WO2010087781A1

WO2010087781A1 - Polyelectrolyte complexes with bound peptides

Info

Publication number: WO2010087781A1
Application number: PCT/SG2010/000026
Authority: WO
Inventors: Andrew Chwee Aun Wan; Jackie Y. Ying; Sau Yin Chin
Original assignee: Agency For Science, Technology And Research
Priority date: 2009-01-28
Filing date: 2010-01-28
Publication date: 2010-08-05

Abstract

A process is described for producing a loaded complex comprising a polyelectrolyte complex bound to a biologically active peptide. In this process, a solution of a polyanion is combined with a solution of a polycation to form the polyelectrolyte complex. The initially formed polyelectrolyte complex is then exposed to the peptide so as to bind the peptide to the initially formed polyelectrolyte complex and form the loaded complex. The process is such that the peptide retains its biological activity while bound to the polyelectrolyte complex.

Description

Polyeiectrolyte complexes with bound peptides

Technical Field

The present invention relates to polyeiectrolyte complexes with peptides bound thereto, and to processes for making said complexes.

Background of the Invention

The development of biomaterials for clinical application has often involved an empirical, trial-and-error approach. The rationale for selecting biomaterials is frequently based on general considerations, such as (i) inherent biocompatibility of naturally derived materials (e.g. ECM components such as collagen and hyaluronic acid, and biopolymers such as alginic acid and chitosan), and (ii) biodegradability and manufacturability of synthetic polymers (e.g. poly (L-lactic-co-glycolic acid) (PLGA) and polycaprolactone (PCL)). In addition, bioactivity can be conferred by incorporating growth factors that are covalently bound or in a controlled release form (e.g. microspheres).

The chemistry for functionalization of a polyeiectrolyte complex (PEC) membrane using a 3-layered configuration has previously been developed (Wan A.C.. Tai B.C.. Schumacher K.M., Schumacher A., Chin S. Y.. Ying J. Y., Polyeiectrolyte complex membranes for specific cell adhesion, Langmuir. 24(6) (2008), 2611-2617). This strategy is related to the layer-by-layer (LBL) electrostatic self-assembly of polyelectrolytes, as popularized by Decher (Decher G., Fuzzy nanoassemblies: toward layered polymeric multicomposites. Science, 277 (5330) (1997), 1232-1237) and others. The disadvantages of the multilayer approach are the number of steps needed to achieve an appreciable coating thickness and the loss of polyeiectrolyte at each layering step. These factors would impede the commercial use of the LBL process for the coating of biomedical implants.

There is therefore a need for a more efficient method for preparing biomaterials for binding and optionally sustained release of biological molecules such as peptides.

Object of the Invention

It is the object of the present invention to substantially overcome or at least ameliorate one or more of the above disadvantages.

Summary of the Invention

In a first aspect of the invention there is provided a process for producing a loaded complex comprising a polyeiectrolyte complex bound to a biologically active peptide, said process comprising: (a) combining a solution of a polyanion with a solution of a polycation to form the polyelectrolyte complex, and

(b) exposing the polyelectrolyte complex produced in step (a) to the peptide so as to bind said peptide to said polyelectrolyte complex and form the loaded complex.

The process may be such that the peptide retains its biological activity while bound to the polyelectrolyte complex. Step (b) may be conducted under conditions such that the peptide retains its biological activity while bound to the polyelectrolyte complex. It may be conducted under conditions that do not denature the peptide. It may be conducted under conditions of temperature and pH that do not denature the peptide. It may be conducted in the absence of chemical compounds that denature the peptide. It may be conducted under conditions of temperature and pH that do not denature the peptide and in the absence of chemical compounds that denature the peptide.

The following options may be used hi conjunction with the first aspect, either individually or hi any suitable combination.

Step (a) may be as described hi the thirteenth aspect (see below).

One or both of the polyanion and the polycation may comprise a functional group capable of binding the peptide. The polyanion may comprise a functional group capable of binding the peptide. The polycation may comprise a functional group capable of binding the peptide. The functional group may be a sulfate group. It may be a carboxyl group. It may be an acetamido group, hi some embodiments one or both of the polyanion and the polycation comprises more than one (e.g. 2, 3, 4 or 5) functional groups capable of binding the peptide. In some embodiments a combination of functional groups (e.g. sulfates, acetamido groups, carboxyl groups) on the polycation and on the polyanion may be capable of binding the peptide.

The process may comprise the step of allowing the polyelectrolyte complex to form particles. The so formed particles may be (and may optionally remain stably) hi suspension hi the combined solvents of the two solutions. They may settle or precipitate from the combined solvents. Step (b) may be conducted after formation of the particles of the polyelectrolyte complex.

The polyanion may comprise a polysaccharide. It may comprise a chitosan derivative. It may comprise a mixture of at least two polyanionic components. It may for example comprise chitosan sulfate, carboxymethylchitin or a mixture of these. It may be water soluble. The polycation may comprise a cationic polysaccharide. It may be, or may comprise, chitosan or a partially hydrolysed or deacetylated chitin. It may be water soluble. It may be water soluble chitin. It may be, or comprise, chitin which has been deacetylated about 30 to about 70%, e.g. about 50%.

The process ma}' comprise crosslinking the polycation prior to step (a). In some embodiments the polycation comprises water soluble chitin and the step of crosslinking comprises exposing said chitin to a crosslinker. Suitable crosslinkers include genipin, formaldehyde, diisocyanates and glutaraldehyde.

The solution of the polyanion and the solution of the polycation may each independently, or both, be aqueous solutions.

At least one, optionally both, of the solution of the polyanion and the solution of the polycation may comprise a dissolved inorganic salt. Each independently, or both, may comprise PBS or a concentrated or diluted form thereof. The concentration of the dissolved organic salt (or of more than one dissolved organic salt, e.g. of the PBS. if used) in the solution of the polyanion or in the solution of the polycation or in both of these may be, or may be controlled, so as to obtain a desired size of said particles.

The process may comprise at least partially drying the polyelectrolyte complex so as to form a film of the polyelectrolyte complex. Step (b) may be conducted after forming the film of the polyelectrolyte complex.

The peptide may be, or may comprise, a growth factor, a cytokine, an anticoagulant protein, a hormone, a cell adhesion peptide, a cell targeting peptide or some other type of protein. It may be angiotensin III, a peptide of the fibroblast growth factor family, a peptide of the vascular endothelial growth factor family or a peptide of the platelet derived growth factor family.

The polyanion and the polycation may be such that the loaded complex is capable of releasing the peptide into an aqueous solution. They may be such that the loaded complex is capable of releasing the peptide into an aqueous solution in a sustained manner. It may be released in an approximately linear manner over time. It may be released over a period of greater than about 1 day, or over a period of about 1 day to about 1 year. In some embodiments the polyanion and the polycation are such that the loaded complex does not substantially release the peptide into the aqueous solution. The biological activity of the peptide in such loaded complexes is retained. For example a loaded complex comprising an anticoagulant or cell adhesion peptide may have the polyanion and polycation selected such that the anticoagulant or cell adhesion peptide does not substantially release (i.e. remains substantially bound to the polyelectrolyte complex) when said loaded complex is implanted in vivo. In this event, the loaded complex may still retain the anticoagulant or cell adhesion property of the bound peptide. In the event that the loaded complex is capable of releasing the peptide into an aqueous solution, the polyanion, the polycation and the conditions used in forming the loaded complex may all be such that the peptide retains its biological activity following its release into the aqueous solution.

The process may be such that the peptide retains its biological activity while bound to the polyelectrolyte complex. The polyanion and the polycation may be such that the peptide retains its biological activity while bound to the polyelectrolyte complex. The conditions used in the process may be such that the peptide retains its biological activity while bound to the polyelectrolyte complex, hi particular they may be such that they do not damage, denature or deactivate the peptide. In particular the process may be conducted at a temperature below the denaturation temperature of the peptide. It may be conducted at about ambient temperature. It may for example be conducted at about 10 to about 3O⁰C, or about 10 to 20, 20 to 30 or 15 to 25⁰C, e.g. about 10, 15. 20, 25 or 3O⁰C.

The process may produce the loaded complex of the second aspect (see below). The invention also encompasses a loaded complex made by the process of the first aspect.

In an embodiment there is provided a process for producing a loaded complex comprising a polyelectrolyte complex bound to a biologically active peptide, said process comprising:

(a) combining a solution of a polysaccharide polyanion with a solution of a polysaccharide polycation to form the polyelectrolyte complex; and

(b) exposing the polyelectrolyte complex produced in step (a) to the peptide so as to bind said peptide to said polyelectrolyte complex and form the loaded complex; such that the peptide retains its biological activity while bound to the polyelectrolyte complex.

In another embodiment there is provided a process for producing a loaded complex comprising a polyelectrolyte complex bound to a biologically active peptide, said process comprising:

(a) combining a solution of a polysaccharide polyanion with a solution of a polysaccharide polycation to form the polyelectrolyte complex;

(a') at least partially drying the polyelectrolyte complex so as to form a film of the polyelectrolyte complex; and (b) exposing the film to the peptide so as to bind said peptide to said polyelectrolyte complex and form the loaded complex in the form of a film; such that the peptide retains its biological activity while bound to the polyelectrolyte complex.

In a second aspect of the invention there is provided a loaded complex comprising:

• a polyelectrolyte complex comprising a polycation complexed with a polyanion; and

• a biologically active peptide bound to said polyelectrolyte complex; wherein the peptide retains its biological activity while bound to the polyelectrolyte complex.

The following options may be used in conjunction with the second aspect, either individually or in any suitable combination.

The peptide may be a growth factor, a cytokine, an anticoagulant protein, a hormone, a cell adhesion peptide, a cell targeting peptide or a mixture of any two or more of these, or may be, or comprise, some other type of protein.

The polyanion may be, or may comprise, an anionic polysaccharide. The polycation may be, or may comprise, a cationic polysaccharide. The polycation may be crosslinked. It may be crosslinked by a species other than the polyanion. The polyanion may be, or may comprise, chitosan sulfate, carboxymethylchitin or a mixture of these. The polycation may be, or may comprise water soluble chitin or partially hydrolysed or deacetylated chitin, and optionally crosslinked, e.g. by genipin.

The polyanion and the polycation may be such that the peptide is releasable from said loaded complex. They may be such that the peptide retains its biological activity following release from the loaded complex.

The polyelectrolyte complex may be as described in the twelfth aspect (see below).

The loaded complex may be in the form of particles. The particles may have a mean diameter of less than about 5 microns.

The loaded complex may be in the form of a film.

The loaded complex may be made by the process of the first aspect.

In an embodiment there is provided a loaded complex in the form of a film, said complex comprising:

• a polyelectrolyte complex comprising a cationic polysaccharide complexed with an anionic polysaccharide; and

In another embodiment there is provided a loaded complex in the form of a particles having a mean diameter of less than about 5 microns, said complex comprising:

In another embodiment there is provided a loaded complex, said complex comprising:

• a polyelectrolyte complex comprising a cationic polysaccharide crosslinked with genipin and complexed with an anionic polysaccharide; and

• a biologically active peptide bound to said polyelectrolyte complex; wherein the peptide retains its biological activity while bound to the polyelectrolyte complex. hi a third aspect of the invention there is provided a device suitable for implantation into a living subject, said device comprising a loaded complex according to the second aspect or made by the process of the first aspect. The loaded complex may be such that it is not in the form of particles. The peptide of the loaded complex may be an anticoagulant peptide. hi a fourth aspect of the invention there is provided a process for making a device suitable for implantation into a living subject, said process comprising forming a loaded complex according to the second aspect or made by the process of the first aspect. The forming may comprise moulding, compressing, evaporating a solution or suspension of the loaded complex or some other forming process. hi a fifth aspect of the invention there is provided a process for making a device suitable for implantation into a living subject, said process comprising at least partially coating a device to be implanted into a living subject with a loaded complex according to the second aspect or made by the process of the first aspect, said complex being in the form of a film.

In a sixth aspect of the invention there is provided a method of delivering a biologically active peptide to a subject, said method comprising introducing a loaded complex according to the second aspect or made by the process of the first aspect into said subject, wherein the loaded complex comprises said peptide bound to a polyelectrolyte complex. The peptide may be releasably bound to the polyelectrolyte complex. This method may represent a method of treating a condition in a patient. In this case the condition is one for which the peptide is indicated.

In a seventh aspect of the invention there is provided a method of delivering a biologically active peptide to a liquid, said method comprising introducing a loaded complex according to the second aspect or made by the process of the first aspect into said liquid, wherein the loaded complex comprises said peptide bound to a polyelectrolyte complex. The peptide may be releasably bound to the polyelectrolyte complex. The liquid may be a biological liquid. It may be an aqueous liquid. It may be a non-biological liquid. The method may be for treatment of a condition in a patient. The patient may be a human patient, or may be a non-human patient, e.g. a non-human mammal or non-human primate. The method may be a purpose other than treatment or diagnosis of a condition hi a human, or for a purpose other than treatment or diagnosis of a condition in a human or animal.

In an eighth aspect of the invention there is provided use of a loaded complex according to the second aspect or made by the process of the first aspect for delivering a biologically active peptide to a subject. The peptide may be releasably bound to the polyelectrolyte complex.

In a ninth aspect of the invention there is provided use of a loaded complex according to the second aspect or made by the process of the first aspect for delivering a biologically active peptide to a liquid. The peptide may be releasably bound to the polyelectrolyte complex.

In a tenth aspect of the invention there is provided use of a loaded complex according to the second aspect or made by the process of the first aspect for the manufacture of a medicament for treatment of a condition in a subject, wherein the biologically active peptide is indicated for said condition and wherein the loaded complex is not in the form of a film. The peptide may be releasably bound to the polyelectrolyte complex.

In an eleventh aspect of the invention there is provided a medicament for treatment of a condition in a subject, said medicament comprising a loaded complex according to the second aspect or made by the process of the first aspect and a pharmaceutically acceptable carrier, wherein the biologically active peptide is indicated for said condition and wherein the loaded complex is not in the form of a film. The peptide may be releasably bound to the polyelectrolyte complex.

In a twelfth aspect of the invention there is provided a polyelectrolyte complex capable of binding a target peptide, said complex comprising a polyanion complexed with a polycation wherein one or both of the polyanion and the polycation comprises a functional group capable of binding the target peptide or wherein a combination of functional groups on the polyanion and the polycation is capable of binding said target peptide. Otherwise stated, this aspect provides a polyelectrolyte complex capable of binding a target peptide, said complex comprising a polyanion complexed with a polycation wherein one or more functional groups on the polyanion and/or on the polycation are either individually or in combination capable of binding said target peptide.

The following options may be used in conjunction with the twelfth aspect either individually or in any suitable combination.

The polyanion and the polycation may each, independently, be a polysaccharide. They may each, independently, be a chitin derivative or a chitosan derivative.

The target peptide may be biologically active. It may be angiotensin III, a peptide of the fibroblast growth factor family, a peptide of the vascular endothelial growth factor family or a peptide of the platelet derived growth factor family. It may be a growth factor, a cytokine, an anticoagulant protein, a hormone, a cell adhesion peptide or a cell targeting peptide.

The functional group(s) (i.e. the chemical nature thereof) will depend on the nature of the target peptide. It may be for example sulfate, acetamido or carboxyl. There may be more than one functional group capable of binding the peptide, e.g. 2, 3, 4 or 5 such functional groups. There may be more than one functional group, e.g. 2, 3, 4 or 5, which, in combination, are capable of binding the peptide.

The polyanion may comprise a single polyanionic component. It may comprise more than one, e.g. 2, 3 or 4, polyanionic components. Each, independently, may comprise the functional group(s) capable of binding the peptide. At least one of the polyanionic components may comprise a second functional group different to the previously mentioned functional group and capable of binding the peptide. Each polyanionic component, independently, may be a polysaccharide. They may each, independently, be a chitin derivative or a chitosan derivative. Thus for example the polyanion may comprise a first polyanionic component comprising a polysaccharide comprising sulfate groups and a second polyanionic component comprising a polysaccharide comprising carboxyl groups. In some instances the first polyanionic component may additionally comprise carboxyl groups and/or other groups capable of binding the peptide and/or the second polyanionic component may additionally comprise sulfate groups and/or other groups capable of binding the peptide.

The polycation may comprise a partially deacetylated chitin. It may comprise a water soluble partially deacetylated chitin.

The polyelectrolyte complex may be in the form of particles. It may be in the form of a film or layer. It may be in some other form. The dimensions of the polyelectrolyte complex may be substantially the same as for the loaded complex, as described elsewhere herein. For example the particle diameter (in the event that the complex is in the form of particles) may be less than about 5 microns, or about 50 to about 5000nm.

In an embodiment there is provided a polyelectrolyte complex capable of binding a target peptide, said peptide being selected from the group consisting of angiotensin III, peptides of the fibroblast growth factor family, peptides of the vascular endothelial growth factor family and peptides of the platelet derived growth factor family, said complex comprising:

• a polycation comprising a water soluble partially deacetylated chitin; and

• a polyanion complexed with the polycation and comprising a polysaccharide (optionally a chitin derivative) comprising functional groups capable of binding to said target peptide.

In another embodiment there is provided a polyelectrolyte complex capable of binding a target peptide, said peptide being selected from the group consisting of angiotensin III, peptides of the fibroblast growth factor family, peptides of the vascular endothelial growth factor family and peptides of the platelet derived growth factor family, said complex comprising:

• a polycation comprising a a water soluble partially deacetylated chitin; and

• a polyanion complexed with the polycation and comprising a first polysaccharide {optionally a chitin derivative) comprising sulfate groups and a second polysaccharide (optionally a chitin derivative) comprising carboxyl groups.

The polyelectrolyte complex of the twelfth aspect may represent the complex made in step (a) of the first aspect. It may be the polyelectrolyte complex of the second aspect. It may be made by the process of the thirteenth aspect. In a thirteenth aspect of the invention there is provided a process for making a polyelectrolyte complex capable of binding a target peptide, said process comprising combining a solution of a polyanion with a solution of a polycation so as to form the polyelectrolyte complex, wherein one or both of the polyanion and the polycation comprises a functional group capable of binding the target peptide.

The combining may result in complexation of the polyanion and the polycation so as to form the polyelectrolyte complex. The polyanion, polycation and peptide may be as described elsewhere herein, in particular above in the twelfth aspect. The polyanion and polycation may comprise functional groups which, following formation of the polyelectrolyte complex, are capable in combination of binding the target peptide. The process of the thirteenth aspect may represent step (a) of the first aspect. The options provided in the first aspect that relate to step (a) of that aspect apply equally to the thirteenth aspect. In particular, the process may comprise allowing the resulting polyelectrolyte to form particles. The polyelectrolyte complex (commonly in the form of particles) may be at least partially dried so as to form a film of the polyelectrolyte complex. The process may make the polyelectrolyte complex of the twelfth aspect.

In an embodiment there is provided a process for making a polyelectrolyte complex capable of binding a target peptide, said peptide being selected from the group consisting of angiotensin III, peptides of the fibroblast growth factor family, peptides of the vascular endothelial growth factor family and peptides of the platelet derived growth factor family, said process comprising combining:

• an aqueous solution of a polycation comprising a water soluble partially deacetylated chitin; and

• an aqueous solution of a polyanion comprising a first polysaccharide (optionally a chitin derivative) comprising sulfate groups and a second polysaccharide (optionally a chitin derivative) comprising carboxyl groups; so as to form the polyelectrolyte complex.

In another embodiment there is provided a process for making a polyelectrolyte complex capable of binding a target peptide, said peptide being selected from the group consisting of angiotensin III, peptides of the fibroblast growth factor family, peptides of the vascular endothelial growth factor family and peptides of the platelet derived growth factor family, said process comprising:

• combining an aqueous solution of a polycation comprising a water soluble partially deacetylated chitin; and an aqueous solution of a polyanion comprising a first polysaccharide (optionally a chitin derivative) comprising sulfate groups and a second polysaccharide (optionally a chitin derivative) comprising carboxyl groups; and

• allowing the resulting polyelectrolyte complex to form particles of the polyelectrolyte complex.

• at least partially drying the resulting polyelectrolyte complex so as to form a film of the polyelectrolyte complex.

Brief Description of the Drawings

A preferred embodiment of the present invention will now be described, by way of an example only, with reference to the accompanying drawings wherein: Figure 1 shows FTIR spectra of (A) chitin, (B) WSC, and (C) CMC. The peaks (1732 and 1558 cm^"1) may be used to estimate the degree of carboxymethylation for CMC are indicated by arrows. Degree of carboxymethylation is proportional to A₁₇₃₂ZA_15Sg. Figure IA shows a calibration curve for determining degree of carboxymethyl substitution for CMC, constructed by addition of monochloroacetic acid to chitin. Figure 2 shows FTIR spectra of (A) chitosan and (B) CS. The main peaks associated with sulfation are indicated by arrows.

Figure 3 shows particle size of WSC-CS complexes as a function of PBS concentration used in their synthesis. WSC (0.5 mg/mL) was added to CS (1 mg/mJL) at a volume ratio of l:l.

Figure 4 shows anti-coagulant activities of WSC, CMC, CS, PECl = WSC (0.5 mg/mL) + CS (1 mg/mL), PEC2 = WSC (0.5 mg/mL) + CS (1 mg/mL) + CMC (0.25 mg/mL), and PEC3 = WSC (0.5 mg/mL) + CS (1 mg/mL) + CMC (0.5 mg/mL). Figure 5 shows (A) chemical structure of WSC containing acetamido groups that are not present in CS, but present in HS (X and Y are variable functional groups); (B) the structure of the oligosaccharide identified as the domain for anti-coagulant activity of heparin.

Figure 6 shows anti-coagulant activity of various PECs. The concentrations of WSC (W), CMC (C) and CS (S) are noted in parenthesis in mg/mL.

Figure 7 shows a QCM trace showing the areal masses (solid lines) of WSC, CS, CMC and FGF-2 sequentially deposited on the quartz crystal, as calculated by Sauerbrey's Equation from the corresponding changes in resonant frequency (dotted lines). Figure 8 shows normalized proliferation of MC-3T3 cells from Day 1 to 2 in wells containing films of various formulations (see Table 1): W = WSC; C = CMC; S = CS. Figure 9 shows sustained release of FGF-2 from WSC-CS PECs. Method A (diamond symbols): addition of FGF-2 to CS, followed by WSC addition to form PEC. Method B (square symbols): addition of WSC to CS to form PEC, followed by FGF-2 addition. Detailed Description of the Preferred Embodiments

Abbreviations used in the present specification include the following: ATIII: angiotensin HI

CMC: carboxymethylchitin (sometimes referred to as carboxymethylchitosan) CS: chitosan sulphate ECM: extracellular matrix FGF: fibroblast growth factor HS: heparan sulphate LBL: layer by layer PBS: phosphate buffered saline PCL: polycaprolactone PDGF: platelet derived growth factor PEC: polyelectrolyte complex PLGA: poly lactic-co-glycolic acid QCM: quartz crystal microbalance VEGF: vascular endothelial growth factor WSC: water soluble chitin/chitosan

All references cited in this specification are incorporated herein by cross reference. In the present specification, a peptide may be a protein, or it may be an antisense protein, or a natural polypeptide or a synthetic polypeptide or a natural oligopeptide or a synthetic oligopeptide. The peptide may comprise more than one individual peptides, each of which may, independently, be as described above. The peptide, or at least one of the peptides, ore each peptide independently, may be for example a growth factor, a cytokine, an anticoagulant protein, a hormone, a cell adhesion peptide, a cell targeting peptide or some other type of protein.

The inventors have found that a PEC may be prepared as an active (e.g. hi terms of its biological activity such as anti-coagulant property) molecular complex with a peptide or protein and that it is possible to process such a PEC into other forms such as membranes which retain the biological activity of the peptide or protein.

It has also been found that complexes may be prepared with a higher binding activity than would be expected from a simple additive effect of the component polyions (poly electrolytes). Such complexes with unproved binding activity may be designed by incorporating appropriate functional groups into the polyions, by combining the functional groups available on two or more polyelectrolytes. For example, heparan sulphate (HS), which is known to bind ATIII well, possesses sulfate, acetamido and carboxyl groups. A combination of polyelectrolytes may be chosen to contribute the different types of functional groups, hi examples provided herein, chitosan sulphate (CS) has been used to contribute the sulfate groups, water soluble chitrn/chitosan (WSC) to contribute the acetamido groups and carboxymethylchitin (CMC) to contribute the carboxyl groups, hi some cases therefore, the polyanion may comprise two separate polyanic species (hi the example above, CS and CMC), or more than two. These may be hi any desired ratio. For example the ratio between one of the polyanions and the other (e.g. of CS to CMC) may be between about 10:1 and about 1:1 on a weight or mole basis, or about 10:1 to 2:1, 10:1 to 5:1, 7:1 to 1 :1, 5:1 to 1:1, 3:1 to 1:1, 7:1 to 3:1, 7:1 to 5:1 or 5:1 to 3:1, e.g. about 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5. 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5 or 1 to 1. This ratio may be used to tailor the affinity of the PEC for the peptide. Similarly, mixtures of polycationic species in desired ratios may also be used as the polycation.

It is considered that this combinatorial effect might also be useful for combinatorial studies to identify complexes that have a high binding affinity towards a particular protein/peptide and may be applicable to drug discovery. The inventors have demonstrated that the protein-binding ability of a particular polyelectrolyte (e.g. binding of ATIII by heparin) may be retained upon complexation of the polyelectrolyte with a second oppositely-charged polyelectrolyte. The binding of the polyelectrolyte complex to the peptide may be due to specific functional groups on the polycation and/or on the polyanion. In some instances the functional group of the polyanion and polycation may not bind the peptide separately but may do so only upon combination. Furthermore, the binding may be due to more than one type of interaction with the molecule. Suitable groups which may promote binding, either individually or in combination, include sulfate, acetamido and carboxyl.

The process described herein for making polyelectrolyte complexes comprises combining a solution of a polyanion with a solution of a polycation so as to produce a polyelectrolyte complex (PEC). The polyanion may be capable of binding the peptide. The PEC forms particles dispersed within the combined solvents obtained from the polyanion solution and the polycation solution. The formation of these particles is at least partly governed by the electrostatic attraction between the polyanion and the polycation. In the process of the present invention, the peptide may not be present in either the solution of the cation or the solution of the anion. Thus the binding of the peptide is to the polyelectrolyte complex rather than to either of the polyelectrolytes prior to formation of the polyelectrolyte complex. This contrasts with certain prior art processes in which a peptide was bound to a polyelectrolyte and the polyelectrolyte then complexed with an oppositely charged polyelectrolyte to form a peptide loaded complex. The inventors have surprisingly found that in certain cases the affinity of a peptide for a polyelectrolyte complex is greater than for either of the individual polyelectrolytes used to make the polyelectrolyte complex. Thus the presently disclosed process can lead to a higher peptide loading and/or more tightly bound peptide relative to coacervation of a peptide loaded polyelectrolyte with a polyelectrolyte of opposite charge. This is a surprising result, since at first sight it might be thought that formation of a polyelectrolyte complex would reduce the available charge that could be used for binding the peptide (by coupling those charges with charges from the oppositely charged polyelectrolyte to make the complex), and consequently it might be thought that binding a peptide to a preformed polyelectrolyte complex might be less efficient rather than (as presently found) more efficient.

The ratio of polycation to polyanion in the process may be between about 3 and about 0.3 (i.e. between about 3:1 and about 0.3:1), or about 3 and 0.5, 3 and 1, 3 and 2, 2 and 0.3, 1 and 0.3, 0.5 and 0.3, 0.5 and 2, 0.5 and 1 or 1 and 2, e.g. about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5 or 3. These ratios may be on a weight basis or they may be on a mole equivalent basis, i.e. on the basis of moles of ionic charges. For example a 1:1 ratio on a mole equivalent basis would have a ratio such that there are equivalent numbers of positive charges on the polycation as negative charges on the polyanion. The actual weight ratio in this basis (or mole ratio, based on the molecular weights of the polycation and polyanion) may vary depending on the relative molecular weights and charge densities of the polycation and the polyanion. The concentrations of the polycation and the polyanion in the solutions thereof may be, independently, about 0.05 to about lmg/ml, or about 0.1 to 1, 0.1 to 0.5, 0.5 to I₅ 0.3 to 1, 0.1 to 0.8 or 0.3 to 0.7mg/ml, e.g. about 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or lmg/ml. The polyanion solution may be added to the polycation solution or the polycation solution may be added to the polyanion solution, or both solutions may be added to a diluent liquid either simultaneously or sequentially. It/they may be added dropwise. It/they may be added at about 0.5 to about 5ml/min, or about 0.5 to 2, 0.5 to 1, 1 to 5, 2 to 5 or 1.5 to 3ml/min, e.g. about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or Sml/min./The solution to which it/they is/are being added may be agitated during the addition. It may be stirred, vortexed, shaken, swirled, sonicated or otherwise agitated.

The solvents for the polyanion solution and the polycation solution (independently) may be aqueous. They may be ionic solutions. They may be for example phosphate buffered saline (PBS). Ix PBS contains 3.2 mM Na₂HPO₄, 0.5 mM KH₂PO₄, 1.3 mM KCl and 135 mM NaCl at pH 7.4. The solutions may, independently, be nxPBS, where n is between 1 and about 20, or about 1 to 10, 1 to 5, 2 to 20, 5 to 20, 10 to 20 or 2 to 5, e.g. about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. The ionic strength of either one or both of the solutions, or the concentration of a particular salt or combination of salts in said solutions, may be used to control the particle size of the loaded complex which is formed. For example, as demonstrated by an example provided herein, a more dilute PBS solution may lead to a larger particle size and a more concentrated PBS solution may lead to a smaller particle size.

During and/or after combination of the polyanion and the polycation solutions, the polyanion and polycation combine to form particles. Sufficient time should be allowed for formation of these particles. A suitable tune is generally between about 0.1 and 2 hours, or about 0.1 to 1.5, 0.1 to 1, 0.1 to 0.5, 0.5 to 2, 1 to 2 or 0.5 to 1.5 hours, e.g. about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 14, 1.5; 1.6, 1.7, 1.8, 1.9 or 2 hours, although less than 0.1 or greater than 2 hours may be appropriate on occasions. The particles may remain in suspension, or they may settle or precipitate spontaneously.

The polyanion may comprise, or may be, a saccharide, e.g. an oligosaccharide or a polysaccharide. It may be, or may comprise, a chitosan derivative. The anionic groups on the polyanion may be for example sulfates, carboxylates, phosphates, sulfonates etc. In some instances a polyanion may comprise more than one of these in the same molecule. In other instances mixtures of polyanionic components may be used. In the latter case, each polyanion may comprise a different anionic group, or they may all have the same anionic group or some may be the same and some different. Suitable polyanions, or polyanionic components, include chitosan sulfate and carboxymethylchitin. The carboxyl groups may be present as carboxyalkyl groups, e.g. carboxymethyl or carboxyethyl groups. The polyanion may be a water soluble polyanion. It may be soluble in PBS solution.

The polycation may comprise, or may be, a saccharide, e.g. an oligosaccharide or a polysaccharide. It may be, or may comprise, a chitosan derivative. The polycation may be crosslinked or it may be uncrosslinked. The process may comprise crosslinking the polycation prior to the step of combining the solution of the solution of the polyanion and the solution of the polycation. This may improve the physical properties (e.g. strength, resilience, elasticity, modulus etc.) of the PEC. The cationic group of the polycation may be an amino group. It may be a substituted amino group e.g. an alkylamino group or a dialkylamino group. The cationic group may be such that it is ionized (e.g. protonated) at the pH at which the polyanion is combined with the polycation. Each of the solutions (the solution of the polycation and the solution of the polyanion) and the combined solutions may independently have a pH of about 6 to about 8, or about 6 to 7, 7 to 8 or 6.5 to 7.5, e.g. about 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, €.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8. The polycation may be, or may comprise, for example WSC: It may be, or may comprise, an at least partially deacetylated chitin. The chitin may be deacetylated about 20 to about 90%, or about 20 to 70, 20 to 50, 40 to 90, 50 to 90, 70 to 90, 30 to 70 or 40 to 60%, e.g. about 20, 30, 40, 50, 60, 70, 80 or 90%, or it may be 100% deacetylated. Thus in an example the WSC may be about 50% deacetylated, i.e. about 50% of its nitrogen atoms may be present as amine groups and about 50% as acetamide groups. As discussed above, the polycation may be crosslinked. This may comprise exposing the polycation to a crosslinking agent. Suitable crosslinking agents for polyamines are well known, and include genipin, formaldehyde, diisocyanates and glutaraldehyde. Of these, genipin is generally preferred due to its lower toxicity.

In some embodiments both the polycation and the polyanion are chitosan derivatives. Thus the loaded complex may comprise an optionally crosslinked chitosan- based polyelectrolyte complex having a peptide bound thereto. Commonly at least one, preferably both, of the solution of the polyanion and the solution of the polycation is aqueous. In many embodiments one or both of these solutions contains one or more dissolved inorganic salts. The concentration of the salt(s) may be used to control or affect the particle size of the PEC produced by the process. A suitable aqueous medium for these solutions is PBS (as described above). This may be at the standard concentration or may be diluted or may be more concentrated than standard. In the case of dilution or increased concentration, it may still retain the same ratio of dissolved salts as the standard concentration of PBS. PBS provides both a suitable salt concentration and an appropriate buffering to a desired pH.

One aim of the present invention was to provide a PEC bound to a peptide. These are referred to herein at tunes as "loaded" PEC complexes. Such materials may be used for controlled, or sustained, release of the peptide, either in vivo or in vitro. In the present context, where the peptide is described as being "bound" to a particular species (either the PEC or the polyanion), the peptide may be releasably bound to said species. It may be bound electrostatically. It may be bound by ionic attraction. It may be bound, optionally releasably bound, in some other manner.

In contrast with some prior art processes, the PEC of the present invention may be preformed and then exposed to the peptide (optionally to a solution of the peptide) so as to bind the peptide to the PEC. The preformed PEC may be in the form of particles, optionally suspended in a solvent (e.g. an aqueous solvent) or it may be in the form of a film, which may be formed from such particles.

The initially formed PEC particles (with or without the peptide bound thereto) may be formed into a film or layer. This may comprise centrifuging or ultracentrifuging a suspension of the PEC particles so as to sediment them. The supernatant may be removed e.g. by decantation and the resulting slurry of particles may be. dried to form the film. Alternatively the film may be formed from the suspension without separation of a concentrated particle slurry, simply by drying the suspension of PEC particles. It is thought that on drying, at least some of the charge pairs of the PEC complex (e.g. ammonium-carboxylate pairs) may react to form covalent linkages, thereby crosslinking the PEC particles. Such crosslinks are thought to improve the physical properties of the film. The drying may be at reduced pressure or it may be at atmospheric pressure. It may be at less than about 10 Torr, or less than about 5, 2 or 1 Torr, or about 0.1 to about Torr, or about 0.1 to 5, 0.1 to 1, 1 to 10, 1 to 5 or 5 to 10 Torr, e.g. a bout 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 Torr. It may be at elevated temperature, provide that the temperature is sufficiently low as to not adversely affect the PEC or the bound peptide. The elevated temperature may be about 25 to about 15O⁰C, or about 25 to 60. 25 to 40, 25 to 30, 30 to 100, 50 to 100, 30 to 60, 40 to 60, 40 to 50, 100 to 150, 100 to 120, 120 to 150, 50 to 150, 80 to 120 or 100 to 12O⁰C, e.g. about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100⁰C. The drying may be at about or below about ambient temperature so as to protect the peptide. Thus it may be conducted at about 10 to about 3O⁰C, or about 10 to 20, 20 to 30 or 15 to 25⁰C, e.g. about 10, 15, 20, 25 or 3O⁰C. The drying may be for sufficient time for the desired degree of drying, e.g. for drying to the point of mechanical integrity of the film or for achievement of a suitable degree of crosslinking. This tune will depend on the degree of mechanical integrity required, the nature of the PEC, the thickness of the film, the degree (if any) of preconcentration (e.g. by centrifugation/decantation etc.) etc. It may be for example about 10 minutes to 2 weeks, or about 10 to 60 minutes, 10 to 30 minutes, 30 to 60 minutes, 15 to 45 minutes, 1 to 14 days, 1 to 7 days, 7 to 14 days, 5 to 10 days, 1 to 24 hours, 1 to 12 hours, 1 to 6 hours 6 to 24 hours, 12 to 24 hours or 6 to 18 hours, e.g. about 10, 20, 30 4o or 50 minutes, 1, 2. 3, 4, 5, 6, 9, 12, 15, 18 or 21 hours, 1, 2, 3, 4, 5, 6, 7. 8, 9, 10, 11, 12, 13 or 14 days or some other time.

Suitable peptides which may be bound to the PECs of the present invention include a variety of growth factors, hormones, cell adhesion peptides, cell targeting peptides etc. Examples of possible uses of loaded PECs provided by the invention include:

• therapeutic delivery of angiogenic growth factors e.g. VEGF, for ischaemia;

• therapeutic delivery of anti-cancer or anti-fibrotic drugs;

• therapeutic delivery of factors for tissue regeneration to treat tissue defects;

• medicine or biomaterials for anticoagulation.

For any or all of the above, targeted delivery can potentially be achieved by binding a cell targeting peptide.

Examples of particular properties which may be incorporated into a biomaterial using the process of the invention include:

• anti-coagulant property;

• cell-adhesion;

• binding of peptides (growth factors) to specific glycosaminoglycans (GAGs) such as the heparan sulfates (HS) can help to stabilize as well as potentiate the action of the peptides, by helping to present them to the cells. In certain instances, the peptide may be capable of binding the cell receptor in the form of a GAG-peptide complex, so as to induce the signaling cascade within the cell and thus affect cellular processes (e.g. proliferation, differentiation). The PEC-peptide complex may be used to substitute for the GAG-peptide complex.

The loaded PEC complex provided herein may be capable of releasing a bound peptide in a sustained manner. It may be capable of releasing the peptide over at least about 1 day, or at least about 2, 3. 4, 5 or 6 days, or at least about 1, 2 or 3 weeks, or at least about 1, 2, 3, 4, 5, 6, 7, 8. 9, 10 or 11 months, or at least about 1, 2, 3, 4 or 5 years. It may be capable of releasing the peptide over a period of about 1 day to 5 years, or about 1 day to 1 year, 1 to 200 days, 1 to 100 days, 1 to 50 days, 1 to 10 days, 1 to 5 days, 1 week to 5 years, 1 month to 5 years, 1 to 5 years, 3 to 5 years, 1 to 52 weeks, 1 to 25 weeks, 1 to 10 weeks 1 to 5 weeks, 1 to 12 months, 1 to 6 months or 6 to 12 months. It may be capable of releasing the peptide over a period of about 1, 2, 3, 4, 5 or 6 days, or about 1, 2 or 3 weeks, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 months, or about 1, 2, 3, 4 or 5 years. The release rate may be dependent on the nature of the polyelectrolyte complex (hi particular on the nature of the polyanion and of the polycation and on the ratio between these and on whether the polyanion is crosslmked) and may be dependent on the nature of the peptide. In some embodiments the loaded PEC complex does not substantially release the bound peptide. The bound peptide may retain its biological activity while bound to the PEC complex. It may retain at least about 50% activity, or at least about 60, 70, 80 or 90% of its activity or may retain substantially all of its activity. If the peptide is releasable from the loaded complex, the released peptide may retain its biological activity following release from the PEC complex. It may retain at least about 50% activity, or at least about 60, 70, 80 or 90% of its activity or may retain substantially all of its activity.

The binding of the peptide to the PEC may be a reversible binding. It may be a releasable binding. It may be an irreversible binding. It may be a non-releasable binding. It may be an electrostatic binding. It may be some other type of binding. The polyelectrolyte complexes may have adsorbed thereto up to about 20ng/cm" of the peptide, or up to about 30, 40, 50, 60, 70, 80, 90, 100, 150 or 200 ng/cm² of the peptide, or about 5 to about 200 ng/cm², or about 10 to 200, 50 to 200, 100 to 200, 5 to 100, 5 to 50, 5 to 20, 10 to 100, 10 to 50, 50 to 100 or 50 to 70 ng/cm², e.g. about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 ng/cm².

As discussed earlier, the peptide may comprise more than one individual peptides. In this event, each may be releasably bound to the PEC, or each may be non-releasably bound to the PEC, or at least one may be releasably bound to the PEC and at least one may be non-releasably bound to the PEC. In the last case, the functional groups on the polyanion and the polycation may be carefully selected to achieve this state, with reference to the different functional groups on the different peptides (i.e. the peptide(s) to be releasably bound and the peptide(s) to be non-releasably bound). Thus the invention also encompasses a PEC capable of releasably binding a first peptide and non-releasably binding a second peptide, and to corresponding processes of making loaded complexes therefrom and methods of using said loaded complexes as will be clear from the present specification. In the above, both releasably and non-releasably bound peptides retain their biological activity when bound to the PEC in a loaded complex. The releasably bound peptide may also retain its biological activity following release from the loaded complex.

The polyelectrolyte complex may be in the form of particles. These may have a mean diameter of less than about 5 microns, or less than about 2 or 1 microns, or less than about 500, 400, 300 or 200nm, or about 50 to about 5000nm, or about 50 to 2000, 50 to 1000, 50 to 500, 50 to 200, 50 to 100, 100 to 5000, 200 to 5000, 500 to 5000, 1000 to 5000, 200 to 2000, 200 to 1000, 200 to 500 or 500 to lOOOnm, e.g. about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500 or 5000nm. The particles may have a narrow polydispersity or may have a broad polydispersity. They may be substantially monodispersed. Alternatively, the polyelectrolyte complex may be in the form of a film. The film may have a thickness of about 5nm to about 5mm, or about 5nm to about 100 microns, 5nm to 50 microns, 5nm to 10 microns, 5 to lOOOnm, 5 to 500nm, 5 to 200nm, 5 to lOOnm, 5 to 50nm, 5 to 20nm, lOOnm to 100 microns, 1 to 100 microns, 10 to 100 microns or 1 to 10 microns or about 0.1 to about 5mm, or about 0.1 to 2, 0.1 to 1. 0.1 to 0.5, 0.5 to 5, 1 to 5, 2 to 5, 0.5 to 2, 0.5 to 1 or 1 to 2mm, e.g. about 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800 or 900nm or about 1, 2, 5, 10, 20 or 50 microns or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5_* 2, 2.5, 3, 3.5, 4, 4.5 or 5mm. The film may have a homogeneous thickness. It may have a heterogeneous thickness.

The polyelectrolyte complexes of the invention may be suitable for implantation into a subject. This may be for the purpose of delivering a bound peptide to the subject. It may be for the purpose of utilizing the biological activity of the bound peptide (e.g. an anticoagulant activity) to improve the .performance of the implanted item relative to a similar item with no bound peptide. The complex may have a bound peptide which renders the complex more biocompatible or which reduces adverse responses (e.g. clot formation) in the subject. The subject may be a human subject or may be a non-human subject. The subject may be a non-human mammal e.g. a non-human primate. It may be an ape, a monkey, a dog, a cat a cow, a horse, a pig, a goat, a dear, a buffalo or some other type of animal. It may be a domestic animal or may be a wild animal. The loaded complex may be in the form of an implantable device. It may be in the form of a prosthesis or of a coating for a prosthesis. It may be in the form of an implantable drug release device.

In the present work, a rationale for the design of biomaterials invoked growth factor binding and release. Proteins, specifically cytokines and growth factors, mediate the response of cells towards the biomaterial. If these signalling molecules could be presented to the cells in the appropriate manner, the desired cellular response could be evoked. This holds true, whether the biomaterials are employed in the form of tissue engineering scaffolds or as coatings to improve the biocompatibility of implants.

As discussed earlier, a known method for preparing a functionalised polyelectrolyte membrane uses the layer-by-layer (LBL) approach. An alternative strategy to the LBL process for the formation of functionalized biomaterials is to form polyelectrolyte complex (PEC) coacervates with the right chemistry that allows for growth factor binding and sustained release. To exemplify this approach, the inventors selected chitin/chitosan as a template polymer, which was further derivatized to produce polyelectrolytes, some of which possessed growth factor binding properties. One of the derivatives used was chitosan sulphate (CS), a heparan sulphate (HS) mimetic capable of binding growth factors, such as FGF-2 and vascular endothelial growth factor (VEGF). It was considered that binding of growth factors might help to stabilize and potentiate the action of the growth factors through presenting them to the cells. In the examples presented herein, the inventors show that the PEC formed between the HS mimetic (CS) and a suitable polycation is capable of retaining protein binding activity. Additionalfy, the combinatorial effect of polycation and polyanion led to complexes with an even higher protein binding activity than would be expected from a simple additive effect.

The inventors have developted a polyelectrolyte complex comprising a combination of functionalized polysaccharides which exhibits a measurable binding affinity for a target peptide. Suitable target peptides include angiotensin III (ATIII), peptides of the fibroblast growth factor (FGF) family, peptides of the vascular endothelial growth factor (VEGF) family and peptides of the platelet derived growth factor (PDGF) family. The functionalised polysaccharides have functional groups which are capable of providing the measurable binding affinity. They may have functional groups which are capable of binding the target peptide.

The measurable binding affinity is implied or demonstrated by the examples provided herein. For ATIII, the affinity is implied by the anti-coagulant function of the resulting peptide-bound complex. For FGF-2 (basic fibroblast growth factor), the affinity is demonstrated by the degree of peptide-binding by the combined layers of water-soluble chitin, carboxymethylchitin and chitosan sulfate. The affinity may vary depending on the combination of polyelectrolytes within the complex. The polyelectrolyte complex was postulated to bind VEGF and PDGF due to its similarity and commonality of functional groups with the heparan sulfates, which is known to bind VEGF and PDGF.

The polyelectrolyte complex as described above is exemplified herein by the combination of carboxymethylchitin, water soluble chitin and chitosan sulfate. However, these may be substituted with other functionalized polysaccharides possessing the appropriate functional groups. For example, chitosan sulfate may be substituted with other sulfated polysaccharides such as dextran sulfate, chitin sulfate, cellulose sulphate etc. Carboxymethylchitin may be substituted with other carboxymethylated polysaccharides such as carboxymethyldextran, carboxymethylchitin. carboxymethylcellulose, carboxyethylated polysaccharides such as carboxyethyldextran, carboxyethylchitin, carboxyethylcellulose, or other polysaccharide derivatives containing carboxyl groups. Water-soluble chitin may be substituted with other N-acetylated polysaccharides such as the various derivatives of chitin and chitosan.

The inventors have found that the PECs prepared according to the present application may be engineered into insoluble matrices of the desired shape and form, e.g. particles and films. They therefore possessed a wide range of utility for biomedical applications, hi addition, various crosslinking processes have been employed to improve the mechanical integrity of the PEC membranes.

Thus particles and membranes of PEC have been synthesized based on complexation of polyanionic and polycationic derivatives of chitin/chitosan. These polyelectrolyte complex particles and membranes were endowed with specific protein binding activity, primarily by including chitosan sulfate (CS)₅ a polymer that mimics the heparan sulfate (HS) present in the biological extracellular matrix (ECM). The degree of protein binding was found to depend on the specific combinations and ratios of polyelectrolytes used in forming the complexes, and was quantified with an anticoagulant assay (ATIII and Factor X binding) and quartz crystal microbalance (QCM) studies (basic fibroblast growth factor (FGF-2) binding). This combinatorial property was also observed when a pre-osteoblast cell line, MC-3T3 was cultured on membranes formed from the polyelectrolyte complexes, where specific combinations of polyelectrolytes yielded higher MC-3T3 viability in combination with FGF-2 treatment.

FGF-2, when incorporated into pellets formed from the particulates, was released in a sustained fashion, presumably due to binding with the sulfated component of the complex. Sustained release was observed even when FGF-2 was added after the polyelectrolyte complex had been formed, indicating that pre-formed polyelectrolyte complex could effectively bind and release growth factors.

Examples

Materials and methods

Preparation of PEC nanoparticles and membranes

WSC-CS particles

WSC (water soluble chitin/chitosan) and CS (chitosan sulphate) were separately dissolved in «^χ phosphate buffered saline (PBS) (n = 1, 2, 4, 5, 6, 8, 10). The CS solution was then added dropwise to the WSC solution under magnetic stirring at a rate of 1.8 rnL/min in a 1:1 volume ratio. For the particle size analysis, 0.5 mg/mL of WSC and 1 mg/mL of CS were used. For the anti-coagulant assay, various concentrations of polyelectrolyte were prepared in 1 * PBS. These were characterized as will be described later. WSC-CMC-CS particles

WSC, CS and CMC (carboxymethylchitin) were separately dissolved in l ^χ PBS. A polyanionic solution was first formed by mixing the solutions of the two polyanions (CS and CMC). This polyanionic solution was then added dropwise to the WSC solution under magnetic stirring at a rate of 1.8 mL/min and a WSC:CMC:CS volume ratio of 1:1:2. The concentration of each polyelectrolyte was varied, and their anti-coagulant activities were characterized as will be described later. PEC membrane preparation for pre-osteoblast (MCSTS) adhesion study

PECs were formed from solutions of WSC, CMC and CS at volumes and concentrations summarized in Table 1. For samples 1, 2, 3 and 4, WSC was first placed in a Falcon™ tube. The polyanionic solution (CMC or CS) was then added to the tube and vortexed for about 1 h. For samples 5, 6, 7 and 8, CMC and CS were first mixed in a separate Falcon™ tube before being added to WSC. After vortexing, the PEC suspensions were decanted into polypropylene molds and centrifuged at 2400 rpm for 10 min. The supernatants were removed, and the polypropylene molds containing the sedimented PECs were placed in a vacuum oven at 70 ⁰C for 1 h or until the PECs formed dry membranes.

Table 1. Formulation of chitin derivatives for PEC membranes.

After cooling to room temperature, each membrane was cut with a 6-mm surgical bore to form discs. Each disc was placed in a separate well in a 96-well plate. The samples were then sterilized by filling each well with 70% aqueous ethanol and placing the plates under germicidal UV light (wavelength 253.7nm) for 1 h. The ethanol was then aspirated, and the discs were rinsed twice with deionised water. Two discs of each membrane type were then incubated with 0.5 μg/ml of FGF-2 hi tris(hydroxymethyl)aminomethane (TRIS) pH 8 buffer for 3 h (designated as "FGF- treated"). Two discs of each membrane type were incubated for the same tune period in TRIS pH 8 buffer alone, i.e. with no FGF-2 (designated as "non-FGF-treated"). The discs were rinsed with Dulbecco^"s Modified Eagle Medium (DMEM) and seeded with MC3T3 cells. Crosslinked PEC membranes

In one variation, genipin (GP) was used to form crosslinked PEC membranes. WSC (2 mg/mL) was added to GP (5 mg/mL) at a volume ratio of 9:1. The reaction was allowed to proceed under magnetic stirring for 6-12 h. 6 mL of the WSC-GP reaction mixture were then added to 6 mL of CS, resulting in the formation of PEC precipitate. The precipitate was transferred to a polypropylene mold, and centrifuged at 2500 rpm for 5 min, following which the supernatant was removed. The sediment was then air dried for 1 h, and dried in vacuo at 50 °C for 30 min.

In another variation, dry PEC membranes were prepared as described above {PEC membrane preparation for pre-osteoblast (MCSTS) adhesion study), but subjected to an additional crosslinking step in a vacuum oven (under 30 in Hg vacuum) at 110 ⁰C for 1 week.

Characterization of PECs, and protein binding and release Anti-coagulant assay

The anti-coagulant activity of the synthesized compounds and PEC particles (prepared as described earlier) were assessed using a Stachrom^® Heparin colorimetric assay. This assay examined the inhibition of Factor Xa in the coagulation cascade via the binding of antithrombin III. Binding of antithrombrn HI would change its conformation, allowing it to bind to Factor Xa, resulting in the anti-coagulant effect. This anti-coagulant or anti-Xa activity of the compound was inversely proportional to the measured absorbance at 405 nm (A₄J₀₅). and was normalized against the measured activity of a phosphate buffered saline (PBS) blank:

Anti-coagulant Activity (Normalized against PBS) = (1/A₄₀₅ sample) / (1/A₄₀₅ Blank) Particle sizing

The hydrodynamic radii of the PEC particles (prepared as described above: WSC- CS particles) were measured using a Brookhaven ZetaPALS® particle sizer. The hydrodynamic radii were plotted as a function of the PBS concentration used in synthesizing the particles. QCM

Polyelectrolyte solutions were first prepared by dissolving the chitin derivatives in PBS. A FGF-2 solution (5 μg/ml) was also prepared in PBS. Quartz crystal resonators were plasma-treated to generate hydroxyl groups in order to create a negatively charged surface. This crystal was then placed in a Q-Sense E4 Quartz Crystal Microbalance, and various polyelectrolyte solutions were introduced to the crystal surface while real-time changes in the crystal's resonant frequency was measured. Firstly, a polycationic WSC solution was introduced to the surface of the crystal and the change in the crystal^'s resonant frequency was measured. The surface of the crystal was then rinsed with PBS to remove excess, unbound WSC. A polyanionic solution (CS, CMC or a mixture of both) was then introduced onto the surface of the crystal. The surface of the crystal was then rinsed again with PBS before the introduction of FGF-2. The areal masses of the polyelectrolytes and FGF-2 that were bound to the surface of the crystal were then calculated from the changes in the crystal^'s resonant frequency using the Sauerbrey equation.

FGF-2 release from PEC Method A: Addition of FGF-2 to CS, followed by PEC formation

3 mL of CS solution (4 mg/mL) were added to a 15-mL centrifuge tube. This was followed by the addition of 100 μL of recombinant human FGF-2 (10 μg/mL). The tube was mildly agitated, and allowed to stand for 5 min. 3 mL of WSC solution (4 mg/mL) were then added to the same tube, upon which a white precipitate was rapidly formed and sedimented. After 0.5 h, the precipitate was centrifuged at 1000 rpm for 5 min, and the supernatant was removed. The precipitate clump was washed with 2 mL of DI water, transferred to a well of a 96-well plate, and packed into a dense, wet pellet using spatula/forceps. 200 μL of PBS were added to the well. After 2 h, about 200 μL of the supernatant were transferred to a labelled vial, and stored at 4 °C. Each well was replaced with 200 μL of fresh PBS. The sampling procedure was repeated after 6 h, and 1, 2, 3, 4, 6, 8, 10, 12 and 14 days. ELISA was performed to establish the FGF-2 release profile. Method B: Formation of PEC, followed by FGF-2 addition

2 mL of a CS solution (4 mg/mL) in pH 8 PBS was added to a 15-mL centrifuge tube. This was followed by 2 mL of WSC solution (4 mg/mL) in pH 8 PBS. A PEC was formed that sedimented to give an almost clear supernatant after 5 min. 4 mL of the supernatant were discarded. 100 μL of FGF-2 (10 μg/mL) were added, and the tube was gently agitated. The tube was centrifuged at 1000 rpm for 5 min. The precipitate clump was washed with 2 mL of DI water, transferred to a well of a 96-well plate, and packed into a dense, wet pellet using spatula/forceps. 200 μL of PBS was added to each well. The sampling procedure was performed as in Method A. Results and discussions

Synthesis and characterization ofchitin derivatives

Methods for the preparation of WSC [Sannan T, Kurita K, Iwakura Y. Studies on chitin. Makromolecular Chemistry, 111 (1976), 3589-3600], CMC [Tokura S, Nishimura S, Nishi N. Studies on chitin VHI. Some properties of water soluble chitin derivatives. Polymer Journal, 15 (6) (1983), 485-489; Chen X, Park H. Chemical characteristics of O- carboxymethyl chitosans related to the preparation conditions. Carbohydrate Polymers 53 (2003), 355-359; Liu XF, Guan YL, Yang DZ, Li Z. Yao KD. Antibacterial action of chitosan and carboxymethylated chitosan. Journal of Applied Polymer Science, 79 (2000), 1324-1335] and CS [Wolfrom ML, Shen-Han TM. Sulfonation of chitosan, Journal of the American Chemical Society, 81 (1959), 1764-1766] were based on those found in literature. The principle behind the synthesis of WSC was about 50% deacetylation of chitin by alkali, upon which the chitin became water soluble. The degree of deacetylation for our WSC product was measured by potentiometric titration to be 0.488. The Fourier- transform infrared (FTIR) spectrum of CMC showed the development of a strong carboxyl peak at 1600 cm^"1 due to the carboxymethyl group (Figure 1C). From the ratio of absorbances at 1732 and 1558 cm^"1, the degree of carboxymethylation was estimated to be 0.435 from a calibration curve (Figure IA). Elemental analysis for CMC (Na salt): C, 36.83%; H, 6.69%; N, 5.05%; Na, 2.47%. The FTIR spectrum of CS showed the major peaks associated with the sulfate groups at 1240 cm^"1 (S=O) and 800 cm^"1 (C-O-S). (Figure 2). Elemental analysis for CS (Na salt): C, 21.18%; H, 4.21%; N, 3.11%; S, 12.46 %; Na, 6.92%. ATIII and Factor Xa binding by PECs

HS or the corresponding HS mimetic has previously been covalently conjugated to surfaces to bind growth factors and mediate the cellular response. The latter requires functionalization that may affect bioactivity of the HS, and often influences the surface instead of. the bulk properties of the biomaterial. In contrast, the present approach involves the formation of PECs whereby the HS mimetic would be uniformly present throughout the biomaterial. An alternative approach that would not require covalent immobilization of heparin has been employed by Sakiyama-Elbert and Hubbell (Sakiyama-Elbert SE, Hubbell JA. Controlled release of nerve growth factor from a heparin-containing fibrin-based cell ingrowth matrix. Journal of Controlled Release, 69(1) (2000), 149-158). whereby a heparin-binding peptide was immobilized in a fibrin matrix for binding heparin. Binding of growth factors/cytokines to HS and HS mimetics involves specific functional groups of heparin that interact with the corresponding groups in the heparin- binding domain of the protein concerned. In forming a PEC that binds growth factors by combining the HS mimetic (polyanion) with a polycation, a very fundamental question arises, i.e. would the functional groups of the HS-mirnetic be available for binding with the protein since these same groups are used in the polyelectrolyte complexation.

To better understand and quantify protein binding by the HS mimetics, the binding of ATπi and Factor Xa was examined. The binding of heparin to ATIII changed the conformation of the latter, thus allowing Factor Xa to bind. This was the proposed mechanistic explanation of the anti-coagulant activity of heparin. The inventors synthesized the PECs in the particulate form so as to allow direct application of the anticoagulant assay. In addition, particles could be quite easily translated to other forms.

The PEC particles of the present invention were formed by controlled introduction of one polyelectrolyte to the other using a peristaltic pump. The sizes of the particles were dependent on the ionic strength (PBS concentration) of the polyelectrolyte solutions used. Higher ionic strength solutions stabilized the particles by the counter ion effect, leading to smaller particle sizes (Figure 3).

CS, complexed with WSC in the form of particles, was shown to exhibit anticoagulant activity (Figure 4). The anti-coagulant activity of the PEC was unexpectedly higher than that of CS alone. This suggested that not only was the CS accessible for binding to the protein (ATIII) in the form of a complex, but also the presence of WSC was advantageous. WSC consisted of acetamido groups that were not present in CS These groups were a common repeating unit in HS (Figure 5). When different WSC:CMC:SC ratios were used, higher anti-coagulant activities were obtained at higher WSC concentrations (Figure 6), pointing to a possible combinatorial effect.

Carboxyl groups, which are also featured in HS, were provided to the complex in the form of CMC. Figure 4 shows that the anti-coagulant activity was higher for PEC2 and PEC3, which consisted of CMC. FGF-2 binding and release by PECs

The PEC membranes of the present invention were essentially PEC particles that had been allowed to aggregate in a mould and dried completely to form a film. PEC membranes were cast from solutions containing large or aggregated PEC particles. The suspensions of large particles were prepared in PBS, cast onto polypropylene moulds and left to dry overnight. Alternatively, for the controlled release experiments, the particle suspensions were centrifuged, congealed in water, and packed into pellets. The films were used for growth factor binding studies.

As the first step, binding of growth factor by the chitin polyelectrolytes was demonstrated with QCM. The polyelectrolytes were obtained by alternating the deposition of oppositely charged polyelectrolytes with a PBS rinse in between. Again, a combinatorial effect of the polyelectrolytes towards FGF-2 binding was observed. The WSC/FGF-2 sequence yielded 24 ng/cm² of bound FGF-2. In contrast, the WSC/CS/FGF- 2/rinse sequence yielded 30 ng/cm² of bound FGF-2. When CS was combined with CMC in a 2:1 mass ratio as the second coating (i.e. WSC/CS+CMC/FGF-2), 60 ng/cm² of FGF- 2 was bound (Figure 7). A combinatorial effect clearly existed, as for the binding of ATIII by PEC particles described above.

In the cell culture studies, MC-3T3, a pre-osteoblast cell line was cultured in wells containing non-crosslinked, FGF-2-treated PEC films composed of WSC, CS and CMC. The alamarBlue™ assay revealed higher cell viabilities for certain cases where FGF-2 was added, especially for films synthesized using equimolar concentrations of WSC and CS (3W3S) (Figure 8). FGF-2 would have been released in either the free form, or bound to PEC particles resulting from the dissolving membrane.

Some applications may require a more stable, less swellable form of the PEC film. For such applications, genipin crosslinking of WSC prior to the addition of CS and/or CMC was used to improve the mechanical integrity and stability of the PEC membranes in tissue culture media. Genipin could form crosslinks by reacting with the amino groups of WSC, effectively increasing the molecular weight of WSC prior to. the complexation reaction. Genipin could also crosslink WSC to the available, albeit lower concentrations of, amino groups on CS and CMC during the compexation process and membrane casting. This secondary crosslinking would further strengthen the PEC membrane as it would allow for covalent crosslinking between PEC particles.

Another crosslinking method that effectively stabilized the membrane was dehydration under vacuum at elevated temperatures, which, it is thought, promotes the formation of covalent bonds between the ammo and , carboxyl groups of the polyelectrolytes .

The ability of the PECs to bind growth factor would also be useful hi the sustained release of the growth factor, based on the equilibrium between the bound and free forms of the growth factor. Two methods were employed to bind growth factor to the PECs. hi Method A, FGF-2 was first introduced to a solution of CS to activate binding. This was followed by addition of WSC, which resulted in the formation of PEC particles and presumably 'encapsulation' of the growth factor. In Method B, the PEC was formed first, by addition of WSC to CS. This was followed by FGF-2 addition to the nascent precipitate. Logically, Method A would be the method of choice to incorporate FGF-2. However, the inventors' anti-coagulant and QCM studies showed that FGF-2 would be bound equally well via the two methods, if not better by the pre-formed PEC particles. This was confirmed by the sustained release experiments. FGF-2. when incorporated into the complex via Method A, was released in a sustained fashion due primarily to binding with the sulfated component of the complex (Figure 9). Sustained release was also observed when FGF-2 was incorporated via Method B (i.e. after the PEC has been formed), indicating that the PEC could effectively bind growth factor.

While the rate of FGF-2 release from the complex prepared by Method A was sustained or slightly increasing from Day 6 to 14, the release of FGF-2 from that by Method B seemed to level off during Day 6 to 14. In Method B where FGF-2 was added only after the complex has formed, the binding of FGF would presumably occur only on the surface of the PEC particles. Thus, the amount of FGF-2 bound in a specific manner to CS and which would be released more slowly in the long term, would be limited. For Method B, it was expected that by changing the particle size (which could be achieved by varying the solution concentrations), a longer period of sustained release could be attained.

An advantage of forming the PEC particles first (as in Method B) would be the possibility of lyophilizing the particles to obtain a powder form. The powder could then be re-swollen in water or a suitable buffer for growth factor binding. The PEC-growth factor complex could subsequently be molded into insoluble forms, such as membranes and pellets, as desired in the final applications.

Claims

Claims:

1. A process for producing a loaded complex comprising a polyelectrolyte complex bound to a biologically active peptide, said process comprising:

(a) combining a solution of a polyanion with a solution of a polycation to form the polyelectrolyte complex, and

2. The process of claim 1 wherein one or both of the polyanion and the polycation comprises a functional group capable of binding the peptide or wherein a combination of functional groups on the polyanion and the polycation is capable of binding said peptide.

3. The process of claim 1 or claim 2 comprising allowing the polyelectrolyte complex to form particles.

4. The process of claim 3 wherein step (b) is conducted after formation of the particles of the polyelectrolyte complex.

5. The process of any one of claims 1 to 4 wherein the polyanion comprises a polysaccharide.

6. The process of any one of claims 1 to 5 wherein the polyanion comprises a mixture of at least two polyanionic components.

7. The process of any one of claims 1 to 6 wherein the polyanion comprises chitosan sulfate, carboxymethylchmn or a mixture of these.

8. The process of any one of claims 1 to 7 wherein the polycation comprises a cationic polysaccharide.

9. The process of claim 8 wherein the cationic polysaccharide is water soluble chitin.

10. The process of any one of claims 1 to 9 comprising crosslinking the polycation prior to step (a).

11. The process of claim 10 wherein the polycation comprises water soluble chitin and the step of crosslinking comprises exposing said chitin to a crosslinker selected from the group consisting of genipin. formaldehyde, a diisocyanate and glutaraldehyde.

12. The process of any one of claims 1 to 11 wherein the solution of the polyanion and the solution of the polycation are both aqueous solutions.

13. The process of any one of claims 1 to 12 wherein at least one of the solution of the polyanion and the solution of the polycation comprises a dissolved inorganic salt.

14. The process of any one of claims 1 to 13 wherein a concentration of the dissolved organic salt in the solution of the polyanion or in the solution of the polycation or in both of these is controlled so as to obtain a desired size of said particles.

15. The process of any one of claims 1 to 14 comprising at least partially drying the polyelectrolyte complex so as to form a film of the polyelectrolyte complex.

16. The process of claim 15 wherein step (b) is conducted after forming the film of the polyelectrolyte complex.

17. The process of any one of claims 1 to 16 wherein the peptide is a growth factor, a cytokine, an anticoagulant protein, a hormone, a cell adhesion peptide or a cell targeting peptide.

18. The process of any one of claims 1 to 17 wherein the polyanion and the polycation are such that the loaded complex is capable of releasing the peptide into an aqueous solution in a sustained manner.

19. A loaded complex comprising:

• a biologically active peptide bound to said polyelectrolyte complex: wherein the peptide retains its biological activity while bound to the polyelectrolyte complex.

20. The loaded complex of claim 19 wherein the peptide is selected from the group consisting of a growth factor, a cytokine, an anticoagulant protein, a hormone, a cell adhesion peptide, a cell targeting peptide and mixtures of any two or more of these.

21. The loaded complex of any one of claim 19 or claim 20 wherein the polyanion comprises an anionic polysaccharide.

22. The loaded complex of any one of claims 19 to 21 wherein the polycation comprises a cationic polysaccharide.

23. The loaded complex of any one of claims 19 to 22 wherein the polycation is crosslinked by a species other than the polyanion.

24. The loaded complex of any one of claims 19 to 23 wherein the polyanion comprises chitosan sulfate, carboxymethylchitin or a mixture of these, and the polycation comprises water soluble chitosan.

25. The loaded complex of any one of claims 19 to 24 wherein the polyanion and the polycation are such that the peptide is releasable from said loaded complex and retains its biological activity following release from the loaded complex.

26. The loaded complex of any one of claims 19 to 25 in the form of particles.

27. The loaded complex of claim 26 wherein the particles have a mean diameter of less than about 5 microns.

28. The loaded complex of any one of claims 19 to 25 in the form of a film.

29. A device suitable for implantation into a living subject, said device comprising a loaded complex according to any one of claims 19 to 25 or 28.

30. The device of claim 29 wherein the peptide of the loaded complex is an anticoagulant peptide.

31. A process for making a device suitable for implantation into a living subject, said process comprising forming a loaded complex according to any one of claims 19 to 28, or made by the process of any one of claims 1 to 18, into said device.

32. A process for making a device suitable for implantation into a living subject, said process comprising at least partially coating a device to be implanted into a living subject with a loaded complex according to claim 28 or made by the process of claim 15 or claim 16.

33. A method of delivering a biologically active peptide to a subject, said method comprising introducing a loaded complex according to any one of claims 19 to 28 or made by the process of any one of claims 1 to 18 into said subject, wherein the loaded complex comprises said peptide bound to a polyelectrolyte complex.

34. A method of delivering a biologically active peptide to a liquid, said method comprising introducing a loaded complex according to any one of claims 19 to 28 or made by the process of any one of claims 1 to 18 into said liquid, wherein the loaded complex comprises said peptide bound to a polyelectrolyte complex.

35. . Use of a loaded complex according to any one of claims 19 to 28. or made by the process of any one of claims 1 to 18 for delivering a biologically active peptide to a subject.

36. Use of a loaded complex according to any one of claims 19 to 28 or made by the process of any one of claims 1 to 18 for delivering a biologically active peptide to a liquid.

37. Use of a loaded complex according to any one of claims 19 to 27 or made by the process of any one of claims 1 to 14 for the manufacture of a medicament for treatment of a condition in a subject, wherein the biologically active peptide is indicated for said condition.

38. A medicament for treatment of a condition in a subject, said medicament comprising a loaded complex according to any one of claims 19 to 27 or made by the process of any one of claims 1 to 14 and a pharmaceutically acceptable carrier, wherein the biologically active peptide is indicated for said condition.

39. A polyelectrolyte complex capable of binding a target peptide, said complex comprising a polyanion complexed with a polycation wherein one or both of the polyanion and the polycation comprises a functional group capable of binding the target peptide or wherein a combination of functional groups on the polyanion and the polycation is capable of binding said target peptide.

40. The polyelectrolyte complex of claim 39 wherein polyanion and the polycation are each a polysaccharide.

41. The polyelectrolyte complex of claim 39 or claim 40 wherein at least one of the polyanion and the polycation is a chitin derivative or a chitosan derivative.

42. The polyelectrolyte complex of any one of claims 39 to 41 wherein the target peptide is a growth factor, a cytokine, an anticoagulant protein, a hormone, a cell adhesion peptide or a cell targeting peptide.

43. The polyelectrolyte complex of any one of claims 39 to 42 wherein the functional group is sulfate, acetamido or carboxyl.

44. The polyelectrolyte complex of any one of claims 39 to 43 wherein the polyanion comprises more than one polyanionic components.

45. The polyelectrolyte complex of any one of claims 39 to 44 wherein the polycation comprises a water soluble partially deacetylated chitin.

46. The polyelectrolyte complex of any one of claims 39 to 45 in the form of particles.

47. The polyelectrolyte complex of any one of claims 39 to 45 in the form of a film or layer.

48. A process for making a polyelectrolyte complex capable of binding a target peptide, said process comprising combining a solution of a polyanion with a solution of a polycation so as to form said complex, wherein one or both of the polyanion and the polycation comprises a functional group capable of binding the target peptide or wherein the polyanion and polycation comprise functional groups which, following formation of the polyelectrolyte complex, are capable in combination of binding the target peptide.

49. The process of claim 48 additionally comprising allowing the resulting polyelectrolyte complex to form particles.

50. The process of claim 48 or claim 49 comprising at least partially drying the resulting polyelectrolyte complex so as to form a film of the polyelectrolyte complex.

51. The process of any one of claims 48 to 50 wherein the target peptide is selected from the group consisting of a growth factor, a cytokine, an anticoagulant protein, a hormone, a cell adhesion peptide or a cell targeting peptide.

52. The process of any one of claims 48 to 51 wherein the polycation comprises a water soluble partially deacetylated chitin.

53. The process of any one of claims 48 to 52 wherein the polyanion comprises an anionic polysaccharide or more than one anionic polysaccharide.