NANOSTRUCTURES SUITABLE FOR DELIVERY OF AGENTS

Field of the Invention

The present invention relates to novel nanostructures suitable for delivery of pharmaceutical agents and complexes comprising the same. The present invention further extends to methods of making the nanostructures and use of such nanostructures to deliver pharmaceutical agents for use in a range of diagnostic, prophylactic, therapeutic, nutritional and/or research applications.

Background of the Invention

Many pharmaceutical agents, particularly protein drugs and vaccines are administered by parenteral delivery such as injection. The disadvantages associated with this form of administration include a potential lack of patient compliance, discomfort, expense, the need for training for administration and potential adverse reactions at the site of injection. A more desirable administration route for pharmaceuticals is an oral delivery system, however there are major challenges associated with oral delivery of pharmaceuticals. Successful oral delivery via the gastrointestinal tract of a host is hindered by a number of physical and chemical barriers, including degradation of the pharmaceutical agent by enzymes present in the gastrointestinal tract and poor permeability of pharmaceutical agents across the intestine to reach the systemic circulation and sites of action. Thus, an effective oral delivery system must be able to withstand the attack of endogenous enzymes, gastric acidity and intestinal alkalinity without losing activity, as well as being able to penetrate and cross the gastrointestinal mucosa and enter into the blood stream, thereby delivering the pharmaceutical to the site where activity is required. Moreover,

the delivery must take place at an appropriate rate to ensure the correct therapeutic dosage is delivered.

Targeted oral delivery of pharmaceutical agents such as peptides and proteins has in the past utilized various solid particulate systems in which the pharmaceutical is entrapped during particle preparation. For many proteins entrapment within such systems often leads to degradation of the pharmaceutical due to the use of organic solvents, cross- linking of the pharmaceutical to the particle matrix, or desolvation of the pharmaceutical.

In addition, the solid particles only passively and non-specifically entrap the pharmaceutical agent. These particles are also highly polydisperse meaning that some of the particles may be too big for uptake via intestinal epithelial cells, or alternatively some particles may be too small to effectively encapsulate the drug to be delivered. As a result, an accurate uptake of the pharmaceutical agent by a subject may not be readily determined, rendering the particles unsuitable for clinical application. The reason for this is that many drugs exhibit a narrow pharmaceutical index, meaning that an under supply of drug results in no pharmaceutical effect, while an over delivery of the drug results in a toxic dose. For example, an overdose of insulin will result in hypoglycemic shock, whereas an under supply results in ineffective control of blood sugar levels.

There is therefore a need for particles capable of carrying pharmaceutical agents that overcome or substantially ameliorate at least some of the deficiencies associated with known particles. Recently, nanotechnology has provided tools of constructing biostructures in a nano scale and such nanostructures can be applied in a variety of areas. In particular there is a need for improved nanostructures that can include the delivery of increased or amplified amounts of pharmaceutical agents per particle.

Suφrisingly, the inventors have produced multi-layer nanostructures which can entrap and deliver pharmaceutical agents without the use of solvents or covalent cross-linking agents during the process of entrapment. The nanostructures of the invention involve the use of water-in-oil microemulsions to form pharmaceutical agent-containing core particles that are associated with a polymer coating or cover. Diagnostic agents and pharmaceutically active agents, in particular proteins and peptides, are able to be entrapped at room temperatures allowing for the incorportation of heat-sensitive proteins. The size of the structures of the invention is particularly suitable for oral delivery and also for intravenous injection as their size is small enough for uptake into the endosomes of the intestinal epithelial cells following enteral administration, and sufficiently small to prevent non-specific trapping in the lung following parenteral injection.

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Summary of the Invention

Suφrisingly the present invention provides nanostructures capable of carrying a pharmaceutical agent. The nanostructures have a core comprising a particle which includes a pharmaceutical agent and a cover comprising a polymer. The nanostructures of the invention also have a desired size and PDI. Suφrisingly, the present invention also provides methods for the preparation of the nanostructures of the invention involving micro-emulsion technology for the formation of the nanostructures containing the pharmaceutical agent.

According to an aspect of the invention there is provided a nanostructure capable of carrying a pharmaceutical agent, the nanostructure having a core comprising a particle which includes the pharmaceutical agent and a cover comprising a polymer.

In an embodiment, the core particle comprises a chelated pharmaceutical agent.

In another embodiment, the pharmaceutical agent is a peptide, protein, polysaccharide, nucleic acid, oligonucleotide, sugar, nutrient, vitamin, mineral or antioxidant.

In yet another embodiment, the pharmaceutical agent is insulin, a nucleic acid or an anti- TNF protein.

In another embodiment, pharmaceutical agent is chelated by zinc, calcium, iron, magnesium, manganese or selenium, preferably zinc (Zn ²⁺ ) or calcium (Ca ²⁺ ).

In yet another embodiment, the pharmaceutical agent is a protein or peptide.

In another embodiment, the core particle comprises a protein or peptide precipitated with zinc hydroxide.

In yet another embodiment, the polymer is a polysaccharide.

In another embodiment, the polysaccharide is dextran, alginate, chitosan or a derivative thereof, and preferably the dextran is carboxy methyl dextran.

In a further embodiment, the polymer is associated with the core particle.

In another embodiment, one or more targeting moieties capable of being bound by, or binding to an intestinal epithelium of the subject are associated with the polymer or the nanostructure.

In a further embodiment, the targeting moiety is covalently bound to the polymer either directly or through a spacer or linker compound.

hi another embodiment, the targeting moiety interacts with the nanostructure by non- covalent means with or without linkers, spacers or other derivatisation.

hi a further embodiment, the targeting moiety is attached to the nanostructure via a pendant side chain of the nanostructure.

In another embodiment, the targeting moiety is biotin, vitamin Bi ₂ , riboflavin, folate or a derivative thereof.

Li a further embodiment, the nanostructure has a polydispersity index (PDI) from about 0.1 to about 0.4.

In another embodiment, the nanostructure has an average cross-sectional length of about 20 - 500 nm.

In a further embodiment, the nanostructure according to the invention is for oral delivery.

In another aspect of the present invention there is provided a process for preparing nanostructures capable of carrying a pharmaceutical agent, said method comprising:

(a) preparing separate mixtures comprising:

(i) a microemulsion including a chelating agent; and (ii) a microemulsion including a pharmaceutical agent; and

(b) combining mixtures (i) and (ii) from step (a) to form mixture (iii);

(c) preparing a further mixture comprising

(iv) a microemulsion including a polymer;

(d) combining mixtures (iii) and (iv) to form mixture (v) including the nanostructures; and

(e) separating the nanostructures from mixture (v).

In still another aspect of the present invention there is provided a process for preparing nanostructures capable of carrying a pharmaceutical agent, said method comprising: (a) preparing separate mixtures comprising:

(i) a microemulsion comprising a charged surfactant and a non-ionic surfactant and including the pharmaceutical agent and a multivalent cation; and

(ii) a microemulsion comprising a non-ionic surfactant and including a base; and (b) combining mixtures (i) and (ii) from step (a) to form within mixture (iii) core particles; (c) preparing a further mixture comprising

(iv) a microemulsion including a polymer;

(d) combining mixtures (iii) and (iv) to form within mixture (v) the nanostructures; and

(e) separating the nanostructures from mixture (v).

In a preferred embodiment to the aspect above, the pharmaceutical agent is a protein or peptide.

In another preferred embodiment, the charged surfactant is an alkyl betaine.

In still another preferred embodiment, the multivalent cation is zinc (Zn ²⁺ ).

In another preferred embodiment, the base is sodium hydroxide or potassium hydroxide.

In an embodiment, the polymer is associated with the core particle to form the nanostructure.

In another embodiment, the nanostructures may further comprise surface modification by one or more reagents to provide one or more pendant side chains.

In a further embodiment, the microemulsions are water-in-oil microemulsions.

In another embodiment, one or more targeting moieties capable of being bound by, or binding to an intestinal epithelium of the subject are associated with the polymer or nanostructure.

In a further embodiment, the targeting moieties allow uptake and internalisation of the nanaostructure.

In another embodiment, the one or more targeting moieties are covalently bound to the polymer either directly or through a spacer or linker compound.

In a further embodiment, the targeting moieties interact with the nanostructure by non- covalent means with or without linkers, spacers or other derivatisation.

In another embodiment, the targeting moieties are attached to the nanostructure via a pendant side chain of the nanostructure.

According to another aspect of the invention there is provided a nanostructure prepared by a process of the invention.

According to still another aspect of the invention there is provided a pharmaceutical composition comprising a nanostructure of the invention in association with a pharmaceutically acceptable carrier and/or diluent.

According to another aspect of the invention there is provided a method of delivering a pharmaceutical agent to a subject in need of such pharmaceutical agent, said method comprising administering of a therapeutically effective amount of the nanostructure of the invention.

In an embodiment, the pharmaceutical agent is insulin, a chemotherapeutic agent, an anti- inflammatory agent, an imaging or diagnostic agent, a monoclonal antibody, an Fc-fusion protein or a nucleic acid.

In another embodiment, the pharmaceutical agent is a nucleic acid, a protein or a peptide.

In a further embodiment, the pharmaceutical agent is delivered orally.

In further embodiments, the nanostructures can also be for subcutaneous delivery, intravenous delivery or pulmonary delivery.

According to another aspect of the invention there is provided a method for the treatment of diabetes comprising the step of administering a nanostructure of the invention to a subject in need of such treatment.

According to yet another aspect of the invention there is provided a method for the treatment of cancer comprising the step of administering a nanostructure of the invention to a subject in need of such treatment.

According to still another aspect of the invention there is provided the use of a nanostructure of the invention in the manufacture of a medicament.

Further aspects relate to the use of the complex of the invention for the manufacture of a medicament, and in particular medicaments for the treatmens of cancer and proliferative disorders, inflammation, cardiovascular diseases, diabeties and as an imaging or diagnostic agent or the delivery of monoclonal antibodies and Fc-fusion proteins.

Advantages of the nanostructures of the invention include the delivery of increased or amplified amounts of pharmaceutical agents per particle. It is also thought that the polymer coating provides some protection to the pharmaceutical agent in the core particle.

These and other aspects, embodiments and preferments of the invention are further detailed in the description below and the claims that follow.

BRIEF DESCRIPTION OF THE FIGURE

Figure 1 graphically represents the mean particle size and distribution of a nanostructure containing insulin prepared according to Example 11.

Figure 2 graphically represents the mean particle size and distribution of a nanostructure containing etanercept prepared according to Example 15.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

It is to be understood that unless otherwise indicated, the subject invention is not limited to specific formulations, manufacturing methods, diagnostic methods, assay protocols, nutritional protocols, or research protocols or the like as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in the field of endeavour.

All cited references and publications referred to herein are incorporated by reference.

In the context of this specification, the terms "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

The term "nanostructure" or "nanoparticle" as used herein generally referrs to particles on the nano scale having a diameter of from about 20 to 500 nm.

As used herein, the term "non-polar phase" refers to a phase comprising a solvent with a dipole moment below 1.35 Debye and a dielectric constant below 6.0. In certain

embodiments, such solvents make up at least 10%, preferably at least 50%, more preferably at least 80% or even at least 97%, of the non-polar phase.

As used herein, the term "polydispersity index" (PDI) refers to an index of the ratio of the standard deviation of the size of the nanostructures to the mean of the size of the nanostructures. Preferably the polydispersity index is in the range of 0.1 to 0.4, and more preferably the PDI is between 0.15 and 0.3

The physical characteristics of the nanostructures of the present invention can be evaluated using one or more of the following systems.

Mean particle size of the nanostructures can be determined by dynamic light scattering (DLS). DLS is particularly suited to determining small changes in mean diameter such as those due to adsorbed layers on particle surfaces or slight variations in manufacturing processes. Cumulative measurements can then be used to determine polydispersity index.

Surface morphology of nanostructures can be visualized by scanning electron microscopy. Typically, freeze-dried samples of nanostructure are coated by platinum prior to observation using an SEM. Various measurements can then be performed via image analysis using standard techniques.

DLS and SEM measurements can be used to infer parameters such as average cross- sectional length, the sum average of two or more linear measurements taken through the nanostructure.

Differential scanning calorimetry (DSC) can be used to study the effects of heat on the polymeric structure of the nanostructures. Thermal transitions, including glass transitions of a polymer can be determined. DSC can be used to understand the different

characteristics of nanostructures formed by various chelating metal ions such as Zn ²⁺ , Ca ²⁺ and Ni ²⁺ .

Surface charge of the nanostructures can be determined using zeta potential measurements. The zeta potential may be useful to study the effects of different charged pendant functional groups of the nanostructure polymers, the interaction between the nanostructure polymers and targeting agents, and the effect of pH on the retention characteristics of pharmaceutical agent release from the nanostructure structure.

Infrared spectroscopy can be used to further characterise particles such as nanostructures by the comparison of different particle spectra. Variables that alter infrared spectra include the presence of pendant side chains such as carboxylate groups, the interaction between the pendant side chains of the nanostructure with one or more pharmaceutical agents and the interaction between the pendant side chains of the nanostructure and a metal chelating agent.

UV-visible spectroscopy (UVS) can be used to further characterise nanoparticles such as nanostructures by the comparison of different particle spectra. In particular, UVS can help determine aggregation states of pharmaceutical agent loaded within the nanostructures.

The term "co-ordination bonding" as used herein is a type of bond in which one atom supplies both electrons. This term excludes a covalent bond in which each atom supplies one electron.

The terms "chelation" and "chelated" as used herein are understood to mean the formation of a heterocyclic complex between a ligand and a metal ion, wherein the bonding between the ligand and the metal ion is co-ordination bonding.

The term "cross-linked" as used herein means that covalent chemical linkages are introduced within and/or between constituent polymers of the nanostructures.

As used herein, the term "average cross-sectional length" refers to the sum average of two or more linear measurements taken through the nanostructure.

As used herein, the terms "active agent", "chemical agent", "pharmaceutical agent", "pharmacologically active agent", "medicament", "active" and "drug" are used interchangeably to refer to a chemical compound and in particular a protein or a peptide. In addition, the pharmaceutical agent may be an enzyme, a hormone, a toxin or an immunogen. The terms also encompass pharmaceutically acceptable and pharmacologically active ingredients of those active agents specifically mentioned herein including but not limited to salts, esters, amides, prodrugs, active metabolites, analogs and the like. When the terms "active agent", "chemical agent", "pharmaceutical agent", "pharmacologically active agent", "medicament", "active" and "drug" are used, then it is to be understood that this includes the active agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs, etc.

As used herein, the terms "pharmaceutical agent", "pharmacologically active agent", "active agent", "chemical agent", "medicament", "active" and "drug" include combinations of two or more active agents such as two or more proteins. A "combination" also includes multi-part such as a two-part composition where the agents are provided separately and given or dispensed separately or admixed together prior to dispensation.

The term "subject" as used herein refers to a vertebrate host.

The term "therapeutically effective amount" of a complex or a pharmaceutical composition as used herein is intended to include within its meaning a non-toxic but sufficient amount of the complex or the pharmaceutical composition of the invention which comprises an amount of the pharmaceutical agent which produces the desired therapeutic effect. The exact therapeutically effective amount will vary depending on factors such as the type of disease, the age, sex, weight of the subject and mode of administration. Dosage regimen can be adjusted to provide the optimum therapeutic response. For example, several divided doses can be administered daily, or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation.

The term "treating" or "treatment" as used herein refers to any and all uses which remedy a disease state or symptoms, prevent the establishment of disease, or otherwise prevent, hinder, ameliorate, retard or reverse the progression of disease or other undesirable symptoms in any way whatsoever.

As used herein, the term "targeting molecules" are molecules that are capable of being bound by, or binding to the intestinal epithelium of a subject, thereby allowing the uptake of itself and any conjugated moieties by the circulation or lymphatic drainage system of the subject. Targeting molecules also include those molecules for which there are natural receptors in the body, epithelium or cancer.

The present invention provides multi-layer nanostructures capable of carrying a pharmaceutical agent, the nanostructure having a core comprising a particle which includes the pharmaceutical agent and a cover comprising a polymer.

Polymers suitable for use in the nanostructures may be any biodegradable polymer. The polymer may be natural, synthetic, semi-synthetic, native or modified.

The polymer may be selected from the group consisting of, but not limited to: proteins, poly-amino acids such as polyglutamic acid, polyaspartic acid, and polylysine; polyacrylamide; poly N-acylhydroxypropine esters; polysebacic acid; polyfumaric acid; polylactic acid; polyglycolic acid; polylactic-co-glycolic acid; polymers formed from hydroxyethylmethacrylate; polymers formed from ethylene bismethacrylate; carboxymethylcellulose; gum arabic; agarose; alginate; polyphosphate; heparin; gelatin; guaran; copolymers of sebacic acid and fumaric acid; copolymers of biscarboxyphenoxypropane and sebacic acid; poly(carboxyphenoxyacetic) acid; poly(carboxyphenoxyvaleric) acid; poly-ε-caprolactone and related polyesters; poly-ε- caprolactone-co-δ-valerolactone; poly-ε-caprolactone-co-DL-lactic acid; hyaluronic acid; chitin; chitosan; dextran; dextrin, carboxy-dextran, diethylaminoethyl dextran, aminoethyl dextran and dextran sulfate; collagen; albumin; fibrinogen; and other naturally occurring polymers, such as collagen, starch, amylose, carboxymethyl amylose, hydroxyethyl amylose, hydroxyethyl amylose, and cellulose, carboxylmethyl cellulose, hydroxyethyl cellulose; agarose, pectic acid, alginic acid, gum Arabic, galactomannan, levan, hyaluronic acid; derivatives or mixtures thereof.

In one embodiment, the polymer may be a polysaccharide. In a preferred embodiment, the polymer is dextran or a derivative thereof. In another embodiment the polymer is alginate. In another embodiment carboxy alkyl dextrans are associated with the core particles to form the nanoparticle. In another embodiment chitosan is associated with the core particles to form the nanoparticle.

PDI is a measure of the variation of the sizes of the nanostructures in a preparation. A high PDI value, such as 0.7 to 1, suggests that there is a large variation in the size of nanostructures in a preparation and hence the nanostructures exhibit "polydispersity". A highly polydisperse preparation results in some structures being too big for uptake via

intestinal epithelial cells, or alternatively some structures being too small to effectively encapsulate the drug to be delivered, such that an accurate uptake of the pharmaceutical agent by the subject may not be readily determined, leading to variable bioavailability.

A PDI of less than 0.3 indicates a state of near monodispersity, i.e. the state of uniform particle size in a preparation. The nanostructures of the present invention have a PDI of between about 0.1 to about 0.4, and preferably between about 0.12 to about 0.35. A more uniform sized dose provides better uptake.

The nanostructures preferably have a diameter of about 20 -500 nm, more preferably in the range of between about 55 nm and 400 nm, and still more preferably between 75 and 300 mm. Larger diameter particles have been found to be unsuitable for intravenous injection due to hepatic and pulmonary clearance. Larger particles are also unsuitable for oral administration as these are either trapped in the intestinal mucous and as such do not reach the intestinal wall, or they are too big to enter the enterocyte, or the endosome that is used for uptake. Particles greater than 1 μm have very low bioavailabilities whilst particles that are very small such as less than 20 nm have very low pay-loads.

In an embodiment, the polymers interact or associate with the core particle. The interaction may be selected from one or more of the following: ionic interactions, hydrogen bonding, hydrophobic interactions, chelation, co-ordination bonding or pi-pi interactions. Typically, the interactions are fascilitated by functional groups on the polymer which are usually located on side chains on the polymer.

Pendant side chains of the polymer capable of having ionic interaction with the core particle may be moieties comprising functional groups of the following, including but not limited to: amino acids, including non-conventional amino acids, primary amines, secondary amines, tertiary amines, quaternary amines, amidines, aziridines, azetidines,

carboxylates, dithioacids, primary imines, furans, guanidines, hydrazines, hydrazones, isocyantes, isothiocyanates, isothiazoles, imidazoles, nitro groups, oxalones, oxazoles, oxetanes, oximes, phosphodiesters, phosphoranes, pyrazoles, pyroles, pyridyls, pyrolidines, pyridines, pyridazines, pyrimidines, pyrazines, thioacids, thiazoles, thiocyanates, thiophosphoranes, thiranes, triazoles, 1,3,5-triazines, 1 ,2,4-triazines at a selected pH. Table IA displays the amino acids that may provide functional groups for having ionic interaction with the pharmaceutical agents. Table 2A displays non- conventional amino acids that may provide functional groups for having ionic interaction with the pharmaceutical agents. However, it will be apparent to one skilled in the art that art that other moieties not listed herein but capable of having ionic interaction with the core particle may be included in the nanostructures.

For example, pendant side groups capable of having ionic interaction with the core particle may be provided by the α-carboxyl group of any amino acids or the α-amino group of any amino acids. Additionally, aspartic acid and glutamic acid may provide additional negatively charged moieties capable of having ionic interaction with the core particle. Alternatively, lysine, arginine and histidine may provide positively charged moieties capable of having ionic interaction with the core particle.

TABLE IA - Amino acids

TABLE 2A - Codes for non-conventional amino acids

α-aminobutyric acid Abu L-N-methylalanine Nmala α-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgln

carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucine Nmile

D-alanine Dal L-N-methylleucine Nmleu

D-arginine Darg L-N-methyllysine Nmlys

D-aspartic acid Dasp L-N-methylmethionine Nmmet

D-cysteine Dcys L-N-methylnorleucine Nmnle

D-glutamine DgIn L-N-methylnorvaline Nmnva

D-glutamic acid DgIu L-N-methylornithine Nmorn

D-histidine Dhis L-N-methylphenylalanine Nmphe

D-isoleucine DiIe L-N-methylproline Nmpro

D-leucine Dleu L-N-methylserine Nmser

D-lysine Dlys L-N-methylthreonine Nmthr

D-methionine Dmet L-N-methyltryptophan Nmtrp

D-ornithine Dorn L-N-methyltyrosine Nmtyr

D-phenylalanine Dphe L-N-methylvaline Nmval

D-proline Dpro L-N-methylethylglycine Nmetg

D-serine Dser L-N-methyl-t-butylglycine Nmtbug

D-threonine Dthr L-norleucine NIe

D-tryptophan Dtrp L-norvaline Nva

D-tyrosine Dtyr α-methyl-aminoisobutyrate Maib

D-valine Dval α-methyl-γ-aminobutyrate Mgabu

D-α-methylalanine Dmala α-methylcyclohexylalanine Mchexa

D-α-methylarginine Dmarg α-methylcylcopentylalanine Mcpen

D-α-methylasparagine Dmasn α-methyl-α-napthylalanine Manap

D-α-methylaspartate Dmasp α-methylpenicillamine Mpen

D-α-methylcysteine Dmcys N-(4-aminobutyl)glycine NgIu

D-α-methylglutamme Dmgln N-(2-ammoethyl)glycine Naeg

D-α-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn

D-α-methylisoleucine Dmile N-amino-α-methylbutyrate Nmaabu

D-α-methylleucine Dmleu α-napthylalanine Anap D-α-methyllysine Dmlys N-benzylglycine Nphe

D-α-methylmethionine Dmmet N-(2-carbamylethyl)glycine NgIn

D-α-methylornithine Dmorn N-(carbamylmethyl)glycine Nasn

D-α-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine NgIu

D-α-methylproline Dmpro N-(carboxymethyl)glycine Nasp D-α-methylserine Dmser N-cyclobutylglycine Ncbut

D-α-methylthreonine Dmthr N-cycloheptylglycine Nchep

D-α-methyltryptophan Dmtφ N-cyclohexylglycine Nchex

D-α-methyltyrosine Dmty N-cyclodecylglycine Ncdec

D-α-methylvaline Dmval N-cylcododecylglycine Ncdod D-N-methylalanine Dnmala N-cyclooctylglycine Ncoct

D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro

D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund

D-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm

D-N-methylcysteine Dnmcys N-(3 ,3 -diphenylpropyl)glycine Nbhe D-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine Narg

D-N-methylglutamate Dnmglu N-( 1 -hydroxyethyl)glycine Nthr

D-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine Nser

D-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine Nhis

D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine Nhtrp D-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate Nmgabu

N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet

D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen

N-methylglycine NaIa D-N-methylphenylalanine Dnmphe

N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro

N-(l-methylpropyl)glycine Nile D-N-methylserine Dnmser

N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr D-N-methyltryptophan Dnmtφ N-( 1 -methyl ethyl)glycine Nval

D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap

D-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acid GABA N-(p-hydroxyphenyl)glycine Nhtyr

L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine Pen

L-homophenylalanine Hphe L-α-methylalanine Mala

L-α-methylarginine Marg L-α-methylasparagine Masn

L-α-methylaspartate Masp L-α-methyl-t-butylglycine Mtbug

L-α-methylcysteine Mcys L-methylethylglycine Metg L-α-methylglutamine MgIn L-α-methylglutamate MgIu

L-α-methylhistidine Mhis L-α-methylhomophenylalanine Mhphe

L-α-methylisoleucine Mile N-(2-methylthioethyl)glycine Nmet

L-α-methylleucine Mleu L-α-methyllysine Mlys

L-α-methylmethionine Mmet L-α-methylnorleucine MnIe L-α-methylnorvaline Mnva L-α-methylornithine Morn

L-α-methylphenylalanine Mphe L-α-methylproline Mpro

L-α-methylserine Mser L-α-methylthreonine Mthr

L-α-methyltryptophan Mtφ L-α-methyltyrosine Mtyr

L-α-methylvaline Mval L-N-methylhomophenylalanine Nmhphe N-(N-(2,2-diphenylethyl) Nnbhm N-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl)glycine carbamylmethyl)glycine l-carboxy-l-(2,2-diphenyl- Nmbc

ethylamino)cyclopropane

Pendant side chains of the polymer capable of hydrogen bonding with the core particle may be selected from functional groups of the following, including but not limited to: amino acids, alcohols, aldehydes, primary amides, secondary amides, tertiary amides, primary amines, secondary amines, tertiary amines, amidines, anhydrides, azos, aziridines, azetidines, carboxylates, carbonates, carbamates, cyanates, dithioacids, disulfides, hydroxylamines, ketones, imides, primary imines, secondary imines, ethers, esters, epoxides, furans, guanidines, hydrazines, hydrazones, isocyantes, isothiocyanates, isothiazoles, isoxazoles, imidazoles, nitriles, nitro groups, nitroso groups, oxalones, oxazoles, oxetanes, oximes, oxiranes, peroxys, phosphodiesters, phosphites, phosphates, phosphanes, phosphonates, phosphoranes, pyrazoles, pyroles, pyridyls, pyrolidines, pyridines, pyridazines, pyrimidines, pyrazines, sulfones, sulfonamides, sulfites, sulfonates, thioacids, thiazoles, thiocyanates, thiocarbamates, thiocarbonates, thioethers, thioethanes, thiols, thioketones, thiophosphites, thiophosphates, thiophosphonates, thiophosphoranes, tetrahydro-thiophenes, thiophenes, thiranes, triazoles, 1,3,5-triazines, 1,2,4-triazines. However, it will be apparent for a skilled person in the art that other moieties not listed herein but capable of having hydrogen bonds with the pharmaceutical agents may be included in the nanostructures of the present invention.

Pendant side chains of the polymer capable of having hydrophobic interaction with the core particle may be selected from the following, including but not limited to: alkanes, alkenes, alkynes, oxiranes, phenyls, pyridyls, pyrolidines, pyridines, pyridazines, pyrimidines, pyrazines, 1,3,5-triazines, 1,2,4-triazines, hydrophobic amino acids (such as, but not limited to alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, tyrosine), lipids. However, it will be apparent for a skilled person in the art

that other moieties not listed herein but capable of having hydrophobic interaction with the core particle may be included in the nanostructures of the present invention.

Pendant side chains of the polymer capable of co-ordination bonding to form chelates may be moieties comprising functional groups capable of donating a pair of electrons (Lewis bases). Such moieties are capable of chelating with metal ions, which in turn may act as ligands for binding to the core particle.

Pendant side chains of the polymer capable of chelating with metal ions, which in turn act as a ligand for binding to the core particle may be moieties including but not limited to: alcohols, aldehydes, primary amides, secondary amides, tertiary amides, primary amines, secondary amines, tertiary amines, amidines, anhydrides, azos, aziridines, azetidines, carboxylates, carbonates, carbamates, cyanates, dithioacids, di-sulfides, hydroxylamines, ketones, imides, primary imines, secondary imines, ethers, esters, epoxides, furans, guanidines, hydrazines, hydrazones, isocyantes, isothiocyanates, isothiazoles, isoxazoles, imidazoles, nitriles, nitro groups, nitroso groups, oxalones, oxazoles, oxetanes, oximes, oxiranes, peroxys, phosphodiesters, phosphites, phosphates, phosphanes, phosphonates, phosphoranes, pyrazoles, pyroles, pyridyls, pyrolidines, pyridines, pyridazines, pyrimidines, pyrazines, sulfones, sulfonamides, sulfites, sulfonates, thioacids, thiazoles, thiocyanates, thiocarbamates, thiocarbonates, thioethers, thioethanes, thiols, thioketones, thiophosphites, thiophosphates, thiophosphonates, thiophosphoranes, tetrahydro- thiophenes, thiophenes, thiranes, triazoles, 1,3,5-triazines, 1,2,4-triazines. However, it will be apparent for a skilled person in the art that other moieties not listed herein but capable of chelation with the core particle via metal ions may be included in the nanostructures of the present invention.

Additional pendant side chains of the polymer capable of chelating with metal ions, which in turn act as a ligand for binding to the core particle may include but are not

limited to molecules such as: EDTA, iminodiacetic acid, o-hydroxy-benzyl-iminodiacetic acid, 3-aminobenyliminodiacetic acid, 6-Amino-l,4,8,l l-tetraazacyclotetradecane, hydrazinonicotinamide (HYNIC), DTPA (N-diethylenetriaminopentaacetic acid), DOTA (l,4,7,10-tetraazacyclododecane-ν,ν',ν",ν'"-tetraacetic acid), TETA (1,4,8,11- tetraazacyclotetradecane-l,4,8,-l l-tetraacetic acid), NOTA (1,4,7-triazacyclononane- 1 ,4,7-triacetic acid), histidine, 2-nitroimidazole, picolinic acid, imidazole carboxylic acid, 2,4 dipicolinic acid, (2-picolylamine-N,N-diacetic acid), Dichloro-[4-(methyleneimino- diacetic acid)phenyl (2,3'-diaminopropionamide), TRODAT, N-[[[(2- mercaptoethyl)amino]carbonyl]methyl]-N-(2-mercaptoethyl)-6-a minohexanoic acid, deferoxamine, ν-bis(2-pyridylmethyl)-L-lysine, aspartic acid and glutamic acid, aminomalonic acid, 1,4, 8, 11-tetraazacyclotetradecane (CYCLAM), bisaminothiols, dithiocarbamates, N-His, for chelation of metals such as ^99m -Tc, 2,3- diaminopropionamide, or 2,3-diaminopropionic acid for chelation of metals such as platinum.

Metal ions for use in chelation include but are not limited to: divalent or trivalent cations of iron, manganese, chromium, cobalt, lead, silver, mercury, bismuth, plutonium, cadmium, germanium, ruthidium, gold, indium, technetium, copper, zinc, gallium, rhodium, palladium, platinum, nickel, gadolinium, erbium, europium, dysprosium, yttrium, promethrium, lutetium and holmium and radioactive isotopes thereof, wherein the metal ions may be complexed with one or more core particle. Examples of pharmaceutical agents suitable for loading onto a metal chelated nanostructure include proteins and peptides such as insulin or nucleic acids including siRNA.

In one embodiment, the pendant side chains may comprise chelating moieties to which are bound metal ions, wherein the metal ions act as ligands for binding to one or more pharmaceutical agents. Metal ions within the core particle include, but are not limited to divalent or trivalent cations of iron, manganese, chromium, cobalt, lead, silver, mercury,

bismuth, plutonium, cadmium, germanium, ruthidium, gold, indium, technetium, copper, zinc, gallium, rhodium, palladium, platinum, nickel, gadolinium, erbium, europium, dysprosium, yttrium, promethrium, lutetium and holmium and radioactive isotopes thereof, wherein the metal ions may be complexed with one or more pharmaceutical agents. Particularly preferred metals suitable for polymer chelation are magnesium, manganese, selenium, calcium and zinc, more preferably is calcium and zinc, and still more preferably is zinc.

Chelation of metals within the pendant side chains of the nanomatrix may be tailored to introduce specific charges to the chelated pendant side chain. For instance, chelation of metals via two, four or six carboxyl groups leads to a negatively charged pendant side chain. In contrast, chelation of metals via two, four or six amino groups leads to a positively charged pendant side chain. Whilst chelation via amino and carboxyl groups can lead to a neutral chelate.

Pendant side chains of the polymer capable of pi-pi interaction with the core particle may be selected from the following, including but not limited to alkenes, alkynes, furans, isothiazoles, isoxazoles, imidazole, oxalone, oxazoles, phenyl, pyrazoles, pyrole, pyridyl, pyridine, pyridazines, pyrimidines, pyrazines, thiophenes, triazoles, 1,3,5-triazine, 1,2,4- triazine. However, it will be apparent for a skilled person in the art that other moieties not listed herein but capable of pi-pi interaction with the core particle may be included in the nanostructures of the present invention.

Suitable pharmaceutical agents that may be carried by the nanostructures include any water soluble solute, including, but not limited to peptides, proteins, polysaccharides, oligonucleotides, salts, sugars, nutrients, vitamins, minerals, acids, antioxidants, or any biological active compounds for administration to a subject, such as a human, animal or other mammal. The pharmaceutical agent is selected based upon the intended application

or therapy, wherein the effect of the pharmaceutical agent is suitable to treat a particular condition. The pharmaceutical active agent needs to be able to form a particle with the chelating agent in a manner and form that does not destroy the activity of the pharmaceutical agent.

Suitable peptides, proteins and nucleic acids that can be delivered via the nanostructures of the present invention include protein molecules, protein chimeric molecules, other chimeric molecules or fragments that have the capacity to associate with complexing agents such as metal cations and in particular divalent metal cations.

In particular, suitable pharmaceutical agents include anti-diabetic peptide drugs including insulin and analogs thereof; glucagon-like peptides (GLPs) including GLP-I, GLP-2, oxyntomodulin, exenatide and pramlintide. Other suitable agents include thyrotropin/ thyroid-stimulating hormone (TSH), thyrotropin-releasing hormone (TRH), follicle- stimulating hormone (FSH), luteinising hormone (LH), prolactin (PL), growth hormone (GH) and calcitonin.

The pharmaceutical agent may be a chimeric molecule comprising an isolated protein or a fragment thereof, such as an extra-cellular domain of a membrane bound protein, linked directly to the constant (Fc) or framework region of a human immunoglobulin via one or more protein linkers. Such a chimeric molecule is also referred to herein as protein-Fc. Other chimeric molecules contemplated include the protein or protein-Fc or a fragment thereof, linked to a lipid moiety such as a polyunsaturated fatty acid molecule. Such lipid moieties may be linked to an amino acid residue in the backbone of the molecule or to a side chain of such an amino acid residue. The human immunoglobulin may be selected from IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgM, IgE, IgD.

The pharmaceutical agent may further include a chimeric molecule comprising an isolated protein or a fragment thereof, such as an extra-cellular domain of a membrane bound protein, linked directly to the constant (Fc) or framework region of a mammalian immunoglobulin via one or more protein linker. In another embodiment, the mammal Fc or framework region of the immunoglobulin is derived from a mammal selected from the group consisting of primates, including humans, marmosets, orangutans and gorillas, livestock animals (e.g. cows, sheep, pigs, horses, donkeys), laboratory test animals (e.g. mice, rats, guinea pigs, hamsters, rabbits, companion animals (e.g. cats, dogs) and captured wild animals (e.g. rodents, foxes, deer, kangaroos). In another embodiment the Fc or framework region is a human immunoglobulin. In a particular embodiment the mammal is a human. Such a chimeric molecule is also referred to herein as protein-Fc. Other chimeric molecules contemplated by the present invention include the protein or protein-Fc or a fragment thereof linked to a lipid moiety such as a polyunsaturated fatty acid molecule. Such lipid moieties may be linked to an amino acid residue in the background of the molecule or to a side chain of such an amino acid residue.

Suitable peptides and proteins that can be delivered via the nanoparticles of the present invention include protein molecules, protein chimeric molecules, other chimeric molecules or fragments selected from the TNF superfamily (including but not limited to TNF-a, TNFRl, TNFR2, BAFF, OX-40, Lymphotoxin-a, Fas-ligand); chemokines (including but not limited to MCP-I, MIP-Ia, MIP-Ib, RANTES, IL-8 and viral like chemokine antagonist MC 148); interleukins, interleukin receptors and antagonist (including but not limited to IL-Ia IL-Ib, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-1 1, IL-12, IL-13, IL- 15, IL- 16, IL- 18, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, their respective receptors including but not limited to IL-IRa, IL-2Ra, IL-2Rb, IL- 2Rg, IL-3Ra, IL-4Ra, IL-5Ra, IL-6Ra, IL-7Ra, IL-IORa, IL-I lRa, IL- 13Ra, IL- 15Ra as well as IL-IR Antagonist); the interferon family (including but not limited to IFN-a2B, IFN-bl, IFN-g, IFN-y, IFN-aR2, IFN-aRa, IFN-aRb, IFN-gRa, IFN-gRb); lectins

(including but not limited to CD209 type I and II, E-Selectin, L-Selectin, P-Selectin, Langerin); growth factors and their receptors (including but not limited to Amphiregulin, Angiopoietin, BDNF, beta-cellulin, BMPs (including but not limited to BMP-2, BMP-4, BMP-7), CNTF, cripto, ECGF-I, EGF, EGFR, EPO, FGFs and their receptors (including but not limited to FGF-I, FGF-2, FGF-5, FGF-7, FGF-9, FGF-11, FGF- 12, FGF- 13, FGF- 14, FGF-14 FLAG, FGF-18, FGF-19, FGF-21, FGFRl, FGFRl, FGFR4, FGFR5), Flt3- Ligand and its receptor (including but not limited to Flt3), G-CSF, GDNF, GM-CSF, GM- CSF-R, hGH and its receptor (including but not limited to hGHR), IGF-I, IGFBP-3, M- CSF, Neuregulin, NGFs and its receptor (including but not limited to NGF-b, NGFR, NGFR), NT-3, PDGFs, TGFs and their receptors (including but not limited to TGF-a, TGF-b, TGFbR2), Trk-A, Trk-B , TPO, VEGFs and their receptors (including but not limited to VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF- 165, VEGFR); embryonic growth factors (including but not limited to Noggin, Nodal, SCF, Wnts, Wnt-2, Wnt-3, Wnt-3A, Wnt-4, Wnt-5A, Wnt-5A-FLAG-C, Wnt-5B, Wnt-6, Wnt-7A, Wnt-7B, Wnt- 1OA, Wnt-IOB, Wnt-1 OB-FLAG-C, WnM l); adhesion molecules (including but not limited to adiponectin, ICAM), other cytokines and proteins, such as LIF, OSM, transferring and its receptor, hormones (including but not limited to insulin, calcitonin, adrenocorticotropin (ACTH), glucagon, somatostatin, somatotropin, thymosin, parathyroid hormone, pigmentary hormones, somatomedin, lutenizing hormone, chorionic gonadotropin, hypothalmic releasing factors, antidiuretic hormones, thyroid stimulating hormone, endorphins, enkephalins, biphalin and prolactin.); antibiotics (including but not limited to gentamycin, amikacin), enzymes (including but not limited to activin A asparaginase, adenosine deaminase, BACE-I, caspase-1, fucosyltransferase, furin, mTACE, sialyltransferase ) Factor VIII, LH-RH analogues, and other biopharmaceuticals such as heparin and vaccines (for instance, vaccines for Hepatitis 'B' surface antigen, typhoid and cholera vaccines), plasminogen activator inhibitors and fusion proteins including BAFFR-Fc.

In particular, suitable chimeric proteins that can be delivered via the nanoparticles of the present invention include but is not limited to TNFRl-Fc, TNFR2-Fc, OX-40-Fc, MC148- Fc, IL-IRa-Fc, IL-2Ra-Fc, IL-2Rb-Fc, IL-2Rg-Fc, IL-3Ra-Fc, IL-4Ra-Fc, IL-5Ra-Fc, IL- 6Ra-Fc, IL-7Ra-Fc, IL-IORa-Fc, IL-I IRa-Fc, IL- 13Ra-Fc, IL- 15Ra-Fc, IFN-aRa-Fc, IFN- aRb-Fc, IFN-gRa-Fc, IFN-gRb-Fc, CD209L-Fc, E-Selectin-Fc, L-Selectin-Fc, P-Selectin- Fc, Langerin-FLAG, EGFR-Fc, FGFRl-Fc, FGFR4-Fc, FGFR5-Fc, Flt3-Fc, hGHR-Fc, NGFR-Fc, TGFbR2-Fc, Trk-A-Fc, Trk-B-Fc, VEGFR-Fc, Wnt-5 A-FLAG-C, Wnt-IOB- FLAG-C and BAFFR-Fc.

Additional suitable proteins that can be delivered via the nanoparticles of the present invention include monoclonal and polyclonal antibodies, single-chain antibodies, other antibody fragments, analogs and derivatives thereof. Polynucleotides, including antisense oligonucleotides, aptamers and therapeutic genes can also be delivered using the methods and compositions of the present invention.

Anticoagulants, such as heparin, also can be delivered using the nanoparticles of the invention. Still other suitable therapeutic agents for use in the present invention include bioactive molecules, such as anticancer drugs, e.g., doxorubicin, epirubicin and daunorubicin, vincristine, cisplatin, carboplatin, oxaliplatin, methotrexate, paclitaxol, taxol, camptothecin and camptothecin analogs, antibiotics, antipsychotics, antidepressants, and drugs for diabetes and cardiovascular disease.

Examples of pharmaceutical agents suitable for delivery by metal chelated nanoparticles include insulin, which may be complexed with zinc, or arginine; and other metallodrugs, such as cisplatin, carboxplatin, oxaliplatin, DACH-platinum. Additional suitable pharmaceutical agents include the anthracyclines, daunomycin, doxorubicin and epirubicin.

It is thought that complexing peptides or nucleic acids with metals can extend the pharmaceutical agent half-life in-vivo thereby possibly providing an additional advantage of a (favourable) sustained release profile. This is particularly important where the agent has a short half-life.

Preparation of the Nanostructures of the Present Invention

According to another aspect, the present invention provides a process for preparing multilayer nanostructures suitable for delivery of pharmaceutical agents. Whilst there are a number of methods for the preparation of nanostructures of the present invention, preferred methods include the use of thermodynamically stabilized water-in-oil microemulsions.

In general, the method of preparing the nanostructures of the present invention involves: (a) preparing separate mixtures comprising:

(i) a microemulsion including a chelating agent; and

(ii) a microemulsion including a pharmaceutical agent; and

(b) combining mixtures (i) and (ii) from step (a) to form mixture (iii);

(c) preparing a further mixture comprising (iv) a microemulsion including a polymer;

(d) combining mixtures (iii) and (iv) to form mixture (v) including the nanostructures; and

(e) separating the nanostructures from mixture (v).

This method is generally applicable to pharmaceutical agents, and in particular proteins, that show substantial affinity to chelating agents. This is particularly true for proteins and peptides with amino acid sequences that have specific domains or sites for metal binding.

In another embodiment, the method for preparing nanostructures capable of carrying a pharmaceutical agent involves:

(a) preparing separate mixtures comprising:

(i) a microemulsion comprising a charged surfactant and a non-ionic surfactant and including the pharmaceutical agent and a multivalent cation; and

(ii) a microemulsion comprising a non-ionic surfactant and including a base; and

(b) combining mixtures (i) and (ii) from step (a) to form within mixture (iii) core particles; (c) preparing a further mixture comprising

(iv) a microemulsion including a polymer;

(d) combining mixtures (iii) and (iv) to form within mixture (v) the nanostructures; and

(e) separating the nanostructures from mixture (v).

In the above method, the pharmaceutical agent precipitates with the multivalent cation. Preferably the pharmaceutical agent typically is any protein or peptide and the cation gives rise to zinc hydroxide. These precipitate combinations are generally not protein specific. Agglomeration of the pharmaceutical agent/cation precipitate is thought to be avoided or at least minimised by use of a charged surfactant in the microemulsions. The charged surfactant acts at the droplet interface thereby supporting the microemulsion.

The polymer is associated with the core particle. Without being limited by theory, it is thought that the polymer is associated with the core particle by ionic interactions, hydrogen bonding, hydrophobic interactions, chelation, co-ordination bonding, pi-pi interactions or combinations thereof.

Preferred aqueous phase ingredients may comprise water and any acceptable water- soluble components in water, including one or more pharmaceutical agents.

Preferred non-polar phase ingredients may comprise natural oils derived from plants or animals, such as vegetable oils, sunflower oils, coconut oils, almond oils; purified synthetic or natural di or triglycerides (such as Crodamol GTCC and Capmul MCM); phospholipids and their derivatives (such as lecithin or lysolecithin); fatty acid esters (such as isopropyl myristate, isopropyl palmitate, ethyl oleate, oleic acid ethyl ester); hydrocarbons (such as hexane, the n-decane through n-octadecane series); glycerolysed fats and oils (such as glyceryl monooleate, glyceryl monocaprylate, glycerol monocaprate, propylene glycol monocaprylate, propyleme glycol monolaurate).

Other non-polar phase ingredients include, but are not limited to, Labrafil M 1944 CS, benzene, tetrahydrofuran, and n-methyl pyrrolidone, or halogenated hydrocarbons, such as methylene chloride, or chloroform. In a particular embodiment, the oil phase comprises Crodamol GTCC and Capmul MCM, at 3:1 ratio.

The non-polar component is either used alone or in combination with other non-polar components. For example, an oil or unique mixture of oils may require a different surfactant or mixture of surfactants or surfactants and co- surfactants to form an emulsion or a microemulsion with the acqueous phase, as can routinely be determined by those of skill in the art.

Surfactants used according to the invention are known surfactants in the art that reduce the interfacial tension between the non-polar and acqueous phases sufficiently to allow the formation of emulsions or microemulsions. Typically, surfactants are organic compounds that are amphiphatic, containing both hydrophobic groups and hydrophilic groups. Preferred surfactants include, but are not limited to, anionic surfactants such as fatty acid

soaps (including sodium oleate, sodium palmitate, sodium myristate, sodium sterate); alkyl sulfates (including sodium dodecyl sulfate); alkyl benzenesulfonates; alkyl sulfonates; alkyl phosphates; acyl sulfates; or acyl sulfosuccinates; cationic surfactants, such as alkyl primary, secondary, tertiary, or quaternary amines; alkyl pyridinium and quaternary ammonium salts; zwitterionic surfactants, for example, betaines, such as dodecyldimethylammonium acetate, tetradecyldimethylammonium acetate, hexadecyldimethylammonium acetate, alkyldimethylammonium acetate wherein the alkyl group averages about 14.8 carbon atoms in length, dodecyldimethylammonium butanoate, tetradecyldimethylammonium butanoate, hexadecyldimethylammonium butanoate, dodecyldimethylammonium hexanoate, hexadecyldimethylammonium hexanoate, tetradecyldiethylammonium pentanoate, tetradecyldipropyl ammonium pentanoate, coco- betaine, sulfobetaines (or sultaines), fatty acid ethanolamides such as cocamide monoethanolamide and other zwitterionic surfactants such as 3-(N,N-dimethyl-N- hexadecylammonio)-propane- 1 -sulfonate; 3-(N,N-dimethyl-N-hexadecylammonio)-2- hydroxypropane-1 -sulfonate; N,N-dimethyl-N-dodecylammonio acetate; 3-(N ,N- dimethyl-N-dodecylammonio)propionate; 2-(N,N-dimethyl-N-octadecylammonio)ethyl sulfate; 3-(P,P-dimethyl-P-dodecylphosphonio)propane- 1 -sulfonate; 2-(S-methyl-S-tert- hexadecylsulfo)ethane-l -sulfonate; 3-(S-methyl-S-dodecylsulfonio)propionate; N ₅ N- bis(oleylamidopropyl-N-methyl-N-carboxymethylammonium betaine; N ₅ N- bis(stearamidopropyl)-N-methyl-N-carboxymethylammonium betaine; N-

(stearamidopropyl)-N-dimethyl-N-carboxymethylammonium betaine; 3-(N-4-n- dodecylbenzyl-N,N-dimethylammonio)propane- 1 -sulfonate; 3-(N-dodecylphenyl-N,N- dimethylammonio)-propane-l -sulfonate; nonionic surfactants, for example, alcohol ethoxylate, alkylphenol ethoxylate, alkyl polyglycosides, mono-, di- or glyceride esters, polyglycerols, polyglycerol esters, phospholipids (such as lecithin), mono- or diglyceride esters of citric acid, tartaric acid and lactic acid, sorbitan fatty acid esters (such as sorbitan monostearate NF, sorbitan monooleate NF, sorbitan isosterate, sorbitan monolaurate), polyoxyethylene sorbitan fatty acid esters (polysorbates) (such as polyoxyethylene

sorbitan monooleate, polysorbate 20 NF, polysorbate 20 NF, EP, polysorbate 60 NF, polysorbate 80 NF, polysorbate 80 NF, EP, JP, PEG-20 Sorbitan Isostearate), polyethoxylated esters of acyl acids, copolymers of polyethylene oxide and polypropylene oxide, polyoxyethylene fatty ethers (such as polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols, polyoxyethylene (4) lauryl ether, polyoxyethylene (23) lauryl ether, polyoxyethylene (2) cetyl ether, polyoxyethylene (10) cetyl ether, polyoxyethylene (20) cetyl ether, polyoxyethylene (2) stearyl ether, polyoxyethylene (10) stearyl ether, polyoxyethylene (20) stearyl ether, polyoxyethylene (2) oleyl ether, polyoxyethylene (10) oleyl ether, polyoxyethylene (20) oleyl ether, polyoxyethylene (21) stearyl ether), Cremophor. Surfactants listed herein may be used alone or in combination of each other.

Co-surfactants used according to the invention are known surface-active agents in the art that act, in addition to surfactants, to further lower the interfacial energy of an emulsion or a microemulsion. Preferred co-surfactants include, but are not limited to non-toxic amphiphilic molecules; alcohols (including aliphatic alcohols, shorter chain alcohols, such as ethanol); fatty acid alcohols (such as n-alkane-l,2-diols); acids (such as acetic acid); esters (such as butyl lactate); any surfactants as herein listed or mixtures thereof.

In a particular embodiment, an aqueous phase may be prepared by mixing a suitable polymer and a non-ionic surfactant (for example Tween 80) in water. The ratio of the amount of surfactant added per gram of polymer may be between about 0.01 and 2 grams, or between about 0.05 and 1.5 grams, or between about 0.075 and 1.25 grams or between about 0.1 and about 1.0 grams or between about 0.1 and 0.75 grams or between about 0.1 and 0.5 grams. In one embodiment, the amount of surfactant added may be about 0.15 grams per gram of polymer.

An non-polar phase may be prepared by mixing a mineral oil and a non-ionic surfactant (for example Span 80). The ratio of the amount of surfactant added per gram of polymer may be between about 0.01 and 2 grams, or between about 0.05 and 1.5 grams, or between about 0.075 and 1.25 grams or between about 0.1 and about 1.0 grams or between about 0.1 and 0.75 grams or between about 0.1 and 0.5 grams. In one embodiment, the amount of surfactant added may be about 0.16 grams per gram of polymer.

In a general method, the aqueous phase and the non-polar phase are combined and homogenized until a stable emulsion is formed. Formation of the nanostructure core particle is achieved by mixing separate emulsions, one containing the chelating agent and the other emulsion containing the pharmaceutical agent whilst the emulsion mixture formed is maintained with stirring. The pharmaceutical agent and chelating agent form a particle within the emulsion. The resultant emulsion containing the particle is added to a further emulsion containing the polymer. The polymer associates with the core particle and this is followed by precipitation of the nanostructure so formed by breaking the emulsion. The nanostructures are allowed to settle, washed in an organic solvent, followed by distilled water and then lyophilized.

In a preferred embodiment, thermodynamically stabilized water-in-oil microemulsions are used in the preparation of the nanostructures of the present invention. Microemulsions are quaternary systems composing of an non-polar phase, a water phase, surfactant/s and a co-surfactant. These systems are thermodynamically stable and form spontaneously upon mixing. They also possess specific physicochemical properties such as transparency, optical isotropy, low viscosity and thermodynamic stability. Thus, in contrast to standard emulsions which are opaque, microemulsions have an observable transparency, which is due to the fact that the maximum size of the droplets of the dispersed phase is not larger than one-fourth of the wavelength of visible light —

approximately 150 nm. Thus the droplet diameter in stable microemulsions is usually within the range of 10-250 nm. The water droplet size in the microemulsions can be controlled by altering the amount of co-surfactant in the microemulsions. Alternatively, the percentage of the water phase can be varied.

The microemulsion used to form the nanostructures of the present invention may comprise an non -polar phase of between 2 and 98% by weight, most preferably between 10 and 90% by weight; a water phase of between 2 and 98% by weight, most preferably between 10 and 90 by weight; 0.1 to 90% by weight surfactant, preferably 1 to 90% by weight surfactant. The microemulsion may further comprise 0.1 to 90% by weight cosurfactant or cosolvent; preferably 1 to 90% by weight cosurfactant or cosolvent.

The microemulsion used to form the nanostructures of the present invention may further comprise solvents or other agents to enhance emulsion formation or stability. Other agents may be introduced to provide functions such as pH, ionic content, polymerisation, viscosity.

The microemulsions may also be generated using any suitable synthetic plastic or polymeric, monomelic or hybrid colloidal material.

In one instance, microemulsions were formed by mixing an oil phase composed of caprylic/capric triglyceride (Crodamol GTCC; Croda) and medium chain mono- and di- glyceride (Capmul MCM C8; Croda) at a 3:1 ratio. A surfactant/co-surfactant mixture containing polysorbate 80 (Crillet 4; Croda; surfactant) and Crill 4 (Span 80; co- surfactant) at a 3:2 ratio was prepared. The oil-surfactant phase was prepared by mixing 7.6 g Crodamol/Capmul oil mixture with 1.4 g Crillet/Crill surfactant/co-surfactant mixture.

In another instance microemulsions were formed by mixing an oil phase composed of Crodamol GTCC and Capmul MCM C8 in a 3:1 ratio. A surfactant/co-surfactant mixture of a 3:2:0.75 ratio of Crillet 4 (Tween 80) to Crill 4 (Span 80) to AB30 (coco-betaine) was prepared. The oil-surfactant phase was then prepared by mixing the oil and surfactant mixtures at a ratio of 7.6: 1.4.

Targeting agents according to the invention are selected from molecules which are known to be internalized by cells, or which have been shown to be transcytosed by cells, such as those of the gastro-intestinal epithelium.

Suitable targeting agents include but are not limited to water soluble vitamins such as vitamin C, analogues or derivatives thereof; folic acid and analogues or derivatives thereof (including but not limited to methotrexate, aminopterin, 10-deazaminopterin, 10- ethyl-10-deazaaminopterin, 5, 10-dideazatetrahydro folate, folinic acid, 7- hydroxyaminopterin); niacin (nicotinic acid, vitamin B3) and analogues or derivatives there of (including but not limited to beta-hydroxybutyrate, acipimox, niceritrol, nicotinamide (niacin)); thiamine (vitamin B-I), analogues and derivatives thereof; riboflavin (vitamin B2)= and its analogues or derivatives thereof (including but not limited to 7-nor-7-chlororiboflavin, 8-nor-8-chlororiboflavin, 7-nor-7-bromoriboflavin, 8- nor-8-bromoriboflavin, 7α-methylriboflavin, 8α-methylriboflavin, 7α,8α- dimethylriboflavin, 7-nor-7-bromo-8α-methylriboflavin, 7α-methyl-8-nor-8- bromoriboflavin, 7-nor-7-chloro-8α-methylriboflavin, 7α-methyl-8-nor-8- chlororiboflavin, 8-nor-8-fluororiboflavin, 7-nor-7-chloro-8-nor-8-chlororiboflavin, 8- nor-8-aminoriboflavin, N(3)-methylriboflavin and 5-deaza-5-carbariboflavin; pyridoxine (vitamin B6), analogues or derivatives thereof; cyanocobalamin (vitamin Bi ₂ ), analogues or derivatives thereof, pantothenic acid (vitamin B5), analogues or derivatices thereof, and biotin, analogues or derivatives thereof.

Other targeting agents include viral haemmaglutinins, bacterial adhesins, transferrin, immunoglobulins, and immunoglobulin Fc fragments either derived therefrom or synthesized separately, bacterial invasins, toxins, and binding sub-units thereof, lectins, and the sugar binding moieties thereof, membrane transduction sequences thereof. Addtionally fusion proteins of the aforementioned targeting agents can also be used.

Riboflavin (Rf) derivatives may be prepared by reaction of the primary OH group of Rf with diacids, di- or tri-carboxylic acids, or diamine acids.

Diacids acids suitable for linkage to Rf derivatives of the invention include, but are not limited to:- glutamic acid, aspartic acid, hydroxylglutamic acid, hydroxyisophthalic acid, 2, 4-diamino-pentanedioic acid, 2-amino-pentanedioic acid, 2-aminohexanedioic acid, mesoxalic acid, oxomalonic acid, ketomalonic acid, and alanosin.

Di-carboxy and tricarboxylic acids suitable for linkage to Rf derivatives of the invention include, but are not limited to:- agaricic acid, 2-hydroxy-l,2,3-nonadecanetricarboxylic acid, Cis, cis-l,3,5,Trimethylcyclohexane-l,3,5-tricarboxylic acid, Cis-Aconitic acid cis- Propene-l,2,3-tricarboxylic acid, Trimellitic acid, Benzene- 1,2,4-tricarboxylic acid, Trimesic acid, Benzene- 1, 3, 5-tricarboxylic acid, Cyclopropane- 1,2,3 -tricarboxylic acid, Carboxyaspartic acid, Carboxyglutamic acid (gamma-carboxy-D-glutamic acid, Malic acid (hydroxysuccinic acid). ^"

Diamine acids suitable for linkage to Rf derivatives of the invention include, but are not limited to:- lysine, ornithine 2,5-diaminopentanoic acid NH ₂ (CH ₂ ) ₂ CH(NH ₂ )COOH, diamino benzoic acid, 2,4,-diaminophenol, 2,3-diaminopropionic acidRf derivatives may be prepared by reaction of the primary OH group of Rf with diacids, di- or tri-carboxylic acids, or diamine acids. Diacids acids suitable for linkage to Rf derivatives of the invention include, but are not limited to:- 2,3-diaminopropionic acid, malonic acid,

maleic acid, succinic acid, glutaric acid, methyl malonic acid, glutamic acid, aspartic acid., hydroxylglutamic acid, hydroxyisophthalic acid, hydroxyglutaric acid, 2,4- diamino-pentanedioic acid, 2-amino-pentanedioic acid-dimethyl ester, 2- aminohexanedioic acid, mesoxalic acid, oxomalonic acid, ketomalonic acid, HOOCCOCOOH and alanosin. Di-carboxy and tricarboxylic acids suitable for linkage to Rf.

Biotin Analogues suitable for chelating include 3-(N-Maleimido-propionyl)biocytin: a thiol-specifϊc biotinylating reagent, alpha-dehydrobiotin, Z- and E-4,5- dehydrodethiobiotin, norbiotinamine, dl-4 xi-(4-carboxybutyl)-5-carbethoxy-cis- hexahydropyrrolo (3,4-d)imidazol-2-one (N-carbethoxyazabiotin), dl-4xi-(2- carboxyethyl)-cis-hexahydropyrrolo-[3,4-d]imidazol-2-one (bisnorazabiotin), bis- allyloxycarbonyl biotin aldehyde, carboxybiotin and methyl biotin.

For the purpose of the invention vitamin Bj ₂ includes vitamin B) ₂ or analogues thereof, such as described below.

In one embodiment, the targeting molecule is Vitamin Bi ₂ or an analogue thereof or a derivative of vitamin Bi ₂ or analogues thereof. Analogues of Vitamin B _]2 that may derivatized and thereby be used as targeting molecules in the complexes of the present invention include, but are not limited to: cyanocobalamin, aquocobalamin, adenosylcobalamin, glutathionylcobalamin, methylcobalamin, hydroxycobalamin, cyanocobalamin carbanalide, 5-O-methylbenzylcobalamin, and the desdimethyl, monoethylamide and methylamide analogues of all of the preceding analogues, as well as coenzyme Bi ₂ , 5'-deoxyadenosylcobalamin, chlorocobalamin, sulfϊtocobamin, nitrocobalamin, thiocyanatocobalamin, 5,6-dichlorobenzimadazole, 5- hydroxybenzimidazole, trimethylbenzimidazole, adenosylcyanocobalamin, cobalamin lactone, cobalamin lactam, and analogues in which the cobalt is replaced by zinc or

nickel or the corrin ring is replaced by a substituent which does not affect the binding capacity of the analogue to Castle's intrinsic Factor.

Derivatives of Vitamin B _]2 or analogues thereof, for use as targeting molecules include but are not limited to: anilides, ethylamides, monocarboxylic and dicarboxylic acid derivatives of Vitamin Bi ₂ and its analogues, and also tricarboxylic acids or proprionamide derivatives of Vitamin Bi ₂ or its analogues thereof, molecules in which alterations or substitutions had been performed to the Corrin ring, or where cobalt has been replaced by another metal ion, or various anionic or alkyl substituents have been added to the corrin ring such that the binding capacity of the molecule to Castle's intrinsic factor is unaffected.

Other derivatives of VBi ₂ or analogues thereof that may be used in the complexes of the present invention include 5'0-substituted VBi ₂ derivatives such as: hexyl-5'O-VBi ₂ , dodecyl-5'O-VB, ₂ , tetradecyl-5'O-VB, ₂ , hexadecyl-5 ¹ O-VB , ₂ , octadecyl-5 ¹ O-VB ₁₂ , aminoethyl-5'O-VBi ₂ , aminobutyl-5 ¹ O-VBi ₂ , /-butyl-Phe-5'O-VBi ₂ , aminohexyl-5'0- VBi ₂ , aminododecanyl-5'O-VBi ₂ , succinylhydrazidyl-5'O-VBπ, adipylhydrazidyl-5'0- VB ₁₂ , phenylalanyl-5'O-VB ₁₂ , glycyl-5'O-VBi ₂ , HO-G ly-5 ¹ O-VBi ₂ , VB, ₂ -5'O- phenylalanine, VB ₎₂ -5'O-lysine, VB _]2 -glycine acid. In one embodiment, the group attached to the 5'0 position may constitute the pendant group of a nanostructure.

Additional VBj ₂ derivatives of the invention include, but are not limited to:- agaricic acid, 2-hydroxy-l,2,3-nonadecanetricarboxylic acid, cis-l,3,5,Trimethylcyclohexane- 1,3,5-tricarboxylic acid, Cis-Aconitic acid cis-Propene-l,2,3-tricarboxylic acid, Trimellitic acid, Benzene- 1,2,4-tricarboxylic acid, Trimesic acid, Benzene- 1,3,5- tricarboxylic acid, Cyclopropane- 1,2,3-tricarboxylic acid, Carboxyaspartic acid, Carboxyglutamic acid (gamma-carboxy-D-glutamic acid, Malic acid (hydroxysuccinic acid).

Upon binding of the nanaostructure of the invention to Castle's intrinsic factor, mucosal epithelial cells take up the intrinsic factor-complex and trans-epithelially transport the complex into the circulation or lymphatic drainage system where the pharmaceutical agent can be released. The nanostructures may degrade in vivo to release the pharmaceutical agent.

Metals for chelation include aluminum, antimony, arsenic, beryllium, bismuth, cadmium, calcium, cobalt, copper, gadolinium, gallium, gold, iron, lanthanum, lead, magnesium, manganese, mercury, nickel, platinum, organic tin, samarium, thallium, zirconium and zinc, and dietary supplemental chelates. More preferably, the chelates are calcium, magnesium, iron and zinc, most preferably zinc.

Amino acids suitable for substitution of the polymers include amino-glycine, Diaminopropionic acid, Diaminobutyric acid, Dehydroalanine, Fluoro-alanine, Chloro- alanine, Azetidine-2-carboxylic acid, aminobutyric acid, cyanoalanine, amino-isobutyric acid, homocysteine, methylene-glutamic acid, hydroxy-glutamic acid, ornithine, difluoromethyl-ornithine, cyclopropyl-alanine, tert-butyl-alanine, propargyl-glycine, 2- allyl-glycine, tert-butyl-glycine, alla-threonine, Ureido-alanine, pyroglutamic acid, carboyglutamic acid, 2,6-Diamino-4-hexynoic acid, 4-amino-piperidine-4-carboxylic acid, B-(l-cyclopentenyl)-alanine, B-Cyclopentyl-alanine, thioproline, 3, 4-dehydro- proline, methyl-valine, hydroxyproline, citrulline, thiocitrulline, aminoadipic acid, 4,5- dehydro-lysine, 6-hydroxy-lysine, B-cyclohexyl-alanine, B,B-dicyclohexyl-alanine, 4- fluoro-proline, methyl-proline, norvaline, 6-diazo-5-oxo-norleucine, homocitrulline, 5- methyl-thiocitrulline, 2-aminoheptanedioic acid, Hydroxy-norarginine, homoarginine, penicillamine, 4,5-dehydro-leucine, allo-isoleucine, pipecolic acid, cyclohexyl-glycine, B-(2-thoazolyl)-alanine, B-(1 ,2,4-triazol- 1 -yl)-alanine, B-(2-thienyl)-serine, aminosuberic acid, amino-arginine, methyl-arginine, 1-amino-cyclopentanecarboxylic

acid, methyl-leucine, Norleucine, octahydroindole^-carboxylic acid, methyl- phenylalanine, 2-mercapto-histidine, 4-nitro-phenylalanine, B-(3,4-dihydroxy-phenyl)- serine, 4-carboxy-phenylalanine, methyl-histidine, 2,5-diido-histidine, B-(2- thienyl)alanine, B-(3-benzothienyl)-alanine, phenylglycine, 4-bromo-phenylalanine, homophenylalanine, 4-azido-phenylalanine, 4-cyano-phenylalanine, 3,5-dinitro-tyrosine, 3,5-dibromo-tyrosine, 1 -methyl-histidine, 3 -methyl-histidine, I ₃ 2,3,4- tetrahydroisoquinoline-3-carboxylic acid, B-(l-Naphthyl)-alanine, 4-iodo-phenylalanine, 3-Fluoro-phenylalanine, 4-tert-butyl -phenylalanine, a-methyl-tryptophan, 3-iodotyrosine, 3-nitro-tyrosine, 3,5-diiodo-tyrosine, B-(2-pyridyl)-alanine, B-(3-pyridyl)alanine, Spinacine, B-(2-naphthyl)-alanine, 3,4-dichloro-phenylalanine, 4-chloro-phenylalanine, 4-chloro-phenylalanine, 4-fluoro-phenylalanine, 4-methyl-tryptophan, 5-methyl- tryptophan, 7-hydroxy-l,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, Thyronine, B- (2-quinolyl)-alanine, 3-aminotyrosine, 4-amino-phenylalanine, B,B-diphenyl-alanine, 4- methyl-phenylalanine, 2,3,4,5,6-pentafluoro-phenylalanine, 4-benzoyl-phenylalanine, l,2,3,4-tetrahydronorharman-3-carboxylic acid and 3-hydroxymethyl-4-isopropylidene- tyrosine.

Additional reagents for modification of the hydroxyl groups of VBi ₂ or riboflavin include chloroacetic acid, 3-Chloropropylamine.HCl, 4-Nitrobenzyl chloroformate, Sodium Chloroacetate, 4-chlorobenzoic acid, 2-chlorobenzoic acid, 3-chlorobenzoic acid, 3- chloropropyl amine, Epichlorohydrin, Ethylchloroacetate, Bromo-benzyl bromide, Bromoethylammonium bromide, 3-bromo-l,2-propandiol, Bromoisobutyric acid, Bromoisovaleric acid, Bromophenacylbromine, Bromophenylhydrazine,

Bromophenylisocyanate, Bromopropionic acid, Bromosuccinic acid, 6-bromocaproic acid, 6-bromohexanoic acid, Bromoacetylbromide, Bromoacetic acid, Ethyl-4- bromobutyrate, 1,3-dibromopropane, 1 ,4-dibromobutane, 2-Bromobenzylbromide, 2- Bromoethylammonium bromide, Iodoacetic acid, Iodobenzoic acid and Iodopropionic acid.

Suitable halides of alkylcarboxylates include but are not limited to the halide derivatives of succinic acid, maleic acid, malonic acid, tartartic acid, such as Bromoacetic acid (C ₂ H ₃ BrO ₂ ), Bromosuccinic acid, (NOOCCH ₂ CHBrCOOH), Bromopropionic acid (BrCH ₂ CH ₂ COOH), Bromocaproic acid (CH ₃ (CH ₂ ) ₃ CHBrCOOH, Bromoisobutyric acid, Bromoisovaleric acid, Bromoaniline, Bromoanthranilic acid, Bromobenzoic acid, Bromoacetylbromide, Bromoacetylchloride, 4,4'-difluoro-3 '3 '-dinitrodiphenylsulfone, toluenediisocyanate, Hexamethylenediisocyanate, Bisoxiranes such as 1,4-butanediol diglycidyl ether (l,4-bis(2,3-eopoxypropoxy)butane), Heterocyclic Halides, s-Triazines, such as cyanuric chloride, 2-amino-4,5-dichloro-s-triazine, 2-carboxymethylamino-4,6- dichloro-s-triazine, and 2-carboxymethyloxy-4,6-dichloro-s-triazine, fluoropyrimidine, Vinyl sulfone, p-benzoquinone, Hydroxy-S-triazole

Hydrazidyl Derivatives Hydrazidyl derivatives that may be linked to the targeting agent or the polymer include, but are not limited to adipic acid dihydrazide, carbohydrazide, ethylmalonic acid dihydrazide, glutaric acid dihydrazide, isophthalic acid dihydrazide, maleic acid dihydrazide, malonic acid dihydrazide, naphthalene dihydrazide, oxalic acid dihydrazide, pimelic acid dihydrazide, sebacic acid dihydrazide, suberic acid dihydrazide, succinic acid dihydrazide, ^lO-dihydro-^lO-ethanoanthracene-l l^-dicarboxylic acid dihydrazide, terathalic acid dihydrazide, thiocarbazide, 2,3,4,5-tetrahydrophthalic acid dihydrazide, 4,4'-oxy-bis(benzenesulfonyl hydrazide).

In addition, hydrazidyl compounds may be made from derivatisation of the carboxyl group(s) on amino acids. Dihydrazides may formed from various diacids including aspartic acid, glutamic acid, or diacids of the general formula NH ₂ NH-CO-R-CO- NHNH ₂ , where R can equal any alkyl or aryl containing group, or where CO-R-CO could be replaced by CO or CS.

Additionally, targeting agents may be chelated to the nanostructure by reaction of a targeting agent with the alpha amine of an amino acid, followed by modification of the free carboxyl group with hydrazine to form a terminal hydrazide. Suitable amino acids include, but are not limited to 6-aminonicotinic acid, 4-amino-3-phenylbutyric acid, p- aminophenylacetic acid, 3-amino-2-naphthoic acid, 3-amino-4-hydroxybutyric acid, p- amino-hippuric acid, aminobutyric acid, aminocaproic acid, l-aminoanthraquinone-2- carboxylic acid, aminoadipic acid, m-aminobenzoic acid, o-aminobenzoic acid, anthranilic acid, aminohexanoic acid, aminonicotinic acid, aminoproprionic acid, aminooctanoic acid, aminooxoacetic acid, 7-amino-4-methyl-3-coumarinylacetic acid, Aminoadipic acid, glycine, proline, hydroxyproline, alanine, valine, glutamine, glutamic acid, asparagine, aspartic acid, phenylalanine, tyrosine, threonine, serine, tryptophan, histidine, leucine, isoleucine, cystine, methionine, ornithine, arginine, and lysine. Alternative amino acids include Amino-Glycine, Diaminopropionic acid, Diaminobutyric acid, Dehydroalanine, Fluoro-alanine, Chloro-alanine, Azetidine-2-carboxylic acid, aminobutyric acid, cyanoalanine, amino-isobutyric acid, homocysteine, methylene- glutamic acid, hydroxy-glutamic acid, ornithine, difluoromethyl-ornithine, cyclopropyl- alanine, tert-butyl-alanine, propargyl-glycine, 2-allyl-glycine, tert-butyl-glycine, alla- threonine, Ureido-alanine, pyroglutamic acid, carboyglutamic acid, 2,6-Diamino-4- hexynoic acid, 4-amino-piperidine-4-carboxylic acid, B-(l-cyclopentenyl)-alanine, B- Cyclopentyl-alanine, thioproline, 3, 4-dehydro-proline, methyl -valine, hydroxyproline, citrulline, thiocitrulline, aminoadipic acid, 4,5-dehydro-lysine, 6-hydroxy-lysine, B- cyclohexyl-alanine, B,B-dicyclohexyl-alanine, 4-fluoro-proline, methyl-proline, norvaline, 6-diazo-5-oxo-norleucine, homocitrulline, 5-methyl-thiocitrulline, 2- aminoheptanedioic acid, Hydroxy-norarginine, homoarginine, penicillamine, 4,5- dehydro-leucine, allo-isoleucine, pipecolic acid, cyclohexyl -glycine, B-(2-thoazolyl)- alanine, B-(l,2,4-triazol-l-yl)-alanine, B-(2-thienyl)-serine, aminosuberic acid, amino- arginine, methyl-arginine, 1-amino-cyclopentanecarboxylic acid, methyl-leucine,

Norleucine, octahydroindole-2-carboxylic acid, methyl-phenylalanine, 2-mercapto- histidine, 4-nitro-phenylalanine, B-(3,4-dihydroxy-phenyl)-serine, 4-carboxy- phenylalanine, methyl-histidine, 2,5-diido-histidine, B-(2-thienyl)alanine, B-(3- benzothienyl)-alanine, phenylglycine, 4-bromo-phenylalanine, homophenylalanine, 4- azido-phenylalanine, 4-cyano-phenylalanine, 3,5-dinitro-tyrosine, 3,5-dibromo-tyrosine, 1 -methyl-histidine, 3 -methyl-histidine, l,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, B-(l-Naphthyl)-alanine, 4-iodo-phenylalanine, 3-Fluoro-phenylalanine, 4-tert-butyl- phenylalanine, a-methyl-tryptophan, 3-iodotyrosine, 3-nitro-tyrosine, 3,5-diiodo-tyrosine, B-(2-pyridyl)-alanine, B-(3-pyridyl)alanine, Spinacine, B-(2-naphthyl)-alanine, 3,4- dichloro-phenylalanine, 4-chloro-phenylalanine, 4-chloro-phenylalanine, 4-fluoro- phenylalanine, 4-methyl-tryptophan, 5-methyl-tryptophan, 7 -hydroxy- 1,2,3, 4- tetrahydroisoquinoline-3-carboxylic acid, Thyronine, B-(2-quinolyl)-alanine, 3- aminotyrosine, 4-amino-phenylalanine, B,B-diphenyl-alanine, 4-methyl-phenylalanine, 2,3,4,5,6-pentafluoro-phenylalanine, 4-benzoyl-phenylalanine, 1 ,2,3,4- tetrahydronorharman-3-carboxylic acid, 3-hydroxymethyl-4-isopropylidene-tyrosine.

Nanostructures of the invention may be synthesized by conjugating the targeting molecule via one or more pendant groups of the polymer. The pendant groups may be free reactive groups on the cross-linking agents that remain after the cross-linking procedure has been completed in the preparation of the nanostructures, for example a bifunctional molecule such as an amino carboxylic acid, a diamine, a dithiol or a dicarboxylic acid. Further examples of cross-linking agents that may be used for conjugation to the targeting molecules include: N-(4-azidophenylthio)-phthalimide, 4,4'- dithiobisphenylazide, dithio-bis-(succinimidyl-propionate), dimethyl-3,3'-dithio-bis- propionimidate.2HCl, 3,3'-dithio-bis-(sulfosuccinimidyl-propionate), ethyl-(4- azidophenyl)- 1 ,3 '-dithiopropionate, sulfo-succinimidyl-2-(m-azido-o-nitrobenzamido)- ethyl- 1 ,3 '-dith.iobutyrimidate.HCl, N-succinimidyl-(4-azido-phenyl)- 1 ,3 'dithiopropionate, sulfo-succinimidyl-2-(m-azido-o-nitro-benzamido)-ethyl- 1,3 '-dithiopropionate, sulfo-

succinimidyl-2-(p-azido-salicylamido)-ethyl- 1 ,3'-dithiopropionate, N -succinimidyl-3-(2- pyridylthio)propionate; sulfosuccinimidyl-(4-azidophenyidithio)-propionate, 2- iminothiolane, disuccinimidyl tartrate and bis-[2-(succinimidyloxycarbony-ethyl]- sulfone.

In one embodiment, where the targeting molecule is VBi ₂ , the targeting molecule may be attached to the polymer via a pendant group as follows:

EDAC Polymer-NH-C(O)(CH ₂ ) _n -COOH + VB, ₂ -NH ₂ → Polymer-NH-C(O)(CH ₂ ) _n -C(O)-NH-VB, ₂

In one embodiment, the targeting molecule is linked to the polymer via an ester linkage.

In an alternative embodiment, the VBi ₂ targeting molecule may be attached directly to the polymer via reaction of a compound having the formula VBj ₂ -X, wherein X is a leaving group, for example a halide, a tosylate or a mesylate, with a polymer comprising a nucleophilic group. An example of this embodiment is given below:

Polymer-OH + VBi ₂ -Br -» polymer-O-VBi ₂

In a further alternative embodiment, the nucleophilic group may be located on the VBi ₂ molecule, and the leaving group may be located on the polymer.

The present invention further provides a pharmaceutical composition comprising the nanostructures of the invention and a pharmaceutically acceptable carrier and/or diluent. Pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, coatings, anti-bacterial and anti-fungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art and except insofar as any conventional media or agent

is incompatible with the modulator; their use in the pharmaceutical compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

As such, the present invention provides a method of orally delivering a pharmaceutical agent, such as those described in the present invention, to a subject in need of such pharmaceutical agent, said method comprising administering of a therapeutically effective amount of a complex of the invention, or a pharmaceutical composition thereof comprising such pharmaceutical agent.

In one embodiment, the pharmaceutical agent in the complexes or the pharmaceutical composition of the invention is insulin. Accordingly, the present invention is directed to a method for treating diabetes (including diabetes mellitus) in a subject in need of said treatment, said method comprising administration of a therapeutically effective amount of the complex or the pharmaceutical composition of the invention, wherein the pharmaceutical agent is insulin. Such treatment may be used alone or in conjunction with another drug or therapy.

The present invention is further described by the following non-limiting examples.

EXAMPLES

EXAMPLE 1

Preparation of 5'Lysyl-VBπ Vitamin Bi ₂ (Sigma) was dissolved at 100 mg/ml in DMSO. Solid l,l'-carbonyl-di( 1,2,4- triazole was added to a final concentration of 25 mg/ml and reacted for 30 minutes. The activated derivative was precipitated with ethyl acetate and the supernatant decanted. The pellet was then dissolved in a solution of DMSO containing 50 mg/ml Lysyl-copper- lysine and 100 mg/ml TEA. The reaction was allowed to proceed overnight. The product was precipitated with acetone, and the resultant pellet dissolved in distilled water. The product was purified by chromatography on S-Sepharose. The eluted material was concentrated on XAD- 16 resin, eluted with methanol, rotary evaporated to remove methanol and lyophilized. Mass spectral analysis of the lysyl-VBi ₂ (K-VBi ₂ ) showed the presence of the product of Molecular Weight 1527.5 and a half mass of 764.

EXAMPLE 2

Preparation of Lysyl-e-VBn

The eVB _]2 -carboxylic acid derivative (1 gm) (Wockhardt) was dissolved at 100 mg/ml in

DW. NHS (0.5 gm) was dissolved at 92 mg/ml in DMF and added to the VB, ₂ . Solid EDAC at ~ 4-fold excess (3.1 gm) was added as powder to the NHS/eVBi ₂ mix, while stirring rapidly. The reaction was allowed to proceed for 20 mins. At this stage complexes of Lysyl-Copper-Lysine at 100 mg/ml in 1% NaHCO3 (5.8 gm in 58 ml) was added to eVB _I2 -NHS and reacted overnight. The reaction mix was precipitated with acetone to 70% and then the pellet was dissolved in 100 ml 50 mM EDTA. The soluble material was loaded onto Dowex 1 X 2 resin. All of the Lys-Cu-Lys stuck to the column. The Lysyl-eVBi ₂ (K-e VBi ₂ ), which appeared in the flow through was loaded onto XAD- 16 resin and washed with DW. Residual Lys-Cu-Lys washed through. The product was eluted with 4 x 100 ml 75% MeOH. After which the product was rotary evaporated,

resuspended in distilled water and lyophilized.

EXAMPLE 3

Preparation of glutathionyl-VBπ Hydroxy-cobalamin (Aventis) was dissolved at 100 mg/ml in 2.5% acetic acid. Solid zinc was added as a powder to a final concentration of 10 mg/ml and allowed to reduce Colli to CoI for 5 minutes. An equal weight of glutathione as a powder was added and reacted overnight. The product, glutathionyl-VBi ₂ (GSVBi ₂ ) was purified by Reverse Phase HPLC. Mass spectral analysis of GSVBi ₂ showed the presence of the product of Molecular Weight 1636 and a mass of 818. Alternatively, GSVB] ₂ is manufactured by alternative methods known in the art, for example, that disclosed in US Patent No. 7,030,105.

EXAMPLE 4 Preparation of cysteinyl-VB^

Hydroxy-cobalamin was dissolved at 100 mg/ml in 2.5% acetic acid. Solid zinc was added as a powder to a final concentration of 10 mg/ml and allowed to reduce Colli to CoI for 5 minutes. An equal weight of cysteine as a powder was added and reacted overnight. The product was purified by Reverse Phase HPLC.

EXAMPLE 5

Preparation of Carboxymethyl Dextran

Dextran T70 (100 gm) of average molecular weight of 70,000 Daltons was dissolved at

50 mg/ml in 1 M Na ₂ CO ₃ and was reacted with 200 gm sodium chloroacetate. The reaction was heated to 80 ⁰ C and allowed to proceed for 3 hours. The carboxymethyl dextran (CMD) was then purified by positive pressure dialysis against DW via tangentional flow filtration and lyophilized. The carboxymethyl pendant side chains comprise carboxylic acid moieties that may be used to interact with pharmaceutical

agents, or may be used to conjugate to targeting agents, or are available for chelation. Alternatively, CMD was obtained from a commercial source such as Fluka. The presence of carboxy groups on the CMD was verified by a negative zeta potential using standard instrumentation, for example, a Zetasizer (NanoZS90, Malvern, UK).

The ability of CMD ability to chelate copper was tested by adding copper chloride to a solution of 50 mg/ml CMD. Chelation was verified by the observance of a spectral change in the 600-900 nm range using standard instruments.

EXAMPLE 6

Preparation of VB^-targeted polysaccharide

Polysaccharide, for example, CMD (see Example 5 above) was dissolved at 50 mg/ml in distilled water. N-Hydroxysuccinimide was dissolved at 100 mg/ml in DMF, and added at 1:2 w/w to CMD. EDAC added as powder at an equal weight to CMD. Activation proceeded for 30 minutes after which Lysyl-vitamin B ₎₂ (K-VB ₁₂ ) or lysyl-eVB _]2 (K- eVBi ₂ ), dissolved at 25 mg/ml in 10% NaHCO ₃ was added 1 :3 w/w to CMD. The solution was reacted overnight and the K-VBi ₂ -CMD was dialysed exhaustively against distilled water and lyophilized. A

Alternatively, a VBi ₂ -targeted polysaccharide, for example, a VB ₎₂ -targeted CMD was formed using non-covalent bonding interactions, for example, using a suitable chelatable VBi ₂ derivative such as GSVBi ₂ (see Example 3 above) and in combination with a suitable chelating agent such as ZnCl ₂ .

EXAMPLE 7

Preparation of VBi2-targeted polysaccharide

Alginate is dissolved at 50 mg/ml in distilled water. N-Hydroxysuccinimide is dissolved at 100 mg/ml in DMF and added at 1:2 w/w to the alginate solution. EDAC is added as

powder at an equal weight to alginate. Activation proceeds for 20-60 minutes after which Lysyl-vitamin Bi ₂ (K-VBi ₂ ) or lysyl-eVB] ₂ (K-eVBi ₂ ), dissolved at 25 mg/ml in 10% NaHCO ₃ is added 1:3 w/w to CMD. The solution is reacted overnight and the VBi ₂ -ALG was dialysed exhaustively against distilled water and lyophilized.

Alternatively, a VB _]2 -targeted polysaccharide, for example, a VB] ₂ -targeted alginate can be formed using non-covalent bonding interactions, for example, using a suitable chelatable VBi ₂ derivative such as GSVBi ₂ (see Example 3 above) and in combination with a suitable chelating agent such as ZnCl ₂ .

EXAMPLE 8

Preparation of an Oil-Surfactant phase suitable for the formation of a Water-in-Oil

Microemulsion

An oil mixture consisted of caprylic/capric triglyceride (Crodamol GTCC; Croda) and medium chain mono- and di-glyceride (Capmul MCM C8; Croda) at a 3:1 ratio was made. A surfactant/co-surfactant mixture contained polysorbate 80 (Crillet 4; Croda; surfactant) and Crill 4 (Span 80; co-surfactant) was made at a 3:2 ratio. The surfactant mixture was heated to 40-50 degrees Celcius during formation. The oil-surfactant phase was prepared by mixing 7.6 g Crodamol/Capmul oil mixture with 1.4 g Crillet/Crill surfactant/co-surfactant mixture.

EXAMPLE 9

Preparation of an Oil-Surfactant phase suitable for the formation of a Water-in-Oil

Microemulsion An oil mixture of a 3:1 ratio of Crodamol GTCC to Capmul MCM C8 was prepared. A surfactant/co-surfactant mixture of a 3:2:0.75 ratio of Crillet 4 (Tween 80) to Crill 4 (Span 80) to AB30 (coco-betaine) was prepared. An oil-surfactant phase was then prepared by mixing the oil and surfactant mixtures at a ratio of 7.6: 1.4.

EXAMPLE 10

Preparation of nanoparticles containing insulin

ZnC12 solution (2 ml; 1.0M) was added to 48 ml of oil-surfactant phase, the formation of which is described above in Example 9 and stirred at 250 rpm for 30 minutes whilst avoiding bubble formation to form a microemulsion ("zinc ME"). Recombinant human insulin (0.3g, Wockhardt) was dissolved at lOOmg/ml in 0.1N HCl (3.0ml) and the pH was adjusted to 3.1. Insulin solution (3.0 ml) was added to 50ml of oil-surfactant phase, the formation of which is described above in Example 8, and stirred at 250 rpm whilst avoiding bubble formation to form a second microemulsion ("insulin ME"). Zinc ME (50 ml) and freshly made insulin ME (53 ml) were mixed and briefly stirred at 250 rpm whilst avoiding bubble formation to form a third microemulsion ("insulin/zinc ME").

VBi ₂ -CMD (2.Og; see Example 6, above) was added to 0.02 M NaOH (20 ml) and stirred for 10 to 20 minutes to allow for complete dissolution. VB] ₂ -CMD solution (20 ml) was added to 300 ml of oil-surfactant phase, the formation of which is described above in Example 8 and then stirred at 250 rpm for 30 minutes whilst avoiding bubble formation to form a fourth microemulsion ("VBi ₂ /CMD ME").

The freshly made insulin/zinc ME was immediately added to 320 ml of CMD/VBπ ME and stirred for 10 to 20 minutes at 250 rpm for whilst avoiding bubble formation to form a fifth microemulsion ("CMD/VBi ₂ / insulin/zinc ME").

CMD/VBi ₂ / insulin/zinc nanoparticles were precipitated by the addition of the CMD/VBi ₂ / insulin/zinc ME to 600 ml of ethanol whilst rapidly stirring. The resulting mixture was spun at 2,000 rpm for 1 minute, decanted and the pellet resuspended in 600 ml of ethanol to wash off residual ME oils. Nanoparticles were pelleted again by spinning at 2,000 RPM for 1 minute. Resuspended particles were lyophilized using standard

techniques and stored at room temperature.

For the purposes of nanoparticle characterisation, some batches of freshly made insulin/zinc ME are precipitated, spun, resuspended, washed, re-pelleted and lyophilized using the above-mentioned procedure, then re-suspended in a suitable volume of distilled water to determine the poly-dispersity of the "core" particles using methods described below in Example 15.

EXAMPLE Il Preparation of nanoparticles containing insulin

ZnCl ₂ (0.27 g) was added to 30 ml of oil-surfactant phase, the formation of which was described above in Example 9 and stirred at 250 rpm for 30 minutes whilst avoiding bubble formation to form a microemulsion ("zinc ME"). Recombinant human insulin (0.3g, Wockhardt) was dissolved at lOOmg/ml in 0.1N HCl (3.0ml) and the pH was adjusted to 3.0. Insulin solution (3.0 ml) was added to 70ml of oil-surfactant phase, the formation of which was described above in Example 8, and stirred at 250 rpm whilst avoiding bubble formation to form a second microemulsion ("insulin ME"). Zinc ME (30ml) and freshly made insulin ME (70ml) were mixed and briefly stirred at 250 rpm whilst avoiding bubble formation to form a third microemulsion ("insulin/zinc ME").

Carboxymethyldextran (CMD; 75 mg/ml; see Example 5 above) and glutathionylcobalamin (GSVB) ₂ ; 25 mg/ml; see Example 3 above) was dissolved in 0.02 M NaOH and stirred for 10 to 20 minutes to allow for complete dissolution. GSVBi ₂ - CMD solution (20 ml) was added to 300 ml of oil-surfactant phase, the formation of which was described above in Example 8 and then stirred at 250 rpm for 30 minutes whilst avoiding bubble formation to form a fourth microemulsion ("GSVB] ₂ /CMD ME").

The freshly made insulin/zinc ME was immediately added to 320 ml of GSVBi ₂ /CMD

ME and stirred for 10 to 20 minutes at 250 rpm for whilst avoiding bubble formation to form a fifth microemulsion ("GSVB ₁₂ /CMD/insulin/zinc ME").

GSVB^/CMD/insulin/zinc nanoparticles were precipitated by the addition of the GSVBi ₂ /CMD/insulin/zinc ME to 600 ml of ethanol whilst rapidly stirring. The resulting mixture was spun at 2,000 rpm for 1 minute, decanted and the pellet resuspended in 600 ml of ethanol to wash off residual ME oils. Nanoparticles were pelleted again by spinning at 2,000 RPM for 1 minute. Resuspended particles were lyophilized using standard techniques and stored at room temperature. '

EXAMPLE 12

Preparation of nanoparticles containing insulin

A suitable volume of ZnCl ₂ solution, for example, 2 ml of a 1.0 M ZnCl ₂ solution, is added to a suitable volume of oil-surfactant phase, for example, 45-50 ml of oil-surfactant phase, the formation of which is described above in Example 9. The resultant mixture is stirred at 250 rpm for 30 minutes whilst avoiding bubble formation to form a microemulsion ("zinc ME"). A suitable protein with known zinc-binding characteristics, for example, recombinant human insulin (0.3g, Wockhardt), is dissolved at 75-125 mg/ml in 0.1N HCl (3.0ml final volume) and the pH adjusted to 3.1. The insulin solution is added to a suitable volume of oil-surfactant phase, for example 45-60 ml of oil-surfactant phase, the formation of which is described above in Example 8, and stirred at 250 rpm whilst avoiding bubble formation to form a second microemulsion ("insulin ME"). A suitable volume of zinc ME, for example, 50 ml of zinc ME, and a suitable volume of the freshly made insulin ME, for example, 50-55 ml of insulin ME, are mixed and briefly stirred at 250 rpm whilst avoiding bubble formation to form a third microemulsion ("insulin/zinc ME").

A suitable weight Of VBi ₂ -ALG (see Example 7, above) is solubilized in a weak solution

of NaOH by stirring. A suitable volume of VBi ₂ -ALG solution, for example, 15-24 ml, is added to a suitable volume of oil-surfactant phase, for example 250-320 ml of oil- surfactant phase, the formation of which is described above in Example 8. The resultant mixture is stirred at 250 rpm for 30 minutes whilst avoiding bubble formation to form a fourth microemulsion ("VB , ₂ /ALG ME").

The freshly made insulin/zinc ME is immediately added to 320 ml of VBi ₂ /ALG ME and stirred for 10 to 20 minutes at 250 rpm for whilst avoiding bubble formation to form a fifth microemulsion ("VBi ₂ /ALG/insulin/zinc ME").

VB i ₂ / ALG/ insulin/zinc nanoparticles are precipitated by addition of the VBi ₂ /ALG/ insulin/zinc ME to an excess volume of 100% ethanol whilst rapidly stirring. The resulting mixture is spun at 2,000 rpm for 1 minute, decanted and the pellet resuspended in 600 ml of ethanol to wash off residual ME oils. Nanoparticles are pelleted again by spinning at 2,000 RPM for 1 minute. Resuspended particles are lyophilized using standard techniques and stored at room temperature.

EXAMPLE 13

Preparation of nanoparticles containing insulin ZnCl ₂ (0.27 g) is added to 30 ml of oil-surfactant phase, the formation of which is described above in Example 9 and stirred at 250 rpm for 30 minutes whilst avoiding bubble formation to form a microemulsion ("zinc ME"). Recombinant human insulin (0.3g, Wockhardt) is dissolved at lOOmg/ml in 0.1N HCl (3.0ml) and the pH is adjusted to 3.0. Insulin solution (3.0 ml) is added to 70ml of oil-surfactant phase, the formation of which is described above in Example 8, and stirred at 250 rpm whilst avoiding bubble formation to form a second microemulsion ("insulin ME"). Zinc ME (30ml) and freshly made insulin ME (70ml) are mixed and briefly stirred at 250 rpm whilst avoiding bubble formation to form a third microemulsion ("insulin/zinc ME").

Alginate (ALG; 75 mg/ml) and glutathionylcobalamin (GSVBi ₂ ; 25 mg/ml; see Example 3 above) is dissolved in 0.02 M NaOH and stirred for 10 to 20 minutes to allow for complete dissolution. GSVB ₎₂ /ALG solution (20 ml) is added to 300 ml of oil-surfactant phase, the formation of which is described above in Example 8 and then stirred at 250 rpm for 30 minutes whilst avoiding bubble formation to form a fourth microemulsion ("GSVB, ₂ /ALG ME").

The freshly made insulin/zinc ME is immediately added to 320 ml of GSVBi ₂ / ALG ME and stirred for 10 to 20 minutes at 250 rpm for whilst avoiding bubble formation to form a fifth microemulsion ("G SVB , ₂ /ALG/insul in/zinc ME").

GSVBi ₂ /ALG/insulin/zinc nanoparticles are precipitated by the addition of the GSVBi ₂ / ALG/insulin/zinc ME to 600 ml of ethanol whilst rapidly stirring. The resulting mixture is spun at 2,000 rpm for 1 minute, decanted and the pellet resuspended in 600 ml of ethanol to wash off residual ME oils. Nanoparticles are pelleted again by spinning at 2,000 RPM for 1 minute. Resuspended particles are lyophilized using standard techniques and stored at room temperature.

EXAMPLE 14

Preparation of nanoparticles containing nucleic acid

A suitable volume of solution containing calcium ions, for example, CaCl ₂ solution, for example, 2 ml of a 1.0M CaCl ₂ solution, is added to a suitable volume of oil-surfactant phase, for example, 45-50 ml of oil-surfactant phase, the formation of which is described above in Example 9. The resultant mixture is stirred at 250 rpm for 30 minutes whilst avoiding bubble formation to form a microemulsion ("Ca ME"). A suitable nucleic acid, such as a specific oligonucleotide species or and si RNA species, is dissolved at 20-125 mg/ml in a suitable buffer and the pH adjusted to 3.1. An example of an siRNA species is

a Fas-siRNA species as described by Song et al. {Nat Med 9:347 - 351, 2003). The nucleic acid solution is added to a suitable volume of oil-surfactant phase, for example 45-60 ml of oil-surfactant phase, the formation of which is described above in Example 8, and stirred at 250 rpm whilst avoiding bubble formation to form a second microemulsion ("NA ME"). A suitable volume of Ca ME, for example, 50 ml of Ca ME, and a suitable volume of the freshly made NA ME, for example, 50-55 ml of NA ME, are mixed and briefly stirred at 250 rpm whilst avoiding bubble formation to form a third microemulsion ("Ca/NA ME").

A suitable VBi ₂ -labelled polysaccharide preparation, for example, either VBi ₂ /CMD (2.Og; see Example 6, above) or VBi ₂ /ALG (see Example 7, above) is solubilized in a weak solution of NaOH by stirring. A suitable volume of VBi ₂ -labelled polysaccharide solution, for example, 15-30 ml, is added to a suitable volume of oil-surfactant phase, for example 250-320 ml of oil-surfactant phase, the formation of which is described above in Example 8 to form a fourth microemulsion ("VB ₁₂ /POLY ME").

Alternatively, a suitable polysaccharide, for example, carboxymethyldextran (CMD) or alginate and a suitable VBi ₂ derivative capable of being linked to a polysaccharide via chelation, for example, glutathionylcobalamin (see Example 3 above) are dissolved at a suitable concentrations in a suitable solution, for example, 0.02 M NaOH and stirred for 10 to 20 minutes to allow for complete dissolution. The VBi ₂ /POLY solution (20 ml) is added to 300 ml of oil-surfactant phase, the formation of which is described above in Example 8 and then stirred at 250 rpm for 30 minutes whilst avoiding bubble formation to form a fourth microemulsion ("VB, ₂ /POLY ME").

The freshly made Ca/NA ME is immediately added to a suitable volume of VB] ₂ /POLY ME, for example, 265-345 ml of VB, ₂ /POLY ME and stirred for 10 to 20 minutes at 250 rpm whilst avoiding bubble formation to form a fifth microemulsion

("VB, ₂ /POLY/Ca/NA ME").

VB _I2 -POL Y/Ca/NA nanoparticles are precipitated by the addition of the VBi ₂ - POLY/Ca/NA ME to an excess volume of 100% ethanol whilst rapidly stirring. The resulting mixture is spun at 2,000 rpm for 1 minute, decanted and the pellet resuspended in 600 ml of ethanol to wash off residual ME oils. Nanoparticles are pelleted again by spinning at 2,000 RPM for 1 minute. Resuspended particles are lyophilized using standard techniques and stored at room temperature.

EXAMPLE 15

Preparation of nanoparticles containing etanercept (Enbrel)

ZnCl ₂ (0.08 M) was dissolved into low pH coco-betaine (pH = 4.0) to a final concentration of 0.15 M. The low pH coco-betaine/Zn solution (0.5 ml) was added to 10 ml of oil-surfactant phase, the formation of which is described above in Example 8, to form a microemulsion (CB/Zn ME).

Protein was dissolved in distilled H2O (500 μl), the pH adjusted to 4.0 and added to CB/Zn ME from above to form a second microemulsion (protein/CB/Zn ME).

Sodium hydroxide (1 ml, 0.2 M) was added to 10 ml of oil-surfactant phase, the formation of which is described above in Example 8, to form a microemulsion (NaOH ME).

The NaOH ME was added to the protein/CB/Zn ME with mixing to precipitate protein/CB/ZnOH particles in the combined microemulsion, whch was immediately added to a pre-made CMD/AlgVBi ₂ ME according to Example 7 above with mixing to form AIgVB i ₂ /CMD/protein/Zn nanoparticles in microemulsion.

The resultant AIgVB i ₂ /CMD/protein/Zn nanoparticles were precipitated by addition into ethanol (120 ml) with rapid stirring. The resulting mixture was spun at 1,500 rpm for 1 minute, decanted and the pellet resuspended in 100 ml of ethanol to wash off residual ME oils. Nanoparticles were again pelleted by spinning at 2,500 RPM for 5 minutes. Resuspended particles were resuspended in distilled water (10 ml), filtered (0.45 micron filter; Millipore) then lyophilized using standard techniques and stored at room temperature.

In the general procedure above, the protein employed was etanercept (Enbrel) (10 mg). The resultant nanoparticles were a light pink powder, 32 mg of having a mean particle size of 253 nm.

EXAMPLE 16

Preparation of nanoparticles containing ALST2 Low pH coco-betaine (pH = 4.0) (3 ml) was added to 30 ml of oil-surfactant phase, the formation of which is described above in Example 8, to form a 10:1 vol/vol MExoco- betaine microemulsion (CB ME).

Protein was dissolved in distilled H2O (33 mg/ml) and the resultant protein solution (20 μl) was added to CB ME (200 μl) from above to form a second microemulsion (protein/CB ME).

ZnCl ₂ (0.5 M) was dissolved into sodium hydroxide (0.1 M) and 10 μl of the resultant solution was added to 200 μl of oil-surfactant phase, the formation of which is described above in Example 8, to form a 10:0.5 ratio vol/vol microemulsion (Zn/NaOH ME).

The protein/CB ME was added to the Zn/NaOH ME with mixing to precipitate protein/CB/Zn/NaOH particles in the combined microemulsion, whch was immediately

added to a pre-made CMD/Alg-GSVB _!2 ME (10 mg alginate-glutathionyl-VB _]2 , 40 mg carboxymethyldextran (CMD) in 1 ml if 0.02 M NaOH) according to Example 7 above with mixing to form Alg-GSVBi ₂ /CMD/protein/Zn nanoparticles in microemulsion.

The resultant AIgVB ₁₂ /CMD/protein/Zn nanoparticles were precipitated by addition into ethanol (1 ml) with rapid stirring. The resulting mixture was spun at 1,500 rpm for 1 minute, decanted and the pellet resuspended in 1 ml of ethanol to wash off residual ME oils. Nanoparticles were again pelleted by spinning at 2,500 RPM for 5 minutes. Resuspended particles were resuspended in distilled water (100 μl), filtered (0.45 micron filter; Millipore) then lyophilized using standard techniques and stored at room temperature.

In the general procedure above, the protein employed was ALST2 (10 mg), a 150 KD protein that acts as a TNF-blockeretanercept (Enbrel) (10 mg). The resultant nanoparticles were a light pink powder, 32 mg of having a mean particle size of 253 nm.

EXAMPLE 17

Mean nanoparticle size and polydispersity index (PDI)

Mean nanoparticle size and size distribution of nanoparticle preparations are analysed using dynamic light scattering (DLS) using standard methods. For example, aliquots of lyophilized nanoparticle preparations, the production of which is described in Example

11 , were re-suspended in DI water prior to DLS measurement using an on a Data Sizer

NanoZS (Malvern). A mean nanoparticle size (Z-average) of 182 nm and a polydispersity index of 0.234 were obtained from cumulative measurements (see Figure 1). These resulted indicate that the preparation contained particles of an average size less than 200 nm and no corresponding evidence of agglomeration.

EXAMPLE 18

Mean nanoparticle size and polydispersity index (PDI)

According to the method of Example 17, the lyophilized nanoparticle preparation containing etanercept described in Example 15 above were re-suspended in DI water prior to DLS measurement using an on a Data Sizer NanoZS (Malvern). A mean nanoparticle size (Z-average) of 253 nm was obtained from cumulative measurements (see Figure 2).

EXAMPLE 19 Zeta potential Zeta potential of various nanoparticle preparations are determined by phase analysis light scattering using standard instruments, for example, a Zetasizer (NanoZS90, Malvern, UK). Aliquots of lyophilized nanoparticle preparations are re-suspended in DI water prior to measurement. Zeta potential is calculated from the electrophoretic mobility using standard methods, for example, the Smoluchowski approximation approach.

EXAMPLE 20

Scanning Electron Microscopy

Surface morphology of nanoparticle preparations are visualized by scanning electron microscopy using standard methods. Freeze-dried samples of re-suspended nanoparticle preparations are coated by platinum prior to scanning. Standard image analysis methods are employed to determine nanoparticle shape and size.

EXAMPLE 21 Infrared spectroscopy IR spectra of nanoparticle preparations are characterised by infrared spectroscopy using standard methods. IR profiles of various nanoparticle preparations are compared to determine presence and interactions of pendant side chains such as carboxylate groups, interactions between the pendant side chains of the nanoparticle with one or more

pharmaceutical agents and interactions between the pendant side chains of the nanoparticle and a metal chelating agent.

EXAMPLE 22 UV-visible spectroscopy (UVS)

UV-vis spectra of nanoparticle preparations are characterised by UV-vis spectroscopy using standard methods. UV-vis spectral profiles of various nanoparticle preparations are compared to determine aggregation states of pharmaceutical agent loaded within the nanoparticles.

EXAMPLE 23

X-ray photoemission spectroscopy (XPS)

X-ray photoemission spectroscopy (XPS) is utilized to obtain information on elements and on their chemical bonds, allowing the identification of the different chemical compounds that is possible to find on nanoparticle surfaces. For example, XPS spectroscopy is able to distinguish the extent of VBi ₂ moieties upon the nanoparticle surface.

EXAMPLE 24 Differential scanning calorimetry (DSC)

Polymeric structure of nanoparticle preparations are characterised by DSC using standard methods. Thermal transition characteristics of various nanoparticle preparations are compared to determine the resulting glass transitions as a result of using different types and/or ratios of chelating metal ions.

EXAMPLE 25

Entrapment Efficiency and Dissolution studies

Nanoparticle preparations (Examples 10-16) are prepared by re-constituting lyophilized

nanoparticles in solution, for example distilled water or one of a variety of buffer soltions. Samples of solution are taken at various time points following re-constitution, for example, at 5, 10, 20, 30 45, 60, 120, 240, 360, 720, and 1200 minutes follwing re- constitution, and then spun or filtered to remove particles and analysed for free protein using a suitable method, for example, a BCA protein assay method, and/or a more specific protein assay such as an anti-human insulin ELISA. Nanoparticle stability over time is thus determined. Variables that can be altered within the course of the experiment include solution pH, temperature.

EXAMPLE 26

VBπ-mediated transcytosis of nanoparticles across cell monolayers

A cell line suitable for the modelling of gut endothelial transport of particles, for example, Caco-2 cells or HT-29 cells, are grown to confluence on filter membrane in transwell plates using known culturing techniques. For example, CaCo-2 cells are pre- seeded in Costar 12 well plates at 350000/well (50OuL per well; DMEM + 10% FCS) and grown for 21 days (media refreshed every 2 days).

Nanoparticle preparations, as described in Examples 10 to 16, are made by re-constituting lyophilised nanoparticles in water, followed by further dilution in culture medium. Intrinsic factor (IF), which, when bound with VB ₎₂ , is the complex that is transported across the gut endothelium. IF is added at suitable dilutions to wells. Controls include one or more of the following: the pre-incubation of luminal surface cell monolayers with an excess VB _]2 to block the VBi ₂ receptor; VB) ₂ nanoparticles incubated without IF; nanoparticles formulated using a polysaccharide to which no VBi ₂ is bound; and free payload (i.e. protein or nucleic acid without nanoparticles).

Plates are incubated at 37°C. Fluid samples are taken at regular intervals from the basolateral sections of each well, spun and analysed using an appropriate ELISA test or

nucleic acid analysis method. Results indicate the rate of transcytosis of nanoparticles and can be used to optimise variables associated with nanoparticle performance, including the effect of nanoparticle size distribution on cell uptake and the least mean number of VBj ₂ moieties per nanoparticle required for transcytosis.

EXAMPLE 27

Reduction in Blood Serum Glucose following sub-cutaneous administered nanoparticles containing insulin

Female Wistar or Lewis rats are made diabetic via a sub-cutaneous injection of 25-50 mg/kg streptozocin. Individual rats are regarded as diabetic when blood glucose levels rose to twice the normal level. Nanoparticle formulations are prepared by dissolving the lyophilised particles, as described in Examples 10 to 13, into sterile water (Baxter). Final particle concentrations varied between 20-40 mg/ml. Free human insulin (recombinant human insulin; Wockhardt). Rats received a sub-cutaneous injected dose of human insulin (dosage range 200 - 500 ug/kg) either alone or entrapped within nanoparticles (as described in Examples 10 to 13).

Blood glucose levels are taken at regular intervals after the insulin injection using Acucheck glucose monitor and plotted as a percentage of pre-administration levels.

It is anticipated that data will demonstrate that insulin administered in nanoparticles will results in sustained lower serum glucose levels, contrast to the shorter time course effect of free insulin.

EXAMPLE 28

Reduction in Blood Serum Glucose following sub-cutaneous administered nanoparticles containing insulin: Pharmacokinetic studies of SC insulin in Diabetic Rats

Recombinant human insulin (Wockhardt) is radiolabelled with ¹²⁵ I via the chloramine T method. Nanoparticles are prepared as described in Examples 10 to 13 using the radiolabelled ¹²⁵ I -insulin.

Female in-bred Wistar or Lewis rats are streptozotocin injected SC in order to induce diabetes, as described above in Example 22. Nanoparticles are sub-cutaneous injected into normal and diabetic rats. Separate groups of animals received ¹²⁵ I -labelled insulin alone, and ¹²⁵ I -labelled insulin in combination with VB ^-polysaccharide, as appropriate, for example, VBi ₂ -CMD (Example 6) or VBi ₂ -ALG (Example 7) which is not formulated into nanoparticles. Post injection blood is obtained from the tail vein of rats at regular intervals after the insulin injection and separated for calculation of recombinant human insulin concentration (by radioactivity) and biological activity by blood glucose measurement using an AcuCheck glucose meter. Data is represented as cpm, blood glucose, or the ratio of (serum insulin): (blood glucose reduction).

Comparison of serum levels of ¹²⁵ I -labelled with reduction in serum glucose levels is anticipated to demonstrate that nanoparticles containing insulin results in a delayed appearance of insulin in blood serum, which correlates with reduction in levels of serum glucose.

EXAMPLE 29

Reduction in Blood Serum Glucose following orally administered nanoparticles containing insulin

Nanoparticle formulations are prepared by dissolving the lyophilised particles, as described in Examples 10-13, into sterile water (Baxter). Final nanoparticle concentration varied between 20-40 mg/ml.

Female in-bred Wistar or Lewis rats are streptozotocin injected SC in order to induce

diabetes, as described above in Example 24. Diabetic rats, for example, 4 per group, received a 0.5 ml dose of nanoparticle dissolved in water by oral gavage. Controls are administered 0.5ml of water. Rats are fasted for a suitable period prior to administration of nanoparticles and allowed food and water ad lib post-oral gavage. Blood samples are taken at at regular intervals after nanoparticle administration

Results are anticipated to show that diabetic rats orally administered nanoparticles display significant and sustained lowering of blood glucose levels relative to controls.

EXAMPLE 30

Reduction in Cellular Apoptosis in a Mouse Model of Autoimmune Hepatitis following orally administered nanoparticles containing siRNA targeting Fas

Nanoparticle formulations are prepared by dissolving lyophilised particles, for example, nanoparticles containing Fas siRNA, as described in Example 14, into sterile water (Baxter). Final nanoparticle concentration varied between 20-40 mg/ml.

A suitable model of autoimmune hepatitis in mice, for example, a ConA induced liver fibrosis model or a Fas-specific antibody induced hepatitis model, is used.

Nanoparticles are orally administered into mice that exhibited autoimmune hepatitis by oral gavage (for example, 4 per group; 0.5 ml dose of nanoparticles dissolved in water). Controls are administered 0.5 ml of water.

Results are anticipated to show that mice with induced autoimmune hepatitis and subsequent orally administered nanoparticles display significant and sustained lowering of Fas mRNA and protein levels in hepatocytes compared with controls. Further, it is anticipated that significant numbers of animals will survive 10 days post-treatment, in contrast to the controls, most of which are expected to succumb by 3 days post-treatment.

EXAMPLE 31

In Vivo Studies Using a Pharmaceutical Composition Comprising Insulin- Containing Nanoparticles of the Present Invention The individual subjects of the in vivo studies described herein are warm-blooded vertebrate animals, which includes humans. In particular, volunteers have a diagnosis of either type I or type II diabetes. The clinical trial is subjected to rigorous controls to ensure that individuals are not unnecessarily put at risk and that they are fully informed about their role in the study.

Preferably to account for the psychological effects of receiving treatments, the trial is conducted in a double-blinded fashion. Volunteers are randomly assigned to placebo or a pharmaceutical composition comprising insulin-containing nanoparticle treatment groups. The pharmaceutical composition contains insulin-containing nanoparticles, the manufacture of which is described in Examples 10 to 13, at a final concentration of between 20-100 mg/ml. Furthermore, the relevant clinicians are blinded as to the treatment regime administered to a given subject to prevent from being biased in their post-treatment observations. Using this randomization approach, each volunteer has the same chance of being given either the pharmaceutical composition or the placebo.

Volunteers receive either the pharmaceutical composition or placebo for an appropriate period with biological parameters associated with type I or type II diabetes being measured at the beginning (baseline measurements before any treatment), end (after the final treatment), and at regular intervals during the study period. Such measurements include the levels of insulin in body fluids, tissues or organs compared to pre-treatment levels. Other measurements include, but are not limited to, indices of diabetes such as the incidence of blood glucose daily glycemic excursions, serum titers of pharmacologic indicators of disease such as C-peptide levels in response to meal challenges, hemoglobin

AIc levels or toxicity as well as ADME (absorption, distribution, metabolism and excretion), body weight and blood pressure measurements.

Information recorded for each patient includes age (years), gender, height (cm), family history of disease state or condition (yes/no), motivation rating (some/moderate/great) and number and type of previous treatment regimens for diabetes.

Volunteers taking part in this study are adults aged 18 to 65 years and roughly an equal number of males and females participate in the study. Volunteers with certain characteristics are equally distributed for placebo and the insulin-containing nanoparticle treatment. In general, at the conclusion of the study, the volunteers treated with placebo have little or no response to treatment, whereas the volunteers treated with the pharmaceutical composition of insulin-containing nanoparticles show positive trends in one or more of the following measures, including but not limited to: significant reductions in the average blood glucose daily glycemic excursions; significant increases in C-peptide levels in response to meal challenges; reduced hemoglobin AIc levels.

EXAMPLE 32

In Vivo Studies Using a Pharmaceutical Composition Comprising siRNA- Containing Nanoparticles of the Present Invention

The individual subjects of the in vivo studies described herein are warm-blooded vertebrate animals, which includes humans. In particular, volunteers have a diagnosis of hepatitis. The clinical trial is subjected to rigorous controls to ensure that individuals are not unnecessarily put at risk and that they are fully informed about their role in the study.

Preferably to account for the psychological effects of receiving treatments, the trial is conducted in a double-blinded fashion. Volunteers are randomly assigned to placebo or a pharmaceutical composition comprising siRNA-containing nanoparticle treatment

groups. The pharmaceutical composition contains Fas siRNA-containing nanoparticles, the manufacture of which is described in Example 14, at a final concentration of between 20-100 mg/ml. Furthermore, the relevant clinicians are blinded as to the treatment regime administered to a given subject to prevent from being biased in their post-treatment observations. Using this randomization approach, each volunteer has the same chance of being given either the pharmaceutical composition or the placebo.

Volunteers receive either the pharmaceutical composition or placebo for an appropriate period with biological parameters associated with hepatitis being measured at the beginning (baseline measurements before any treatment), end (after the final treatment), and at regular intervals during the study period. Such measurements include the levels of Fas protein in body fluids, tissues or organs compared to pre-treatment levels. Other measurements include, but are not limited to, indices of hepatitis such as serum titers of pharmacologic indicators of disease such as increased activities of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatise; bilirubin-related indices such as distribution of direct bilirubin as a percentage of total bilirubin, peak bilirubin and total bilirubin; coagulation tests such as prothrombin time; frequency of jaundice as well as ADME (absorption, distribution, metabolism and excretion), body weight and blood pressure measurements.

Information recorded for each patient includes age (years), gender, height (cm), family history of disease state or condition (yes/no), motivation rating (some/moderate/great) and number and type of previous treatment regimens for hepatitis.

Volunteers taking part in this study are adults aged 18 to 65 years and roughly an equal number of males and females participate in the study. Volunteers with certain characteristics are equally distributed for placebo and the Fas siRNA-containing nanoparticle treatment. In general, at the conclusion of the study, the volunteers treated

with placebo have little or no response to treatment, whereas the volunteers treated with the pharmaceutical composition of Fas siRNA-containing nanoparticles show positive trends in one or more of the following measures, including but not limited to: significant reductions in activities of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatise; bilirubin-related indices such as distribution of direct bilirubin as a percentage of total bilirubin, peak bilirubin and total bilirubin; decreased prothrombin time; reduced frequency of jaundice.

EXAMPLE 34 In Vivo Studies Using a Pharmaceutical Composition Comprising Etanercept (Enbrel) -Containing Nanoparticles of the Present Invention

The individual subjects of the in vivo studies described herein are warm-blooded vertebrate animals, which includes humans. In particular, volunteers suffer from inflammatory disorders generally treated by agents that act as tumor necrosis factor (TNF) blockers. People with an immune disease, such as rheumatoid arthritis, juvenile idiopathic arthritis, ankylosing spondylitis, psoriatic arthritis, or psoriasis are candidates for treatment. The clinical trial is subjected to rigorous controls to ensure that individuals are not unnecessarily put at risk and that they are fully informed about their role in the study.

Preferably to account for the psychological effects of receiving treatments, the trial is conducted in a double-blinded fashion. Volunteers are randomly assigned to placebo or a pharmaceutical composition comprising insulin-containing nanoparticle treatment groups. The pharmaceutical composition contains insulin-containing nanoparticles, the manufacture of which is described in Examples 15 to 16, at a final concentration of between 20-100 mg/ml. Furthermore, the relevant clinicians are blinded as to the treatment regime administered to a given subject to prevent from being biased in their post-treatment observations. Using this randomization approach, each volunteer has the

same chance of being given either the pharmaceutical composition or the placebo.

Volunteers receive either the pharmaceutical composition or placebo for an appropriate period with biological parameters associated inflammatory disorders measured at the beginning (baseline measurements before any treatment), end (after the final treatment), and at regular intervals during the study period. Measurements include levels of TNF in body fluids, tissues or organs compared to pre-treatment levels. Other measurements include, but are not limited to, indices of immune and inflammatory responses.

Information recorded for each patient includes age (years), gender, height (cm), family history of disease state or condition (yes/no), motivation rating (some/moderate/great) and number and type of previous treatment regimens for inflammatory disorders.

Volunteers taking part in this study are adults aged 18 to 65 years and roughly an equal number of males and females participate in the study. Volunteers with certain characteristics are equally distributed for placebo and the insulin-containing nanoparticle treatment. In general, at the conclusion of the study, the volunteers treated with placebo have little or no response to treatment, whereas the volunteers treated with the pharmaceutical composition of TNF blocker-containing nanoparticles show positive trends in _, one or more of the following measures, including but not limited to: significant reductions in TNF production.

Those skilled in the art will appreciate that the invention described herein the susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in the specification, individually or collectively, and any and all combinations of any two or more of said steps of features.