of biomolecules, e.g., a plurality of different biomolecules. For a silica shell comprising a plurality of biomolecules, each biomolecule may be separately encapsulated in silica within the silica shell. In an example described herein, the silica shell comprises two different proteins (e.g., lysozyme and BSA). In some embodiments, the particle comprises 0.5 wt.% to 60 wt.% biomolecule, preferably 10 wt.% to 50 wt.% biomolecule. Protein In some embodiments, the biomolecule can be a protein. In some embodiments, the protein may be a globular protein, a fibrous protein or a membrane protein. In some embodiments, the protein may be an antibody, a contractile protein, an enzyme, a hormonal protein, a structural protein, a storage protein or a transport protein. In some embodiments, the biomolecule can be an enzyme. In some embodiments, the enzyme is an enzyme drug. In some embodiments, the enzyme is an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase or a ligase. In some embodiments, the enzyme is a digestive enzyme. In some embodiments, the enzyme is an amylase, lipase or protease. In an example disclosed herein, the enzyme is a lysozyme. In some embodiments, the biomolecule can be an antibody or fragment thereof. Antibodies includes fragments of antibodies and immunoglobins, as well as any molecule that contains an antibody (i.e., an antibody-drug conjugate). Fragments of antibodies may include single chain Fv antibodies (scFv), Fab fragments, F(ab’)2 fragments, Fc fragments, monospecific Fab2, dispecific Fab2, trispecific Faba, monovalent IgG, diabodies, bispecific diabodies, trispecific triabodies, scFv-Fv or minibodies. In some embodiments, the antibodies are monoclonal antibodies. In some embodiments, the antibody is an antibody drug, i.e., a therapeutic antibody. In some embodiments, the antibody is an antibody vaccine, i.e., a monoclonal antibody vaccine. In some embodiments, the antibody is selected from an IgA, IgG, IgE or IgM antibody. In some embodiments, the biomolecule can be a polypeptide or peptide. In some embodiments, the biomolecule can be a polypeptide or peptide is a drug. In some embodiments, the polypeptide is an antibiotic. In some embodiments, the biomolecule can be a plasma protein. In an example disclosed herein, the protein is bovine serum albumin. In some embodiments, the biomolecule is a recombinant protein, i.e., a synthetic protein made using an engineered DNA sequence to synthesis the protein. In some embodiments, the protein is a protein drug. In some embodiments, the protein is a protein vaccine. In some embodiments, the biomolecule may be a protein subunit. In some embodiments, the biomolecule may be a protein complex, e.g., a metalloprotein complex. The protein may have any suitable isoelectric point as defined elsewhere herein. In some embodiments, the surface of the protein is net positively charged. In other embodiments, the surface of the protein is net negatively charged. The examples show the applicability of the method to ensilicate proteins having different isoelectric points, as well as proteins of different shapes, types and sizes. Nucleic acid and Nucleic acid Lipid Nanoparticles (LNP) In some embodiments, the biomolecule can be a nucleic acid. The nucleic acid may be combined with a carrier to form a complexed nucleic acid. The nucleic acid may comprise or consist of RNA or DNA, or one or more chemically modified bases, including but not including LNA, 2’-MOE, or 2’MOE. The nucleic acid may be single-stranded or doublestranded. The nucleic acid bases may be linked by phosphodiester or phosphorothioate linkages. The nucleic acid may be a nucleic acid drug. In some embodiments, the nucleic acid drug may be an ASO. In some embodiments, the biomolecule can be a nucleic acid lipid nanoparticle. In some embodiments, the nucleic acid lipid nanoparticle is an mRNA-lipid nanoparticle. In some embodiments, the lipids may comprise ionizable lipids, preferably an ionizable cationic lipid, and more preferably an amino cationic lipid. In some embodiments, the lipids may comprise phospholipids. In some embodiments, the lipids may comprise ionizable phospholipids. In some embodiments, the lipids may further comprise cholesterol and / or a stabilising lipid (e.g., a PEG lipid / pegylated lipid). In some examples, the lipid comprises 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester, SM-102. In some embodiments, the nucleic acid may be an mRNA drug. In some embodiments, the nucleic acid may be a nucleic acid vaccine, e.g., an mRNA vaccine. The nucleic acid, nucleic acid complex or nucleic acid LNP may have any suitable isoelectric point as defined elsewhere herein. In some embodiments, the surface of the nucleic acid is net negatively charged. The examples show the applicability of the method to ensilicate nucleic acid LNP nanoparticles. In such systems, the nucleic acid is stabilised by the lipids through charge balance where the lipids act upon the hydrophobicity of the carbon chain. For nucleic acid LNPs formed from ionisable lipids, the ionisable head groups can have a positive net charge that can interact well with silica during an ensilication process. The nucleic acid typically comprises regions of negative charge due to the nucleic acid phosphate backbone. The ensilication method described herein can preferably interact with the surface charge provided by the ionisable lipids and / or can also interact with the negative charge of any exposed internal nucleic acid. Virus and Virus-like particles In some embodiments, the biomolecule can be a virus. As defined herein the term “virus” can be used interchangeable with a virion. A virus is a biological structure that comprises at least genetic material (i.e., nucleic acid) and a capsid (i.e., a protein coat) which surrounds the genetic material. The virus may further comprise an outside envelope of lipids. In some embodiments, a virus (e.g., a phage) may further comprise a collar, tail, baseplate or a combination thereof. In some embodiments, the virus may further comprise fibres (e.g., whiskers or legs). The term “virus” is different and distinguished from a “virus-like particle” because a virus contains nucleic acid. For the avoidance of doubt, the term “virus” does not include a virus-like particle. In some embodiments, the particles described herein may comprise a single virus or a plurality of viruses. In some embodiments, the particle comprises a single virus. In some embodiments, the particle comprises two or more, or three or more, or four or more, or five or more viruses. In some embodiments, the virus is a drug. In some embodiments, the virus is a vaccine. The virus may have any suitable shape. The virus may be a filamentous virus, an isometric (i.e., icosahedral) virus, a rod-shaped virus, a bottle-shaped virus, a lemon-shaped virus, a pleomorphic virus, or a head and tail virus. In some examples, the virus is a head and tail virus, or an isometric virus. The virus may be enveloped or non-enveloped. In some embodiments, the virus is a vaccine or part of a vaccine (i.e., or the mammalian virus is suitable for use as a vaccine to establish immunity in humans, mammals or birds). In some embodiments, the virus is an inactivated / dead virus. In other embodiments, the virus is a live virus, preferably a live attenuated virus. In some embodiments, the virus is a viral vector vaccine. In some embodiments, the virus may be of the family selected from flaviviridae, coronaviridae (i.e., coronavirus), adenoviridae, herpesviridae, poxviridae, parvoviridae, reoviridae (i.e., including rotavirus), retroviridae, togaviridae, orthomyxoviridae (i.e., including the influenza virus), hepadnaviridae, paramyxoviridae, aranaviridea, anteriviridea, bunyaviridea, papillomaviridea, astroviridea, calciviridea, circoviridea, parvoviridae, picornaviridea, adeno-associated virus (AAV). In some embodiments, the virus is a mammalian virus. For embodiments in which the virus is a mammalian virus, the host cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell and the virus is a human virus which can infect and be transmitted in humans. In some embodiments, the mammalian cell is a cow cell, sheep cell, pig cell, goat cell, dog cell, cat cell, donkey cell, deer cell, horse cell or rodent cell (e.g., mouse cell or guinea pig cell), fox cell, wolf cell, bat cell, coyote cell, raccoon cell, skunk cell, ferret cell, monkey cell, primate cell, hare cell, rabbit cell, bear cell, mongoose cell. In some embodiments, the virus is an avian virus. In some embodiments, the virus may be an avulaviridae which is a subfamily of the family paramyxoviridae. For embodiments in which the virus is an avian virus, the host cell is an avian cell. In some embodiments, the avian cell is a poultry cell, e.g., a chicken cell, geese cell, guinea fowl cell, duck cell, turkey cell and the virus is capable of infecting and replicating in poultry. In some embodiments and examples, the virus is Newcastle disease virus. Newcastle disease virus is a negativesense single-stranded RNA virus of the subfamily Avulavirinae. In some embodiments and examples, the virus is an inactivated Newcastle disease virus. The virus described herein contains genetic material. In some embodiments, the virus is an RNA virus (i.e., comprising RNA genetic material). The RNA virus may comprise singlestranded or double-stranded RNA. In some examples, the RNA virus comprises singlestranded RNA, which may be positive sense RNA or negative sense RNA. In some examples, the RNA virus comprises positive-sense single stranded RNA. The RNA virus may comprise linear or circular RNA, e.g., linear dsRNA, linear ssRNA, circular dsRNA or circular ssRNA. In some embodiments, the virus is a DNA virus (i.e., comprising DNA genetic material). The DNA virus may comprise single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA). The DNA virus may comprise linear or circular DNA, e.g., linear dsDNA, linear ssDNA, circular dsDNA or circular ssDNA. In some examples, the DNA virus comprises doublestranded linear DNA (e.g., bacteriophage K). In some embodiments, the virus is viable and / or shows no or minimised loss in biological activity, (i.e., after release from the silica shell). In some embodiments, the virus is capable of infecting a host cell (i.e., after release of the virus from the silica shell). In some embodiments, the virus is capable of replication in a host cell (i.e., after release of the virus from the silica shell). In some embodiments, the virus is inactive. In some embodiments, the virus is attenuated. In some embodiments, the biomolecule is a viral vector (i.e., a modified virus designed to deliver genetic material to cells) but wherein all viral genes have been removed. The viral vector may be a gene therapy drug. In some embodiments, the virus is a phage or bacteriophage (i.e., a virus that is capable of infecting and replicating within bacteria and archaea). In some embodiments, the phage may be used as an antimicrobial. In some embodiments, the virus (i.e., bacteriophage) is of the order belfryvirales, caudovirales, halopanivirales, haloruvirales, kalamavirales, ligamenvirales, mindivirales, norzivirales, petitvirales, primaviriales, timlovirales, tubulavirales, vinavirales or durnavirales. In some embodiments, the biomolecule can be a virus-like particle. Virus-like particles comprise viral proteins but are absent of the viral genetic material. The virus may have any suitable isoelectric point as defined elsewhere herein. In some embodiments, the viral capsid is net negatively charged. In some embodiments, the viral capsid is net positively charged. The examples show the applicability of the method to ensilicate negatively-charged viral capsid, such as NDV. Cell In some embodiments, the biomolecule is a cell. The cell may have a cell wall or may not have a cell wall. In some embodiments, the cell may be a bacterial cell. In some embodiments, the bacterial cell is a live bacteria cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a cultured cell-line. Carbohydrate or Polysaccharide In some embodiments, the biomolecule can also be a carbohydrate or a polysaccharide. The carbohydrate to be enveloped with silica according to the present invention can be a carbohydrate vaccine or carbohydrate based vaccine, optionally the carbohydrate is a bacterial polysaccharide, such as a bacterial capsular polysaccharide, an example of which is pneumococcal polysaccharide vaccine (PPV). Vaccines In some embodiments, the biomolecule is a vaccine. The biomolecule vaccine may be a viral vaccine, virus-like particle vaccine, protein vaccine, polypeptide vaccine, carbohydrate, polysaccharide vaccine, or a nucleic-acid vaccine (e.g., mRNA vaccine or nucleic acid LNP vaccine) which may ultimately be administered in the form of a nucleic acid lipid nanoparticle. The vaccine may be suitable for humans or animals. In some embodiments, the animals are mammals. In some embodiments, the animals are livestock animals. In some embodiments, the animals are birds. In some embodiments, the animals are companion animals, e.g., pets. Particle formed by the methods disclosed herein The method of the invention produces a particle comprising a biomolecule that is encapsulated in an amorphous silica shell which is deposited about the surface of the biomolecule. An amorphous silica shell refers to a silica shell that is non-crystalline. The silica shell is preferably a polymerised matrix of silica, i.e., covalently bonded silica. The amorphous silica shell is continuous about and / or around the biomolecule. The amorphous silica shell protects the internal biomolecule. Since the silica is deposited about the biomolecule surface, the interior surface of the silica shell typically resembles or substantially resembles the shape of biomolecule. Since, the particle is formed from a combination of functionalised and non-functionalised silica precursors, at least a portion of the surface of the silica shell (i.e., including the interior surface of the silica shell) is functionalised and comprises a Si-C bond, i.e., at least a portion of the silica in the silica shell is functionalised with a functional group, wherein the silica is attached to the functional group via carbon. The particles described herein may comprise a single biomolecule or a plurality of biomolecules. Typically, the silica shell is functionalised with a functional group that forms a non-covalent interaction (e.g.., an electrostatic interaction or H bonding interaction) with the surface of the biomolecule. In some embodiments, the silica is functionalised with a functional group that promotes growth of the silica shell in an ensilication method. In some embodiments, the silica shell is functionalised with a basic or positively charged group, optionally wherein the basic or positively charged group forms a non-covalent interaction (e.g., an electrostatic interaction or H bonding interaction) with a negative charge on the surface of biomolecule. In some embodiments, (i.e., wherein the silica shell is formed from a mixture of silica precursors comprising a first silica precursor functionalised with a basic or positively charged group and a second non-functionalised silica precursor), the deposition of silica is nucleated by non-covalent interactions between the functionalised silica precursor and negatively charged surfaces of the biomolecule, and non-covalent interactions between the non-functionalised silica precursors and the positively charged surface of the biomolecule. A negative surface of the biomolecule may include, for example, negatively charged amino acid side chains or the phosphate backbone of a nucleic acid. A positive surface of the biomolecule may include positively charged amino acid side chains. In some embodiments, at least a portion of the silicon atoms in the silica shell have the following structure l / FG 'W'Si n where n is 0 to 4, preferably 1 to 3, more preferably 2 and wherein FG is a functional group, preferably a basic or positively charged group. In some embodiments, the FG is an amino group, preferably a primary amine. In some embodiments, the FG is an ammonium group or an ammoniumalkyl group. The silica shell comprises a mixture of functionalised and non-functionalised silicon atoms. Functionalised silicon atoms are defined as silicon atoms bonded to a functional group via an Si-C bond (but wherein the remaining bonds to the silicon atom are Si-0 bonds). Non-functionalised silicon atoms are defined as those which do not comprise an Si-C bond (i.e., where all the Si bonds are to oxygen). It is believed that the ratio of functionalised to non-functionalised silicon atoms can reflect the ratio of functionalised to non-functionalised silica precursors used in the methods to make the particles described herein. For example, the ratio of functionalised to non-functionalised silicon atoms in the silica shell may be between 1:1 to 1:1000, or from 1:4 to 1:800, or from 1:5 to 1:300, or any other ratio range for the functionalised : non-functionalised silica precursors described herein. In some embodiments, the silica shell is functionalised with an alkylamine (e.g., when an amino-alkyl alkoxide silica precursor is used as the functionalised silica precursor). In some embodiments, the silica shell is functionalised with propylamine (e.g., when (3-Aminopropyl)triethoxysilane (APTES) is used as the functionalised silica precursor). The alkylamine group is attached to the silica of the silica shell by a Si-C bond. In some embodiments, at least a portion of the silicon atoms in the silica shell have the following structure 1 / NH2 'W'Si n where n is 0 to 4, preferably 1 to 3, more preferably 2 In some embodiments, at least a portion of the silicon atoms in the silica shell have the following structure 'w's i —n H2 . In some embodiments, at least a portion refers to at least 0.1% of silica atoms, or at least 0.5% of silica atoms, or at least 1% of silica atoms, or at least 5% of silica atoms, or at least 10% of silica atoms. In some embodiments, the silica shell is functionalised with an alkylammonium (e.g., when an ammonium-alkyl alkoxide silica precursor is used as the functionalised silica precursor). The alkylammonium group is attached to the silica of the silica shell by a Si-C bond. In some embodiments, at least a portion of the silicon atoms in the silica shell have the following structure I wherein n is 0 to 4, preferably 1 to 3, more preferably 2 wherein Ri is methyl, ethyl, propyl, isopropyl, butyl, preferably methyl, and wherein R2 is methyl, ethyl, propyl, isopropyl, butyl, preferably methyl, and wherein R3 is a C1-C25 alkyl, optionally a C1-C20 alkyl, further optionally Cis alkyl, preferably wherein the alkyl is a straight unbranched alkyl. In some embodiments, at least a portion refers to at least 0.1% of silica atoms, or at least 0.5% of silica atoms, or at least 1% of silica atoms, or at least 5% of silica atoms, or at least 10% of silica atoms. In some embodiments, at least a portion of the silicon atoms in the silica shell have the following structure some embodiments, at least a portion refers to at least 0.1% of silica atoms, or at least 0.5% of silica atoms, or at least 1% of silica atoms, or at least 5% of silica atoms, or at least 10% of silica atoms. The biomolecule may alternatively be described as being enveloped or caged by continuous silica. The silica shell can shield, separate or protect the biomolecule from one or more external conditions. The silica shell comprises covalently bonded amorphous silica deposited around the surface of the biomolecule, in one or more layers. The silica shell does not bond to, i.e., there is no covalent interaction between the biomolecule and the silica shell, however, the silica of the silica shell may interact with the biomolecule non-covalently, e.g., electrostatically. In some embodiments, the silica of the silica shell interacts with at least one positively charged group on the biomolecule surface, e.g., a positively charged amino acid on the biomolecule surface. In products formed by the method of the present invention, the surface of the silica shell of particles produced by the present method is functionalised with a functional group. The functional group may alternatively or additionally interact non-covalently with the surface of the biomolecule. For example, an amino functional group may interact non-covalently with negatively-charged regions of the biomolecule surface. Non exhaustive examples include a negatively-charged surface of a protein and / or the phosphate backbone of a nucleic acid or nucleic acid complex. The resulting particles may have any suitable particle size. The particle size of the particles will generally be dependent on the size of the biomolecule that is encapsulated. In some embodiments, the particle has a median particle size of 4 nm to 1000 nm or from 4 nm to 500 nm. In some embodiments, the particle has a median particle size of 10 nm to 500 nm, or 50 nm to 500 nm, or 100 nm to 500 nm. Particle size may be determined by any suitable method, e.g., FE-SEM. A variable range of particle size reflects the various different sizes of biomolecules which can be encapsulated in a silica shell in accordance with the present invention. In some embodiments, the silica shell prevents denaturation of the biomolecule encapsulated within. The silica envelope prevents unfolding, or loss of shape, or loss of structure of the biomolecule such as preventing loss of quaternary, or tertiary, or secondary structure. For protein biomolecules denaturation of the biomolecule can include unfolding of quaternary 4° structure involving several proteins folded together, can include unfolding of tertiary structure involving the folded shape, or can include unfolding of secondary structure such as an alpha helix or a beta sheets in proteins. The silica shell can physically prevent the vaccine protein from denaturing (loss of shape and unfolding). In the particle disclosed herein, the biomolecule may be protected from surrounding conditions. In some examples, the biomolecule may be protected from temperature. In some examples, the virus may be protected from one or more acid, e.g., hydrochloric acid, e.g., 0.01 M to 20 M acid. In some examples, this includes protection from 0.1M HCI, 1 M HCI and 10 M HCI. Within the particle disclosed herein, the biomolecule can be thermally stable, or at least the biomolecule has improved thermal stability as compared with a native biomolecule. In some examples, the particle is stable after heating at 95 °C for at least 5 minutes, or at least 10 minutes, or at least 20 minutes, or at least 30 minutes, or at least 1 hour. In some examples, the particle is stable after storage at 25 °C for at least 24 hours, or at least 48 hours, or at least 1 week, or at least 1 month, or at least 3 months, or at least a year. In some examples, the particle is stable after storage at 4 °C for at least 1 week, or at least 1 month, or at least 3 months, or at least a year. The measurement of stability can be determined by any suitable method depending on the biomolecule. For example: for viruses, the measurement of stability can be determined by any suitable method, including a plaque assay, i.e., wherein after release, the virus is substantially intact and substantially functional to replicate and / or form a plaque. For example: for proteins, the measurement of stability can be determined by enzymatic activity (i.e., for enzymes) and / or differential scanning calorimetry and / or chase methods (e.g., pulse-chase method or bleach-chase method), or for antibodies, circular dichroism could be used. For antigens, ELISA-based methods can be used. For example, for nucleic acids, the measurement of stability can be determined by gel electrophoresis methods. For particles, dynamic light scattering (DLS) may be used. Various methods of determining stability could be used which would be readily apparent to someone skilled in the art. Within the particle disclosed herein, the biomolecule may be stable to acid. A particle disclosed herein can be subjected to low pH such as <pH 4.0, <pH 3.5 or <pH 3.0. When subsequently released from particle, the biomolecule is substantially intact and substantially functional. By contrast a biomolecule that is not ensilicated is more likely to be denatured and lose its function in these conditions. Methods of characterization Isoelectric Point The isoelectric point, e.g., of a biomolecule, can be determined using any suitable method. In some embodiments, the isoelectric point of the biomolecule is determined before step a) of the method, and the acidifying step if present. The isoelectric point may be determined by any suitable method. In some embodiments, the isoelectric point of the biomolecule is determined using electrophoretic light scattering (ELS) (e.g., as defined herein), Phase Analysis Light Scattering (PALS), or Isoelectric focusing (IEF) gels Si-C bond The presence of the Si-C bond defined herein may be determined or detected using any suitable method as may be known in the art. This includes chromatographic and / or spectroscopic methods. The presence of an Si-C bond may be used to identify a functionalised silica precursor in according with this disclosure. The presence of an Si-C bond may be used to identify a particle in accordance with this disclosure, where a biomolecule is encapsulated in a silica shell which is functionalised and comprises an Si-C bond. The presence of an Si-C bond in the silica shell may be determined by analysing the particle (i.e., while the biomolecule is encapsulated within the silica shell). Alternatively, the presence of an Si-C bond in the silica shell may be determined after breaking down the silica shell, e.g., by contacting the particle with a release solution (i.e., after de-ensilication). Method Disclosed herein is a method of a) hydrolysing a mixture of silica precursors, said mixture comprising a functionalised silica precursor comprising a Si-C bond and a non-functionalised silica alkoxide precursor b) directly contacting the hydrolysed precursors with an aqueous solution comprising the biomolecule and c) encapsulating the biomolecule in a silica shell to form a particle, such that at least a portion of the silica on the interior surface of the silica shell is functionalised and comprises an Si-C bond. In preferred embodiments, step a) of the method comprises mixing the functionalised silica precursor with the non-functionalised silica precursor, before hydrolysing one or more of the precursors. In preferred embodiments, step a) of the method comprises mixing the functionalised silica precursor with the non-functionalised silica precursor, before hydrolysing the non-functionalised silica precursor (e.g., TEOS). In some embodiments, step a) comprises ai) mixing a functionalised silica precursor comprising a Si-C bond, with a non-functionalised silica alkoxide precursor to form a mixture of precursors aii) hydrolysing the mixture of precursors. In some embodiments, wherein the functionalised silica precursor is a basic functionalised silica precursor, the basic functionalised silica precursor is acidified and optionally hydrolysed before mixing with and hydrolysing the non-functionalised silica alkoxide precursor. In some embodiments, the acidification of the basic silica precursor occurs at a pH of between 1 and 5, preferably between 1 and 2 Mixture of Silica Precursors In some embodiments, the functionalised silica precursor and non-functionalised silica precursor are mixed in a ratio from 1:1 to 1:2000, optionally from 1:4 to 1:800, or from 1:5 to 1:300, or from 1:10 to 1:200. Any suitable non-functionalised silica precursor may be used in the method, including those described herein. In some embodiments, the non-functionalised silica precursor is an alkoxide silica precursor (e.g., TEOS or TPOS). Any suitable functionalised silica precursor may be used in the method, including those described herein. In some embodiments, the functionalised silica precursor is an aminoalkylalkoxide silica precursor (e.g., APTES), which has preferably been acidified (see acidifying step). In some embodiments, the APTES solution is provided as a 50% aqueous APTES solution. In some embodiments, the functionalised silica precursor is an ammoniumalkyl-alkoxide silica precursor (e.g., C18-TMS). The ratio of the two precursors can be optimised depending on the type of biomolecule and the isoelectric point of the molecule. In some embodiments, when the biomolecule has an isoelectric point of less than 6.5, the functionalised silica precursor and non-functionalised silica precursor are mixed in a ratio from 1:1 to 1:250, preferably from 1:5 to 1:225, preferably from 1:10 to 1:200, and in some examples, from 1:10 to 1:50, for example 1:25. In some embodiments, when the biomolecule has an isoelectric point of less than 6.5, the functionalised silica precursor and non-functionalised silica precursor are mixed in a ratio of 1:<250 (e.g., 1:1 to 1:250, or 1:10 to 1:250), or 1:<225 (e.g., 1:1 to 1:225, or from 1:10 to 1:225), or 1:<200 (e.g., 1:1 to 1:200, or from 1:20 to 1:200), or 1 :<100 (e.g., 1:1 to 1:100, or from 1:10 to 1:100) or 1:<50 (e.g., 1:1 to 1:50, or from 1:10 to 1:50). In some embodiments, when the biomolecule has an isoelectric point of less than 6.5, the functionalised silica precursor and non-functionalised silica precursor are mixed at a ratio of 1 :>1, or 1: >2, or 1: >3, or 1: >4, or 1: >5, most preferably 1: >10 (since while ratios below 1:10 work, there is some effect of steric hindrance) In one example, two biomolecules (e.g., Newcastle disease virus and BSA) have an isoelectric point between 2 and 6.5, or between 2.5 and 6, and the ratio of functionalised silica precursor and non-functionalised silica precursor are preferably mixed in a ratio of 1:4 to 1:250, more preferably 1:10 to 1:200, or 1:10 to 1:50, for example, 1:25. In some embodiments, when the biomolecule has an isoelectric point of greater than 6.5, or greater than 7.5, the functionalised silica precursor and non-functionalised silica precursor are mixed in a ratio from 1:10 to 1:1000, preferably from 1:20 to 1:750, or from 1:25 to 1:500, or from 1:30 to 1:250. In some embodiments, when the biomolecule has an isoelectric point of greater than 7.5, the functionalised silica precursor and non-functionalised silica precursor are mixed in a ratio of 1:>10, or 1:>20, or 1 :>30, or 1:>40, or 1:>50 In one example, the biomolecule (e.g., lysozyme) has an isoelectric point between 8 and 10 and the ratio of functionalised silica precursor and non-functionalised silica precursor are preferably mixed in a ratio of 1:100 to 1:1000, or from 1:100 to 1:300 (e.g., 1:200) In some embodiments, when the biomolecule is a nucleic acid LNP, the functionalised silica precursor and non-functionalised silica precursor are mixed in a ratio of 1:1 to 1:2000, optionally 1:10 to 1:1400, or from 1:250 to 1:1000, e.g., 1:800. In some embodiments, the mixing step takes place for at least 10 seconds, or at least 30 seconds, or at least 1 minute, or at least 5 minutes, or at least 10 minutes, and / or until the mixture becomes homogenous, preferably for between 10 minutes and 180 minutes, for example, 30 and 60 minutes. In some embodiments, the mixture may be stirred. The mixture may be stirred at any suitable speed, such as 100 rpm to 1000 rpm, e.g., when the mixture is added to a 50 ml glass beaker. In some embodiments, the mixing step of the non-functionalised silica precursor and the functionalised silica precursor occurs at an acidic pH, preferably wherein the pH is less than 6, optionally less than 4, preferably between 2 and 4. In some embodiments, the mixing step comprises addition of an acid, e.g., HCI, in an example, 32% HCI. The mixture can be added in any suitable order. In some embodiments, the non-functionalised silica precursor is added to a solution comprising the functionalised silica precursor. When a basic silica precursor is used (e.g., APTES), the non-functionalised silica precursor may be added to an acidified solution comprising the functionalised silica precursor (e.g., using an acidifying step, as detailed below). In some embodiments, further acid may be added to the mixture of functionalised silica precursor and non-functionalised silica precursor. Acidifying step (for basic functionalised silica precursors) In some embodiments, i.e., when the functionalised silica precursor is a basic silica precursor, the method may further comprise acidifying the basic silica precursor before step a). In some embodiments, the acidifying step occurs at an acidic pH, preferably below 5, more preferably between 1 and 5, and even more preferably between 1 and 2. In preferred embodiments, the basic silica precursor is an alkoxy amine silica alkoxide precursor. In some embodiments, the basic silica precursor is APTES. In some embodiments, the acidifying comprises contacting the basic silica precursor with an acid, e.g., HCI acid, more specifically 32% HCI acid. In some examples, the basic silica precursor is mixed with water, prior to acidification, to form an aqueous solution . In some embodiments, the basic silica precursor is APTES and the aqueous APTES solution is 10% to 75% APTES solution. In some examples, the aqueous APTES solution is a 50% APTES solution. Acidification of a basic functionalised silica precursor, such as APTES, is found to reduce agglomeration and precipitation of the silica precursor prior to mixing with the nonfunctionalised silica precursor. Hydrolysing The mixture of functionalised and non-functionalised silica precursors are hydrolysed before contacting with the biomolecule. Generally, the silica precursors are mixed before hydrolysing at least one of the silica precursors. In some embodiments, the silica precursors are co-hydrolysed (i.e., hydrolysed together). In some embodiments, an optionally hydrolysed functionalised silica precursor is added to the non-functionalised silica precursor before hydrolysing the non-functionalised silica precursor. Hydrolysing the mixture of silica precursors produces a mixture of hydrolysed silica precursors. Hydrolysing the silica starting precursors is performed at acidic pH. The acidic pH can be less than or equal to 5, 4.5, 3.5, 3.0, 2.5 or 2.0, preferably wherein the pH is between 2 and 4. In some methods the step of hydrolysing a silica starting material (the pre-hydrolysis step) is performed at pH 3.0 or below, or at about pH 2.0. Any suitable acid can be used to catalyse the hydrolysis of the silica precursors. In some preferred embodiments and examples, the acid used is HCI. In some embodiments, the hydrolysis step is or occurs for about 20 to 120 minutes, or about 40 to 80 minutes. In some embodiments, the hydrolysis step is or occurs for at least 20 minutes, or at least 40 minutes, or for at least or equal to 60 minutes. In some embodiments, the hydrolysis step is or occurs for less than 120 minutes, or less than 90 minutes, or less than or equal to 60 minutes. The hydrolysed silica precursors comprise a mixture of non-functionalised and functionalised silica monomers which are capable of polymerising to form an amorphous silica shell. The non-functionalised silica monomers can include SiO2, or_O-Si-O'. At the microscopic level each silicon atom is generally coordinated by four oxygen atoms and the hydrolysed silica precursor can be tetrahedrally coordinated silica, such as Si(OH)4 or O' O Ss o The hydrolysed non-functionalised silica precursor is preferably a hydrolysed alkoxysilane, and more preferably hydrolysed TEOS or preferably hydrolysed TPOS. TEOS or TPOS are generally preferred as it is less toxic than other precursors, e.g., such as TMOS which has a methanol as opposed to an ethanol or propanol byproduct. The functionalised silica monomers include silica coordinated to a functional group. At the microscopic level each silica atom is generally coordinated by three oxygen atoms and the carbon atom of the functional group. o- o- In some embodiments, i.e., wherein the functionalised silica precursor is an aminoalkylalkoxysilane, the functionalised silica monomers have the following structure: where n is 0-4, preferably 1-3. more preferably wherein the functionalised silica precursor is APTES, the functionalised silica monomers have the following structure: O’ The hydrolysing step takes place in the absence of the biomolecule. In some embodiments, i.e., wherein the functionalised silica precursor is an ammoniumalkyl-alkoxysilane, the functionalised silica monomers have the following structure: where n is 0-4, preferably 1-3, wherein Ri and R2 and R3 are as defined elsewhere herein. In preferred embodiments, i.e., wherein the functionalised silica precursor is an ammoniumalkyl-alkoxysilane, the functionalised silica monomers have the following structure: Contacting The hydrolysed silica precursors are contacted with the biomolecule to precipitate or grow the silica envelope around the biomolecule. Forming a silica envelope is a modified sol-gel process in which a silica gel is prevented from forming. Once hydrolysed silica precursor and biomolecule are contacted, silica grows a network and rapidly precipitates. In the absence of a biomolecule as a template this silica would gel and then glass if left for long enough. On contacting the mixture of hydrolysed silica precursors with the aqueous solution comprising the biomolecule, the silica precursor precipitates about the biomolecule to form a covalently bonded amorphous silica shell about the biomolecule. In preferred embodiments, the contacting occurs at a pH in the range of between 4 and 10, optionally in the range of 5 to 8. In some embodiments, the aqueous solution comprising the biomolecule and the buffer can have a pH in the range of 6.0 <pH >9. The hydrolysed silica precursor can rapidly polymerise and / or precipitate around the biomolecule. Shorter times for contacting the hydrolysed silica precursor with biomolecule will form individual nanoparticles. A nanoparticle comprising a biomolecule enveloped with silica. Longer times for contacting the hydrolysed silica precursor with biomolecule will allow for nanoparticle agglomeration. In preferred embodiments, the contacting takes place for a period of at least 1 second, or at least 10 seconds, or at least 30 seconds. In some embodiments, the contacting takes place for a period from 30 seconds to 120 minutes, more preferably from 5 to 60 minutes, and even more preferably between 10 and 30 minutes. The contacting step may be carried out using any suitable ratio of hydrolysed silica prescursor:biomolecule in aqueous solution, e.g., buffer. In preferred embodiments, the ratio of hydrolysed silica to the biomolecule in buffer is from about 1:5 to 1:200, for example, about 1:10 to 1:125. In some embodiments, the ratio of hydrolysed silica to the virus in buffer 1:> 10, or 1: >25, or 1: >50, or 1: >75, or 1: >100. The contacting step may be carried out at any suitable temperature. In some embodiments, the contacting may be carried out at a temperature between 4 and 40 °C, or between 4 and 25 °C, or between 15 and 25 °C, or between 15 and 20 °C. In preferred embodiments, the biomolecule is added in aqueous buffer, optionally wherein the buffer comprises one or more salts containing one or more of a magnesium salt, a calcium salt, or a sodium salt, or a potassium salt and further optionally wherein the buffer is an SM buffer, a phosphate buffer, a modified SM buffer, PBS, Tris, an imidazole buffer, a sucrose buffer, or bis-tris buffer. In preferred embodiments, the biomolecule is reconstituted in buffer. In some embodiments, the buffer comprises one or more salts, and preferably wherein the one or more salts comprises a dicationic or monocationic metal ion. In some embodiments, the one or more salts may comprise a dicationic metal ion. In some embodiments, the one or more salts may comprise one or more of magnesium or calcium. In some embodiments, the one or more salts may comprise a monocationic metal ion, wherein the monocationic metal ion may comprise sodium. In some examples, the one or more salts may comprise magnesium sulfate and / or sodium chloride. In some embodiments, the buffer comprises one or more dicationic metal salts at a concentration of between 0.1 mM and 50 mM, or between about 1 mM to 25 mM, or between about 5 mM to 11 mM, or about 8 mM. In some embodiments, the buffer comprises one or more dicationic metal salts at a concentration of greater than 0.5 mM, or greater than 1 mM, or greater than 2 mM, or greater than 3 mM, or greater than 4 mM, or greater than 5 mM, or greater than 6 mM, or greater than 7 mM, or greater than about 8 mM. In some embodiments, the dicationic metal salt preferably comprises magnesium and more preferably comprises magnesium sulfate. In some embodiments, the buffer comprises a sodium salt at a concentration of from 0.1 mM to 500 mM, or between about 50 mM to 200 mM, or about 100 mM. In some embodiments, the buffer comprises one or more dicationic metal salts at a concentration of greater than 0.1mM, or greater than 1 mM, or greater than 5 mM, or greater than 10 mM, or greater than 25 mM, or greater than 50 mM, or greater than 75 mM, or about 100 mM. In preferred embodiments, the sodium salt is preferably sodium chloride. In some embodiments, the buffer may comprise one or more small molecules. In some embodiments, the buffer comprises one or more amino acids. In some embodiments, the buffer comprises glycine. In some embodiments, the buffer comprises an amino acid such as glycine at a concentration of at least 1 mM, or at least 5 mM, or at least 10 mM, or at least 25 mM, or at least or equal to 50 mM. In some embodiments, the buffer comprises glycine at a concentration of between 1 mM and 100 mM, or between about 10 mM to 80 mM, or between about 25 mM to 75 mM, or between about 40 mM to 60 mM, or about 50 mM. In some embodiments, the buffer may comprise one or more stabilizers. In some embodiments, the buffer may comprise gelatin. In some embodiments, the buffer may comprise at least 0.01 g / mL of gelatin, or at least 0.5g / mL, or about 0.1 g / mL of gelatin. The aqueous solution comprising the biomolecule can have the biomolecule at a concentration less than that which causes spontaneous aggregation of the biomolecule. The concentration of a biomolecule at which spontaneous aggregation may occur is dependent on the biomolecule and on the conditions. A person skilled in the art can determine the concentration below which to perform the present invention. In preferred embodiments, in the contacting step, the biomolecule is present in the aqueous solution at a concentration of 0.1 mg / ml to 100 mg / ml. In some examples, the biomolecule is present at a concentration less than 100 mg / ml, or less than 50 mg / ml, or less than 10 mg / ml. In some examples, the biomolecule is at a concentration of greater than 0.25 mg / ml (e.g., from 0.25 mg / ml to 100 mg / ml, or from 0.25 mg / ml to 10 mg / ml), or greater than 0.5 mg / ml (e.g., from 0.5 mg / ml to 100 mg / ml, or from 0.5 mg / ml to 10 mg / ml), or greater than 0.75 mg / ml (e.g., from 0.75 mg / ml to 100 mg / ml, or from 0.75 mg / ml to 10 mg / ml) or greater than or equal to 1 mg / ml (e.g., from 1 mg / ml to 100 mg / ml, or from 1 mg / ml to 10 mg / ml). In some embodiments when a virus is used as the biomolecule, the virus in buffer is present at a concentration greater than 1 x 107PFU / ml, more preferably greater than 1 x 108 PFU / ml, more preferably greater than 1 x 109PFU / ml, more preferably in a range of froml x 108 PFU / ml to 1 x 1010 PFU / ml, or from 1 x 109 PFU / ml to 1 x 1010 PFU / ml. In some embodiments, when the cell is used as a biomolecule, the cells in buffer are present at a concentration of greater than 1 cell / pl, or greater than 10 cell / pl, or greater than 50 cell / pl, or greater than 75 cell / pl. In some embodiments, when the cell is used as a biomolecule, the cells in buffer are present at a concentration in a range from 1 cell / pl to 10000 cell / pl, optionally from 10 cell / pl to 1000 cell / pl, or from 25 cell / pl to 500 cell / pl. The contacting may be carried out for any suitable time. In some embodiments, the contacting is carried out for at least 5 minutes, preferably at least 10 minutes. In some embodiments, the contacting is carried out for less than 60 minutes, or less than 45 minutes, or less than 20 minutes, or less than 10 minutes, or less than 5 minutes, or less than 2 minutes, or less than 1 minute, or less than 30 seconds. In some embodiments, the contacting is carried out for between 10 and 20 minutes. The silica is directly deposited about the surface of molecule. As defined herein the term “directly deposited” refers to the silica being deposited and in contact with at least a portion of the surface on the biomolecule, for example, the protein capsid of a virus, as opposed to being deposited on an intermediate polymer layer between the biomolecule and the silica shell. In preferred embodiments, the method is free of a templating step (i.e., where the biomolecule is first coated with an intermediate layer for example intermediate layers comprising polylysine, polyethyleneimine chitosan, polylactic, protamine or spermine or combinations thereof. In other words, the biomolecule is not cloaked or modified for the ensilication process. In preferred embodiments, the particle formed by the method of the invention is free of polylysine, polyethyleneimine chitosan, polylactic, PLGA, protamine or spermine or combinations thereof. In some embodiments, the method further comprises a step of purifying, filtering or enriching the biomolecule prior to contacting the biomolecule with the mixture of hydrolysed silica precursors. The enriching or purifying step may be via any suitable enrichment or purification method. In some embodiments, the enriching or purifying is selected from a PEG-based enrichment, gradient centrifugation, size exclusion chromatography, ion exchange chromatography chloroform purification, or filtration methods. In preferred embodiments, e.g., wherein the biomolecule is a virus, the enriching or purifying is from a virus in a lysate (i.e., virus in growth buffer). In some embodiments, the enriching or purifying is a PEG-based enrichment, where PEG is polyethylene glycol. In some examples, the PEG has an average molecular weight between 3000 and 12000 g / mol, or between 3500 and 10000 g / mol, or between 4000 and 80000 g / mol, or between 5000 and 7000 g / mol, or about 6000 g / mol. In some examples, the PEG-based enrichment comprises PEG 6000, i.e., PEG with an average molecular weight of 6000 g / mol. In some examples, the PEG-based enrichment comprises adding between 1 and 15 % w / w, or from 5 to 12.5% w / w, or from 7 to 10 % w / w PEG to the biomolecule (e.g., virus lysate). In some examples, the PEG-based enrichment comprises adding at least 5% w / w, or at least 7% w / w, or at least 10% w / w PEG to a biomolecule (e.g., virus lysate). In some embodiments, the enriching or purifying comprises a chloroform purification. In some embodiments, the enriching or purifying comprises gradient centrifugation. In some embodiments, the gradient centrifugation utilizes a gradient medium selected from sucrose, cesium chloride, iodixanol, sorbitol, Dextran or 5-(N-2,3-Dihydroxypropylacetamido)-2,4,6-triiodo-N,N'-bis(2,3-dihydroxypropyl)isophthalamide (e.g., Histodenz). In some embodiments, the enriching or purification comprises size exclusion or ion exchange chromatography. In some embodiments, the biomolecule may be dialysed prior to contacting. This is to remove any cross-reactivity and to create a pure solution. In some embodiments, the biomolecule that is dialysed is a protein, and in some embodiments, a recombinant protein. This is because some proteins, more specifically recombinant proteins may be provided as a crude lysate or multiple host cell proteins). Dialysis may be performed using any suitable method. In some embodiments, dialysis is performed using buffer-exchange, e.g., using size exclusion chromatography and / or buffer exchange using columns or passive membranes with a molecular weight cut-off (MWCO). In some embodiments, the biomolecule is filtered prior to contacting, e.g., through a syringe filter. In some embodiments, the biomolecule in buffer (e.g., virus, protein, or any other biomolecule defined herein) has a polydispersity index of less than 0.2 as determined using DLS analysis, or less than 0.15. In some embodiments, the polydispersity index is determined using dynamic light scattering. In some embodiments, the polydispersity index is determined using a Brookhaven DLS Zeta Plus with Pals) or using Malvern Panalytical Zetasizer software and is used to indicate the purity of the samples. Values closer to 0 are purer while particles closer to 1 are impure and contain a mixture of many different particle types. In preferred embodiments, the enriching comprises concentrating the concentration of biomolecule (i.e., such that the biomolecule is increased in concentration or PFU / mL). In some embodiments, the concentrating comprises centrifuging the biomolecule in aqueous solution (e.g., buffer). The centrifuging may take place at any suitable speed, for example, from 5,000 x G to 200,000 x G . In some embodiments, the centrifuging takes place at a speed of less than 25000 x g, or preferably less than 16000 x g. In some embodiments, the biomolecule has been pre-purified (i.e., obtained commercially in a purified state). In some embodiments, wherein the biomolecule is a virus, the method comprises propagating the virus prior to contacting the biomolecule with the mixture of hydrolysed silica precursors. In some embodiments, the propagating is solid-phase or liquid-phase propagation. Ensilication or Encapsulating step Encapsulating or encapsulation herein may otherwise be referred to as ensilicating or ensilication. In some embodiments, the silica shell is formed from a mixture of silica precursors comprising a functionalised silica precursor functionalised with a basic or positively charged group and a second non-functionalised silica precursor, and wherein the deposition of silica is nucleated by non-covalent interactions between the first silica precursor and negatively charged surfaces of the biomolecule, and non-covalent interactions between the non-functionalised silica precursors and positively charged surface of the biomolecule. In some embodiments, the resultant particles precipitate out. In some embodiments, the method further comprises a vacuum filtering step, i.e., wherein the particles are vacuum filtered. In some embodiments, the particles may be further subjected to drying, e.g., airdrying. Methods of Release Disclosed herein is also a method for releasing the biomolecule from the particle of the invention. In one embodiment, the method comprises contacting the particle with a release solution, which may be referred to as a de-ensilication solution elsewhere herein. The biomolecule within the silica shell may be released from its silica shell prior to administration to a subject. This may be achieved by any suitable release method, including a release method of de-ensilication described herein. A number of biomolecules, including some vaccines, require administration by injection. The present invention further embraces a method comprising administration to a subject in need thereof, a biomolecule, wherein the method comprises releasing the biomolecule from a silica shell as described herein prior to administration to the subject. The biomolecule may be reformulated and / or added to one or more excipients after release. In some embodiments, the method is a method of vaccination, and the biomolecule is a vaccine, wherein the vaccine is released from the silica shell prior to administration (e.g., injection). In some embodiments, the release solution may be an alkaline aqueous solution. In some embodiments, the release solution may be at a pH of above 10, or equal to or greater than 11. In some embodiments, the release solution comprises an alkali carbonate and alkali bicarbonate, for example, sodium carbonate and sodium bicarbonate. In alternative embodiments, the release solution may comprise fluoride ions at acidic pH. The release solution comprises buffer, fluoride ions (for example from NaF) and is adjusted to acidic pH (for example with HCI). It has been identified that acidic conditions alone do not allow release of the biomolecule, and fluoride ions alone do not allow release of the biomolecule. The method may further comprise agitation of the mixture. The release stage of contacting the biomolecule and its envelope of silica with a release solution comprising fluoride ions at acidic pH, optionally with agitation, may be from about 10 minutes to about 2 hours, about 30 minutes to about 90 minutes, or about an hour. Biomolecules enveloped with silica may be in powder form when contacted with release solution. Sufficient fluoride ions are required to achieve good release of biomolecule. The release solution can comprise a fluoride ion concentration of from about 75mM to about 150mM, optionally the fluoride ion concentration is from about 75mM to about 125mM, further optionally the fluoride ion concentration is from about 85mM to about 115mM. Removal of any excess fluoride ions can be achieved by addition of a salt, such as a calcium salt, leading to precipitation of a fluoride salt such as calcium fluoride. Removal of excess fluoride ions may be performed following the release stage, such as immediately following the release stage. In some embodiments, the release solution may comprise buffer which can protect the released biomolecule. Examples For the following examples the following definitions apply: Material % - The percentage of entity that has been encapsulated during the ensilication process, determined from supernatant analysis Load% - The percentage of the ensilicated material that is the entity Ensilication Efficiency - The percentage of the starting amount of entity that could be extracted from the ensilicated material. Determined from de-ensilication data. Example 1: Methods of Ensilication using a functionalised silica precursor The present inventors sought to develop a modified ensilicated method which enabled ensilication of a wider range of biomolecules. In development, attempts to ensilicate Newcastle disease virus (NDV) had been poor-yielding using the prior ensilication method using TEOS only, which uses a non-functionalised silica precursor. After a substantial amount of research effort, some of which is discussed in Example 12 below, the present inventors reasoned that using a functionalised silica precursor, the functional group could be selected to interact favourably with the biomolecule surface. Since NDV has a negatively charged surface, a silica precursor was chosen with a basic functional group (e.g., APTES). It was found that using a functionalised silica precursor, in combination with a nonfunctionalised silica (e.g., TEOS), led to ensilication of NDV with much higher yields (see Figure 3) The following exemplary method was used: A 40% APTES solution, pH 2 was prepared (1ml APTES, 1ml DI H2O and 0.5ml of 32% HCI), this was used prepare the hydrolysed silica at a 4:1 TEOS:APTES ratio by adding 300ul of the APTES solution to 1200ul TEOS and 1500ul DI H2O, with 32% HCI and mixed until homogenous. A 1mg / ml stock of NDV-NIBSC in 50mM Tris-HCI, pH 7 was prepared. Using a 24 well plate 0.5 ml of stock was added to a well and the hydrolysed was added at a 1:50 ratio. The plate was rocked for 20 minutes before being diluted with 1ml of DI H2O. 1ml was sampled and centrifuged at 17,000g for 2 minutes before the stock concentration and the remaining NDV concentration was determined using a BCA assay - protocol followed as per manufacturer's instructions. The data was then used to determine the material %. The new method was found to be incredibly versatile and tuneable and the present inventors found that this could also be used to ensilicate a wide range of different biomolecules. Example 2: Ensilication of Virus - Newcastle Disease Virus (NDV) Supplier Details WHO International Standard The 1st International Standard for Newcastle Disease Vaccine (Inactivated) 1963. NIBSC code: NVIA. Material arrives in glass ampules sealed under vacuum with an average weight of lyophilized material of 527.4mg Reconstitution - 50mM Tris-HCI pH 6.5 - 7.5 - Stock • Dissolve 3.33g of Trizma base in 550ml H2O and adjust pH as required using 32% and 3.2% HCI. This is Tris-HCI • Snap 1 vial of the international standard into a glass beaker and add 50-500 ml of 50mM Tris-HCI - depending on required concentration. Stir at 200rpm for 20 minutes before syringe filtering the material into a new beaker -material is then ready for use. Isoelectric point The isoelectric point was determined to be pH 3.31. The isoelectric point was determined using the NDV stock prepared as above at pH 7.5 in 500ml of Tris-HCI and dialysed for 3 hours before use. Initially the pH of 2ml samples of the stock were adjusted in pH to cover a broad range of values (pH 1.3 --> 13) using HCL (3.2 &32%), Tris (2M) and NaOH (2M). A broad range was covered to identify the pH range in which the IEP fell. The PALS (Phase-analysis light scattering) program on the Brookhaven Nanobrook 90PlusPALS instrument was used to measure the zeta potential of each sample. These were plotted vs the pH and this indicated the IEP fell between pH 2-4. Samples were then prepared to fall between pH 2-4 and the zeta potential was again determined for each using the PALS feature. These were plotted vs the pH and IEP was determined to be pH 3.31. Isoelectric point determination is demonstrated in Figure 4. Ensilication Method The silica (1:25 APTES:TEOS) was prepared by adding 400pl of 50% APTES, 5ml water, 5ml Tetraethyl orthosilicate (TEOS) and 10ul of 32%HCI were stirred at 500rpm for 1 hour at room temperature until homogeneous. Stirring was reduced to 80rpm for 20 minutes before being added to 50mls of the NDV-Stock (1mg / ml, pH 7) at a 1:50 ratio (1ml). After 20 minutes DI water was added ata 1:1 ratio and the stirring stopped. Samples of the supernatant were collected and centrifuged for analysis, the rest of the mixture was vacuum filtered and left to dry in the fume hood overnight before being weighed, milled and collected. Material was stored in a climate chamber (25°C, 60% RH) or used in analysis. De-ensilication and analysis The Milled material is de-ensilicated following the general de-ensilication method disclosed herein. The following methods were used to confirm the successfulness of the ensilication and de-ensilication and confirm the functionality of the NDV is maintained. BCA Protein assay The protein concentration of the stock, supernatants and De-ensilicated material was measured using a BCA protein assay. Pierce BCA Protein Assay Reagent A (Thermo Fisher) was used, and protocol was followed as per manufactures instructions. This data was used to calculate the material % (Total incorporation of virus into the silica using the stock concentration and the supernatant concentration), Load % (The percentage of the ensilicated material that is virus) and the ensilication efficiency (Total incorporation of virus based off of the De-ensilication concentration). The results are shown in Figure 5 and the Table below. Sample Measured § Dilution Actual Material Load % Ensilication Cone., Factor Cone., % efficiency % § mg / ml mg / ml J Stock A 0.48 y-2-“ 0.96 - - J Stock B 0.47 § 2 0.95 - - ; Stock C 0.45 § 2 0.89 - - ; SUP A 0.20 § 1 0.20 58.7 - ; SUP B 0.18 1 0.18 61.4 - ; SUPC 0.21 § 1 0.21 53.1 - De-ENS Q 1587 | 1 016 - 31.7 A De-ENS 0 1461 1 1 015 - 28.3 43.4 B DeENS 0.14985 I 1 0.15 - 29.0 49 / 3 C 1 SDS-PAGE Samples were prepared in SDS sample buffer and 1M DTT and loaded on a 4-20% precast polyacrylamide gel (BioRad). Protein bands were visualised with PageBlue protein staining solution. Image taken using the Chemidoc. Stock bands are present in the de-ensilicated material but not in the supernatants indicating near to complete ensilication. The same species is present in the stock and the de-ensilicated material providing proof of ensilication. The results are shown in Figure 6. Western Blot Samples were prepared in SDS sample buffer and 1M DTT and loaded on a 4-20% precast polyacrylamide gel (BioRad). They were then transferred to a Trans-Blot Turbo Mini 0.2um Nitrocellulose Membrane using a trans turbo blotter. The membrane was washed briefly in Tris Buffered Saline (TBS / T) blocked with a 3% BSA in TBS / T (Blocking solution) overnight at 4° C. The following day the membrane was washed three times with 3x 50ml TBS / T for 5-minute periods. It was then incubated for 2 hours, at room temperature, with 50mls of Primary antibody, Anti Newcastle disease serum (NIBSC, NDS), diluted 1:300 in blocking solution. The membrane was then washed a further three times with 3x 50ml TBS / T for 10 minutes periods. Then incubated for 1 hour at room temperature with the secondary antibody, Goat Anti-Chicken IgY (H+L) Horseradish peroxidase conjugate, ata 1:10,000 in blocking solution. The membrane was then washed for the final time in 3x 50mls for 10-minute periods before being incubated with ECL substrate for 5 minutes. The membrane was visualised using Chemidoc and using the blots application. Stock bands are present in the de-ensilicated material but not in the supernatants -indicating near to complete ensilication. The same species is present in the stock and the de-ensilicated material providing proof of ensilication. The results are shown in Figure 7. ELISA Stock NDV, De-ensilicated NDV and the ensilication supernatants were loaded at20ug / ml in column 1 of a 96 well plate. They were then serially diluted across the plate 2-fold across the plate in ELISA coating buffer (pH 9.6); leaving column 12 blank. 100 pl of the dilution was transferred onto a 96 well flat bottom plates (High binding plate, Greiner) and incubated overnight at 4°C. The following day the plates of the were washed with PBS and blocked with 1mg / ml BSA in PBS supplemented with 0.05% Tween20 (PBS-T) for 2 hours. The primary antibody, Anti Newcastle disease serum (NIBSC, NDS) - reconstructed in PBS, was prepared at a 1:300 ratio in PBS-T and 10OpI added to each well after the plate was washed three times with PBS. This was incubated for 1 hour at room temperature and washed again three times with PBS before being incubated with 100 pl of the secondary antibody, Goat Anti-Chicken IgY (H+L) Horseradish peroxidase conjugate, ata 1:10000 in PBS-T for 1 hour. The plate was then washed four times with PBS and were developed using TMB substrate and the reaction stopped with 1M H2SO4. The absorbance was measured at 450nm using the Varioskan Lux. The results are shown in Figures 8 and 9. De-ensilicated material shows similar activity to that of the stock material, indicating the virus has not been damaged during the ensilication process. There is little to no activity in the supernatants indicating near to complete ensilication. Example 3: Ensilication of a Protein (Lysozyme) Supplier details Lysozyme from chicken egg, Sigma Aldrich, Cat. No: 62971-50G-F Isoelectric point The isoelectric point was determined to be 8.6. The isoelectric point was determined with a 300ml solution of 1.5mg / ml lysozyme in 50mM Tris-HCI, pH 7,5 was prepared. Aliquots were adjusted to different pH values between 7.5 and 12. The PALS program on the Brookhaven Nanobrook 90PlusPALS was used. The zeta potential was measured at each pH and then plotted against each other to determine the IEP. This is shown in Figure 10. Ensilication Method A 1mg / ml lysozyme stock was made by dissolving 350mg of lysozyme in 350ml of 50mM Tris-HCI, pH 8. TEOS was prepared in triplicate (5ml TEOS, 5ml H2O and 10pl 32% HCI). 50% APTES was made in triplicate (3ml APTES, 1.5ml H2O and 1.5ml 32% HCI) and then used to make triplicate silica solutions with APTES:TEOS ratios of 1:200 (50pl 50% APTES, 5ml TEOS, 5ml H2O and 10pl 32% HCI). Ensilications were performed by adding 1ml of silica to 50ml of lysozyme stock and stirring at 80rpm for 20 minutes before 50ml of H2O was added and the stirring stopped. Samples of the supernatant were taken, and the remaining protein concentration determined by UV-vis and this data was used to determine the material %. The rest of the material was vacuum filtered and left to dry overnight. The following day the material was weighed, milled and collected. Thermal stability testing, de-ensilication analysis and proof of ensilication A sample of each ensilicated material was heated at 95°C for 1 hour alongside a sample of the stock material. After the hour the material was removed and allowed to cool. 5mgs of each material, heated and non-heated was weighed out into 15ml falcon tubes, 10mls of de-ensilication buffer was added to each and rotated for 1 hour. After the hour was completed, 1ml of each was removed and centrifuged at 17,000g. The protein concentration was then determined by UV-Vis, this data was used to determine the load% and ensilication efficiency. An EnzChek® Lysozyme Assay Kit from fisher scientific was used and performed following the manufacturer’s instructions. All de-ensilicated material has similar activity showing that both methods provide good thermal stability, and the presence of APTES does not decrease the technologies' ability to protect the entity. This is shown in Figure 12. SDS-PAGE Samples were prepared in SDS sample buffer and 1M DTT and loaded on a 4-20% precast polyacrylamide gel (BioRad). Protein bands were visualised with PageBlue protein staining solution. Image taken using the Chemidoc. As can be seen in Figure 13, stock bands are present in the de-ensilicated material but very faint in the supernatants - indicating near to complete ensilication. Same species is present in the stock and the de-ensilicated material providing proof of ensilication. Conclusions An APTES:TEOS ratio of 1:200 can be used to ensilicate lysozyme at a comparable efficiency to that of the TEOS only method. The use of APTES during the ensilication process does not impact the loading of lysozyme or the thermal protection the ensilication process provides. Example 4:Ensilication of Bovine Serum Albumin (Protein) Supplier details Bovine Albumin (BSA), Sigma Millipore, Code: 81-053-3, Lot# 73 Isoelectric point: The isoelectric point was determined to be pH 4.39. 500mg of BSA was dissolved in 500ml of 50mM Tris, pH 10.3. 50ml at a time the pH was adjusted, using 3.2% and 32% HCI, ranging between pH 2.5 and 9; there was a focus between pH 3.5 &5. The PALS program on the Brookhaven Nanobrook 90PlusPALS was used. The zeta potential was measured at each pH and then plotted against each other to determine the IEP. The isoelectric point determination is shown in Figure 14. Ensilication Method A 1mg / ml solution of BSA was made by dissolving 350mg of BSA in 350ml 50mM Tris-HCI, pH 7. TEOS was prepared in triplicate (5ml TEOS, 5ml H2O and 10pl 32% HOI). 50% APTES was made in triplicate (3ml APTES, 1.5ml H2O and 1.5ml 32% HOI) and then used to make triplicate silica solutions with APTES:TEOS ratios of 1:25 (400pl 50% APTES, 5ml TEOS, 5ml H2O and 10pl 32% HOI). Ensilications were performed by adding 1ml of silica to 50ml of BSA stock and stirring at 80rpm for 20 minutes before 50ml of H2O was added and the stirring stopped. Samples of the supernatant were taken, and the remaining protein concentration determined by UV-vis; this data was used to determine the material %. The rest of the material was vacuum filtered and left to dry overnight. The following day the material was weighed, milled and collected. No material was formed using the pure TEOS. De-ensilication analysis and analysis 5mg of each material was added to a 15ml falcon tube and 10ml of 0.1 M de-ensilication buffer added; these were rotated at 15rpm for 1 hour. After the hour 1 ml of each material was removed and centrifuged at 17,000g for 2 minutes. The concentration of protein was determined by UV-Vis, which was used to determine the load % and ensilication efficiency, as demonstrated in Figure 15, and the identity of the de-ensilicated protein was confirmed on the SDS-PAGE gel. This is demonstrated in Figure 16. SDS-PAGE Samples were prepared in SDS sample buffer and 1M DTT and loaded on a 4-20% precast polyacrylamide gel (BioRad). Protein bands were visualised with PageBlue protein staining solution. Image taken using the Chemidoc. Stock bands are present in the de-ensilicated material but very faint in the supernatants of the APTES ensilications - indicating near to complete ensilication. However, there are strong bands in the TEOS supernatants indicating the BSA has not been ensilicated. The same species is present in the stock and the de-ensilicated material providing proof of ensilication. This is demonstrated in Figure 16. Example 5: Comparative data between the prior ensilication process (nonfunctionalised precursor only, e.q., TEOS) vs. the new ensilication process (comprising functionalised and non-functionalised precursors, e.g., TEOS and APTES) for NDV and lysozyme. APTES &TEOS were prepared at various ratios; TEOS, 1:200, 1:160, 1:80, 1:40 &1:20 Stocks of Lysozyme (positive entity) &NDV (negative entity) at 1mg / ml were prepared in 50mM Tris-HCI, pH 7. The silica was added ata 1:50 ratio and mixed for 20 minutes. The volume was then doubled. Each ensilication was performed in duplicate. Samples of the supernatant were taken from each, centrifuged at 17,000g for 2 minutes. The protein concentration of the stocks and supernatants was determined by BCA assay. The data was used to calculate the percentage incorporation - material %. The results are shown in Figures 17 and 18. The virus with a negative charge is ensilicated with a TEOS / APTES mixture but not with pure TEOS, which only ensilicates lysozyme. In contrast, the lysozyme is ensilicated with a pure TEOS precursor and TEOS / APTES mixture where the transition happens at a ratio of 1:80 50% APTES:TEOS. These results demonstrate the tuneability of the new APTES:TEOS ensilication method and the applicability to all biomolecules. Using the new ensilication method, a combination of a more acidic (e.g. TEOS) and a more basic (e.g. APTES) precursor can be selected to ensilicate biomolecules with a wider range of isoelectric points and importantly without the need for any other surface modification of the target (e.g. with protamine, PGLA, chitosan or polylysine). Example 6: Ensilication of an enzyme protein - HRP Supplier Details Sigma Aldrich - 77332 - 100mg - Peroxidase from horseradish -150U / mg, Mw 40 kDa. Reconstitution 50mM Potassium Phosphate pH 6 100mg of peroxidase form horseradish, Sigma Aldrich, was dissolved in 10ml of 50mM potassium phosphate buffer. This was gently stirred until homogeneous. Initial microplate ensilication - comparing TEOS and APTES / TEOS TEOS only silica solution was prepared (5ml TEOS, 5ml H2O and 10pl 32% HOI). 50% APTES was prepared and mixed with TEOS at different ratios: 1:5, 1:25, 1:50, 1:100 and 1:200. All silica solutions were stirred at 500rpm until homogeneous and reduced to 80rpm for 20 minutes before use. A 1mg / ml HRP in 50mM potassium phosphate, pH 6, was prepared by diluting 1ml of the 10mg / ml stock in 9ml of buffer. A 24 well clear flat bottom plate was used. 1ml of 1mg / ml HRP stock was added to 6 wells. Silica was added to each well at a 1:50 ratio and rocked for 20 minutes before 1ml of DI H2O was added to each well and rocking stopped. 500pl of each well was sampled and centrifuged at 17,000g. This is shown in Figure 19. BCA Assay The protein concentration of the stock and supernatants was measured using a BCA protein assay. Pierce BCA Protein Assay Reagent A (Thermo Fisher) was used, and protocol was followed as per manufactures instructions. This data was used to calculate the material % (Total incorporation of HRP into the silica using the stock concentration and the supernatant concentration). From this is as decided that APTES / TEOS 1:25 would be scaled up to allow for de-ensilication analysis. Sample Measured Concentration, mg / ml Material % TEOS SUP 0.25 44 1:200 SUP 0.08 82 1:100 SUP 0.05 90 1:50 SUP 0.04 91 1:25 SUP 0.03 93 1:5 SUP 0.05 89 Stock 0.88 Larger Ensilication Method with APTES / TEOS 1:25 with silica 1:50 14ml of 1mg / ml HRP in 50mM potassium phosphate stock was prepared from 10mg / ml HRP and mixed until homogeneous. 12ml of this stock was added to a glass beaker and stirred at 80rpm. The 1:25 APTES / TEOS solution was added at a 1:50 ratio and continuously stirred for 20 minutes. After the 20 minutes 12ml of water was added and the stirring stopped. 1ml of the supernatant was sampled and centrifuged at 17,000g for 2 minutes for later protein analysis via BCA assay. The rest of the material was vacuum filtered and left to dry overnight in the fume-hood. The follow day the material was weighed, milled and collected. De-ensilication and analysis 5mg of the milled material was added to a 15ml falcon tube and 10ml of de-ensilication buffer was added. This was rotated at 15rpm for 1 hour. After the hour 1ml of the de- ensilicated material was sampled and centrifuged at 17,000g for 2 minutes. De-ensilicated material was analysed via BCA assay and SDS-PAGE BCA assay The protein concentration of the stock, supernatants and De-ensilicated material was measured using a BCA protein assay. Pierce BCA Protein Assay Reagent A (Thermo Fisher) was used, and protocol was followed as per manufactures instructions. This data was used to calculate the material % (Total incorporation of HRP into the silica using the stock concentration and the supernatant concentration), Load % (The percentage of the ensilicated material that is HRP) and the ensilication efficiency (Total incorporation of HRP based off of the De-ensilication concentration) Material % 92.3 Load % 40 Ensilication Efficiency % 110 *Material was still wet when weighed* SDS-PAGE Samples were prepared in SDS sample buffer and 1M DTT and loaded on a 4-20% precast polyacrylamide gel (BioRad). Protein bands were visualised with PageBlue protein staining solution. Image taken using the Chemidoc. Stock bands are present in the de-ensilicated material but not in the supernatants -indicating near to complete ensilication. Same species is present in the stock and the de-ensilicated material providing proof of ensilication. This is shown in Figure 20. Example 7: Ensilication of a bacterial cell Source Details: BL21 E. Coli - Thermo Scientific™ BL21(DE3) Competent Cells, Catalog No. 16440054 Ensilication Method 70ml of BL21 E. coli was grown in Lysogeny Broth (LB) &icillin media in an overnight culture 37° C, shaking at 250rpm. The OD600, optical density at a wavelength of 600 nanometres (nm), was measured, following the overnight growth, at 1.32. 50% APTES was prepared and used to make the silica mixture which had and APTES:TEOS ratio of 1:20 (500pl 50% APTES, 5ml TEOS, 5ml H2O and 10pl 32% HCI). This was stirred at 500rpm until homogeneous and then the stirring reduced to 60rpm and left for 20 minutes before use. This silica was added to 70ml of BL21 E. coli culture at a 1:20 ratio and stirred at 60rpm for 1 hour. After the hour the solution was diluted 1:1 with PBS and the stirring stopped. The material was left to settle for 10 minutes before the top 50% of supernatant was discarded and the remaining material was vacuum filtered and left to dry overnight. The following day the material was collected and stored at room temperature. Regrowth of ensilicated BL21 E. Coli - Day 5 post ensilication • Preparation of ensilicated material: 20mg of the ensilicated material was milled into a fine powder, 5mg of this was weighed out in duplicate into 7ml bijous. 5mg of the un-milled material was also weighed out in duplicate into 7ml bijous. 5mg of milled and un-milled material were also added to bijous with 10OpI of 70% I PA and gently shaken to kill any bacteria that may be stuck to the outside of the ensilicated material. • Preparation of LB &Ampicillin media &growth: 600ml of LB broth media was mixed with 600pl 100mg / ml ampicillin (Amp) to make the LB + Amp media (100pg / ml Amp). 5ml of the media was added to each bijou and covered with foil. Holes were poked in the top to allow for aeration and were incubated at 37° C overnight. • OD600 ~ 24 hours of growth After incubation, standing at 37° C for 24 hours, the OD600 was measured for each ensilicated BL21 sample. The OD was measured using (SPC001) VWR V-1200 Spectrometer. 4mI of each was transferred to a quartz cuvette. The blank from the LB + AMP media was subtracted from each value and data used to determine the level of growth from each ensilicated material. This is shown in Figure 21 Sample Name OD600 Blank Subtracted OD600 LB + Amp Media 0.059 0 Milled 1 0.454 0.395 Milled 2 0.502 0.443 Flakes 1 0.437 0.378 Flakes 2 0.413 0.354 IPA Washed Milled 0.263 0.204 I PA Washed Flakes 0.260 0.201 CFU / ml 200pl from each milled and un-milled ensilicated E. coli culture was transferred to a 96-clear flat bottom welled plate; one sample per row in column 1. 180pl of fresh LB + Amp media was added to columns 2-12 of each row containing a sample. 20pl from column 1 was added to column 2 and mixed through pipetting up and down 6 times. This was repeated across the plate. 10pl of each dilution was loaded onto LB + Amp agar plates. Plates were incubated at 37° C overnight and assessed for growth the following day. The dilution factor of 106 was used to calculate the CFu / ml, this was found to be 9x108 and 12x108 for the milled and un-milled material respectively. Conclusions BL21 E. Coli can be successfully ensilicated using an APTES / TEOS silica mixture and regrowth is still seen 5 days post ensilication. Material that was washed with I PA also had a lower degree of growth and suggests there is residual bacterial left on the silica matrix. Example 8: Ensilication of mammalian cells - human lung carcinoma cell line A549 Supplier details A549 cells passage 13 (P13), ATTC, Code: CCL-185, Lot# 70047545 Ensilication method Each silica precursor was made as follows; TEOS (10ml TEOS, 10ml Ultra Pure H2O and 20pl 32% HCI), 50% APTES (3ml APTES, 1.5ml Ultra Pure H2O and 1.5ml 32% HCI). Range of 1:10 to 1:800 ratios of APTES:TEOS was prepared by mixing 2.5ml TEOS, 2.5ml Ultra Pure H2O with either: 500, 100, 50, 25, 12.5, 6.25ul of 50% APTES (for 1:10 to 1:800 ratio respectively) and 5ul 32% HCI was added. Each was mixed at 500rpm until homogeneous and stirring was reduced for 20 minutes before use. All silica solutions were filtered using 0.2um syringe filters. A549 cell at P13 were set up on four 24-well plates at density of 50 000 cell / well in F-12K complete cell media (supplemented with 10% FBS and Penicillin / Streptomycin) in a 500ul volume / well. Soon after addition of cells to each well, 12.5ul of each previously prepared APTES:TEOS ratios was added to Row A (silica ratio 1:10) and 50ul of APTES:TEOS ratio to Row B (silica ratio 1:40). 1:10 to 1:320 TEOS ratios were achieved by adding 50, 25, 12.5, 6.25, 3.11, 1,56ul of TEOS respectively to each well. Cells plus silicas were then stirred on orbital shaker at 300rpm for 20 minutes. After the 20 minutes the stirring was stopped and 500ul of Ultra Pure H2O was added. Solution was left to settle for 5min at room temperature and supernatant was removed. Next, 500ul of PBS / well was added to wash the wells from an unreacted silica, settled for 5min and removed. 5 After that, another 500ul of PBS / well was added and ensilicated material was subjected to four different processes, according to the process layout below. Plate # Ensilication conditions Post-ensilication treatment of cells Storage condition of ensilicated cells Cell regrowth time (h) 1 APTES:TEOS or TEOS Reconstitution in PBS buffer Overnight incubation at 4°C 24 2 APTES:TEOS or TEOS Reconstitution in PBS buffer Overnight incubation at room temperature 24 3 APTES:TEOS or TEOS Dried in room temperature overnight De-ensilication 24 4 APTES:TEOS or TEOS Reconstitution in media Overnight incubation at 37°C 24 Ensilication is visible in a 24-well plate after addition of silica to A459 cell line, as shown in 10 Figure 22 Post-ensilication treatment, de-ensilication and analysis As stated in the table above ensilicated cells were subjected to 4 different processes: 15 Plate 1- ensilicated cells were reconstituted in PBS and stored at 4°C overnight, after which ensilicated cells were transferred into another 24-well plate for further 24h re-growth time. Plate 2- ensilicated cells were reconstituted in PBS and stored at room temperature overnight, after which ensilicated cells were transferred into another 24-well plate for further 24h re-growth time. Plate 3- ensilicated cells were de-ensilicated with 100ul of de-ensilication buffer (0.1M sodium carbonate pH11 filtered) / welI for the total of 20min at orbital shaker at 300rpm. After that time 1ml of cell media was added to each well and de-ensilicated cells were added onto 24-well plate and incubated for 24h in 37°C. Plate 4- ensilicated cells were reconstituted in media and incubated at 37°C for 24h, after which cells were collected and transferred into another 24-well plate for further 24h regrowth time. Pictures of ensilicated cells From all the condition of post-ensilication processes and type of reacting silica, the best and most substantial cell re-growth was seen after ensilication with 1:320 APTES:TEOS ratio, reconstitution of ensilicated cells in PBS, followed by 24h incubation in room temperature and further 24h re-growth time. This is shown in Figure 23. Example 9: Ensilication with C18-TMS C18-TMS is a different functionalised silica precursor to APTES. The full name of C18-TMS is Dimethyloctadecyl[3-(trimethylsilyl)propyl]ammonium chloride solution, with structure shown in Figure 24. This was sourced from Sigma-Aldrich, 435694-1 OOML (42% in methanol &pH 5) Initial Testing Two protein stocks were prepared by dissolving 50mg of lysozyme and BSA separately in 50ml of 50mM Tris-HCI pH 7. TEOS was prepared (5ml TEOS, 5ml H2O and 10pl 32% HOI) as well as 1:25 &1:200 APTES:TEOS solutions using pre-prepared 50% APTES (400pl &50 pl 50% APTES respectively plus 5ml TEOS, 5ml H2O and 10pl 32% HCI).The 42% C18-TMS and TEOS were mixed together, with water and 32% HOI in the following ratios 1:25, 1:50, 1:100 &1:200; amounts used are shown in the table below Ratio C18:TEOS TEOS, ml 42%C18, pl Water, ml HCI, pl 1:25 3 285.6 3 6 1:50 3 142.8 3 6 1:100 3 71.4 3 6 1:200 3 35.7 3 6 All silica solutions were stirred at 500rpm for one hour until homogeneous and then the stirring was reduced to 80rpm for 20 minutes before use. Utilising 6 well plates 5mls of each stock was added to 7 wells. Silica was added at a 1:50 ratio and rocked for 20 minutes. 5ml of DI H2O was then added and the rocking stopped. 1ml of each well was sampled and centrifuged at 17,000g for 2 minutes. The remaining protein concentration and starting stock concentration was determined using UV-Vis. This data was used to calculate the total protein incorporation in the ensilicated material - material %. The results are shown in Figure 25. This demonstrates that C18-TMS can also be used as a functionalised silica precursor with TEOS at ratios 1:25, 1:50, 1:100 and 1:200 with lysozyme and BSA at pH 7. Lysozyme had a -90% efficiency at all ratios whereas BSA had preferred ratio of 1:25. Ensilication Method with C18- TMS- Scale-up The silica precursor, made in triplicate, was prepared by mixing C18-TMS and TEOS together at a 1:25 ratio (285.6pl 42% C18-TMS, 3ml TEOS, 3ml H2O and 6pl 32% HOI) until homogeneous. 1mg / ml stocks of lysozyme and BSA in 50mM Tris-HCI, pH 7 were made. Each stock was used for three 50ml ensilications; the silica was added at a 1:50 ratio and stirred at 80rpm for 20 minutes. After the 20 minutes the stirring was stopped and 50ml of DI H2O added. 1ml of each supernatant was sampled and centrifuged at 17,000g for 2 minutes before UV-Vis was used to determine the stock and remaining protein concentration; this data was used to determine the total protein incorporation in the ensilicated material - material %. The rest of the material was vacuum filtered and left to dry overnight. The following day the material was weighed, milled and collected. De-ensilication and analysis 5mg of each material was weighed out into an eppendorf. 1ml of 1M NaOH was added to each and vortexed until clear. These were then centrifuged at 17,000g for 2 minutes. UV-Vis was then used to determine the protein concentration in the de-ensilicated material; this data was used to determine the load % and the ensilication efficiency. The results are shown in Figure 26. SDS-Page Samples were prepared in SDS sample buffer and 1M DTT and loaded on a 4-20% precast polyacrylamide gel (BioRad). Protein bands were visualised with PageBlue protein staining solution. Image taken using the Chemidoc. BSA and Lysozyme bands are seen in de- ensilicated material indicating it is possible to ensilicate both positively and negatively charged proteins using C18-TMS as an alternative to APTES. This is shown in Figure 27. Conclusions C18-TMS was successfully able to ensilicate both lysozyme and BSA at different C18-TMS / TEOS ratios. The ability of C18-TMS to more effectively ensilicate lysozyme at a ratio of 1:25 is likely due to the length and flexibility of the organic chain compared to that of APTES which provides steric hinderance. The method can successfully be scaled up and material can be de-ensilicated. Example 10: Ensilication with TPOS and APTES An alternative non-functionalised silica precursor to be used in combination with a nonfunctionalised silica precursor was Tetrapropyl orthosilicate TPOS Source Sigma Aldrich, 235741-100G, pH 5-6 (same as TEOS) Initial ensilication method Four silica solutions were prepared; TEOS (5ml TEOS, 5ml H2O and 10pl 32% HOI), APTES:TEOS (5ml TEOS, 400pl 50% APTES, and 10pl 32% HOI), TPOS (5ml TPOS, 5ml H2O and 10pl 32% HOI) and TPOS:APTES (5ml TPOS, 5ml H2O, 400pl 50% APTES and 10pl 32% HOI) - silica solutions using pre-prepared 50% APTES. These were stirred at 500rpm for 1 hour after which those made using TEOS had become homogeneous but those made using TPOS were still immiscible. Isopropanol was then used as a homogenising agent. Three ratios were investigated; TPOS A (5ml TPOS, 2.5ml H2O, 2.5ml I PA &10pl 32% HOI), TPOS B (3ml TPOS, 3ml H2O, 3ml I PA &32% HOI) and TPOS C (2ml TPOS, 2ml H2O, 4ml IPA &10pl 32% HOI). These were all stirred at 50rpm for 1 hour, after which TPOS C had become homogeneous. The four silica solutions were then prepared: ; TEOS (5ml TEOS, 5ml H2O and 10pl 32% HOI), APTES:TEOS (5ml TEOS, 400pl 50% APTES, and 10pl 32% HOI), TPOS (2ml TPOS, 2ml H2O, 4ml IPA and 1Opl 32% HOI) and TPOS:APTES (4ml TPOS, 2ml H2O, 4ml IPA, 160pl 50% APTES and 10pl 32% HOI), these were stirred at 500rpm for 1 hour before the stirring was reduced for 20minutes before use. Two protein stocks were prepared by dissolving 50mg of lysozyme and BSA separately in 50ml of 50mM Tris-HCl. Utilising 6-well plates, 5mls of each stock was added to four wells. Silica was added to each well 10OpI for TEOS based and 200pl for TPOS based. These were rocked for 20 minutes, 5mls of H2O was then added and rocking stopped. 1ml of each was sampled and centrifuged at 17,000g for 2 minutes. The remailing protein concentration and starting stock was then determined by UV-Vis. This data was used to calculate the total protein incorporation in the ensilicated material - material % Material % SILICA LYZ BSA TEOS 95.1 5.3 APTES:TEOS 91.7 85.0 TPOS 91.6 -3.4 APTES:TPOS -1.6 95.6 Ensilication Method - Scale-up Each silica precursor was made in triplicate; TEOS (5ml TEOS, 5ml H2O and 10pl 32% HOI), APTES:TEOS (1:25) (5ml TEOS, 400pl 50% APTES, and 10pl 32% HOI), TPOS (2ml TPOS, 2ml H2O, 4ml IPA and 10pl 32% HOI) TPOS:APTES (1:25) (4ml TPOS, 2ml H2O, 4ml IPA, 160pl 50% APTES and 10pl 32% HOI). Each was mixed at 500rpm until homogeneous and stirring was reduced for 20 minutes before use. 1mg / ml stocks of lysozyme and BSA in 50mM Tris-HCI, pH 7.5 were prepared in triplicate. Each ensilication was 50ml of stock; lysozyme stocks were used for TEOS &TPOS and BSA stock was used for APTES:TEOS &APTES:TPOS. The silica was added at a 1:50 for TEOS based silicas and 1:25 TPOS silicas and stirred at 80rpm for 20 minutes. After the 20 minutes the stirring was stopped and 50ml of H2O was added. 1ml of each supernatant was sampled and centrifuged at 17,000g for 2 minutes before UV-Vis was used to determine the total protein incorporation in the ensilicated material - material %. The rest of the material was vacuum filtered and left to dry overnight. The following day the material was weighed, milled and collected. De-ensilication and analysis 5mgofeach material was weighed out into a 15ml falcon tube. 10ml of 0.1 M De-ensilication buffer was then added, these were rotated at 15rpm for 1 hour at 25° C. After the hour 1ml of each was centrifuged at 17,000g for 2 minutes. UV-Vis was then used to determine the protein concentration in the de-ensilicated material; this data was used to determine the load % and ensilication efficiency. This is shown in Figure 28. SDS-PAGE Samples were prepared in SDS sample buffer and 1M DTT then loaded on a 4-20% precast polyacrylamide gel (BioRad). Protein bands were visualised with PageBlue protein staining solution. Image taken using Chemidoc. BSA and Lysozyme bands seen in all de-ensilicated material confirming TPOS can be used in place of TEOS in the ensilication and successful de-ensilication of both positively and negatively charged entities. This is shown in Figures 29 (Lysozyme) and Figure 30 (BSA). Enzchek We used Enzchek Lysozyme Assay Kit from fisher scientific and performed the assay following the manufacturer’s instructions. This was performed on the de-ensilicated lysozyme material that had been ensilicated with TEOS and TPOS. Figure 31 demonstrates that ensilicated material made with TPOS showed similar activity to that made with TEOS showing that TPOS can be used as an alternative to TEOS and does not impact the protection the ensilication process provides. Example 11: Dual Ensilication Process In this example, two different biomolecules, a lysozyme and BSA were ensilicated within the same mixture. Ensilication Method 50% APTES was made and used to prepare a 1:200 APTES:TEOS mixture in triplicate (50ul 50%APTES to 5ml TEOS &5ml H2O + 10pl 32% HCI) - Silica A, B &C. 50mM Tris-HCI pH 7 was prepared in triplicate by dissolving 605.5mg Trizma base in 100ml DI H2O and reducing the pH using 32% HCI. To each buffer 50mg of BSA and 50mg of Lysozyme was added and mixed until dissolved; with a final total protein concentration of 1mg / ml -stocks A, B and C. Samples of stocks were taken before 2ml of Silica A,B and C was added to Stocks A,B and C respectively and stirred at 80rpm for 20 minutes. After the 20 minutes the solutions diluted with 100ml of DI H2O and stirring stopped. Samples of the supernatants were taken and the rest of the material was vacuum filtered and left to dry overnight. The following day the material was weighed, milled and collected. De-ensilication and analysis 5mg of each material was weighed out into a 15ml falcon tube 10ml of 0.1 M De-ensilication buffer was then added, these were rotated at 15rpm for 1 hour at 25° C. After the hour 1ml of each was centrifuged at 17,000g for 2 minutes. BCA Protein assay The protein concentration of the stock, supernatants and De-ensilicated material was measured using a BCA protein assay. Pierce BCA Protein Assay Reagent A (Thermo Fisher) was used, and protocol was followed as per manufactures instructions. This data was used to calculate the material % (Total incorporation of virus into the silica using the stock concentration and the supernatant concentration), Load % (The percentage of the ensilicated material that is protein) and the ensilication efficiency (Total incorporation of protein based off of the De-ensilication concentration). This is demonstrated in Figure 32. SDS-PAGE Samples were prepared in SDS sample buffer and 1M DTT and loaded on a 4-20% precast polyacrylamide gel (BioRad). Protein bands were visualised with PageBlue protein staining solution. Image taken using the Chemidoc. This is shown in Figure 33. Example 12: Development of Ensilication Process and Comparative Examples Comparative Example 1: Use of functionalised silica precursor (e.g., APTES) alone In optimisation, ensilication was first tested with pure hydrolysed APTES. APTES alone is used in various chemical applications alone to silane surfaces such as metals, metal oxides and glasses. It was rationalised that the functional group of the silica precursor could interact with the biomolecule surface. However, ensilication with pure APTES was found not to work and does not react with the biomolecule surface once hydrolysed and added to neutral buffer. Without wishing to be bound by theory, it is hypothesised that pure APTES does not work due to too much steric hindrance. Comparative Example 2: Combination of functionalised and non-functionalised silica precursor hydrolysed separately It was next tested whether APTES and TEOS could be used in combination, where TEOS precursors and APTES precursors were hydrolysed separately before contacting with the biomolecule. Details of the test method can be found below. Test Method 2ml of NDV-NIBSC stock (3mg / ml) was defrosted at room temperature. A protamine sulphate solution was prepared as a 5ml mixture of 10mg / ml protamine sulphate in 50mM Tris-HCI pH 7.50. A BCA assay was set up following the manufactures instructions to measure the protein concentration of both of these. From this data two stocks (2.5ml) were prepared in 50mM Tris-HCI pH 7; One with 1mg / ml of NDV present and the other with 1mg / ml NDV present and 0.2 mg / ml of protamine. These were mixed for 20 minutes before use. The TEOS solution was prepared by mixing TEOS, Water &32% HCI on a 1:1:0.02 ratio at 500 rpm for 1 hour, until homogeneous, before reducing the speed to 60rpm for 20 minutes before use. The APTES solution was prepared by mixing APTES, water &32% HCI at a 1:1:0.02 ratio in an Eppendorf until homogeneous. Both are prepared separately. The APTES &TEOS solutions were mixed together at various percentages of APTES 100% a 0% in a 96 well plate. The stocks were added to a row each, 200ul per well, a blank row of 50mM Tris-HCI pH 7.5 was also added. 2ul of each APTES &TEOS mixture was added to each well and left for 20 minutes. After the 20 minutes the turbidity was measured on the Varioskan Lux at 600nm. Turbidity was plotted against the percentage of APTES present. The results are shown in Figure 34. The lower turbidity indicates that less ensilicated material made. The lack of turbidity in any of the wells comprising pure NDV indicates that combining APTES with pre-hydrolysed TEOS does not allow the virus to be ensilicated, without protamine, using this method. The data shows that for the NDV + protamine mixture, it is the pure TEOS which creates ensilicated material and that APTES does not aid in the ensilication proceed when combined with TEOS in this way. Without being bound by theory, it was reasoned that hydrolysis of the precursors separately makes two separate sets of silica chains which do not interact with each other once contacted with the biomolecule. Hydrolyzation of the mixture of functionalised and non-functionalised silica precursor On the contrary, as demonstrated by the Examples above, it was found that hydrolysing the non-functionalised silica precursor (e.g., TEOS, TPOS) after mixing with the nonfunctionalised silica precursor (e.g., APTES, C18-TMS) led to successful ensilication. Without wishing to be bound by theory, a co-hydrolysation condensation process enables silica chains to form that contain enough functionalised precursor (e.g., APTES, C18-TMS) to be attracted to the biomolecule surface but enough non-functionalised precursor (e.g., TEOS, TPOS) to have the flexibility to form a macro-structure around the biomolecule. For the basic functionalised silica precursor APTES, it was found that prior acidification of the APTES precursor prior to mixing with TEOS reduced the tendency of the mixtures to form a gel or a slurry, which prevented the mixture from reaching a fully usable hydrolysed state. By testing the pH of TEOS and APTES in water, it was found that TEOS has a pH or 2 and APTES has a pH of 12. Without wishing to be bound by theory, it is believed that, without prior acidification, the APTES silica precursors are hydrolysed in a pH region of rapid aggregation and are therefore more likely to precipitate out. This contrasts with pure TEOS, which has a lower pH, and which is therefore hydrolysed in a metastable region, which only rapidly aggregates when brought into contact with the biomolecule section. This is consistent with what is shown and described in Sol Gel Science, The Physics and Chemistry of sol-gel processing, C. Jeffrey Brinker &George W. Scherer, Academic Press Inc, 1990, page 104. Acidification was not required for the C18-TMS functionalised silica precursor. The following exemplary method can be used to form a homogenous mixture of APTES and TEOS. 1 ml of APTES was added to 1 ml of H2O in a 50ml glass beaker 32% HCI was added until the pH was between 1-2. The pH was measured using pH paper. 500pl of 32% HCI was needed , but wherein the HCI was added 100 pl at a time and swirled until the reaction had stopped. (This is an exothermic reaction) TEOS (pure) and the 50% APTES solution were then mixed in a 4:1 ratio (1200pl + 300pl), 1500pl H2O was added and 30pl HCI. Example 13 - Ensilication of nucleic acid lipid nanoparticle (LNP) Supplier details: eGFP mRNA LNP #U618F685G0-1 / MA 1157, Gene Script - eGFP mRNA Modification: N1-Methvl-pseudoU Lipid Type:SM102 eGFP mRNA-LNP stands for enhanced green fluorescent protein (eGFP) mRNA is encapsulated within lipid nanoparticles (LNPs). This is used for efficient delivery of mRNA into cells, allowing for the expression of eGFP, which emits a bright green fluorescence. This is useful for tracking gene expression and transfection efficiency in various cell types. Ensilication method: 50% APTES was made by mixing 3ml APTES, 1.5ml Ultra Pure H2O and 1.5ml 32% HCI and pH was confirmed with pH paper to be between 1 and 2. 1:800 ratio of APTES:TEOS was prepared by mixing 5ml TEOS, 5ml Ultra Pure H2O with 12.5ul of 50% APTES and 10ul 32% HCI was added. Solution was mixed at 500rpm until homogeneous and stirring was reduced for 20 minutes before use. Silica solutions were filtered using 0.2um syringe filters. EGFP mRNA-LNP was ensilicated in 3 different wavs: A) ensilicated and left in solution at 4C for stability testing at DAY 1 / 4 / 7 / 14 / 28 133ul (20ug) of eGFP was diluted with 266ul ultra pure water and ensilicated with 44ul of 1:800 APTES:TEOS ratio (1:10 silica ratio) for20min on ice, while shaking on orbital shaker set to 300rpm. After 20min, 440ul of ultra pure water was added as a quencher, then solution was transferred to RNase free tube. Next, 500ul of ultra pure water was used to wash each well and remaining ensilicated material was added to the tube and centrifuged for 2min at 12000g, after which supernatant was collected. Pellet of ensilicated material was resuspended in 1000ul of PBS with 8.7% sucrose. Ensilicated material was distributed across 5 crimp vials by adding 200ul into each vial (4000ng of material / vial). Vials were stored in 4C (fridge) until needed. B) ensilicated and left in solution at 4C for further de-ensilication and stability testing at DAY 1 / 4 / 7 / 14 / 28 160ul (26ug) of eGFP was diluted with 320ul ultra pure water and ensilicated with 53ul of 1:800 APTES:TEOS ratio (1:10 silica ratio) for20min on ice, while shaking on orbital shaker set to 300rpm. After 20min, 530ul of ultra pure water was added as a quencher, then solution was transferred to RNase free tube. Next, 500ul of ultra pure water was used to wash each well and remaining ensilicated material was added to the tube and centrifuged for 2min at 12000g, after which supernatant was collected. Pellet of ensilicated material was resuspended in 1200ul of PBS with 8.7% sucrose. Ensilicated material was distributed across 6 crimp vials by adding 200ul into each vial (4000ng of material / vial). Vials were stored in 4C (fridge) until needed. C) ensilicated and left dry at room temperature for further de-ensilication and stability testing at DAY 1 / 4 / 7 / 14 / 28 6x 27ul (24.3ug) of eGFP was added to 6 crimp vials and diluted with 54ul ultra pure water per vial and ensilicated with 9uI of 1:800 APTES:TEOS ratio (1:10 silica ratio) for 20min on ice, while shaking on orbital shaker set to 300rpm. After 20min, 90ul of ultra pure water was added as a quencher and solution was left to settle for 5min before supernatant was collected. Each vial was washed with 90ul of PBS to reduce the amount of unreacted silica, then solution was left to settle again for 5min and supernatant was discarded. Vials were left to dry in the desiccator overnight. Next, ensilicated material was stored at room temperature until needed. Proof of ensilication is demonstrated in Figure 35. Post-ensilication treatment and de-ensilication - stability study DAY1 Each time one crimp vial of ensilicated material was opened in the biosafety hood, solution was transferred into RNase free tube. Next, vial was washed with 200ul of PBS, added to the tube and centrifuged at 12,000g for 2min at 4° C. 96-well plate with A549 cells, seeded the day before at a density of 10,000cell / well was transfected with 400ng of ensilicated eGFP mRNA-LNP per well for 48h. Two other crimp vials were de-ensilicated in either 0.01 M or 0.05M sodium carbonate with sucrose by adding 10.6mg or 53mg of sodium carbonate respectively to 10ml ultra pure water with 870mg of sucrose. pH of the solution was adjusted to 11 with 0.2M sodium bicarbonate. Contents of the vials with ensilicated material in solution were transferred into RNase free tube, centrifuged at 12,000g for 3min at 4° C and supernatant was discarded. 200ul of de-ensilication buffer was added to each pellet and tubes were rotated at 15rpm for 1 hour at 4° C. After that, A549 cell were transfected with 400ng of de-ensilicated and neutralised eGFP mRNA-LNP per well for48h. Another two crimp vials with dried ensilicated material were de-ensilicated in either 0.01 M or 0.05M sodium carbonate with sucrose by adding 200ul of de-ensilicated solution / vial. Material was broken down by pipetting and transferred into an RNase free eppendorf tube. Tubes were rotated at 15rpm for 1 hour at 4° C. After that, A549 cell were transfected with 400ng of de-ensilicated and neutralised eGFP mRNA-LNP per well for 48h. 250ng / well of non-ensilicated eGFP mRNA-LNP was added as a positive control and wells set up with only cells were used as negative control. Fluorescent pictures of the A549 cells transfected with ensilicated eGFP-mRNA LNP are shown in Figure 36 (day 1), Figure 37 (Day 4), Figure 38 (Day 7) or Figure 39 (Day 14). The overall fluorescence intensity measured after 48h transfection time. 96-well plate was read on a plate reader with 488 nm excitation filter and 513nm emission filter. Figure 40 shows a significant increase of fluorescence after transfection with de-ensilicated mRNA-LNP in solution (graph represents day 4, similar pattern observed in subsequent timepoints). Cell viability using CyQUANT™ MTT Cell Viability Assay (Invitrogen) was assessed after fluorescent pictures were taken. Figure 41 shows no significant effect of either ensilicated and de-ensilicated eGFP mRNA-LNP on cell viability (graph represents day 4, similar pattern observed in subsequent timepoints). Example 14 - Protection of biomolecules from acidic pH This experiment was designed to test whether material ensilicated with APTES:TEOS is protected from acidic conditions. Method: 0.1M, 1M and 10M HCI were prepared from 32% HCI, VWR 20254.401 P. Lysozyme that was ensilicated, 4 months prior and had been stored, milled, in a climate chamber at 25°C with 60% relative humidity, was used. At the time of ensilication 1mg / ml lysozyme in Tris-HCI, pH 8 was prepared in triplicate. Silica was also prepared in triplicate at a 1:200 APTES:TEOS ratio using 50% APTES. The silica was then used to ensilicate the lysozyme at a 1:50 ratio, the material - A, B &C was vacuum filtered, dried overnight, milled and stored in the climate chamber. For each concentration of acid, 3mg of each material (A, B &C) was weighed out into an Eppendorf and 1ml of acid added. These were rotated at room temperature for 1 hour at 15rpm. The material was then centrifuged for 2 minutes at 17,000g and the supernatant discarded. The pellet was then re-suspended in 1ml DI H2O and re-centrifuged for 2 minutes at 17,000g and supernatant discarded - this was repeated a further two times. The pellet was then re-suspended in 1ml 0.1M de-ensilication buffer and transferred to a 15ml falcon tube with 5ml 0.1M de-ensilication buffer in. These were rotated at 15rpm for 1 hour at 25°C. Alongside these, 3mgs of un-treated material A, B and C were weighed into a 15ml falcon tube with 0.1M De-ensilication buffer. After the hour 1ml of each de-ensilication was sampled and centrifuged at 17,000g for 2 minutes and UV-Vis was used to determine the lysozyme concentration. We used an EnzChek® Lysozyme Assay Kit from fisher scientific and performed the assay following the manufacturer’s instructions. The results are shown in Figure 42. The data suggests that using APTES:TEOS to ensilicate provides good protection from acidic environments. Materials and Methods Preparation of de-ensilication buffer • Dissolve 1060mg of sodium carbonate in 80ml of H2O (A) • Dissolve 840mg of sodium bicarbonate in 50ml of H2O (B) • Add B to A until A is pH 11 and top the volume up of A to 100ml • Weigh out X mg of ensilicated material and add 2X ml of A • Rotate for 1 hour at 15rpm at 25°C • Centrifuge material at 17,000g for 2 minutes - it is then ready for the required analysis methods e.g. protein assays, DLS, SDS PAGE, Western Blot &ELISA Preparation of 50% APTES (same for all biomolecules) Add 3ml of 3-Aminopropyl-triethoxysilane (APTES) to 1.5ml of H2O. To this add 1.5ml of 32% HCI and stir until homogeneous. This is referred to as 50% APTES elsewhere herein. BCA Protein assay The protein concentration of the stock, supernatants and de-ensilicated material was measured using a BCA protein assay. Pierce BCA Protein Assay Reagent A (Thermo Fisher) was used, and protocol was followed as per manufactures instructions. This data was used to calculate the material % (Total incorporation of entity into the silica using the stock concentration and the supernatant concentration), Load % (The percentage of the ensilicated material that is the entity) and the ensilication efficiency (Total incorporation of the entity based off of the De-ensilication concentration). SDS-PAGE Samples were prepared in SDS sample buffer and 1M DTT and loaded on a 4-20% precast polyacrylamide gel (BioRad). Protein bands were visualised with PageBlue protein staining solution. Images of gels were taken using the Chemidoc. Western Blot Samples were prepared in SDS sample buffer and 1M DTT and loaded on a 4-20% precast polyacrylamide gel (BioRad). They were then transferred to a Trans-Blot Turbo Mini 0.2um Nitrocellulose Membrane using a trans turbo blotter. The membrane was washed briefly in Tris Buffered Saline (TBS / T) blocked with a 3% BSA in TBS / T (Blocking solution) overnight at 4° C. The following day the membrane was washed three times with 3x 50ml TBS / T for 5-minute periods. It was then incubated for 2 hours, at room temperature, with 50mls of Primary antibody, (e.g., Anti Newcastle disease serum (NIBSC, NDS), diluted 1:300 in blocking solution for analysis of Newcastle disease virus). The membrane was then washed a further three times with 3x 50ml TBS / T for 10 minutes periods. Then incubated for 1 hour at room temperature with the secondary antibody, Goat Anti-Chicken IgY (H+L) Horseradish peroxidase conjugate, at a 1:10,000 in blocking solution. The membrane was then washed for the final time in 3x 50mls for 10-minute periods before being incubated with ECL substrate for 5 minutes. The membrane was visualised using Chemidoc and using the blots application. ELISA Stock biomolecule (e.g., stock NDV), De-ensilicated biomolecule (e.g., de-ensilicated NDV) and the ensilication supernatants were loaded at 20ug / ml in column 1 of a 96 well plate. They were then serially diluted across the plate 2-fold across the plate in ELISA coating buffer (pH 9.6), leaving column 12 blank. 100 pl of the dilution was transferred onto a 96 well flat bottom plates (High binding plate, Greiner) and incubated overnight at 4°C. The following day the plates of the were washed with PBS and blocked with 1mg / ml BSA in PBS supplemented with 0.05% Tween20 (PBS-T) for 2 hours. The primary antibody, (e.g., Anti Newcastle disease serum (NIBSC, NDS) - reconstructed in PBS, prepared at a 1:300 ratio in PBS-T and 10OpI added to each well after the plate was washed three times with PBS), was incubated for 1 hour at room temperature and washed again three times with PBS before being incubated with 100 pl of the secondary antibody, Goat Anti-Chicken IgY (H+L) Horseradish peroxidase conjugate, at a 1:10000 in PBS-T for 1 hour. The plate was then washed four times with PBS and were developed using TMB substrate and the reaction stopped with 1M H2SO4. The absorbance was measured at 450nm using the Varioskan Lux. Measuring the protein concentration For some entities (e.g., BSA &Lysozyme) it is possible to measure the protein concentration using UV-Vis due to the presence of conjugated bonds. The IMPLEN 5 NanoPhotometer N60 is used. The Protein UV appliance is opened and set to the entity of interest (e.g., Lysozyme or BSA). 2pl of sample is used for each reading and are loaded on to the sample window and wiped off between samples using a lint free tissue. Firstly, the machine is blanked using the buffer in use. Three reading of each sample are then taken and the average of these is then used within calculations. 10 As an alternative to UV-Vis, or if UV-Vis is not suitable, protein concentration assays such as BCA protein assay or Bradford protein assay can also be used following the supplier’s instructions.

Claims

1. A method of encapsulating a biomolecule in a silica shell to form a particle, themethod comprising:a) hydrolysing a mixture of silica precursors, said mixture comprising afunctionalised silica precursor comprising a Si-C bond and a non-functionalised silica alkoxide precursor,b) directly contacting the hydrolysed precursors with an aqueous solutioncomprising the biomolecule andc) encapsulating the biomolecule in a silica shell to form a particle, such that at leasta portion of the silica on the interior surface of the silica shell is functionalised and comprises an Si-C bond.

2. The method of claim 1, wherein the functionalised silica precursor is a basic silicaprecursor, and wherein the method comprises acidifying the basic silica precursor before step a), optionally at a pH of between 1 and 5, and more preferably at a pH between 1 and 2.

3. The method of any preceding claim, wherein the functionalised silica precursoris functionalised with an aminoalkyl group, preferably wherein the functionalised silica precursor is an amino-alkyl alkoxide silica precursor, more preferably with the formula:wherein n is 0-4, and / or R is selected from methyl, ethyl, propyl, isopropyl, butyl.

4. The method of any preceding claim, wherein the functionalised silica precursoris aminoalkyl-alkoxysilane is (3-Aminopropyl)triethoxysilane (APTES) or a Dimethyloctadecyl[3-(trimethylsilyl)propyl]ammonium salt, preferably aminoalkyl-alkoxysilane is (3-Aminopropyl)triethoxysilane (APTES).

5. The method of any preceding claim, wherein the functionalised silica precursoris functionalised with an ammoniumalkyl group, preferably wherein the functionalised silica precursor has the formula:ORwherein n is 0-4, and / orR is selected from methyl, ethyl, propyl, isopropyl, butyl.Ri is selected from methyl, ethyl, propyl, isopropyl, butyl, preferably methylR2 is selected from methyl, ethyl, propyl, isopropyl, butyl, preferably methyl, andRais a C1-C25 alkyl, optionally a C1-C20 alkyl, further optionally Cis alkyl, andfurther preferably wherein the functionalised silica precursor is C18-TMS.

6. The method of any preceding claim, wherein the non-functionalised silicaprecursor is a silica alkoxide, more preferably tetraethylorthosilicate (TEOS) or tetrapropylorthosilicate (TPOS), even more preferably tetraethylorthosilicate (TEOS).

7. The method according to any preceding claim, wherein the functionalised silicaprecursor and non-functionalised silica precursor are mixed in a ratio from 1:1 to 1:2000, preferably from 1:4 to 1:8008. The method of any preceding claim, wherein the isoelectric point of thebiomolecule is determined before step a) of the method.

9. The method according to any preceding claim, wherein the biomolecule isselected from a protein, polypeptide, virus, viral vector, virus-like particle, cell, nucleic acid, or a nucleic acid lipid nanoparticle (LNP).

10. The method according to any preceding claim, wherein the biomolecule is avaccine, optionally wherein the vaccine is selected from a live virus vaccine, live attenuated virus vaccine, an inactivated vaccine, a nucleic acid (e.g., mRNA) vaccine, a polypeptide vaccine, heat-killed vaccine, subunit vaccine, a viral vector vaccine, or recombinant vaccine.

11. The method according to any preceding claim, wherein the isoelectric point ofthe biomolecule is between 0.5 and 12, and optionally wherein the isoelectric point of the biomolecule is less than 6.5.

12. The method according to claim 11, wherein the isoelectric point of thebiomolecule is determined using electrophoretic light scattering (ELS), Phase Analysis Light Scattering (PALS), or Isoelectric focusing (IEF) gels13. The method of any preceding claim, wherein the mixing step takes place for atleast 10 minutes, and / or until the mixture becomes homogenous, preferably for between 30 and 60 minutes14. The method of any preceding claim, wherein the mixing and / or hydrolysing stepoccurs at an acidic pH, preferably wherein the pH is between 2 and 4.

15. The method of any preceding claim, wherein the contacting takes place atwherein the contacting occurs at a pH in the range of between 4 and 10, optionally in the range of 5 to 8.

16. The method of any preceding claim, wherein the contacting takes place for aperiod of 30 seconds to 120 minutes, preferably from 5 to 60 minutes, and more preferably between 10 and 30 minutes.

17. The method of any preceding claim, wherein the ratio of hydrolysed silicaprecursor to the biomolecule in buffer is from about 1:5 to 1:200, for example, about 1:10 to 1:100.

18. The method of any preceding claim, wherein the biomolecule is added inaqueous buffer, optionally wherein the buffer comprises one or more salts containing one or more of a magnesium salt, a calcium salt, or a sodium salt, or a potassium salt and further optionally wherein the buffer is an SM buffer, a modified SM buffer, PBS, Tris, an imidazole buffer, a sucrose buffer, or bis-tris buffer.

19. The method of any preceding claim, wherein the biomolecule is provided as aformulation comprising one or more adjuvants, carriers or preservatives.

20. The method of any preceding claim, wherein the method is free of a templatingstep where the biomolecule is first coated with an intermediate layer, for example, intermediate layers comprising polylysine, polyethyleneimine chitosan, polylactic, protamine or spermine or combinations thereof.

21. The method of any preceding claim, wherein the silica shell is formed from amixture of silica precursors comprising a first silica precursor functionalised with a basic or positively charged group and a second non-functionalised silica precursor, and wherein the deposition of silica is nucleated by non-covalent interactions between the first silica precursor and negatively charged surfaces of the biomolecule, and non-covalent interactions between the non-functionalised silica precursors and positively charged surface of the biomolecule.

22. The method of any preceding claim, wherein in the contacting step, thebiomolecule is present in the aqueous solution at a concentration of 0.1 mg / ml to 100 mg / ml.AMENDMENTS TO THE CLAIMS HAVE BEEN FILED AS FOLLOWS16 02 26Claims1. A method of encapsulating a biomolecule in a silica shell to form a particle, the method comprising:a) hydrolysing a mixture of silica precursors, said mixture comprising a functionalised silica precursor comprising a Si-C bond and a non-functionalised silica alkoxide precursor, b) directly contacting the hydrolysed precursors with an aqueous solution comprising the biomolecule andc) encapsulating the biomolecule in a silica shell to form a particle, such that at least a portion of the silica on the interior surface of the silica shell is functionalised and comprises an Si-C bond,wherein the functionalised silica precursor is a basic precursor or a positively charged silica precursor, with the proviso that when the functionalised silica precursor is a basic silica precursor, the method comprises acidifying the basic silica precursor before step a)2. The method of any preceding claim, wherein the basic silica precursor is an aminoalkyl alkoxide silica precursor and the positively charged silica precursor is an ammonium alkyl alkoxide silica precursor.

3. The method of claim 1, wherein the functionalised silica precursor is a basic silica precursor and wherein the method comprises acidifying the basic silica precursor before step a) at a pH of between 1 and 5.

4. The method of any preceding claim, wherein the functionalised silica precursor is functionalised with an aminoalkyl group5. The method of any preceding claim, wherein the functionalised silica precursor is aminoalkyl-alkoxysilane is (3-Aminopropyl)triethoxysilane (APTES) or a Dimethyloctadecyl[3-(trimethylsilyl)propyl]ammonium salt6. The method of any one of claims 1-3, wherein the functionalised silica precursor is functionalised with an ammoniumalkyl group7. The method of any one of claims 1-3, wherein the functionalised silica precursor is C18-TMS.16 02 268. The method of any preceding claim, wherein the non-functionalised silica precursor is tetraethylorthosilicate (TEOS) or tetrapropylorthosilicate (TPOS).

9. The method according to any preceding claim, wherein the functionalised silica precursor and non-functionalised silica precursor are mixed in a ratio from 1:1 to 1:2000.

10. The method according to any preceding claim, wherein the biomolecule is selected from a protein, polypeptide, virus, viral vector, virus-like particle, cell, nucleic acid, or a nucleic acid lipid nanoparticle (LNP).

11. The method according to any preceding claim, wherein the biomolecule is a vaccine, optionally wherein the vaccine is selected from a live virus vaccine, live attenuated virus vaccine, an inactivated vaccine, a nucleic acid (e.g., mRNA) vaccine, a polypeptide vaccine, heat-killed vaccine, subunit vaccine, a viral vector vaccine, or recombinant vaccine.

12. The method according to any preceding claim, wherein the isoelectric point of the biomolecule is between 0.5 and 12.

13. The method according to any proceeding claim, wherein the isoelectric point of the biomolecule is less than 6.5.

14. The method according to claims 12 or 13, wherein the isoelectric point of the biomolecule is determined using electrophoretic light scattering (ELS), Phase Analysis Light Scattering (PALS), or Isoelectric focusing (IEF) gels15. The method of any preceding claim, wherein the isoelectric point of the biomolecule is determined before step a) of the method.

16. The method of any preceding claim, wherein the mixing step takes place for at least 10 minutes, and / or until the mixture becomes homogenous, preferably for between 30 and 60 minutes17. The method of any preceding claim, wherein the mixing and / or hydrolysing step occurs at an acidic pH.

18. The method of claim 1, wherein the mixing and / or hydrolysing step occurs at a pH between 2 and 4.16 02 2619. The method of any preceding claim, wherein the contacting takes place at wherein the contacting occurs at a pH in the range of between 4 and 10.

20. The method of any preceding claim, wherein the contacting takes place for a period of 30 seconds to 120 minutes.

21. The method of any preceding claim, wherein the ratio of hydrolysed silica precursor to the biomolecule in buffer is from 1:5 to 1:200.22 The method of any preceding claim, wherein the biomolecule is added in aqueous buffer.

23. The method of any preceding claim, wherein the biomolecule is provided as a formulation comprising one or more adjuvants, carriers or preservatives.

24. The method of any preceding claim, wherein the method is free of a templating step where the biomolecule is first coated with an intermediate layer.

25. The method of any preceding claim, wherein the silica shell is formed from a mixture of silica precursors comprising a first silica precursor functionalised with a basic or positively charged group and a second non-functionalised silica precursor, and wherein the deposition of silica is nucleated by non-covalent interactions between the first silica precursor and negatively charged surfaces of the biomolecule, and non-covalent interactions between the non-functionalised silica precursors and positively charged surface of the biomolecule.

26. The method of any preceding claim, wherein in the contacting step, the biomolecule is present in the aqueous solution at a concentration of 0.1 mg / ml to 100 mg / ml.s

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