The present invention relates to novel nanoparticle conjugates and methods for manufacturing the same. In particular, the present invention relates to insulin- nanoparticle conjugates and methods for manufacturing the same. The present invention relates to insulin-nanoparticle conjugates wherein nanoparticles are formed within an insulin molecule.

Insulin is an important biologically active polypeptide hormone secreted by the islets of Langerhans in pancreas. Insulin is composed of two peptide chains referred to as the A chain and B chain. The A and B chains are linked together by two disulfide bonds. An additional disulfide is formed within the A chain. In most species, the A chain consists of 21 amino acids and the B chain of 30 amino acids. Insulin regulates the glucose levels in the blood by facilitating the entry of glucose into mainly muscle, liver and adipose tissues. Any impairment of insulin handling by the body can result in metabolic aberrations, which can precipitate insulin resistance and other sequelae associated with diabetes mellitus.

The present invention aims to provide a non-toxic nanoparticle conjugate. The present invention also aims to provide a biocompatible nanoparticle conjugate. The present invention also aims to provide a water-soluble nanoparticle conjugate. The present invention aims to provide an insulin-nanoparticle conjugate in which the biological activity of insulin is maintained. The present invention aims to provide a therapeutic agent and a diagnostic agent. The present invention also aims to provide an inexpensive method of manufacturing an insulin-nanoparticle conjugate. The present invention also aims to provide an environmentally-friendly method of manufacturing an insulin-nanoparticle conjugate. The present invention also aims to provide a method of manufacturing an insulin-nanoparticle conjugate in which mild reaction conditions are employed.

The present invention solves all or some the above aims.

According to one aspect, the present invention provides an insulin-nanoparticle conjugate comprising: insulin or an insulin derivative and at least one nanoparticle constrained in association with the insulin or insulin derivative.

According to another aspect, the present invention provides a method of manufacturing an insulin-nanoparticle conjugate, wherein the method comprises: a) providing a first nanoparticle precursor or a first nanoparticle, and optionally reacting the first nanoparticle precursor to form a first nanoparticle as necessary; b) optionally providing a second nanoparticle precursor or a second nanoparticle, and optionally reacting the second nanoparticle precursor to form a second nanoparticle as necessary; c) providing insulin or an insulin derivative; d) optionally providing at least one reagent to react with the first and/or second nanoparticle precursor to form a first and/or second nanoparticle; and performing one of the following steps: e1 ) combining the insulin or insulin derivative with the first nanoparticle precursor and reacting the first nanoparticle precursor with the reagent to form a nanoparticle which, together with the insulin or insulin derivative, forms the insulin-nanoparticle conjugate; or e2) combining the insulin or insulin derivative with the first nanoparticle to form the insulin-nanoparticle conjugate; or e3) combining the insulin or insulin derivative with the first and second nanoparticle or nanoparticle precursor, and optionally reacting the at least one reagent with the first and/or second nanoparticle precursor to form a first and/or second nanoparticle, to form the insulin-nanoparticle conjugate; or e4) combining the insulin or insulin derivative with the first nanoparticle or nanoparticle precursor to form a mixture comprising insulin or insulin derivative and the first nanoparticle or nanoparticle precursor, and combining the second nanoparticle or nanoparticle precursor with the mixture and optionally reacting the at least one reagent with the first and/or second nanoparticle precursor to form a nanoparticle, to form the insulin-nanoparticle conjugate; or e5) combining the first and second nanoparticle or nanoparticle precursor to form a mixture comprising the first and second nanoparticle or nanoparticle precursor, and combining the insulin or insulin derivative with the mixture, optionally providing at least one reagent to react with the first and/or second nanoparticle precursor to form a nanoparticle, to form the insulin-nanoparticle conjugate.

According to another aspect, the present invention provides a use of insulin as a template in the manufacture of a metal, metal oxide or semiconductor nanoparticle. According to another aspect, the present invention provides an insulin-nanoparticle conjugate for use as a medicament.

According to another aspect, the present invention provides an insulin-nanoparticle conjugate for use as a contrast enhancer for biomedical imaging.

In one variant of the method of the present invention, apart from the final step, the method comprises steps a) and c). In another variant of the method of the present invention, apart from the final step, the method comprises steps a), b) and c). In another variant of the method of the present invention, apart from the final step, the method comprises steps a), c) and d). In another variant of the method of the present invention, apart from the final step, the method comprises steps a), b), c) and d).

The final step of the method of the present invention comprises one of the steps e1 ), e2), e3), e4) or e5). Of these possibilities, one of e1 ), e3) or e5) is preferred.

The term "constrained" as used above means physically trapped, sterically hindered, chemically bonded, e.g. covalently bonded, electrostatically attracted, e.g. through ionic bonding, hydrogen bonded or held by any other polar interaction, or held via Van der Waals forces.

The term "insulin-nanoparticle conjugate" as used herein means a molecule of insulin (or an insulin derivative) with at least one nanoparticle either chemically or physically bound thereto. Insulin effectively acts as a template for the nanoparticles to cluster around and/or within. The conjugate can be thus regarded as an insulin-templated nanoparticle conjugate.

The insulin or insulin derivative described herein may be any type of insulin. The insulin may be chemically or physically modified in some way, e.g. synthetically modified by e.g. conjugating to the insulin, an amino acid or peptide. The skilled person will appreciate how insulin might be modified and used in the conjugated nanoparticles of the present invention. For example, United States Patent 4840897 describes a novel modified human insulin and its use as starting material for the preparation of human insulin.

Other examples of modified insulin or insulin derivatives that have been described in the arl include insulin derivatives that are used as in vivo imaging agents. For example, radio labelled insulin derivatives where a radiolabel moiety suitable for detection by SPECT or PET is attached to the insulin. Such modified insulins are described in the prior art and can also be used in the conjugate of the present invention. The skilled person will thus be aware of methods foi preparing these materials as well as their use in in vivo imaging methods.

It is also contemplated that the insulin may be a mixture of more than one, for example two or more, or three or more different types of insulin of natural or synthetic origin. For example, two premixed insulin analogue formulations are currently available: insulin lispro 75/25 (75% insulin lispro protamine suspension and 25% insulin lispro) and biphasic insulin aspart 70/30 (BIAsp 70/30; 70% insulin aspart protamine suspension and 30% insulin aspart). Materials such as these can also be used in the conjugate of the present invention.

The insulin may be bovine, porcine, feline or human insulin, or the insulin may be lyspro insulin. The insulin can be readily obtained from a number of different sources and is sold under a number of different brand names e.g. Humalog (EIi Lilly); Lantus (Sanofi Aventis); Levemir (Novo Nordisk); Actrapid (Novo Nordisk); Novorapid (Novo Nordisk); Velosulin (Novo Nordisk); Humulin M3 (Lilly); Hypurin (Novo Nordisk); lnsuman (Sanofi Aventis); lnsulatard (Novo Nordisk); Mixtard (Novo Nordisk). In an embodiment, the insulin described herein is bovine insulin. In an alternative embodiment, the insulin described herein is human insulin. In an alternative embodiment, the insulin described herein is porcine insulin.

In an embodiment, the nanoparticle of the insulin-nanoparticle conjugate is a metal nanoparticle comprising at least one metal. In an embodiment, the metal is a transition metal or a lanthanoid metal. Preferably the metal is a transition metal. Thus in one embodiment, the metal is a d-block element. In an alternative embodiment, the metal is an f-block element. Preferably, the metal is a d-block element and more preferably is a group VIII element. In one embodiment, the metal is a metal used in X-ray or MRI contrast agents, e.g. Fe, Au, Mn or Gd. Preferably the metal is Au. In another embodiment, the metal is selected from the group comprising: Au, Pt, Pd, Co, and Fe. In another embodiment, the metal is selected from the group comprising: Ag, Cu, Cr, Nb, Ta and Zr.

In another embodiment, the nanoparticle of the insulin-nanoparticle conjugate is a compound containing one or more metals and one or more non-metals. In an embodiment, the nanoparticle comprises a metal and a non-metal. The metal may be as defined above. The non-metals are typically C, O, N, S, P, Se and Si.

A preferred group of nanoparticles are metal oxides (including both simple binary oxides and complex metal oxides). The metal oxide may be a conductive metal oxide or a non- conductive metal oxide. The conductive oxide is preferably a binary conductive oxide or a ternary conductive oxide. The binary conductive oxide is preferably selected from the group consisting of RuO _x , PdO _x , IrO _x , PtO _x , OsO _x , RhO _x , ReO _x and ZnO _x , and the ternary conductive oxide is preferably selected from the group consisting of SrRuO ₃ , ln _1-x Sn0 ₃ , NaxW _1-x 0 ₃ , Znx(AI, M n) _1-x O and La ₀ -SSr ₀ .5CoO ₃ . The non-conductive oxide is preferably a binary non-conductive oxide or a ternary non-conductive oxide. The binary nonconductive oxide is preferably selected from the group consisting Of AIO _x , TiO _x , TaOx, HfO _x , BsO _x , VO _x , MoO _x , SrO _x , NbO _x , MgO _x , SiO _x , FeO _x , CrO _x , NiO _x , CuO _x and ZrO _x , and the ternary nonconductive oxide is preferably selected from the group consisting of SiTiO ₃ , BaTiO ₃ , Al _x Ti _1-x 0 _y , HfSii _-x 0y, HfAI _1-x O _y , Ti _x Sii _-x 0 _y and LaTiO ₃

In an embodiment, the nanoparticle of the insulin-nanoparticle conjugate is a magnetic nanoparticle comprising at least one metal. In an embodiment, the magnetic nanoparticle is a metal oxide. Preferably the magnetic nanoparticle is an iron oxide and more preferably Fe ₃ O ₄ . Alternatively, the metal oxide may be NiOFe ₂ O ₃ , CuOFe ₂ O ₃ or MgOFe ₂ O ₃ . In another embodiment, the magnetic nanoparticle comprises more than one metal, preferably two metals. The magnetic nanoparticle may be a simple mixture of metals, a compound comprising more than one metal or an alloy. In an embodiment, the magnetic nanoparticle is FeCo.

In an embodiment, the nanoparticle of the insulin-nanoparticle conjugate is a semiconductor nanoparticle. In an embodiment, the semiconductor nanoparticle comprises a core and a shell. The core and/or the shell of the semiconductor nanoparticle may be a semiconductor material comprising a group Il element and a group Vl element, e.g. ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, HgTe, MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe or BaTe. Alternatively, the core and/or shell of the semiconductor nanoparticle may be a semiconductor material comprising a group III element and a group V element, e.g. GaN, GaP, GaAs, GaSb, InN, InP, InAs or InSb. Preferably, the core and/or shell materials are CdS and ZnS. In an embodiment, the semiconductor nanoparticle is CdSe/ZnS. In an embodiment, the semiconductor nanoparticle comprises a core but does not comprise a shell. In an embodiment, the semiconductor nanoparticle is CdSe. In an alternative embodiment, the semiconductor nanoparticle is TiC> ₂ .

In an embodiment, the nanoparticle of the insulin-nanoparticle conjugate has a size in the range of 1 to 100 nm. Preferably, the nanoparticle has a size in the range 1 to 50 nm. More preferably, the nanoparticle has a size in the range 1 to 20 nm. The person skilled in the art will immediately realise that the size of the nanoparticle is dependent on the intended function of the conjugate and dependent on the type of nanoparticle. For example, when the nanoparticle is a semiconductor nanoparticle, the size of the nanoparticle will be in the range of 2 to 5 nm, preferably 2.5 to 3 nm. When the nanoparticle is a metallic nanoparticle, the size of the nanoparticle will be in the range of 2 to 6 nm, preferably 3 to 5 nm. When the nanoparticle is a magnetic nanoparticle, the size of the nanoparticle will be in the range of 2 to 20 nm, preferably 10 to 15 nm.

As mentioned above, the nanoparticle conjugate of the present invention utilises insulin as a template to stabilise the metal, metal oxide or semiconductor nanoparticles. Not meaning to be bound by theory, it is thought that the nanoparticles are grown within the insulin template. In other words, it is thought that the nanoparticles are grown in situ. The insulin matrix is used to control the size and the size distribution of the grown nanoparticles. Again, not meaning to be bound by theory, it is thought that the disulfide bonds within insulin stabilise the nanoparticles. This theory is illustrated in Figure 1. The figure on the left illustrates a small section of insulin; the central figure illustrates the formation of inorganic nanoparticles within insulin; the figure on the right illustrates a formed insulin-stabilised nanoparticle.

In an embodiment, in the method of manufacturing the insulin-nanoparticle conjugate, the insulin or insulin derivative is provided in solvent. Preferably the solvent is water or is aqueous based.

In another embodiment, the first nanoparticle or first nanoparticle precursor is provided in a solvent. Preferably the solvent is water or is aqueous based.

In another embodiment, the second nanoparticle or second nanoparticle precursor is provided in a solvent. Preferably the solvent is water or is aqueous based.

Preferably, the solvent for the first nanoparticle or first nanoparticle precursor is the same as the solvent for the second nanoparticle or second nanoparticle precursor. Preferably, the solvent for the insulin or insulin derivative is the same as the solvent for the first nanoparticle or first nanoparticle precursor and/or the solvent for the second nanoparticle or second nanoparticle precursor.

In an embodiment, the method of manufacturing the insulin-nanoparticle conjugate is performed in aqueous media. In an embodiment, the method is performed in a phosphate buffered saline solution. In an embodiment, the method is performed in a TRIS containing buffer solution e.g. TAE or TBE. The benefit of such media is that they are immediately compatible with biological systems. Other media can be used instead, for example, the method of manufacturing the insulin-nanoparticle conjugate may be performed in non-aqueous media, e.g. an organic solvent such as THF, DMF or DMSO. However, if the conjugate is to be used in a biological application, it is preferred that the conjugate be manufactured in aqueous media.

In an embodiment, the method of manufacturing an insulin-nanoparticle conjugate is performed at a temperature of less than 40 ⁰ C, so as to avoid denaturing the insulin. Usually the lower limit is 0 ⁰ C. Preferably the method is performed at about room temperature, e.g. in the range of about 18 to about 28°C.

In an embodiment, the first nanoparticle precursor is a metal precursor. In an embodiment, the metal precursor is a transition metal precursor. The metal precursor can be a metal salt, such as a metal halide (e.g. FeCI ₂ , FeCI ₃ , BaI ₂ or CoCI ₂ ), a chloro metallic acid (e.g. HAuCI ₄ or H ₂ PtCIe), metal hydroxide, metal alkoxide (e.g. Nd(OCH(CH ₃ ) ₂ ) or metal acetate (e.g. [Pd(CH ₃ COO) ₂ ] ₃ or [Pt(CH ₃ COO) ₂ )J ₄ ). In an embodiment, the metal precursor is an Au, Pt, Pd, Co, and Fe precursor. In an embodiment, the metal precursor is an Au, Mn or Fe precursor. In an embodiment, the first nanoparticle precursor is HAuCI ₄ . Other metal precursors that can be used include: molybdenum(VI) oxide, iron(lll) acetylacetonate, chromium(O) hexacarbonyl, bismuth(lll) bromide bis(methylcyclopentadienyl)nickel(ll), antimony(lll) propoxide and aluminum isopropoxide.

In an embodiment, the first nanoparticle precursor and/or second nanoparticle precursors are reacted to form a nanoparticle. In another embodiment, the first nanoparticle precursor and second nanoparticle precursor are reacted together to form a nanoparticle. In either embodiment, this may require the presence of one or more reagents. Typical reagents include oxidants, reductants, acids and alkalis. The term "acids" includes both organic acids and inorganic acids. Examples of organic acids include carboxylic acids such as Ci-io alkyl or aryl carboxylic acids and halogenated derivatives thereof, and C _MO alkyl or aryl sulfonic acids and halogenated derivatives thereof. Examples of inorganic acids include H ₂ SO ₄ , HCI, HNO ₃ and H ₃ PO ₄ . The term "alkali" includes both organic alkalis and inorganic alkalis. Examples of organic alkalis include aliphatic or aromatic amines, such as C _MO alkyl amines. Examples of inorganic alkalis include hydroxides, carbonates, bicarbonates of group I or Il metals or ammonium.

In an embodiment, the nanoparticle precursor is reduced before or after the precursor is combined with insulin. In another embodiment, the nanoparticle precursor is reduced at the same time as the precursor is combined with insulin. In other words, a reducing agent may be added at the same time as, or before or after, the insulin and nanoparticle precursor are combined. In embodiments when the nanoparticle precursor is a metal precursor and the metal precursor is reduced, the resulting insulin-nanoparticle conjugate comprises a metallic nanoparticle and insulin, e.g. an insulin-gold nanoparticle conjugate.

Generally, reducing agents include organic and inorganic reducing agents. Suitable reducing agents include: borane dimethylamine, lithium aluminium hydride (LiAIH ₄ ), sodium borohydride (NaBH ₄ ), stannous chloride (SnCI ₂ ), hydrazine and diisobutylaluminum hydride (DIBAH). Other reducing agents include Lindlar catalyst, oxalic acid (C ₂ H ₂ O ₄ ), formic acid (HCOOH), citric acid and nascent hydrogen.

In an embodiment, the first nanoparticle precursor is a metal oxide precursor. Suitable precursors for the metal oxides described above will be apparent to the skilled person. For example, in one embodiment, the metal oxide precursor is a metal halide (e.g. FeCI ₂ , FeCI ₃ , NiCI ₂ , MgCI ₂ or CuCI ₂ ). In such embodiments, the metal oxide precursor is combined with the insulin in an alkali solution.

In an embodiment, the method further comprises combining a second nanoparticle precursor. Again, in such embodiments, metal oxide precursors are combined with insulin in an alkali solution and the resulting metal oxide co-precipitated therefrom. Preferably, the first and second nanoparticle precursors are different. In an embodiment, the first nanoparticle precursor is FeCI ₂ and the second nanoparticle precursor is FeCI ₃ . In an alternative embodiment, the first nanoparticle precursor is CuCI ₂ , NiCI ₂ or MgCI ₂ and the second nanoparticle precursor is FeCI ₃ . Suitable alkali solutions include solutions of e.g. sodium hydroxide, potassium hydroxide, magnesium hydroxide or ammonium hydroxide. In an embodiment, the alkali solution is ammonium hydroxide.

In an embodiment, the method of manufacturing the insulin-nanoparticle conjugate comprises: combining a first nanoparticle precursor, insulin a second nanoparticle precursor. In an embodiment, the first and second nanoparticle precursors are semiconductor nanoparticle precursors.

Examples of first nanoparticle precursors include: cadmium and zinc halides (e.g. CdCI ₂ , CdBr ₂ , CdI ₂ , ZnCI ₂ , ZnBr ₂ , ZnI ₂ ), cadmium and zinc oxide, cadmium and zinc hydroxide, cadmium and zinc acetates (e.g. Cd(CH ₃ COO) ₂ and Zn(CH ₃ COO) ₂ ). Other transition metal halides and acetates may also be used.

Examples of second nanoparticle precursors include: metal selenides, metal tellurides (e.g. Li ₂ Se, Na ₂ Se, K ₂ Se, MgSe, CaSe, SrSe, Li ₂ Te, Na ₂ Te, K ₂ Te, MgTe, CaTe and SrTe), metal hydrogen selenides and metal hydrogen tellurides (e.g. NaHSe, NaHTe, LiHSe, LiHTe).

In an embodiment, the method further comprises a purification step to remove excess nanoparticles from the insulin-nanoparticle conjugate. For example, this step may involve size exclusion chromatography.

The size and/or the size distribution of the insulin-nanoparticle conjugate can be tailored by a number of means, for example, by adjusting the ratio of insulin : metal. This is demonstrated in, for illustration purposes, example 1. As shown, the method of the present invention provides a reasonable degree of control over the size and size distribution of the generated nanoparticles.

Generally the physical properties of nanoparticles are fully dependent on the size and the size distribution. For example, small Au-nanoparticles of less than 5 nm show interesting optical properties at around 520 nm. The optical properties of semiconductors nanoparticles (such as CdSe nanoparticles) are totally dependent on the particle size, even having a direct relation to the particles size. The magnetic properties of magnetic nanoparticles (such as Fe ₃ O ₄ nanoparticles) exhibit superparamagnetism when the nanoparticles are under approximately 20 nm. As discussed above, one of the aims of the invention is to provide a non-toxic nanoparticle conjugate. It has been surprisingly found that a number of different types of nanoparticles, including metallic, metal oxide, magnetic and semiconductor nanoparticles can be synthesised within insulin and that the resulting insulin-nanoparticle conjugates have substantially no deleterious effect on living cells. In other words, the insulin- nanoparticle conjugates of the present invention have been demonstrated to be nontoxic. This is a significant technical advantage of conjugates of the present invention. This is discussed below in Example 4. As illustrated in Example 4, a range of concentrations has substantially no toxic effect on living cells.

Furthermore, it has also been found that the nanoparticles do not leach from the insulin- nanoparticle conjugates. This is another technical advantage because the conjugates remain stable and retain their integrity for an extended period of time. Although not necessary, it is within the scope of the invention that the insulin-nanoparticle conjugates may be coated with a coating layer. A number of suitable coating materials will be apparent to the skilled person which are compatible with the insulin-nanoparticle conjugate. Suitable coating materials include, for example, poly-L-lactide (PLLA) or poly- lactic-co-glycolic acid (PLGA).

Another technical advantage of the invention is that they are biocompatible nanoparticle conjugates. Surprisingly, it has been found that the conjugates of the present invention are capable of being absorbed by cells. This is achieved without disrupting the cells. This is a further significant advantage, which opens up many therapeutic and diagnostic uses for the conjugates of the invention. This is also discussed in Example 4.

Another technical advantage of the invention is that the activity of insulin is maintained in the insulin-nanoparticle conjugate. As shown in Figure 1 , it is thought that nanoparticles interact with the insulin through disulfide bonds within the insulin. Such an interaction would inevitably cause a weakening or even breaking of at least one disulfide bond within insulin. It would be expected that such a modification of the structure of insulin would affect the activity of insulin. However, despite the inclusion of the nanoparticle within insulin it has been found that the insulin remains active and still performs its function in vivo.

Standard procedures The morphological characterization was performed by Tapping Mode™ scanning force microscopy in air using a Dimension IVa Nanoscope (Digital Instruments). Standard silicon cantilevers were used with a resonance frequency of about 330 kHz, a spring constant of 45 N/m and a tip radius of less than 10nm (Pointprobe SPM Cantilevers, Nanoworld). The operating frequency was chosen to be on the repulsive side of the resonance frequency to increase scanning performance and stability.

For the TEM measurements, thin films were prepared on carbon coated copper grids (400 mesh / AGAR Scientific) by spin-coating the particular solution using a solid substrate support. The copper grid was then peeled off the substrate and analysed in a TECNAI Biotwin (FEI Ltd) transmission electron microscope at 100keV. The instrument was operated at very low beam intensities to prevent electron damage of the polymer samples.

The invention is illustrated by the following Figures, in which:

Figure 1 : the figure on the left illustrates a small section of insulin; the central figure illustrates the formation of inorganic nanoparticles within insulin; the figure on the right illustrates a formed insulin-stabilised nanoparticle.

Figure 2: illustrates typical absorption behaviour of Au-NPs.

Figure 3: (a) is a topographic AFM image of a thin film of insulin : Au = 1 :1 ; (b) is a TEM micrograph of insulin : Au = 1 :1 ; (c) is a particle size distribution of insulin : Au = 1 :1.

Figure 4: (a) is a topographic AFM image of a thin film of insulin : Au = 1 :3; (b) is a TEM micrograph of insulin : Au = 1 :3; (c) is a particle size distribution of insulin : Au = 1 :3.

Figure 5: (a) is a topographic AFM image of a thin film of insulin : Au = 1 :6; (b) is a TEM micrograph of insulin : Au = 1 :6; (c) is a particle size distribution of insulin : Au = 1 :6.

Figure 6: A, B and C illustrate the variation in size and size distribution of the insulin-gold nanoparticle conjugates as the ratio of insulin : gold is varied from 1 :1 , 1 :3 and 1 :6.

Figure 7: show UV-Vis spectra of CdSe nanoparticle conjugates. Figure 8: (a) is a topographic AFM image of a thin film of insulin-CdSe; (b) is a TEM micrograph of insulin-CdSe; (c) is a particle size distribution of insulin-CdSe.

Figure 9: (a) is a TEM micrograph of insulin-stabilized Fe ₃ O ₄ nanoparticles; (b) is corresponding histogram of the particle size distribution.

Figure 10: (a) is the control, (b) insulin : Au 1 :1 , (c) insulin : Au 1 :6, (d) gold stock 0.05M, (e) insulin : Fe ₃ O ₄ 1 :1 , (T) insulin : Fe ₃ O ₄ 1 :6, (g) FeCI ₃ 0.1 M, (h) insulin: CdSe 1 :1 and (i) insulin : CdSe 1 :3.

Figure 1 1 : shows a histogram of the average cell-counts over 22 samples for different types of insulin-stabilized nanoparticles. The data, left to right, follows the same order as the figure legend top to bottom.

Figure 12: shows relative cell counts (for 3T3 fibroblast cells) as a function of particle dose of insulin-stabilized nanoparticles compared to a particle free sample.

Figure 13: are micrographs of sections through 3T3 fibroblast cells after exposure to insulin-stabilized CdSe nanoparticles.

Figure 14: is a topographic AFM image of insulin spin-cast on silicon oxide substrates; the inset shows a section across the AFM image.

The invention will now be illustrated by the following Examples which are intended to demonstrate specific embodiments of the invention but which are not to be construed as limiting.

Example 1 - Insulin-gold nanoparticles conjugates

A gold precursor (HAuCI ₄ ) (3 mg; 9 mg; and 17.8 mg) was added to an aqueous insulin solution (50 mg insulin in 8 ml H ₂ O) in the ratio 1 :1 ; 1 :3 and 1 :6 with respect to the insulin to provide a 1 :1 Au:insulin solution; a 1 :3 Au:insulin solution and a 1 :6 Au:insulin solution. The insulin solution was stirred for 2 hours. Subsequently, the gold precursor was reduced to metallic gold by adding NaBH ₄ (5 times excess to HAuCI ₄ ; for the 1 :1 Au:insulin solution (0.05 ml of 1 M NaBH ₄ ); for the 1 :3 Au:insulin solution (0.13 ml of 1 M NaBH ₄ ); and for the 1 :6 Au:insulin solution (0.26 ml of 1 M NaBH ₄ )). The reaction was conducted at room temperature.

The particle size and size distribution was investigated by means of UV-Vis spectroscopy, dynamic light scattering (DLS) as well as transmission electron-(TEM) and atomic force microscopy (AFM).

The UV-Vis spectra in Figure 2 show typical absorption behaviour of Au-NPs; the characteristic absorption band due to the surface plasmon resonance (SPR) depends on the particle size and composition as well as the nature of the stabilizer. The insulin- stabilized gold nanoparticles show similar absorption behaviour with SPR bands around 530nm. A light red-shift can be observed for higher gold contents, indicating the presence of larger Au-NPs.

The particle size and size distribution was further verified by TEM analysis. Additionally, the morphology of spin-coated thin films was investigated with respect to the insulin/Au ratio. Figure 3-5 show AFM and TEM data of insulin-stabilized Au-NPs at different insulin/Au ratios respectively.

Figure 3 (a) is a topographic AFM image of a thin film of insulin : Au = 1 :1. Figure 3 (b) is a TEM micrograph of insulin : Au = 1 :1. Figure 3 (c) is a particle size distribution of insulin : Au = 1 :1.

Figure 4 (a) is a topographic AFM image of a thin film of insulin: Au = 1 :3. Figure 4 (b) is a TEM micrograph of insulin : Au = 1 :3. Figure 4 (c) is a particle size distribution of insulin : Au = 1 :3.

Figure 5 (a) is a topographic AFM image of a thin film of insulin : Au = 1 :6. Figure 5 (b) is a TEM micrograph of insulin : Au = 1 :6. Figure 5 (c) is a particle size distribution of insulin : Au = 1 :6.

Spherical aggregates with a non-regular structure are obtained, when the gold nanoparticles were generated within the insulin. The size of the aggregates increases with the ratio Au : insulin. The AFM images indicate that, consistent with the UV-Vis data, larger particles are formed with increasing metal content, leading to rougher films. The TEM micrographs in figures 3-5 show insulin stabilised Au-NPs at different Au : insulin ratios as well as the corresponding particle size distribution. At an insulin : Au ratio of 1 :1 , a mean particle diameter of 3nm was determined. At an insulin : Au ratio of 1 :3, a mean particle diameter of 4.5nm was determined. At an insulin : Au ratio of 1 : 6, a mean particle size of 3.6nm was determined.

Dynamic light scattering can be used to determine size and size distributions of particles in solution. Figures 6A, 6B and 6C illustrate the variation in size and size distribution of the insulin-gold nanoparticle conjugates as the ratio of insulin : gold is varied from 1 :1 , 1 :3 and 1 :6.

Example 2 - Insulin-CdSe nanoparticle conjugates

Insulin stabilized CdSe nanoparticles are prepared in aqueous solution at room temperature through the reaction between Cd(CH ₃ COO) ₂ and NaSeH under N ₂ atmosphere. Upon addition of NaSeH (1 :1 0.44 ml of 0.01 M NaHSe )/(1 :6 2.6 ml of 0.01 M NaHSe )to aqueous solutions of Cd(CH ₃ COO) ₂ (1 :1 2.3 mg/ 1 :6 14 mg) in the presence of insulin (1 mM), the colour changes to bright yellow indicating the formation of small CdSe nanoparticles, which are stabilized by the insulin. Although the reaction may proceed fairly quickly revealing a colour change from colourless to yellow on adding NaHSe, in practice, it was better to wait for 15 mins after adding the NaHSe to ensure that the reaction had been completed.

The UV-Vis spectra of these particles (see Figure 7) show two characteristic shoulders at λ= 550 nm and λ= 405 nm respectively, signifying the existence of semiconductor nanoparticles in nanoparticles having an insulin : CdSe ratio of 1 :6. The optical properties of the insulin-stabilized CdSe nanoparticles were further investigated by photoluminescene (PL) spectroscopy. The results signify the fluorescent properties of the CdSe particles. As shown in Figure 7, a strong and narrow emission band is observed at λ _E = 600 nm with FWHM = 53nm. The PL emission band is slightly asymmetric, which may indicate asymmetric particle size distribution, e.g. larger particles.

However, the TEM micrograph (Figure 8 (b)) and corresponding histogram of the particle size distribution shows CdSe nanoparticles with a narrow size distribution and a mean particle diameter of 2.8 nm. The measurement effectively considers the dominant particle size whose mean diameter average is 2.8 nm. A few large particles are excluded from the histogram calculations but this is not considered significant.

Spin-coated thin films of insulin-CdSe conjugates show a highly compact structure of spherical nanoparticles (Figure 8 (a)). Figure 8 (a) is a topographic AFM image of a thin film of insulin-CdSe. Figure 8 (b) is a TEM micrograph of insulin-CdSe. Figure 8 (c) is a particle size distribution of insulin-CdSe.

Example 3 - InSuMn-Fe ₃ O ₄ nanoparticle conjugates

Magnetic Fe ₃ O ₄ nanoparticles were fabricated by pipetting aqueous solutions of FeCI ₃ (ratio 1 :1 , 4.7 mg and ratio 1 :6, 28 mg) and FeCI ₂ (ratio 1 :1 , 1.7mg and ratio 1 :6, 10.2 mg) (molar ratio 2:1 ) into de-oxidised aqueous solutions of sodium hydroxide 500 ml of 2M Na ₄ OH in the presence of insulin (50 mg insulin 50 ml H ₂ O). The reaction was conducted at room temperature. Upon mixing the components, the originally colourless solution turns yellow-brown signifying the formation of nanoparticles.

Figure 9 (a) is a TEM micrograph of insulin-stabilized Fe ₃ O ₄ nanoparticles and (b) corresponding histogram of the particle size distribution

The TEM micrograph in figure 9 (a) shows insulin-stabilized Fe ₃ O ₄ nanoparticles, which self-assemble into hexagonal patterns. We believe this arrangement is driven by the magnetic interaction among the nanoparticles. The size-distribution histogram, displayed in figure 9 (b), indicates a relatively wide size-distribution with a mean particle diameter of 17nm and a standard deviation of 5.7nm.

Example 4 - Cytotoxicity and Metabolic Activity

Cytotoxicity tests were performed using 3T3 fibroblast cells stained with propidium iodine. Fluorescence microscopy allows cell counts for living cells in the presence of insulin-stabilized nanoparticles to evaluate their toxicity. Figure 10 shows selected confocal microscopy images of living 3T3 fibroblast cells 24h after the insertion of insulin- stabilized nanoparticles.

Figure 10 shows a number of cytotoxicity test images of 3T3 cells in the presence of insulin-stabilized nanoparticles. Figures 10 (a) - (i) are described next: (a) is the control, (b) insulin : Au 1 :1 , (c) insulin : Au 1 :6, (d) gold stock 0.05M, (e) insulin : Fe ₃ O ₄ 1 :1 , (f) insulin : Fe ₃ O ₄ 1 :6, (g) FeCI ₃ 0.1 M, (h) insulin: CdSe 1 :1 and (i) insulin : CdSe 1 :3. 60μg of nanoparticles were placed into each cell culture well plate (concentration of 1x10 ⁴ cells per ml).

Markedly, most of the tested nanoparticle systems show only a weak reduction in cell count after 24h, whereas in the case of metal precursors (Figure 10 (d) and (g)) no living cells can be observed. Hence the insulin-stabilized nanoparticles show less toxicity than the metal precursors. For the CdSe nanoparticles (Figures 10 (h) and (f)) the cell count is reduced significantly; however, little difference is observed for higher dose.

A histogram of the average cell-counts over 22 samples for different types of insulin- stabilized nanoparticles is shown in Figure 11. The cell count of all nanoparticle systems reduced by less than 50% of the control sample, whereas the cell counts for precursors reduces by more than 80%. Figure 1 1 illustrates the cell count of the cytotoxicity test of insulin-stabilized nanoparticles using 3T3 fibroblast cells. The error bars show the standard deviation of the distribution averaged over 22 samples.

The metabolic activity of 3T3 fibroblast cells in the presence of insulin-stabilized nanoparticles was evaluated by measuring the proliferation rate over 24h using the Alamar Blue assay test.

The Alamar Blue (AB) assay is a simple, non-toxic, one-step procedure, quite amenable to high throughput, whereby metabolic activity of the cells results in the chemical reduction of AB, which is a water soluble dye. AB is reduced by FMNH2, FADH2, NADH and NADPH, i.e. cellular reduction equivalents. Alamar blue both fluoresces and changes colour in response to chemical reduction, and the extent of the conversion is a reflection of cell viability since continued growth maintains a reduced environment while inhibition of growth maintains an oxidized environment.

Figure 12 shows relative cell counts (for 3T3 fibroblast cells) as a function of particle dose of insulin-stabilized nanoparticles compared to a particle free sample.

Commonly for all types of nanoparticle, the cell count decreases with increasing nanoparticle dose, signifying a negative proliferation rate. Markedly, at very low particle doses for Au- and CdSe-NPs, the cell count is significantly higher than the control sample. This observation gives rise to the conclusion that insulin-stabilized nanoparticles at low doses can be considered non-toxic. The low toxicity of insulin-stabilized nanoparticles may enable their use as novel delivery or labelling systems. In order to investigate the potential of towards such applications, nanoparticle uptake on living cells was studied using TEM microscopy to locate the nanoparticles within the cells. The micrographs are shown in figure 13 which show sections through 3T3 fibroblast cells after exposure to insulin-stabilized CdSe nanoparticles.

The nanoparticles are mainly located at the outer rim of the cell membrane; only few nanoparticles were found within the cells. These preliminary results show that the insulin-stabilized nanoparticles are taken up in the cell; hence, the key function of the insulin is not altered by the nanoparticles.

Example 5 - Insulin

Aqueous solutions of insulin (1 mM bovine pancreas, C254H377N65O75S6,) were prepared at pH 2.6. The AFM height image in figure 14 shows the topography of thin insulin films spin-coated on native oxidised silicon substrates. Thin films of insulin show porous morphologies with a non-regular structure when spin-cast on SiC> ₂ from aqueous solution. Figure 14 is a topographic AFM image of insulin spin-cast on silicon oxide substrates; the inset shows a section across the AFM image.