WO2010040173A1

WO2010040173A1 - A method of delivering functional agents across the blood-brain barrier

Info

Publication number: WO2010040173A1
Application number: PCT/AU2009/001325
Authority: WO
Inventors: Kerry Ann Breheney; Frank Caruso; Andrew David Price; Brigitte Maria Stadler; Elvira Tjipto; Kim Louise Wark; Alexander N. Zelikin; Remy Michel Robert
Original assignee: Commonwealth Scientific And Industrial Research Organisation; The University Of Melbourne
Priority date: 2008-10-08
Filing date: 2009-10-06
Publication date: 2010-04-15

Abstract

The present invention provides a method of delivering a functional agent across the blood brain barrier so that the functional agent is delivered into the brain of the subject, the method comprising the step of administering a polymeric capsule associated with said functional agent to the subject whereby the polymeric capsule crosses the blood brain barrier and wherein the polymeric capsule is from 300 nm to 2000 nm in diameter and comprises a polymeric shell defining an internal cavity. Further provided is a method of delivering a polymeric capsule across the blood brain barrier. The present invention also provides a polymeric capsule, or a polymeric capsule associated with a functional agent, which is suitable for use in a method of the present invention.

Description

A METHOD OF DELIVERING FUNCTIONAL AGENTS ACROSS THE

BLOOD-BRAIN BARRIER

FIELD OF THE INVENTION

The present invention provides a method of delivering a functional agent across the blood brain barrier using polymeric capsules of an unusually large size (300 - 2000 nm). The method has applications in the treatment of diseases of the brain and in brain imaging.

BACKGROUND TO THE INVENTION

The blood brain barrier (BBB) is a highly specialised arrangement of microvascular endothelial cells at the interface between the blood and brain which serves to maintain homeostasis of the central nervous system (CNS). It is composed of a microvascular endothelium, astrocytes, basement membrane, pericytes and neurons, all contributing to the regulation of the BBB.

The primary function of the BBB is to protect the brain by preventing foreign and or toxic substances present in the blood from entering, whilst allowing the passage of nutrients such as glucose and other substances (eg. oxygen) to cross. It also filters harmful substances from the brain back to the bloodstream.

In addition to providing a barrier, the endothelial cells allow transport of nutrients, receptor mediated signalling, leukocyte trafficking and osmoregulation.

The endothelial cells lining the brain micro vessels are packed very closely together, such that they form tight junctions [~10nm in diameter]. This endothelial barrier exhibits a high electrical resistance [~2000Ωcm^"2] through which most molecules can not pass. Molecules which do cross the BBB do so either by passing through the endothelial cell or between endothelial cells (ie. through the tight junctions). Integral transmembrane proteins expressed on the surface of the endothelial cells such as junction adhesion molecules (JAMS) are thought to play a role in this process. The breakdown of the BBB is considered a key step in many neurological diseases. Vascular endothelial cells and associated pericytes are often abnormal in tumours and the blood-brain barrier may not always be intact in patients with brain tumours.

Other disease states where BBB dysfunction is implicated include neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease; and neuroinflammatory diseases such as stroke, multiple sclerosis, infections and vascular dementia. Traumatic injury is also associated with BBB dysfunction.

The majority of transport across the BBB requires movement across 2 membranes, the luminal and abluminal membranes of the capillary endothelium, separated by 200 nm of endothelial cytoplasm. 98% of small molecules do not cross the BBB.

There are two mechanisms of transport: passive diffusion and active transport.

In general, molecules with a molecular weight less than 400 -500 Da [~100A] and with high lipid solubility (less than 8 hydrogen bonds) are capable of passive diffusion. However, it is not these attributes alone that predict and, in practice, allow a molecule to cross the BBB.

Active transport across the BBB may be classified into three categories:

1. Carrier mediated e.g. glucose, amino acids, choline, adenosine and adenine.

2. Active efflux transport (of molecules in brain to blood direction) e.g. p- glycoprotein.

3. Receptor mediated transport

(i) Bidirectional RMT e.g. transferrin receptor,

(ii) Reverse RMT e.g. neonatal Fc receptor.

(iii) Receptor mediated endocytosis e.g. type I scavenger receptor - mediates uptake of low density lipoprotein. Whilst other large molecules, viruses, and cells do cross the BBB, this is often during infection, after inflammation or in disease states which create a 'leaky barrier' through which they either pass through endothelial cells or between cells.

Certain cells are able to cross the blood brain barrier with or without infection. For instance, leukocytes (8-20μm in diameter) can transmigrate through the tight junction by a mechanism called diapedesis (diapedesis of monocytes is associated with MMP- mediated occludin disappearance in brain endothelial cells). This transmigration involves some receptors (example: ALCAM expressed on BBB) and counterpart receptors on both the brain endothelial surface and the leukocytes surface. In addition, circulating B cells (8-12 μm) enter the CNS as part of normal immune surveillance and in pathologic states.

Certain viruses are also able to cross the BBB. For instance, human immunodeficiency virus (HIV) (100-150 nm diameter) can cross directly and can also cross after infection of monocytes. The Tat protein can selectively disrupt tight junctions in brain microvascular endothelial cells. It is hypothesized that West Nile Virus (4-50 nm diameter) can also cross the BBB in one or more of three ways: i) it may directly infect microvascular endothelial cells, ii) it may cross endothelial cell junctions and or iii) it may migrate within infected leukocytes that enter the CNS. Other viruses that can cross the BBB include Eastern equine encephalitis virus (55-70 nm diameter) and Herpes simplex virus (95-105 nm diameter).

The permeability of the BBB may be increased by physical and biological/chemical methods. Physical techniques include ultrasound-induced MRI-guided BBB disruption techniques and osmotic BBB disruption (hyperosmotic mannitol administration). Available biological/chemical methods include the use of VEGF (angiogenesis) which can increase the permeability of the BBB. Bradykinin has been shown to selectively and transiently increase the permeability of the BBB, and MCP-I induces a significant increase in the BBB permeability (which in fact triggers an inflammatory reaction). Other inflammatory cytokines or inflammatory mediators (e.g. nitric oxide) can also be used. In normal healthy patients, mannitol is used to disrupt the BBB so that Mangnevist® (an imaging agent) can enter the brain in order for an MRI scan to be conducted. Physical and biological/chemical methods are not ideal because they can either result in permanent damage to the BBB or allow entry of other non-desirable molecules such as toxics or foreign pathogens during the transient period in which the tight junctions are relaxed.

The delivery of functional therapeutic and diagnostic agents to the brain is severely limited by an intact and healthy BBB. One proposed delivery mechanism is the use of nanoparticles or nanocapsules (such as micelles) that are associated, in some fashion, with the agent of interest. However, to date, the nanoparticles and nanocapsules used for this purpose have tended to be relatively small (less than 300 nm) and/or solid and are therefore limited in their ability to carry and or deliver suitable amounts of the functional agent. Larger nanoparticles and nanocapsules tend to be unable to cross the BBB due to their increased size. Although some larger nanoparticles and nanocapsules have been shown to be capable of crossing the BBB, these nanoparticles and/or nanocapsules rely on having surfactants on the outer surface which disrupt the BBB.

SUMMARY OF THE INVENTION

The present invention provides a method of delivering polymeric capsules across the blood brain barrier. The polymeric capsules may be associated with functional agents which are optionally released after the polymeric capsule has crossed the blood brain barrier, either intracellularly or after the polymeric capsule has been taken up into a cell.

Accordingly, in one embodiment, the present invention provides a method of delivering a functional agent across the blood brain barrier so that the functional agent is delivered into the brain of the subject, the method comprising the step of administering a polymeric capsule associated with said functional agent to the subject whereby the polymeric capsule crosses the blood brain barrier and wherein the polymeric capsule is from 300 nm to 2000 nm in diameter and comprises a polymeric shell defining an internal cavity. In another embodiment, the present invention provides the use of a functional agent associated with a polymeric capsule in the preparation of a medicament for delivering the functional agent across the blood brain barrier of a subject, wherein the polymeric capsule is from 300 nm to 2000 nm in diameter and comprises a polymeric shell defining an internal cavity.

In a further embodiment, the present invention provides a method of delivering a polymeric capsule across the blood brain barrier so that the polymeric capsule is delivered into the brain of the subject, the method comprising the step of administering the polymeric capsule to the subject whereby the polymeric capsule crosses the blood brain barrier and wherein the polymeric capsule is from 300 nm to 2000 nm in diameter and comprises a polymeric shell defining an internal cavity.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Fluorescence of 1 μm PMA HC incubated with FITC-labelled IgG (oom and ooh refer to anti-mouse and anti-human IgG respectively; IgG' is a Fab specific antibody). Bare PMA HC support adsorption of a minimal amount of proteins from their PBS solution and the capsules exhibit negligible fluorescence. PMA HC with mlgG and hlgG (mouse and human IgG, respectively) pre-adsorbed from their 50 mM MES solution, pH 6, exhibit high fluorescence when incubated with the correct secondary antibody.

Figure 2. Cell viability was measured by an MTT assay. Increasing concentrations of polymer were incubated with CHO-Kl cells (left panels) and CHOtGFP (right panels) and either after filtration (upper panels) or before filtration (lower panels) of the polymers. The cell viability was measured in presence of the polymers (red line with squares). PBS was used in each experiment as a positive control (blue line with triangles). Each experiment was performed in triplicate.

Figure 3. Uptake level exhibited by the panel of mammalian cells towards internalization of PMA HC, 1 μm (left) and 500 nm (right) in size. Uptake levels were ascertained using Alexa Fluor 633 labeled capsules and flow cytometry as means of analysis. The raw histograms were further analysed using Flow Jo analysis software to yield numerical values corresponding to the fraction of cells with internalized capsules.

Figure 4. Uptake level exhibited by CHO cells towards 500 nm PMA HC in cell culture media at 4 C (B) and 37 C (C-E) in the presence (B, C) or absence (D, E) of serum proteins. For experiment (E), the capsules were pre-incubated with serum proteins prior administration onto cultured cells.

Figure 5. Normalized uptake level (top row) and population mean fluorescence (bottom row) of cells incubated with PMA HC in the presence of specific inhibitors of cellular uptake mechanisms: chlorpromazine (10 μM; inhibitor of clathrin-mediate endocytosis), fϊlipin (1 mg/L; inhibitor of the caveolae-mediated uptake), amiloride (50 μM, macropinocytosis inhibitor). Left: uptake and mean fluorescence for different mammalian cells incubated with 500 nm PMA HC; right: uptake and mean fluorescence of CHO cells incubated with PMA HC of differed size. In each case the uptake level and mean population fluorescence were normalized using the data for the respective cells/capsules in absence of inhibitors; the presented data are mean ± SD of at least 2 runs, 6 samples each run, analysed using a one way ANOVA test ( *** : p < 0.001; ** ; p < 0.01; * : p < 0.05 ; non-significant unless marked otherwise).

Figure 6. Internalization of PMA HC, PSS/PAH polyelectrolyte capsules and PMA- coated silica particles, all ~ 500 nm, by CHO cells in the presence of inhibitors of endocytosis. All conditions and data analyses as in Example 7 and Figure 5.

Figure 7. Cellular uptake pathway. Mean fluorescence of CHO cells and A549 cells after exposure to fluorescently labeled PMA capsules or capsosomes (C_L i and C_L3) in the presence of different inhibitors. The data is normalized to the mean fluorescence of cells due to the uptake of the fluorescently labeled assemblies in the absence of any inhibitor. Chlorpromazine showed significant reduced uptake of the assemblies for both cell lines suggesting clatherin-mediated endocytosis (** p < 0.01 and * p < 0.05). DETAILED DESCRIPTION OF THE INVENTION

The present inventors have identified a class of biocompatible polymeric, hollow capsules (with flexible attributes) of unexpectedly large size (300nm-2000nm) which are able to cross the blood brain barrier (BBB) and enter into brain tissue. The crossing of the BBB appears to occur without any apparent biological/chemical or physical disruption of the BBB. The polymeric capsules can migrate to different parts of the brain (e.g. cortex, striatum, hippocampus) as well as enter brain cells (e.g. neurones).

The class of polymeric capsules is known and is described, for instance, in Zelikin et al, Angew. Chem. Int. Ed. 2006, 45, 7743-7745, Zelikin et al, Biomacromolecules 2006, 7, 27-30, Zelikin et al, ACSNano, 2007, 1, 63-69, and Zelikin et al, Chem. Mater., 2008, 20, 2655-2661. It is, however, surprising that the larger of these capsules would cross the BBB, given the teaching of the prior art.

The polymeric capsules have the capacity to be loaded with a cargo of functional agents (nucleic acids, peptides, proteins, earth metals and their derivatives, chemical molecules and hybrid molecules thereof) which can be released once the capsules are within a cell. The size of the capsules allows a high loading capacity compared to other brain delivery systems. In addition to protecting the cargo from physiological attacks which normally would degrade the cargo or elicit an undesirable immune response in the host, this capsule system also decreases potential side effects otherwise resulting from cargo being exposed to other organs in the body.

The system can be further improved by attaching targeting molecules to the surface of the capsule which recognise molecular markers on the blood brain barrier and/or specific molecules either expressed on the surface of specific brain cells or in between cells. This includes molecules which are expressed in normal healthy individuals and also those associated with a particular central nervous system (CNS) disease (e.g. Alzheimer's Disease, Parkinson's disease) including infectious diseases affecting the brain (e.g. encephalitis caused by Japanese encephalitis virus, WestNile virus, Henipah viruses). In this case, targeting to the BBB and specific molecules in brain tissues allows applications in imaging and drug delivery. It also decreases the effective dose of a pharmaceutical or imaging reagent required, producing a safer diagnostic or therapeutic entity.

The pathway by which these polymeric capsules cross the BBB is unknown. Without being bound by theory, it is believed that the "flexibility" or softness of the polymeric capsules of the present invention is of importance in allowing the polymeric capsules to be transported across the BBB via pathways or mechanisms which are not available to less flexible or soft particles of comparable size.

One indication of flexibility is the ability of the polymeric capsule to change diameter depending on ambient conditions.

The inventors have demonstrated that polymeric capsules enter cells in culture via a clathrin dependant endocytotic pathway. However, it is not yet clear whether this is the pathway which is responsible for transport of the polymeric capsules across the BBB.

Clathrin dependant endocytosis is a well described transcellular pathway. It is described in the literature as responsible for the transport of transferrin (ie. iron) across the BBB. Initially the transferrin ligand binds to the extracellular portion of the Trf R (transferrin receptor) expressed on the the BBB endothelial cells. The receptor-iron- transferrin complexes are then clustered together and localized in the so- called clathrin coated pits which eventually bud to form coated vesicles. Once the vesicle has formed, the clathrin coat is lost (perhaps via a chaperone protein of the heat shock protein 70 family). The loss of the coat is an energy requiring process. After the coat is lost, the vesicles join with other vesicles to form endosomes or receptosomes.

Clathrin dependant endocytosis typically requires the transported molecules to be less than 150 nm in size. The fact that this pathway may be associated with the transport of polymeric capsules of a much larger size is totally unexpected.

The following table provides an overview of the potential advantages that the methods of the present invention may provide over the methods of the prior art:

Accordingly, in one embodiment, the present invention provides a method of delivering a functional agent across the blood brain barrier so that the functional agent is delivered into the brain of the subject, the method comprising the step of administering a polymeric capsule associated with said functional agent to the subject whereby the polymeric capsule crosses the blood brain barrier and wherein the polymeric capsule is from 300 nm to 2000 nm in diameter and comprises a polymeric shell defining an internal cavity.

In another embodiment, the present invention provides the use of a functional agent associated with a polymeric capsule in the preparation of a medicament for delivering the functional agent across the blood brain barrier of a subject, wherein the polymeric capsule is from 300 nm to 2000 nm in diameter and comprises a polymeric shell defining an internal cavity.

Certain of the polymeric capsules, or of the polymeric capsules associated with a functional agent, described herein will be novel and inventive over the prior art.

Accordingly, the present invention also provides a polymeric capsule, or a polymeric capsule associated with a functional agent, which is suitable for use in a method of the present invention.

In one embodiment, the polymeric capsule crosses the blood-brain barrier by clathrin dependant endocytosis.

Preferably, the polymeric capsule is from 300-1000 nm in diameter, more preferably 400 to 800 nm in diameter, yet more preferably 450 to 650 nm in diameter, even more preferably 500 nm in diameter. In another embodiment, the polymeric capsule is from 1000-2000 nm in diameter.

In another embodiment, the polymeric capsule is 500 nm or greater in diameter and less than 1000 nm in diameter. In one embodiment, the diameter of the internal cavity is from 50 to 97%, more preferably 70 to 97%, yet more preferably 80% to 95%, even more preferably 86 to 90%, of the diameter of the polymeric capsule. In another embodiment, the thickness of the wall of the polymeric shell is from 15 to 50 nm, preferably 20 to 40 ran, more preferably 30 nm.

In measuring the thickness of the walls of polymeric capsules of this type the margin for error is typically around 10 nm.

The polymeric capsules of the present invention may demonstrate pronounced swelling and change their size in response to external conditions. The diameter of a polymeric capsule may vary from 5 to 50 % of its original diameter. See, for instance, Zelikin et al, Chem. Mater. 2008, 20, 2655-2661.

Accordingly, in one embodiment, the diameter of the polymeric capsule varies by greater than 5% of its original diameter depending on ambient conditions, more preferably greater than 10%, yet more preferably greater than 20%, even more preferably greater than 30%, and even more preferably greater than 40%. In another embodiment, the diameter of the capsule may vary by 50% or greater depending on the external conditions.

In a preferred form, the polymeric capsule is prepared by a layer-by-layer methodology around a template, as set out below. The polymeric capsule may be constructed of any number of layers and may, for instance, consist of two layers or one complemetary bilayer. Preferably, the capsule is preferably formed of three to seven, more preferably four to six, even more preferably five, alternating layers and the template is preferably from 400 to 800 nm in diameter, more preferably 450 to 650 nm in diameter, yet more preferably 500 nm in diameter. After formation of the capsule, the template is removed and, optionally, one of the sets of alternating layers.

Preferably, the polymeric capsule crosses the blood brain barrier without significantly disrupting the blood brain barrier. Although not bound by theory, the present inventors believe that the relative thinness of the walls of the polymeric shell is a factor in improving the flexibility of the capsule thereby facilitating its movement across the blood brain barrier. Furthermore, the thinness of the walls results in a large internal cavity which allows for the delivery of large amounts of encapsulated functional agent.

The polymeric capsule may have a charged outer surface. The charge may be positive or negative. Preferably, the charge is negative.

In a preferred form, the polymeric shell of the polymeric capsule does not comprise a lipid.

In a preferred form, the polymeric shell of the polymeric capsule does not comprise a surfactant.

The polymeric capsule can be made from any suitable material. In one embodiment, the polymeric capsule includes a polymer selected from the group consisting of chain growth polymers, step growth polymers, polyelectrolytes, proteins, polypeptides, polysaccharides, polynucleotides, deoxyribonucleic acid and ribonucleic acid. In one embodiment, the polymeric capsule includes poly(methacrylic acid) crosslinked with disulfide linkages. In another embodiment, the polymeric capsule includes poly(lysine) and poly(glutamic acid). In yet another embodiment the polymeric capsule includes poly(sodium styrene sulfonate) and pol(allylamine hydrochloride). In one embodiment the polymeric capsule is biodegradable. In another embodiment, the polymeric capsule is substantially resistant to degradation in an extra-cellular environment and degrades in an intra-cellular environment.

In a preferred form, the polymeric capsule is formed by the layer-by-layer methodology. That is, by depositing alternating layers of two different polymers on a template, preferably a silica or other suitable template, and optionally crosslinking between and/or within the layers, followed by removing the template, and optionally removing the set of layers formed by one of the polymers, by dissolution or the like. The resulting polymeric capsule may therefore consist of a number of layers of the same polymer, as is the case for the PMA_SH polymeric capsules discussed in the Examples, or it may consist of alternating layers of two different polymers, as is discussed for the PSS/PAH polymeric capsules of the Examples.

Suitable polymers for use in layer-by-layer systems are described in US 7101575 and WO 2005/032512.

In one embodiment, one or more of the layers is internally cross-linked. In another embodiment, two or more of the layers are cross-linked to one another. The cross- linking is achieved by the reaction of cross-linking groups incorporated within a polymer of the layer or layers. In a preferred form, the percentage of cross-linking groups incorporated within the polymer is from 10 to 25 mol%, preferably 15 to 20 mol%, more preferably 17 to 19 mol%, even more preferably 18%.

The degree of cross-linking can influence the flexibility of the polymeric capsule.

Preferably, the cross-linking group is a thiol and the cross-link is a disulphide group.

In a preferred form, the polymeric capsule is a polymeric shell comprising layers thiolated poly(methacrylic acid) PMA_SH wherein the PMA_SH layers are cross-linked by disulphide bonds.

The preparation of PMA_SH (having 18 mol% thiol containing units) by conjugation of PMA with cysteamine in the presence of EDC/NHS is disclosed in Zelikin et al, Biomacromolecules 2006, 7, 27-30.

Preferably, 10 to 25 mol%, preferably 15 to 20 mol%, more preferably 17 to 19 mol%, yet more preferably 18 mol % of the thiolated poly(methacrylic acid) is functionalized with thiol groups.

Preferably, the outer layer of the polymeric capsule is PMA_SH-

The present inventors have found that these polymeric capsules readily disperse in aqueous solution which improves their ease of administration. Preferably, there are three to seven, more preferably four to six, even more preferably five, layers of PMASH-

Similarly, polymeric capsules formed of alternating layers of poly(styrene sulfonate) (PSS) and poly(allylamine) hydrochloride (PAH) may be formed by a similar layer-by- layer methodology. In the case of these systems, the two different types of layers may be retained in the final polymeric capsule.

Accordingly, in another embodiment, the polymeric capsule is a polymeric shell comprising alternating layers of poly(styrene sulfonate) (PSS) and poly(allylamine) (PAH)hydrochloride.

Preferably, there are five layers of PSS and four layers of PAH such that PSS forms the outer layer.

Methods of preparing such polymeric capsules by depositing layers of PVP and PMA_SH on a silica template using a layer-by-layer method are described, for instance, in Zelikin et al, Angew. Chem. Int. Ed. 2006, 45, 7743-7745, Zelikin et al, Biomacromolecules 2006, 7, 27-30, Zelikin et al, ACS Nano, 2007, 1 , 63-69, and Zelikin et al, Chem. Mater., 2008, 20, 2655-2661. This procedure forms capsules which have a large internal cavity and correspondingly high flexibility. This flexibility is demonstrated by the swellability of this type of capsules and their tendency to change size depending on external conditions. The present inventors have found these polymeric capsules are able cross the blood brain barrier at unexpectedly large sizes.

The functional agent may be associated with the polymeric capsule by any suitable means. For instance, the functional agent may be chemically bonded or physically adsorbed to an external or internal surface of the polymeric shell. In another embodiment, when the polymeric shell is formed by the layer-by-layer method, the functional agent is incorporated between one or more pairs of the polymer layers in the shell. Alternatively, and preferably, the agent is encapsulated in the internal cavity. Encapsulation allows for a high loading capacity compared to other brain delivery systems. For example, 1000 copies of a 800 base pair double stranded nucleic sequence or 10 000 copies of a 20 mer single stranded nucleic sequence can be encapsulated into a 1 um capsule. In addition, encapsulation protects the functional agent from physiological attacks which normally would degrade the functional agent or elicit an undesirable immune response in the host. Encapsulation also decreases potential side effects otherwise resulting from cargo being exposed to other organs in the body.

In the case of polymeric capsules prepared by depositing the layers of PVP and PMASH on a silica template, the functional agent may be encapsulated in the internal cavity of the polymeric capsule by the methods described in Zelikin et al, Angew. Chem. Int. Ed. 2006, 45, 7743-7745, Zelikin et al, Biomacromolecules 2006, 7, 27-30, Zelikin et al, ACSNano, 2007, 1, 63-69, and Zelikin et al, Chem. Mater., 2008, 20, 2655-2661. That is, by adsorption of the functional agent to a silica template, followed by depositing the layers of PVP and PMA_SH, cross-linking of the thiols of the PMA_SH layers to form disulphide bonds and removal of the template and the PVP layers.

The functional agent is any suitable agent, such as a molecule or composition, for administration to cells or regions of the brain where it performs, for example, a therapeutic or diagnostic function. The functional agent may be selected from the group consisting of nucleic acids, peptides, proteins, MRI contrast reagents such as magnevist and iron oxide, PET reagents, pharmaceuticals, such as an antidepressant, and hybrid molecules thereof.

As an example of a therapeutic functional reagent, therapeutic RNAs (double and single stranded) can be either loaded into the capsule and or alternatively in a preferred approach synthesised inside the capsule. The advantages of the latter method allow a high concentration of the 'therapeutic' to be delivered to the target site and protect it from degradation prior to reaching its target tissue. This type of medicament is especially suited for infectious diseases which affect the brain, as they are, pathogen and strain specific (ie. increased efficacy) and can be synthesised very rapidly; particularly in the event of a new strain outbreak once the sequence is known. The major barrier for applying this type of treatment previously has been gaining access to the brain. The polymeric capsules offer a new method. As an example of an imaging functional reagent, PET (positron emission tomography) reagents involve a biologically active molecule labelled with a positron emitting radionuclide that is introduced (usually by way of injection) into the body. The material accumulates in the organ or area of the body being examined, where it gives off a small amount of energy in the form of gamma rays. A scanner detects this energy and using tailored software creates a three-dimensional image of both the structure and functional processes in the body. The higher loading capacity of the capsules will increase the sensitivity and also decrease the overall dosage of radiolabel required.

Diagnostic functional agents include contrast imaging agents selected from the group comprising Gd-DTPA, magnevist, iron oxide and other Tl weighted agents.

The timing of biodegradation of the polymeric capsule can be tuned for the application for which it is required. For example, if the polymeric capsule is intended to deliver a cargo it can be tuned to enable a quick release of the therapeutic active but for imaging reagents a slow degradation of the capsule may be required so that at the time of application the imaging reagent is still held in the capsule and it is only released and broken down after imaging for clearance from the body

In one embodiment, the functional agent is released from the polymeric capsule. Preferably, the functional agent is released from the polymeric capsule under appropriate physiological conditions.

In one embodiment, the polymeric capsule releases the functional agent after the polymeric capsule has crossed the blood brain barrier. The functional agent may be released in the extracellular matrix or after the polymeric capsule is internalised in a brain cell. Preferably, the functional agent is released after the polymeric capsule is internalised in a brain cell.

The person skilled in the art would be aware of a number of suitable release mechanisms. For instance, where the polymeric shell includes cross-linking disulphide bonds, the polymeric shell is stable under normal physiological conditions but is disrupted when in a reducing environment, such as a cell, thus releasing encapsulated reducing agent. Thiol-disulphide exchange mechanisms, which also occur in cells, can also disrupt the cross-linking disulphide bonds.

There may also be situations where it is preferred that the polymeric capsule does not release the functional agent. For instance, in the delivery of contrast imaging agents across the BBB.

As would be understood by the person skilled in the art, the method of the present invention allows for the treatment of diseases and disorders associated with regions or cells of the brain including Alzheimer's disease, Parkinson's disease, Huntington's disease, A.L.S, Multiple sclerosis, brain cancer, stroke, brain trauma, autism, lysosomal storage disorder, fragile X syndrome, inherited ataxias, depression, schizophrenia, chromic pain and epilepsy.

The polymeric capsule may comprise one or more targeting molecules associated with the surface of the polymeric capsule. The one or more targeting molecules target the polymeric capsule to a specific molecule expressed either on the surface of an endothelial cell lining the brain micro vessels or a specific brain cell type and therefore allow for more precise control in delivering the functional agent once the polymeric capsule has crossed the blood brain barrier.

The targeting molecule can be an antibody or a portion thereof or a peptide. Examples of suitable antibodies include WO2, which recognises the A-beta protein involved in Alzheimer's disease, anti -transferrin receptors, anti -human insulin receptor, and anti- epidermal growth factor receptor.

Association of the targeting molecule with the outer surface of the polymeric capsule may be carried out by any suitable means. For instance, the association may be due to electrostatic forces between the outer surface and the targeting molecule. Alternatively, the association may be by means of a covalent bond between the targeting molecule and the outer surface. Examples include using classic amine chemistry or "click chemistry". For example, the targeting molecule can be modified by placing a free thiol through the introduction of a cysteine residue thus facilitating conjugation to maleimide residue on the surface of the capsules. Similarly, when the targeting molecule is an antibody, a non canonical amino acid residue can be introduced in the antibody sequence for conjugation to the capsules by "click chemistry".

The polymeric capsules may be administered in the form of a composition comprising a pharmaceutically acceptable diluent, excipient or carrier. The composition may be administered in any way known in the art. For instance, the composition may be administered parenterally either subcutaneously, intramuscularly or intravenously, or alternatively nasally.

In order that the nature of the present invention may be more clearly understood preferred forms thereof will now be described by reference to the following non- limiting Examples.

EXAMPLES

EXAMPLE 1: Demonstration of 500 nm capsules crossing blood brain barrier.

In order to demonstrate that the capsules crossed the blood brain barrier (that is moved from the blood, through the wall of the blood vessel and into the brain tissue), immunohistochemical methods were employed. This allowed the visualisation of endothelial cells lining the brain micro vessels as well as markers for neurones and microglial cells. The capsules were fluorescently labelled with a different colour fluorophore for identification and their location in three different areas of the rat brain; the cortex, striatum and hippocampus was studied.

Capsules comprising 5 layers of alternating polymers, poly(vinylpyrrolidone) PVP and poly(methacrylic acid) PMA_SH were synthesised on a 500 nm monodisperse spherical silica template. The template and the PVP layers were removed upon completion of the synthesis to form a polymeric capsule comprising five layers of PMASH- The hollow capsules measured 500nm in diameter and were labelled with Alexa fluorophore 633 using the techniques described in Zelikin et al, Angew. Chem. Int. Ed. 2006, 45, 7743- 7745, Zelikin et al, Biomacromolecules 2006, 7, 27-30, Zelikin et al, ACSNano, 2007, 1, 63-69, and Zelikin et al, Chem. Mater., 2008, 20, 2655-2661. The homogeneity of the capsules per size and their aggregation state (dispersed / aggregated) were determined by fluorescent microscopy. Few aggregates were observed. The capsules were suspended in phosphate buffered saline (PBS) at a final concentration of

Three adult male Sprague Dawley rats weighing 250-35Og each received 0.2 ml of the capsule solution via an intra-arterial injection administered under anaesthesia (initially 5% reduced to 2-2.5 % isofluorane). A polyethylene catheter filled with the capsule suspension was inserted 1.5 cm into the common carotid artery and the capsules were slowly injected. Once the catheter was removed the carotid was ligatured and the skin sutured. Animals were recovered and monitored. None of the animals displayed any physical adverse reactions.

Following 24 hours post injection with the capsules, the rats were killed with sodium pentobarbitone via an intraperitoneal injection (100mg/kg) and perfused with 300ml of pre chilled 0.1M PBS. The brains were removed and snap frozen over liquid nitrogen and stored at -8O⁰C. Frozen coronal sections of the brains were serially cut at 50 μm from the caudate putamen through to the caudal end of the hippocampus. Sections were then collected onto 1% gelatinised, 1% chrome alum microscope slides and stored at -80C until immunohistochemistry was performed.

Brain tissue from a rat which did not receive the capsules was processed in the same manner as previously described except that, after perfusing with PBS, the animal was also perfused with 4% paraformaldehyde prior to freezing. A standard protocol was used to prepare the slides for staining consisting of ; air dry for 30 mins, treat with 3% hydrogen peroxide in 90% methanol for 5 mins, rinse with PBS for 5 mins, block with PBS,0.3% Triton X-100 and 10% normal goat serum for 30 mins.

Micro-vascular endothelial cells in the brain and neuronal nuclei were identified by immunoreactivity with the glucose transport 1 receptor (Glut-1) and neuron-specifc nuclear protein (NeuN) respectively. Resident and activated microglia were labelled with antibodies against a cluster of differentiation molecule l ib (CDl Ib). Sections were incubated with a primary antibody for 48 hours at 4⁰C, washed three times in PBS prior to the addition of the secondary antibody conjugated to Alexa fluorophore 488. Slides were incubated at room temperature in the dark for 4 hours, then washed in PBS three times. Sections were coverslipped with DAKO cytomation Fluorescent mounting media and stored at 4⁰C.

Sections were examined using a confocal scanning laser system (Olympus FVlOOO) coupled to an inverted 1X81 motorised microscope. The fluorophores Alexa 488 and Alex 633 were detected sequentially using a 473 nm diode laser. The laser intensity and PMT voltage was selected to minimise background signal present in negative control samples. For each image, one channel was pseudo colour green and excited at 473nm with the signal detected from 485-585nm corresponding to Alexa Fluor 488 conjugated to secondary antibodies which recognise primary antibodies against Glut-1, NeuN or CDl Ib markers. A second channel was pseudo colour red and excited at 473 nm with the signal detected from 600-700nm to image Alexa Fluor 633 conjugated to the capsules. These settings to detect the labelled capsules were unexpected but the Alexa Fluor 633 conjugated to the capsules was found to have a shifted excitation maximum which was best detected at 473nm and not 633 nm. Images were captured with Olympus FVlO ASW software (V 1.07).

An intensity profile of the confocal images of the capsules was used to confirm the size of the capsules in the captured images. A line was drawn through the centre of the capsules and a full width half maximum measurement was taken as a representative measurement of the capsule diameter. Capsules were measured at around 500 nm.

Results :

i) Detection of endotheial cells which line the blood micro vessels of BBB

Brain sections from an animal that received the capsule solution and which were imniunoreacted with a goat anti-mouse secondary antibody labelled with Alexa Fluor 488 exhibited a faint non-specific labelling which was considered the background level thereafter. The glucose transporter 1 which is highly expressed on the surface of endothelial cells lining the brain micro vessels (and has minimal if any expression in neurons or glial cells) was detected using a polyclonal serum against GLUT 1 in all three rat brains tested from animals that received the capsule solution. The intensity of staining was above that of the background defined by the control and therefore considered specific.

In the cortex, capsules were visible within the blood vessels endothelium. Most of the capsules were within the lumen of the vessels and the epithelium. Two animals of the three which received the capsule solution also had capsules present within the extracellular cortical parenchyma.

In the hippocampus and striatum capsules were also observed within the lumen of the vessels, epithelium and within the extracellular matrix in all three animals.

As expected, capsules were not detected in brain sections from an animal that did not receive any capsule solution. As seen previously, these negative control sections showed a very faint background level of staining when reacted with the secondary antibody only, labelled with Alexa Fluor 488.

ii) Detection of nucleus of neuronal cells

Pyramidal neurons (CAl) also known as projection neurons were detected by immunoreacting tissue sections from animals which received the capsules solution with an antibody against a neuron-specific nuclear protein (NeuN). In the hippocampus region, the nuclei of the CAl cells immunoreacted intensely compared to the surrounding cytoplasm. The specificity of this staining was verified by the observation that neuronal nuclei were not labelled when these sections when incubated with the secondary antibody alone and no background staining was observed. Capsules were detected in the cytoplasm of CAl cells and also in the surrounding tissue.

Capsules were not observed in tissue sections of the hippocampus from an animal that did not receive the capsule solution. However, as expected, the CAl cells were labelled in this section. Consistent with the result above, the secondary antibody control did not produce any background staining on this negative control tissue. iii) Detection of CBl 1 marker specific to microglial cells

Microgial cells are the main resident immunological cells in the central nervous system. When activated, their presence is indicative of an inflammatory response. Activated microglial cells were detected in positive control tissue sections from the striatum of a rat with brain lesions. Interestingly, staining of tissue sections from the cortex of a rat which received the capsule solution did not show any significant microglial labelling, indicating that the capsules did not induce an inflammatory response. However, these results are preliminary and would need to be repeated using other measures to substantiate this claim. Treatment with the secondary antibody alone on tissue sections from the striatum of a rat which did not receive the capsules produced faint background staining.

iv) Detection of cell type nuclei

The histological stain 4',6-diamidino-2-phenylindole (DAPI) binds DNA highlighting cell nuclei. Tissue sections from the brain cortex of a rat which received the capsule solution, stained with DAPI showed the presence of capsules in the cytoplasm of cells and the surrounding tissue. These sections did not display any non-specific staining with DAPI.

Conclusions:

Twenty four hours post carotid injection of fluorescently labelled capsules, using immunohistochemical analysis and confocal microscopy, capsules were found to have passed through the blood brain barrier. Capsules were located in all areas examined including the cortex, striatum and hippocampus. The capsules were also detected in and around brain capillaries. Furthermore they were found within the cytoplasm of neuronal cells providing definitive evidence of capsules having passed through the BBB. EXAMPLE 2: Encapsulating deoxyribonucleic acid (DNA) into capsules

The example provided in the publication by Zelikin et .al. (2007), ACS Nano, 1, (1), 63-69) describes the encapsulation of DNA only, however the general principal upon which the method works is equally applicable to encapsulating ribonucleic acid (RNA).

EXAMPLE 3: Encapsulating a protein into the capsule

The example provided in the publication by Zelikin et al (2006) Biomacromolecules 2006, 7, 27-30 describes the encapsulation and release of a model fluorescently labelled protein, transferrin.

EXAMPLE 4: Antibody attachment onto the capsule.

For the purposes of targeting the capsule to a specific molecule expressed either on the surface of an endothelial cell lining the brain micro vessels or a specific type of brain cell, it was necessary to demonstrate the attachment of antibodies to capsules. This example focuses only on one method of attachment which relies on the electrostatic forces between the capsule outer surface and antibody itself.

Capsules comprising 5 bilayers of alternating polymers, poly(vinylpyrrolidone) PVP and poly(methacrylic acid) PMA_SH were deposited onto 1 μm spherical silica template particles using fluorescently labelled sample of PMA_SH (Alexa Fluor 633 dye). The particles with deposited multilayered polymer film were then washed with 50 mM 2-( N -morpholino)ethanesulfonic acid (MES) buffer pH 6 once by pelleting at 600-80Og, removing supernatant and resuspending in buffer. The resulting capsules consist only of PMA_SH, as the PVP polymer is removed by raising the pH.

The WO2 monoclonal antibody which recognises the A-beta protein involved in Alzheimer's disease was produced in culture and purified using protein sepharose A column and is isotope IgG2a. A polyclonal antibody against whole human immunoglobulin (IgG) was purchased from Zymed Laboratories. Each antibody (MgG at 2 g/L; WO2 at 0.6 g/L) was diluted with 10 ul of MES pH 6 buffer and used to suspend the pellet of the particles with adsorbed multilayered thin film. A control sample was included which had no antibodies but was processed with the same protocol. The reaction was shaken for 30 minutes at room temperature after which time the particles were washed once with PBS and then incubated with respective secondary antibodies, diluted 1 in 40 (final concentration 25 ug/mL) for 15 minutes. Two FITC conjugated secondary antibodies were directed against human and mouse heavy and light chains, whilst the third was against a mouse Fab'2 component. These secondary antibodies will only detect primary antibodies which have absorbed onto the capsule surface via the Fc portion and are therefore orientated in the correct direction to behave as capsule targeting molecules.

After 15 minutes incubation the fluorescence of the particles was quantified on a Becton Dickson FACS calibur flow cytometer using an excitation wavelength of 488 nm.

Results :

Results are presented in Figure 1.

The control with capsules but no primary antibody failed to show binding with any of the secondary antibodies tested indicating that the surface of the capsule in this environment is non-fouling. As expected the WO2 monoclonal antibody only reacted with the mouse secondary antibodies and not the anti-human secondary antibody.

Significant amounts of WO2 antibody was detected on the capsule surface.

As expected, the primary antibody against human IgG was only detected using the human secondary antibody raised against the heavy and light chains further indicating that the secondary antibodies were specific.

Conclusions: Antibodies can be successfully absorbed using electrostatic forces onto the surface and the capsules for targeting experiments. This is only one way of attaching antibodies to the capsule surface. Furthermore, whether this form of attachment is sufficient for the antibody to remain on the capsule for in vivo studies remains to be tested. For this purpose it is more likely that antibodies attached using covalent methods will be more reliable (i.e. the antibody will not 'fall off the capsule in physiological conditions such as blood). Such methods involving covalent linkages involve using classic amine chemistry or "click chemistry". For example, antibody can be modified by placing a free thiol through the introduction of a cysteine residue thus facilitating conjugation to maleimide residue on the surface of the capsules. Similarly, a non canonical amino acid residue can be introduced in the antibody sequence for conjugation to the capsules by "click chemistry".

PROPHETIC EXAMPLE 1: Determining the optimum concentration of MRI contrast loaded capsules in the rat brain for imaging.

In order to determine the optimum concentration of capsules loaded with an MRI Tl contrast reagent (magnevist) required to give a detectable and reproducible MRI signal, different concentrations of the capsules will be injected directly into the lateral brain ventricle by an intracerebroventricular (I. C. V) injection into a catheter. This bypasses the blood brain barrier and provides the capsules access to a large surface area of the brain. It will also eliminate artefacts which sometimes occur if a direct intracranial injection is administered. Rats will be scanned for approximately one hour using multiple Tl weighted (TlW) MR imaging sequences at different repetition times (TR) to confirm the presence of magnevist loaded capsules in the ventricles immediately after injection, and re-scanned using the same sequences, 24 hours post injection. Images will be acquired using standard spin echo sequences and analysed with custom software.

Capsules will be prepared by the Layer-by-Layer assembly method described in Zelikin et al (2006) Biomacromolecules 2006, 7, 27-30 inter alia. Porous 400 nm silica spheres will be used as templates, and coated with poly-1-lysine (PLL), poly(methacrylic acid) (PMA) and again with PLL then cross-linked. The contrast MRI reagent, gadolinium will be chelated with the ligand, diethylene triamine pentaacetic acid (DTPA) separately and tested for the presence of free gadolinium. An excess of DTPA will be used to ensure complete chelation. Gd-DTPA will be incubated with coated particles overnight in a mixture of pH 6 buffer and THF at room temperature. After washing steps using MES buffer at pH 6, the particles will be coated with 4 bilayers of (PMAsh/PVP), cross-linked with chloramine T and the cores and the PVP layers dissolved with hydrofluoric acid to yield the final preparation. The capsules will be resuspended in phosphate buffered saline (pH 7) and quantitfied using flow cytometry, ICP-AES (inductively coupled plasma atomic emission spectroscopy) for gadolinium content.

Five adult male Sprague Dawley rats weighing 250-35Og will each receive a specified concentration of capsules loaded with magnevist in a final volume of ~0.5ml via an intracerebroventricular injection administered under anaesthesia (4% Chloral hydrate (lml/lOOg body weight)). Five different concentrations of magnevist loaded capsules will be tested in triplicate; including 2.5 x 10^Λ8 to 2.5 x 10^Λ12. The amount of magnevist in the total capsules injected will be greater than 0.6umol/kg (standard dose is 0.1mmol/kg).

The rat will be placed in a sterotaxic apparatus and the skull levelled between bregma and lambda. A glass micropipette will be lowered into the lateral brain ventricle, (bregma -0.9mm; lateral 1.6mm; depth 3.4mm according to the coordinates of Paxinos and Franklin (The Rat Brain in Stereotaxic Coordinates 6th Edition (httρ://books.google.com/books?id=4OBQ8wpK0usC&pg=PR14&lpg=PR14&dq=Paxi nos+and+Franklin+co-ordinates&source=web&ots=F46QXfClEJ&sig=^:- vLHpO AXMAw WMGkJmDezSA5Jo&hl=en&sa=X&oi=book result&resnum=l &ct= result#PPPl.,Mlϊ), and the magnevist loaded capsules will be injected using a picospritzer. The micropipette will be left in place for 5 minutes after the administration of the capsules. Once the injector is removed, the wound will be sutured with cotton/nylon, and an analgesia (meloxicam, 2 mg/kg) administered subcutaneously.

Whilst still anaesthetised, each rat will be placed in an MRI compatible head holder and scanned for a ten minute MR imaging Tl weighted sequence to confirm the presence of the magnevist loaded capsules in the ventricles on a Bruker BioSpect 4.7T small animal MRI scanner.

The rat will be removed from the MRI machine, recovered and transferred to the holding facility. At 24 hours post the initial injection each rat will be anaesthetised (5% Isoflurane), placed in an MRI compatible head holder with anaesthetic maintained at 1.5 - 2.5% isoflurane and scanned. High resolution TlW spin echo sequences will be acquired with several TR. The field of view will be set up so as to cover the entire brain. Imaging processing and analysis will be performed using Matlab (The Mathworks, Natwick, MA) to quantify the MR signal. Relaxivity curve will be fitted to the data for each pixel and Tl values will be computed. The two series of images (after injection, and 24 hours later) will be registered under rigid body transformation with the MILX view software (http://www.aehrc.com/biomedical imaging/milx.html) so as to compare signal change and Tl change between the different acquisitions and time points. Total scanning is less than 2 hours. After the final scan the rat will be killed with Lethabarb (100mg/kg body weight, i.p.) whilst still under isoflurane anaesthesia.

Because all the images will be registered to the initial one, signal comparison will be possible and expressed as a percentage signal increase/decrease from the first scan. In order to remove signal variation due to coil sensitivity (since the animal will be moved between tO and t24), we will compute parametric maps at tO and t24. Absolute Tl values will then be compared on a pixel by pixel basis, as well as using region of interest manually delineating whole brain areas if noise is too important.

Expected Results:

It is expected that the images acquired immediately after injection (tO) will show significant enhancement in the ventricle due to the contrast agent, and no change in the brain parenchyma. After 24h (t24), it is expected that the contrast agent will diffuse throughout the brain tissue (giving rise to some hyper-intensity) and that none will be left in the cerebrospinal fluid (CSF) of the ventricle due to natural physiological clearance. Therefore, we expect to measure large hyper-intensity at tO in the ventricle (due to Tl shortening of the agent) and slight hyper-intensity in the brain paremchyma at t24.

PROPHETIC EXAMPLE 2: Targeting capsules in vitro

To target the capsules to specific cell types, an antibody against a particular receptor expressed on the surface of the cell will be absorbed onto the capsule surface. By binding to an epitope on the receptor this is expected to trigger receptor mediated endocytosis, a process which will enable the capsule to internalise and enter the cell.

Cells : All cell lines will be incubated at 37⁰C, 5% CO₂.

Chinese hamster ovary (CHO) cells stably expressing an amyloid precursor protein (APP) on the cell surface which consists of the A-beta protein implicated with Alzheimer's Disease will be grown and maintained in RPMl 1640 medium supplemented with penicillin (5U/ml) & streptomycin (5μg/ml), 1 % L-glutamine, 5 % bovine calf serum and puromycin (final concentration 7μg/ml) for several passages. The CHO wildtype cell line (ATCC : CCL61), which does not express APP will be cultured under the same conditions, with the exception of puromycin, which will be excluded from the growth medium.

Stable CHO T cells expressing the human insulin receptor will be obtained from Dr Tim Adams (CSIRO) and grown in alpha MEM medium supplemented with penicillin (5U/ml) & streptomycin (5ug/ml) and 5% bovine calf serum.

RAMOS RAl cells (ATCC CRL-1596) are B lymphocytes originating from a Burkitt' s lymphoma. These cells express an antigen called CD 19 (Cluster of Differentiation 19) on their surface which is unique to B cells and follicular dendritic cells. These cells will be grown in medium containing RPMl 1640 and 10% bovine calf serum.

Ml 7 cells (ATCC: CRL-226) originate from a human neuroblastoma. The cells will be cultured in OptiMEM medium supplemented with penicillin (5LVmI) & streptomycin (5μg/ml) 1% sodium pyruvate , non-essential amino acids, 5 % bovine calf serum. Antibodies: Antibodies which targeted receptors known to undergo receptor mediated endocyotosis will be selected for targeting.

Hybridomas WO2 and FMC63 (will be provided by Drs. Roberto Cappai, Bio21, VIC and Peter Macardle, Flinders Medical Centre, SA. respectively) will be grown in serum free hybridoma medium will be purified on a protein A sepharose column and concentrated using a microsep™ centrifugal device (PALL Life Sciences). The WO2 monoclonal antibody recognises the A-beta protein whilst the FMC63 mAb is against CD 19 antigen.

Capsules : This example will describe 500nm diameter capsules, however the same protocol will apply for using 300nm and 1 um capsules.

PMA_SH capsules will be synthesised by the methodology described in the above examples. Whole antibodies will be absorbed onto the fluorescently labelled capsule surface using methods described in Example 4. The presence of antibody on the capsule surface will be confirmed by methods described in Example 4.

Cells will be plated at 4 x 10^Λ4 cells per well in a 96 well Nunc plate with 250 μl of their respective growth medium. Plates will be incubated overnight at 37⁰C, 5% CO₂ to allow adherent cells to attach and assume normal morphology.

Capsules will be diluted in phosphate buffered saline (PBS) pH 7 to a concentration that allows a total volume of 40 μl to be added to each well and is equivalent to a capsule to cell ratio of 1 : 1 , 1:10, 1:100. Diluted capsules will be added to wells ~ 24 hours after initial plating. Each ratio will be tested in six replicates. Treatments will include : i) no capsules, cells only representing each cell type ii) capsules without any antibody attached (i.e. naked) and each cell type; iii) capsules with antibody that recognise a surface marker on a specific cell type with their cognate cell type and iv) capsules with antibody that recognises a surface marker on a specific cell type with cell types which do not express that specific surface marker. Plates will be incubated on a shaker moving at 90 revolutions per minutes (rpm) at 37⁰C, 5% CO₂ for ~ 3 hours. In the case of adherent cell lines, the medium will be aspirated from each well using a glass pipette and cells will be washed twice in 200 μl of pre-warmed PBS then harvested by adding 180 μl of 10 mM of pre-warmed ethylenediaminetetraacetic acid (EDTA). Plates will be incubated with rocking for ~ 5 minutes to detached cells from the wells. Cells will be transferred to 1.5 ml eppendorf tubes and stored on ice.

In the case of suspension cell lines, the medium and cells will be transferred to 1.5ml eppendorf tubes. The wells will be washed with 180 μl of pre-warmed PBS and the contents of each well will be transferred to their respectively tubes. Next, cells will be pelleted at 600 g for 8 minutes, the supernatant removed and discarded. Cells will be washed once in pre warmed PBS and finally resuspended in 180 μl of PBS, then stored on ice.

Approximately 800 μl of PBS will be added to all cell samples and cells transferred to tubes for analysis with Becton Dickson FACS calibur flow cytometer using an excitation wavelength of 488 nm. In each sample, the live population of cells will be gated and analysed.

Results:

All cells lines will exhibit a low level of background fluorescence. Likewise, it is expected that all cell types will display a low percentage of cells which have non- specifically taken up the capsules (treatment in which capsules without any antibody were incubated with each cell type separately). This percentage will vary between cell types and also follow a dose dependency related to the ratio of capsules to cells; with the highest ratio (1:1) yielding a higher percentage of cells with capsules and the lowest ratio (1:100) yielding the least.

For samples where capsules with an antibody against a specific marker were incubated with their cognate cell line expressing that marker, a significantly higher percentage of cells will be fluorescent compared to their corresponding treatments involving capsules without antibody and the same targeted capsules incubated with cells which do not express the marker to which the antibody is directed. The percentage of cells which take up capsules with antibody on the surface that is not directed to a marker on the cells will vary between different cell types as well. For example, M 17, CHO wildtype, and CHO T cells do not express the A-beta protein that is recognised by the WO2 antibody, so when capsules with the WO2 antibody are incubated with CHO wildtype versus Ml 7, the percentage of cells with capsules will be different. Given that this represents non-specific uptake, the same trend between cell types will be observed as was seen for 'naked' capsules without antibody (e.g. CHO wildtype > CHO T > Ml 7).

Conclusion: In vitro targeting of capsules to specific cells can be demonstrated using flow cytometric analyses. The degree of targeting exhibited (ie. above non-specific background levels of uptake) is dependant on the cell line, amount of antibody on the capsule surface and ratio of capsules to cells. The level of targeting observed may also be enhanced if the non-specific uptake level was decreased. This may be achieved by the addition of non-fouling compounds such as PEG which alter the charge and physical surface of the capsule.

In vitro targeting of capsules to cell types can be further demonstrated using this protocol by co-culturing different cell types together and co-staining for a another marker which is specific to the targeted cell type. This will confirm that only cell type A and not B or C were targeted. Confocal microscopy of immunohistological stained cells grown in vitro would offer an alternative method to investigating the targeting. In this case the protocol would remain the same as described but cells would not be harvested from the wells, instead immunohistochemistry would be performed directly on the cells in wells.

PROPHETIC EXAMPLE 3: Determination of capsules crossing BBB via a tail vein injection in a rat model

To assess if the capsules reach the brain 24 hours following administration via a tail vein injection, two separate techniques will be employed. The first will allow detection of the capsules loaded with a contrast reagent using MRI. This will enable the location of the capsules in the brain to be determined and also the signal quantified. Capsules will be prepared using the Layer-by-Layer assembly method. Porous 500 nm silica spheres will be used as templates, and coated with poly-1-lysine (PLL), poly(methacrylic acid) (PMA) and again with PLL then cross-linked. The contrast MRI reagent, gadolinium will be chelated with the ligand, diethylene triamine pentaacetic acid (DTPA) separately and tested for the presence of free gadolinium. An excess of DTPA will be used to ensure complete chelation. Gd-DTPA will be incubated with coated particles overnight in a mixture of pH 6 buffer and THF at room temperature. After washing steps using MES buffer at pH 6, the particles will be coated with 4 bilayers of (PMAsh/PVP), cross-linked with chloramine T and the cores dissolved with hydrofluoric acid to yield the final preparation. The capsules will be resuspended in phosphate buffered saline (pH 7) and quantified using flow cytometry, ICP-AES (inductively coupled plasma atomic emission spectroscopy) for gadolinium content.

Two adult male Sprague Dawley rats weighing 250-350g will be anaesthetised and placed in an MRI compatible head holder with anaesthetic maintained (1.5-2.5% isoflurane). The rat brain will be then scanned for about 60 minutes using MR imaging Tl weighted sequence on a Bruker BioSpect 4.7T small animal MRI scanner. This scan will be referred to herein as the baseline scan. The rat in the holder will be transported, whilst still anaesthetised to the surgery table, and the tail warmed. Each rat will each receive 180-200ul μl of at least 2.33 x 10¹⁰capsules loaded with magnevist to give a final total concentration of with -0.8 mM -1 mM in PBS via a tail vein. This concentration will be equivalent to at least 0.6 μmol/kg. A third rat will be injected with the equivalent dose of free magnevist as a positive control. The capsule solution will be injected directed into the tail vein and the rat in holder will be returned to scanner for further brain image acquisition using the same TlW protocol. This scan will be referred to hereinafter as scan 1.

Approximately 24 hours post the first injection the rat will be anaesthetized again, placed in the scanner holder and re-scanned using the same MR protocol for another 60 minutes. Upon completion of this final scan, the rat will be killed by administering sodium pentobarbitone (80 mg/kg) by an intraperitoneal injection (i.p). Results:

For the baseline scan, scan 1, and scan 2, several TlW sequences (10 min each) with different repetition times will be used ranging from 350ms to 3000ms. This will allow us to image with the best parameter depending on the relaxivity of the agent mixed in the different tissues, and to compute parametric maps of absolute Tl values that will be compared directly between the two scans avoiding any intensity inhomogeneity artefacts due to the receiver coils different positioning.

All the image volume for a given rat will be co-registered to the baseline scan under rigid body transformation with the MILX view software (http://www.aehrc.com/biomedical imaging/milx .html) , so as to compare signal change and Tl change between the different acquisitions and time points. Because all the images will be registered to the initial one, signal comparison will be possible and expressed as a percentage signal increase/decrease from the baseline scan. Parametric maps of Tl values will be compared on a pixel by pixel basis, as well as using region of interest manually delineating whole brain areas if noise is too important.

It is expected that a weak MR signal will be detected above background noise level in brain tissue. The signal will be present in different parts of the brain. This MR signal will be further validated by evidence of the capsules containing magnevist in cortical tissue using confocal microscopy. Capsules will be identified by their size and immunohistochemical means. Capsules will be detected in and around brain capillaries indicating they have crossed the blood brain barrier.

EXAMPLE 5: Capsule toxicity in vitro

To assess the cytotoxicity of the capsules in vitro a standard colorimetric assay which measures the activity of mitochrondrial enzymes was employed. The MTT assay is widely accepted as a test to determine the toxicity of potential medicinal agents and other novel materials. In living cells, mitochrondrial enzymes convert the substrate 3- (4,5-dimethylthiazol -2-yl)-2,5-diphenyltetrazolium bromide (MTT) which is yellow to a purple coloured product, formazan. The absorbance of this colored solution can be quantified by measuring at a wavelength between 500 and 600 nm. The toxicity of a test material is obtained by comparing the amount of purple formazan produced by cells treated with material to untreated cells. The ability of the test material to cause cell death, or change the metabolism of cells, can be deduced through the production of a dose response curve.

Cells :

Chinese hamster ovary cells stably expressing a green fluorescent protein (CHOtGFP) and the wildtype line which does not express GFP (CHO Kl, ATCC : CCL61) grown and maintained in alpha MEM medium supplemented 5 % bovine calf serum at 37°C with 5% CO₂. Cells (CHO Kl passage 11 and CHOtGFP passage 31 ) were plated at 3000 cells per well in a Nunc 96 well plate and incubated for 48 hours. Cells were -40-60% confluent when the capsules were added.

Capsules :

PMA_SH capsules consisting of 4 bilayers of alternating polymers, polyvinylpyrrolidone) PVP and poly(methacrylic acid) PMA_SH were constructed using the layer by layer method described in the previous Examples.

Given that the capsules were prepared in a non sterile laboratory, preparations were initially pre-treated with UV light in the tissue culture hood overnight then either added directly to cells or filtered through a 0.22 μm filter prior to their addition. Capsules were diluted in PBS to represent a range of concentrations and were added to wells containing growth medium, yielding a final volume of 200 μl. Each concentration was tested in duplicate. An equivalent amount of PBS was added to each well as a control. Next cells were incubated at 37⁰C, 5% CO₂ for 48 hours.

To perform the MTT assay, growth medium was removed and 100 μl of fresh medium containing MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (500 ug/ml) was added to each well. This substrate was incubated at 37⁰C, 5% CO₂ for 4 hours after which the plates were centrifuged, the medium was removed and 100 μl of 0.1 M HCl in isopropanol was added to each well to dissolve the crystals. Plates were read at 570 nm and 630 nm. The final data plotted represents the mean of readings taken at 570 nm subtracted from those at 630 nm.

Results:

Results are presented in Figure 2.

Across a test range of concentrations, the amount of formazan produced in cells treated with the capsules compared to cells treated with PBS only was similar (i.e. the dose response curves almost overlay each other). This observation was independent of cell line. Perhaps not surprisingly, more variation was observed between replicate readings for capsules which were not filtered as opposed to those that were filtered.

Conclusion: Together these data suggested that the capsules at concentrations up to 1000 ng/ μl are not toxic to cells.

In addition, animal experiments with rats injected with a total of 4 x 10^Λ9 capsules did not display any adverse affects in a 24 hour period

EXAMPLE 6: Non-specific uptake in different types of cells with lum and 500nm PMA_SH capsules.

The size, morphology and biological surfaces of different cell types are expected to influence the efficiency with which a particular cell type can non-specifically take up the capsules. Similarly, the size of the capsule and length of incubation time with cells may also affect the level of uptake observed in different cell types. To investigate these hypotheses fluorescently labelled capsules were incubated with six cells types representative of different tissues (e.g. kidney, ovary, muscle, brain) for either 3 or 24 hours.

Cells : All cell lines were incubated at 37⁰C, 5% CO₂.

Chinese hamster ovary (CHO) cells stably expressing an amyloid precursor protein (APP) on the cell surface which consists of the A-beta protein implicated with

Alzheimer's Disease. These cells were grown and maintained in RPMl 1640 medium supplemented with penicillin (5U/ml) & streptomycin (5ug/ml), 1 % L-glutamine, 5 % bovine calf serum and puromycin (final concentration 7ug/ml) for several passages. The CHO wildtype cell line (ATCC : CCL61), which does not express APP was cultured under the same conditions, with the exception of puromycin, which was excluded from the growth medium.

Ml 7 cells (ATCC: CRL-226) originate from a human neuroblastoma. The cells will be cultured in OptiMEM medium supplemented with penicillin (5U/ml) & streptomycin (5ug/ml) 1% sodium pyruvate , non-essential amino acids, 5 % bovine calf serum.

HEK 293T cells (ATCC : CRL 11268) are a human embryonic kidney cell line which also stably expresses the simian virus 40 (S V40) large T antigen. Morphologically, they are epithelial adherent cells. Cells were grown in DMEM medium supplemented with penicillin (5U/ml) & streptomycin (5ug/ml) and 5% bovine calf serum.

MA 104 cells are African monkey kidney cells which highly express the integrins alpha V beta 3 on their surface. These adherent epithelial cells were obtained from Dr. Barbara Coulson (University of Melbourne). They were grown in DMEM medium supplemented with penicillin (5U/ml) & streptomycin (5ug/ml), 1 % L-glutamine, HEPES buffer (final concentration 2mM) and 10% bovine calf serum.

NR6 cells were obtained from Dr. Tim Adams (CSIRO Molecular Health Technologies). These cells are mouse fibroblasts which appear diverse in their morphology. They were grown in DMEM medium supplemented with penicillin (5LVmI) & streptomycin (5ug/ml) and 5% bovine calf serum.

Capsules :

PMA_SH capsules composed of 4 bilayers were synthesised using the layer by layer method and fluorescently labelled with Alexa 633 described previously.

All cells were plated at 4 x 10^Λ4 cells per well in a 96 well Nunc plate with 250 μl of their respective growth medium. Plates were incubated overnight at 37⁰C, 5% CO2 to allow adherent cells to attach and assume normal morphology. Capsules were diluted in phosphate buffered saline (PBS) pH 7 to a concentration that allowed a total volume of 40 μl to be added to each well and was equivalent to a capsule to cell ratio of 100 to 1. Diluted capsules were added to wells and plates were incubated on a shaker moving at 90 revolutions per minutes (rpm) at 37⁰C, 5% CO₂ for either 3 or 24 hours. Samples were tested in six replicates in at least three independent repeat experiments.

In the case of adherent cell lines, the medium will be aspirated from each well using a glass pipette and cells will be washed twice in 200 μl of pre- warmed PBS then harvested by adding 180 μl of 10 raM of pre- warmed ethylenediaminetetraacetic acid (EDTA). Plates were incubated with rocking for ~ 5 minutes to detached cells from the wells. Cells will be transferred to 1.5ml eppendorf tubes and stored on ice.

In the case of suspension cell lines, the medium and cells were transferred to 1.5 ml eppendorf tubes. The wells were washed with 180 μl of pre-warmed PBS and the contents of each well will be transferred to their respectively tubes. Next , cells were pelleted at 600 g for 8 minutes, the supernatant removed and discarded. Cells were washed once in pre warmed PBS and finally resuspended in 180 μl of PBS, then stored on ice.

Approximately 800 μl of PBS was added to all cell samples and cells transferred to tubes for analysis with Becton Dickson FACS calibur flow cytometer using an excitation wavelength of 488 nm. In each sample the live population of cells was gated and analysed.

Results :

Results are presented in Figure 3.

For all cell lines tested, a longer incubation period of 24 hours led to a higher percentage of cells in the population which took up capsules compared to the 3 hour time point. This result was independent of the capsules size (1 μm or 500 nm). Even after 3 hours capsules were detected in a proportion of cells for all cell types, regardless of capsule size. These data indicate that all cell types tested were capable of internalising both 500 nm and lμm capsules.

Differences in the percentage of cells with capsules amongst cell types were observed at both 3 and 24 hours for both 1 μm and 500 nm capsules. Interestingly, there was not a trend was between cell types at 3 or 24 hours. For example, if comparing cell lines at 24 hours the number of cells with 1 μm capsules followed the order NR6 >WT CHO>APP>HEK>MA104>M17. However, for the same size capsules at 3 hours the order changed to NR6>APP>HEK>M17>WT CHO MAl 04. The same observation was made for the 500 nm capsules. In many cases the differences between cell types are small and not all are expected to be statistically significant. It is likely, that given the high ratio of 100 capsules to one cell, a saturation point is reached after 24 hours. In spite of this, the M 17 and MA 104 cell lines both exhibited a lower percentage of cells with capsules compared to other cell lines independent of incubation time or capsule size. This suggested that some cell types are more efficient at taking up the capsules compared to others.

The difference between Ml 7 cells and other cell lines is more obvious with 1 μm capsules compared to 500 nm capsules at both time points. This indicated that at least for this cell line, the size of the capsule influences the number of cells which take up the capsules. This is further suggested by the fact that even after 24 hours with 1 μm only a low percentage of the population have capsules, unlike that for 500 nm.

Conclusions:

Different cell types do exhibit different efficiencies in non-specifically taking up capsules. The incubation time also affects the efficiency of uptake as well as the size of the capsule.

In terms of targeting capsules to specific cell types in future applications, it would be important to select those cells which are less efficient at non-specifically internalising capsules. The human neuroblastoma cells serve as good example for targeting given their disease state (brain tumour cells) and also natural low uptake, provided these results were confirmed using primary culture of brain tumour cells.

Other ways of decreasing the non-specific uptake which is considered background noise when targeting, would be to lower the capsule to cell ratio (e.g. 1:10 or 1:100) and or add non fouling compounds (e.g. PEG) onto the surface of the capsule such that it is no longer "attractive" to many cell types. These approaches combined with attaching antibody molecules onto the capsule surface are expected to increase targeting.

Example 7

Several molecular pathways by which natural and synthetic molecules as well as pathogens enter cells have been well documented. These can be broadly divided into (i) energy dependant and (ii) energy independent routes which are also referred to as active or passive transport respectively. Moreover, other studies have suggested that the charge on the outer surface of a compound such as a nanoparticle is responsible for its interaction with the cell membrane either directly, or indirectly via absorption of serum proteins thus assisting entry into the cell. The PMA_SH capsules are negatively charged and would be expected to interact more readily with positively charged serum proteins than with the negatively charged cell membrane. Serum proteins represent natural ligands for receptors expressed on the surface of cells and can be used to activate receptor mediated endocytosis into the cell.

To determine i) which type of transport pathway the untargeted capsules use to enter cells and ii) if serum proteins absorb to the capsule surface and facilitate entry into cells, fluorescently labelled 500 nm capsules were incubated either at 4⁰C or 37⁰C and in the presence of absence of serum in the cell medium. The same methodology as described for the uptake assay in example 6 was employed to investigate these parameters. Results :

At low temperatures, the metabolic activity of the cell is slowed down and internalisation of extracellular molecules is less efficient. Consistently, less CHO cells took up labelled capsules at 4⁰C compared with those incubated at 37⁰C, indicating that capsule uptake is an energy dependant process.

Interestingly, no significant differences in capsule uptake were observed in the presence or absence of serum or when capsules were pre-incubated with serum prior to their addition to cells cultured in serum free medium.

Results are presented in Figure 4.

Conclusion : Together these data suggest a low level of interaction between capsules and serum proteins (i.e. the capsules are low fouling) and, more importantly, suggest that the capsules are not necessarily using receptors for serum ligands to enter cells as a dominant mechanism of entry.

Example 8

The pathways by which molecules are actively transported into or across a cell are collectively referred to as phagocytic and endocytotic pathways. Endocytotic pathways include phagocytosis, macropinocytosis, clathrin dependant endocytosis, caveolin dependant endocytosis and both clathrin and caveolae independent endocytosis.

These pathways have been well characterised and are generally size restricted. For example molecules up to -100-150 nm generally utilise a clathrin dependant pathway whereas molecules ~200-300 nm use a caveloe dependant pathway . Larger molecules

500 - 2000 nm or greater generally enter cells via phagocytosis or macropinocytosis. In order, to elucidate the exact mechanism by which the untargeted capsules entered the cells, fluorescently labelled capsules were incubated with cells in the presence or absence or specific inhibitors which block individual entry pathways. For each inhibitor two concentrations representative of an upper and lower limit were tested. These were concentrations typically used in the literature and preliminary experiments confirmed these were not toxic to cells (data not shown).

In the first instance, 500 nm PMA_SH capsules were used in a variety of cell lines with inhibitors to investigate if the capsules used the same entry pathway in different cell types (eg. neuronal, ovary, macrophage and B lymphocyte). Secondly, different sized PMASH capsules were tested in the same cell line (CHO) to investigate if the size of the capsules influenced the predominant entry pathway. The same methodology as described for the uptake assay in example 5 was employed to investigate these parameters. Results were expressed either as the proportion of the cells with capsules or as the mean fluorescence of the total cell population (i.e. an average number of capsules internalized by each cell).

Data points represent the mean ± standard deviation of at least two independent experiments each with six replicates. All data was normalised to the respective controls of cells with no inhibitor. Whilst the latter analysis exhibited a greater response, the conclusions drawn from both analyses were similar. Data was statistically analysed using a one way ANOVA test in which the inhibitor data was directly compared to that without the inhibitor.

Results are presented in Figure 5. In the case of 500 nm PMA_SH capsules, filipin and amiloride which inhibit caveolae dependant endocytosis and macropinocytosis respectively, had no significant effect on the capsule uptake indicating that alternate pathways were being ulitised for capsule entry. This trend was common in all cell types tested despite differences in absolute values obtained between cell types. Conversely, capsule uptake was significantly affected in the presence of chlorpromazine which blocks clathrin dependant endocytosis in all cell types except for B lymphocytes. Statistically the effect of this inhibitor was most evident in ovary and macrophage cells (p< 0.001) and to a lesser degree in neuronal cells (p < 0.01). Interestingly, capsule uptake in B lymphocytes was not affected by chlorpromazine, however nor was it greatly affected by filipin or amiloride.

When different sized PMA_SH capsules were tested in CHO cells, a similar trend was observed, in that chlorpromazine significantly decreased the capsule uptake, whilst filipin and amiloride had either no or little effect in all cell lines tested. This data supported the previous results, which indicate that the capsules are predominantly entering most cell types through the clathrin dependent endocytotic pathway.

Conclusion : Together these data suggest that the predominant entry pathway for capsules into ovary (CHO), neuronal (M 17) and macrophage cells is through clathrin dependant endocytosis. This is a significant observation because normally the clathrin pathway is size restricted to molecules of 100-150 nm or less. Hence, it was totally unexpected that capsules of sizes 300 - 2000 nm would be able to enter through this pathway. Moreover, this remains the predominant pathway despite the size of capsule. This observation is also not in line with the previously characterised molecular pathways which traditionally have been described in terms of size.

Example 8

The structure of PMASH capsules is that of a volume of water surrounded by only a ~ 30 nm thick highly swollen polymer membrane, which makes them inherently soft and deformable. To investigate whether it is these properties which allow the large sized PMASH capsules to enter cells via a clathrin dependant pathway through which normally, smaller biological molecules (less than -150 run) pass, a comparative study using PMA_SH capsules both hollow (as tested in all previous examples) and also with the solid core- shell silica particle (ie. the same surface chemistry but no flexibility). In addition, similar sized - 500 nm capsules constructed using the same methodology of layer by layer as previously described, but consisting of a different polymer, namely PAH/PSS (a two component polyelectrolyte system) were also tested. The PAH/PSS were expected to be more rigid than the swollen single component PMA_SH capsules but less rigid than the solid core- shell silica particle coated in PMA polymer.

The PSS/PAH polymeric capsules were prepared by the following method. Poly(styrene sulfonate), (PSS, 70 KDa), was purchased from Sigma- Aldrich and used as received. Poly(allylamine) hydrochloride, (PAH, 35 KDa), was purchased from Sigma- Aldrich and fluorescently labeled via a reaction with Alexa Fluor 633 succimidyl ester (a solution of 40 mg of polymer in 1 mL of 0.1 carbonate buffer, pH 8.3 was charged with 40 μg fluorescent dye and incubated for 2 h after which time the polymer was isolated via size exclusion chromatography). Polyelectrolyte capsules composed of PSS and PAH were assembled using 500 nm template silica particles which were alternately incubated in 2 g/L solutions of PSS and PAH for 10 minutes with periodic sonication to prevent particles aggregation. Poly(ethyleneimine) was used as a priming layer deposited onto the silica particles, followed by PSS/PAH multilayers. A total of 5 layers of PSS and 4 layers of PAH were deposited, after which time the silica core particles were removed by hydrofluoric acid. The capsules were counted using a flow cytometer with an absolute volume counting and stored in distilled water.

All capsules were fluorescently labelled with Alexa Fluorophore 633 as previously described and tested on CHO cells. The same methodology as described for the uptake assay in example 6 was employed to investigate these parameters. Capsules uptake was measured and expressed as the mean fluorescence of the cell population. Data points represented the mean ± standard deviation of two independent experiments each with six replicates. All data was normalised to the respective controls of cells with no inhibitor. Data was statistically analysed using a one way ANOVA test in which the inhibitor data was directly compared to that without the inhibitor.

Results :

Results are presented in Figure 6.

Interestingly, the uptake of solid PMASH coated silica particles was not significantly decreased in the presence of chlorpromazine, indicating that clathrin dependant endocytosis is not the major pathway these particle use to enter the cells. In contrast, the uptake of hollow, PMASH capsules were significantly affected by chlorpromazine treatment compared to the solid format (p< 0.01). This clearly demonstrated that the same sized entities with the same surface chemistry are utilising different entry pathways into cells in terms of the predominant entry mechanism. Thus the apparent softness and flexibility of the hollow capsules is a contributing factor how they enter cells.

Importantly, the uptake of similar sized PAH/PSS capsules was also affected by chloropromazine treatment but to a lesser degree than the PMASH capsules (p<0.05). This data indicated that the PAH/PSS did use clathrin as a dominant entry pathway into cells but also utilised other entry pathway/s.

Conclusion : Together these data indicate that a combination of the polymer and softness or flexibility of the capsules is important in determining the entry pathway into cell. The pathway associated with entry into the CHO cells in not necessarily indicative of the pathway by which the polymeric nanocapsules pass the BBB. However, the fact that the entry into cells of the PAH/PSS nanocapsules also seems to be associated with the clathrin pathway is indicative that these capsules will also be able to cross the BBB.

Example 9

To further address the issue of capsule size and its affect on entry pathway into cells, 1000 nm PMASH capsules with the core removed were compared to PMA_SH capsules containing liposomal subcompartments (capsosomes). These capsosomes consisted of either one (Cu) or three (C_L3) layers of liposomes, surrounded by the PMA_SH hydrogel. Cu capsosomes contain ~ 800 liposomes per capsule whilst CL₃ capsosomes have ~ 2000 liposome per capsule.

Capsosomes Preparation :

A suspension of 1.11 μm SiO₂ particles (5 wt%) in HEPES buffer was incubated with the polymer precursor layer PLL_C, (1 mg mL^"1, 15 min) and washed three times (1060 g, 30 s). Liposomes (1.25 mg mL^"1, 40 min) were allowed to interact with the polymer- coated particles, washed three times, and the two polymer separation layers, PMA_C and PLL, were subsequently adsorbed (1 mg mL^'1, 15 min). The adsorption steps of liposomes and polymer separation layer(s) were repeated until the required number of layers was deposited (1 or 3 layers), followed by the adsorption of a PMA_C capping layer (1 mg mL^"1, 15 min). The fluorescence of the particles after each liposome adsorption step was analyzed by flow cytometry. After the assembly of the PMA_C capping layer, the solution was changed to NaOAc buffer and five bilayers of alternating PVP (1 mg mL^*1, 10 min) and PMA_SH (1 mg mL^"1, 10 min) were sequentially deposited. The thiols within the polymer layers were cross-linked with chloramine T in MES buffer (2.5 mM, 1 min). Capsosomes were obtained by dissolving the silica core using a 2 M HF solution for 2 min, followed by multiple centrifugation (4500 g, 3 min)/NaOAc buffer washing cycles.

Cells : Both cell lines were incubated at 37⁰C, 5% CO₂

CHO wildtype cell line (ATCC : CCL61) was grown and maintained in RPMl 1640 medium supplemented with penicillin (5LVmI) & streptomycin (5μg/ml), 1 % L- glutamine, 5 % bovine calf serum.

A549 (ATCC CCL- 185) a lung cancer cell line was maintained in RPMl 1640 medium supplemented with penicillin (5U/ml) & streptomycin (5μg/ml), 1 % L-glutamine, 10% bovine calf serum for several passages.

The same methodology as described for the uptake assay in example 6 was employed to investigate these parameters. Capsules uptake was measured and expressed as the mean fluorescence of the cell population. Data points represented the mean ± standard deviation of two independent experiments each with six replicates. All data was normalised to the respective controls of cells with no inhibitor. Data was statistically analysed using a one way ANOVA test in which the inhibitor data was directly compared to that without the inhibitor.

Results

Results are presented in Figure 7.

Both cell lines, CHO and A549, produced similar results in that trends observed for capsule uptake did not differ between the three compositions tested (ie. PMASH hollow capsules and capsosomes of one or three layers of liposomes). In all instances, uptake was not significantly affected by inhibitors filipin and amiloride suggesting that caveolae mediated endocytosis and macropinocytosis are not major pathways of entry for these materials. Conversely, chloropromazine which inhibits clathrin mediated endocytosis significantly decreased the uptake of hollow PMASH capsules and capsosomes independent of the number of liposome layers.

Conclusion : These results indicate that the composition of the polymer plays an important role in mediating cell entry via a clathrin dependant endocytotic pathway. Together with data from example 8, these observations suggest that this pathway preference is independent of size and more reliant on the polymer attributes which afford a softness and or flexibility allowing capsosomes and capsules of a large size (500 and lOOOnm) to unexpectedly enter cells via a pathway that is normally size restricted to molecules of ~ 150 nm. This study also confirms that a polymeric capsule carrying a cargo is able to be transported into cells.

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

All publications mentioned in this specification are herein incorporated by reference. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia or elsewhere before the priority date of each claim of this application.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims

1. A method of delivering a functional agent across the blood brain barrier so that the functional agent is delivered into the brain of the subject, the method comprising the step of administering a polymeric capsule associated with said functional agent to the subject whereby the polymeric capsule crosses the blood brain barrier and wherein the polymeric capsule is from 300 nm to 2000 nm in diameter and comprises a polymeric shell defining an internal cavity.

2. A method according to claim 1 wherein the polymeric capsule is from 300 to 1000 nm in diameter, more preferably 400 to 1000 nm in diameter, yet more preferably 450 to 650 nm in diameter, and even more preferably 500 nm in diameter.

3. A method according to any one of claims 1 to 2 wherein the diameter of the internal cavity is from 50 to 97%, more preferably 70 to 97%, yet more preferably 80% to 95%, even more preferably 86 to 90%, of the diameter of the polymeric capsule.

4. A method according to any one of claims 1 to 3 wherein the thickness of the wall of the polymeric shell is from 15 to 50 nm, preferably 20 to 40 nm, more preferably 30 nm.

5. A method according to any one of claims 1 to 4 wherein the polymeric capsule crosses the blood brain barrier without significantly disrupting the blood brain barrier.

6. A method according to any one of claims 1 to 5 wherein the polymeric capsule is a polymeric shell comprising layers of thiolated poly(methacrylic acid) (PMA_SH) wherein the PMA_SH layers are cross-linked by disulphide bonds.

7. A method according to claim 6 wherein 10 to 25 mol%, preferably 15 to 20 mol%, more preferably 17 to 19 mol%, yet more preferably 18 mol % of the thiolated poly(methacrylic acid) is functionalized with thiol groups.

8. A method according to claim 6 or claim 7 wherein, there are three to seven, more preferably four to six, even more preferably five, layers of PMA_SH-

9. A method according to any one of claims 1 to 5 wherein the polymeric capsule is a polymeric shell comprising alternating layers of poly(styrene sulfonate) and poly(allylamine) hydrochloride.

10. A method according to any one of claims 1 to 9 wherein the functional agent is selected from the group consisting of nucleic acids, peptides, proteins, MRI contrast reagents such as magnevist and iron oxide, pharmaceuticals, such as an antidepressants, and hybrid molecules thereof.

1 1. A method according to claim any one of claims 1 to 10 wherein the functional agent is released from the polymeric capsule.

12. A method according to any one of claims 1 to 10 wherein the polymeric capsule does not release the functional agent.

13. A method according to any one of claims 1 to 12 wherein the polymeric capsule comprises one or more targeting molecules associated with the surface of the polymeric capsule.

14. A method of delivering a polymeric capsule across the blood brain barrier so that the polymeric capsule is delivered into the brain of the subject, the method comprising the step of administering the polymeric capsule to the subject whereby the polymeric capsule crosses the blood brain barrier and wherein the polymeric capsule is from 300 nm to 2000 nm in diameter and comprises a polymeric shell defining an internal cavity.

15. A method according to claim 14 wherein the polymeric capsule is associated with a functional agent.