AU2007202988A1 - Multilayer polymer films - Google Patents

Description

AUSTRALIA

Patents Act COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: Name of Applicant: The University of Melbourne Actual Inventor(s): Francesco Caruso, Georgina Such, Angus Johnston, Elvira Tjipto, Heng Pho Yap Address for Service and Correspondence: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Invention Title: MULTILAYER POLYMER FILMS Our Ref: 805497 POF Code: 475682/466907 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): -1- MO6 SMULTILAYER POLYMER FILMS FIELD OF THE INVENTION r"c 5 The present invention relates to multilayer polymer assemblies, particularly covalently crosslinked multilayer polymer assemblies, to processes for the oO 00 preparation of such assemblies and to core-shell particles comprising the N assemblies.

BACKGROUND

Multilayer polymer materials have been prepared using a variety of different techniques. Layer-by-layer (LbL) assembly is one technique that has been used to fabricate tailored multilayer thin films of diverse composition. The majority of work in LbL assembly has focused on the construction of polyelectrolyte (PE) films by either electrostatic or hydrogen bonding interactions. Such films however can be susceptible to disassembly under varying solution conditions that disrupt the electrostatic or hydrogen bonds.

Covalently bound films offer the advantage of increased stability compared to electrostatic or hydrogen bonded films due to the presence of covalent crosslinks between layers of polymer films. However, some covalent crosslinking reactions may be limited by electrostatic, steric or thermodynamic considerations, which can adversely impact on the efficiency of covalent bond formation. In addition, covalent crosslinking reactions may require conditions such as exposure to radiation or high temperature to generate the crosslinks. Such conditions are not compatible with a number of polymeric materials.

It would be desirable to provide new covalently crosslinked multilayer polymer assemblies as well as new methods of making such assemblies.

The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these Y UOum Jebourme UnhSp~a eS 04747W0547_spede AU Soc L matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of g each claim of this application.

SUMMARY

It has been found that new, stable multilayer polymer materials may be afforded oo oO by using click reactions to covalently crosslink layers of polymer films assembled C using a LbL approach. The present invention is applicable to the preparation of a N wide variety of multilayer polymer assemblies of different composition and controlled physical properties.

In accordance with one aspect, the present invention provides a multilayer polymer assembly comprising: a plurality of polymer layers, the polymer layers forming one or more pairs of adjacent polymer layers; and (ii) a plurality of crosslinks between at least one pair of adjacent polymer layers, wherein each of the crosslinks comprise a cyclic moiety formed by a cycloaddition reaction.

In accordance with another aspect, the present invention provides a core-shell particle comprising a core and a shell material, wherein the shell material comprises:(i) a plurality of polymer layers, the polymer layers forming one or more pairs of adjacent polymer layers; and (ii) a plurality of crosslinks between at least one pair of adjacent polymer layers, wherein each crosslink comprises a cyclic moiety formed by a cycloaddition reaction.

In accordance with a further aspect, the present invention provides a process for the preparation of a multilayer polymer assembly comprising: providing a polymer layer; (ii) depositing a further polymer layer to form a pair of adjacent polymer layers; and YxLolsaWdbownme U .Spedole%747805497_spedO AU.OC 0 (iii) forming a plurality of crosslinks between the pair of adjacent 0 c polymer layers, wherein each crosslink comprises a cyclic moiety formed Sby a cycloaddition reaction.

5 BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows a scheme illustrating the crosslinking of layer by layer (LbL) 00 00 assembled polymer multilayer films using click chemistry.

SFigure 2 shows UV-vis absorption spectra for (PAA-Az/PAA-Alk) multilayer films assembled on quartz with the arrow indicating increasing bilayer number.

c Absorbance as a function of bilayer number at 240 nm is shown in inset.

Figure 3 shows RAS-FTIR absorption spectra of (PAA-Az/PAA-Alk) multilayer films assembled on gold substrates with increasing bilayer number with the arrow indicating increasing bilayer number (bottom to top: bilayers 1, 2, 3, 4 and Reversible change in the RAS-FTIR peak at 1700 cm-1 with a change in pH fora (PAA-Az/PAA-Alk) film is shown in inset.

Figure 4 shows AFM images of (PAA-Alk-PAA-Az)4 (z scale of 10nm) and (b) (PAA-Alk-PAA-Az)8 multilayer films assembled on silicon (z scale of 25 nm).

Figure 5 shows fluorescence intensity of (PAA-Az/PAA-Alk)-coated silica particles as a function of layer number, as measured by flow cytometry after deposition of each PAA-Alk layer, which was fluorescently labeled with rhodamine and fluorescence microscopy image of silica particles coated with (PAA-Az/PAA-Alk) 6 where the PAA-Alk is fluorescently labeled with rhodamine.

Figure 6 shows fluorescence microscopy images of (PAA-Az/PAA-Alk)-coated silica particles functionalized with Rh-Az and non-specifically adsorbed Rh.

Figure 7 shows TEM and AFM images of (PAA-Az/PAA-Alk) 6 capsules.

The thickness of the capsule wall, determined by AFM, is -5 nm.

Y:UsOer elboume Uni\SpeieskhO474T%5497speCie AU.doc Figure 8 shows the differential interference contrast (DIC) microscopy images of

O

O 5 pm (PAA-Az/PAA-Alk) 6 core-shell particles and capsules.

s Figure 9 shows fluorescence microscopy images of (PAA-AzlPAA-Alk)6 click capsules after addition of pH 2 solution and pH 10 buffer and a graph showing reversible pH response of the (PAA-Az/PAA-Alk) 6 capsules.

00 00 oO a Figure 10 shows a scheme illustrating the formation of capsules comprising click Scrosslinked PAA multilayers (PAA-Az/PAA-Alk).

DETAILED DESCRIPTION Various terms that will be used throughout the specification have meanings that will be well understood by a skilled addressee. However, for ease of reference some of these terms will now be defined.

As used herein the term "layer-by-layer" refers to the sequential deposition of successive layers of polymer material in an overlapping manner.

As used herein reference to molecular weight for a polymer refers to number average molecular weight unless otherwise specified.

As used herein the terms "polyelectrolyte" or "polyelectrolyte material" refers to a material that either has a plurality of charged moieties or has the ability to carry a plurality of charged moieties. A number of polyelectrolyte materials are known in the art. Polyelectrolyte materials may be a positively charged (or have the ability to be positively charged), negatively charged polyelectrolyte (or have the ability to be negatively charged) or have a zero net charge. The term polyelectrolyte may also include macromolecules which have the ability to carry a plurality of charges, including bio-macromolecules such as such as proteins, enzymes, polypeptides, peptides, polyoligonucleotides, polysaccharides, polynucleotides, DNA, RNA and the like.

YVA outsW3 bOunO UnJASpees'ao474797_pedte AUdOC The LbL approach has conventionally been used to construct multilayered polyelectrolyte films by sequential deposition and self-assembly of oppositely Scharged polyelectrolyte materials. Such self-assembled structures rely on Selectrostatic or hydrogen bonding interactions to maintain a coherent multilayer C 5 structure. However, electrostatic and hydrogen bonds are susceptible to disruption and disassembly under varying solution conditions, which leads to oo oo destruction of the polyelectrolyte films.

0 C Intermolecular covalent bonding between individual polymer layers of a multilayer polymer assembly can impart improved stability to the assembly by crosslinking cN the layers of the polymer assembly together. In the present invention, 'click chemistry' is used to form covalent bonds that crosslink the layers of a multilayered polymer assembly.

The term 'click chemistry' is used to describe covalent reactions with high reaction yields that can be performed under extremely mild conditions. A number of 'click' reactions involve a cycloaddition reaction between appropriate functional groups to generate a stable cyclic structure. The most well documented click reaction is the Cu(l) catalyzed variant of the Huisgen 1,3-dipolar cycloaddition of azides and alkynes to form 1,2,3-triazoles. Many click reactions are thermodynamically driven, leading to fast reaction times, high product yields and high selectivity in the reaction.

The preparation of multilayer polymer films using a combination of click chemistry and LbL assembly offers a number of significant advantages over prior techniques. Firstly, the click reactions generally proceed with high yields in mild conditions, and it is particularly efficient in water. Secondly, cyclic groups such as triazole groups, produced from the click reaction can have excellent physicochemical properties and are extremely stable to hydrolysis, oxidation or reduction. Thirdly, the click reactions are applicable to a wide range of materials, from polymers and proteins to nanoparticles, dye molecules and biological systems. Finally, the use of click chemistry to bond together individual polymer YIImGWeLbourr UrASpeoeSN474'B05497_sDpece AU.O c layers enables the multilayer polymer assemblies to be prepared from a single 0 component polymer, which is not possible using conventional LbL assembly.

Multilayer Polymer Assemblies The present invention relates in one aspect to a multilayer polymer assembly.

The polymer assembly comprises a plurality of polymer layers. The polymer o0 layers are prepared by sequential deposition of two or more polymer layers in a SLbL approach, which results in at least a portion of a polymer layer overlapping N with at least a portion of another polymer layer.

CN The multilayer polymer assembly may comprise any number of polymer layers.

In one embodiment, the assembly preferably comprises at least two polymer layers. The polymer assembly may comprise up to any maximum number of polymer layers, and the maximum number of layers may in part be dictated by the end use application of the polymer assembly. In one embodiment, the polymer assembly may comprise from between two to twelve polymer layers.

The polymer layers of the assembly may comprise any suitable polymer material.

The person skilled in the art would understand that the present invention is widely applicable to a range of polymer materials and that the choice of polymer material would depend on the intended end use application. Examples of suitable polymer materials include polymers, copolymers, polyelectrolyte polymers, polyethers such as polyethylene glycol, polyesters such as poly(acrylates) and poly(methacrylates), polyalcohols such as poly(vinyl alcohol), polyamides such as poly(acrylamides) and poly(methacrylamides), biocompatible polymers, biodegradable polymers, polypeptides, polynucleotides, polycarbohydrates and lipopolymers. The person skilled in the art would be able to select an appropriate polymer material suitable for an intended application. In one embodiment, the same polymer material may be used in each polymer layer. Alternatively, the polymer layers may comprise different polymer materials. The use of different polymer materials in different layers of the assembly may advantageously enable the properties of the polymer assembly to be tailored for specific applications, such as for controlled or sustained drug release applications.

Y:VuiselWlboe Un0SpeaesO4747%5497_ spede AU doc Q In one embodiment, the polymer layers comprise a polyelectrolyte material.

SExamples of suitable polyelectrolyte material may comprise any suitable polyelectrolyte polymer, including but not limited to those selected from C 5 polyglycolic acid (PGA), polylactic acid (PLA), polyacrylic acid (PAA), polyamides, poly-2-hydroxy butyrate (PHB), gelatins, B) polycaprolactone (PCL), poly o0 (lactic-co-glycolic acid) (PLGA), flourescently labelled polymers, conducting Spolymers, liquid crystal polymers, photoconducting polymers, photochromic Cpolymers; poly(amino acids) including peptides and S-layer proteins; peptides, 0 10 glycopeptides, peptidoglycans, glycosaminoglycans, glycolipids, C lipopolysaccharides, proteins, glycoproteins, polypeptides, polycarbohydrates such as dextrans, alginates, amyloses, pectins, glycogens, and chitins; polynucleotides such as DNA, RNA and oligonucleotides; modified biopolymers such as carboxymethyl cellulose, carboxymethyl dextran and lignin sulfonates; polysilanes, polysilanols, poly phosphazenes, polysulfazenes, polysulfide and polyphosphate and mixtures thereof. Poly(acrylic acid) and poly(L-lysine) are particularly preferred polyelectrolyte materials. At least one polymer layer, and preferably, each polymer layer of the multilayer polymer assembly may comprise a polyelectrolyte material. In one embodiment, each polymer layer comprises a polyelectrolyte material. The polyelectrolyte materials may be of the same charge or have no charge.

In another embodiment of the invention, the polymer layers of the multilayer polymer assembly may comprise uncharged polymer materials. Preferred uncharged polymer materials are those that are compatible with biological systems. Particularly preferred polymer materials are polyethers, such as poly(ethylene glycol).

The material used in each polymer layer may be of any suitable size or molecular weight. In a preferred embodiment, the material used in the polymer layers has a molecular weight of at least 100, and preferably a molecular weight of 100 to 1,000,000.

Y U."m sAlb., UnrtSpean474T5BO 97_spdee AU doc 1 The plurality of polymer layers of the multilayer polymer assembly form one or more pairs of adjacent polymer layers. The term "adjacent" is used herein to Srefer to a polymer layer that lies next to and preferably at least partially overlaps Swith another polymer layer. For example, where the multilayer polymer assembly c1 5 comprises two polymer layers, the two polymer layers will be adjacent to one another and will form one pair of adjacent polymer layers. In addition, where the 00oo 00 polymer assembly comprises three or more polymer layers, a first polymer layer will be adjacent a second polymer layer, while the second polymer layer will be (cN adjacent to both the first polymer layer and a third polymer layer, and so on.

0 10 Consequently, one pair of adjacent polymer layers is formed between the first 0 (N and second polymer layers, while another pair of adjacent polymer layers is formed between the second and third polymer layers, and so on.

The multilayer polymer assembly of the invention comprises a plurality of crosslinks between at least one polymer layer and an adjacent polymer layer.

The crosslinks each comprise a cyclic moiety formed by a cycloaddition reaction.

In one embodiment, each polymer layer of the assembly is crosslinked via a plurality of crosslinks to each polymer layer adjacent to it.

In another embodiment, at least one polymer layer of the assembly is not crosslinked to each polymer adjacent to it. In this instance, the uncrosslinked polymer layer may be bound to adjacent polymer layers by other interactions such as electrostatic or hydrogen bonding interactions, rather than by covalent bonds.

Examples of different multilayer polymer assemblies according to the invention are shown in Scheme 1: Y:V Mzuiaboeme UV\Speae6%BO4747\h5497_spO8 AU Soc JWW/VV V x TVVWJWnfVAA

Y

(B)

x

Y

~AJ /vvV\ ~Avv\ z

(C)

x

(D)

x

(E)

Scheme 1 Multilayer polymer assemblies comprising adjacent polymer layers bonded with one type of crosslink, adjacent polymer layers bonded with two different types of crosslinks, adjacent polymer layers bonded with three different types of crosslinks, (D)and at least one pair of adjacent polymer layers bonded with a crosslink and at least one pair of adjacent polymer layers that is not crosslinked.

Each crosslink of the multiplayer polymer assembly may comprise any suitable cyclic moiety formed from a cycloaddition reaction. In a preferred embodiment, the cyclic moiety is selected from the group consisting of tetrazoles, triazoles and oxazoles. Preferably, the cyclic moiety is a 1,2,3-triazole. Each crosslink of the plurality of crosslinks between a pair of adjacent polymer layers may comprise the same cyclic moiety. Alternatively, the crosslinks may comprise different cyclic moieties. As a result, the plurality of crosslinks between one pair of adjacent polymer layers may comprise two or more different cyclic moieties.

YL ws8m4eboume UnASPesBG474780549speae AU.oc In another embodiment, the plurality of crosslinks between one pair of adjacent 0 polymer layers of the multilayer assembly may comprise a different cyclic moiety Sto that of the plurality of crosslinks between another pair of adjacent polymer layers. Accordingly in this embodiment, the multiplayer polymer assembly l"- C 5 comprises two or more pairs of adjacent polymer layers, wherein the cyclic moieties of the plurality of crosslinks between one pair of adjacent polymer layers 00oo 00 is different to the cyclic moieties of the plurality of crosslinks between another pair c of adjacent polymer layers. In this regard, different pairs of adjacent polymer c layers may therefore be covalently bound together by different types of 0 10 crosslinks, where each type of crosslink comprises a different cyclic moiety.

The crosslinks comprising the cyclic moiety may be formed by any cycloaddition reaction known in the art. In one embodiment, the crosslinks are formed by a cycloaddition reaction involving appropriate functional groups extending from, and between, a pair of adjacent polymer layers.

The functional groups are selected from those adapted to undergo cycloaddition reactions. In this manner, the participation of the functional groups in cycloaddition reactions contributes to the formation of the plurality of crosslinks between a pair of adjacent polymer layers to covalently bond the polymer layers together. The crosslinks each comprise a cyclic moiety, which is formed by the reaction of a functional group of each of the adjacent polymer layers in the cycloaddition reaction.

The functional groups may be incorporated into the polymer material of the polymer layers by any suitable method. Suitable methods may involve the copolymerization of appropriately functionalized monomers during polymer preparation or the post-polymerization functionalisation of the polymer material.

The functional groups may be present in any concentration. Preferably, the functional groups are present in an amount of from about 0.01 to 99% of the polymer. A linking group may also be present to connect the functional groups to the polymer material of the polymer layers. When the functional groups have Y:Ubuisarelb Uff\Spea 8O4747Yt5497_peae AU doc 11 covalently reacted in the cycloaddition reaction, the linking group becomes a part 0 of the crosslink bonding adjacent polymer layers together.

;1 The functional groups of the adjacent polymer layers are selected from any of c 5 those adapted to undergo cycloaddition reactions. Preferably, the functional groups are independently selected from the group consisting of alkenes, alkynes, 00 azides, nitriles, nitrile oxides, cycloalkenes, heterocycloalkenes, maleimide, canthracene and maleic anhydride. Particularly preferred functional groups are c alkynes, azides, nitriles, nitrile oxides, anthracene and maleimide. Each 0 10 individual polymer layer may comprise the same type of functional groups, or they cN may comprise a mixture of types of functional groups. A mixture of functional groups on the same polymer layer may be advantageous where the controlled selectivity of covalent reactions is desired.

Upon covalent reaction of the functional groups of the opposed polymer layers in a cycloaddition reaction, a cyclic moiety is formed. Preferably, the cyclic moiety is selected from the group consisting of tetrazoles, triazoles and oxazoles. More preferably, the cyclic moiety is a 1,2,3-triazole. In one embodiment, the polymer layers in a pair of adjacent polymer layers may each individually comprise different types of functional groups. Where the polymer layers comprise types of different functional groups, it is preferred that the functional groups be complementary functional groups. In this sense, the term "complementary" is used to refer to those the functional groups that are capable of directly reacting with one another in the cycloaddition reaction to generate the cyclic moiety. The crosslink comprising the cyclic moiety is therefore formed as a direct result of the reaction between the different types of functional groups extending between the pair of adjacent polymer layers.

In one embodiment, the pair of adjacent polymer layers comprises a first polymer layer comprising one type of functional group and a second polymer layer comprising another type of functional group that is complementary to the functional groups of the first polymer layer. Preferably, the first polymer layer comprises alkyne functional groups while the second polymer layer comprises YV MlIwoeMlbue Ur S000e 50474TVM5497_zpede AU.doc 12 azide functional groups. The alkyne and azide functionalities react with each C other in the variant of the Huisgen 1,3-dipolar cycloaddition to form a 1,2,3- Striazole moiety, which covalently bonds the first and second polymer layers together. The person skilled in the art would appreciate that the order to the arrangement of the functionalities may be reversed, that is, the first polymer layer may comprise the azide functionalities while the second polymer layer may 0o comprise the alkyne functionalities, and still obtain the same 1,2,3-triazole moiety.

SAn example of the covalent cycloaddition reaction of complementary functional cgroups to form crosslinks in the polymer assembly is shown in Scheme 2: S H H H N N N N N, c c c c N N 7 H H H H I II I II

C

Scheme 2 Formation of crosslinks in the assembly of polymer multilayers Other complementary functional groups may be paired in similar manner between adjacent polymer layers in order to from the crosslink comprising the cyclic moiety. In addition to the alkyne-azide functional pair, examples of other complementary paired functional groups are alkyne-nitrile oxide, nitrile-azide and maleimide-anthracene. Each of the paired complementary functional groups gives rise to cyclic moieties when they directly react with one another in a covalent cycloaddition reaction. The person skilled in the art would be able to select other functional group pairings capable of participating in cycloaddition reactions that satisfy the requirements of click chemistry.

Y:\oulslelMboume UrnSpeiesA074785497 _spece AU doc In another embodiment, the polymer layers of a pair of adjacent polymer layers Smay each comprise the same type of functional groups. In this instance, the Sfunctional groups may not be capable of directly reacting with one another to 1 5 generate the crosslink. Accordingly in this embodiment, a crosslinking agent may be covalently reacted with the functional groups of the adjacent polymer layers in 00 00 a cycloaddition reaction, to from the crosslink comprising a cyclic moiety.

c The crosslinking agent comprises at least two reactive groups adapted to 0 10 undergo cycloaddition reactions with the functional groups of the polymer layers.

N The crosslinking agent may comprise any number of reactive groups, however it is preferred that the crosslinking agent comprise two reactive groups. Any suitable crosslinking agent of appropriate composition may be used provided that the functional groups of the crosslinking agent are complementary to the functional groups of each of the adjacent polymer layers. The complementary arrangement of the functional groups of the polymer layers and crosslinking agent is such that when the crosslinking agent is reacted with the respective functional groups of the each polymer layer in a pair of adjacent polymer layers, a cyclic moiety is formed between the crosslinking agent and the respective polymer layers.

The functional groups of the crosslinking agent and the adjacent polymer layers are selected from those adapted to undergo cycloaddition reactions with the functional groups of the adjacent polymer layers. Preferably, the adjacent polymer layers and the crosslinking agent each comprise functional groups independently selected from the group consisting of alkenes, alkynes, azides, nitriles, nitrile oxides, cycloalkenes, heterocycloalkenes, maleimide, anthracene and maleic anhydride. Particularly preferred reactive groups are alkynes, azides, nitriles, nitrile oxides, anthracene and maleimide.

Where a pair of adjacent polymer layers each comprise the same type of functional group, the crosslinking agent will preferably also comprise complementary functional groups having the same type of functionality. As an Y:Mo",LseWebow UMSpeaeSO%"741TMO497-SpeSC AUODC example, where the polymer layers each comprise alkyne functional groups, the Scrosslinking agent may therefore comprise azide functionalities as the Scorresponding complementary reactive groups. The alkyne and azide Sfunctionalities of the polymer layers and the crosslinking agent respectively, may then covalently react in a cycloaddition reaction to form the crosslink between the polymer layers.

00 00 SIn a further embodiment of the invention, where the pair of adjacent polymer Slayers comprises different types of functional groups, a crosslinking agent may also be employed to covalently bond the polymer layers together. In this N embodiment, the functional groups of the crosslinking agent would be of differential functionality, and each functional group of the crosslinking agent would be complementary with a respective functional group of a selected polymer layer. As an example, where a first polymer layer comprises azide functional groups and a second polymer layer comprises alkyne functional groups, a crosslinking agent having both azide and alkyne reactive groups may be used.

Where a crosslinking agent comprising at least two different types of functional groups is used, one of the types of functional group may be selectively protected using an appropriate protecting group in order to avoid undesired reactions occurring with the selected functional group. The protecting group may then be removed prior to the desired cycloaddition reaction to form the crosslink.

In addition to the functional groups described, the pair of adjacent polymer layers may also comprise other functional groups, such as carboxylic functional groups, as seen in Scheme 3. These functional groups typically do not participate in the cycloaddition reactions that covalently bond the polymer layers, and remain within the multilayer polymer assembly. The additional functional groups may be introduced by any method known in the art. For example, the functional groups may be present within the polymer material of a given polymer layer, or they may be introduced by modification of the polymer material. Such functional groups may be useful to impart a charge to the polymer assembly or for further functionalisation of the polymer assembly.

Y:M sseMbo m UrASpa ee%8O74rMO6sdedo AUdcc

H

C H H w ll O O H H 2 C n CI 0 C c

H

1. PAA-Azide Cu(l)

H

o^O N- H H, H, H H, -N O 0 c ,,c0 C H

H

H

c H N'N H 0 N H H 2. PAA-Alkyne Cu() 1.2 Cu(l)

H

C H H H H 111 O 0 NC C C 2 f 0O N 1 0^ 0 SN/ H

H

H, I H, H, 0 C 0~-HJ\H H H, H H N 0. 0 C C' cH C o4 C o C H

H

Scheme 3 Crosslinked multilayer polymer assembly comprising carboxylic functional groups In one embodiment of the invention, the crosslinks of the multilayer polymer assembly may comprise a cleavable moiety which is adapted to undergo selective degradation under pre-determined conditions. The cleavable moiety may be any suitable moiety that undergoes selective degradation. Preferably, the cleavable moiety degrades under hydrolytic, thermal, enzymatic, proteolytic or photolytic conditions. In a preferred embodiment, the cleavable moiety is selected from the group consisting of a disulfide, an ester, an amide, a photocleavable link and bio-fragments such as proteins. The cleavable moiety may be introduced by a crosslinking agent that has covalently reacted with the functional groups of the opposed polymer layers. Alternatively, the cleavable moiety may be present in a linking group that is used to connect the functional group to the polymer material of a polymer layer. As described above, the linking Y: odwddbo UnNSpoes\84747805497_spemo AU.doc 16 groups become a part of the crosslink once the functional groups of the adjacent Spolymer layers have reacted in a cycloaddition reaction. The degradation of the Scleavable moiety provides a convenient route for the selective disassembly of the Smultilayer polymer structure under controlled conditions.

Process for Preparation of Multilayer Polymer Assembly 0o In accordance with another aspect of the invention there is provided a process for Sthe preparation of a multilayer polymer material. The process of the invention N utilizes a layer-by-layer (LbL) approach of depositing successive polymer layers to construct the polymer assembly. The present invention offers particular appeal c for systems that cannot be fabricated using traditional LbL assembly, such as non-charged, non H-bonding polymers. Further, it is particularly well suited to biological systems as a result of the extremely mild reaction conditions employed to covalently bond the polymer layers.

In accordance with one aspect, the present invention relates to a process for the preparation of a multilayer polymer assembly comprising: providing a polymer layer; (ii) depositing a further polymer layer to form a pair of adjacent polymer layers; and (iii) forming a plurality of crosslinks between the pair of adjacent polymer layers, wherein each crosslink comprises a cyclic moiety formed by a cycloaddition reaction The provision of a polymer layer in the first step of the process may be achieved by any suitable method. In one embodiment, the provision of the polymer layer is achieved by forming the polymer layer on a substrate. The polymer layer may be bound to the substrate by covalent, electrostatic or hydrogen bonding interactions. Any suitable substrate may be used. In one embodiment, the substrate is a planar substrate. In another embodiment, the substrate is a particulate template. Examples of particulate templates include colloidal particles, nanoparticles, microspheres, crystals, and the like. A preferred particulate template is a colloidal particle. The substrate may comprise any Y:UojWelbocn UNSp~edns8O474T%80517_sp AU dOC 17 suitable material. Preferably, the substrate comprises a material selected from Sthe group consisting of silicon, gold, quartz, polymeric materials such a Sdegradable polymer, for example, polyesters and silica. The substrate may also be provided by micelles, emulsion droplets, air, bubbles or any other surface or C 5 material that provides a phase interface. The substrate may be optionally coated with a coating material. An example of a suitable coating material is 00 oo polyethyleneimine (PEI). In a preferred embodiment, the substrate is removable.

SIn this regard, the substrate may be removed by exposing the substrate to Sappropriate conditions that destroy the substrate but do not adversely affect the 010 polymers used in the preparation of the assembly. In one embodiment, the c substrate is removed by exposure to hydrofluoric acid. It is preferred that the hydrofluoric acid has a concentration of from 0.01 to 10 M, more preferably from 1 to 10 M, most preferably about 5 M. The substrate may also be removed by disrupting any covalent, electrostatic or hydrogen bonding interactions between the substrate and the polymers used in the preparation of the multilayer assembly, and thereafter liberating the substrate from the polymers.

The substrate may be dipped into the solution comprising the polymer material to form the polymer layer on the substrate. In this manner, the polymer material is dispersed as a layer on the substrate. The person skilled in the art would appreciate that other methods may be used to form the polymer layer. Where the substrate is a particulate template, the polymer solution may be dispersed on the surface of the template to provide a polymer layer that typically surrounds the entire template. The polymer solution may comprise the polymer material in any suitable concentration. Typically, the solution may have a concentration of the polymer material from about 0.001 to 100 mg mL 1 more preferably from about 0.1 to 30 mg mL- 1 most preferably from 0.5 to 10 mg mL 1 The process of the invention then involves the step of depositing a further polymer layer to form a pair of adjacent polymer layers. The further polymer layer may be deposited using any suitable technique. In one embodiment, the further polymer layer may be deposited by contacting a substrate carrying a polymer layer with a solution comprising a suitable polymer material. In a preferred Y: ~meedbo UNSpeoes80 74?O549 _Sped1 AU doc 18 embodiment, the substrate carrying the polymer layer is dipped into a solution Scomprising a polymer material to deposit the further polymer layer and thereby Sform a pair of adjacent polymer layers.

c 5 After deposition of the further polymer layer, the mixture thus formed is typically incubated to allow the further polymer layer to be adsorbed. This can be done for oo any suitable length of time but it is typically found that the solution is incubated Sfrom 15 minutes to 24 hours, more preferably from 2 hours to 20 hours, even cmore preferably from 4 hours to 12 hours, most preferably about 6 hours. During incubation, the solution may also be agitated to assist in the deposition of the c further polymer layer.

The process of the invention subsequently involves the step of forming a plurality of crosslinks between adjacent polymer layers, wherein each crosslink comprises a cyclic moiety formed by a cycloaddition reaction. Each crosslink may comprise any suitable cyclic moiety formed from a cycloaddition reaction. In a preferred embodiment, each crosslink may comprise a cyclic moiety independently selected from the group consisting of tetrazoles, triazoles and oxazoles.

Preferably, the cyclic moiety is a 1,2,3-triazole. Each crosslink of the plurality of crosslinks between the adjacent polymer layers may comprise the same cyclic moiety. Alternatively, the crosslinks may comprise different cyclic moieties.

The crosslinks comprising the cyclic moiety may be formed by any cycloaddition reaction known in the art. Typically, the crosslinks are formed by a cycloaddition reaction involving appropriate functional groups extending from, and located between, the adjacent polymer layers.

The functional groups of the polymer layers may be selected from any of those adapted to undergo cycloaddition reactions. Preferably, the functional groups are independently selected from the group consisting of alkenes, alkynes, azides, nitriles, nitrile oxides, cycloalkenes, heterocycloalkenes, maleimide, anthracene and maleic anhydride. Particularly preferred functional groups are alkynes, azides, nitriles, nitrile oxides, anthracene and maleimide.

Y:urnise4M3eboum ur UnNpsdkOm%4747%6497&specie AUdoc O As discussed above, the functional groups of the polymer layers may be Sintroduced by incorporating appropriate functionalities into the polymer material used to prepare the polymer layers. The introduction of the functional groups may be achieved using any technique, such as through the copolymerization of appropriately functionalized monomers during preparation of the polymer material 0o or by post-polymerization functionalisation of the polymer material. The Sfunctional groups may be present in any concentration. In one embodiment, the cfunctional groups are present in an amount of from about 0.01 to 99% of the 0 10 polymer. A linking group may also be present to connect the functional groups to CN the polymer material of the polymer layers. The linking group becomes a part of the crosslink bonding adjacent polymer layers together once the functional groups have reacted to form the crosslink.

In one embodiment, the plurality of crosslinks is formed by a cycloaddition reaction between complementary functional groups extending from the pair of adjacent polymer layers. In this regard, the polymer layers in the pair of adjacent polymer layers may each individually comprise different types of functional groups, which are complementary to each other and are capable of directly covalently reacting with one another in a cycloaddition reaction to form the crosslink comprising the cyclic moiety in between the polymer layers. As an example, a first polymer layer may have alkyne functional groups while an adjacent second polymer layer has azide functional groups. The alkyne and azide functionalities react with each other in the variant of the Huisgen 1,3-dipolar cycloaddition to form a 1,2,3-triazole moiety, which covalently bonds the first and second polymer layers together. In addition to the alkyne-azide functional pair, the pair of adjacent polymer layers may comprise other complementary paired functional groups capable of participating in click reactions. Examples of other complementary pairs of functional groups include alkyne-nitrile oxide, nitrile-azide and maleimide-anthracene.

In another embodiment, the plurality of crosslinks is formed by a cycloaddition reaction between functional groups extending from the pair of adjacent polymer Y M OwIe4bW UnPSpeaes5%8747aO5497_pWOe AU doc layers and a crosslinking agent. This may be desirable where the adjacent polymer layers each have the same type of functional groups. When a Scrosslinking agent is used, the crosslinking agent comprises at least two reactive functional groups.

N In one embodiment, the crosslinking agent may covalently react with the oO oO functional groups of each polymer layer in the pair of adjacent polymer layers in a N cycloaddition reaction tothereby form the crosslink comprising a cyclic moiety.

c The functional groups of the crosslinking agent are therefore complementary with the functional groups of each of the adjacent polymer layers. As an example, N where the polymer layers each comprise alkyne functional groups, the crosslinking agent may therefore comprise azide functionalities as the corresponding complementary reactive groups. The alkyne and azide functionalities of the polymer layers and the crosslinking agent respectively covalently react in a cycloaddition reaction to form the crosslink between the polymer layers.

In another embodiment, at least one of the functional groups of the crosslinking agent is adapted to undergo cycloaddition reactions with the functional groups of one of the pair of adjacent polymer layers, while the remaining functional groups of the crosslinking agent participate in different covalent bonding interactions with functionalities present in the other polymer layer of the pair of adjacent polymer layers.

Preferably, the crosslinking agent comprises at least two functional groups, wherein each functional group is adapted to undergo cycloaddition reactions.

Preferably, the functional groups of the crosslinking agent are independently selected from the group consisting of alkenes, alkynes, azides, nitriles, nitrile oxides, cycloalkenes, heterocycloalkenes, maleimide, anthracene and maleic anhydride. Particularly preferred functional groups are alkynes, azides, nitriles, nitrile oxides, anthracene and maleimide.

Y:U tdLweVj4ftmxm U PSpes O4747MSO54QS7poe AU doe I In one embodiment, the crosslinking agent is of general formula (I) SY-Q2-Z (I) c 5 where Q2 is a linking group, and o0 Y and Z are functional groups that may be the same or different, and at Sleast one of Y and Z is selected from the group consisting of alkenes, Salkynes, azides, nitriles, nitrile oxides, cycloalkenes, heterocycloalkenes, maleimide, anthracene and maleic anhydride.

The plurality of crosslinks between one pair of adjacent polymer layers of the multilayer assembly may comprise a different cyclic moiety to that of the plurality of crosslinks between another pair of adjacent polymer layers. Accordingly, where the multiplayer polymer assembly comprises two or more pairs of adjacent polymer layers, the cyclic moieties of the plurality of crosslinks between one pair of adjacent polymer layers may be different to the cyclic moieties of the plurality of crosslnks between another pair of adjacent polymer layers. In this regard, different pairs of adjacent polymer layers may therefore be covalently bound together by different types of crosslinks, where each type of crosslink comprises a different cyclic moiety.

The plurality of crosslinks may comprise a cleavable moiety adapted to undergo selective degradation under pre-determined conditions. Preferably, the cleavable moiety is selected from the group consisting of a disulfide, an ester, an amide a photocleavable link and bio-fragments such as proteins. The cleavable moiety may be provided by a crosslinking agent or alternatively, it may be provided by a linking group that is used to connect the functional group to the polymer material of a polymer layer. As discussed above, the linking group forms part of the crosslink once the functional groups of the adjacent polymer layers have reacted in a cycloaddition reaction.

YMouiseVeM~bonlm UmVSpednSO474TOS49lsped9 AU.doc 22 The cycloaddition reactions are preferably performed in the presence of a catalyst, which enhances the rate of the reaction. Preferably, the catalyst g comprises a metal selected from the group consisting of Au, Ag, Hg, Cd, Zr, Ru, Fe, Co, Pt, Pd, Ni, Cu, Rh and W. More preferably, the catalyst comprises a c 5 metal selected from the group consisting of Ru, Pt, Ni, Cu and Pd. Even more preferably, the catalyst comprises Cu(I). The presence of a catalyst however is o00 not essential, and the covalent reaction may be performed in the absence of a catalyst. The use of high temperature or pressure reaction conditions or Sirradiation such as by microwaves, may eliminate the need to use a catalyst.

010 c The multilayer polymer assembly thus formed in accordance with the process of the invention comprises at least two polymer layers. In its simplest form, the multilayer assembly comprises only two polymer layers. However, the person skilled in the art that would appreciate that the multilayer assembly may comprise any number of polymer layers. Theoretically, there is no upper limit to the number of polymer layers in the multilayer assembly, although for some practical purposes, the assembly may comprise from between two and ten polymer layers.

If it is desired for the multilayer polymer assembly to comprise more than two polymer layers, one or more further polymer layers may be subsequently deposited. Accordingly, the process of the invention may comprise a further step of depositing a polymer layer. The polymer layer may be deposited by any suitable process. In one embodiment, the polymer layer is deposited by a process selected from the group consisting of: depositing a polymer layer to form a pair of adjacent polymer layers, and forming a plurality of crosslinks between the pair of adjacent polymer layers, where each crosslink comprises a cyclic moiety formed by a cycloaddition reaction; and depositing a polymer layer, wherein said polymer layer is not crosslinked to the polymer layer it is deposited on. The further step of depositing a polymer layer may be repeated a plurality of times, depending on the number of polymer layers desired in the final assembly. In this regard, the person skilled in the art would understand that the process of the invention allows successive polymer layers to be deposited in the Y:.M.saVaetbou" UnASpede, ON47T80497_sed AU doc 23 construction of the multilayer polymer assembly using a layer by layer approach, 0 0 to form additional pairs of adjacent polymer layers.

;Z

In process the polymer layer is deposited on an existing polymer layer of the c 5 assembly to form a pair of adjacent polymer layers and crosslinked. The crosslinking may be performed prior to the deposition of a succeeding polymer 00oO oO layer. Alternatively, a plurality of further polymer layers may be firstly deposited, Sthen crosslinked.

0 10 In process the polymer layer is not crosslinked to the polymer layer it is c deposited on. In a preferred embodiment, the further polymer layer may be bound to the polymer layers adjacent to it by other interactions, such as electrostatic or hydrogen bonds.

Preferred processes and as described above for the further deposition of polymer layers may be performed in any order during the construction of the multilayer polymer assembly. Accordingly, the further deposition of the plurality of polymer layers may proceed by depositing the polymer layers in accordance with process followed by process or vice-versa. In addition, each of process and may be repeated a plurality of times in any order. In this manner, the multilayer polymer assembly may therefore comprise a combination of crosslinked and uncrosslinked polymer layers. Furthermore, either of process or process may be used alone to further deposit polymer layers, and each of process or process may be repeated a plurality of times.

The further deposition of a polymer layer may be achieved using any suitable technique. Preferably, the further deposition is achieved by contacting a substrate carrying the polymer layers with successive solutions comprising a polymer material, such as by dipping. After the further deposition of each polymer layer, the assembly thus formed may be typically incubated to allow the polymer layers to be adsorbed. This can be done for any suitable length of time but it is typically found that the solution is incubated from 15 minutes to 24 hours, more preferably from 2 hours to 20 hours, even more preferably from 4 hours to Y:V Wui 5\Mtl,6 USpkocO474TV605497 .spece AU.doc 24 12 hours, most preferably about 6 hours. During incubation, the solution may Salso be agitated to assist in the deposition of the polymer layer. In this manner, Sseveral polymer layers may be assembled together in a layer-by-layer approach.

The person skilled in the art would recognize that in theory any number of further C 5 polymer layers may be deposited, and that the total number of polymer layers may depend on the end use of the polymer assembly. In a one embodiment, 0 from two to twelve further polymer layers are deposited. The growth of the polymer layers in the multilayer polymer assembly, including the thickness of the clayers, may be monitored using any suitable technique. Examples of suitable 0 10 techniques include UV-vis and IR spectroscopy, ellipsometry and atomic force cN microscopy.

Any suitable polymer material may be used to prepare the polymer layers. The person skilled in the art would understand that the present invention is widely applicable to a range of polymer materials and that the choice of polymer material would depend on the intended end use application. Examples of suitable polymer materials include polymers, copolymers, polyelectrolyte polymers, polyethers such as polyethylene glycol, polyesters such as poly(acrylates) and poly(methacrylates), polyalcohols such as poly(vinyl alcohol), polyamides such as poly(acrylamides) and poly(methacrylamides), biocompatible polymers, biodegradable polymers, polypeptides, polynucleotides, polycarbohydrates and lipopolymers. In one embodiment, the same polymer material is used in each individual polymer layer. Alternatively, the polymer layers may comprise different polymer materials.

The polymer material may be dissolved in a suitable solvent to form a solution comprising the material. The solution may then used to form polymer layers in a LbL approach.

In a one embodiment, a polyelectrolyte material is used to prepare the polymer layers. The polyelectrolyte material may be comprise any suitable polyelectrolyte polymer, including but not limited to those selected from the group consisting of polyglycolic acid (PGA), polylactic acid (PLA), polyacrylic acid (PAA), polyamides, Y:VMulw elbm UrASpoaesVO4747 h 497_sped.e AU.mc poly-2-hydroxy butyrate (PHB), gelatins, B) polycaprolactone (PCL), poly 0 (lactic-co-glycolic acid) (PLGA), flourescently labelled polymers, conducting Spolymers, liquid crystal polymers, photoconducting polymers, photochromic Spolymers; poly(amino acids) including peptides and S-layer proteins; peptides, c 5 glycopeptides, peptidoglycans, glycosaminoglycans, glycolipids, lipopolysaccharides, proteins, glycoproteins, polypeptides, polycarbohydrates 00oo oo such as dextrans, alginates, amyloses, pectins, glycogens, and chitins; Spolynucleotides such as DNA, RNA and oligonucleotides; modified biopolymers Ssuch as carboxymethyl cellulose, carboxymethyl dextran and lignin sulfonates; polysilanes, polysilanols, poly phosphazenes, polysulfazenes, polysulfide and CN polyphosphate or a mixture thereof. Poly(acrylic acid) and poly(L-lysine) are particularly preferred polyelectrolyte materials. At least one polymer layer, and preferably, each polymer layer of the multilayer polymer assembly may comprise a polyelectrolyte material. In a one embodiment, each polymer layer comprises a polyelectrolyte material of the same charge or no charge.

In another embodiment, uncharged polymer materials are used in the polymer layers. Preferred uncharged polymer materials are those that are compatible with biological systems. Particularly preferred polymer materials are polyethers, such as poly(ethylene glycol).

The polymer material used to form the polymer layers may be of any suitable size or molecular weight. It is preferred that the material used in each polymer layer have a molecular weight of at least 100, and preferably a molecular weight of 100 to 1,000,000.

The multilayer polymer assembly prepared in accordance with the present invention may possess free functional groups. The free functional groups occur as not all the functional groups that are present in the polymer layers participate in cycloaddition reactions to form crosslinks between the polymer layers. The free functional groups may be used to modify the polymer assembly with other compounds or materials, such as polymers, biomacromolecules, or other functional compounds to thereby enhance the ability to use the polymer V UUutM.Webou Un1%Spesoe8D04747 RQ7O5.seC AUAdO 26 assemblies in a range of applications, by the use of click reactions. This may be Sachieved by further reacting at least one functional group of a polymer layer with Sa modifying compound that comprises a complementary 'click' functional group Sthat is adapted to undergo a cycloaddition reaction with the functional group of c 5 the polymer layer.

oo00 Thus in another embodiment, the process of the present invention further 00 Scomprises the step of modifying the multilayer polymer assembly by reacting at Sleast one functional group of the polymer assembly with a compound selected from the group consisting of antifouling agents, antimicrobials, chelating Scompounds, fluorescent compounds, antibodies, scavenging compounds, and physiologically active compounds. Such compounds would generally comprise a complementary functional group adapted to undergo a cycloaddition reaction with the functional group of the polymer layer. The compounds may modify the surface of the polymer assembly, or may infiltrate the layers of the polymer assembly and react with functional groups within the polymer assembly.

The ability to modify the multilayer polymer assembly with such compounds is useful given that click chemistry is a simple technique performed under mild conditions with high efficiency, making it broadly compatible with biological systems. This would permit biofunctionalization of the multilayer polymer assemblies for application in areas such as targeted drug delivery, biosensing and biocatalysis.

Core-Shell Particles When the multilayer polymer assembly of the invention is formed on a particulate template, such as a colloidal particle, as a substrate, a core-shell particle may be formed. In this instance, the particulate template assists to define the core while the shell is comprised of a multilayer polymer assembly.

Thus in another aspect of the invention there is provided process for the preparation of a core-shell particle comprising a core and a shell material, the process comprising the steps of providing a particulate template and (b) Y:V 'J4eWf UmSpeoe%8U47%5D5O49 5Pede AU Ooc 27 forming a shell material comprising a multilayer polymer assembly on the Sparticulate template, wherein the multilayer polymer assembly comprises: a Splurality of polymer layers, the polymer layers forming one or more pairs of Sadjacent polymer layers; and (ii) a plurality of crosslinks between at least one pair of adjacent polymer layers, and wherein each crosslink comprises a cyclic moiety formed by a cycloaddition reaction. The formation of the multilayer polymer oO0 assembly may be achieved by the process of the invention as described herein.

SThe particulate template used to prepare the core-shell particle may comprise any suitable material and be of any suitable form. Examples of particulate Stemplates include colloidal particles, nanoparticles, microspheres, crystals, and the like. A preferred particulate template is a colloidal particle. Examples of suitable materials include silicon, gold, quartz, polymeric materials and silica. A preferred material is silica. The particulate template may also be of any suitable size and form. It is most preferred that the particulate template produces a spherical or substantially spherical core-shell particle. It will be convenient to describe the invention in terms of a spherical material, but it shall be kept in mind that the core-shell particle produced by the process of the invention may be of any form, depending on the form of the particulate template used. Thus in general the final shape of the core-shell particles produced by the process of the invention will take the general shape or form of the particulate template used in their synthesis. Thus for example if the template is spherical then the final product will typically be spherical. In one aspect of the invention, the particulate template is a solid particle.

In another aspect of the invention, the particulate template is a porous particle.

The porous particle may be used to provide core-shell particles having an interconnected network of pores. The pores may take a number of different shapes and sizes however it is preferred that the porous particle is a mesoporous template. Mesoporous templates are templates in which there are at least some pores, preferably a majority of pores having a pore size in the range 2 to 50 nm.

The mesoporous template may be made of a number of suitable materials. In a Y: M iewMleboum UrSpeOsVRDM74?%64O7_spde~ AU doc 28 r- preferred embodiment, the mesoporous template is made of a material that Sallows for its subsequent removal, such as for example a mesoporous silica Smaterial. In general, the mesoporous silica material may have a bimodal pore structure, that is, having smaller pores of about 2-3 nm and larger pores from about 10-40 nm. The template may take any suitable form and may be for example in the form of powder particles or spheres. It is preferred that the oo00 template is spherical or substantially spherical. 0o Where a porous particle is used as a particulate template, porous core-shell materials may be formed. The pores are formed in the core-shell material as a result of the ability to construct the multilayer polymer assembly within the pores of the particulate template. It is a preferred embodiment of the invention that the porous particulate template be removal in order to form a hollow core-shell particle having a porous structure. The pores in the core-shell material may be of a wide variety of sizes however the material preferably includes pores with a pore size of from 5 to 50 nm, even more preferably 10 to 50 nm. In a particularly preferred embodiment the pores of the core-shell material are interconnecting to produce an interconnected porous network. It is an advantage of the invention that the porous core-shell particles of the invention are self-supporting in that the pores do not collapse under the weight of the polymer material after template removal.

In addition, where a porous particle is used, the exposed surface of the pores of a porous particulate template may be modified prior formation of the core-shell particle to enhance the interaction of the particulate template with the polymer layers of the multilayer polymer assembly in the shell material. An example of a suitable process to modify the pores of the porous particulate template is described in International patent application no PCT/AU2005/001511 (W02006/037160), the disclosures of which is herein incorporated by reference.

A skilled worker in the area will generally have little difficulty in choosing a functional moiety to introduce onto the pores of the colloidal particle to Y:VwseMlbo, UPR.Spmeaes%044780547_sgspece AU doc 29 complement the first polymer layer deposited onto the porous particulate template Sto form the shell material.

In each of the above aspects, the particulate template may be removable. The person skilled in the art would understand that the ability to remove the template 00oo would depend upon the nature of the template material. It is aspect of the 00o invention that the template is capable of being removed from the core of the coreshell material under conditions that do not disrupt the multilayer polymer assembly that constitutes the shell material. A range of removable particulate templates would be known the person skilled in the art. A preferred removal particulate template is a silica particle. The removal of the particulate template from the core gives rise to a core-shell material having a hollow core. The hollow core-shell particles may be regarded as nanoparticles or capsules. The removal of the particulate template may be carried out by any suitable method and the person skilled in the art would understand that the method would depend upon the nature of the particulate template and/or the shell material of the core-shell particle. Preferably, the removal of the particulate template is carried out by exposure to hydrofluoric acid. It is preferred that the hydrofluoric acid has a concentration of from 0.01 to 10 M, more preferably from 1 to 10 M, most preferably about 5 M.

In another aspect of the invention there is provided a core-shell particle comprising a core and a shell material, wherein the shell material comprises: a plurality of polymer layers, the polymer layers forming one or more pairs of adjacent polymer layers; and (ii) a plurality of crosslinks between at least one pair of adjacent polymer layers, wherein each crosslink comprises a cyclic moiety formed by a cycloaddition reaction.

The core may be a hollow core. In this instance, the core-shell particle may therefore be a hollow nanoparticle or capsule.

Y:IAboolbou UnNSpeoesffi747AM;0497_pede AU doc The core-shell particle may also be a porous particle. Preferably, the porous Sparticle is a nanoporous capsule comprising a hollow core.

SApplications C 5 The multilayer polymer assemblies of the invention are stable and robust systems that may be used in a variety of applications, including biomaterials, drug 00 00 delivery, chelating, targeting, anti-fouling, scavening and bio-sensing applications.

c Depending on the nature of the polymer material used to prepare the multilayer rassembly, a range of useful physical characteristics may be obtained. As an N example, multilayer polymer assemblies formed with poly(acrylic acid) (PAA) have been found to be stable over a wide pH range and in a range of organic solvents (ethanol, acetone and dimethylformamide).

The multilayer polymer assembly of the invention has also been found to be useful in the preparation of well-defined core-shell particles. The core-shell particle comprises a core and a shell material formed from the multilayer polymer assembly. In one preferred embodiment, the core-shell particle is a capsule that comprises a hollow core and a shell material comprising the multilayer polymer assembly of the invention.

Core-shell particles prepared with poly(acrylic acid) as a multilayer shell material have been found to exhibit pH-responsive behaviour. For example, as shown in Figure 9, incubation of PAA capsules in pH 10 and pH 2 solutions resulted in reversible swelling and shrinking of the capsules, respectively (Figure 9a and Figure 9b). The capsule diameter oscillated between about 5 and 8 pm in acidic and basic conditions, respectively (Figure 9c). The capsules were observed to deform when swollen under basic conditions (Figure 9b), but reverted to their original spherical shape when exposed to acidic conditions (Figure 9a). Without being limited by theory, it is believed that the swelling is due to ionization of the carboxylic acid groups of the PAA at higher pH, while the deformation may be explained by the cross-linking between the layers, which causes the capsules to resist greater swelling, leading to buckling/deformation. Such pH-responsive Y:lmnsumaV 'ub UrSpdesO84747=O6497_spede AU doc 31 behavior could be exploited to load and concentrate drugs inside the capsules.

SThe core-shell particles of the invention may therefore be used in a variety of Sdifferent applications, including for example, in drug delivery, as adsorbents and as micro-reactors.

N The stable and responsive properties afforded in multilayer polymer assemblies

O

oO of the invention together with ability to selectively post-functionalize the C assemblies enables the polymer assemblies to serve as a versatile platform for N designing advanced and responsive structures for use in a range of applications.

0 N EXAMPLES The present invention is described with reference to the following examples. It is to be understood that the examples are illustrative of and not limiting to the invention described herein.

Materials and Methods Materials High-purity (Milli-Q) water with a resistivity greater than 18 MQ cm was obtained from an in-line Millipore RiOs/Origin water purification system. Acrylic acid was purified by vacuum distillation and propargyl acrylate was purified by filtration through neutral alumina (70-230 mesh) immediately prior to use. Silica particles (diameter -5 pm) were obtained from Microparticles GmbH (Germany). All other chemicals were purchased from Sigma-Aldrich and used without further purification.

Quartz slides used as substrates were cleaned with Piranha solution (70/30 v/v% sulfuric acid: hydrogen peroxide). The slides were then sonicated with 50:50 (isopropanol water) for 15 min and finally heated to 60 "C for 20 min in RCA solution (5:1:1 water ammonia hydrogen peroxide). After each step the slides were washed thoroughly with Milli-Q water. Silicon wafers used for both ellipsometry and AFM were prepared using the above procedure without the Piranha treatment. Gold surfaces for IR measurement were cleaned by immersion in Piranha solution twice and then washed thoroughly with Milli-Q Y: UxDeiaoV.bou UnPSpwns%4747\8O0497_spede AkU.doc 32 water. All substrates were immersed in poly(ethyleneimine) (-25 KDa) with 0.5 M SNaCI for 20 minutes before assembly of the click functionalized polymers. The Ssubstrates were then washed with Milli-Q water and dried with a stream of i nitrogen. pH measurements were taken with a Mettler-Toledo MP220 pH meter, c- 5 and the pH values were adjusted with 0.1 M HCI and 0.1 M NaOH.

S3-chloropropan-l-ol (8.27 mL), triethylamine (17.57 mL) and hydroquinone (0.1 g) c were added to dichloromethane (50 mL) and stirred for 10 min. Acryloyl chloride 0 10 (9.53 mL) was then added drop-wise under argon at 0 The reaction was left to Ci stir at 0 "C for 60 min and then at room temperature overnight. The reaction was purified by washing with 100 mL water (twice), 0.5 M HCI, 100 mL water (twice) and then dried with magnesium sulfate (MgSO 4 The crude product was purified by rotary evaporation and then distilled. 2.23 mL (33.5% conversion) of clear liquid was produced. 1 H NMR (D 2 2.10 CH 2 3.59 CH 2 4.27 O-

CH

2 5.79 =CH 2 6.07 =CH) 6.35 =CH 2 ppm.

Synthesis of dodecyl 1-phenylethyl carbonotrithioate Dodecane thiol (4.8 carbon disulphide (3.6 triethylamine (4.8 and dichloromethane (15 mL) were added to a round-bottom flask and stirred overnight. 1-bromoethyl benzene (3.6 g) in a further 10 mL dichloromethane was added and then the reaction was stirred again overnight. The purity of the reaction was confirmed using TLC (diethyl ether:hexane The product was washed several times with water, brine and then dried over magnesium sulfate.

The product was rotary evaporated to produce 6.21 g (83.8% conversion) of yellow solid material. 1 H NMR (CDCI 3 0.85 CH 2

CH

3 1.23 CH2CH 2 1.36

CH

2

CH

3 1.65 CH 2

CH

2 1.72 CH3CH), 3.32 CH 2 5.30 (CH3CH), 7.29 benzylic CH).

Polymer synthesis.

Poly(acrylic acid) with azide functionality (PAA-Az) was synthesized with the following procedure: initial reactants were mixed at a 270:30:1 molar ratio of acrylic acid (0.932 3-chloropropyl acrylate (0.236 and RAFT agent (dodecyl Y: OuIseftume UnN aesn\O84747\805497-gedeA U.OC 33 r- 1-phenylethyl carbonotrithioate (0.018 10 wt Azobisisobutyronitrile (0.7 mg) 0 relative to the RAFT agent was also added. The solution was purged by bubbling g with nitrogen for 45 min and then polymerized at 60°C in a constant temperature Soil bath (2 The product was dialyzed for 24 h to remove excess monomer. The c 5 polymer was then stirred overnight with sodium azide at 60°C (0.29 The final product was then dialyzed again for 24 h and freeze dried. 1 H NMR (D 2 1.24oO 0o 1.84 CH 2 (polymer), 1.88-2.50 CH pendant CH 2

CH

2

CH

2 (polymer), 3.25-3.61 c pendant CH 2

N

3 (polymer), 3.84-4.18 pendant OCH 2 (polymer). The yellowish c polymer obtained had a molecular weight of 86000 (Mw) with a polydispersity of 2.21. This high polydispersity implies the RAFT agent used is not ideal for this N system, however good living characteristics are not required for this application.

Poly(acrylic acid) with alkyne functionality (PAA-Alk) was synthesized using the same procedure as above. However, the molar ratio used was 300:1 acrylic acid to RAFT agent. The polymer was heated at 60 °C in a constant temperature oil bath for 3 h. The polymer was stirred overnight with propargyl amine (0.10 molar equivalents) in the presence of 1-[3'-(dimethylamino)propyl]-3-ethylcarboimide methoiodide (0.15 molar equivalents). The product was dialyzed for 7 days and then freeze dried. 1 H NMR (D20): 0.96-1.78 CH 2 (polymer), 1.86-2.52 CH pendant alkyne CH (polymer), 3.62-3.87 pendant NHCH 2 (polymer). The yellowish polymer obtained had a molecular weight of 61000 (Mw) with a polydispersity of 1.52.

Characterization Methods.

UV Spectrophotometry. UV-visible spectra were collected from multilayer films assembled on quartz substrates using a Varian 4000 double-beam UV-visible spectrophotometer. An air blank was used for all measurements Ellipisometry: Measurements were performed on a UVISEL spectroscopic ellipsometer from Jobin Yvon. Spectroscopic data was acquired between 400 and 800 nm with a 2 nm increment, and thicknesses were extracted with the integrated software by fitting with a classical wavelength dispersion model.

Y:Ua0user bou e Un[SpeesW474Ti8M497_specO AU doc 34 Atomic Force Microscope: AFM images were acquired of air-dried multilayer films 0 on silicon wafers with a MFP-3D Asylum Research instrument. Typical scans Swere conducted in AC mode with ultrasharp SiN gold coated cantilevers (NT- MDT) over 5 Om 2 Multilayer thicknesses were determined by scratching the multilayer with a razor blade exposing the substrate, and measuring the step height difference.

00 00 IR Spectroscopy: Measurements were taken using a Varian 7000 FT-IR C( Spectrometer with a variable angle reflectance attachment. The incident angle for the measurements was 70 Films were deposited on reflecting substrates (glass cN slides coated with chromium (10 nm) and then gold (150 nm) using an Edwards Auto 306 thermal deposition chamber).

Gel Permeation Chromatography. SEC on a Shimadzu modular LC system comprising a DGU 12A solvent degasser, an LC-10AT pump, an SIL-10AD auto injector, an SIL-10A controller, an SPD-10AVP UV-Vis detector, an refractive index detector, a Polymer Lab aquagel-OH 50 x 7.5mm guard column and three 300 x 7.5mm aquagel-OH columns (30, 40, 50) with a 8pm particle size. The mobile phase/eluent used is made up of water (distilled H20 0.02% NaN3). The system was calibrated with sodium polystyrene sulphonate standards (4,600 -400,000).

X-ray Photoelectron Spectroscopy (XPS): XPS enabled characterization of the surface composition of the films. A KRATOS Analytical AXIS-HIS spectroscopic instrument with a monochromated Al Ka radiation source operated below 5 x 10 8 mbar with an analysis area of approximately 0.8 mm 2 was used to measure three locations per sample.

DIC and Fluorescence Microscopy. An inverted Olympus IX71 microscope equipped with a DIC slider (U-DICT, Olympus) with a 40x objective lens (Olympus UPLFL20 /0.5 W.D. 1.6) was used to view the core-shell and hollow particles. A CCD camera (Cool SNAP fx, Photometrics, Tucson, AZ) was mounted on the left hand port of the microscope. Transmission and DIC images YNZ s.WelbWm UrASpe wR4747\64g7_5POOee AU doc were illuminated with a tungsten lamp, and the fluorescence images were Silluminated with a Hg arc lap, using a UF1032 filter cube.

Flow Cytometry. Flow cytometry was performed on a Becton Dickinson FACS N 5 Calibur flow cytometer. 5 pL of the particle suspension was diluted in 250 pJL of 0.1 M HCI solution. Measurements were acquired by triggering on the forward oo oo scatter detection (EO detector) with a threshold of 400. Rhodamine fluorescence cN was monitored on the FL2 (570-610 nm) parameter with a PMT voltage of 600 V.

N Flow cytometry data analysis was performed with Summit v. 3.1 software (Cytomation Inc., Colorado, USA). The mean fluorescence intensity was obtained cN from the fluorescence intensity histograms.

Transmission Electron Microscopy: Air-dried hollow capsules were characterized with a Philips CM120 BioTWIN TEM operated at 120 kV.

Example 1 Poly(acrylic acid) with either azide (PAA-Az) or alkyne functionality (PAA-Alk) was synthesized using living radical polymerization in accordance with the procedure desribed above. NMR analysis showed that PAA-Az (Mw 86,000) and PAA-Alk (Mw 61,000) contained at least -10% of the respective functional groups for cross-linking. Infrared spectroscopy showed the characteristic azide peak at 2100 cm-1 for PAA-Az and a fingerprint weak alkyne peak at 2120 cm-1 for PAA-Alk, confirming functionalization of the polymers.

The azide and alkyne functionalised PAA polymers were assembled in a layer-bylayer approach with Cu(l) as a catalyst in accordance with the scheme shown in Figure 1.

LbL assembly was performed by sequentially exposing a quartz, silicon or gold substrate to PAA-Az and PAA-Alk solutions containing copper sulfate and sodium ascorbate for 20 min, with water rinsing after deposition of each layer. Dipping solutions were prepared from the following stock solutions: PAA-Az (0.83 mg mL-1), PAA-Alk (0.83 mg mL-1), MilliQ water (pH copper sulfate Y V\o,,woW.dbo UnfSpe8s0r474TrO5497_spee AU.OmC 36 (0.36 mg mL-1), and sodium ascorbate (0.88 mg mL-1). The pH of each solution was adjusted to pH 3.5 using 0.1 M HCI. Polymer dipping solutions were F made up in a constant volume ratio of 3 (a or The aqueous wash Ssolutions were made up in a similar ratio, however, using solution in place of c 5 or To prevent oxidation of the copper, new copper stock solutions were prepared after deposition of each PAA-Az/PAA-Alk bilayer.

00 00 oO CLbL assembly of the PAA-Az/PAA-Alk multilayers was first monitored by UV-vis C spectroscopy. As shown in Figure 2, linear growth of the film was observed by 0 10 monitoring the peak at 240 nm, which corresponds to the complex formation 0 N between copper and the PAA. A control system of PAA without click groups (PAA/PAA), used for comparison, showed a plateau in absorbance after only two bilayers, indicating that the click groups are essential for the deposition of consecutive PAA layers and the formation of PAA multilayers.

The prepared multilayer films were further characterized by reflection-absorption Fourier transform infrared spectroscopy (RAS-FTIR). The carboxylic acid peak at -1700 cm-1 from the PAA multilayers was used to monitor the film build-up on a gold surface (Figure The arrow shown in Figure 3 indicates increasing bilayer number (bottom to top: bilayers 1, 2, 3, 4 and The film absorbance was observed to increase regularly with bilayer number, in accordance with the UV-vis data (Figure 2).

The 5-bilayer film assembly was prepared in accordance with the above procedure was demonstrated to be stable to pH cycling (Figure 3 inset). The peak at 1700 cm-1 could be reversibly switched between protonated and deprotonated forms by immersion of the film in alternating solutions of pH 3.5 and (the peak height is given as zero as it disappears into the bulk spectra at basic pH and is too low to assign). The peak height at pH 3.5 remained essentially constant, indicating negligible polymer desorption during the cycling experiments. This result provides further evidence that the film is constructed using covalent interactions, as PAA films assembled using hydrogen bonding Y:tAmWebourne UnNSpeaes%8O4747=4O697_sp6de AU doc 37 interactions would disassemble under these basic conditions. The film stability is Sattributed to the triazole cross-links between the layers of polymer.

;1 Example 2 c 5 PAA-Az/PAA-Alk multilayer film assemblies of 4- and 8-bilayers were prepared on poly(ethyleneimine) (PEI)-coated silicon substrates in accordance with the 00oo oo method described in Example 1. The initial PAA-Az layer was adsorbed onto the c substrate using electrostatic interactions. The obtained multilayer films were then N air-dried.

0 N The thickness of air-dried PAA-Az/PAA-Alk multilayer films and 8-bilayers) was determined by spectroscopic ellipsometry. Film thicknesses of 25±6 nm and 38±4 nm were calculated for the 4- and 8-bilayer systems, respectively. Taking into account the thickness of the PAA-Az prelayers (4 nm), we obtain PAA- Az/PAA-Alk average bilayer thicknesses of approximately 4.6 nm, or a PAA layer thickness of about 2.3 nm.

The morphology of the air-dried click PAA multilayer films was examined by atomic force microscopy (AFM). The resulting images are shown in Figure 4.

Surface roughness over 5 x 5 lm2 was approximately 4 and 6 nm for the 4- and 8-bilayer films, respectively.

Thickness of the multilayer films was also determined by scratching the surface and measuring the step increment with the AFM. Thickness values (for films comprising the PEI/PAA-Az prelayers) consistent with those measured by ellipsometry were obtained: 22±4 nm and 43±7 nm for the 4- and 8-bilayer films, respectively.

Example 3 Poly(acrylic acid) containing -10% of either the alkyne (PAA-Alk) or azide (PAA- Az) functional groups was synthesized using living radical polymerizationin accordance with the procedure described above. To monitor multilayer growth Y: MC Wefstome UnrSpeaesBU4747M5497_speoe AU.doc 38 via fluorescence intensity changes, the PAA-Alk was also modified with an azide- 0 functionalized rhodamine dye (Rh-Az).

STo prepare PAA-Alk polymer modified with the Rh-Az functional compound, PAAc 5 Alk (40 mg) was added to a round bottom flask with copper sulfate (5.6 mg) and sodium ascorbate (12.8 mg) in 20 mL of water. A stock solution of azide- 3OO OO functionalized rhodamine dye (tetramethylrhodamine 5-carbonyl azide) was made c up of 0.1 mg of material dissolved in 1 mL dimethyl sulfoxide (DMSO). An aliquot N of 0.1 mL of the dye solution was then added to the round bottom flask and stirred for 16 h. The pink solution obtained was dialyzed for several days and ci then freeze-dried.

LbL assembly of the polymer materials on colloids was performed by sequentially exposing -5 um poly(ethyleneimine) (PEI)-coated silica particles to PAA-Alk and PAA-Az solutions (0.83 mg mL-1) containing copper sulfate (1.8 mg mL-1) and sodium ascorbate (4.4 mg mL-1) at pH 3.5. The particles were incubated for min in each PAA solution to deposit the polymer. The particles were then centrifuged and washed three times with water.

The growth of the PAA-Az/PAA-Alk click multilayers was monitored using flow cytometry. This approach is based on recording the fluorescence intensity of tens of thousands of individual particles after deposition of fluorescently labeled materials, comprising polymer layers. As seen in Figure 5 the increase in fluorescence intensity of the dye (Rh-Az)-functionalized PAA-Alk, and thus the mass of each PAA-Alk layer, was shown to be linear to at least a total of 12.

(PAA-Az/PAA-Alk) layers. This suggests linear growth of the click multilayers.

The relatively large fluorescence intensity observed for the first PAA-Alk layer (layer compared with subsequent layers, is attributed to electrostatic association of the first PAA layer with the PEI primer layer on the silica particle.

Fluorescence microscopy confirmed that the polymer multilayer coating on the particles was uniform.

Y: iasoWebibm UmSpedwsM47%80547_spede AU doc Example 4 O Core-shell particles comprising a multilayer polymer film as the shell material was Sprepared by LbL assembly on a colloid particle. The multilayer polymer assembly was prepared according to the following procedure: Approximately 200 pL of c 5 wt% silica particles and 1.5 mL of water were added to a 2 mL centrifuge tube.

The particles were first washed 3 times with water. The tube was agitated with a 00oo oo vortex mixer and then centrifuged at 1000 g for 1 min. This resulted in a pellet c forming at the bottom of the tube. Approximately 1.5 mL of the supernatant was

O

c removed and replaced with water. This was repeated twice prior to polyelectrolyte O 10 coating. After the last wash, approximately 1.5 mL of poly(ethyleneimine) (PEI) N solution (1 mg mL 1 0.5 M NaCI) was added to establish a PEI layer on the particles. This dispersion was allowed to incubate for 15 min, followed by 3 washing steps with Milli-Q water.

The following stock solutions were made: PAA-Az (0.83 mg PAA-Alk (0.83 mg mL- 1 copper sulfate (1.8 mg mL' 1 and sodium ascorbate (4.4 mg mL- 1 The pH of each solution was adjusted to 3.5 using 0.1 M HCI. The final PAA solutions for adsorption were made up in a constant volume ratio of 3 (a or To prevent oxidation of the copper, new copper stock solutions were prepared after deposition of each PAA-Az/PAA-Alk bilayer.

After pre-coating with PEI, 1.5 mL of PAA-Az solution (containing copper and ascorbate) were added and allowed to incubate for 15 min. After adsorption, the particles were washed 3 times with water (centrifugation speed of 100 g for 2 min to prevent particle aggregation). This was followed by adsorption of PAA-Alk, followed by the same washing steps with water. The process was repeated until the desired number of layers was deposited.

The (PAA-Az/PAA-Alk) 4 -coated silica particles were modifed with azidefunctionalized rhodamine dye (Rh-Az) according to the following procedure: pL of 0.1 mg mL of Rh-Az was diluted in 1.5 mL of Milli-Q water. 0.6 mL of this solution was mixed with 0.2 mL of copper sulfate (1.8 mg mL 1 and 0.2 mL (4.4 mg mL of sodium ascorbate solutions. This mixture is then added to the (PAA- Y:VUauiwMbome UMNSPOdOM SN747 97 9spe AU dm Az/PAA-Alk) 4 -coated silica particles and allowed to incubate for 30 min. Then the Sparticles were washed with pH 3.5 water three times and 50/50 v/v DMSO/water times to remove any unbound dye. The particle suspension was then dialyzed Sexhaustively against DMSO/water solution and washed another 10 times with t'- C 5 DMSO/water solution. As a control, rhodamine dye that has not been click functionalized was used in place of Rh-Az. In the adsorption solution, pH 0 other procedures were the same as above.

C 10 Both the Rh-Az and non-functionalized Rh (Rh) were used to demonstrate the N specificity of coupling of Rh-Az to the free Alk click groups in the multilayers. Rh showed some level of non-specific binding to the (PAA-Az/PAA-Alk)-coated particles (possibly due to the interaction of the Rh and PEI as the first layer on the particles). However, after the particles were subjected to multiple washing steps in both 50/50 v/v dimethyl sulfoxide (DMSO)/water solution (to remove unbound Rh) and acidic water (to remove copper) and finally extensive dialysis, the particles exposed to Rh-Az showed significantly higher fluorescence (a factor of than those exposed to unmodified Rh (Figure This indicates that the click-functionalized Rh-Az dye was specifically clicked onto the (PAA-Az/PAA- Alk) multilayers assembled on the particles.

Example The core-shell particles prepared in Example 4 were treated with ammonium fluoride-buffered hydrofluoric acid (HF) at pH 5 remove the silica particle core.

The silica core was dissolved by mixing 1 pL of the polymer-coated particle suspension with 1 pL of ammonium fluoride (8 M) buffered HF (2 M) at pH 5 and the capsules were visualized in situ. Dissolution of the silica core occurred after less than 1 min. The particles were imaged on an Olympus IX71 fluorescence microscope.

The resulting hollow spherical capsules were characterized with transmission electron microscopy (TEM) and atomic force microscopy (AFM). After drying, the Y:Uulw~sclMAbm UnFSD qO4aN74Th5497_specOO AU doc 41 capsules were observed to collapse and folds were visible from TEM and AFM Simages (Figure 7).

SAFM was used to determine the thickness of the capsule walls by taking a cross- N 5 sectional profile of the capsules where they folded only once and then halving the thickness. The wall thickness of the 12-layer (PAA-Az/PAA-Alk)6 capsules 00 oo (comprising a PEI primer layer from the substrate) was calculated to be 4.8 nm.

This corresponds to less than 0.4 nm per PAA layer.

The formation of capsules was also verified by using differential interference N contrast (DIC) microscopy. The results are shown in Figure 8. This technique distinguishes materials based on changes in refractive index instead of light absorption and, as such, capsules appear distinctly different to core-shell particles. This confirmed that the solid silica core was dissolved and that single component PAA capsules were prepared.

The effect of pH on the PAA capsules was investigated, pH-induced swelling and shrinkage of the capsules was performed by alternately adding 1 pL of HCI at pH 2 and 1 pL of 10 mM sodium carbonate (NaHCO 3 Na 2

CO

3 buffer solution at pH 10 directly to the click capsule solution on the microscope slide. The shrinkage or swelling of the capsules occurred within less than 1 min of adding the acidic or basic solution. The size of the capsules quoted is the average of about capsules.

The capsules were alternately incubated in pH 10 and pH 2 solutions, resulting in reversible swelling and shrinking of the capsules, respectively (Figure 9a and Figure 9b). The capsule diameter oscillated between about 5 and 8 pm in acidic and basic conditions, respectively (Figure 9c). The capsules were observed to deform when swollen under basic conditions (Figure 9b), but reverted to their original spherical shape when exposed to acidic conditions (Figure 9a). The swelling is attributed to ionization of the carboxylic acid groups at higher pH, while the deformation may be explained by the cross-linking between the layers, which Y:VUume~bowfl UnNSpeoes%8O4747%05497_spede AU doc 42 causes the capsules to resist greater swelling, leading to buckling/deformation.

0 Size measurements were performed only on the capsules that had not deformed.

Example 6 Poly(acrylic acid) containing -10% of either the alkyne (PAA-Alk) or azide (PAA- Az) functional groups was synthesized using living radical polymerisation.

oo 00 oo SAssembly of nanoporous silica spheres was performed by sequentially exposing c -7.5 im amine modified silica particles to PAA-Alk and PAA-Az solutions (0.83 0 10 mg mL-1) containing copper sulfate (1.8 mg mL-1) and sodium ascorbate (4.4 mg c mL-1) at pH 3.5. The particles were incubated overnight in each PAA solution to deposit the polymer. The particles were then centrifuged and washed three times with water.

Alternatively PAA-Alk was infiltrated into -7.5 (pm amine modified silica particles overnight and was then subsequently cross-linked with Bis-[b-(4- Azidosalicylamido)ethyl]disulfide (BASED) dissolved in ethanol (0.83 mg mL-1) containing copper sulfate (1.8 mg mL-1) and sodium ascorbate (4.4 mg mL-1).

It is understood that various other modifications and/or alterations may be made without departing from the spirit of the present invention as outlined herein.

vY:LoutsIterowne UnNSpeden\80477\8075O4g7_spe AU doc

Claims (44)

1. A multilayer polymer assembly comprising: a plurality of polymer layers, the polymer layers forming one or c 5 more adjacent polymer layers; and (ii) a plurality of crosslinks between at least one pair of adjacent 00 polymer layers, wherein each of the crosslinks comprise a cyclic moiety formed by a Scycloaddition reaction. S2. An assembly according to claim 1 wherein each polymer layer of the assembly is crosslinked via a plurality of crosslinks to each polymer layer adjacent to it.

3. An assembly according to claim 1 wherein at least one polymer layer of the assembly is not crosslinked to each polymer layer adjacent to it.

4. An assembly according to any one of claims 1 to 4 wherein the cyclic moiety is selected from the group consisting of tetrazoles, triazoles and oxazoles. An assembly according to claim 4 wherein the cyclic moiety is a 1,2,3- triazole.

6. An assembly according to any one of claims 1 to 5 wherein each crosslink comprises the same cyclic moiety.

7. An assembly according to any one of claims 1 to 6 wherein the crosslinks are formed by a cycloaddition reaction between complementary functional groups in the pair of adjacent polymer layers.

8. An assembly according to claim 7 wherein the complementary functional groups are independently selected from the group consisting of alkenes, Y:V odw"Ww UrAspedw%8D4747=54o7_spde AU doc 44 r- alkynes, azides, nitriles, nitrile oxides, cycloalkenes, heterocycloalkenes, O maleimide, anthracene and maleic anhydride. S9. An assembly according to claim 7 wherein the complementary functional groups are paired functional groups selected from the group consisting of alkyne-azide, alkyne-nitrile oxide, nitrile-azide and maleimide-anthracene. 00 00 An assembly according to any one of claims 1 to 6 wherein the crosslinks are formed by a cycloaddition reaction between functional groups in the pair of adjacent polymer layers and a crosslinking agent.

11. An assembly according to claim 10 wherein the pair of adjacent polymer layers and the crosslinking agent each comprise functional groups independently selected from the group consisting of alkenes, alkynes, azides, nitriles, nitrile oxides, cycloalkenes, heterocycloalkenes, maleimide, anthracene and maleic anhydride, and wherein the functional groups of the crosslinking agent are complementary with the functional groups of the adjacent polymer layers.

12. An assembly according to any one of claims 1 to 11 wherein the crosslinks further comprises a cleavable moiety adapted to undergo selective degradation under pre-determined conditions.

13. An assembly according to claim 12 wherein the cleavable moiety degrades under hydrolytic, thermal, enzymatic, proteolytic or photolytic conditions.

14. An assembly to claim 13 wherein the cleavable moiety is selected from the group consisting of a disulfide, an ester, an amide, a photocleavable link and bio-fragments. An assembly according to any one of claims 1 to 14 wherein each polymer layer comprises a polymer material independently selected from the group consisting of polymers, copolymer, polyelectrolyte polymers, polyethers, YA\Lois ebourn UnNSpedw%804747\5497&pede AU doc I polyesters, polyalcohols, polyamides, biocompatible polymers, biodegradable polymers, polypeptides, polynucleotides, polycarbohydrates Sand lipopolymers. c 5 16. An assembly according to any one of claims 1 to 15 wherein each polymer layer comprises a polyelectrolyte material. 00 00 C

17. An assembly according to any one of claims 1 to 15 wherein each polymer N layer comprises an uncharged polymer.

18. An assembly according to claim 17 wherein the uncharged polymer is a polyether.

19. An assembly according to claim 16 wherein each polymer layer comprises a polyelectrolyte material with the same charge or no charge. An assembly according to claim 16 wherein each polymer layer comprises a polyelectrolyte material independently selected from the group consisting of polyglycolic acid (PGA), polylactic acid (PLA), polyacrylic acid (PAA), polyamides, poly-2-hydroxy butyrate (PHB), gelatins, B) polycaprolactone (PCL), poly (lactic-co-glycolic acid) (PLGA), flourescently labelled polymers, conducting polymers, liquid crystal polymers, photoconducting polymers, photochromic polymers; poly(amino acids) including peptides and S-layer proteins; peptides, glycopeptides, peptidoglycans, glycosaminoglycans, glycolipids, lipopolysaccharides, proteins, glycoproteins, polypeptides, polycarbohydrates such as dextrans, alginates, amyloses, pectins, glycogens, and chitins; polynucleotides such as DNA, RNA and oligonucleotides; modified biopolymers such as carboxymethyl cellulose, carboxymethyl dextran and lignin sulfonates; polysilanes, polysilanols, poly phosphazenes, polysulfazenes, polysulfide and polyphosphate or a mixture thereof. YVlmulseebow, UnFSpaCeSW4747%80549eIde AU.tAdoe 46

21. An assembly according to any one of claims 1 to 20 wherein each polymer Slayer comprises the same polymer material.

22. A core-shell particle comprising a core and a shell material, wherein the shell material comprises: a plurality of polymer layers, the polymer layers forming one or more pairs of adjacent polymer layers; and (ii) a 00 plurality of crosslinks between at least one pair of adjacent polymer layers, wherein each crosslink comprises a cyclic moiety formed by a cycloaddition reaction.

23. A core-shell particle according to claim 22 which is a capsule.

24. A process for the preparation of a multilayer polymer assembly comprising: providing a polymer layer; (ii) depositing a further polymer layer to form a pair of adjacent polymer layers; and (iii) forming a plurality of crosslinks between the adjacent polymer layers, wherein each crosslink comprises a cyclic moiety formed by a cycloaddition reaction. A process according to claim 24 further comprising the step of: (iv) depositing a polymer layer by a process selected from the group consisting of: depositing a polymer to form a pair of adjacent polymer layers, and forming a plurality of crosslinks between the pair of adjacent polymer layers, wherein each crosslink comprises a cyclic moiety from by a cycloaddition reaction; and depositing a polymer layer, wherein said polymer layer is not subsequently crosslinked to the polymer layer it is deposited on.

26. A process according to claim 25 wherein step (iv) is repeated a plurality of times. Y:UxViw lbo e UnPSipeoesW874786497_speds e AU do

27. A process according to claim 26 wherein in repeating step (iv) process (a) O is repeated.

28. A process according to claim 26 wherein in repeating step (iv) both N 5 process and are repeated. o0 29. A process according to any one of claims 24 to 28 wherein the cyclic Smoiety is selected from the group consisting of tetrazoles, triazoles and oxazoles.

30. A process according to claim 29 wherein the cyclic moiety is a 1,2,3- triazole.

31. A process according to any one of claims 24 to 30 wherein each crosslink comprises the same cyclic moiety.

32. A process according to any one of claims 24 to 31 wherein the crosslinks are formed by a cycloaddition reaction between complementary functional groups in the adjacent polymer layers.

33. A process according to claim 32 wherein the complementary functional groups are independently selected from the group consisting of alkenes, alkynes, azides, nitriles, nitrile oxides, cycloalkenes, heterocycloalkenes, maleimide, anthracene and maleic anhydride.

34. A process according to claim 32 wherein the complementary functional groups are paired functional groups selected from the group consisting of alkyne-azide, alkyne-nitrile oxide, nitrile-azide and maleimide-anthracene.

35. A process according to any one of claims 24 to 31 wherein the crosslinks are formed by a cycloaddition reaction between functional groups in the adjacent polymer layers and a crosslinking agent. Y:soWelboe UnNSOOeOesMBOW74T%8S497_sp~d AU.doc 48

36. A process according to claim 35 wherein the adjacent polymer layers and O the crosslinking agent each comprise functional groups independently Sselected from the group consisting of alkenes, alkynes, azides, nitriles, Snitrile oxides, cycloalkenes, heterocycloalkenes, maleimide, anthracene and maleic anhydride, and wherein the functional groups of the crosslinking agent are complementary with the functional groups of the 00 adjacent polymer layers.

37. A process according to any one of claims claim 24 to 36 wherein the crosslink further comprises a cleavable moiety adapted to undergo ci selective degradation under pre-determined conditions.

38. A process according to claim 37 wherein the cleavable moiety degrades under hydrolytic, thermal, enzymatic, proteolytic or photolytic conditions.

39. A process according to claim 38 wherein the cleavable moiety is selected from the group consisting of a disulfide, an ester, an amide, a photocleavable link or a bio-fragment.

40. A process according to any one of claims 24 to 39 wherein the cycloaddition reaction is catalysed by a metal catalyst.

41. A process according to claim 40 wherein the catalyst comprises a metal selected from the group consisting of Au, Ag, Hg, Cd, Zr, Ru, Fe, Co, Pt, Pd, Ni, Cu, Rh and W.

42. A process according to claim 40 wherein the catalyst comprises Cu(l).

43. A process according to any one of claims 24 to 42 wherein the polymer layer of step is provided on a substrate. YAV -WeD o- UrSp. 0=4747'05407_sped AU dmc 49

44. A process according to claim 43 wherein the substrate is selected from the O group consisting of silicon, gold, quartz, a polymeric material, silica, a Smicelle, an emulsion droplet and surfaces having a phase interface.

45. A process according to claim 43 wherein the substrate is a porous particle. 00 oO 46. A process according to claim 43 wherein the substrate is a particulate oO template and wherein the polymer layers are formed around the template.

47. A process according to any one of claims 43 to 46 wherein the substrate is C( removable.

48. A process according to any one of claims 24 to 47 wherein each polymer layer comprises a material independently selected from the group consisting of polymers, polyelectrolyte polymers, polyethers, polyesters, polyalcohols, polyamides, biocompatible polymers, biodegradable polymers, polypeptides, polynucleotides, polycarbohydrates and lipopolymers.

49. A process according to claim 48 wherein each polymer layer comprises a polyelectrolyte material. A process according to claim 48 wherein each polymer layer comprises an uncharged polymer.

51. A process according to claim 50 wherein the uncharged polymer is a polyether.

52. A process according to claim 49 wherein each polymer layer comprises a polyelectrolyte material with the same or no charge. Y:V MLo4.Awourne UnRSpeaiesWOD4747=497_jpede AU.doc

53. A process according to claim 49 wherein each polymer layer comprises a Spolyelectrolyte material independently selected from the group consisting Sof polyglycolic acid (PGA), polylactic acid (PLA), polyacrylic acid (PAA), Spolyamides, poly-2-hydroxy butyrate (PHB), gelatins, B) polycaprolactone (PCL), poly (lactic-co-glycolic acid) (PLGA), flourescently labelled polymers, conducting polymers, liquid crystal polymers, 00 photoconducting polymers, photochromic polymers; poly(amino acids) including peptides and S-layer proteins; peptides, glycopeptides, Speptidoglycans, glycosaminoglycans, glycolipids, lipopolysaccharides, 0 10 proteins, glycoproteins, polypeptides, polycarbohydrates such as dextrans, CN alginates, amyloses, pectins, glycogens, and chitins; polynucleotides such as DNA, RNA and oligonucleotides; modified biopolymers such as carboxymethyl cellulose, carboxymethyl dextran and lignin sulfonates; polysilanes, polysilanols, poly phosphazenes, polysulfazenes, polysulfide and polyphosphate or a mixture thereof.

54. A process according to any one of claims 24 to 53 wherein the polymer layers each comprises the same polymer material.

55. A process according to any one of claims 24 to 54 further comprising the step of modifying the multilayer polymer assembly by reacting at least one functional group of a polymer layer with a compound selected from the group consisting of antifouling agents, antimicrobials, chelating compounds, fluorescent compounds, antibodies, scavenging compounds, and physiologically active compounds. Y:UutueVeslbousn UnRVSpoa U747N805497_spede AU .doc