WO2011156831A1

WO2011156831A1 - Formation of nucleic acid within a polymeric capsule

Info

Publication number: WO2011156831A1
Application number: PCT/AU2010/000756
Authority: WO
Inventors: Kim Wark; Frank Caruso; Andrew Price; Alexander N. Zelikin
Original assignee: Commonwealth Scientific and Industrial Research Organization CSIRO; University of Melbourne
Current assignee: Commonwealth Scientific and Industrial Research Organization CSIRO; University of Melbourne
Priority date: 2010-06-17
Filing date: 2010-06-17
Publication date: 2011-12-22
Anticipated expiration: 2012-12-17

Abstract

The present invention provides method of producing a nucleic acid molecule within a polymeric capsule. The method comprises providing a polymeric capsule having entrapped therein a DNA molecule and mixing the capsule with a polymerase and nucleotides, wherein the polymerase and the nucleotides can diffuse into the capsule, to form a reactant mixture. The reactant mixture is then incubated for a time and under conditions such as to allow the de novo synthesis of a nucleic acid molecule within the capsule. The method is particularly useful for the preparation of encapsulated RNAs such as miRNA.

Description

FORMATION OF NUCLEIC ACID WITHIN A POLYMERIC CAPSULE

FIELD OF THE INVENTION

[0001] The present invention relates to a method for producing a nucleic acid molecule within a polymeric capsule where the nucleic acid molecule is synthesised de novo. The invention further relates to polymeric capsules produced by the method and to the use of these capsules to deliver the nucleic acid to a cell.

BACKGROUND

[0002] Synthetic counterparts to cellular compartments remain far from the complexity of living systems but hold tremendous promise for advancing studies into the synthesis, confinement, and delivery of biomolecules. Considerable effort has also been made in developing the nucleic acid molecules, similar to those that make up our genetic structure, such as DNA and RNA, into effective therapeutics. Of all the biomolecular candidates R A has proven to be a very potent therapeutic as it spans a range of cellular functions including information storage, regulation, and catalysis. However RNA remains highly susceptible to degradation if left unprotected and unmodified and thus could benefit from encapsulation. A practical approach that has been explored to date is that of RNA encapsulation using liposomes and emulsions, yet controlled RNA synthesis and their subsequent use for the cellular internalization of the newly synthesized RNA have not yet been accomplished.

[0003] The recent development of polymer capsules produced via the layer-by-layer (LbL) methodology has provided a novel platform for encapsulated catalysis with a number of advantages, including precise control over the capsule size and permeability based on both the size and/or the charge of the solutes. The degree of permeability may also be influenced by the number of polymeric layers. Successful examples of DNA synthesis, hybridization, and degradation within the confines of polymer capsules highlight the potential of these assemblies as host compartments for biomolecular reactions. In addition, their shape, stability, size and the mechanical properties which these features allow endow them with characteristics suitable for the delivery of molecular therapeutics.

SUMMARY OF THE INVENTION

[0004] The present inventors have used micrometer-sized, polymeric capsules in encapsulated de novo RNA synthesis and subsequent cellular internalization of the capsules carrying the RNA. The capsules act as both microreactors and drug carriers. Unlike other methods of encapsulated RNA transcription, polymeric capsules also allow real-time monitoring of RNA synthesis and, therefore, precise control over the

encapsulated RNA concentration via the reaction time and supply of reactants. In addition to the production of encapsulated RNA, the method developed by the present invention also provides for de novo synthesis of DNA within capsules, enabling DNA directed RNA silencing.

[0005] Furthermore, enzyme-catalyzed synthesis of the nucleic acid within the confines of a drug delivery vehicle has the potential to i) minimize the handling of RNA, bypassing isolation and purification steps, ii) protect the RNA until it reaches its site of action and iii) increase the dosage of therapeutic molecule administered.

[0006] Accordingly, in a first aspect the present invention provides a method of producing a nucleic acid molecule within a polymeric capsule, the method comprising providing a polymeric capsule having entrapped therein a DNA molecule, mixing the capsule with a polymerase and nucleotides, wherein the polymerase and the nucleotides can diffuse into the capsule, to form a reactant mixture, incubating the reactant mixture for a time and under conditions such as to allow the de novo synthesis of a nucleic acid molecule within the capsule.

[0007] In a second aspect the present invention provides a method of producing a nucleic acid molecule within a polymeric capsule, the method comprising providing a polymeric capsule having entrapped therein a DNA molecule and a polymerase, mixing the capsule with nucleotides which can diffuse into the capsule and optionally a polymerase which can diffuse into the capsule, to form a reactant mixture, and incubating the reactant mixture for a time and under conditions such as to allow the de novo synthesis of a nucleic acid molecule within the capsule.

[0008] In a third aspect the present invention provides a polymeric capsule comprising a nucleic acid molecule wherein the capsule is produced according to the method of the first or second aspect of the present invention.

[0009] In a third aspect the present invention provides a method of delivering a nucleic acid to a cell comprising contacting the cell with a polymeric capsule of the third aspect of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 - Assembly of polymer polymeric capsules for encapsulated

transcription. (A) Double-stranded DNA (dsDNA) amplicons are electrostatically absorbed onto positively charged silica colloids (SiO+) at subsaturation coverage followed by electrostatic adsorption of thiol-modified polymethacrylic acid (PMASH). At pH 4.0, hydrogen bonding poly-N-vinylpyrrolidone (PVPON) and PMASH are layered onto the colloids via the layer-by-layer (LbL) technique. After layering, PMASH is crosslinked at the free thiols and the silica cores are dissolved with buffered hydrofluoric acid

(HF NH4F). Raising the pH disrupts hydrogen bonding between PMASH and PVPON, releasing PVPON from the capsules. Diffusion of T7 RNA polymerase (T7Pol) into the capsules initiates transcription of RNA under proper reaction conditions when the T7 ' promoter sequence is present in the amplicons. Synthesized RNA remains entrapped within the capsules. Chemical structures of (B) the polymers used for LbL assembly of the polymeric capsules, (C) 1 ,8-bis-Maleimidodiethyleneglycol, a non-cleavable PEG- bismalemide crosslinking reagent, and (D) 1 ,4 bismaleimidyl-2,3-dihydroxybutane , a bismaleimide crosslinking reagent cleavable by sodium periodate.

[0011] Figure 2. Internalization of RNA-loaded 1.35-pm diameter PMA polymeric capsules by mammalian cells as imaged by CLSM. The cell membranes are stained red and the RNA encapsulated in PMA polymeric capsules is labeled with a green fluorophore. The cell lines are (a) Chinese hamster ovary (CHO) cells and (b) Ramos B lymphocytes.

DETAILED DESCRIPTION OF THE INVENTION

[0012] The present inventors have succeeded in synthesising RNA and DNA within polymeric capsules. The method results in the production of polymeric capsules which include multiple copies of RNA or DNA molecules. The polymeric capsules can then be used to deliver their nucleic acid "payload" to cells.

[0013] The successful synthesis of RNA within the confines of a polymeric capsule represents a robust technique for the controlled capsule synthesis of RNA, en route to a drug-delivery platform for RNA therapeutics. The protection which the capsule provides to the synthesised RNA is of particular value given the susceptibility of RNA to

degradation.

[0014] To synthesise RNA, the presence of two macromolecules within the interior of a polymeric capsule is essential: an RNA polymerase and an at least partially double- stranded DNA template containing a specific promoter sequence required for enzyme binding and the initiation of RNA transcription. Recently, DNA has been encapsulated into poly(methacrylic acid) (PMA) polymeric capsules through LbL assembly (Zelikin et al, ACS Nano, 2007, 1 , 63). The PMA (also referred to as PMASH) polymeric capsules entrap DNA through a combination of size exclusion and electrostatic repulsion. Herein, the present inventors exploit the encapsulated dsDNA to template the transcription of RNA by T7 RNA polymerase (T7Pol) (Figure 1 ). T7Pol has sufficient diffusivity through the walls of PMA polymeric capsules, possibly via the "relay race" mechanism described for protein-polymer interactions. Once inside, the T7Pol binds the promoter sequence in the DNA template and is immobilized inside the capsule. Due to their small size, individual ribonucleotides freely diffuse into the PMA polymeric capsules, where T7Pol assembles them into single-stranded RNA molecules. Similar to the DNA template, the size, shape, and charge of the newly synthesized RNA polymers ensure they remain trapped within the capsules. By appropriate selection of DNA template, polymerases and promoters, a range of functional RNA molecules can be produced.

[0015] In a similar manner PCR can be conducted within the capsule resulting in a capsule which includes multiple copies of the DNA template. As is well understood in the art PCR is conducted using DNA primers, a thermostable DNA polymerase such as Taq polymerase and deoxyribonucleotides. The reaction mixture including the capsule containing the DNA template is the thermocycled.

[0016] As will be appreciated as opposed to encapsulating only the DNA template the capsules may also have provided therein the required polymerase. In such an arrangement contacting the polymeric capsule with the polymerase is obviously optional.

[0017] The preferred type of polymeric capsules is known and is described, for instance, in Zelikin et al, Angew. Chem. Int. Ed. 2006, 45, 7743-7745, Zelikin et al,

Biomacromolecules 2006, 7, 27-30, Zelikin et al, ACSNano, 2007, 1 , 63-69, and Zelikin et al, Chem. Mater., 2008, 20, 2655-2661.

[0018] The system can be further improved by attaching targeting molecules to the surface of the capsule which recognize molecular markers on target cells or tissues.

[0019] The inventors have demonstrated using inhibitor studies that polymeric capsules appear to enter cells in culture. Cellular entry is important for therapeutic applications. The most efficient and effective therapy would be one in which the RNA enters the cell cytoplasm. Efficiency of RNAi silencing for example depends on which step in the silencing pathway the RNA exerts its action. Alternatively, capsules with DNA delivered directly to cells have the potential to make RNAi continuously which alleviates the transiency commonly seen in current RNA delivery systems.

[0020] Accordingly, in a first aspect the present invention provides a method of producing a nucleic acid molecule within a polymeric capsule, the method comprising providing a polymeric capsule having entrapped therein a DNA molecule, mixing the capsule with a polymerase and nucleotides, wherein the polymerase and the nucleotides can diffuse into the capsule, to form a reactant mixture, incubating the reactant mixture for a time and under conditions such as to allow the de novo synthesis of a nucleic acid molecule within the capsule.

[0021] In a second aspect the present invention provides a method of producing a nucleic acid molecule within a polymeric capsule, the method comprising providing a polymeric capsule having entrapped therein a DNA molecule and a polymerase, mixing the capsule with nucleotides which can diffuse into the capsule and optionally a polymerase which can diffuse into the capsule, to form a reactant mixture, and incubating the reactant mixture for a time and under conditions such as to allow the de novo synthesis of a nucleic acid molecule within the capsule.

[0022] In a preferred embodiment wherein the nucleotides are ribonucleotides and the RNA synthesised is single stranded RNA, hairpin RNA or double stranded RNA.

[0023] The synthesised RNA may be a therapeutic molecule selected from the group consisting of microRNA (miRNA), hairpin RNA, shRNA siRNA and ribozymes.

[0024] The polymerase may be any of a wide range of polymerase known in the art including RNA Poll, PolII, PolIII, T7 RNA polymerase and DNA polymerases

[0025] For synthesis of RNA within the capsule the entrapped DNA will comprise at least one promoter. In one embodiment the entrapped DNA comprises a single T7 promoter and the polymerase is T7 RNA polymerase.

[0026] The present invention is particularly advantageous for the production and delivery of miRNA. miRNA is typically readily degraded, however, in the present invention the synthesised miRNA is protected during synthesis and delivery by the polymeric capsule.

[0027] Where it is desired to synthesise double stranded RNA the entrapped DNA will typically include two promoters. These may be in sequence or opposed promoters such that two RNA strands which will hybridise are produced. Where it is desired to synthesise two or more RNA forms and or-sequences, two or more different entrapped DNAs can be used. The resultant R As may or may not hybridise.

[0028] The method of the present invention may also be used to synthesise DNA within the capsule. In this embodiment the reactant mixture will further comprises DNA primers and the polymerase will be a DNA polymerase. As will be appreciated the synthesis of DNA basically means conducting PCR within the capsule. In this embodiment the reactant mixture is thermocycled so as to allow amplification of the DNA within the capsule

[0029] The synthesised DNA may be a sequence which encodes a protein of interest, a therapeutic RNA such as hairpin RNA, shRNA, siRNA or miRNA, or may itself be a functional molecule such as a DNAzyme.

[0030] In a preferred form, the polymeric capsule is prepared by a layer-by-layer methodology around the DNA template which is adsorbed onto positively charged silica colloids. The polymeric capsule may have a charged outer surface. The charge may be positive or negative. Preferably, the charge is negative.

(0031 J In a preferred form, the polymeric shell of the polymeric capsule does not comprise a lipid.

[0032] In a preferred form, the polymeric shell of the polymeric capsule does not comprise a surfactant.

[0033] The polymeric capsule can be made from any suitable material. In one

embodiment, the polymeric capsule includes a polymer^'selected from the group consisting of chain growth polymers, step growth polymers, polyelectrolytes, and polysaccharides. In one embodiment, the polymeric capsule includes poly(methacrylic acid) crosslinked with disulfide linkages. In another embodiment, the polymeric capsule includes poly(Iysine) and poly(glutamic acid). In yet another embodiment the polymeric capsule includes

poly(sodium styrene sulfonate) and pol(allylamine hydrochloride). In one embodiment the polymeric capsule is biodegradable. In another embodiment, the polymeric capsule is substantially resistant to degradation in an extra-cellular environment and degrades in an intra-cellular environment.

[0034] Suitable polymers for use in layer-by-layer- systems are described in US 7101575 and WO 2005/032512. [0035| In one embodiment, one or more of the layers is internally cross-linked. In another embodiment, two or more of the layers are cross-linked to one another. The cross- linking is achieved by the reaction of cross-linking groups incorporated within a polymer of the layer or layers. In a preferred form, the percentage of cross-linking groups incorporated within the polymer is from 10 to 25 mol%, preferably 15 to 20 mol%, more preferably 17 to 19 mol%, even more preferably 18%.

(0036J Preferably, the cross-linking group is a thiol and the cross-link is a disulphide group. Alternatively the cross-linking group may be maleimide.

[0037] In a preferred form, the polymeric capsule is a polymeric shell comprising layers thiolated poly(methacrylic acid) PMASH wherein the PMASH layers are cross-linked by disulphide bonds.

[0038] The preparation of PMASH (having 18 mol% thiol containing units) by conjugation of PMA with cysteamine in the presence of EDC/NHS is disclosed in Zelikin et al, Biomacromolecules 2006, 7, 27-30.

[0039] Preferably, 10 to 25 mol%, preferably 15 to 20 mol%, more preferably 17 to 19 mol%, yet more preferably 18 mol % of the thiolated poly(methacrylic acid) is

functionalized with thiol groups.

[0040] Preferably, the outer layer of the polymeric capsule is PMASH-

[0041] The present inventors have found that these polymeric capsules readily disperse in aqueous solution which improves their ease of administration.

[0042] Preferably, there are three to seven, more preferably four to six, even more preferably five, layers of PMASH.

[0043] Similarly, polymeric capsules formed of alternating layers of poly(styrene sulfonate) (PSS) and poly(allylamine) hydrochloride (PAH) may be formed by a similar layer-by- layer methodology.

[0044] Accordingly, in another embodiment, the polymeric capsule is a polymeric shell comprising alternating layers of poly(styrene sulfonate) (PSS) and poly(allylamine) (PAH)hydrochloride. [0045] Preferably, there are five layers of PSS and four layers of PAH such that PSS forms the outer layer. The selection of the number of layers assists in adjusting the reaction rate.

[0046] Methods of preparing such polymeric capsules by depositing layers of PVP and PMASH on a silica template using a layer-by-layer method are described, for instance, in Zelikin et al, Angew. Chem. Int. Ed. 2006, 45, 7743-7745, Zelikin et al,

Biomacromolecules 2006, 7, 27-30, Zelikin et al, ACS Nano, 2007, 1 , 63-69, and Zelikin et al, Chem. Mater., 2008, 20, 2655-2661. This procedure forms capsules which have a large internal cavity and correspondingly high flexibility.

[0047] As mentioned above the polymeric capsule may comprise one or more targeting molecules associated with the surface of the polymeric capsule. The one or more targeting molecules target the polymeric capsule to a specific molecule expressed either on the surface of a target cell or tissue. As an example the targeting molecule can be an antibody or a portion thereof or a peptide. If the site of delivery is the brain examples of suitable antibodies include W02, which recognises the A-beta protein involved in 20 Alzheimer's disease, anti -transferrin receptors, anti-human insulin receptor, and antiepidermal growth factor receptor. If the site is a cancer site suitable antibodies included those that recognise proteins such as clusterin, insulin-like growth factor binding protein, cell adhesion protein to name but a few.

[0048] Association of the targeting molecule with the outer surface of the polymeric capsule may be carried out by any suitable means. For instance, the association may be due to electrostatic forces between the outer surface and the targeting molecule.

Alternatively, the association may be by means of a covalent bond between the targeting molecule and the outer surface. Examples include using classic amine chemistry. For example, the targeting molecule can be modified by placing a free thiol through the introduction of a cysteine residue thus facilitating conjugation to maleimide residue on the surface of the capsules.

[0049] The polymeric capsules may be administered in the form of a composition comprising a pharmaceutically acceptable diluent, excipient or carrier. The composition may be administered in any way known in the art. For instance, the composition may be administered parenterally either subcutaneously, intramuscularly or intravenously, or alternatively nasally. [0050] In a third aspect the present invention provides a polymeric capsule comprising a nucleic acid molecule wherein the capsule is produced according to the method of the first or second aspect of the present invention.

[0051] In a fourth aspect the present invention provides a method of delivering a nucleic acid to a cell comprising contacting the cell with a polymeric capsule of the third aspect of the present invention.

[0052] Throughout this specification the word "comprise", or variations such as

"comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0053] All publications mentioned in this specification are herein incorporated by reference. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia or elsewhere before the priority date of each claim of this application.

[0054] In order that the nature of the present invention may be better understood preferred forms thereof will now be described by reference to the following Examples.

EXAMPLE 1

Internalisation of Capsules Materials

[0055] Unless stated otherwise, all chemicals and materials were purchased from Sigma- Aldrich and used as received without purification. Si02 particles of 300 nm, 500 nm and 1 μηι diameter were purchased from MicroParticles GmbH (Berlin, Germany).

Poly(methacrylic acid), MW 15 KDa was purchased from Polysciences (USA). Alexa Fluor 488 and 633 maleimides, RPMI-1640 + L-Glutamine, DMEM, L- Glutamine, Opti- MEM 1 reduced serum medium, MEM non essential amino acids, sodium pyruvate, trypsin, penicillin and streptomycin were purchased from Invitrogen. Fetal bovine serum (FBS) was from JRH. Methods

[0056] Flow cytometry measurements were performed using Partec CyFlow Space flow cytometer with an absolute volume counting capability using 488 nm and 633 nm excitation wavelengths. The capsules and cells were imaged using an Olympus 1X71 Digital Wide field fluorescence microscope and a Leica confocal laser scanning microscope.

[0057] Assembly of PMA polymeric capsules was performed as described in Zelikin et al, Chem Mater 2008, 20, 2655-2661. In brief, monodisperse silica template particles were alternately incubated in a 1 g/L solution of poly(vinylpyrrolidone) (PVPON, 10 KDa) and a 1 g/L solution of thiolated PMA (12 mole % of carboxyl groups converted into thiol groups) in 20 niM sodium acetate buffer, pH 4, with intermediate washing via

centrifugation / redispersion cycles. A total of 1 1 polymer layers were deposited (6 layers of PVPON and 5 layers of thiolated PMA), after which time the thiol groups were oxidized using chloramine T. The template particles were removed using dilute hydrofluoric acid, and the resulting capsules were washed using 20 mM sodium acetate buffer, pH 4. When transferred into a buffer with pH > 6.5, ionization of PMA caused the release of PVPON from the capsules walls and yielded single component PMA polymeric capsules stabilized via disulfide linkages.

[0058] For cell uptake experiments, the PMA polymeric capsules were fluorescently labelled with Alexa Fluor 633 using a 1 g/L solution of Alexa Fluor 633 maleimide in DMSO which was added to a dispersion of PMA polymeric capsules in 50 mM MES buffer, pH 6, to a final DMSO content of 5-10 vol %. In a typical labelling experiment, approximately 1010 - 101 1 500 nm PMA polymeric capsules were labelled using 10 - 50 μg of the dye. The reaction between maleimide dye molecules and residual non-oxidized thiol groups within the PMA polymeric capsules capsules wall was allowed to proceed for at least 2 hours, after which time the capsules were washed with fresh buffer via centrifugation and redispersion cycles until the supernatant became non-fluorescent.

[0059] For capsule counting, we used a sample of PVPON synthesized via Reversible Addition-Fragmentation chain Transfer (RAFT) polymerization technique to obtain a polymer sample labelled with Alexa Fluor 488. Briefly, to convert the polymer terminal thioester group into a thiol, an aliquot of the synthesized polymer was dissolved and incubated in a 1 M sodium borohydride solution for 1 h. After this time, excess

borohydride was quenched with concentrated hydrochloric acid, and the solution pH was adjusted to ~ 7. The resulting solution was supplemented with Tris-EDTA buffer to 10 mM and charged with Alexa Fluor 488 maleimide solution (typically 0.1 % to the weight of the polymer). The reaction was allowed to proceed overnight, after which time the polymer was recovered via gel filtration and freeze drying. An aliquot of PMA polymeric capsules was mixed with 10 volumes of PBS, pH 7.4, to release PVPON. An aliquot of the resulting single component capsules was subsequently mixed with an equal volume of the Alexa Fluor 488 labeled PVPON solution in 50 mM sodium acetate buffer, pH 4. The fluorescent polymer, being in excess to that released from the capsules, effectively infiltrates into the PMA polymeric capsules to give rise to highly fluorescent capsules. This procedure has proven effective for labelling of the PMA polymeric capsules with sizes as low as 300 nm and allowed for reliable and reproducible counting of the capsules on a CyFlow Partee Space flow cytometer with absolute volume counting triggering on Alexa Fluor 488 fluorescence.

[0060] Cell Culture The CHO-K1 WT (ATCC: CCL61), NR6 (a 3T3 derivative), HEK293T (ATCC: CRL 1 1268), M17 (ATCC: CRL-2267), P388D1 (ATCC: CCL-.46) and RAMOS (ATCC: CRL-1596) cell lines were maintained according to the American Type Culture Collection (ATCC) protocols. RAMOS cells were grown in suspension at 37°C and 5 % C0₂. All other cells were cultured as monolayers at 37°C and 5% C0₂. CHO and HEK293T cells were grown in RPMI-1640 + L-glutamine supplemented with 5% FBS and antibiotics (penicillin 500U/ml, streptomycin 50μg/ml). P388D1 and RAMOS cells were grown in RPMI-1640 + L-glutamine supplemented with 10% FBS and penicillin 50 U/ml; streptomycin 50 μg/ml. NR6 fibroblasts were grown in DMEM supplemented with 2mM L-glutamine, 5% FBS and penicillin 500U/ml and streptomycin 50 g/ml. Ml 7 cells were grown in Opti-MEM 1 reduced serum medium supplemented with non essential amino acids, sodium pyruvate, 10% FBS and penicillin 500 U/ml, and streptomycin 50 ug/ml.

[0061] PMA polymeric capsules Uptake. Cells were seeded into 96-well plates (Nunc) at a density of 4 ^χ 10⁴ cells per well in complete growth medium and allowed to attach overnight at 37°C and 5% C0₂. After this time, PMA polymeric capsules in PBS were added to the cells to give a final well volume of 290 μΐ and incubated for 24 h at 37°C, 5% C0₂, with shaking at 90 rpm. Prior to analysis, the cells were washed twice with pre- warmed PBS, trypsinized and stored on ice. Cells were then analyzed using a CyFlow Partec flow cytometer. Raw data were analyzed using Flow Jo software with a built-in population comparison algorithm.

[0062] CellTiter-Glo® Cell Viability Assay. Cells were seeded into 96-well opaque tissue culture plates (PerkinElmer) at a density of 4 x 10⁴ cells/well in 100 μΐ growth medium and allowed to attach overnight at 37°C and 5% C0₂. The cells were then exposed to 500 nm PMA polymeric capsules at various capsule: cell ratios and incubated for 24 h. The viability of the cells was assessed using CellTiter-Glo® luminescent cell viability kit from Promega Corporation (Madison, WI) according to the manufacturer's instructions. The assay detected the amount of bioluminescent ATP present, which was directly proportional to the number of viable cells. Luminescence was measured on a FLUOstar Optima (BMG LABTECH).

RESULTS AND DISCUSSION

[0063] Assembly of PMA polymeric capsules relies on the hydrogen bonding-assisted sequential deposition of PMA and poly(vinylpyrrolidone) onto sacrificial template particles. It has been previously shown that this assembly proceeds without aggregation of the.particles, which makes it a convenient starting platform to obtain polymer capsules of different compositions (single component PMA18 or PVPON20 hydrogel capsules). Single component disulfide stabilized PMA polymeric capsules as used in this work were obtained using thiol modified PMA, conversion of thiols into bridging disulfide linkages within the assembled multilayered film, removal of the core particles, and finally increasing pH to release PVPON. This assembly was routinely performed using 500 nm and 1 μπι sized particles without aggregation which ensures a high yield (i.e. minimal particle loss during assembly) and produces a population of capsules monodisperse in size. To assemble PMA polymeric capsules, we used 300 nm, 500 nm and 1 μηι commercial monodisperse silica template particles. The resulting single component hydrogel capsules were larger in size compared to the parent core particles due to the characteristic hydrogel swelling. Herein, capsules sizes are quoted as those of the respective template particles. To ensure accurate and reproducible dosage, the capsules were counted using a flow cytometer with an absolute volume counting function using fluorescence parameter as a trigger.

[0064] Capsule cytotoxity, an essential aspect of drug delivery, was assessed in five different cells lines including kidney (HEK), muscle (NR6), ovary (CHO), neuron (Ml 7) and macrophage (P388D1) over a 24 h period using CellTiter-Glo® cell viability assay. A range of capsule to cell ratios was tested using 500 nm PMA polymeric capsules (Fig. 2). Cell viability was not significantly affected in any of the cell lines, even at a ratio of 1000 capsules to 1 cell, whereas all cell lines treated with 25 % DMSO viability was reduced to less than 20%. The morphology of CHO cells was not affected following treatment with 500 nm capsules as observed by fluorescence microscopy. Confocal laser scanning micrographs confirmed that 500 nm and 1 μπι capsules were internalized by mammalian cells. Together, the data imply that the PMA polymeric capsules do not contain or release toxic products which affect cell viability upon binding and internalization into cells, at least over the period of 24 hours. This puts PMA polymeric capsules in stark contrast with other LbL-derived capsules which were shown to cause cellular toxicity. Indeed, the number of viable C6 glioma and 3T3 fibroblasts cells was decreased upon incubation with capsules assembled using either synthetic or natural polyelectrolytes ( poly(sodium

4-styrenesulfonate) (PSS) / poly(allylamine hydrochloride) (PAH); bovine serum albumin (BSA) / PAH; alginate/polylysine, etc) with sizes from 1 to 10 μιη, even at moderate (10: 1) capsule to cell ratio. Similarly, there was a reduction in the viability of denditric cells when treated with 3 μη capsules assembled from dextran sulphate and poly(arginine). Other reports demonstrate that polyelectrolyte capsules can be non-toxic to cells (e.g. PSS/PAH capsules taken in a 100: 1 ratio to dendritic cells and macrophages) which may be reflective of different length of incubation with cells, capsule. concentration, cell specific effects and possibly batch-to-batch variation in the capsules. However, PMA polymeric capsules have consistently proven to be non-toxic at all concentrations and to all cell types tested.

[0065] To investigate the interaction of capsules with mammalian cells, we used flow cytometric analysis and Alexa Fluor 633 as a fluorescent dye to label the capsules, which was chosen to minimize the background signal resulting from cell autofluorescence. The model CHO cell line was used to develop a protocol for measuring capsule uptake. In optimizing the protocol the following was observed; i) a higher ratio of capsules to cells was desirable to obtain results in a 24 hour time period, ii) in the presence of excess capsules, the concentration of cells was a far less important parameter than the number of capsules and iii) no significant differences were observed between rocking the assay plate or keeping it idle during the incubation period. In all further experiments, unless stated otherwise, a 96 well format, with seeding 4 x 10⁴ cells per well and 100 capsules to 1 cell ratio, was used. After incubation with capsules, the cells were washed twice with PBS, trypsinized and transferred into PBS buffer for subsequent analysis by flow cytometry. The raw fluorescence histograms were compared to the negative population using the Flow Jo analysis software and the built-in population comparison algorithm to obtain a numerical value of the fraction of cells with associated fluorescence, i.e. PMA polymeric capsules. While a fluorescence read out does not distinguish between adsorbed and internalized capsules, confocal laser scanning photographs show that the employed washing protocol effectively removes the capsules bound to the outer surface of the cells, and therefore the fluorescent signal obtained by flow cytometry corresponds to internalized capsules. [0066] Different cell types were assessed for their ability to internalize PMA polymeric capsules after 3 h and 24 h post incubation. Both phagocytic and non-phagocytic cells internalized 500 nm and 1 μιη diameter capsules, indicating that PMA polymeric capsules are amenable for drug delivery applications for all of the cell lines tested. In all but one ce) line, an increased incubation time resulted in a greater fraction of cells with internalized capsules. A notable exception was the RAMOS B cells, which exhibited a greater uptake level at the 3 h time point compared with 24 h for both capsule sizes. The latter observatio suggests either that RAMOS has a different uptake mechanism/s or physiological conditions specific to its cell membrane which differs from the other epithelial cell lines tested. Whilst the Ml 7 cells exhibited a lower level of uptake for the 1 μιη capsules, it doe not imply that the uptake of PMA polymeric capsules by these cells is restricted to smaller capsules, as these cells successfully internalized 1 μηι PMA polymeric capsules at higher capsule to cell ratios.

EXAMPLE 2

RNA Synthesis within the capsule Materials

[0067] Poly(methacrylic acid, sodium salt) (PMA), Mw 15,000, was purchased from Polysciences (USA), and poly(vinylpyrrolidone) (PVPON), Mw 10,000, dithiothreitol (DTT), and spermidine hydrochloride were purchased from Sigma- Aldrich and used as received. Aminated silca colloids (D = 1.11 ± 0.05.μηι and D = 3.25 ± 0.18 μηι) were purchased form Micro Particles GmbH. 1,8-bis-Maleimidodiethyleneglycol (BM(PEG)2) and 1,4 bismaleimidyl-2,3-dihydroxybutane (BMDB) were purchased from Thermo Scientific. Short oligonucleotides were purchased from Geneworks and pPCR-script Amp SK(+) plasmid was purchased from Stratagene. Taq DNA polymerase (recombinant), DNase I (recombinant), Vent polymerase (recombinant), T7 RNA polymerase

(recombinant), T4 polynucleotide kinase, low range ssRNA ladder, and PCR buffer were obtained from New England Biosciences. Deoxynucleotide triphosphates (dNTPs) and ribonucleotide triphosphates (rNTPs) were obtained from Roche Biosciences. QIAquick PCR purification kit was purchased from Qiagen. Alexa Fluor 633 maleimide, Alexa Fluor 488 UTP, Sybr Green II gel stain, and RNaseOUT were purchased from Invitrogen. High- purity water with a resistivity greater than 18 ΜΩ cm was obtained from an in-line Millipore RiOs/Origin system (MilliQ water) and treated with Diethyl pyrocarbonate (DEPC) prior to use. Thiol-modified PMA with 15 mole% of thiol groups (PMASH) was synthesized via carbodiimide chemistry, as described in detail previously.1 Methods.

[0068] Flow cytometry was performed on a Becton Dickson FACS calibur flow cytometer using an excitation wavelength of 488 ran. Particles and cells were imaged on anOIympus 1X71 digital wide-field fluorescence microscope with a fluorescein filter cube and a Leica time-correlated single-photon-counting confocal fluorescence microscope. UV-Vis measurements were performed on a Thermo Scientific NanoDrop-1000 spectrophotometer. PC temperature cycling was carried out in an Applied Biosystems GeneAmp PCR System 9700.

[0069] Synthesis of DNA Amplicons. PCR amplicons were synthesized by PCR using a mixture containing 200 μΜ dNTPs, 10 pmol primers, 2.5 U Taq DNA polymerase, and 100 ng of dsDNA template in a total volume of 20 μΐ, PCR buffer (20 mM Tris-HCl pH 8.4, 50 mM KC1, 1.5 mM MgC12). Cycling conditions were 5 min at 94 °C, 30 cycles of 94 °C for 30 s, 60 °C for 1 min, and 72 °C for 1 min, followed by 72 °C for 5 min. The amplicons synthesized are listed in Table 1 below. +T7DNA and -T7DNA are -800 bp amplicons with or without the 5' T7 promoter sequence on the sense strands respectively. +T7DNA2 is -T7DNA modified to contain the T7 promoter sequence, enabling transcription of -T7DNA. Samples were purified using the QIAquick PCR cleanup kit. A portion of the samples were separated on 1% agarose gel in TAE buffer (40 mM

Trisacetate, pH 8.3, 1 mM EDTA) and stained with ethidium bromide.

Table 1. PCR amplicons.

1 Amplicon < Forward Primer Reverse Primer [ Template

1 +T7DNA ! CGAACGACCTACACCGAACTGAGATACCTA CGTAATACGACTCACTATAGGGCGAATTGG | pPC -script i -T7DNA 1 GAAGTGGTGGCCTAACTACGGCTACACTAG TAACACTGCGGCCAACTTACTTCTGACAAC ; pPCR-script j +T7DNA2 I CGTAATACGACTCACTATAGGGAAGTGGTGGCCTA TAACACTGCGGCCAACTTACTTCTGACAAC ! -T7DNA

[0070] Preparation of Polymeric Capsules. A suspension of 5 mg of aminated particles in 25 mM sodium acetate buffer (pH 4) was prepared with 0.5% solids content. To this suspension, 1.08 μg (3.25 μΐΏ particles) or 1.7 § (1.1 1 μ^ηι particles) of DNA amplicons in 25 mM sodium acetate, pH 4, was slowly added while vortexing the particles. This was followed by three wash steps (collection of the particles via centrifugation, removal of the supernatant, and resuspension in fresh 25 mM sodium acetate buffer, pH 4). The DNA coated particles were then incubated in a 1.0 g L-1 solution of PMASH for 15 min at room temperature. After being washed three times in 25 mM sodium acetate buffer (pH 4), the particles were suspended in a 1.0 g L-1 solution of PVPON (Mw 10,000) for 15 min. PMASH and PVPON were added sequentially until 8 layers (3.25 μπι particles) or 6 layers ( 1.1 1 μιη particles) had been deposited, after which time the PMASH layers were crosslinked by suspending the particles in a 0.5 g L-l solution of either BM(PEG)2 or BMDB in 50 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 6) for 16 h. The particles were washed with fresh buffer and dispersed in 50 μΐ, of 25 mM sodium acetate buffer (pH 4), to which 100 uL of 2 M HF/8 M NH4F (pH ~5) was added. The resulting capsules were washed by centrifugation (4500g for 5 min) and dispersed into 10 mM Tris- HC1, 50 μΜ EDTA, pH 8, which was repeated until the pH of the supernatant was equal to the pH of the fresh buffer solution. After the final wash, the capsules were suspended in 50 μΐ_^ buffer. The capsule concentration was ascertained by counting a small portion of the capsule solution with flow cytometry. Labeling of the capsules was achieved by reaction of 1 μg of Alexa Fluor 633-maleimide with the capsules suspended in 50 mM MES, pH 6.

[0071] Encapsulated Transcription. Approximately 7-10 million capsules (from 3.25 μπ particles) or 200-250 million capsules (from 1.1 1 μπι particles) were suspended into 50 μΐ, of 40 mM Tris-HCl, pH 8, containing the following components: 6 mM Magnesium chloride, 20 mM DTT, 2 mM spermidine, 0.5 mM rNTPs, 10 U RNaseOUT, and 50 U T7 RNA Polymerase. The capsules contained either +T7DNA or -T7DNA amplicons. The reaction was allowed to progress at 37 oC while shaking at 600 rpm. For labeled RNA, the reaction was supplemented with 0.1 nmol Alexa Fluor 488 UTP. To stop the reaction, 5 μΙ_. of 0.1 M EDTA, pH 7.0, was added. To remove the encapsulated DNA amplicons the capsules were suspended into 50 μί of 10 mM Tris-HCl, pH 7.6, containing 2.5 mM Magnesium chloride, 0.5 mM Calcium chloride, and 2 U DNase I and allowed to react for 20 min at 37 oC and 600 rpm.

[0072] Gel Electrophoresis. Capsules were washed 3X into 0.1 M HEPES buffer, pH 7.2 and suspended into a final volume of 10 μΐ_^. To this suspension was added 10 μί of 50 mM sodium periodate in 0.1 M HEPES buffer, pH 7.2. The capsules were reacted in the dark for 30 min at room temperature, cleaving the BMDB crosslinkers and releasing the encapsulant. To this volume, 8 μΐ, of loading buffer ( 0.5 M EDTA pH8, 1 M Tris pH 8, 1% orange G, 60% glycerol) was added and loaded into a 1.5% agarose gel cast in IX TAE buffer. Other lanes were loaded with a low range ssRNA ladder (according to manufacturer's directions) and the product of a non-encapsulated transcription of

+T7DNA. Following electrophoresis in IX TAE, the gels was stained for 30 min with IX Sybr Green II gel stain and imaged using epi-illumination at 254 nm on a Biorad

VersaDoc.Imaging system; [0073] Cellular Uptake: Chinese hamster ovary (CHO) cells and Ramos B lymphocytes were maintained in RPM] 1640 medium (S AFC) supplemented with 1 % t-glutamine, penicillin and streptomycin (50U mL-1, 50μg and 5% bovine calf serum at 37 °C and 5% C02. Cells were seeded at 4 x 10⁴ cells per well into 96 well plates and incubated with fluorescent RNA-filled capsules (mean diameter =1.35 μηι) at a ratio of 1 : 100. The assay was incubated at 37 °C with rocking for 24 h. Ramos cells were harvested and washed twice with pre-warmed phosphate buffered saline solution. CHO cells were washed twice and then trypsinized and harvested. The outer cell membranes were stained using FM 4-64 FX dye according to the manufacturer's instructions. See also the Supporting Information for detailed experimental procedures.

Results and Discussion

[0074] The results clearly demonstrate that newly synthesized RNA was clearly visible in the interior of the PMA polymeric capsules containing DNA with the T7 promoter sequence. The sequence was confirmed to be the expected transcript (758 bases), and this sequence was determined to hybridise to its complimentary sequence. It was also shown that the polymeric capsules including this synthesised RNA were taken up by cells.

[0075] A significant advantage of the presented technology is that PMA polymeric capsules have been developed as colloidal drug carriers and were shown to be effectively internalized by cancerous cells and white blood cells in human blood without any reduction in cell viability from the capsules in the absence of drugs. In the current work the inventors have also found that PMA polymeric capsules with de novo synthesized RNA were internalized by other mammalian cells (Figure2). This aspect, combined with the ability to synthesize a controlled amount of RNA within the capsule interior, establishes a unique biomedical platform en route to the cellular delivery of RNA therapeutics. The complete synthetic nature of the PMA polymeric capsules provides opportunities for engineering its structure, allowing such features as controlled degradability and cellular targeting.

[0076] The strategy outlined in Figure 1 was used to assemble PMA polymeric capsules (diameter of 4.35 + 0.25 p,m), each containing— 9000 copies of a 777-base pair (bp) dsDNA polymerase chain reaction (PCR) product. The PCR products were designed to either include (+T7DNA) or exclude (— T7DNA) the T7 promoter sequence at the 5 '-end of the sense strand. Approximately 107 capsules were added to a 50 pi transcription reaction supplemented with 0.1 nmol of fluorescently labeled uridine5 '-triphosphate (UTP, green) and the mixture was incubated at 37 °C.1131 A concentration of 50 units of T7Pol in the 50 ί reaction was sufficient to enable diffusion of enzyme into the core of the PMA polymeric capsules and initiate transcription. Confocal laser scanning microscopy (CLSM) was used to image_, fluorescently labeled polymeric capsules (red) following 3 h of incubation. Newly synthesized RNA was clearly visible in the interior of the PMA polymeric capsules containing DNA with the T7 promoter sequence, however, no fluorescence was observed in the PMA polymeric capsules containing DNA without the T7 promoter sequence. An identical reaction was performed using 1.35 ± 0.15 p.m PMA polymeric capsules, each containing— 500 copies of +T7DNA. Flow cytometry was used to measure the progress of the reaction within the PMA polymeric capsules (Fig. lc), allowing real-time monitoring of RNA synthesis. Synthesis was slow in the first hour but then increased to a linear rate of synthesis between— 1.5 h and 5 h, and finally leveled off over the next 24 h. The initial slow rate of synthesis possibly reflects the time required for the T7Pol to diffuse into the capsules and bind to the T7 promoter sequences.

[0077] To confirm that the synthesized RNA was the expected transcript (758 bases), the RNA was recovered from the PMA polymeric .capsules. The capsules were first treated with a large excess of DNase I, an endonuclease to degrade the DNA template, as confirmed by gel electrophoresis (data not shown). Recovered RNA was subjected to non- denaturing gel electrophoresis on a 1.5% agarose gel which confirmed that the products of the encapsulated reactions were the same size (758 bases) as the positive control reaction (P) performed in the absence of capsules. Encapsulated reactions that were incubated for longer time periods produced more RNA, verifying the flow cytometry measurements. As expected, no product was observed for the negative-control capsules (N) containing - T7DNA, which were incubated for 5 h.

[0078] To confirm that the synthesized RNA hybridized to its complementary sequence, dot blots were performed. Approximately 2.5 x 10⁸ capsules (mean diameter -1.35 μπι) containing either +T7DNA or -T7DNA templates were degraded following transcription and DNase I treatment and were spotted onto nitrocellulose membranes along with a series of control DNA and RNA sequences. Membranes were incubated with radiolabeled DNA probes specific for either the RNA transcript of +T7DNA (P) or a different RNA transcript (T) transcribed from the same plasmid. Following several washing steps to remove non- hybridized probes the membranes were exposed to film and later developed. The probe hybridized to the RNA transcript, indicating the RNA synthesized was the expected sequence. This is important as binding is necessary for its further use as a reaction component (e.g., reverse transcription) or as a biologically active therapeutic. [0079J The amount of RNA synthesized in the capsules at 5 h of incubation for 2 x 10 capsules (mean diameter of 1.35 μπι) was estimated to be 200 ng or an encapsulated RNA concentration of 775 p,g ml .^"1. These data imply that each of the encapsulated DNA copies were transcribed multiple times to produce de novo synthesized RNA. A significant advantage of the presented technology is that PMA polymeric capsules have been developed as colloidal drug carriers and were shown to be effectively internalized by cancerous cells and white blood cells in human blood without any reduction in cell viability from the capsules in the absence of drugs. In the current work we also found that PMA polymeric capsules with de novo synthesized RNA were internalized by other mammalian cells (Figure2). This aspect, combined with the ability to synthesize a controlled amount of RNA within the capsule interior, establishes a unique biomedical platform en route to the cellular delivery of RNA therapeutics. The complete synthetic nature of the PMA polymeric capsules provides opportunities for engineering its structure, allowing such features as controlled degradability and cellular targeting. In conclusion, we have demonstrated a method to monitor real-time transcription within PMA polymeric capsules, allowing precise control of RNA loading into the capsules via reaction time. Synthesized RNA was recovered from the PMA polymeric capsules and was of the expected size and sequence. RNA-loaded PMA polymeric capsules were also internalized by mammalian cells. This method of encapsulated transcription demonstrates a unique biomedical platform, where polymer polymeric capsules function as both microreactors and biocompatible delivery vehicles for the cellular internalization of the newly

synthesized RNA.

Claims

1. A method of producing a nucleic acid molecule within a polymeric capsule, the method comprising providing a polymeric capsule having entrapped therein a DNA molecule, mixing the capsule with a polymerase and nucleotides, wherein the polymerase and the nucleotides can diffuse into the capsule, to form a reactant mixture, and incubating the reactant mixture for a time and under conditions such as to allow the de novo synthesis of a nucleic acid molecule within the capsule.

2. A method of producing a nucleic acid molecule within a polymeric capsule, the method comprising providing a polymeric capsule having entrapped therein a DNA molecule and a polymerase, mixing the capsule with nucleotides which can diffuse into the capsule and optionally a polymerase which can diffuse into the capsule, to form a reactant mixture, and incubating the reactant mixture for a time and under conditions such as to allow the de novo synthesis of a nucleic acid molecule within the capsule.

3. A method according to claim 1 or claim 2 wherein the nucleotides are

ribonucleotides.

4. A method according to any one of claims 1 to 3 wherein the synthesised nucleic acid molecule is single stranded RNA.

5. A method according to claim 3 wherein the single stranded RNA is miRNA.

6. A method according to claim 1 or claim 2 wherein the synthesised nucleic acid molecule is hairpin RNA.

7. A method according to claim 5 wherein the single stranded RNA is shRNA.

8. A method according to claim 1 or claim 2 wherein the synthesised nucleic acid molecule is double stranded RNA.

9. A method according to claim 7 wherein the single stranded RNA is siRNA.

10. A method according to any one of claims 1 to 3 wherein the synthesised nucleic acid molecule is a ribozyme.

1 1. A method according to any one of claims 1 to 7 wherein the polymerase is T7 RNA polymerase and the DNA comprises a T7 promoter.

12. A method according to claim 1 or claim 2 wherein the reactant mixture further comprises DNA primers and the synthesised nucleic acid molecule is DNA.

13. A method according to claim 12 wherein the synthesised DNA is a DNAzyme.

14. The method according to claim 1, wherein two or more different DNA molecules are entrapped within the polymeric capsule.

15. A method according to any one of claims 1 to 14 wherein the polymeric capsule is a polymeric shell comprising layers of thiolated poly(methacrylic acid) (PMASH) wherein the PMASH layers are cross-linked by disulphide bonds.

16. A polymeric capsule comprising a nucleic acid molecule wherein the capsule is produced according to the method of any one of claims 1 to 15.

17. A method of delivering a nucleic acid to a cell comprising contacting the cell with a polymeric capsule according to claim 16.