Patent Description:
Nucleic acids and analogues thereof have many applications in basic research and both prophylactic and therapeutic interventions. However, prokaryotic and eukaryotic cells are in general impermeable to nucleic acids/analogues. Typically, nucleic acid/analogue uptake has required delivery vehicles, either viral or non-viral, in order to reach their potential. To be effective, the delivery vehicle/nucleic acid or analogue combination or complex must overcome many challenges, for example extracellular degradation, cell membrane barrier penetration and intracellular release, whilst minimising cytotoxicity and antigenicity. Many polypeptides (for example proteins, peptides) and other bioactive molecules also are cell impermeable, but otherwise useful. For example, the activity of many compounds in biochemical assays is much higher than in cellular assays or in vivo. The drop off in cellular activity is believed to be largely due to poor delivery into cells.

Lipofectamine is a widely used example of an agent used in promoting entry of agents, for example nucleic acids, into cells. However, lipofectamine is considered to have toxic effects on mammalian cells in particular, which can, for example, make in vitro experiments difficult to assess and in vivo experiments difficult to perform.

Other proposed entry-promoting agents are described in, for example, <CIT> (Biodegradable cationic polymer gene transfer compositions and methods of use); <CIT> (cationic lipopolymer as biocompatible gene delivery agent); <CIT> (non-viral transfection agent); <CIT> (amphiphilic biguanide derivatives).

There is a need for alternative entry-promoting agents, for example for nucleic acids and other substances where activities are limited by poor cell entry. Described herein are entry-promoting agents and methods. Such entry-promoting agents and methods are considered to be beneficial, for example in providing good entry promotion, poor antigenicity, low cost and/or low toxicity, for example particularly for eukaryotic cells. Such entry-promoting agents and methods may, for example, be useful in areas such as nucleic acid/analogue transfection, for example functional studies; generation of stable cell lines; gene silencing; and DNA vaccination. For example, such entry-promoting agents and methods may, for example, be useful in relation to RNA interference (RNAi) or other antisense technologies, as will readily be appreciated by those skilled in the art. The challenge of delivery of reagents or drugs into cells is not limited to only nucleic acids. Proteins and peptides also enter cells very poorly in general and entry-promoting technology would be useful. Also, many molecules that are described as "small molecules" being less than <NUM> grams/mol enter cells poorly and their usefulness as reagents or drugs would be enhance by improved cell delivery technology.

PHMB (polyhexamethylene biguanide) is known as a safe and effective biocidal agent and is used as a sanitiser and preservative: <CIT>, <CIT>, <CIT>; <CIT>. The present inventors have surprisingly found that PHMB and related molecules are useful entry-promoting agents. It was surprisingly observed that PHMB (for example) itself enters a wide range of cells, including bacteria, fungi and mammalian cells. More surprisingly, PHMB (for example) is able to form nanoparticles with a wide range of molecules and deliver these molecules into such cells. Finally, the delivered molecules ranging from nucleic acids to small molecules were found to be functional inside cells.

<NPL>), describes the synthesis of guanidinylated poly(allyl amine) and its use as a gene carrier.

<CIT> describes the use of nanoparticles of inorganic materials as carriers for biocides in ophthalmic compositions.

<NPL>) describes the interaction between PHMB and various nucleic acids.

In a first aspect of the present invention, there is provided an in vitro or ex vivo method for promoting entry of an agent to be introduced into a cell, the method comprising the step of exposing the cell to the agent to be introduced in the presence of an entry-promoting agent, wherein the entry-promoting agent is selected from the group consisting of: polyhexamethylene biguanide (PHMB), polyhexamethylene monoguanide (PHMG), polyethylene biguanide (PEB), polytetramethylene biguanide (PTMB), polyethylene hexamethylene biguanide (PEHMB), polymethylene biguanides (PMB), poly(allybiguianidnio-co-allylamine), poly(N-vinylbiguanide) or polyallybiguanide, wherein the agent to be introduced is selected from the group consisting of:.

wherein the agent to be introduced and the entry-promoting agent are provided as nanoparticles consisting of the agent to be introduced and the entry-promoting agent, where the nanoparticles are formed at a pH of <NUM> - <NUM>.

Described herein, but not claimed, is a method for promoting entry of an agent into a cell, the method comprising the step of exposing the cell to the introduced agent in the presence of a polymer, wherein the polymer comprises a linear and/or branched polymonoguanide/polyguanidine, polybiguanide, analogue or derivative thereof, for example according to the following formula 1a or formula 1b, with examples given in tables <NUM> and <NUM>, below:
<CHM>
<CHM>
wherein:.

L<NUM> and L<NUM> are the linking groups between the G<NUM> and G<NUM> cationic groups in the polymer. L<NUM> and L<NUM> can independently represent an aliphatic group containing C<NUM>-C<NUM> carbon atoms, for example an alkyl group such as methylene, ethylene, propylene, C<NUM>, C<NUM>, C<NUM>, C<NUM>, C<NUM>, C<NUM> or C<NUM>; C<NUM>-C<NUM>, -C<NUM>, - C<NUM>, -C<NUM>, -C<NUM> -C<NUM>, -C<NUM>, -C<NUM>, -C<NUM>, -C<NUM>, -C<NUM>, -C<NUM>, -C<NUM> or -C<NUM>, alkyl ; or L<NUM> and L<NUM> can (independently) be C<NUM>-C<NUM> (for example C<NUM>, C<NUM>, C<NUM>, C<NUM>, C<NUM>, C<NUM>, C<NUM>, C<NUM>, C<NUM> or C<NUM>; C<NUM>-C<NUM>, -C<NUM>, - C<NUM>, -C<NUM>, -C<NUM> -C<NUM>, -C<NUM>, -C<NUM>, -C<NUM>, -C<NUM>, -C<NUM>, -C<NUM>, -C<NUM> or -C<NUM>), cycloaliphatic, heterocyclic, aromatic, aryl, alkylaryl, arylalkyl , oxyalkylene radicals, or L<NUM> and L<NUM> can (independently) be a polyalkylene radical optionally interrupted by one or more, preferably one, oxygen, nitrogen or sulphur atoms, functional groups as well as saturated or unsaturated cyclic moiety. Examples of suitable L<NUM> and L<NUM> are groups are listed in table <NUM>.

L<NUM>, L<NUM>, G<NUM> and G<NUM> may have been modified using aliphatic, cycloaliphatic, heterocyclic, aryl, alkaryl, and oxyalkylene radicals.

N and G<NUM> are preferably end groups. Typically, the polymers have terminal amino (N) and cyanoguanidine (G<NUM>) or guanidine (G<NUM>) end groups. Such end groups may be modified (for example with <NUM>,<NUM>-diaminohexane, <NUM>,<NUM> di(cyanoguanidino)hexane, <NUM>,<NUM>-diguanidinohexane, <NUM>-guanidinobutyric acid) by linkage to aliphatic, cycloaliphatic heterocyclic, heterocyclic, aryl, alkylaryl, arylalkyl, oxyalkylene radicals. In addition, end groups may be modified by linkage to receptor ligands, dextrans, cyclodextrins, fatty acids or fatty acid derivatives, cholesterol or cholesterol derivatives or polyethylene glycol (PEG). Optionally, the polymer can end with guanidine or biguanide or cyanoamine or amine or cyanoguanidine at N and G<NUM> positions or cyanoamine at N and cyanoguanidine at G<NUM> position or guanidine at N and Cyanoguanidne at G<NUM> positions or L1 amine at G<NUM> and cyanoguanidine at N. G<NUM> can be L<NUM>-amine, L<NUM>-cyanoguanidine or L<NUM>-guanidine. Depending on the number of polymerization (n) or polymer chain breakage and side reactions during synthesis, heterogeneous mixture of end groups can arise as described above as an example. Thus, the N and G<NUM> groups can be interchanged/present as a heterogeneous mixture, as noted above. Alternatively, N and G<NUM> may be absent and the polymer may be cyclic, in which case the respective terminal L<NUM> and G<NUM> groups are linked directly to one another.

In formula 1b, X can be either present or absent. L<NUM>, L<NUM> and X are as noted above for "L<NUM> or L<NUM>". In Thus, L<NUM> and L<NUM> and X are the linking groups between the G<NUM> and G<NUM> cationic groups in the polymer. L<NUM> and L<NUM> and X can independently represent an aliphatic group containing C<NUM>-C<NUM> carbon atoms, for example an alkyl group such as methylene, ethylene, propylene, C<NUM>, C<NUM>, C<NUM>, C<NUM>, C<NUM>, C<NUM> or C<NUM>; C<NUM>-C<NUM>, -C<NUM>, -C<NUM>, -C<NUM>, -C<NUM> -C<NUM>, -C<NUM>, -C<NUM>, -C<NUM>, -C<NUM>, -C<NUM>, -C<NUM>, -C<NUM> or - C<NUM>, alkyl ; or L<NUM> and L<NUM> and X can independently be C<NUM>-C<NUM> (for example C<NUM>, C<NUM>, C<NUM>, C<NUM>, C<NUM>, C<NUM>, C<NUM>, C<NUM>, C<NUM> or C<NUM>; C<NUM>-C<NUM>, -C<NUM>, -C<NUM>, -C<NUM>, -C<NUM> -C<NUM>, -C<NUM>, -C<NUM>, -C<NUM>, -C<NUM>, -C<NUM>, -C<NUM>, -C<NUM> or - C<NUM>), cycloaliphatic, heterocyclic, aromatic, aryl, alkylaryl, arylalkyl , oxyalkylene radicals, or L<NUM> and L<NUM> and X can independently be a polyalkylene radical optionally interrupted by one or more, preferably one, oxygen, nitrogen or sulphur atoms, functional groups as well as saturated or unsaturated cyclic moiety. Examples of suitable L<NUM> and L<NUM> and X are groups are listed in table <NUM>.

"G<NUM>" and "G<NUM>" are cationic moieties and can be same or different. At least one of them is a biguanidine moiety or carbamoylguanidine, and the other moiety may be as above (biguanidine or carbamoylguanidine) or amine. For the avoidance of doubt, in formula 1b, cationic moiety G<NUM> and G<NUM> do not contain only single guanidine groups. For example, G<NUM> and G<NUM> typically do not contain single guanidine groups. Examples of such compounds are polyallylbiguanide, poly(allylbiguanidnio-co-allylamine), poly(allylcarbamoylguanidino-co-allylamine), polyvinylbiguanide, as listed in table <NUM>.

Example of polyallylbiguanide is as shown below
<CHM>.

In case of polyallylbigunidine L<NUM> and L<NUM> are identical, G<NUM> and G<NUM> are similar, thus polyallylbiguanide can be simplified as below.

Example of poly(allylcarbamoylguanidnio-co-allylamine) is as shown below
<CHM>.

The polymers will generally have counter ions associated with them. Suitable counter ions include but are not limited to the following: halide (for example chloride), phosphate, lactate, phosphonate, sulfonate, amino carboxylate, carboxylate, hydroxy carboxylate, organophosphate, organophosphonate, organosulfornate and organosuflate.

The polymers can be either heterogeneous mixtures of polymers of different "n" number or homogenous fractions comprising specified "n" numbers purified by standard purification methods. As indicated above the polymers may also be cyclic and in addition may be branched.

Preferred numbers for "n" include <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM> and <NUM>-<NUM>.

The described entry-promoting agent may comprise linear, branched or dendrimeric molecules. The described entry promoting agent may comprise a combination of linear, branched or dendrimeric molecules. The entry promoting agent may comprise one or any combination of molecules of Formula 1a or formula 1b, for example as described above.

For example, the described entry-promoting agent can comprise one or more of polyhexamethylene biguanide (PHMB), polyhexamethylene monoguanide (PHMG), polyethylene biguanide (PEB), polytetramethylene biguanide (PTMB) or polyethylene hexamethylene biguanide (PEHMB). Some examples are listed in table <NUM> and <NUM>.

The entry-promoting agent may comprise homogeneous or heterogeneous mixtures of one or more of polyhexamethylene biguanide (PHMB), polyhexamethylene monoguanide (PHMG), polyethylene biguanide (PEB), polytetramethylene biguanide (PTMB), polyethylene hexamethylene biguanide (PEHMB), polymethylene biguanides (PMB), poly(allylbiguanidnio-co-allylamine), poly(N-vinylbiguanide), polyallybiguanide.

In the method of the present invention, the entry-promoting agent is selected from the group consisting of: polyhexamethylene biguanide (PHMB), polyhexamethylene monoguanide (PHMG), polyethylene biguanide (PEB), polytetramethylene biguanide (PTMB), polyethylene hexamethylene biguanide (PEHMB), polymethylene biguanides (PMB), poly(allybiguianidnio-co-allylamine), poly(N-vinylbiguanide) and polyallybiguanide.

The compounds can be synthesised in the laboratory by standard procedures or may be obtained from commercial suppliers, as will be well known to those skilled in the art.

PHMB, for example, may also have synonyms poly(hexamethylene)biguanide hydrochloride; polymeric biguanide hydrochloride; polyhexanide; biguanide; CAS Number <NUM>-<NUM>-<NUM>; <NUM>-<NUM>-<NUM>; IUPAC name Poly(iminoimidocarbonyl)iminohexamethylene hydrochloride. PHMB can be synthesised in the laboratory by standard procedures or may be obtained from suppliers, for example, Arch (http://www. archchemicals. com/Fed/BIO/Products/phmb. Typically n = <NUM> to <NUM>, average n:<NUM>, average molecular weight: <NUM>. PHMB is sold as a biocide, for example for use in hygiene products, swimming pool water treatment and wound dressings.

Polyhexamethylene monoguanide (PHMG) can be synthesised in the laboratory by standard procedures or obtained from suppliers, for example from Shanghai Scunder Industry Co. ,Ltd, http://scunder. com/products/info/<NUM>/PHMG.

As will be appreciated by those skilled in the art, the entry-promoting polymer may be a copolymer or heteropolymer i.e. the monomers may not be intended to be identical. However, typically the monomer units may be intended to be identical.

The entry promoting polymer can be used for delivery into both prokaryotic and eukaryotic cells. Thus, the cell may be a prokaryotic cell. Examples of such cells will be well known to those skilled in the art and include Gram negative bacteria; Gram positive bacteria; and mycobacteria or acid fast bacteria, for example Mycobacterium smegmatis. Examples of Gram-negative bacteria include E. enterica, for example S. entericia serovar Typhimurium, Salmonella spp and Campylobacter spp. Examples of Gram-positive bacteria include S.

The cell may alternatively be a eukaryotic cell, for example a fungal cell, for example Aspergillus, for example A. fumigatus; Candida spp; Saccharomyces spp; Pichia spp. The cell may be a mammalian cell (which may be a cell in cell culture, or a cell present in a tissue or organ). The cell may, for example, be a human, mouse, rat, rabbit, bovine or dog (or, for example, any other wild, livestock/domesticated animal) cell. The cell may, for example, be a stable cell line cell, or a primary cell, adherent or suspension cell. As examples, the cell may be a macrophage, osteosarcoma or HeLa cell line cell or a mouse primary cell.

The eukaryotic cell may alternatively be a plant cell (for example a monocotyledonous or dicotyledenous plant cell; typically an experimental, crop and/or ornamental plant cell, for example Arabidopsis, maize); fish (for example Zebra fish; salmon), bird (for example chicken or other domesticated bird), insect (for example Drosophila; bees), Nematoidia or Protista (for example Plasmodium spp or Acantamoeba spp) cell.

The introduced agent may typically be or comprise a bioactive compound, for example a pharmaceutically active substance, or a diagnostic/imaging tool or probe, typically that has poor cell uptake properties. The introduced agent may comprise a nucleic acid or nucleic acid analogue. The nucleic acid or nucleic acid analogue may, for example, be or comprise DNA or RNA or both. The nucleic acid or nucleic acid analogue may typically be an antisense nucleobase oligomer, or a sense nucleobase oligomer (for example encoding a polypeptide or a structural or regulatory nucleic acid). The nucleobase oligomer may be any type of nucleic acid analogue or mimic that retains the capacity for base pairing, as will be well known to those skilled in the art. For example, the nucleic acid/analogue or nucleobase oligomer may be a phosphorothioate, <NUM>'O-methyl nucleic acid, locked nucleic acid, peptide nucleic acid (PNA) oligomer. Alternatively, the nucleic acid/analogue or nucleobase oligomer may be, for example, a phosphorothioate, morpholino oligomer (PMO). The skilled person will readily be able to determine whether a given nucleobase oligomer chemistry is compatible with the nucleobase oligomer retaining the ability to bind specifically to a target nucleic acid, for example to act as a probe, guide polypeptide/nucleic acid expression, or mediate gene silencing (for example through antisense or RNAi, as will be well known to those skilled in the art). The skilled person will also readily be able to determine whether a given nucleobase oligomer chemistry is compatible with promotion of entry by the entry-promoting agent: it is considered that an entry-promoting agent as set out above is useful generally with nucleic acids/analogues/nucleobase oligomers.

PNA and morpholino oligomers typically have uncharged (rather than anionic) backbones. There has previously been very modest success in PNA delivery. Many strategies that work with DNA and RNA do not work with PNA and morpholino oligomers, which are typically uncharged. We have surprisingly found that the entry-promoting agent of the present invention works with such molecules.

The nucleic acid/analogue/nucleobase oligomer may be single stranded or double stranded. The nucleobase oligomer may be able to act as a probe, in guiding polypeptide/nucleic acid expression, or as an RNAi molecule, for example small interfering RNA (siRNA) or microRNA, as an aptamer or ligand as is well known to those skilled in the art. Typically the nucleic acid/analogue/nucleobase oligomer may be single stranded, particularly when the cell is prokaryotic. When the cell is eukaryotic (for example when the cell is a yeast or other fungus, or is a mammalian cell), the nucleobase oligomer may typically be single stranded or double stranded, which may include a mixture of single and double stranded regions.

The nucleobase oligomer can be any molecule that hybridizes by a sequence specific base pairing to a complementary DNA and/or RNA sequence. In the context of this disclosure, "hybridization" means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds.

Further relevant features of nucleobase oligomers, for example antisense nucleobase oligomers, will be well known to those skilled in the art, and are, for example, set out in <CIT>, for example on page <NUM>, lines <NUM> to <NUM> (antisense RNA regulation; PNA preparation, use as an antisense compound; cell uptake); page <NUM>, lines <NUM> to <NUM> (hybridization to target sequence); page <NUM>, lines <NUM> to <NUM> (types of nucleobase oligomer); page <NUM>, line <NUM> to page <NUM>, line <NUM> (length of antisense compounds; types of more on types of nucleobase oligomer); page <NUM>, line <NUM> to page <NUM>, line <NUM> (cell uptake; length considerations); page <NUM>, line <NUM> to page <NUM>, line <NUM> (cell uptake); page <NUM>, line <NUM> to page <NUM>, line <NUM> (synthesis); page <NUM>, line <NUM> to page <NUM>, line <NUM> (linker connection between PNA and peptide).

The nucleobase oligomer may comprise, for example, phosphorothioate, <NUM>'O-methyl, <NUM>'Fluoro, locked nucleic acid (LNA), morpholino, PNA or deoxy nucleotides.

The introduced agent may comprise plasmid or other vector DNA (which may be modified DNA), as will be well known to those skilled in the art. Typically plasmid or vector DNA may encode and/or be suitable for expressing (or promoting expression of, for example by encoding a cellular factor which, when expressed, activates the expression of an endogenous gene) a polypeptide or nucleic acid of interest. Alternatively, the introduced agent may comprise an RNA (which may be modified RNA, for example as discussed above) molecule, for example the introduced agent may comprise an siRNA molecule i.e. a molecule capable of mediating RNA interference, as well known to those skilled in the art.

Successfully transformed or transfected cells, i.e. cells that contain a DNA construct as noted above, can be identified by well-known techniques. For example, one selection technique involves incorporating into the expression vector a DNA sequence (marker) that codes for a selectable trait in the transformed cell. These markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture, and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria. Alternatively, the gene for such selectable trait can be on another vector, which is used to co-transform the desired host cell.

The marker gene can be used to identify transformants but it is desirable to determine which of the cells contain recombinant DNA molecules and which contain self-ligated vector molecules. This can be achieved by using a cloning vector where insertion of a DNA fragment destroys the integrity of one of the genes present on the molecule. Recombinants can therefore be identified because of loss of function of that gene.

Successfully transformed cells, i.e. cells that contain a DNA construct as noted above, can be identified by well-known techniques. For example, another method of identifying successfully transformed cells involves growing the cells resulting from the introduction of an expression construct as noted above can be grown to produce the encoded polypeptide. Cells can be harvested and lysed and their DNA content examined for the presence of the DNA using a method such as that described by Southern (<NUM>) J. <NUM>, <NUM> or Berent et al (<NUM>) Biotech. <NUM>, <NUM>. Alternatively, the presence of the protein in the supernatant can be detected using antibodies as well known to those skilled in the art.

In addition to directly assaying for the presence of recombinant DNA, successful transformation can be confirmed by well-known phenotypic assays, for example immunological methods, when the recombinant DNA is capable of directing the expression of the protein. For example, cells successfully transformed or transfected with an expression vector produce proteins displaying appropriate antigenicity. Samples of cells suspected of being transformed or transfected are harvested and assayed for the protein using suitable antibodies.

Methods for determining whether an antisense reagent has been taken up by a cell will also be well known to those skilled in the art, for example methods similar in concept to those described above; the introduced antisense agent may result in a reduction in endogenous gene function or expression.

The introduced agent may comprise a polypeptide (by which term is included smaller polypeptides or fragments of larger polypeptides, which may be termed peptides, for example of fewer than <NUM> amino acids in length, typically <NUM>-<NUM> amino acid residues in length; as well as larger or full length polypeptides which may be termed proteins, for example of more than <NUM> amino acids in length). org/wiki/Peptide. The term polypeptide is intended to encompass peptidomimetic compounds, as will be well known to those skilled in the art. One example of peptidomimetic compounds is discussed in <NPL>). The polypeptide may be a therapeutic polypeptide, for example a polypeptide that replaces the function of a missing/depleted and/or defective polypeptide; or may be an antigenic polypeptide, for example a polypeptide intended to serve as a vaccine, or may act as a ligand that binds to another protein or peptide. For example, the polypeptide may be an agonist or antagonist of a cellular protein. The delivered peptide or protein may act by either inhibiting or promoting an enzyme's function in a cell to build understanding of the role of the enzyme in a cell or to have a therapeutic or other useful effect in the cell, or in the tissue, organ or body of which the cell may be a part.

Methods for determining whether a polypeptide has been taken up by a cell will also be well known to those skilled in the art. For example the protein may have fluorescence properties, such as green fluorescence protein (GFP) that can be observed and monitored using fluorescence microscopy, flow cytometry or similar methods. Also, uptake may be assessed using antibody-mediated staining. Finally, diverse functional studies can be utilized, according to the known function of the protein. For example, if the protein is a transcription factor, genes regulated by the factor could be profiled to assess changes in expression levels. Alternatively, the protein may be a kinase, and changes in phosphorylation of its known substrates could be measured using phosphorylation assays. Methods for determining whether a peptide has been taken up by a cell will also be well known to those skilled in the art. For example the peptide may be fluorescently labelled, using for example fluorescein, which can be observed and monitored using fluorescence microscopy, flow cytometry or similar methods. Also, uptake may be assessed using antibody-mediated staining. Finally, diverse functional studies can be utilized, according to the known function(s) of proteins and peptides. Alternatively, if the peptide is a ligand, the downstream effects of peptide interaction with a receptor could be assessed. If the protein/peptide is a toxin, the toxic effects could be assayed. Finally, many peptides and proteins are immunogenic and the immune function effects following delivery can be assessed using methods that are well known to molecular and cellular immunologists and others skilled in the art.

The introduced agent may comprise a small drug or bioactive reagent, for example having a molecular weight of less than <NUM> or <NUM> Da. The small drug or bioactive reagent may have poor cell uptake or stability properties in the absence of the entry-promoting agent of the present invention. An example is gentamycin, which is a useful antibiotic, but does not readily enter host cells. A second example is amphotericin B. Negatively charged or highly hydrophobic molecules may be introduced agents that may benefit from the delivery technology of the present invention.

Methods for determining whether a small molecule has been taken up by a cell will also be well known to those skilled in the art. The small molecule may have inherent fluorescent properties allowing uptake to be monitored using the methods described about. Also, radioisotopes can be incorporated, or small fluorophores may be conjugated providing fluorescent properties. For example a DNA ligand small molecule that increases in fluorescence upon binding to DNA provides a convenient assay for delivery, showing both cellular uptake and binding to the target molecule. In many cases small molecule delivery may be assessed through functional analyses of effects caused by binding to its receptor. For example, the small molecule may be an agonist or an antagonist and downstream effects can be measured using methods that are well known to those skilled in the art.

The introduced agent may comprise a cellular imaging probe, for example a contrast agent for in vivo imaging such as quantum dots, superparamagnetic iron oxide; or a receptor ligand, for example for an intracellular receptor, for example a nuclear hormone receptor; or a nucleic acid based imaging probe, as noted above, all of which will be well known to those skilled in the art. Methods used to determine the location of imaging probes are well known to those skilled in the art and include, but are not limited to, fluorescence imaging and radioactivity monitoring.

Described herein is an introduced agent and entry-promoting agent which may be covalently joined. The introduced agent and the entry-promoting agent may be provided as a formulation, for example as a non-covalent complex. The formulation may be prepared by mixing the entry-promoting agent and the introduced agent in appropriate ratios and under appropriate conditions of, for example, pH and salt concentration, for example as set out in the examples. The method may, for example, be performed from up to <NUM> fold molar excess of introduced agent over entry-promoting agent, through using an equal molar concentration of carrier and cargo molecules, to up to <NUM> fold molar excess of entry-promoting agent over introduced agent. For example, an appropriate molar ratio of introduced agent (for example nucleic acid, for example oligonucleotide, for example of <NUM>-<NUM> bases in length and entry-promoting agent may be in the range of <NUM>:<NUM> to <NUM>:<NUM> or <NUM>:<NUM> to <NUM>:<NUM>, for example <NUM>:<NUM> to <NUM>:<NUM> or <NUM>:<NUM>, for example around <NUM>:<NUM>. An appropriate weight:weight ratio of introduced agent and entry-promoting agent may be in the range of <NUM>:<NUM> to <NUM>:<NUM> or <NUM>:<NUM> to <NUM>:<NUM>, for example <NUM>:<NUM> to <NUM>:<NUM> or <NUM>:<NUM>, for example around <NUM>:<NUM>. The formation of complexes is discussed further below. The pH at which the entry-promoting agent and the introduced agent are mixed/incubated is pH <NUM>-<NUM>, as discussed further below.

The method of the first aspect of the invention may be performed in vitro. Examples of situations in which the method of the invention may be useful include large-scale batch transfections and experimental transfections, for example for expressing a valuable protein or to understand the role of a gene or conditions that affect the gene or gene product. Also, the method may be used to characterise a microbiological sample. As noted above, the invention may be useful in, for example, functional studies; generation of stable cell lines; gene silencing; DNA vaccination; and drug delivery.

Further provided herein is a method for making a target polypeptide (which term, as noted above, includes both peptides and proteins, including covalently modified polypeptides, for example glycosylated polypeptides, as appropriate), the method comprising the step of preparing the target polypeptide from a cell culture of a host cell, wherein the host cell is a host cell that has been transformed (or whose progenitor has been transformed) by an exogenous nucleic acid molecule (which may be a copy of an endogenous nucleic acid molecule, but which typically is a nucleic acid molecule that differs from nucleic acid molecules previously present within the cell) so that the cell synthesises the target polypeptide, wherein the transfection comprises exposing the host cell to the exogenous nucleic acid molecule in the presence of an entry-promoting agent as defined herein.

The method may comprise the step of culturing a host cell under conditions for synthesising the target polypeptide, as will be well known to those skilled in the art. Typically, the method may be performed in vitro. Also described, is such a method performed in vivo, for example in a plant or (non-human) animal.

The selection of appropriate host cell and other factors such as culture conditions may readily be performed by the skilled person for the intended target polypeptide.

The method of the first aspect of the invention may be performed ex vivo. For example the method may be performed on a body surface, for example skin or mucosal membrane. The method may be performed on cells that are subsequently introduced or returned to a subject organism, for example mammal, for example human or livestock/companion/laboratory animal.

Further described is an entry-promoting agent as defined herein and an introduced agent as defined herein for use in treating a subject in need of the introduced agent.

Similarly, further described is the use of an entry-promoting agent as defined herein and an introduced agent as defined herein in the manufacture of a medicament for use in treating a subject in need of the introduced agent.

The subject may be, for example, a mammal, for example a human or a livestock/companion/laboratory/wild animal.

The skilled person will readily appreciate that the method described herein may be useful in relation to a wide range of disease or conditions, for example diseases or conditions in which treatment with siRNA reagents or therapeutic or antigenic polypeptides may be useful. As examples, the method may be useful in relation to a mouthwash for treating oral viral infections, for example the common cold, for example with the entry-promoting agent and a nucleic acid against the virus strain in question; for treatment of skeletal related diseases, where the entry-promoting agent is combined with polypeptide or nucleic acid and injected into a joint as an emulsion or suspension to treat arthritis; delivery (for example to the skin) of a DNA vaccine (i.e. DNA encoding an antigenic polypeptide or vaccine against cancer or other disease e.g. analogous to DNA electroporation vaccines into skin (targeting the DC's) but as a topical agent instead; treatment (for example topical treatment) of a skin disease (for example acne, psoriasis) for example a nucleic acid based therapeutic. However, many other uses of the method described herein are envisaged, for example corresponding to the range of situations in which RNAi, DNA or polypeptide therapeutics are considered to be useful, particularly with delivery to a body surface, as noted above.

Further described herein is a kit of parts or composition comprising an entry-promoting agent and an introduced agent as defined herein. The kit of parts may be intended to, or composition may, comprise between a <NUM> fold molar excess of introduced agent over entry-promoting agent, through an equal molar concentration of carrier and cargo molecules, up to <NUM> fold molar excess of entry-promoting agent over introduced agent. For example, an appropriate molar ratio of introduced agent (for example nucleic acid, for example oligonucleotide, for example of <NUM>-<NUM> bases in length and entry-promoting agent may be in the range of <NUM>:<NUM> to <NUM>:<NUM> or <NUM>:<NUM> to <NUM>:<NUM>, for example <NUM>:<NUM> to <NUM>:<NUM> or <NUM>:<NUM>, for example around <NUM>:<NUM>. An appropriate weight:weight ratio of introduced agent and entry-promoting agent may be in the range of <NUM>:<NUM> to <NUM>:<NUM> or <NUM>:<NUM> to <NUM>:<NUM>, for example <NUM>:<NUM> to <NUM>:<NUM> or <NUM>:<NUM>, for example around <NUM>:<NUM>. Preferences for the entry-promoting agent and introduced agent are as set out above. For example, the introduced agent may be siRNA molecules. The formation of complexes is discussed further below. The pH at which the entry-promoting agent and the introduced agent are mixed/incubated may be a high pH, for example <NUM>-<NUM>, as discussed further below.

Further described herein is a kit of parts or composition wherein the kit of parts or composition is pharmaceutically acceptable. Thus, the kit components or composition may comprise (or consist of) pharmaceutically acceptable components, as will be well known to those skilled in the art. Entry-promoting agents described herein are considered to be pharmaceutically acceptable. PHMB, for example, is already used in, for example, wound dressings.

Further described herein is a composition or kit of parts for use in treating a patient in need of the introduced agent.

Further described herein is a composition or kit of parts for use in an imaging or diagnostic method. For example, as discussed above, the introduced agent may be an agent useful in imaging or otherwise detecting an intracellular component, for example a nucleic acid or protein. Thus, for example, the introduced agent may be an agent that binds specifically to an intracellular component. For example, the introduced agent may be an antibody or antibody fragment typically retaining specific binding affinity for a particular antigen, as well known to those skilled in the art; or a nucleic acid that hybridises with the required degree of specificity to a particular nucleic acid sequence. As will be appreciated, the imaging or diagnostic method is typically a medical imaging or diagnostic method and may typically be performed on the subject or may be performed on a sample obtained from a subject or may be performed on some other sample. For example, the sample may be a food or water sample or other environmental sample, as will be well known to those skilled in the art.

Thus, for example, an entry-promoting agent described herein and an introduced agent as defined above, for example a nucleic acid/analogue or nucleobase oligomer, may be used in the detection of an intracellular molecule, for example in the detection of a polypeptide or a nucleic acid sequence, for example by in-situ hybridisation, for example where the delivered nucleic acid is labelled with a fluorophore or other detectable molecule.

The kit of parts or composition described herein may further comprise a cell for receiving the introduced agent. The cell may be, for example, a cell useful in expressing a polypeptide whose expression is encoded or induced by the introduced agent.

The described entry promoting agent and delivered molecule may be provided together in a buffer having a high pH. Thus, the method for promoting entry of an agent (introduced agent) into a cell may comprise the step of exposing the cell to the introduced agent in the presence of an entry promoting agent (all as set out above) wherein the introduced agent and the entry promoting agent have been mixed or incubated at high pH, for example in a buffer having high pH. The term "high pH" will be well known to the skilled person and typically indicates a pH of above <NUM>, for example above <NUM> or above <NUM>, for example between <NUM> and <NUM>. The introduced agent and the entry promoting agent are mixed at high pH to form nanoparticles, before exposing the cell to the introduced agent in the presence of the entry promoting agent.

Specifically, buffers (with or without added salts, for example as commonly used in molecular biology buffers, for example PBS; NaCl; or many others) in the range of pH <NUM> - <NUM> are considered to provide formulations with improved transfection efficiencies (as shown in <FIG>). Resulting complex can be diluted <NUM>:<NUM> to <NUM>:<NUM> in a suitable growth medium, even complex can be added at several time points to cells (repeated multiple transfection) to achieve more efficiency. The procedure involves separate dilution of both the entry promoting agent and the delivered molecule in buffers with high pH and mixing them to form nanoparticles. Ratios and concentrations of the entry promoting agent and the introduced agent may be as discussed above in relation to preparation of a formulation and non-covalent complex; and in relation to the kit of parts.

For example, the following procedure can be used:
Dilute <NUM>-<NUM>µg of the entry promoting agent in buffer, for example <NUM>µl. Dilute <NUM> - <NUM>µg of plasmid DNA in for example <NUM>µl of buffer. Mix these two solutions and incubate, for example at room temperature for <NUM> minute to several hours. This mixture can then be used in transfection reactions, using well know methods. Add an appropriate volume of growth medium to the entry promoting agent / delivered molecule mixture, mix and add to cells growing in culture. Transfection will occur as the cell culture is incubated under appropriate conditions know to those working with cell culture.

Those skilled in the art will appreciate that high pH buffers can be easily prepared using, for example, NaOH or KOH. These buffer conditions provide improve transfection efficiencies when using typical complexation times, for example <NUM> minutes. Therefore, high pH buffers (and the entry promoting agents set out herein) can be easily incorporated into the protocols currently used by researchers.

Further provided herein is a method for preparing a complex comprising an entry promoting agent (for example PHMB) and an introduced agent as defined above, the method comprising incubating the entry promoting agent and the introduced agent in a complexation buffer at a pH of <NUM>-<NUM>. It is considered that nanoparticles are formed comprising the entry promoting agent (for example PHMB) and the introduced agent, for example oligonucleotide polymers (DNA, PNA, siRNA), proteins, peptides and small molecules. Specifically, formation of nanoparticles can be achieved by incubating PHMB and similar molecules as described above with oligonucleotides, proteins, peptides and small molecules in an appropriate buffer prior to use with cells. An appropriate incubation buffer may include water, PBS, and other buffers used commonly in laboratories. High pH buffers are described above. The optimal buffer may depend on the specific identity of both the entry promoting agent and the delivered molecule, as will be apparent to those skilled in the art. Nanoparticle formation and cell delivery typically is achieved by dilution of both partner molecules in complexation buffer prior to mixing the two components. Also, mixing of the two components typically is carried out prior to combination with other excipients or active ingredients and application to cells or use in vivo. Efficient nanoparticle formation is considered to occur within seconds or minutes but the procedure may be carried out over a number of hours. An appropriate ratio for efficient nanoparticle formation varies with different partner combinations. For example, <NUM>-<NUM>:<NUM> (wt:wt) for PHMB:plasmid DNA provides efficient nanoparticle formation. Examples are given above for PHMB:DNA combinations that result in nanoparticle formation. A person skilled in the art will be able to assess nanoparticle formation and delivery efficiencies when using different partner molecule ratios. Nanoparticle formation can be assessed in a number of ways. For example, an individual skilled in the art will be able to assess nanoparticle formation using dynamic light scattering (DLS) and microscopy methods.

Further described herein is a complex comprising an entry promoting agent (for example PHMB) and an introduced agent as defined above, wherein the complex is obtainable (or obtained) by a method comprising incubating the entry promoting agent and the introduced agent in a complexation buffer, for example at a high pH, for example at a pH of <NUM>-<NUM>.

The complexation buffer may in each case alternatively or in addition comprise a crosslinking agent, for example as set out in <FIG>, for example <NUM>,<NUM>-butanediol glycidylether or similar.

A method for promoting entry of an agent (introduced agent) into a cell, the method comprising the step of exposing the cell to the introduced agent in the presence of an entry-promoting agent, wherein the entry-promoting agent comprises a linear and/or branched or cyclic polymonoguanide/polyguanidine, polybiguanide, analogue or derivative thereof according to the following Formula 1a or formula 1b:
<CHM>
<CHM>
wherein:.

The method of paragraph <NUM> wherein the entry-promoting agent according to formula 1a comprises or is composed of one or more of the following.

or the entry promoting agent according to formula 1b comprises or consists of poly(allylbiguanidnio-co-allylamine), poly(N-vinylbiguanide), poly(allylcarbamoylguanidno-co-allylamine) or polyallybiguanide
<NUM>. The method paragraph <NUM> wherein the cell is a prokaryotic cell. The method of paragraph <NUM> wherein the cell is a eukaryotic cell, optionally in a tissue or organ. The method of any one of paragraphs <NUM>, <NUM> or <NUM> wherein the cell is a mammalian cell. The method of any one of paragraphs <NUM> to <NUM> where in the introduced agent comprises a nucleic acid or nucleic acid analogue. The method of paragraph <NUM> wherein the introduced agent comprises plasmid DNA. The method of paragraph <NUM> wherein the introduced agent comprises an RNAi molecule. The method of any one of paragraphs <NUM> to <NUM> wherein the introduced agent comprises a polypeptide, optionally a peptide or protein. The method of any one of paragraphs <NUM> to <NUM> wherein the introduced agent comprises a small drug, bioactive reagent or cellular imaging probe/contrast agent. The method of any one of paragraphs <NUM> to <NUM> where the introduced agent and the entry-promoting agent are covalently joined. The method of any one of paragraphs <NUM> to <NUM> wherein the introduced agent and the entry-promoting agent are provided as a formulation, for example as a non-covalent complex, optionally as nanoparticles. The method of any one of paragraphs <NUM> to <NUM> wherein the method is performed in vitro. The method of any one of paragraphs <NUM> to <NUM> wherein the method is performed in vivo. The method of any one of paragraphs <NUM> to <NUM> wherein the method is performed ex vivo. The method of paragraph <NUM> wherein the method is performed with from up to <NUM> fold molar excess of introduced agent over entry-promoting agent to up to <NUM> fold molar excess of entry-promoting agent over introduced agent. A method for making a target polypeptide, the method comprising the step of preparing the target polypeptide from a cell culture of a host cell, wherein the host cell is a host cell that has been transformed (or whose progenitor has been transformed) by an exogenous nucleic acid molecule so that the cell synthesises the target polypeptide, wherein the transfection comprises exposing the host cell to the exogenous nucleic acid molecule in the presence of an entry-promoting agent as defined in any one of paragraphs <NUM> to <NUM>. An entry-promoting agent as defined in any one of paragraphs <NUM> to <NUM> and an introduced agent as defined in any one of paragraphs <NUM> to <NUM> for use in treating a subject in need of the introduced agent
<NUM>. The use of an entry-promoting agent as defined in any one of paragraphs <NUM> to <NUM> and an introduced agent as defined in any one of paragraphs <NUM> to <NUM> in the manufacture of a medicament for use in treating a subject in need of the introduced agent. A method of treating a subject in need of an introduced agent as defined in any one of paragraphs <NUM> to <NUM>, the method comprising the step of treating the subject with an entry-promoting agent as defined in any one of paragraphs <NUM> to <NUM> and the introduced agent. The use of an entry-promoting agent as defined in any one of paragraphs <NUM> to <NUM> and an introduced agent as defined in any one of paragraphs <NUM> to <NUM> in the detection of an intracellular molecule, optionally by in-situ hybridisation, optionally where the delivered nucleic acid is labelled with a fluorophore or other detectable molecule. A kit of parts or composition comprising an entry-promoting agent and an introduced agent as defined in any one of paragraphs <NUM> to <NUM>, optionally comprising a complex wherein the complex is obtainable (or obtained) by a method comprising mixing or incubating the entry promoting agent and the introduced agent in a complexation buffer, for example at a high pH, for example at a pH of <NUM>-<NUM>. A composition or kit of parts for use in an imaging or diagnostic method. A pharmaceutical composition comprising an entry-promoting agent and an introduced agent as defined in any one of paragraphs <NUM> to <NUM>. A reagent composition an entry-promoting agent and an introduced agent as defined in any one of paragraphs <NUM> to <NUM>. Use of an entry-promoting agent as defined in paragraph <NUM> or <NUM> for promoting the cellular delivery of a bioactive substance. The method of any one of paragraphs <NUM> to <NUM> wherein the entry promoting agent and delivered molecule have been mixed or incubated or are provided together in a buffer having a high pH, optionally a pH of <NUM>-<NUM>. A method for preparing a complex comprising an entry promoting agent as defined in any one of paragraphs <NUM> to <NUM> (for example PHMB) and an introduced agent as defined in any one of paragraphs <NUM> to <NUM>, the method comprising mixing or incubating the entry promoting agent and the introduced agent in a complexation buffer, optionally at a high pH, for example at a pH of <NUM>-<NUM>.

The invention is now described in more detail by reference to the following, Figures and Examples.

We have assessed PHMB and related molecules as carriers for delivery into prokaryotic and eukaryotic cells. No single technology has yet emerged as a practical gene transfer method for the clinic. Desirable qualities of a carrier include:.

We considered that PHMB and related molecules may be able to act as effective carriers. Polyhexamethylenebiguanide (PHMB) is a cationic polymer with broad spectrum antimicrobial agent and less toxic to mammalian cells (Vantocil™ Arch biocides limited, UK). Low toxicity is an unusual and very beneficial property among delivery reagents and the low toxicity observed and safe track record of these compounds in a broad range of applications distinguishes this technology.

n = <NUM> to <NUM>, The weight-average molecular weight (Mw) and polydispersity for Bx529 VANTOCIL <NUM> are <NUM> and <NUM> respectively. Such a Mw corresponds to an 'n' value of around <NUM> (more precisely <NUM>). End groups: amine, guanidine and cyanoguanidine.

Polyhexamethylenemonoguanide (PHMG) is an analogue of PHMB with antimicrobial properties (Zhou et al.

We considered that PHMB may be useful as a carrier, either as a covalent conjugate or as a non-covalent complex, possibly with hydrophobic and/or electrostatic interactions. If this were to be the case, other properties of PHMB may enhance its use as a carrier. The toxicity profile of PHMB is well studied (Muller et al. , <NUM>; Kathryn V. Montague, <NUM>) and PHMB is considered to be a poor allergen (a desirable property) from tests in human patients (Schnuch et al. The antimicrobial properties may also be advantageous in reducing the problem of contamination in cell culture.

Uptake of free PHMB-FITC into three kingdoms was assessed:.

Evidence for interaction of PHMB with nucleic acids in vitro (Essential for a carrier to form a noncovalent complex with cargo and deliver it into cells).

Free PHMB enters into a wide variety of cells. PHMB interacts with nucleic acids. Can PHMB also carry nucleic acids and analogues? Yes: See <FIG>, <FIG>.

Are the delivered nucleic acids available for effective function? Yes: see <FIG> PHMB analogue also possess cell penetrating properties: see <FIG>
Effect on primary cells: See <FIG>.

Analogues: see <FIG> and <FIG>, (Note, the chemical formulae in <FIG> are adopted from <NPL>).

We have shown that PHMB and related cationic polymers can interact with small molecules (for example Nystatin, Rifampicin, curcumin, free FITC, SYTOX Green) and deliver them into mammalian cells, for example HeLa. See <FIG>, <FIG>. Also, we observed synergistic bacterial killing using PHMB/rifampicin complexes that were formed prior to exposure to bacterial cells.

PHMB and analogues were able to deliver protein molecules (for example R-phycoerythrin or alexa <NUM> conjugated IgG antibodies) into mammalian cells such as HeLa cells, see <FIG>.

Our method shows that higher transfection efficiency of nucleic acids can be achieved when carrier and cargo are complexed in buffers with high pH (<NUM>-<NUM>). We tested the amount of time required for complex formation between PHMB and nucleic acids, Our results show that as little as five minutes is sufficient to form complex which can enter mammalian cells or even allowing the complex to stay for hours at room temperature will not compromise the transfection capacity.

We have assessed PHMB and related molecules as a means to form nanoparticles with a range of bioactive molecules. No single technology has yet emerged as a practical nanoparticle formation method for the clinic.

Desirable qualities of a carrier include:.

We observed while investigating the cell delivery properties of PHMB and related molecules that these polymers are able to effectively form nanoparticles with a range of molecules, many of which are bioactive. The use of bioactive molecules may benefit from inclusion within nanoparticles prior to further formulation and application. Such benefits could include improved solubility, improved stability, protection against degradation during use and improve or altered distribution or clearance properties in vivo. Low toxicity is an unusual and very beneficial property among nanoparticle forming reagents and the low toxicity observed and safe track record of these compounds in a broad range of applications over several decades of wide usage distinguishes this technology.

Evidence for interaction of PHMB with nucleic acids in vitro, which is essential for a carrier to form a noncovalent complex and nanoparticle with cargo molecules and deliver it into cells.

Dynamic light scattering (DLS) and fluorescence microscopy were used to measure the formation of nanoparticles. Evidence of nanoparticle formation was assessed using a range of molecules.

Claim 1:
An in vitro or ex vivo method for promoting entry of an agent to be introduced into a cell, the method comprising the step of exposing the cell to the agent to be introduced in the presence of an entry-promoting agent, wherein the entry-promoting agent is selected from the group consisting of: polyhexamethylene biguanide (PHMB), polyhexamethylene monoguanide (PHMG), polyethylene biguanide (PEB), polytetramethylene biguanide (PTMB), polyethylene hexamethylene biguanide (PEHMB), polymethylene biguanides (PMB), poly(allylbiguanidnio-co-allylamine), poly(N-vinylbiguanide) or polyallybiguanide, wherein the agent to be introduced is selected from the group consisting of:
a) an oligonucleotide, plasmid or other vector DNA or an RNAi molecule; or
b) a polypeptide, optionally a peptide; or
c) a small drug having a molecular weight less than <NUM> Da, bioactive reagent or cellular imaging probe/contrast agent; and
wherein the agent to be introduced and the entry-promoting agent are provided as nanoparticles consisting of the agent to be introduced and the entry-promoting agent, where the nanoparticles are formed at a pH of <NUM>-<NUM>.