Methods for promoting entry of an agent into a cell

Inventors

Good, Liam • Chindera, Kantaraja • Gburcik, Valentina

Assignees

Tecrea Ltd

Abstract

A method for promoting entry of an agent (introduced agent) into a cell, the method comprising the step of complexing the introduced agent in the presence of an entry-promoting agent and then exposing to cells, 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 & b. The method also provides a means for formation of nanoparticles formed between the entry promoting agent and the introduced agent. wherein: “n”, refers to number of repeating units in the polymer, and n can vary from 2 to 1000, for example from 2 or 5 to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800 or 900; G1 and G2 independently represent a cationic group comprising biguanide or guanidine, wherein L1, and L2 are directly joined to a Nitrogen atom of the guanide; L1 and L2 are linking groups between the G1 and G2 cationic groups in the polymer and independently represent an aliphatic group containing C1-C40 carbon atoms, for example an alkyl group such as methylene, ethylene, propylene, C4, C5, C6, C7, C8, C9 or C10; C1-C10, -C20, -C30, -C40, -C50-C60 -C70, -C80, -C90, -C-100, -C110, -C120, -C130 or -C140, alkyl; or a C1-C140 (for example C1, C2, C3, C4, C5, C6, C7, C8, C9 or C10; C1-C10, -C20, -C30, -C40, -C50 -C60, -C70, -C80, -C90, -C100, -C110, -C120, -C130 or -C140), cycloaliphatic, heterocyclic, aromatic, aryl, alkylaryl, arylalkyl, or oxyalkylene radical; or a polyalkylene radical optionally interrupted by one or more, preferably one, oxygen, nitrogen or sulphur atoms, functional groups or saturated or unsaturated cyclic moiety: N and G3 are optional end groups; X can be either present or absent; L3, L4 and X are linking groups between the G4 and G5 cationic groups in the polymer and independently represent an aliphatic group containing C1-C140 carbon atoms, for example an alkyl group such as methylene, ethylene, propylene, C4, C5, C6, C7, C8, C9 or C10; C1-C10, -C20, -C30, -C40, -C50 -C60, -C70, -C80, -C90, -C100, -C110, -C120, -C130 or -C140, alkyl; or L3 and L4 and X can independently be C1-C140 (for example C1, C2, C3, C4, C5, C6, C7, C8, C9 or C10; C1-C10, -C20, -C30, -C40, -C50 -C60, -C70, -C80, -C90, -C100, -C110, -C120, -C130 or -C140), cycloaliphatic, heterocyclic, aromatic, aryl, alkylaryl, arylalkyl, oxyalkylene radicals; or 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; “G4” and “G5” are cationic moieties and can be same or different, and at least one of them is a biguanidine moiety or carbamoylguanidine, and the other moiety may be biguanidine or carbamoylguanidine or amine; and cationic moieties G4 and G5 do not contain single guanidine groups. The entry-promoting agent may comprise homogeneous or heterogeneous mixture of one or more of agents arising from formulae 1 a and b, for example 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.

Core Innovation

The invention relates to a method for promoting entry of an introduced agent into a cell by exposing the cell to non-covalently complexed nanoparticles comprising the introduced agent and an entry-promoting agent. The entry-promoting agent comprises polyhexamethylene biguanide (PHMB), and the introduced agent and the entry-promoting agent are mixed together at a pH of 10–13.5.

The document describes cationic polymonoguanide/polyguanidine polymers, including PHMB and related biguanidine/guanidine polymers, forming non-covalent nanoparticles with an introduced agent. It further describes that nanoparticles are characterized and that complexation can occur rapidly and form stable complexes.

The introduced agent is stated to include nucleic acids and nucleic acid analogues, proteins and peptides, and small molecules. The document states that cellular entry is promoted across multiple cell types including prokaryotic, fungal, plant, insect/protist, and mammalian cells, while reporting low cytotoxicity relative to a referenced transfection reagent.

Claims Coverage

This patent document includes one independent claim directed to a method of promoting cellular entry using non-covalently complexed nanoparticles containing an introduced agent and PHMB, with mixing at pH 10–13.5. The dependent claims refine the independent claim by specifying cell categories and categories of introduced agents, and by adding constraints on how the introduced agent and entry-promoting agent are combined.

The claim set is centered on PHMB-containing, non-covalently complexed nanoparticles and mixing the introduced agent with the entry-promoting agent at pH 10–13.5 to promote entry into cells. Dependent claims further narrow the method by specifying mixing or combination conditions and by limiting the method to particular cell categories and classes of introduced agents, including nucleic acids and nucleic acid analogues.

Stated Advantages

Documented Applications