FIELD OF THE INVENTION

The present invention is related to the compounds and methods having entry promoting (broadly referred as drug delivery) properties. It particularly relates to entry promoting agents for delivery of proteins, polypeptides and analogues; nucleic acids and analogues; small molecules (often referred to as drugs including antimicrobials) with applications in research or medicinal/therapeutic use. More specifically it relates to natural polymers or peptides or polypeptides or proteins or synthetic biopolymers decorated with biguanidine. It also relates to the method of synthesis/modification of biomolecules with biguanidine. Further aspect of the invention relates to the compositions and methods for entry promoting (delivery) of therapeutics or prophylactics or imaging agents or agents used in research and therapy.

BACKGROUND OF THE INVENTION

The present invention relates to the field of methods and biocompatible reagents for promoting entry of an agent, for example a nucleic acid, analogues or protein or small molecules into a cell. Entry promoting agent is described as a method that promotes/facilitates entry of a cell impermeable or poorly cell permeable agent into the cell (intracellular compartment), often referred to as drug delivery molecules/agents.

Biomolecules such as proteins, peptides, polypeptides and analogues, small molecules (drugs), nucleic acids and analogues, synthetic compounds, analogues and derivatives thereof (often referred to as cargo molecules in delivery) have many applications in both prophylactic and therapeutic interventions and also in basic research and drug development process and as diagnostics. However, majority of the prokaryotic and eukaryotic cells are primarily impermeable to above listed molecules/derivatives or analogues, including synthetic compounds used for research and prophylactic/therapeutic applications. Delivery of the above mentioned molecules types into a cell are very important to achieve desired target functions both in-vitro and in-vivo. To achieve a maximum functional effects, the vehicle/cargo complexes has to overcome many extracellular and intracellular hurdles such as extracellular cargo degradation, cell membrane barrier which prevents entry into a cell and unloading or release of cargo molecules inside a cell (often involving endosome escape) for effective function with least cytotoxic effects. Additionally, it is desired to use a delivery vehicle, which is biocompatible, biodegradable, and non hazardous to the public health. There is a dire need for a biocompatible, efficient, nontoxic assisted delivery vehicle.

Delivery of the above listed cargo molecules is achieved by using assisted delivery methods, which are broadly grouped into viral (example; lentiviral, retroviral or adenoviral method for nucleic acid delivery, but not suitable for small molecules or protein delivery) and non-viral delivery (lipids/liposomes, carbohydrates, peptides and other synthetic polymers) methods. Example of assisted delivery, non-viral methods: lipid reagents (e.g. liposomes or lipofectamine, lipofectamine 2000) or cationic polymers (e.g. PEI, Chitosan, Polyhexamethylene Biguanide) (WO2013054123 Al, US 5,958,894 WO 02/22174, WO 2009/015143, WO2010/086406). Viral methods; Retroviral or lentiviral or adenoviral vectors for shRNA expression or over expression of target genes.

Though some of the entry promoting agents are not biocompatible and not easily biodegradable (example; Polyhexamethylene biguanidine used in the patent WO2013054123 Al, considered poorly biodegradable (O'Malley LP et al., J Ind Microbiol Biotechnol. 2006 Aug;33(8):677-84. Epub 2006 May 9.) and raises concern on safety (EU 2014 report). A recent European commission ruling that PHMB may be banned for cosmetic applications due to inhalation toxicity (SCCS/1535/14,Revision of 16 December 2014, Scientific Committee on Consumer Safety; SCCS; opinion on the safety of poly (hexamethylene) biguanidine hydrochloride (PHMB http://ec.europa.eu/health/scientific_committees/consumer_sa fety/docs/sccs_o_157.pdf) and is also considered as class 2 carcinogen (Care. 2 H351 - Suspected of Causing Cancer). Hence necessitating alternative novel biodegradable biguanidine based delivery molecule. There are few biocompatible and biodegradable entry promoting agents exists (such as chitosan and chitosan derivatives, other carbohydrate derivatives such as modified hyaluronic acid or dextran) mainly based on amine, imine or guanidine. Though modifications in these carrier molecules are made, poor efficiency and solubility issues are major drawbacks (example; chitosan is poorly water soluble and efficiency is poor). Also majority of the biomolecules are not carriers as such; such as dextran, cellulose, other neutral carbohydrates (bearing no charge; example starch, glycogen, pullulan etc.,), cannot be used as entry promoting agent. It is desirable to modify them with functional groups to enable them to have entry promoting properties.

Majority of the biodegradable cationic molecules are based on active cationic moieties such as amine, imine, imidazole and guanidine molecules. In spite of modifications of biomolecules with above cationic groups, still solubility, efficiency and toxicity remain a major hurdle. There is a huge demand for alternative entry promoting agents or invention of existing biomolecules with new cationic group that would improve their performance.

Modification of existing biomolecules with new cationic groups could confer them with unique properties suitable for improving drug delivery. The present invention identifies decoration of existing class of natural compounds or synthetic polymers with a new cationic moiety (biguanidine) that imparts them or improves their carrier/entry promoting properties. Variety of synthetic molecules having biguanidine has been synthesized for antidiabetic, antimalarial and anti-infective applications, such as metformin, chlorhexidine, alexidine, polybiguanides (polyhexamethylene biguanides; PHMB). (Baker et al., 1987; Rose and Swain, 1956). (Baker et al., 1987).

The present invention describes use of biguanidine-grafted molecules as entry promoting agent. Novel, biguanidine derived molecules described in the present invention have several unique advantages over existing molecules/modifications. Biguanidine offers potent bidentate hydrogen bonding and electrostatic interaction with introduced agents. Such entry-promoting agents and methods are considered to bring added beneficial advantages, for example in providing efficient entry promotion and biocompatibility, biodegradability or low cytotoxicity into a cell. Such entry -promoting agents and methods may, for example, be useful in delivering molecules such as nucleic acids/analogues (often referred to as transfection; for siRNA, miRNA, shRNA, mRNA and pDNA), small molecules (broadly classified as drugs, including antimicrobials; antibacterials, antifungals, antivirals), imaging agents, proteins polypeptides and peptide and derivatives for in-vitro or in-vivo use (for example functional studies; overexpression of a gene of interest, establishing stable cell lines; target gene silencing; and DNA vaccination or treatment of a microbial infection or production of a polypeptide or protein), such applications are easily appreciated by those skilled in the art.

Chitosan is a well-known biopolymer widely explored for variety of applications including as an entry promoting agent (US6184037 B l, US5840341 A). But its solubility and poor performance is a major hurdle. Similarly, other biopolymers such as hyaluronic acid, synthetic molecules such as polyethylene glycols, polyethylene imines (US6013240 A, European Patent 0,770140, US 8318856 B2) are some of the listed biocompatible polymers used as entry promoting agent. Also variety of amine containing natural (example; poly lysine) or synthetic polymers (example; polyethyleneimine or polyallylamine) are used as entry promoting agents. Though many modifications (such as addition of amine, guanidine or cell receptor targeting molecules) in some of the above listed polymers have aided in improving delivery, but not substantially. The present inventor has surprisingly found that use of biguanidine substituted biopolymers or addition of biguanidine moiety in the existing biopolymers (as listed above) or derivatives or analogues thereof has substantially improved solubility, and entry promoting activity and, in some instances this modification added/imparted entry promoting property to modified/existing molecules with no or low cell penetrating property. It was surprisingly observed that these derivatives are useful for delivering variety of cargo molecules into cells. Therefore, the present inventor successfully developed the commercially applicable entry promoting agents which are biguanidine substituted biopolymers along with a suitable method for promoting entry of an agent (introduced agent) into a cell (prokaryotic or eukaryotic) for prophylactic or therapeutic or diagnostic purposes. Mentioning or listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

OBJECTS OF THE INVENTION

The primary object of the present invention is for providing the compounds having entry promoting properties.

The object of the present invention is for biguanidine grafted biodegradable natural polymers with entry promoting properties.

The object of the present invention is for biguanidine grafted natural or synthetic polymers with enhanced entry promoting properties.

Another object of the present invention is for the synthesis of biguanidine-grafted entry promoting agents. Another object of the present invention is for the composition of biguanidine grafted biodegradable biomolecules and their derivatives for delivery of prophylactic or therapeutic or diagnostic or research reagents or molecules.

Further object of the present invention provides a method for promoting entry of prophylactic or therapeutic or diagnostic or research reagents or molecules into a eukaryotic or prokaryotic cells.

STATEMENT OF THE INVENTION

Entry-promoting agent comprises a linear and/or branched or dendrimeric; natural biopolymers such as carbohydrates, protein, polypeptides or peptides; synthetic or semisynthetic polymer having biguanidine moieties. The entry-promoting agent is selected from the group of biguanidine substituted linear and/or branched/cross linked derivatives of natural biopolymers such as carbohydrates including modified carbohydrates or analogues such as chitosan, dextran, pollulan, schizophyllan, inulin, cellulose, and derivatives, hyaluronic acids, alginic acids, protein such as histones. lysine rich proteins, peptide/polypeptide such as polylysine (CAS number 25988-63-0; 27964-99-4; 61686-25-7), e-polylysine (CAS number; 25104-18-1 ), polyornithine (CAS number 27378-49-0; 82682- 33-5; 82682-33-5), Poly diaminopropionic acid, polyspermine, polyspermidine, polyanionic acids such as hyaluronic acid or alginic acid, polyaspartic acid, polyglutamic acid, polybutyric acid, and synthetic biopolymers such as polyallylamine, PEI analogue or derivative thereof, amine derivatised PEG dendrimers, amine bearing dendrimers, such as poly(amidoamine) (PAMAM). The degree of biguanidine substitution varies from 0.01 to 100%. The entry promoting agent is selected from the group of chitosan Biguanide, Dextran biguanide, Pollulan biguanide, cellulose biguanide, biguanidylated biguanide, biguanidylated polyornithine, biguanidylated polyornithine, biguanidylated Poly(L-diaminopropionic acid), biguanidylated e-polylysine, biguanidylated polyornithine, biguanidylated (Poly(gamma-L- diaminobutanoic acid), biguanidylated histones, biguanidylated spermine, biguanidylated spermidine, biguanidylated polyspermine, biguanidylated polyspermidine, biguanidylated polyethyleneimine, biguanidylated polyallylamine, biguanidylated alginate, biguanidine cross linked chitosan, Biguanidine cross linked polyethyleneimine linear, Biguanidine cross linked polyethyleneimine branched, Biguanidine cross linked polylysine, Biguanidine cross linked polyornithine.

A method for promoting entry of an agent and introduced agent into a cell comprising the step of exposing the cell to the introduced agent in the presence of an entry-promoting agent as described above. The introduced agent is selected for the group of nucleic acids such as pDNA, DNA, miRNA or RNAi molecule or analogues; peptides, polypeptides, proteins, small drug molecules including antimicrobials such as antibacterials and antifungals, bioactive reagents and cellular imaging probe/contrast agents. The entry promoting agents are used for assisted delivery into a prokaryotic cell or eukaryotic cell. The method is performed in vitro or in vivo or ex vivo. The method is performed with up to 1000 fold molar or weight excess/excess weight of introduced agent over entry-promoting agent, through using an equal molar concentration of carrier and cargo molecules, to up to 1000 fold molar excess of entry- promoting agent over introduced agent.

A method for making a target polypeptide/protein 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 is carried by exposing the host cell to the exogenous nucleic acid molecule in the presence of an entry- promoting agent.

The use of an entry-promoting agent and an introduced agent 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 comprising the step of treating the subject with an entry-promoting agent and the introduced agent.

A pharmaceutical composition comprising entry -promoting agent and introduced agent as defined.

A reagent composition comprising entry-promoting agent and introduced agent. BRIEF DESCRIPTION OF FIGURES

Figure 1. Confirmation of biguanidine grafting on the biopolymers using UV spectra:

Biguanidylated Chitosan, dextran and polylysine were prepared by reacting with cyanoguanidine under reflux condition as described, purified by dialysis in lkDa cut-off membrane and lyophilised. The resulting compound was dissolved in water, O. lmg/ml, and UV absorbance is recorded. Presence of biguanidine group in the resulting polymer derivative is seen as a peak in absorbance at 230nm, indicating successful grafting of biguanidine on the polymer.

Figure 2: Efficient delivery of plasmid DNA into mammalian cells

Production of a protein/polypeptide in a target cells: Complexes of entry promoting agent (3 μg) and introduced agents (plasmid DNA encoding Green fluorescent protein, pEGFP-cl, 1 μg), were prepared in OPTIMEM medium (100 μΐ), by incubating at room temperature for 30 minutes. The resulting complexes were added on to a culture of 293t cells grown on 12 well plates (transfection). The cells were examined 48 hours after transfection under an inverted fluorescence microscope (Leica), images were recorded. Appearance of green fluorescent cells indicates successful delivery of the introduced agent into cells. Examples of use of biguanidine-grafted chitosan, dextran and PEI are shown. b. Production of recombinant virus: The entry promoting agents described in the present invention are used to produce recombinant lentivirus or retrovirus. Complexes of entry promoting agent (30 μg) and introduced agents (Lenti-CMV-GFP-2A-Puro Vector (5 μg), 3 μg envelope vector pCMV-VSVG, 2 μg packaging vector pCMV dR 8.2) were prepared in lml OPTIMEM, by incubating at Room temperature for 30minutes. The complexes were added on to a culture of 293t cells grown in a 60mm dish. Following 24 hours post transfection, medium (10ml) was changed, at 48 hours supernatant was harvested (48hour virus pool). Again 10ml fresh medium was added, and collected at 72 hours time point (72 hour Virus pool). The virus titre was quantified by ELISA and fold change in virus titer compared to unmodified polymer is shown. The virus pool was used to infect HeLa cells, efficient virus transduction is confirmed by number of GFP positive cells. Indicating the potential use of the entry promoting agents in producing recombinant therapeutic viruses.

Figure 3: Efficient delivery of siRNA into mammalian cells: Complexes of entry promoting agent (3 μg) and introduced agents (siRNA targeted to Vimentin, 20nM Final concentration in the medium), were prepared in OPTIMEM medium (100 μΐ), by incubating at room temperature for 30 minutes. The resulting complexes were added on to a culture of HeLa cells grown on 6 well plates (transfection). Total RNA was harvested 48 hours post transfection and relative levels of vimentin mRNA were quantified by Real Time PCR, using beta actin as reference gene. A drastic reduction in the target mRNA indicates successful delivery of the introduced agent into cells.

Figure 4: Efficient delivery of protein into mammalian cells: Complexes of entry promoting agent (3 μg) and introduced agents (0^g FITC labelled 15mer random peptide or R- Phycoerythrin, were prepared in OPTIMEM medium (100 μΐ), by incubating at room temperature for 30 minutes. The resulting complexes were added on to a culture of HeLa cells grown on 12 well plates (transfection), and incubated for 2 hours. Following incubation cells were processed and observed under a fluorescent microscope. The number of transfected cells was quantified by flow cytometry.

Figure 5: antimicrobial and antifungal delivery: Complexes of entry promoting agent (3 μg) and introduced agents (Ιμΐ of ΙΟΟμΜ berberine) were prepared in PBS (100 μΐ), by incubating at room temperature for 30 minutes. The resulting complexes were added on to a culture of Candida albicans in 200ul volume, and incubated for 2 hours. Following incubation cells were processed and observed under a fluorescent microscope. The number of transfected cells was quantified by flow cytometry using GFP filter. Similarly complexes of entry -promoting agent/gentamycin were prepared. HeLa cells were infected with Salmonella enterica serovar typhimurium or Staphylococcus aureus, and their presence inside the cells was confirmed by DAPI staining and fluorescent microscope. Infected HeLa cells were treated with entry promoting agent/antibiotic complexes and the efficacy of intracellular bacterial killing was confirmed 24 hours post treatment by colony formation assay. Percentage reduction in the colony formation is represented to assess reduction in intracellular load.

Figure 6. Delivery of a DNA probe into mammalian cells: Complexes of entry promoting agent (3 μg) and introduced agents (Ιμΐ of ΙΟΟμΜ FITC labelled 30mer random oligonucleotides) were prepared in OPTEVIEM medium (100 μΐ), by incubating at room temperature for 30 minutes. The resulting complexes were added on to a culture of HeLa cells grown on 12 well plates (transfection), and incubated for 2 hours. Following incubation cells were processed and observed under a fluorescent microscope. The number of transfected cells was quantified by flow cytometry.

Figure 7. Formation of Nanoparticles in entry promoting agent/introduced agent complexes: A mixture of entry promoting agent/introduced agent were prepared as described above, and the mean particle size was measured by dynamic light scattering using Malvern zetasizer.

Figure 8: Low cytotoxicity of entry promoting agents: HeLa cells in 96 well plates (at 25% confluence) were treated with a range of concentrations of entry promoting agents or complexes (as prepared above) for 72 hours. MTT reagent was added, and formation of formazan crystal was quantified.

DETAILED DESCRIPTION OF THE INVENTION

A first aspect of the invention provides 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 or complexes comprising the polymer and cargo molecules, wherein the polymer comprises a biguanidine/biguanide grafted linear and/or branched/cross linked derivatives of natural biopolymers (such as carbohydrates (oligo or polysaccharides)/modified carbohydrates or analogues (e.g chitosan, dextan, pollulan, schizophyllan, inulin, cellulose, and derivatives, hyaluronic acids, alginic acids etc.), protein (e.g.histones. lysine rich proteins) peptide/polypeptide (polylysine (CAS number 25988-63-0; 27964-99-4; 61686-25-7), e- polylysine (CAS number; 25104-18-1 ), polyornithine (CAS number 27378-49-0; 82682-33-5; 82682-33-5), Poly diaminopropionic acid, polyspermine, polyspermidine, polyanionic acids (such as carbohydrates (hyaluronic acid or alginic acid) polyaspartic acid, polyglutamic acid, polybutyric acid)), and synthetic biopolymers (such as polyallylamine, PEI analogue or derivative thereof, Amine derivatised PEG dendrimers, amine bearing dendrimers, such as poly(amidoamine) (PAMAM) etc.)).

The inventor of the present invention has surprisingly found thai when a biomolecule or synthetic molecule (as mentioned above) are decorated or grafted with biguanidine, they have an excellent solubility, low toxicity and entry promoting properties over parent molecule. The present invention describes use of all the above listed class of biguanidine substituted biomolecules as entry promoting agents.

The biomolecules described in the present invention have at least an oligomer of 2 units, and has a biguanidine substitution from 0.01- 100%.

Biguanidine (Formula I) is a cationic molecule at physiological pH, in addition to electrostatic interactions, biguanidine participates in potent bidentate hydrogen bonding. These features add a unique property which earlier groups/molecules describes in the prior art does not have.

Formula I. Chemical structure of a biguanidine

BG represents a cationic group comprising biguanide/Biguanide. The biguanidine are added as a decorative agent, in the polymer, either directly added or as a part of another intermediate (through a linker). Structure of biguanidine-grafted biopolymers are described below.

Biguanic!ylated(biguanic!ine-grafted)protein/poIypeptide/ peptides;

Proteins/polypepiides/peptides/synthetic analogues described in the present invention refers to lysine rich proteins/polypeptides/peptides of natural or synthetic origin (with amine in the side chain or terminal end), where amine is converted to biguanidine. Example of such peptides are, but not limited to (polylysine (CAS number 25988-63-0; 27964-99-4; 61686-25- 7), e-polylysine (CAS number; 25104-18-1 ), polyornithine (CAS number 27378-49-0; 82682-33-5; 82682-33-5) or analogues as described in the general formula below. Such peptides/polypeptide can be linear or branched or dendrimeric form.

X refers to amino acid lysine or lysine analogues or synthetic amino acid analogues with terminal amine.

Y refers to any amino acid or amino acid derivatives, Y can be present or absent,

n ; 0-1000; b; 1-1000

When Y is absent (n=0), the peptide is a homogenous peptide (e.g. polylysine or polyornithine or analogue).

Peptides can be in a lineal- form or multiple antigenic format (MAP) or dendrimeric form as well known to those skilled in the art.

When the terminal amino group of X is converted to biguanidine (either post synthesis modification or during peptide synthesis or using biguanidylated amino acid analogue), then the above formula is represented as

Where BG stands for biguanidine.

When both X and Y are biguanidylated (example if Y is a lysine or lysine analogue or amino acid analogue bearing terminal amine group. Amine is substituted with biguanidine) as shown below. Then the modified peptide is re resented by the below formulae

In some instance, biguanidine attached to Y is absent, if the amino acid at Y is non amine bearing amino acid/analogue. Degree of biguanidine substitution may vary from 0.01-100%. Hence this would give a peptide/polypeptide with amine, guanidine and biguanidine depending on the nature of X and Y. Bui at least has biguanidylated lysine or lysine analogues.

The biguanidine-modified peptides are usually lysine rich peptides, where amine group of lysine is converted to biguanidine. Such biguanidine-modified peptides can be synthesised on solid phase peptide synthesis or modified post synthetically or using natural peptide or polypeptide or protein.

The invention also covers the biguanidine modified polylysine or analogues (alpha or epsilon polylysine). In general polylysine or analogues are represented by the below formula (example but not limited to; Polylysine - CAS number 25988-63-0; 27964-99-4; 61686-25-7 or polyornithine (CAS number 27378-49-0; 82682-33-5; 82682-33-5),

0

n = 0 - 10

b= 2 - 500

When n = 4, the compound is lysine derivative

When n = 3, the compound is ornithine derivative

Polylysine can be in a linear form or dendrimeric form as well known to those skilled in the art. The inventor has shown that amine in the lysine side chain is replaced/substituted into a biguanidine containing bio peptide analogue (as described in the formula below) with improved entry promoting activity. Some of the biguanidine-derived polypeptides are represented in the formula below.

Where n = 0 - 10; b= 2 - 500. When n =4, then it will be a biguanidine derivative of polylysine

n in above listed structure can be as high as 500. In some cases the polypeptide is an epsilon Polylysine (e-polylysine) represented by the formula below, e-polylysine (CAS number; 25104-18-1).

Alternatively, it can be represented as

where n = 2-1000.

The biguanidine derivative of e-polylysine can be represented in the formula below.

Similarly, Poly(L-diaminopropionic acid) is represented

The biguanidine substituted derivative will have below structure

Other example of amine bearing peptide analogues (Poly(gamma-L-diaminobutanoic acid) or (Poly(gamma-L-diamino ropionoic acid) or analogue

Amines in such peptide are easily converted to biguanidine.

In cases where, not all the amine group in the above peptides are replaced by biguanidine, then the resulting polymer is a mixture of amine and biguanidine (at least 0.01% of the amine are substituted with biguanide). Also biguanidylated natural polyamines are covered (histones, spermine, spermidine, polyspermine, poly spermidine).

In some instances, anionic peptide/polypeptide are converted to biguanidylated derivatives. Example of such anionic polypeptide is poly glutamic acid.

Biguanidylated Carbohydrate analogues:

The carbohydrates mentioned in the present invention are usually referred to a oligomeric/polymeric carbohydrate (often referred to as oligosaccharides or polysaccharides) with a molecular weight of at least lOOODa upto TOOOkDa. Such carbohydrate polymers are well known to those skilled in the art. These molecules are broadly classified into a group, which does not have entry promoting agent or have inherent entry promoting agent. Those skilled in the art appreciate that there are several such natural carbohydrate polymers exist. Some of the example (but not limited to) of carbohydrate polymers without entry promoting property are; dextran, amylose, amylopectin, schizophyllan, glycogen, inulin, pullulan, ficoil, cellulose, hemicellulose (xylan, glucuronoxylan, arabinoxylan, glucomannan, and xyloglucan), galacto mannan, arabinogalactan, droxyethyl cellulose, hydroxypropyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl methyl cellulose, hydroxyethyl carboxymethyl cellulose and mixtures, sinistrin/ polyfructosane, anionic polysaccharides (such as alginate or heparin, etc.)

Some of the example (but not limited to) of carbohydrate polymers with inherent entry promoting property are chitin/chitosan, cationic guar gum, starch, dextran/derivatives etc. In one embodiment, the invention describes biguanidine modified cationic carbohydrates, where in the carbohydrate is a chitosan/analogue/derivatives. Those skilled in the art appreciate that chitosan is a naturally occurring cationic polysaccharide, which is used as entry promoting agent, with solubility issues and poor performance.

The present invention describes biguanidine substituted chitosan as a novel entry promoting agent.

Deacetylated chitosan is presented by the formula

Simplified version of chitosan is represented as

The present invention describes the use of biguanidine-grafted chitosan as a new and novel entry promoting agent represented by the following formula

Synthesis of Chitosan biguanidine for other use (antimicrobial applications) has been described in the literature (S20070281904, Japanese Patent 60233102, Chinese patent CN 1944467 A).

general biguanidine modified chitosan is represented as below

X; Can be a biguanidine directly attached to sugar moiety

X; Can be present as a part of the side chain attached to sugar moiety

X; Can be a biguanidine/s group attached to sugar moiety through a linker a described below.

A can be biguanidine

A can be an alkyl chain ending with biguanidine/s as described below.

X can be any molecule as described in below structure.

In some instances both A and B are biguanidine or alkylbiguanidine

In some instances; A is a alkyl group or amine or guanidine and B is a biguanidine moiety, which can be a part of alkyl chain. In some instance; A is a is a biguanidine moiety, which can be a part of alkyl chain and B is an alkyl substituted with amine or guanidine. In some instance a mixture of amine and biguanidine substituted chitosan or a mixture of amine and guanidine and biguanidine substituted chitosan are possible.

Example of such groups are shown below. Molecular weight of biguanidine substituted chitosan vary from 5kDa to 1000 kDa.

When A is guanidine and B is propylbiguanidine (or analogues), then X is represented as below

In another embodiment, the present invention relates to synthesis of biguanidine decorated carbohydrate polymers (but not limited to), such as dextran, pollulan with biguanidine substituted carbohydrates is shown as an example below using dextran (but applies all other carbohydrates). Such modification confer a new entry promoting properties.

Generally, dextran is a neutral polymer of glucose without inherent entry promoting agent (example; dextran as such is unable to delivery nucleic acids), generally represented as below

wherein n; 2 - 160,000

Biguanidylated dextrans (molecular weight lkDa to 2,800kDa) can be prepared in the laboratory. First dextran is oxidised, then reacted with an amino acid/analogue, spermine, alkylamine, alkyldiamine or analogues. The amine derivative dextran analogue (e.g. amino dextran) is converted to biguanidine, either linked to sugar moiety directly (biguanidylating dextran amine or reacting oxidised dextran with aminoalkylbiuganidine or analogue) or through a linker (example biguanidylation of dextran-spermine analogue). A variety of dextran derivatives can be synthesised based on the nature of oxidation and amine substitution, followed by biguanidylation. Example, dextran is amine substituted as below (Journal of Neuroscience Methods, 1994, 51(1):9-21). Then amine groups are converted to biguanidine.

The present inventor has surprisingly shown that biguanidine decorated dextran possess entry promoting properties. Some of the dextran biguanidine are represented by the formulae below.

When amine groups are reacted with biguanidine transfer reagent or cyanoguanidine, then the biguanidine decorated polymer is represented as

stands for biguanidine.

The biguanidylated version is created by reacting amino substituted dextran/analogues or other polysaccharide with cyanoguanidine at >100 °C under reflux conditions.

Alternatively dextran biguanide agents are represented as below

Where X is a biguanidine directly attached to dextran or a linker bearing biguanidine at the end, as shown in some of the structures above.

In some instance, not all the amine group in the carbohydrates are replaced by biguanidine, hence the resulting polymer is a mixture of amine and biguanidine.

Similarly other uncharged carbohydrates are converted to biguanidine derivatives using similar approach (first oxidising and then derivatizing directly or indirectly).

Similarly biguanidylated anionic carbohydrates can be synthesised. Example of biguanidylated alginate is shown below.

Carboxyl groups in alginic acid can be easily converted into a biguanidine derivatives by first converting into a suitable amine derivative. Structure of amine grafted alginic acid is shown below.

then reacting amine derivative (amino acid analogue or alkylamine/diamine analogue) with cyanoguanidine . Either single or both carboxyl groups can be converted to biguanidine bearing molecules.

Where X is a biguanidine attached directly or through a linker

Another example of biguanidylated alginic acid is shown below

Biguanidylated synthetic polymers: The invention also describes biguanidine substituted synthetic polymers; such as biguanidylated polyethyleneimine or amine derivitised PEG dendrimers, amine bearing dendrimers, polyaminodendrimers such as poly(amidoamine) (PAMAM) etc.).

Branched polyethyleneimine can be represented by the formulae below

or

Linear olyethyleneimine is represented by the formulae below.

Where primary or secondary amine in PEI is converted to biguanidine by reacting with cyanoguanidine under reflux conditions. Such compounds offer more potent bidentate hydrogen bonding and less toxicity compared to native PEI.

Not all the amine group in the PEI are replaced by biguanidine, hence the resulting polymer is a mixture of amine and biguanidine, biguanidine substation ranges from 1% to 100%.

Similarly other biguanidylated entry promoting agents are biguanidine substituted polyallylamine, poly(amidoamine) (PAMAM) etc. Similarly biguanidylated polyallylamine (degree of substation 0.011-100 %).

Polyallylamine Polyallylbiguanide

In some case, not all the amine in polyallyamine is substituted with biguanidine; hence the resulting polymer is a mixture of amine and biguanidine, not 100% biguanidine substituted.

The covalent conjugation may occur via any synthetic method known to a person skilled in the art, for example, by reacting a amine group with dicyandiamide or biguanidine transfer.

Synthesis of Biguanidylated protein/po ypeptide/pe tides: This invention also described method to convert polyamino biopolymers (such as polylysine, polyornithine). Amine group in the peptide/polypeptide/protein is converted to biguanidine by reacting with cyanoguanidine under reflux conditions (100 to 120°C in water or 0.1N HC1), preferably using >1 fold molar excess of cyanoguanidine (can be varied from 0.01-10 fold molar excess). Compound is purified by dialysis and lyophilisation. Biguanidylated peptides/polypeptides could also be synthesised by using a biguanidine substituted monomer and by standard solid phase peptide synthesis.

Biguanidylated Carbohydrate analogues; Synthesis of biguanidine modified chitosan (Chitosan-biguanidine) has been described (described in Japanese Patent 60233102, Chinese patent CN1944467A, CN101033264 B and CN101638445A). Briefly, lOOmg of chitosan is dissolved in 0.1N HC1, 2 fold molar excess of cyanoguanidine is added. The mixture is refluxed (100 to 120°C) for 2 hours, compounds is purified by dialysis and lyophilisation. Similarly biguanidine modified dextran, is synthesised by reacting amine derivitised dextran (aminodextran) with cyanoguanidine as above. Other carbohydrate polymers were first converted to amine bearing molecules, and then reacted with cyanoguanidine as described above.

Synthesis of biguanidine cross linked chitosans: In some instance, low molecular weight chitosan are reacted with hexamethylenebiscyanoguanidine under reflux conditions as described above, resulting in a biguanidine crosslinked chitosan molecules.

Biguanidylated synthetic polymers; These compounds are synthesized as described above, by reacting amine bearing/modified synthetic polymers (such as PEI, poly ally 1 amine, Dendrimers, linear and branched amine bearing polymers) with cyanoguanidine under reflux conditions.

Synthesis of biguanidine grafted polyanions;

This invention also describes method to modify polyanionic biopolymers (such as polyglutamic acid, polyaspartic acid, alginic acid). First the anionic polymer is reacted with a diaminoalkylgroup, then reacting resulting amine modified polymer with cyanoguanidine, alternatively modifications can be achieved in one step reaction, by reacting with biguanidinonalkylamine.

Biguanidine cross-linked molecules: Above described amine modified/amine bearing molecules were reacted with hexamethylenebiscyanoguanidine (CAS: 15894-70-9) under reflux conditions as described above, resulting in a biguanidine crosslinked molecules with potent entry promoting properties. Example: Equimolar hexamethylenebiscyanoguanidine (HMBCDA) and PEIVchitosan/polylysine/aminodextran/amine bearing dendrimers were reacted under reflux conditions in 0.1N HC1 or a suitable solvent, and purified. The resulting polymer has biguanidine moieties bridged between native polymers, the resulting polymer has a mixture of biguanidine and amine groups. The extent of crosslinking can be controlled by varying the HMBCDA to polymer ratio.

The biguanidine grafted biomolecule/synthetic molecules/analogues can be characterised by suitable analytical methods (UV, IR, NMR) well know to those skilled in the art (Example of characterisation of biguanidine grafting of the above listed molecule is shown in Figurel). The entry-promoting agent used in the method of the invention may comprise linear, branched or dendrimeric/cross-linked natural, synthetic or semi synthetic biopolymers with biguanide substitution. The entry promoting agent may comprise a combination of linear, branched or dendrimeric/cross-linked molecules described above. The entry-promoting agent may comprise one or any combination of molecules described in this invention. For example, the entry-promoting agent can comprise one or more of biguanidine modified biopolymers such as chitosan-biguanide, Dextran-biguanidine, PEI-biguanidine, Polylysine-biguanide, polyornithine-biguanide, biguanidylated dendrimers, biguanidylated aspartic acid or glutamic acid substituted with biguanide, biguanide substituted poly aspartic or polygulatamic acid or combination of these. Thus, the entry-promoting agent may comprise homogeneous or heterogeneous mixtures of one or more of biguanide modified molecules described above. As will be appreciated by those skilled in the art, the entry-promoting polymer may be a homogenous or heterogeneous.

Description of commercial applications of entry promoting agents

The entry promoting agents in the present invention can be used for delivery into both eukaryotic and prokaryotic cells either in-vitro or in-vivo. Thus, the cell may be a eukaryotic (e.g animal cells; such as fibroblasts, epithelial cells or cells of immune origin, yeast or fungi, a plant cell (for example a monocotyledonous or dicotyledenous plant cell), fish cell (for example Zebra fish, trout), bird cell (for example cells from chicken), insect cell (for example Drosophila, Nematoidia or Protista (for example Plasmodium spp or Acantamoeba spp) or a prokaryotic cells (example bacteria; Staphylococcus aureues, Escherichia coli, Mycobacterium tuberculosis). Examples of prokaryotic and eukaryotic cells are known to those skilled in the art.

The introduced agent (usually called as cargo molecule) may typically comprise a bioactive compounds (such as those used in prophylactic or therapeutic applications or a diagnostic/imaging probes) with poor uptake into cells. The introduced agent may comprise a biomolecules such as proteins, polypeptides, peptides and analogues, small molecules (often referred to as drugs, including antimicrobials including antibacterial, antifungal, antiviral agents), nucleic acids and analogues and synthetic compounds, analogues and derivatives thereof. The nucleic acid or nucleic acid analogue may be DNA or RNA or both for gene expression (encoding a polypeptide or nuclei acid) or such nucleic acids will be well known to those skilled in the art (example plasmid DNA (example;pEGFP~Cl) for encoding green fluorescent protein; antisense nucleic acids such as miRNA, siRNA; synthetic nucleic acid analogues such as peptide nucleic acid, locked nucleic acid, morpholino, phoshporothioates etc.). Use of such nucleic acids, and also nucleic acid modified cells for research, diagnostic or therapeutic use is well understood and well known to the skilled person.

Example of commercial use of entry promoting agent used in the present invention can be explained as below. The entry promoting agent can be used to produce large quantity of recombinant proteins or nucleic acids in the introduced cell for commercial medicinal or research applications. Use of entry promoting agent in bioprocessing industry is well known to those skilled in the art. Entry promoting agent used in the invention can be used to lower the cost of production or recombinant proteins by maximising the target gene expression (for example polypeptide production in cells, (Figure2a: showing production of green fluorescent protein in a mammalian cells by introducing a plasmid DNA encoding the gene, Figure2b: production of a lentivirus, such methods are useful for virotherapy). Entry promoting agent can also be used to introduce a plasmid DNA encoding an antigen to elicit immune response (DNA vaccine) or to modify target cell for anticancer applications. Methods for assessing whether entry promoting agent introduces an introduced agent (cargo molecules; small molecules; nucleic acids or analogue) into a cell is well known to those skilled in the art. The entry promoting agent can be used to knockdown/supress target gene (example; suppression of a gene important in cancer progression as shown in Figure 3).

The introduced agent may also comprise a protein, polypeptide (generally referred to peptides composed of fewer than 50 amino acids in length, some time polypeptides are also referred to as proteins if more than 50 amino acid residue in length, an example of protein delivery is shown in Figure 4), peptidomimetic compounds, these compounds will be well known to those skilled in the art. These proteins/polypeptides or analogues derivatives are used either or therapeutic purpose (example as antigen for vaccine or targeting intracellular processes such as enzyme inhibition for therapeutic usefulness). Methods for assessing whether entry promoting agent introduces an introduced agent (cargo molecules; polypeptides or peptides) into a cell is well known to those skilled in the art.

The introduced agent may also comprise a small drug molecule or bioactive reagent (for molecular weight of less than 1000 or 2000 Da. The introducing agent in the present invention helps small drug or bioactive reagent with poor cell uptake or stability properties to reach targets inside cells (Figure 5, berberine delivery into Candida albicans) (example; introducing agent in the present invention can be used to in combination with antimicrobials such as ampicillin, kanamycin, ciprofloxacin, gentamicin or amphotericin or rifampicin to potentiates their activity to kill bacteria or fungi which reside inside the cell; example killing/clearance of intracellular Staphylococcus aureus inside osteoblast or skin cells or Salmonella enterica typhimurium in other host cells, as shown in Figure 5). The present inventor has shown that the entry promoting agents potentiates antimicrobial activity, thus providing synergistic action, similarly the formulations also potentiates clearing intracellular microbes as shown in Figure 6. Methods for assessing whether entry promoting agent introduces an introduced agent (cargo molecules; small molecules) into a cell is well known to those skilled in the art.

The introduced agent may also comprise a cellular imaging probes (for example nucleic acid/analogue based imaging probe, as shown in Figure 6), well known to those skilled in the art. Methods for assessing whether entry promoting agent introduces an introduced agent (cargo molecules; imaging probe) into a cell is well known to those skilled in the art.

The introduced agent and the entry-promoting agent may be provided as a formulation, for example as a noncovalent complex. The mixing of entry-promoting agent and the introduced agent results in formation of nanoparticles (as shown in Figure 7). The formulation may be prepared by mixing the entry-promoting agent and the introduced agent (cargo molecules) in appropriate ratios (by changing relative concentration of either cargo or entry promoting agent) and under suitable conditions, for example, buffers with varied salt concentration and pH. Noncovalent formulations may be prepared by mixing up to 1000 fold molar or weight excess/excess weight of introduced agent over entry-promoting agent, through using an equal molar concentration of carrier and cargo molecules, to up to 1000 fold molar excess of entry- promoting agent over introduced agent. The ratio can vary from 0.01-1000. For example, an appropriate molar ratio or weight ratio of introduced agent (for example nucleic acid, for example oligonucleotide, protein or small drug molecules, for example molar ratio of introduced agent and entry-promoting agent may be in the range of 1:0.1 to 1:50 or 1:0.5 to 1: 1000, for example 1: 1 to 1: 10 or 1:5, for example around 1: 1.5. The molar or weight/weight ratio of introduced agent and entry -promoting agent can be 1: 1000 to 1000: 1. An appropriate weight:weight ratio of introduced agent and entry-promoting agent may be in the range of 1:0.1 to 1:50 or 1:0.5 to 1: 1000, or viceversa, for example 1: 1 to 1: 10 or 1:5, for example around 1:3. 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 any pH, for example 4-13.

The entry promoting agents of this invention may be performed in-vitro (e.g. siRNA, miRNA, oligonucleotides plasmid DNA, protein transfection) in cell culture or large scale transfection in bioprocess industry. Such experiments are performed to understand gene functional studies; creation of stable cell lines; target gene silencing, also can be used to produce polypeptide of commercial applications through bioprocess industry (Figure 2). Use of entry promoting agents for above mentioned applications is well known to those skilled in the art. Another application of the entry promoting agents in this invention can be used to delivery in-vivo (e.g. DNA vaccination or siRNA delivery or drug formulations such as anticancer, antibacterial, antifungal, anti-inflammatory), transdermal or topical delivery either in animal models or humans, vertebrates or invertebrates, in a range of disease conditions, where the introduced agent needs assisted delivery.

The molar ratio or weight/weight of entry promoting agent (carrier molecule) and introduced agent (cargo) can be 0.01-1000. For example; 2.5 microgram of entry promoting agent and 1 microgram of plasmid DNA encoding GFP (pEGFP-Cl) gives weight ratio 2.5: 1.

Working example of routinely used weight: weight ratio of entry promoting agent and introduced agent (example: plasmid DNA) is 0.1: 1 to 50:1. More often, entry promoting agent is used in excess over introduced agent. In some instance, the entry promoting agent is used lesser concentration than the introduced agent.

The complexes/formulations of entry promoting agent and introduced agent can be prepared by mixing both components in water or any buffers or medium used in laboratory or compatible for invivo use (such as with or without salt; water, phosphate buffer, saline, or salt solutions or growth medium, nutrient broth with varied pH, preferably from 4-13, pH can be adjusted by using either HC1 or NaOH, Well known to those skilled in the art). Usually formulations are used for delivery. Complexes can be prepared at room temperature or on ice or at 37°C. List of examples where the present invention is useful is described in Figures 2-6. The present inventor has shown that the molecules of the present invention are less toxic compared to parent molecules (Figure 8). The inventor has shown that the biguanidine modified molecules are highly water soluble and less toxic, a major advantage for clinical use. Experiment conditions used to perform entry-promoting agents are described in in Figure legends.

The present invention shows that the biguanidine grafted/modified molecules have superior performance over native molecules.

• High solubility compared to native molecules

• Interacts potently with biomolecules and forms nanoparticles

• Confer carrier potential to a biomolecule which is not useful as carrier before (example; dextran alone is not suitable for nucleic acid delivery, whereas dextran- biguanidine is capable of delivering nucleic acids into cells)

• High transfection efficiency for entry promoting activity is an important feature for research, as are low cytotoxicity, and effectiveness for difficult to transfect cells

• Enter on its own and as well as when loaded with biomolecules

• Ability to release biomolecules upon entry

• Low cytotoxic and suitable for in-vivo applications

• Provide synergistic formulations for antimicrobial activity by promoting improved intracellular delivery.

I considered that introduction of biguanidine in the natural or synthetic polymer enables them to efficiently interact with cargo molecules and enable them to act as an efficient carriers with low toxicity. Moreover they help in intracellular delivery of a variety of cargo molecules. Additionally, these modified biomolecules are low toxic, biocompatible, hence they have huge potential for in-vivo use. The inventor has shown that these modified biomolecules have unique properties over native biomolecules, even convert a non carrier molecule into a carrier molecule (such as dextran-biguanide) with several commercial molecules.