Nanoparticle production

Abstract

The present invention provides a process for producing nanoparticles, comprising: providing a first liquid comprising a metal salt and at least one species of ligand having a functional group capable of binding to a metal surface, providing a second liquid comprising a reducing agent; providing at least one liquid droplet generator operable to generate liquid droplets, causing the at least one liquid droplet generator to form liquid droplets of the first liquid, passing the liquid droplets through a gas to contact the second liquid so as to cause the metal salt and the at least one species of ligand to come into contact with the reducing agent, thereby causing self-assembly of nanoparticles, said nanoparticles having a core of said metal and a corona comprising a plurality of said ligands covalently bound to the core. Also provided are nanoparticles produced by the process of the invention and use of such nanoparticles in medicine.

Claims

1. A process for producing nanoparticles, comprising: providing a first liquid comprising a metal salt and at least one species of ligand having a functional group capable of binding to a metal surface, providing a second liquid comprising a reducing agent, wherein the second liquid is provided as a jet passing through a gas and wherein the jet of second liquid is not in contact with any wall or channel for at least part of its length; providing at least one liquid droplet generator operable to generate liquid droplets, said at least one liquid droplet generator comprising a piezoelectric component, causing the at least one liquid droplet generator to form liquid droplets of the first liquid, passing the liquid droplets through the gas to contact the second liquid so as to cause the metal salt and the at least one species of ligand to come into contact with the reducing agent, thereby causing self-assembly of nanoparticles, said nanoparticles having a core of said metal and a corona comprising a plurality of said ligands covalently bound to the core.

2. The process according to claim 1, wherein the metal salt comprises a salt of Au, Ag, Cu, Pt, Pd, Fe, Co, Gd, Eu, or Zn.

3. The process according to claim 2, wherein the metal salt comprises HAuCl.sub.4.

4. The process according to claim 1, wherein the at least one species of ligand comprises a carbohydrate moiety and wherein the carbohydrate moiety comprises glucose, galactose, glucosamine, N-acetylglucosamine, mannose, fucose and/or lactose, or a glycoside thereof.

5. The process according to claim 1, wherein the at least one species of ligand comprises an oligo ethylene glycol or a polyethylene glycol.

6. The process according to claim 1, wherein the at least one species of ligand comprises a therapeutic or bioaffecting agent.

7. The process according to claim 6, wherein the at least one species of ligand comprises a compound selected from the group consisting of: doxorubicin, irinotecan, platinum (II), platinum (IV), temozolomide, chlorotoxin, carmustine, camptothecin, docetaxel, sorafenib, maytansine, a maytansinoid (e.g. maytansinoid DM1 or maytansinoid DM4), monomethyl auristatin E (MMAE) and a histone deacetylase (HDAC) inhibitor.

8. The process according to claim 1, wherein the droplet generators operate in drop-on-demand mode.

9. The process according to claim 1, wherein a signal generator supplies an electric field to the piezoelectric component.

10. The process according to claim 1, wherein the frequency of liquid droplet generation per liquid droplet generator outlet is in the range 0.1 to 100 kHz.

11. The process according to claim 1, wherein said liquid droplets have an individual droplet volume in the range 1 to 100 pL.

12. The process according to claim 1, wherein the process is for forming meta-stable nanoparticles.

13. The process according to claim 1, wherein the process further comprises a step of adding further ligands to a solution comprising the nanoparticles and wherein the further ligands are different from said at least one species of ligand in the first liquid.

14. The process according to claim 1, further comprising collecting the nanoparticles by separating the nanoparticles from the second liquid.

15. The process according to claim 14, further comprising formulating or packaging the nanoparticles into a pharmaceutical composition or delivery form.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows a schematic depiction of an embodiment of the present invention. A solution comprising gold salt (Au.sup.3+) and ligands is added dropwise to a vessel containing an excess of reducing agent (BH.sub.4.sup.−). As Au.sup.3+ is added dropwise it meets an excess of BH.sub.4.sup.−, hence all gold atoms in the droplet should be reduced to completion (without the need for mixing). Providing ligands are available at a suitable concentration, nanoparticles are produced.

(2) FIG. 2 shows a schematic depiction of an embodiment of the present invention employing a piezoelectric droplet generator. The insert panel shows a high-speed photograph of the droplets ejected from the orifice of the piezoelectric droplet generator. A solution of Au.sup.3+ and Glucose-C2 dimer (2′-thioethyl-β-D-glucopyranoside provided as a disulphide dimer) in DMSO was ejected from the piezoelectric droplet generator as a stable stream of droplets (ejection conditions: 44V, 13 μs pulse, 50° C., ν=4000 Hz) into a solution containing NaBH.sub.4.

(3) FIG. 3 shows the absorbance spectrum of gold nanoparticles having a corona of glucose-C2 ligands, produced by the method of the present invention. Optical density (y-axis) is plotted against wavelength in nm (x-axis). The absorbance spectrum shows little or no evidence of a surface plasmon resonance band.

(4) FIG. 4 shows four size distribution traces determined by dynamic light scattering (DLS), in which the percentage (y-axis) by volume of nanoparticles in the population are plotted against the nanoparticle diameter in nm (x-axis). The average diameter was found to be 2.7 nm. The nanoparticles for each of the four traces were produced by a method embodiment of the present invention in which excess (3× mole equivalent) of glucose-C2 disulphide was added to the solution containing the nanoparticles after droplet ejection was complete (20 minutes) and the resulting mixture stirred for 1 hour.

(5) FIG. 5 shows a schematic illustration of a continuous reductant flow embodiment of the method of the present invention. A horizontal jet of a solution containing sodium borohydride is shown from left to right. A solution of gold salt (Au.sup.3+) and ligands is ejected from the piezoelectric droplet generator to form a downward stream of droplets passing through air before making contact with the jet containing sodium borohydride. Reduction of the gold salt and formation of gold nanoparticles with a corona of ligands occurs rapidly, and the jet stream of liquid, now containing formed nanoparticles, enters an elbow joint section of tubing (shown at the right-hand end of the jet) and is directed downwards to a collecting vessel.

(6) FIG. 6 shows a transmission electron microscopy (TEM) image of nanoparticles produced according the process of the present invention. A 50 nm scale bar is shown. The dark spots show generally spherical gold-core nanoparticles. The batch of nanoparticles were produced via the piezoelectric droplet generator that ejected droplets of a solution of Au.sup.3+ and Glucose-C2 dimer in DMSO into a solution containing NaBH.sub.4.

DETAILED DESCRIPTION OF THE INVENTION

(7) In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below.

(8) Nanoparticles

(9) As used herein, “nanoparticle” refers to a particle having a nanomeric scale, and is not intended to convey any specific shape limitation. In particular, “nanoparticle” encompasses nanospheres, nanotubes, nanoboxes, nanoclusters, nanorods and the like. In certain embodiments the nanoparticles and/or nanoparticle cores contemplated herein have a generally polyhedral or spherical geometry.

(10) Nanoparticles comprising a plurality of carbohydrate-containing ligands have been described in, for example, WO 2002/032404, WO 2004/108165, WO 2005/116226, WO 2006/037979, WO 2007/015105, WO 2007/122388, WO 2005/091704 (the entire contents of each of which is expressly incorporated herein by reference) and such nanoparticles may find use in accordance with the present invention.

(11) As used herein, “corona” refers to a layer or coating, which may partially or completely cover the exposed surface of the nanoparticle core. The corona includes a plurality of ligands which generally include at least one carbohydrate moiety, one surfactant moiety and/or one glutathione moiety. Thus, the corona may be considered to be an organic layer that surrounds or partially surrounds the metallic core. In certain embodiments the corona provides and/or participates in passivating the core of the nanoparticle. Thus, in certain cases the corona may include a sufficiently complete coating layer substantially to stabilise the semiconductor or metal-containing core. However, it is specifically contemplated herein that certain nanoparticles having cores, e.g., that include a metal oxide-containing inner core coated with a noble metal may include a corona that only partially coats the core surface. In certain cases the corona facilitates solubility, such as water solubility, of the nanoparticles of the present invention.

(12) Nanoparticles are small particles, e.g. clusters of metal or semiconductor atoms, that can be used as a substrate for immobilising ligands.

(13) Preferably, the nanoparticles have cores having mean diameters between 0.5 and 50 nm, more preferably between 0.5 and 10 nm, more preferably between 0.5 and 5 nm, more preferably between 0.5 and 3 nm and still more preferably between 0.5 and 2.5 nm. When the ligands are considered in addition to the cores, preferably the overall mean diameter of the particles is between 2.0 and 20 nm, more preferably between 3 and 10 nm and most preferably between 4 and 5 nm. The mean diameter can be measured using techniques well known in the art such as transmission electron microscopy.

(14) The core material can be a metal or semiconductor (said semiconductor optionally comprising metal atoms or being an organic semiconductor) and may be formed of more than one type of atom. Preferably, the core material is a metal selected from Au, Fe or Cu. Nanoparticle cores may also be formed from alloys including Au/Fe, Au/Cu, Au/Gd, Au/Fe/Cu, Au/Fe/Gd and Au/Fe/Cu/Gd, and may be used in the present invention. Preferred core materials are Au and Fe, with the most preferred material being Au. The cores of the nanoparticles preferably comprise between about 100 and 500 atoms (e.g. gold atoms) to provide core diameters in the nanometre range. Other particularly useful core materials are doped with one or more atoms that are NMR active, allowing the nanoparticles to be detected using NMR, both in vitro and in vivo. Examples of NMR active atoms include Mn.sup.+2, Gd.sup.+3, Eu.sup.+2, Cu.sup.+2, V.sup.+2, Co.sup.+2, Ni.sup.+2, Fe.sup.+2, Fe.sup.+3, and lanthanides.sup.+3, or quantum dots.

(15) Nanoparticle cores comprising semiconductor compounds can be detected as nanometre scale semiconductor crystals are capable of acting as quantum dots, that is they can absorb light thereby exciting electrons in the materials to higher energy levels, subsequently releasing photons of light at frequencies characteristic of the material. An example of a semiconductor core material is cadmium selenide, cadmium sulphide, cadmium tellurium. Also included are the zinc compounds such as zinc sulphide.

(16) In some embodiments, the nanoparticle or its ligand comprises a detectable label. The label may be an element of the core of the nanoparticle or the ligand. The label may be detectable because of an intrinsic property of that element of the nanoparticle or by being linked, conjugated or associated with a further moiety that is detectable. Preferred examples of labels include a label which is a fluorescent group, a radionuclide, a magnetic label or a dye. Fluorescent groups include fluorescein, rhodamine or tetramethyl rhodamine, Texas-Red, Cy3, Cy5, etc., and may be detected by excitation of the fluorescent label and detection of the emitted light using Raman scattering spectroscopy (Y. C. Cao, R. Jin, C. A. Mirkin, Science 2002, 297: 1536-1539). In some cases, the detectable label may comprise fluorescein isothiocyanate (FITC). In certain cases, the detectable label (e.g. FITC) may be covalently linked to the core of the nanoparticle, e.g. via a linker.

(17) In some embodiments, the nanoparticles may comprise a radionuclide for use in detecting the nanoparticle using the radioactivity emitted by the radionuclide, e.g. by using PET, SPECT, or for therapy, i.e. for killing target cells. Examples of radionuclides commonly used in the art that could be readily adapted for use in the present invention include .sup.99mTc, which exists in a variety of oxidation states although the most stable is TcO.sup.4−; .sup.32P or .sup.33P; .sup.57Co; .sup.59Fe; .sup.67Cu which is often used as Cu.sup.2+ salts; .sup.67Ga which is commonly used a Ga.sup.3+ salt, e.g. gallium citrate; .sup.68Ge; .sup.82Sr; .sup.99Mo; .sup.103Pd; .sup.111In which is generally used as In.sup.3+ salts; .sup.1251 or .sup.131I which is generally used as sodium iodide; .sup.137Cs; .sup.153Gd; .sup.153Sm; .sup.158Au; .sup.186Re; .sup.201Tl generally used as a Tl.sup.+ salt such as thallium chloride; .sup.39Y.sup.3+; .sup.71Lu.sup.3+; and .sup.24Cr.sup.2+. The general use of radionuclides as labels and tracers is well known in the art and could readily be adapted by the skilled person for use in the aspects of the present invention. The radionuclides may be employed most easily by doping the cores of the nanoparticles or including them as labels present as part of ligands immobilised on the nanoparticles.

(18) Actives

(19) As used herein the term “bioaffecting agent” is intended to encompass drugs and pro-drugs that exert an effect on a biological system, preferably a therapeutic effect. Class of active agent contemplated herein include small molecule organic compounds, peptides, polypeptides and nucleic acids. Particular examples include: chemotherapeutic agents (e.g. temozolomide, irinotecan, chlorotoxin, carmustine, platinum(IV), platinum(II), camptothecin, doxorubicin, docetaxel Maytansine, Maytansinoids (e.g. DM1 and DM4), monomethyl auristatin E (MMAE) and/or histone deacetylase (HDAC) inhibitors such as Panobinostat, Vorinostat, Romidepsin and Chidamide); peptides or polypeptides (e.g. insulin, GLP-1, amylin, exenatide, octreotide, teriparatide, glucagon, a cytokine, and/or an antibody); DNA or RNA (including, e.g. siRNA).

(20) The following is presented by way of example and is not to be construed as a limitation to the scope of the claims.

EXAMPLE

Example 1—Piezoelectric Ejection-Based Synthesis of Gold Nanoparticles

(21) A solution containing HAuCl.sub.4 ([Au.sup.3+] of 1 mg/ml), glucose-C2-disulphide (Au.sup.3+: Glucose-C2-disulphide 1:3 mole ratio) in DMSO was ejected from a single piezoelectric droplet generator using the following ejection conditions: 44V, 13 μs pulse, 50° C., n=4000 Hz. The droplets were ejected into a 12 mL vial containing 8 mL of an aqueous solution of NaBH.sub.4 at a concentration of 0.05M.

(22) The resulting nanoparticles were isolated by Amicon ultracentrifugation after droplet ejection was complete. Meta-stable nanoparticles of appropriate size were produced (demonstrated by a very small plasmon band). It was found that these nanoparticles “ripened” overnight with the size increasing to a diameter of >8 nm (red colour observed). The present inventors believe that this indicates that the gold nanoparticle core was not fully capped with ligand at the point when the nanoparticles were isolated (i.e. the nanoparticles were initially meta-stable).

(23) A further batch of gold nanoparticles was then produced using the above-described reactant concentrations and droplet ejection conditions. After ejection was complete (20 minutes), a further 3× mole equivalents of Glucose C2 disulfide was added to the solution containing the nanoparticles. The mixture was stirred for 1 hour.

(24) Stable nanoparticles produced of appropriate size (average diameter of 2.7 nm by dynamic light scattering (DLS)—see FIGS. 3 and 4. These were visualised by transmission electron microscopy—see FIG. 6.

(25) Further experiments were then carried out to investigate the scalability of the process. In particular, increasing Au.sup.3+ and ligand concentration in the first liquid increases Au.sub.(s) yield per unit time. It was found that Au.sup.3+ concentrations of 5 mg/mL, 10 mg/mL, 25 mg/mL and 30 mg/mL (and correspondingly increased ligand concentrations to maintain a 1:3 Au:ligand ratio) produced stable gold nanoparticles with essentially no plasmon resonance band observable. The following calculation provides an estimated yield achievable using a 512 nozzle print-head array at 4 kHz droplet generation frequency:
4000 Hz×512 nozzles=2.05 MHz

(26) $\begin{array}{c}\mathrm{Droplet}\mathrm{volume}=42\mathrm{pL}\\ =0.086\mathrm{mL}\text{/}s\\ =5.16\mathrm{mL}\text{/}\min \\ =309\mathrm{mL}\text{/}\mathrm{hr}\end{array}$

(27) At [Au.sup.3+]=30 mg/mL (and assuming ˜90% yield), this would give 8.34 g Au.sub.(s)/hr.

(28) It should be noted however, that the above calculation provides representative figures and should not be taken as a maximum yield for the process of the present invention.

(29) All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

(30) The specific embodiments described herein are offered by way of example, not by way of limitation. Any sub-titles herein are included for convenience only, and are not to be construed as limiting the disclosure in any way.