WO2019220088A1 - Polymers, nanoparticles formed from the polymers and pharmaceutical compositions comprising an active agent encapsulated in the nanoparticles - Google Patents

Abstract

The present invention relates to a polymer comprising a polymer chain of general formula (I): wherein R1 is H or methyl; R2 is C1-6 alkyl R3 is H or methyl; R4 is -(CH2)p-N+ (CH3)2-(CH2)3-S(O)2O- or -(CH2)p-N(CH3)2; p is an integer of 1 to 4; n is an integer of 15 to 30; and m is an integer wherein the ratio of n:m is from 80:20 to 20:80; provided that at least 90% of the R4 groups are -(CH2)P-N+ (CH3)2-(CH2)3-S(O)2O- The invention also relates to nanoparticles formed from the polymers and to pharmaceutical compositions comprising an active agent encapsulated in the nanoparticles.

Description

POLYMERS, NANOPARTICLES FORMED FROM THE POLYMERS AND PHARMACEUTICAL COMPOSITIONS COMPRISING AN ACTIVE AGENT ENCAPSULATED IN THE NANOPARTICLES

Summary of Invention

The present invention relates to novel polymers, in particular to block copolymers, and more specifically to block copolymers comprising a lipophilic block and a zwitterionic 5 block, which is suitably a polysulfobetaine block. The invention also relates to nanoparticles formed from these polymers, to the nanoparticles loaded with biologically active substances and to the loaded nanoparticles for use in delivering the biologically active substance through a layer of a biopolymer such as mucous or microbial biofilm.

10 Background

One of the problems faced when treating patients with biologically active substances is that of finding a suitable form for delivery of the biologically active substance. In general, patient compliance is increased if a drug is formulated for delivery via a route such as oral, rectal, nasal, bronchial (inhaled), topical (including eye drops, buccal and 15 sublingual), vaginal administration than via a parenteral route, for example subcutaneous, intramuscular, intravenous and intradermal administration.

In many cases, however, effective delivery by a non-parenteral route has been difficult or impossible to achieve due to the mucous barrier lining organs such as the gastro-

20 intestinal tract, the lungs and the genito-urinary tract. For example, only small drug molecules can permeate relatively easily through intestinal mucous, while large molecules like large peptides can be sterically trapped by the mucous. Furthermore, orally administered therapeutic peptides are highly susceptible to degradation in the intestinal mucous layer by various protease enzymes such trypsin, chymotrypsin, and 25 carboxypeptidase.

Similar problems have also been encountered when treating wounds in that it has proved difficult to deliver a biologically active substance through microbial biofilms.

30 Attempts have been made to solve this problem by loading the biologically active substance onto appropriate particles to protect the cargo from mucosal enzymes with the aim of delivering the particles carrying the active substance through mucous membranes. These attempts involve different strategies to improve drug delivery through mucous barrier. For examples of these strategies: (i) muco-adhesion strategy to 35 prolong the residence time of nanoparticles (NPs) at the mucosal barrier was approached by designing particles that can interact with mucous through electrostatic interactions (chitosan, polyethyleneimine, polylysine and polycarbophil), hydrogen bonding or simple van der Waal’s forces (Eudragit). (ii) The strategy of M cells targeting to avoid mucous barrier since these cells which existed on the peyers patches in the ileum are covered with only a 30 nm glycocalyx layer. Various ligands such as lectin, tomato lectin, invasin and wheat germ agglutinin lectin have been used so as to target the NP to M cells through the interaction to the specific carbohydrate residues at the M cells (iii) Slippery particles’ surface strategy to avoid electrostatic or hydrophobic interactions with mucous which was achieved through either neutral charged surface polyelectrolyte NP, or hydrophilic surface NPs such as Pegylated NPs. (iv) Self- microemulsifying drug delivery (SMEDD) strategy where small droplet size with presence of enhancing agents can improve the permeation of drug through mucous barrier (v) Mucolytic strategy where NPs are loaded with mucolytic agent that can disrupt the mucous network upon releasing resulting in high permeation through mucous (vi) Thiomer strategy which is supposed also to have mucolytic activity through the sulfhydryl molecules which interact with mucin disulfide bonds resulting in the destruction of the mucous network. However, most of these approaches showed limited success with the exception of PEG NPs which showed promising improvement of mucous permeation.

Polymers comprising sulfobetaine-modified monomer units are known and have been used to confer anti-bioadherent properties and as coatings to prevent bio-adhesion.

The present inventors have developed a novel block copolymer comprising alkyl (meth)acrylate units and a zwitterionic block comprising betainised monomer units. The polymer can form particles suitable for loading with a biologically active material and for transporting that material through a biopolymer.

Statements of Invention

In one aspect of the invention, there is provided a polymer comprising a polymer chain of general formula (I):

(I)

wherein

R¹ is H or methyl;

R² is C1 -6 alkyl

R³ is H or methyl;

R⁴ is -(CH₂)_P-N⁺(CH3)2-(CH₂)3-S(0)20- or -(CH₂)_P-N(CH₃)2;

p is an integer of 1 to 4;

n is an integer of 15 to 30; and

m is an integer wherein the ratio of n:m is from 80:20 to 20:80;

provided that at least 90% of the R⁴ groups are -(CH₂)_P-N⁺(CH₃)₂-(CH₂)₃-S(0)₂0\

The polymers of the invention are particularly advantageous because they contain a lipophilic block and a zwitterionic hydrophilic block, wherein, in the zwitterionic block, each monomer unit carries both a positive and a negative charge. Moreover, the ratio of lipophilic to zwitterionic monomer residues is such that polymer particles can be formed. The particles formed from the polymers of the present invention are of uniform size, do not aggregate when suspended in a solvent and can be loaded with biologically active molecules. The particles also show excellent penetration of biopolymers such that they have utility for transporting biologically active molecules across layers of mucous or of bacterial biofilms.

Throughout the description and claims of this specification, the words“comprise” and “contain” and variations of the words, for example“comprising” and“comprises”, mean “including but not limited to” and do not exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

All references, including any patent or patent application, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. Further, no admission is made that any of the prior art constitutes part of the common general knowledge in the art.

In the present specification, the term“Ci-e alkyl” refers to a straight or branched chain fully saturated hydrocarbon group having from one to 6 carbon atoms. Examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl, t-butyl, n-pentyl and n-hexyl. Terms such as C1-4 alkyl and C1-3 alkyl have similar meanings but the number of carbon atoms in the alkyl group is different (1 to 4 and 1 to 3 carbons respectively in these examples).

In the present specification the term “(meth)acrylate” refers to an acrylate and a methacrylate.

Suitably, the polymer consists essentially of the polymer chain of general formula (I). In this case, the polymer may be terminated at each end by small organic groups.

For example, the polymer may be of general formula (Iz):

Wherein R¹, R², R³ and R³ are as defined in general formula (I);

X is a residue of a chain transfer agent; and

Y is a residue of a monomer.

In the present specification, the chain transfer agent (CTA) is a reagent suitable for use in a reversible addition-fragmentation chain-transfer (RAFT) polymerisation method. Suitable chain transfer agents include compounds of the structure:

where Z is group which controls the C=S bond reactivity and is, for example, a Ce-ie alkyl group, typically a straight chain alkyl group, for example dodecyl;

R is a free radical leaving group which can re-initiate radical polymerisation, such as Ci-e alkyl optionally substituted with CN or C(0)0H, for example cyanomethyl, 2-cyano-2- propyl, 4-cyano-pentanoic acid, or 2-methylpropionic acid.

Examples of suitable chain transfer agents include 2-cyano-2-propyl dodecyl trithiocarbonate, cyanomethyl dodecyl trithiocarbonate, 4-cyano-4-[(dodecylsulfanylthio- carbonyl)sulfanyl]pentanoic acid and 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid. 2-Cyano-2-propyl dodecyl trithiocarbonate is particularly suitable.

In the polymer of general formula (Iz), X may be represented by the formula:

where - is the link to the polymer and R is as defined above for the CTA.

In the present specification, a“residue of a monomer” refers to the remaining portion of the monomer unit after polymerisation has taken place.

The monomer residue Y may be the residue after polymerisation of a (meth)acrylate monomer of formula (II):

wherein R¹ and R² are as defined for general formula (I). Thus, for example, the monomer may be of formula (lx) or (ly)

wherein R¹ , R², R³ and R⁴ are as defined for general formula (I) and X is as defined for general formula (Iz).

In some suitable polymers of the present invention, R¹ and/or R³ is methyl. More suitably, both R¹ and R³ are methyl.

Suitably, R² is C3-6 alkyl, for example a propyl, butyl or pentyl group and, in particular, a straight chain alkyl group such as n-propyl, n-butyl or n-pentyl. Polymers in which R² is n-butyl are particularly suitable.

Suitably, n is from 15 to 29, for example 20 to 28, 22 to 27.

More suitably, n is from 15 to 28, 15 to 27, 15 to 26 or 15 to 25. In some cases, n is from 18 to 28, 18 to 27, 18 to 26 or 18 to 25. In other cases, n may be from 20 to 28, 20 to 27, 20 to 26 or 20 to 25.

Alternatively, n may be from 22 to 26 or 24 to 26, typically about 25.

The ratio of n:m may be from 30:70 to 70:30 or from 40:60 to 60:40. Example values for the ratio of n:m are 30:70, 40:60, 50:50, 60:40 and 70:30.

In the group R⁴, suitable values for p are 1 to 3, for example 1 or 2 and particularly 2.

In some suitable polymers of the present application, in increasing order of suitability, at least 95%, 96%, 97%, 98%, 99% or 99.5% of the R⁴ groups are -(CH₂)p-N⁺(CH₃)2-(CH₂)3- S(0)₂0\ Most suitably, 100% of the R⁴ groups are -(CH₂)p-N⁺(CH₃)2-(CH₂)3-S(C>)₂C>- such that the polymer comprises a polymer chain of general formula (la):

wherein R¹, R², R³, m, n and p are as defined above for general formula (I). The polymer may consist essentially of the polymer chain of general formula (la). For example, the polymer may have the formula (laz):

wherein R¹, R², R³, m, n and p are as defined above for general formula (I); and X and Y are as defined above for general formula (Iz). A particularly suitable example of a polymer of the present invention is a betainised block copolymer of n-butyl methacrylate with 2-(dimethylamino)ethyl methacrylate comprising a polymer chain of general formula (lb):

wherein R¹, R³, m and n are as defined above for general formula (I) and

R⁴ is -(CH₂)₂-N⁺(CH₃)₂-(CH₂)₃-S(0)₂0- or -(CH₂)2-N(CH₃)2, wherein at least 90%, 95%,

96%, 97%, 98%, 99% or 99.5% of the R⁴ groups are -(CH₂)₂-N⁺(CH₃)₂-(CH₂)₃-S(0)₂0\

The polymer may consist essentially of the polymer chain of general formula (lb). For example, the polymer may be of formula (Ibz):

wherein R¹, R², R⁴, m and n are as defined above for general formula (I); and X and Y are as defined above for general formula (Iz).

Suitably, the polymer is fully betainised and the polymer comprises a polymer chain of formula (lc):

wherein R¹, R³, m and n are as defined above for general formula (I).

Suitably, the polymer consists essentially of the polymer chain of general formula (lc). For example, the polymer may have the formula (lcz)

wherein R¹, R³, m and n are as defined above for general formula (I) and X and Y are as defined above for general formula (Iz).

Particularly suitable X and Y groups for the polymers of formulae (laz), (Ibz) and (lcz) are as discussed above for the polymers of general formula (Iz). For example, X may be a residue of a (meth)acrylate monomer of general formula (II). The polymers of general formula (I) suitably have a narrow size distribution, with a polydispersity index of not greater than 1.4 (low polydispersity) [1 ,2], typically from about 1.1 to 1.4.

Polydispersity index in the polymers is defined by the equation:

—

Where M_w is the weight average molecular weight and M_n is the number average molecular weight.

Polymers comprising a polymer chain of general formula (I) are suitably prepared by RAFT polymerisation. Therefore, in a further aspect of the invention there is provided a process for the preparation of a polymer comprising a polymer chain of general formula (I), the process comprising:

(i) Polymerising a monomer of general formula (II):

Wherein R¹ and R² are as defined for general formula (I);

by RAFT polymerisation in the presence of an initiator and a chain transfer agent (CTA) to form a polymer comprising a polymer chain of general formula (III):

wherein R¹, R² and n are as defined for general formula (I);

(ii) Copolymerising the polymer comprising the polymer chain of general formula (III) with a monomer of general formula (IV):

Wherein R³ is as defined in general formula (I) and

R⁵ is -(CH₂)_P-N(CH₃)₂, where p is as defined in general formula (I);

by RAFT polymerisation to form a polymer comprising a polymer chain of general formula (V):

wherein R¹, R², R³, n and m are as defined for general formula (I) and R⁵ is as defined for general formula (IV)

(iii) Reacting the polymer comprising the chain of general formula (V) with 1 ,3- propane sultone to form a polymer comprising a chain of general formula (I). Suitably, in step (i), the ratio of the monomer of general formula (II) to CTA is from about 15:1 to 35: 1 , more suitably from 20:1 to 30:1 , for example 22:1 to 28:1 , 23:1 to 27:1 or 24:2 to 26: 1 , typically about 25: 11.

Suitable CTAs are as described above.

Any suitable radical polymerisation initiator may be used. One example of such an initiator is 2,2'-azobis(2-methylpropionitrile) (AIBN). The polymerisation of step (i) may be carried out in any suitable organic solvent, for example dioxane, typically under an inert atmosphere, for example under nitrogen. The reaction temperature is suitably about 60-80°C, typically about 70°C.

Suitably, the polymerisation reaction is stopped when n reaches the desired value, typically after about 5 to 7 hours. The value of n may be determined by NMR. In addition or alternatively, gas permeation chromatography (GPC) may be used to determine the molecular weight and polydispersity index of the polymer comprising the polymer chain of general formula (III).

The polymer comprising the polymer chain of general formula (III) may be a polymer of general formula (lllz):

Wherein R¹ and R² are as defined for general formula (I); X is as defined for general formula (Iz) and Y is a residue of the monomer of general formula (II).

Step (ii) is suitably carried out using the dried polymer comprising a chain of general formula (III). The polymer comprising the chain of general formula (III) serves as a macro-CTA for the RAFT polymerisation and therefore it is not necessary to add a CTA at this stage. Trithiocarbonate as a macro-CTA agent was widely reported as a good source for di-block and tri-block control copolymerization [3,4]

The RAFT polymerisation conditions are similar to those used for step (i). The amount of the monomer of general formula (IV) is selected such that the molar ratio of monomer of general formula (II) to monomer of general formula (IV) used to form the polymer comprising the chain of general formula (V) is from 80:20 to 20:80, in order to give a ratio of n:m as defined above for general formula (I). Suitably, the polymer comprising the chain of general formula (V) is a polymer of general formula (Vz)

(Vz)

wherein R¹, R², R³, n and m are as defined for general formula (I); X is as defined for general formula (Iz), Y is a residue of the monomer of general formula (II) and R⁵ is as defined for general formula (IV).

In step (iii), the polymer having the chain of general formula (V) is reacted with 1 ,3- propane sultone. Suitably, the reaction is carried out in an organic solvent such as tetrahydrofuran. The degree of reaction can be determined using IR and NMR spectral analysis and the reaction terminated once betainisation is complete. Typically, the reaction time is 60 to 84 hours, for example about 72 hours.

The polymers of general formula (I) may be used to form nanoparticles. Therefore, in a further aspect of the invention, there is provided a nanoparticle comprising a compound of general formula (I), wherein the particle has an average diameter of about 20 to 70 nm, more suitably 25 to 60 nm, for example about 40-50 nm.

In the present specification, average particle diameter is determined on an intensity basis such that the z-average value is obtained, and is suitably measured using photon correlation spectroscopy. A method for particle size measurement is provided in the examples below. Suitable instruments for making the measurement are well known to those of skill in the art.

The particles are relatively monodisperse, i.e. they have a polydispersity index of about 0.2 to 0.6, more usually about 0.3 to 0.5, typically about 0.4.

The nanoparticles of the invention have a hydrophilic surface which is densely charged and zwitterionic but, because the positive and negative charges are carried on the same monomer unit, the surface has an overall net neutral zeta potential when suspended in an aqueous solvent. In contrast to currently available neutrally charged nanoparticles, the nanoparticles of the present invention exhibit relatively small particle size and show minimal aggregation.

The nanoparticles are bioinert and have also shown an ability to pass rapidly through biopolymers such as mucous layers and bacterial biofilms. Furthermore, the nanoparticles of the invention have the ability to avoid non-specific surface based cellular uptake processes by the body and are therefore suitable as vehicles for the targeted delivery of biologically active molecules to tissues. The nanoparticles may be loaded with a biologically active agent. Suitable active compounds include peptides and proteins such as exenatide, insulin, leucine enkephalin, proapoptotic peptide and cilengitide as well as other active agents such as antimicrobial agents, for example antibacterial agents such as polymyxin B, tobramycin, and benzyl penicillin, anti-viral agents such as zanamivir, sialidases and oseltamivir , anti-fungal agents such as Econazole nitrate, itraconazole and pimaricin or anti-protazoal agents such as nitazoxanide and chloroquine.

In the nanoparticles of the invention, the sulfobetaine serves as the particle shell and the lipophilic polymer (BMA) as a core. Suitably, the biologically active agent is encapsulated in the lipophilic core of the nanoparticle.

The nanoparticles may be formed by a nano-precipitation technique comprising

(i) Solubilising the polymer comprising the polymer chain of general formula (I) in a suitable solvent to form a polymer solution;

(ii) Adding the polymer solution dropwise into an aqueous phase with mixing to allow formation of nanoparticles by self-assembly.

Suitable solvents include methanol, ethanol or mixtures thereof or mixtures of methanol or ethanol with aqueous solvents, for example containing salts.

An example of a suitable solvent is a mixture of aqueous sodium chloride solution and methanol in a ratio which may be determined experimentally and which is dependent on the ratio of n:m in the polymer comprising the chain of general formula (I).

The rate of addition of the polymer solution to the solvent may be from about 10 to 30 pi_¬ per minute, more usually 15 to 25 pL per minute, for example about 20 pL per minute.

The volume of the aqueous phase may be from about 1 to 10 ml_, suitably 3 to 7 ml_, typically about 5 ml_.

The aqueous phase to which the polymer solution is added may have a pH from 5.5 to 7.5, typically pH 6.5 to 7.5, for example pH 6.8 to 7.0. Suitably, a phosphate buffer may be used.

After the addition of the polymer solution to the aqueous phase, mixing may be continued for 10 to 60 minutes to allow evaporation of non-aqueous solvent (methanol).

The process for forming the nanoparticles may include the additional step of dialysing the suspension of nanoparticles against a further aqueous solution, for example phosphate buffered saline at a pH from 5.5 to 7.5, typically pH 6.5 to 7.5, for example pH 6.8 to 7.0.

The process may include the additional step of loading the nanoparticles with a biologically active agent. This may be achieved by modifying step (i) above by solubilising the active agent in the suitable solvent.

For some active agents, a co-solvent may be employed.

As a result of this process, particles are formed in which the biologically active agent is encapsulated in the lipophilic core of the particle.

The nanoparticles of the invention are of use in medicine for delivering biologically active agents to a chosen site which might be less accessible using conventional formulations. In the present context“medicine” comprises both human and veterinary medicine.

Therefore, in a further aspect of the invention, there is provided a nanoparticle of the invention for use in delivering a biologically active agent across a mucous membrane or a bacterial biofilm.

There is also provided the use of a nanoparticle of the invention in the preparation of an agent for use in delivering a biologically active agent across a mucous membrane or a bacterial biofilm.

In addition, the invention provides a method for delivering a biologically active agent across a mucous membrane or a bacterial biofilm, the method comprising administering to a patient in need of such treatment an effective amount of nanoparticle according to the invention.

Suitably, the mucous membrane is the lining of the gastro-intestinal tract, the lungs or the genito-urinary tract.

The bacterial biofilm may comprise a biofilm found in a wound. In this case the biologically active agent is suitably an anti-bacterial agent.

When the mucous membrane is the lining of the gastro-intestinal tract, the lungs or the genito-urinary tract, the biologically active agent may comprise a biologically active peptide or other pharmacologically active molecule which it is desirable to deliver across the mucous membrane.

Examples of biologically active peptides include exenatide, insulin, leucine, enkephalin, proapoptotic peptides and cilengitide and particles loaded with these peptides may be used for treating type II or type I diabetes, pain, or cancer. The nanoparticles may be delivered orally such that they are absorbed through the mucous membrane comprising the lining of the gastrointestinal tract.

When the biologically active agent is delivered across a bacterial biofilm, it may be an anti-bacterial agent, for example an agent suitable for the treatment of a bacterial infection e.g. Pseudomonas aeruginosa infection. The nanoparticles may be delivered topically to a wound or lesion infected with the bacteria.

Therefore, in a further aspect of the invention there is provided a nanoparticle according to the invention for use in medicine wherein:

the biologically active agent is exenatide or insulin and the nanoparticle is for use in the treatment of diabetes; or

the biologically active agent is leucine encephalin and the particle is for use in the treatment of pain; or

the biologically active agent is a proapoptotic peptide or cilengitide and the nanoparticle is for use in the treatment of cancer.

In further aspect of the invention, there is provided a particle according to the invention loaded with exenatide or insulin for use in the treatment of diabetes.

There is also provided the use of a nanoparticle according to the invention loaded with exenatide or insulin in the preparation of an agent for the treatment of diabetes.

Furthermore, there is provided a method for the treatment of diabetes, the method comprising administering to a patient in need of such treatment an effective amount of exenatide or insulin encapsulated in a particle according to the invention. In these cases, the particles of the invention are suitably formulated for oral administration.

In some cases, the biologically active agent may be an anti-microbial agent, e.g. an anti bacterial such as polymyxin B, tobramycin, and benzyl penicillin, anti-viral such as Zanamivir, sialidases and oseltamivir , anti-fungal such as econazole nitrate, itraconazole and pimaricin or anti-protazoal agent such as nitazoxanide and chloroquine.

Therefore, in a further aspect of the invention there is provided a particle according to the present invention loaded with an anti-microbial agent for use in treating a microbial infection.

There is also provided the use of a particle according to the invention loaded with an anti-microbial agent in the preparation of an agent for the treatment of a microbial infection.

There is further provided a method for the treatment of a microbial infection, the method comprising administering to a patient in need of such treatment an effective amount of a particle according to the present invention loaded with an anti-microbial agent.

The microbial infection may be a bacterial, viral, fungal or protozoal infection and the antimicrobial agent will be selected accordingly.

Suitably, the microbial infection is a topical infection or an infection of a wound. In this case, the nanoparticles may be formulated for topical administration.

The infection may be a bacterial infection, for example an infection of a bacteria which produces a bacterial biofilm, for example Pseudomonas aeruginosa.

In this case, the particles of the present invention may be formulated for topical administration to a wound or lesion infected with the bacteria.

The particles will generally be formulated as part of a pharmaceutical composition and therefore in a further aspect of the invention there is provided a pharmaceutical composition comprising a particle of the present invention loaded with a biologically active agent. The term“pharmaceutical composition” refers to a composition for use either in human or veterinary medicine. The composition may therefore be adapted for administration to humans or animals, more suitably mammals.

Suitable biologically active agents are as outlined above.

In some cases, the composition will be formulated for oral, rectal, nasal, bronchial (inhaled), topical (including eye drops, buccal and sublingual) or vaginal administration. The particular route of administration selected will depend on the nature of the biologically active agent.

For example, when the biologically active agent is insulin or exenatide, it may be appropriate to formulate the composition for oral administration. On the other hand, when the biologically active agent is an antibacterial agent, the composition may be formulated for administration by the most convenient route to reach the site of infection. This may be, for example topical administration.

The composition may be prepared by bringing into association the above defined particles with the carrier. In general, the formulations are prepared by uniformly and intimately bringing into association the particles with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product. The invention extends to methods for preparing a pharmaceutical composition comprising bringing particles of the present invention in conjunction or association with a pharmaceutically acceptable carrier or vehicle.

Formulations for oral administration in the present invention may be presented as: discrete units such as capsules, sachets or tablets each containing a predetermined amount of the active agent; as a powder or granules; as a solution or a suspension of the active agent in an aqueous liquid; or as a bolus etc.

For compositions for oral administration (e.g. tablets and capsules), the term“acceptable carrier” includes vehicles such as common excipients e.g. binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone (Povidone), methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sucrose and starch; fillers and carriers, for example corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid; and lubricants such as magnesium stearate, sodium stearate and other metallic stearates, glycerol stearate, stearic acid, silicone fluid, talc waxes, oils and colloidal silica. Flavouring agents such as peppermint, oil of wintergreen, cherry flavouring and the like can also be used. It may be desirable to add a colouring agent to make the dosage form readily identifiable. Tablets may also be coated by methods well known in the art.

A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active agent in a free flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active agent.

Other formulations suitable for oral administration include lozenges comprising the active agent in a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the active agent in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active agent in a suitable liquid carrier.

For topical application to the skin, the particles may be made up into a cream, ointment, jelly, solution or suspension etc. Cream or ointment formulations that may be used for the drug are conventional formulations well known in the art, for example, as described in standard text books of pharmaceutics such as the British Pharmacopoeia.

Particles loaded with an appropriate biologically active agent may be used for the treatment of the respiratory tract by nasal, bronchial or buccal administration of, for example, aerosols or sprays which can disperse the particles loaded with pharmacologically active ingredient in the form of a powder or in the form of drops of a solution or suspension. Pharmaceutical compositions with powder-dispersing properties usually contain, in addition to the active ingredient, a liquid propellant with a boiling point below room temperature and, if desired, adjuncts, such as liquid or solid non-ionic or anionic surfactants and/or diluents [5] Pharmaceutical compositions in which the pharmacological active ingredient is in solution contain, in addition to this, a suitable propellant, and furthermore, if necessary, an additional solvent and/or a stabiliser. Instead of the propellant, compressed air can also be used, it being possible for this to be produced as required by means of a suitable compression and expansion device. The invention will now be described with reference to the following examples and to the drawings in which:

FIGURE 1 is a schematic representation of the method used to synthesise the polymer and form the nanoparticles.

FIGURE 2 is the NMR spectrum of butyl methacrylate (BMA) polymer polymerised for 6 hours.

FIGURE 3 is the GPC profile of BMA polymer showing the molecular weight and the polydispersity index (PDI) of the BMA polymerized for 6 hours.

FIGURE 4 is the ¹H-NMR spectrum of the BMA:DMAEMA di-block copolymer in which the peaks showing the ratios of number of units of BMA to DMAEMA was highlighted. (A) (P1) BMA:DMAEMA (70:30). (B) (P2) BMA:DMAEMA (60:40). (C) (P3) BMA:DMAEMA (50:50). (D) (P4) BMA: DMAEMA (40:60). (E) (P5) BMA:DMAEMA (30:70). (Solvent: CDC ).

FIGURE 5 is an infra-red (IR) spectrum showing the structural formation of BMA- sulfobetaine step by step where step (A) shows the functional groups of the BMA, step (B) shows the functional groups of BMA-DMAEMA while step (C) shows the functional groups of BMA-sulfobetaine. (Note: only the main functional groups were highlighted each IR spectrum).

FIGURE 6 is a histogram comparison of diffusivities of sulfobetaine NPs (defined by PXS abbreviation) as compared to 113 other NPs comprising various surface chemistries and permeation strategies. (A) <Deff> of various sulfobetaine NPs. (B) % ratio <Deff>/D° of various sulfobetaine NPs.

FIGURE 7 is a plot showing the in vitro release profile of Lumogen® red from sulfobetaine NPs (P2S and P5S). (A) Percent of Lumogen® release. (B) Cumulative amount of Lumogen® release in pg.

FIGURE 8 is a series of plots showing the in vitro release profile of exenatide by the dialysis method in PBS buffer pH 6.8 at 37 °C (8A) Cumulative release profile in pg of exenatide from sulfobetaine NPs (P2S, P3S, P4S and P5S) for 8 hours [lower panel] Cumulative release profile in pg of free exenatide versus its release from sulfobetaine NPs over the first 2 hours. (8B) Release profile (%) for 8 hours of exenatide from the sulfobetaine NPs. [lower] Percent release profile (%) of free exenatide versus its release from sulfobetaine NPs over the first 2 hours.

FIGURE 9 is a series of plots showing the in vitro release profile of exenatide acetate from P5S NPs assessed by the dialysis method in which the first 2 hr release was conducted in buffer pH 1.2 and remaining 2 hr to 24 hr release in PBS buffer pH 6.8 at 37 °C. (9A) Cumulative release profile in pg of exenatide from P5S over 24 hours [lower panel] cumulative release of exenatide from P5S over the first 2hrs comparing pH 1.2 and 6.8. (9B) Cumulative % release of exenatide from P5S NPs over 24 hours [Lower panel] % release of exenatide.

FIGURE 10 is a schematic representation showing the treatment arms for the in vivo studies on exenatide in rats.

FIGURE 11 is a plot showing the blood glucose level of various exenatide routes and systems versus no exenatide medication at time 0 of the glucose challenge test. Statistical analysis was carried out with (n=3). (* means significant difference compared with no medication glucose challenge test, NS means non-significant difference).

FIGURE 12 is a plot showing absolute blood glucose level following glucose challenge test of various exenatide routes and systems versus GCT alone. * Represents significant difference (P < 0.05) compared with glucose challenge test alone. (n=3).

FIGURE 13 is an exenatide plasma concentration-time curve for orally administered exenatide sulfobetaine NP versus S.C exenatide solution. Bioavailability is presented for exenatide oral NPs versus S.C exenatide solution. (n=3, SEM).

FIGURE 14 is a plot showing MTT cytotoxicity study of sulfobetaine NPs with the range of concentration between 1000 pg/ml to 10 ng/ml (n=3). Study was carried out in 96 well plate (MDCK cell line, 5 X 10⁴ cells in each well plate), 1% Triton was used as positive control to express (0% viability).

FIGURE 15 is a plot showing the lactate dehydrogenase (LDH) cytotoxicity study of sulfobetaine NPs according to the invention (n=3, concentration range is 0.01-650 pg/ml). (A) Shows the cytotoxicity on porcine proximal tubule cells (LLC-PK1 cell line); (B) shows the cytotoxicity on hepatocarcinoma cells (Hep G2 cell line). The percentage of LDH leakage versus different NPs concentrations incubated with cells for 48 hrs is shown. Both Figure 1A and 1 B show no change in cell membrane integrity of both LLC- PK1 and Hep G2 cell lines correspondingly. These data are in agreement with our in house cytotoxicity MTT test.

FIGURE 16. Minimum Inhibitory Concentration (MIC) of polymyxin on non-mucoidal P. aeruginosa (black bars) versus mucoidal P. aeruginosa (grey bars) grown overnight and treated with (A) Free polymyxin (8 pg/ml - 0.007813 pg/ml); (B) Polymyxin loaded into sulfobetaine NPs (8 pg/ml - 0.007813 pg/ml) and (C) Free NPs (unloaded) at the same concentrations range of NPs used to load polymyxin in (B). NPs alone had no effect on bacteria and bacterial biofilm growth. Importantly, polymyxin loaded into sulfobetaine NPs had substantial impact on the P. aeruginosa compared with free polymyxin especially with mucoidal P. aeruginosa where MIC was significantly different for polymyxin loaded into NP versus free polymyxin at concentrations of 0.25, 0.5 and 1 pg/ml

Example 1 - Synthesis of Polymer and Formation of Nanoparticles The polymer was synthesised and nanoparticles formed according to the method shown schematically in Figure 1.

1.1. Synthesis of the of the BMA polymer

The first step of the synthesis of the zwitterionic polymer was the polymerization of the lipophilic block, which was carried out as shown in Scheme 1 , where, in the BMA-CTA polymer Y is the residue of the butyl methacrylate monomer and CTA is the residue of the chain transfer agent.

Scheme 1

CTA AIBN BMA-CTA polymer

RAFT technique was used to control the molecular weight and the chain length of the BMA polymer. With aim to produce BMA polymer with 25 monomers per chain, BMA monomer was used at the ratio of (25:1) to the chain transfer agent (CTA) (2-Cyano-2- propyl dodecyl trithiocarbonate). 2,2'-Azobis(2-methylpropionitrile) (AIBN) was used as the initiator at a ratio of (1 :250) of the BMA monomer and dioxane as the solvent of reaction. The ingredients were dissolved in Dioxane and the solution was flushed with nitrogen gas for 30 min before the reaction; the container was kept under the nitrogen flushing during the polymerization process. Polymerisation was carried out under heating (70 °C) and stirring (100 rpm). Reaction was stopped after 6 hrs by exposing the reaction to the air to stop the polymerization process. BMA polymer was precipitated in excess cold methanol and centrifugation at 3500 rpm for 20 min. The precipitate was washed twice with cold methanol and the supernatant was removed and the precipitated polymer was dried under vacuum at room temperature. The dried polymer was used for further analysis and copolymerization with DMAEMA monomer in the next step.

The NMR spectrum (Figure 2) was used to detect the number of BMA units (24 units) in each polymer chain by comparing the peak signal of the BMA monomer (highlighted with symbol A) to the peak signal of the CTA agent.

GPC technique was used to detect the molecular weight and the polymer polydispersity index (PDI) of the BMA polymer which were 3988 and 1.28 respectively (Figure 3).

1.2. Synthesis of the amphiphilic block polymer BMA-b-DMAEMA

In this step, the lipophilic block polybutylmethacrylate (PBMA) was copolymerized with 2- (dimethylamino)ethyl methacrylate (DMAEMA) monomer by RAFT control polymerization. Herein, BMA polymer holding CTA agent served as a macro-initiator for the Raft polymerization as shown in Scheme 2, where Y and CTA are as defined in Scheme 1.

Scheme 2

BMA-CTA polymer DMAEMA monomer BMA-b-DMAEMA polymer

The reaction was carried out under the same condition for the synthesis of BMA polymer with the exception that BMA-DMAEMA copolymer was precipitated in cold hexane. Table 1 shows the ratios at which BMA and DMAEMA were copolymerized with the symbols for each BMA: DMAEMA ratio. The PBMA: DMAEMA ratio was calculated based on the number of BMA monomer units in the block BMA polymer (24 units in this case) versus the number of molecules of DMAEMA monomer. Table 1 : Calculation of BMA-DMAEMA ratio in mole percent.

Figure 4 shows the BMA-DMAEMA amphiphilic copolymers at the ratios of (P1) 70:30, (P2) 60:40, (P3) 50:50, (P4) 40:60 and (P5) 30:70. It can be seen that the synthesized ratios were similar to the calculated ones indicating a successful control polymerization.

Table 2 shows the calculated versus the detected by the NMR ratios of BMA: DMAEMA (n:m).

Table 2: Calculated and detected ratios of BMA block polymer to DMAEMA block polymer and the molecular weights of each BMA-DMAEMA di-block copolymer.

1.3. Synthesis of BMA-DMAEMA-Sulfobetaine block copolymer (Betainisation of

DMAEMA)

The synthesis was carried out in THF where BMA-DMAEMA and 1 ,3 propane sultone were reacted in (1 :3) molar ratio (Scheme 3). The reaction was stopped after 72 hours and the precipitate was washed twice with acetone. Here, each ratio of BMA-DMAEMA copolymers (P1 , P2, P3, P4 and P5) was betainised by propane sultone to form the relative BMA-Sulfobetaine where BMA: Sulfobetaine ratios symbolised as follows: (P1S) 70:30, (P2S) 60:40, (P3S) 50:50, (P4S) 40:60 and (P5S) 30:70.

Scheme 3

Y and CTA are as defined in Schemes 1 and 2.

BMA-Sulfobetaine polymer

The synthesis of BMA-sulfobetaine was confirmed by IR and NMR spectra analysis. Indeed, IR spectral analysis was used to identify the structural transformation of the BMA polymer (Figure 5A) to BMA-DMAEMA copolymer (Figure 5B) then to BMA-sulfobetaine (Figure 5C) through identifying the main functional groups in each polymer. The complete betainisation (100% betainisation of DMAEMA amino group) was confirmed (in the next step) by the measurement of the zeta potential of sulfobetaine NPs which showed a slightly negatively charged NPs surface (~ -2) indicated a complete betainisation of the DMAEMA to form electrically neutral betainised block polymer. The yield of P1S (BMA (70: 30) Sulfobetaine) was very low and it was excluded from further studies (nanoparticles formation).

1.4. Formation of Zwitterionic sulfobetaine nanoparticles

Sulfobetaine nanoparticles (NPs) were formed by the nano-precipitation technique where the polymer is solubilised in a proper solvent then added dropwise into excess aqueous phase with mixing to allow the formation of nanoparticles by self-assembly. The solubilisation media consisted of solution of 2 M NaCI + methanol where different ratios of each solvent was used to dissolve each of the BMA-Sulfobetaine polymers (P2S, P3S, P4S and P5S) (l.e., the ratio of methanol to 2 M NaCI was varied depending on the ratios of BMA to the Sulfobetaine in each copolymer). Table 3 shows the ratios of solvents solubilisation where 5 g of each polymer was dissolved in 500 pi solubilisation media then added dropwise at a rate of 20 mI/min into a 5 ml pH 6.8 phosphate buffer. The media was further mixed for 45 minutes to allow the methanol evaporation then NPs suspension was dialysed using dialysis tube (molecular weight cut off (MWCO): 20000) against 500 ml PBS 6.8 for 4 hrs. For the purpose of particles tracking in mucous by the MPT technique, Lumogen® red dye was loaded into the NPs following the same nano precipitation method except that Lumogen® red was dissolved in methanol during the polymer solubilisation. After preparation, these NPs was freeze dried for clinical use and the particles size and zeta potential were measured.

Particle Size Measurement

Particle size was measured by photon correlation spectroscopy (Malvern Zetasizer NANO ZS, UK) with data collected in uni-modal setting. The instrument was standardised prior to each experiment by use of calibration standards. The Malvern system allows defining particle size distribution in form of intensity, volume and number distributions. In Table 4, particle sizes represent the Z-average particle sizes which are the intensity-based overall average sizes while PDI represents the polydispersity. Z- average particle size is the standard method to present particle sizes since the size distribution by intensity is obtained from an entirely different fitting scheme (instead of a force fit to one average size) where a simple Gaussian distribution is obtained in which the Z-average is the mean and the PDI is related to the width of this simple distribution. The PDI value depends on the type of nanoparticle and method of preparation with most nanoparticles falling within the range of 0.1- 0.6. A PDI value greater than 0.7 indicates sample has a very broad size distribution (ISO 13321 :1996, ISO 22412:2008 and ISO 22412:2017). The Malvern Zetasizer NANO ZS system identifies the quality of results as good if the polydispersity is acceptable. The Malvern system allows the distribution to be shown in volume or number form to obtain more detail about the sample.

It can be seen from Table 4 that PDI for these particles is around 0.4, this is mainly due to the mechanism of formation of these particles by the nano-precipitation technique where the solubilised polymer is added drop-wise into the external phase followed by rapid diffusion of solubilising media into the external phase leaving the polymer to undergo fast precipitation. This precipitation is very quick with low homogeneity leads to the formation of nano-sized particles with 0.4 polydispersity [6] Moreover, Table 4 shows these NPs have very small particle sizes. These small particles sizes are consistent with previous studies which showed that zwitterionic sulfobetaine NPs had particle sizes smaller than 50 nm [7,8] This finding can be interpreted depending on the chemical nature of the synthesized di-block copolymer. That is, the size and type of NPs formed from pre-synthesized di-block copolymers is highly affected by the molecular weight of the lipophilic and the hydrophilic block polymers [9] If the lipophilic block polymer is smaller than 9000 Da, micelles-like NPs are formed which are characterised by particle sizes as small as micelles [10,11] This is in agreement with the synthesized BMA- sulfobetaine in this study in which the molecular weight of the BMA lipophilic block polymer is 3500 Da (much less than 9000 Da). Hence, these sulfobetaine NPs should have small particle sizes (less than 50 nm).

Zeta Potential Measurement

A Malvern Zetasizer NANO ZS (Malvern, UK) was used to measure the zeta potential of the NP samples. The instrument was standardised prior to each experiment by use of calibration standards.

Table 4 shows the particle size and zeta potential of each sulfobetaine NPs in phosphate buffer pH 6.8 and in response to loading of Lumogen® red, freeze drying and storage of NP suspension up to 6 hrs after formation at 25 °C and 37 °C. It can be seen that all particles showed sizes lower than 50 nm with slightly negative to neutral surface charge. Also, the particle sizes and zeta potential did not change for all particles throughout the storage time scale up to 6 hours at room temperature and 37 °C indicating the stability of these particles for the time enough to exert biological action after oral administration. Also, this table shows that the freeze dried NPs retain their physicochemical properties after re-suspension in phosphate buffer aqueous phase indicating the re-suspendability of these particles and ease of storage of these NPs for future use and testing. Moreover, loading of Lumogen® red showed no impact on the particle sizes and zeta potential of sulfobetaine NPs (More loading studies were carried out in Examples 4 and 5.1).

Table 4: Particle sizes and zeta potentials of sulfobetaine NPs at external phase PBS pH6.8 and after Lumogen® loading, freeze drying, at PBS pH 7.4, and after 6 hr aggregation studies at 37 °C and 25 °C.

Example 2 - Diffusion coefficient of Sulfobetaine NPs through the mucous Barrier

Diffusion coefficient of particles was measured by the multiple particle tracking (MPT) technique. We first implemented the MPT assay, viewed in the field as the superior approach for the in-vitro assessment of NP mucous permeation. Herein, simply, Epifluorescence microscopy is used to capture the videos of the movements of the fluorescently labelled particles (Lumogen® red) inoculated into the mucous. Videos were analysed by special software (ImageJ) and particles’ movements were tracked and these trajectories were converted into mathematical values representing the displacements of particle in pixel. These displacements were converted into metric value to allow the measurement of particles diffusion in cm² per seconds. Table 5 shows the calculated diffusion coefficient in water by Stock-Einstein equation (D°), ensemble effective diffusion coefficients in mucous (<Deff>), percent ratio of diffusion coefficient in mucous to that freely diffusion in water (% ratio of <Deff> / D°) and the percent of diffusive particles of various formulae of BMA-sulfobetaine NPs.

Table 5: Diffusion coefficients in mucous, ratio of diffusion coefficient in mucous to that freely diffusion in water and the percent of diffusive particles of various formulae of BMA- sulfobetaine NPs. (D° is diffusion in water, <Deff> is ensemble diffusion coefficient in mucous).

It can be seen that these NPs showed direct correlation between diffusivity through the mucous and the degree of the shell hydrophilicity of these NPs represented by the ratio of the hydrophilic sulfobetaine to lipophilic BMA core (Table 5). I.e., while these NPs have close zeta potential and particle size (~ 0 mV, < 50 nm), their diffusivities are ranked based on the ratio of the muco-inert highly hydrophilic sulfobetaine to BMA. Moreover, the diffusivity across mucous barrier of these sulfobetaine NPs was compared with >150 different NP types ( in-vitro MPT mucous permeation assay) produced over four years by the ALEXANDER consortium, which comprises 15 of the leading oral NP pharmaceutical scientists across Europe (Figure 6). Depending on the state of art for NPs used in mucous delivery field, European partners synthesized around 150 NPs classified into six groups depending on the strategy adopted to enhance NP diffusion through the mucous barrier. These are (i) Slippery-PEGylated strategy: involving particles coated or copolymerized with PEG, (ii) Slippery-Amphiphilic polymer strategy: involving particles comprising amphiphilic polymers with the hydrophilic polymer at the surface and the lipophilic polymer at the core, (iii) Slippery polyelectrolyte strategy: involving particles comprising +ve and -ve charged polymers, (iv) Self-microemulsifying drug delivery (SMEDD) systems: involving microemulsion systems in which the effects of the various ingredients were studied, (v) Mucolytic NPs strategy: involving particles loaded with mucolytic agents, (vi) Thiolated NPs strategy: involving particles loaded with thiomers.

Our NPs were superior to all NPs representing the strategies that were adopted by the other partners in the Alexander consortium.

Specifically, Figure 6 shows that sulfobetaine NPs out-performed by up to x100-fold all of the current‘gold-standard’ particles, i.e. pegylated solid NPs or nanoemulsions. The capacity of sulfobetaine NP to rapidly penetrate the rate-limiting intestinal mucous layers will facilitates the delivery of cargo directly to the intestinal absorption surface (underlying epithelial surface). Besides that, sulfobetaine NPs were synthesised to mimic some muco-inert viruses with highly dense, hydrophilic and zwitterionic (electrically neutral) surface completely devoid of hydrophobic domains so the particles can slip through mucous in a non-destructive manner. These NPs should induce a relatively high bioavailability of the orally administered peptide/cargo if these cargos are well protected in the core of NPs. Hence, loading and release of various molecules were studied in the coming sections.

Example 3 - BMA-Sulfobetaine NPs to Treat Chronic Wound Infections (Microbial Biofilm) Following the successful performance of these novel NPs in mucous barrier, sulfobetaine NPs were tested as a delivery system through microbial biofilm for clinical unmet condition of chronic wound infection. Specifically, MPT technique was used where the diffusion coefficients of the all particles were confirmed to be measured within the bacterial biofilm by staining the biofilms and tracking the particles within the biofilm. Our novel NPs diffusion was examined across biofilm barrier of gram negative Pseudomonas aeruginosa bacterial biofilm (Hoiby biofilm model) and gram positive Methicillin-Resistant Staphylococcus aureus (MRSA) bacterial biofilm. This study was carried out comparatively with 40, 100, 200 and 500 nm negatively charged carboxylate fluosphere, and 200 nm positively charged amine fluosphere. Indeed sulfobetaine NPs was freely diffusive across the Hoiby biofilm with biofilm diffusion of only 12 times slower than its free diffusion in water. Moreover, our sulfobetaine NPs was faster by 1100 times compared with the +ve charged amino polystyrene NPs (Table 6) and 2.5 times faster than the 40 nm negatively charged fluosphere. This small difference between our novel NPs and negatively charged particles is due to the negative nature of the Pseudomonas aeruginosa biofilm leading to the high repulsion with the negatively charged fluosphere and apparent high diffusion of these particles. Accordingly, the diffusion coefficient of sulfobetaine NPs through the highly dense multi-resistant Staphylococcus aureus (MRSA) biofilm is shown in Table 7 where it is illustrated in comparison with 200 nm +ve charged amino fluosphere and different sizes -ve charged carboxylate fluosphere. It can be seen that the diffusion of our novel NPs is profoundly faster than the relatively small size (40 nm) -ve charged carboxylate fluosphere (6 times in MRSA compared with 2 times in Pseudomonas aeruginosa). The superior diffusion properties of sulfobetaine NPs across various bacterial biofilms makes these NPs a promising delivery system for medication in clinical conditions associated with bacterial biofilms like cystic fibrosis and chronic wound.

Table 6: Diffusion coefficient of 40, 100, 200 and 500 nm negatively charged carboxylate fluosphere, positively charged amine fluosphere and neutrally charged sulfobetaine NPs in water calculated by Stoke-Einstein equation versus their effective diffusion coefficients through Pseudomonas aeruginosa bacterial biofilm (PA01) measured by the MPT technique.

Table 7: Diffusion coefficient of 40, 100, 200 and 500 nm negatively charged carboxylate fluosphere, positively charged amine fluosphere and neutrally charged sulfobetaine NPs in water calculated by Stoke-Einstein equation versus their effective diffusion coefficients through Methicillin-Resistant Staphylococcus aureus (MRSA) bacterial biofilm measured by the MPT technique.

Example 4 - Loading and Release of Lipophilic Agent (Lumogen® Red) into Sulfobetaine NP

The very large lipophilic molecules (Lumogen® red) with molecular weight 1079 gm was selected to study the maximum loading capacity of sulfobetaine NPs to lipophilic cargo. The loading method was carried out as described in the NPs formation section on sulfobetaine NPs with the smallest hydrophilic sulfobetaine ratio (40% P2S) and NP with the biggest hydrophilic sulfobetaine ratio (70%, P5S) with aimed loading capacity of 50%. I.e. , The Lumogen® red weight is 50% of the NPs weight so 500 pg Lumogen® red was used for each 1mg polymeric NPs. The entrapment efficiency (EE%) and the loading capacity (LD%) were calculated by the following equations:

Table 8 shows EE% and LD% of these NPs toward the Lumogen® red (i.e., the capacity of incorporation of the lipophilic agent into the BMA core. While P2S showed very high EE% (82.26%) and LD% (40.33%), P5S showed lower EE% and LD% for Lumogen® red (4.53 % and 2.26 % respectively). It can be seen that loading capacity for sulfobetaine NPs was associated with the ratio of the lipophilic BMA core, i.e., the loading capacity increased from 2.26% to 40.33% when the BMA ratio increased from 30% to 60%. The NPs with high lipophilic BMA content (P2S) showed a very high loading capacity to load the lipophilic cargo due to the larger content of the BMA lipophilic core. Accordingly, P5S showed acceptable loading capacity toward a large lipophilic molecule like Lumogen® red indicating the suitability of these novel NPs for loading of lipophilic cargo.

Table 8: Entrapment efficiency and loading capacity of sulfobetaine NPs to Lumogen® red lipophilic dye at concentrations of 0.1 % and 50%.

Accordingly the in vitro release of the Lumogen® red was studied using the dialysis method. Briefly, freeze dried NPs loaded with Lumogen® red was re-suspended in phosphate buffer pH 6.8 (1 mg NPs per 1 ml buffer). Then, suspension was divided into 1 ml aliquots, each aliquot was added into dialysis tube (1 ml, MWCO: 20000) then dialysed against 500 ml PBS 6.8. In vitro release was studied up to 24 hrs where samples were collected at time intervals of 30 min, 1 , 2, 3, 4, 6, 8, 16 and 24 hr.

Figure 7 shows the in vitro release profile of Lumogen® red from formulae P2S and P5S where 7A shows the percent of release while 7B shows the cumulative amount in pg. Based on Figure 7A, both particles exhibited incomplete release profile of Lumogen® red after 24 hr, where P5S and P2S showed 80% and 48% release after 24 hr. P5S NP exhibited fast release (33%) within the first 2 hr followed by a gradual release within the time intervals between 2 and 8 hr. Oppositely, P2S showed a gradual release reaching to 30% after 8 hr. Both formulae showed almost a plateau release profile after 8 hr up to 24 hr. On the other hand, Figure 7B shows that the total cumulative release of Lumogen® red from P5S was 18 pg after 24 hrs which is equal to the amount released within the first 30 minutes for P2S. The observed huge difference between the release profiles in the 2 figures is associated with the high content of Lumogen® red in P2S (40.33) versus the content in P5S (2.26). The slow release profile of Lumogen red from these NPs is related to the high solubility of the lipophilic dye in the lipophilic core of the NP versus the low solubility in the hydrophilic external medium. This slow release is required for the delivery of drug through various routes since it gives prolonged protection of drug throughout the delivery process. The loading and in vitro release data show the suitability of these novel particles for the delivery of lipophilic agents.

Example 5 - Oral Delivery of Exenatide Using Sulfobetaine NPs

Based on the above mentioned MPT in-vitro studies, the effectiveness of these novel NPs for oral delivery of peptides was intended for further investigation. The candidate peptide was exenatide which is a hormone used for the treatment of type 2 diabetes (non-insulin dependent) which global prevalence is ca 7-8%. Due to the very low stability in the intestinal environment, exenatide is administered in injectable form to control the blood glucose level and promote central satiety leading to weight loss benefits. Hence, patient adherence to medication is critical and will be enhanced by a proper oral medication. Thus, these NPs were studied for their effectiveness to improve the in vivo pharmacokinetic and pharmacodynamics characteristics of exenatide. This technology should be applicable to a wide range of alternative markets demanding the oral delivery of peptides.

5.1. Loading of Exenatide (Hydrophilic peptide) into Sulfobetaine NPs

We dismissed the strategy of loading the hydrophilic exenatide solely into the outer hydrophilic shell. While this approach is readily achievable, it can be accompanied with a very fast in vitro release (a‘burst effect’) and NP surface-associated peptide lacks the necessary protection against the environment, e.g. acid conditions of stomach.

As an alternative, exenatide was modified into a more lipophilic molecule by ion-pairing with sodium dodecyl sulphate (SDS) to generate lipophilic exenatide laurate, where the sulphate ion of SDS have four potential ion-pairing sites within the exenatide molecule, (i.e. amino acids Hi, R20, Ki2,K27). At pH of 3.0 or 4.0 (dilute glacial acetic acid solutions), SDS and exenatide acetate were mixed at molar ratios of 3:1 , 4:1 and 5:1. As can be seen in Table 9, a molar ratio of 4:1 (SDS:exenatide) at a media pH 4.0 showed the highest yield of exenatide laurate (88.47%, ±0.29) which was accepted as the selected ion-pairing conditions for future work. Table 9 shows the effect of molar ratio (SDS:exenatide), pH of the media and the concentration of exenatide in the solution. (± s.d)

Table 9: The % of exenatide laurate yield as a result of ion pairing with SDS.

The encapsulation of the lipophilic exenatide laurate into the BMA lipophilic core of the NPs was carried out similarly to the method of Lumogen® red loading where exenatide laurate was dissolved in methanol aliquot prior to the mixing with 2M NaCI: methanol to solubilise the BMA-sulfobetaine polymer. Here, based on the visual appearance of the solution of 2M NaCI: methanol containing exenatide laurate and the polymer, 15% loading capacity was aimed i.e., 150 pg exenatide laurate per 1mg BMA-sulfobetaine polymer was dissolved in the solution of 2M NaCI: methanol since any increase in the concentration of the peptide resulted in unclear solution indicating dissolubility issues with exenatide laurate. Solubilised polymer/exenatide laurate mix was then added dropwise into an aqueous phase (PBS pH 6.8) to allow self-assembly of the NPs with incorporation of the exenatide into the NP core. The NP suspensions were then dialysed (dialysis tubing, MWCO: 20000) against PBS 6.8 for 4 hr and the exenatide EE% and LD% were measured. Table 10 shows the entrapment efficiency (EE%) of the NPs which exceeded 90% and loading capacity (LD%) exceeding over 13.5%.

Table 10: Chemical composition, codes, particle sizes, zeta potentials Entrapment efficiency and loading capacity of sulfobetaine NPs to the lipophilic exenatide laurate (n=3)

The loading capacity is significantly in excess of the acceptable industrial level (ca. 5%) and is an indication of the commercial potential of the NPs. Further data obtained from the in-vitro dialysis release studies showed no burst release of the exenatide into the external media (Figure 8) indicating the suitability of these NPs for the oral delivery of these peptides.

5.2 In vitro release of exenatide from Sulfobetaine NPs

Two series of in vitro release studies were conducted on these NPs; release over 8 hr in pH 6.8 buffer and release over 24 hr where the first two hrs was in buffer pH 1.2 (KCL, HCL) after which the NPs were transferred to pH 6.8 buffer (PBS) between 2 and 24 hr.

1. In vitro release of exenatide from NP over 8 hrs at pH 6.8

Studies on the in vitro release of exenatide from the various NP formulations, P2S, P3S, P4S and P5S, (and indeed the transfer of free exenatide acetate across the dialysis tubing) were carried out by the dialysis tube method (MWCO: 20 KDa) at 37 °C. All experiments involved 150 pg exenatide as the initial drug mass (either free in solution as exenatide acetate or loaded within NPs ca 1 mg of NP mass) in 1 ml dialysis tube. All these initial dialysis experiments were carried out over 8 hr duration with external media of PBS buffer pH 6.8.

Figure 8 shows the in vitro release profile of exenatide either free or from NPs (pH 6.8 throughout). Figure 8A shows the 8 hr cumulative release (pg) profile for P2S, P3S, P4S and P5S versus time with the lower panel showing the release over the first 2 hrs. The release rates over the first 8 hr varied between 4.21 to 5.08 pg/hr for formulations P2S to P4S while the NP formulation with the smallest BMA hydrophobic core, P5S, showed a significantly greater release rate of 8.85 pg/hr. By 8hr the formulations had released, respectively 17 to 22 pg exenatide for NPs P2S to P4S, while 40 pg exenatide was released by P5S. Figure 8B shows the corresponding data represented as % of loaded exenatide released. Over 8 hr the NPs P2S to P4S released 10-14% of the loaded material, while P5S had released 26% of the original exenatide loaded into the NPs (Figure 8B). The lower panels in both Figures 8A and 8B also show the transfer of free exenatide across the dialysis tubing over the first 2 hrs with recovery of approximately 90% of the material by this time.

2. In vitro release of exenatide from NP over 24 hrs with varying pH conditions of 1.2 and 6 3

Extended studies on exenatide from P5S formula were carried out for 24 hours where the first 2 hours was carried out in buffer pH 1.2 and from 2 to 24 hours in PBS pH 6.8 (Figure 9). The P5S formula was selected based on the high loading capacity and superior release of exenatide.

Comparing the data in Figures 8 and 9, a few conclusions can be drawn. Firstly, all the tested sulfobetaine NPs exhibited controlled progressive releases of exenatide with a lag time of 4 hrs prior to the more linear pseudo-steady-state release of exenatide. This non instantaneous release (burst effect) indicates the encapsulation of exenatide into the core rather than surface adsorption which is of great importance to prolog the protection of peptide from the intestinal environment.

Sulfobetaine P5S NP formulation showed an extended release throughout the 24 hr period. This release profile was characterised by a clear lag phase of ca 4 hr prior to the more linear pseudo-steady-state release of exenatide with only 1.2 pg (0.8% of loaded material) released after 2 hours and overall release of 43% after 24 hrs. Moreover, Exposing the P5S NP formulation to pH 1.2 for the first 2 hrs of release showed no significant difference to release at pH 6.8 indicating no effect of low pH (1.2) upon exenatide release characteristics. Thus, based on the loading and release data, P5S formulation was the most efficient carrier for exenatide oral delivery.

5.3 In vitro stability of exenatide to model intestinal enzymes

This protection in the lipophilic core of the NPs is particularly important for exenatide since our assessment of the intestinal enzymatic stability of this peptide showed abrupt degradation of exenatide when exposed to model intestinal enzymes. Incubation (thermo-mixer at 37 °C) of exenatide in the enzymatic solutions (PBS buffer) of 9.35 Ili/mL trypsin, 7.16 BTEE U/mL a-chymotrypsin and 0.29 I U/mL of elastase revealed: complete (100%) degradation of exenatide after only 2.5 mins in the chymotrypsin solution, with 33% degradation in the trypsin solution after 180 minutes.

5.4 PK-PD implications for in-vivo efficacy studies

PK-PD studies involve four treatment arms (Figure 10). PD studies include the measurement of glucose level in response to intraperitoneal (I.P) glucose challenge test (GCT) where 2 g/kg glucose is administered by I.P into rats then blood glucose level is measured up to 5 hrs after glucose administration by Glucometer AccuCheck Active. On the other hand, PK studies involve the measurement of exenatide plasma glucose level up to 10 hrs after exenatide administration without glucose administration (No GCT). The 4 arms in the PK-PD studies are (Figure 10): (i) I.P GCT PD studies alone (no treatment), (ii) PD studies in response to GCT and S.C. administration of exenatide solution and PK studies after S.C. administration of exenatide without glucose administration (No GCT), (iii) PD studies in response to GCT and oral administration of sulfobetaine NPs loaded with exenatide laurate and PK studies after oral administration of these NPs without glucose administration (No GCT), (iv) PD studies in response to GCT and oral administration of exenatide solution and PK studies after oral administration of exenatide solution without glucose administration (No GCT).

1. Pilot in-vivo PK-PD Studies

The aim of the pilot studies was to adjust the timing (lag time) between glucose I.P administration and exenatide administration and to identify the best quantification protocol for PK-PD studies. 1. Arm 1:“IP. GCT” PD study alone :

Pilot study was conducted to confirm the suitability of the measurement technique to detect the glucose level at time intervals of 0, 30 min, 1 hr, 1.5 hr, 2 hr, 3hr, 4 hr and 5 hr after I.P administration of 2 g/kg glucose to the experimental rats. This study showed that a volume of 40 pi blood samples was appropriate to measure glucose levels by Glucometer AccuChek® Active. Moreover, pilot study showed that the peak glucose level was reached 20 min after the I.P administration of glucose. The peak glucose level is important to identify the time at which exenatide should be administered orally or S.C. ii. Arm 2:“SC exenatide + I.P. GCT” PK and PD study:

Exenatide was administered S.C. at a dose of 20 pg at different time before the GCT. Pilot studies showed exenatide S.C. dosing can show significant effect on the glucose level if it is administered 10 min before the I.P glucose challenge test. Thus, a 10 min delay between injecting exenatide SC and the IP administration of glucose was set for the following full-scale studies. iii. Arm 3:“Oral Cardiff NPs with exenatide + I.P. GCT” PK and PD study.

Pilot study was conducted to detect the best lag time between oral administration of sulfobetaine NPs loaded with exenatide and the I.P GCT. 150 pg exenatide loaded into NPs was administered into a group of rats with lag time of 1 , 2, 3 and 4 hrs before the GCT. Lag time of 4 hrs was identified to have a significant effect on the glucose level after the GCT. iv. Arm 4:“Oral solution of exenatide + I.P. GCT” PK and PD study:

Similar to study in arm 3, lag times between 1-4 hrs were tested and 4 hrs lag time was selected between the administration of exenatide oral solution (150 pg) and the I.P GCT.

2. Full-Scale In-vivo PK-PD Studies on Rats

These studies were carried out based on the lag time and doses that were identified to be the best in the pilot studies. Glucose levels were measured using the technique (Glucometer AccuChek® Active) identified in the pilot studies. i. Arm 1:“I.P. GCT” PD study alone:

PD studies were performed on rats (n= 4). As was mentioned in the pilot studies, 40 mI blood samples were collected at time intervals of 0, 0.16, 0.5, 1 , 1.5, 2, 3, 4 and 5 hrs after glucose administration to immediately measure the blood glucose level by Glucometer AccuChek® Active. ii. Arm 2:“SC exenatide + I.P. GCT” PK and PD study:

PD studies were carried on n= 4 rats following the same abovementioned method with lag time of 10 minutes between exenatide and glucose administration. PK studies were carried out separately on another group of rats (n=3) where exenatide was given S.C. and 200mI blood samples were collected at time intervals of 0 min, 0,5 hr, 1 hr, 1 ,5 hr, 2 hr, 3,5 hr, 6 hr, 10 hr followed by plasma separation and exenatide plasma level was measured by luminescent immunoassay ELISA kit (Phoenix Pharmaceuticals Inc, linear range 1-10000 pg/ml, 50 mI plasma sample). iii. Arm 3:“Oral Cardiff NPs with exenatide + I.P. GCT” PK and PD study:

PD studies were carried on n= 4 rats following the same abovementioned method with lag time of 4 hrs between exenatide and glucose administration. PK studies were carried out similarly to arm 2. iv. Arm 4:“Oral solution of exenatide + I.P. GCT” PK and PD study :

PD and PK studies were performed following the same procedures in arm 3.

5.5 Glucose Measurement (PD) Studies:

The data obtained from glucose measurement were used to indicate the effect of different routes and formulations on the pharmacological effect of exenatide. Firstly, comparing the blood glucose level at time zero of the 4 treatment arms revealed that oral administration of sulfobetaine NPs containing exenatide reduced the blood glucose level significantly compared with GCT alone with no treatment (Figure 11). I.e., Cardiff oral NPs loaded with exenatide reduced blood glucose level before the administration of IP glucose GCT. This indicates that 4 hrs lag time was too long where our NPs could deliver enough amount of exenatide to reduce blood glucose level.

Figure 12 shows the absolute blood glucose levels at time interval 0, 0.16, 0.5, 1 , 1.5, 2, 3, 4 and 5 hrs of rats subjected to the 4 treatment arms (time 0 is the time immediately before glucose administration by I.P route, while remaining time intervals are post glucose administration). It can be seen that exenatide oral solution given 4 hrs before the GCT (green line) showed no effect on the glucose levels compared with the control GCT alone (blue line). On the other hand, both of S.C. exenatide solution (red line) and sulfobetaine exenatide NPs (black line) showed significant effect on the blood glucose levels compared with GCT alone. Thus, comparing blood glucose levels following sulfobetaine exenatide NPs and GCT alone exhibit significant difference from time intervals 1.5 hrs up to 5 hrs. Similarly, S.C.. exenatide solution reduced significantly blood glucose level from time intervals 1 hr up to 5 hrs compared with GCT alone. This indicates the efficiency of sulfobetaine particles to significantly improve the pharmacological effect of exenatide compared with the no treatment and oral solution of exenatide. I.e., Oral administration of exenatide sulfobetaine NPs showed significantly efficient reduction of blood glucose level compared with the S.C. dose.

Relative Bioavailability of oral exenatide nanoparticles versus exenatide S.C. dose was calculated by the following equation:

In this equation, glucose AUC was used to identify the pharmacological effect of orally administered sulfobetaine exenatide NPs compared with the commercial S.C. route. The relative bioavailability was 12.3% which is much higher than the industrially acceptable bioavailability (5%) This is another indication about the effectiveness of these particles in oral delivery of peptides.

5.6 Plasma Exenatide Measurement (PK) Study

As was described above, exenatide plasma concentration was measured for orally administered exenatide sulfobetaine NP versus S.C exenatide solution to calculate exenatide bioavailability and to add value and substantiate the data obtained from PD studies. Following exenatide administration, blood samples were collected at time intervals of 0, 0.5, 1 , 1.5, 2, 3.5, 6 and 10 hrs. Blood samples were processed to separate plasma for measurement of exenatide and stored at -70_°C. Extraction of exenatide from plasma samples was carried out following the protocol from phoenix pharmaceuticals, where plasma samples were acidified by equal volume of acidic buffer (supplied by Phoenix) and centrifuged for 20 minutes at 17,000 x g (4°C). Supernatant was collected and loaded into C18 column which was pre-treated with the same buffer. Exenatide was eluted from the C18 column using another buffer supplied by phoenix pharmaceuticals. Then, exenatide buffer solution was freeze dried and exenatide concentration was measured by Chemiluminescent Elisa technique (phoenix pharmaceuticals, inc). The recovery of exenatide from the column extraction method was found to be 40.8%. This recovery % was used to measure the actual concentration of exenatide in plasma. Following the assessment procedures, exenatide plasma levels from rats’ blood samples were measured. The concentration-time curve was plotted and AUCs were calculated by trapezoidal rule for all exenatide systems. Relative bioavailability was measured by the following equation:

([AUC]oral * [Dose]S.C /[AUC]S.C * [Dose]oral) * 100.

The relative bioavailability of exenatide sulfobetiane NPs versus S.C exenatide solution was 12.99%. Figure 13 shows the exenatide concentration-time curves and bioavailabilities of oral exenatide Cardiff NPs versus S.C exenatide solution.

Example 6. Cytotoxicity study of Sulfobetaine NPs

6.1 MTT Cytotoxicity Study

The superb properties of sulfobetaine NPs were shown through the loading, in vitro and in vivo studies on oral delivery of exenatide peptide. The safety of these NPs for biological application is important requirement to be proven by studying the cytotoxicity of sulfobetaine NP.

The cytotoxicity of sulfobetaine NP was tested in epithelial Madin-Darby canine kidney (MDCK) cell line using MTT viability assay. MDCK Cells (passage 7) were grown in standard conditions (humidified incubator at 37°C with 5% C02) using DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin. MDCK cells were seeded in 96-well plates at a density of 5*10⁴ cells/well (100 pi culture medium per well) and cultured for 24 hrs. Next day, well plate was examined under microscopy for cells confluency. Freeze dried sulfobetaine NPs sample was re-suspended in the same MDCK culture medium and diluted serially to prepare a concentration range of NPs suspensions between 1 mg/ml to 10 ng/ml. 100 mI of each NPs suspension was added in triplicate by replacing the 100 mI medium in each well. Besides NPs, three controls were used, these are: (i) Cells without NP to determine the 100% cells viability (ii) Medium without cells or NP (to determine the background) (iii) Cells without NP but with 1% Triton (positive control for 0% cells viability).

After the addition of NPs suspensions and controls, well plate was incubated for 24 hrs. After the incubation period, 10 mI of the MTT labelling reagent (Sigma-Aldrich) was added into each well and the well plate was incubated for 4 hrs (Only viable cells can enzymatically cleave the MTT reagent into insoluble formazen crystals). This step is followed by the addition of 100 mI solubilisation buffer solution (Sigma-Aldrich) into each well followed by incubation for 24 hrs to solubilise the purple formazan crystals. Lastly, the absorbance of well plate was measured at wave length between 550 and 600 nm and reference wavelength of 650 nm using Elisa reader (LT-5000 MS, Taiwan). The absorbance value represents the concentration of formazen solution that is formed by viable cells. The % of viable cells for each NPs concentration versus cells alone (the 100% viable cells) was determined by the following equation:

% -

Where CNP represents the cells with NPs suspension, M is the media only without cells or NPs to subtract the background, C is the cells with media (no particles) representing 100% viability.

Figure 14 shows the viability of cells in response to a range of NPs concentrations versus no particles (cells only, 100% viability) and 1 % triton (0% viability). The maximum NPs concentration was selected depending on the maximum possible concentration that these NPs can reach in the body. It can be seen that there is no significant difference in cell viability among all the concentrations of NPs versus the 100% cells viability (no particle in the media) indicating that these NPs are nontoxic and can be administered safely.

6.2 LDH Cytotoxicity Study

The cytotoxicity of the novel sulfobetaine NPs were further studied by the European Nanomedicine Characterisation Laboratory (EUNCL) following their in vitro preclinical characterization cascade. EU-NCL carried out lactate dehydrogenase (LDH) assays which measures the membrane integrity since LDH enzyme is released into the cytoplasm upon cell lysis. LDH assay was performed on 2 cell types: hepatocarcinoma cells (Hep G2) (Liver carcinoma cell line is commonly used as model for liver toxicity) and porcine proximal tubule cells (LLC-PK1). Briefly, both cell types were seeded in 96- well plates (100 pi of 2.5-5 x 10⁵ cells /ml and incubated for 24 hr at 5% C02, 37°C and 95% humidity). Cells were treated with sulfobetaine NPs for up to 48 hrs by adding 100 mI of NPs suspensions at range concentrations of 0.01-650 pg/ml (cytotoxicity was measured for up to 48 hrs). After the incubation period, 100 mI was transferred from each well plate into another 96 well plates to which LDH reagent was added and incubated for 20 minutes followed by reading the absorbance (plate reader) at 490 nm using a reference wavelength of 680 nm. In this test, 0.1 % Triton was used as positive control and toxicity was measured using the following equation: Figure 15 shows the % of LDH leakage versus different NPs concentrations incubated with cells for 48 hrs. Both Figure 1A and 1 B show no change in cell membrane integrity of both LLC-PK1 and Hep G2 cell lines correspondingly. These data are in agreement with our in house cytotoxicity MTT test.

Example 7. Loading and release of a hydrophilic agent across bacterial biofilms

The ability to load a hydrophilic agent into the shell of the sulfobetaine NPs and the ability to deliver across bacterial biofilms were investigated. Polymyxin B is an antibacterial agent used as a model for treatment of bacterial biofilms in chronic wounds. This hydrophilic agent consists of 9 amino acid with a molecular weight of 1 ,301 g/mole which makes it a proper candidate to study the loading capacity of sulfobetaine NPs’ shell toward hydrophilic agents and their delivery across biological barriers. To load this agent into the shell, a different loading strategy was selected to enable the separation of loaded polymyxin from unloaded free agent (i.e. the strategy was selected to employ the centrifugation method to separate the unloaded polymyxin from the centrifuged NPs loaded with polymyxin). Alternatively, all trials to load polymyxin into sulfobetaine NPs using dialysis tubes or ultra-centrifugation method resulted in failure to separate unloaded polymyxin since polymyxin is small peptide that can stick to the dialysis membranes and ultra-centrifugation filter. Hence, in this study, sulfobetaine polymer was dispersed firstly in 2 M NaCL (1 mg/50 pi) then overnight in PBS (1 mg/ml) to form clear suspension of NPs. Formed NPs was mixed with excess pre-weighed polymyxin for 4 hrs then NPs were centrifuged at 14000 rpm for 30 minutes. Unloaded polymyxin was measured in the supernatant solution using the bicinchoninic acid (BCA) assay. The LD% of NPs towards polymyxin was calculated to be (11.74% ±0.98, n=3). The LD% of particles to load hydrophilic agents into the shell is in accordance with capacity of these particles to encapsulate the modified lipophilic agent (exenatide laurate) into the core.

7.1 Antibacterial activity of loaded polymyxin versus unloaded polymyxin:

Delivery of loaded polymyxin into sulfobetaine NPs across bacterial biofilms

The antibacterial activity of loaded polymyxin versus free polymyxin was carried out by SINTEF (Norway) where the minimum inhibitory concentration (MIC) was calculated. In this study, measuring the antibacterial activity of polymyxin reflects its delivery across the bacterial biofilm since bacteria were grown overnight which is enough time for bacteria to grow biofilms. Thus this study reflects the antibacterial/anti-biofilm activity of loaded polymyxin into the NPs. The studied bacterial strains were P. aeruginosa ATCC15692 (PA01 , non-mucoid) and mucoidal P. aeruginosa ATCC39324. The strains were grown over night in Tryptic soy broth (TSB) medium then these pre-cultures were diluted to an optical density of 0,1 at wavelength 600 nm (OD600) in TSB, before another dilution step of 40x in Muller-Hinton medium. 30 pi were then inoculated to 96-well plates containing 120 mI Muller-Hinton medium with free polymyxin, nanoparticles and nanoparticles loaded with polymyxin.

Serial dilutions of polymyxin for both free and loaded polymyxin were made starting at 8 pg/ml as the highest concentration down to 0.007813 pg/ml as the lowest concentration. As a control, empty nanoparticles in the same concentration were diluted and used to investigate whether the nanoparticles alone influenced on bacterial growth. Accordingly, a control without polymyxin was included giving information of free growth of the bacteria. After inoculating the different media with bacteria, the plates were incubated at 37°C in plastic bags for 19 hours. OD600 were measured and the influence of the different conditions on bacterial growth determined (Figure 16).

Figure 16A, 16B and 16C shows the effect of free polymyxin, loaded polymyxin into sulfobetaine NPs and unloaded NPs correspondingly on non-mucoidal (black bars) versus mucoidal P. aeruginosa (grey bars). Figure 16 shows NPs alone had no effect on bacterial and bacterial biofilm growth. Importantly, polymyxin loaded into sulfobetaine NPs had substantial impact on the P. aeruginosa compared with free polymyxin especially with mucoidal P. aeruginosa where MIC was significantly different for polymyxin loaded into NP versus free polymyxin at concentrations of 0.25, 0.5 and 1 pg/ml. This indicates that sulfobetaine NPs improved the permeation of loaded polymyxin across the mucoidal biofilm of P. aeruginosa versus free polymyxin which resulted in lower MIC for loaded polymyxin versus free polymyxin.

References:

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Claims

1. A nanoparticle comprising a polymer, wherein the nanoparticle has an average diameter of about 20 to 70 nm, determined on an intensity basis, and a polydispersity index of 0.

2 to 0.6, and wherein said polymer comprises a polymer chain of general formula (I):

wherein

R¹ is H or methyl;

R² is Ci-e alkyl

R³ is H or methyl;

R⁴ is -(CH₂)_P-N⁺(CH3)2-(CH₂)3-S(0)20- or -(CH₂)_P-N(CH₃)2;

p is an integer of 1 to 4;

n is an integer of 15 to 30; and

m is an integer wherein the ratio of n:m is from 80:20 to 20:80;

provided that at least 90% of the R⁴ groups are -(CH₂)_P-N⁺(CH₃)₂-(CH₂)₃-S(0)₂0\ 2. A nanoparticle according to claim 1 , wherein said polymer consists essentially of the polymer chain of general formula (I) as defined in claim 1 and terminated at each end by small organic groups.

3. A nanoparticle according to claim 1 or claim 2, wherein said polymer is of general formula (Iz):

wherein R¹, R², R³, R⁴, m and n are as defined in claim 1 ;

X is a residue of a chain transfer agent; and

Y is a residue after polymerisation of a (meth)acrylate monomer of formula (II):

wherein R¹ and R² are as defined in claim 1.

4. A nanoparticle according to claim 3, wherein X is represented by the formula:

where - is the link to the polymer and R is as defined above for the CTA.

5. A nanoparticle according to any one of claims 1 to 4 wherein, independently or in any combination:

R¹ is methyl;

R³ is methyl;

R¹ and R³ are methyl;

R² is C3-6 alkyl;

n is from 20 to 28;

the ratio of n:m is from 30:70 to 70:30; in the group R⁴, p is 1 to 3.

6. A nanoparticle according to any one of claims 1 to 5 wherein R² is n-butyl.

7. A nanoparticle according to any one of claims 1 to 6 wherein n is from 22 to 26.

8. A nanoparticle according to any one of claims 1 to 7 wherein in the group R⁴, p is

In the group R⁴, p is 2.

9. A nanoparticle according to any one of claims 1 to 8, wherein at least 99% of the

R⁴ groups are -(CH₂)_P-N⁺(CH₃)₂-(CH₂)₃-S(0)₂0 , wherein p is as defined in claim 1.

10. A nanoparticle according to claim 9 wherein 100% of the R⁴ groups are -(CH2) - N⁺(CH3)2-(CH2)3-S(0)20 such that the polymer comprises a polymer chain of general formula (la):

11. A nanoparticle according to any one of claims 1 to 10, wherein said polymer is: a polymer comprising a polymer chain of general formula (lb):

wherein R¹ , R², R³, R⁴, m and n are as defined in claim 1 ; and

R⁴ is -(CH₂)₂-N⁺(CH₃)₂-(CH₂)₃-S(0)₂0- or -(CH₂)2-N(CH₃)2, wherein at least 90% of the R⁴ groups are -(CH₂)₂-N⁺(CH₃)₂-(CH₂)₃-S(0)₂C>-; or a polymer comprising a polymer chain of formula (lc):

wherein R¹ , R², R³, m and n and are as defined in claim 1.

12. A nanoparticle according to any one of claims 1 to 11 , wherein said polymer has a polydispersity index of not greater than 1.4, wherein polydispersity index is defined by the equation:

where M_w is the weight average molecular weight and M_n is the number average molecular weight.

13. A nanoparticle according to any of claims 1 to 12 loaded with a biologically active agent.

14. A nanoparticle according to claim 13, wherein the biologically active agent is selected from: Peptides, proteins and antimicrobial agents.

15. A nanoparticle according to claim 13 or claim 14 wherein the biologically active agent is encapsulated in the lipophilic core of the nanoparticle.

16. A polymer for use in a nanoparticle according to any one of claims 1 to 12.

17. A process for the preparation of a polymer according to claim 16 comprising:

(i) polymerising a monomer of general formula (II):

wherein R¹ and R² are as defined in claim 1 ;

by RAFT polymerisation in the presence of an initiator and a chain transfer agent (CTA) to form a polymer comprising a polymer chain of general formula (III):

wherein R¹, R² and n are as defined for general formula (I); (ii) copolymerising the polymer comprising the polymer chain of general formula (III) with a monomer of general formula (IV):

wherein R³ is as defined in general formula (I) and

R⁵ is -(CH₂)_P-N(CH₃)2, where p is as defined in claim 1 ;

by RAFT polymerisation to form a polymer comprising a polymer chain of general formula (V):

wherein R¹, R², R³, n and m are as defined in claim 1 and R⁵ is as defined for general formula (IV); and (iii) reacting the polymer comprising the chain of general formula (V) with 1 ,3- propane sultone to form a polymer comprising a chain of general formula (I) as defined in claim 1.

18. A process according to claim 17 wherein, independently or in any combination: a. in step (i), the ratio of the monomer of general formula (II) to CTA is from about 15:1 to 35:1 ; b. in step (i) the radical polymerisation initiator is 2,2'-azobis(2-methylpropionitrile) (AIBN); c. step (ii) is carried out using the dried polymer comprising a chain of general formula (III); d. in step (ii), the amount of the monomer of general formula (IV) is selected such that the molar ratio of monomer of general formula (II) to monomer of general formula (IV) used to form the polymer comprising the chain of general formula (V) is from 80:20 to 20:80, in order to give a ratio of n:m as defined in claim 1.

19. A process for the preparation of a nanoparticle according to any one of claims 1 to 15 comprising the steps of:

(i) solubilising a polymer recited in any one of claims 1 to 12 and, optionally, a biologically active agent in a suitable solvent to form a polymer solution;

(ii) adding the polymer solution dropwise into an aqueous phase with mixing to allow formation of nanoparticles by self-assembly.

20. A pharmaceutical composition comprising a nanoparticle according to any one of claims 1 to 15 and a pharmaceutically acceptable carrier or vehicle.

21. A nanoparticle according to any one of claims 1 to 15 or a pharmaceutical composition according to claim 20 for use in delivering a biologically active agent across a mucous membrane or a bacterial biofilm.

22. Use of the nanoparticle according to any one of claims 1 to 15 or a pharmaceutical composition according to claim 20 in the preparation of an agent for use in delivering a biologically active agent across a mucous membrane or a bacterial biofilm.

23. A method for delivering a biologically active agent across a mucous membrane or a bacterial biofilm, the method comprising administering to a patient in need of such treatment an effective amount of nanoparticles according to any one of claims 1 to 15 or a pharmaceutical composition according to claim 20.

24. A nanoparticle or composition according to claims 20-21 , a use according to claim 22 or a method according to claim 23 wherein:

the mucous membrane is the lining of the gastro-intestinal tract, the lungs or the genito urinary tract and the biologically active agent comprises a biologically active peptide; or the bacterial biofilm is found in a wound and the biologically active agent is an antibacterial agent.

25. A nanoparticle or composition for use, a use or a method according to claim 24 wherein:

the active agent is exenatide or insulin and the nanoparticle is for use in the treatment of diabetes; or

the active agent is leucine encephalin and the nanoparticle is for use in the treatment of pain; or

the active agent is a proapoptotic peptide or cilengitide and the nanoparticle is for use in the treatment of cancer.

26. A nanoparticle or composition for use, a use or a method according to claim 24 or 25 wherein the nanoparticle or pharmaceutical composition is formulated for oral administration.

27. A nanoparticle or composition for use, a use or a method according to claim 24 wherein the active agent is an anti-microbial agent and the nanoparticle is for the treatment of a microbial infection.

28. A nanoparticle or composition for use, a use or a method according to claim 27 wherein the nanoparticle or pharmaceutical composition is formulated for topical administration.

29. A process for preparing a pharmaceutical composition comprising bringing a nanoparticle according to any one of claims 1 to 15 in conjunction or association with a pharmaceutically acceptable carrier or vehicle.