WO2020240176A1

WO2020240176A1 - Dispersion

Info

Publication number: WO2020240176A1
Application number: PCT/GB2020/051278
Authority: WO
Inventors: Houzheng WU; Tao Sun
Original assignee: Loughborough University
Priority date: 2019-05-28
Filing date: 2020-05-27
Publication date: 2020-12-03
Also published as: GB201907468D0

Abstract

There is provided the use of a virus to disperse particles, such as nanoparticles, in a liquid medium; a process for dispersing particles; and a composition comprising a plurality of particles and a plurality of virus; wherein at least one virus is bonded to an outer surface of a particle. The process may comprise providing a first liquid medium comprising the particles; providing a second liquid medium comprising the virus; and combining the first liquid medium and the second liquid medium to yield the mixture having the particles suspended therein. The first liquid medium may be an aqueous medium having a pH of from 5 to 9. The particles may comprise alumina and / or calcium carbonate particles. The virus may be a bacteriophage of the Myoviridae and/or Inoviridae family.

Description

DISPERSION

The present invention relates to a process for dispersing particles, in particular a process for dispersing nanoparticles in a liquid medium, and compositions comprising liquid-dispersible particles.

Background to the invention

There is an ongoing desire to use small particles, such as nanoparticles, in a range of applications such as advanced ceramics, coatings, adhesives and polymeric nanocomposites. It would be very useful to employ such particles in a liquid medium during processing, or in a final product. However it is challenging to keep small particles in suspension since they have a tendency to agglomerate, leading to sedimentation.

Particles in suspension experience attractive forces (e.g. van der Waals forces) and repulsive forces (e.g. electrostatic interactions). If the repulsive forces exceed the attractive forces then a stable system results. The tendency of small particles to agglomerate is thought to be due to their high specific surface area, i.e. surface area per unit mass / volume. Small particles have a high ratio of surface free energy relative to bulk free energy and agglomeration reduces the surface free energy relative to the bulk free energy.

Conventional methods to reduce or retard sedimentation include sonication i.e. the use of sound energy to agitate particles within a sample; increasing the viscosity of a dispersion medium; and the use of a dispersant, such as a surfactant.

Summary of the Invention

According to a first aspect of the invention there is provided the use of a virus (such as bacteriophage) to disperse particles in a liquid medium.

The present inventors recognised that current methods to avoid agglomeration and sedimentation were unsatisfactory. The inventors have determined that a virus (such as a bacteriophage) can be used for this novel purpose and thereby provide a stable suspension of particles in the liquid medium. This is a completely different approach from the usual mechanical and chemical methods used to disperse particles and thereby keep them in suspension.

A virus is a small infectious agent that replicates only inside the living cells of an organism. For example, the virus may be an animal virus, a plant virus, a bacterial virus or an archaeal virus. While not inside an infected cell or in the process of infecting a cell, viruses exist in the form of independent particles. These viral particles, also known as virions, consist of: (i) the genetic material made from either DNA or RNA, long molecules that carry genetic information; (ii) a protein coat, called the capsid, which surrounds and protects the genetic material; and in some cases (iii) an envelope of lipids that surrounds the protein coat. The virus may be in the form of a virion.

Animal viruses are viruses that infect animals, including vertebrates (such as humans) and invertebrates (such as insects).

The virus may be a vertebrate virus, such as adenovirus. Adenoviruses have long been a popular viral vector for gene therapy.

The virus may be an insect-specific virus (ISV), such as baculovirus. An ISV is incapable of infecting vertebrates.

Plant viruses are viruses that infect plants. Plant viruses are not generally considered harmful to humans. Tobacco mosaic virus (TMV) is a positive-sense single stranded RNA virus in genus Tobamovirus that infects a wide range of plants, especially tobacco and other members of the family Solanaceae. TMV was the first virus ever to be discovered. The virus may be a plant virus, such as TMV.

The virus may be a wild-type virus or a mutated (genetically modified) virus.

The virus may be an attenuated virus or a killed virus. An attenuated virus is live, but has reduced virulence. A bacteriophage (also known as a phage) is a virus that infects and replicates within bacteria and archaea. A schematic diagram of typical phage 10 is shown in figure 1. The phage 10 has a head or capsid 12 which is a protein shell often in the shape of an icosahedron. The capsid contains the viral genome (e.g. DNA) 14. Bacteriophages, unlike most other viruses, remain attached to the outer surface of a cell during infection and have a tail to deliver the genome to the host. The tail can comprise a tail sheath 16 and tail fibres 18.

Without being bound by theory, the inventors propose that the virus (e.g. phage) interacts with the outer surface of the particles and thereby prevent the particles from agglomerating. Moreover where agglomeration is already underway, the inventors believe that the virus (e.g. phage) can fit in spaces between particles causing the agglomerate to separate.

WO2010/007723 describes a peptide capable of binding to ceramic material. The peptide can be presented by a phage clone and is said to bind specifically to barium titanate (BT) particles. It will be appreciated that there is no disclosure of the use of bacteriophage to disperse particles in a liquid medium. The abstract does not mention dispersion and the phage clone is employed simply to display the peptide.

According to a second aspect of the invention there is provided a process for dispersing particles in a liquid medium, the process comprising

combining a virus (e.g. bacteriophage) and particles in a liquid medium to form a mixture having the particles suspended therein.

The virus (e.g. bacteriophage) may be a wild-type virus.

The bacteriophage may be of the Myoviridae and/or Inoviridae family.

Combining the virus (e.g. bacteriophage) and the particles in a liquid medium may comprise providing a first liquid medium comprising the particles;

providing a second liquid medium comprising the virus (e.g. bacteriophage); and combining the first liquid medium and the second liquid medium to yield the mixture having the particles suspended therein. The first liquid medium, the second liquid medium and/or the mixture may be sonicated. For example, ultrasonic frequencies may be applied by means of an ultrasonic bath or an ultrasonic probe.

The process may additionally comprise isolating the particles from the suspension. The resulting particles having virus (e.g. bacteriophage) bonded thereto may be liquid- dispersible.

The first liquid medium may be the same as, or different from the second liquid medium. Both the first and second liquid media may be aqueous.

According to a third aspect of the invention there is provided a composition comprising a plurality of particles and a plurality of virus (e.g. bacteriophage), wherein at least one virus (e.g. bacteriophage) is bonded to an outer surface of a particle.

A bacteriophage has a head that contains the viral genome and a tail to deliver the genome to the host. The bacteriophage may be bonded to the outer surface of the particle by means of its head or its tail. Multiple bacteriophage may be bonded to the outer surface of the particle.

The composition of the third aspect may be obtained by the process of the second aspect.

The composition may additionally comprise a liquid medium and the particles may be suspended in the liquid medium.

The particles may be isolated particles having the virus (e.g. bacteriophage) bonded thereto.

Detailed Description of the Invention

Particles The particles for use in the invention are solid and insoluble in the liquid medium at standard ambient temperature and pressure (SATP, 25°C, l OOkPa) . By insoluble, we mean that less than O. lg of the solute (i.e . particle material) is soluble in 100ml of the solvent (i.e . the liquid medium) at SATP.

The particles may be inorganic particles, such as ceramic particles. Ceramic particles are heat-resistant and made from both metallic and non-metallic compounds.

The particles may comprise an oxide, a nitride, a carbonate and/or a carbide, e.g. alumina (A1₂0₃), aluminium oxynitride, silica (Si0₂), titanium oxide (Ti0₂), zinc oxide (ZnO), zirconium oxide (Zr0₂), iron oxide, boron nitride (BN), barium titanate (BaTi0₃), calcium carbonate (CaC0₃), boron carbide, silicon carbide or silicon nitride.

For example, the particles may comprise alumina, aluminium oxynitride, silica, boron nitride, calcium carbonate, boron carbide, silicon carbide and/or silicon nitride .

The particles may comprise or consist of an oxide, such as alumina, titanium oxide, zinc oxide, zirconium oxide, and/or iron oxide . For example, the particles may comprise one or more oxides such as alumina and/or iron oxide.

The particles may comprise or consist of alumina particles. Alumina nanoparticles are thermodynamically stable over a wide range of temperatures. They are used in electronics, optoelectronics, armours, thin-film coating, waste-water treatment, catalysis, nanocomposites, reinforcement, abrasive materials, absorbents, polymer modification, heat-transfer fluids and in biological applications, such as drug delivery, biosensors and biofiltration. Alumina nanoparticles have a strong tendency to agglomerate extensively because of their high specific surface area and grain growth.

The particles may comprise or consist of a carbonate, such as calcium carbonate particles.

No treatment of the particles is required, e .g. to change surface properties such as zeta potential etc. The particles may be untreated, e.g. the particles may not be treated with an aminosilane. The particles may be described with reference to their particle size. The particle size may be a geometric weight mean value for the particle size (appropriate for the approximately log normal distribution which is often found with such particles).

The particle size may alternatively be determined by laser diffraction and may be measured using a laser diffraction machine, such as those available from Malvern Instruments Ltd, e.g. a Mastersizer 3000 machine, optionally with Hydro SV dispersion unit. The standard ISO 13320 :2009 “Particle Size Analysis - Laser Diffraction Methods” may be employed.

The particle size may alternatively be determined by X-ray sedimentation and may be measured using a X-ray disc centrifuge, such as those available from Brookhaven, e.g. a BI-XDC machine.

The particles may have a particle size (e.g. D90) of 100 micron ( 100 pm) or less, 10 micron or less or 5 micron or less and/or the particles may have a particle size (e.g. D90) of 1 micron or more, or 10 micron or more. The invention is particularly useful for nanoparticles, since smaller particles are more likely to agglomerate. Hence the particles may have a particle size (e.g. D90) of l OOOnm or less, 800nm or less, 600nm or less, 400nm or less, 200nm or less or l OOnm or less and/or the particles may have a particle size (e.g. D90) of 5nm or more, l Onm or more, 50nm or more, l OOnm or more, 200nm or more, 300nm or more or 400nm or more. For example, the particles may have a particle size (D90) of 5 to l OOnm. The examples include particles having a particle size of less than l OOnm.

Without being bound by theory, the inventors propose that multiple phage attach to each particle. As such, the particles are typically larger than the virus (phage). For example, the particles may be l Onm or more .

The particles may be described with reference to their specific surface area. The specific surface area may be determined using the Brunauer, Emmett and Teller method (BET method) as described in J. Am. Chem. Soc., 1938, 60, 309. The particles may have a BET specific surface area of 5m²/g or more, such as from 10 to 20m²/g. The examples include alumina particles having a BET of 15m²/g.

Bacteriophage

Bacteriophage (phage) are viruses that cannot infect mammalian cells and specifically target bacteria. There are many different phages, each able to infect one species host bacteria or in some cases a single strain of bacterium. Examples of bacteriophage families are listed below ds = double stranded, ss = single stranded

The phage may comprise a phage of the Myoviridae family, such as T4 phage. Escherichia virus T4 is a species of bacteriophage that infects Escherichia coli bacteria.

The phage may comprise a phage of the Inoviridae family, such as M13. M13 is a virus that infects the bacterium Escherichia coli. It is composed of a circular single- stranded DNA molecule encased in a thin flexible tube made up of about 2700 copies of a single protein called P8 a major coat protein. The ends of the tube are capped with minor coat proteins. Infection starts when the minor coat protein P3 attaches to the receptor at the tip of the F pilus of the bacterium.

The phage may comprise a phage of the Myoviridae or Inoviridae family. The phage may be a wild-type phage or a mutated (genetically modified) phage. T4 and M13 are examples of wild-type phage. E4 is an example of a genetically modified phage. A wild-type phage is the non-mutated, i.e. naturally occurring strain. A wild-type phage is different from a phage obtained by phage display.

A mutated phage may be obtained by phage display. In this technique, a gene encoding a protein of interest is inserted into a phage coat protein gene, causing the phage to "display" the protein on its outside while containing the gene for the protein on its inside.

The dispersion effect is demonstrated by both wild-type and mutated phage. As such, it is clear that the ability to disperse particles is not reliant on peptides displayed on the outer surface of the bacteriophage. Without being bound by theory, the inventors propose that the main reason that the phages can be used as dispersion agents is phage-particle interactions. Such interactions are dependent on the outer-surface properties of the phages, rather than their structures. In this research phages belonging to both the Myoviridae and Inoviridae families (T4 and Ml 3) provide a dispersion effect despite having different structures.

Mixture

The particles are dispersed in a liquid medium to form a mixture having the particles suspended therein, i.e. the mixture may be described as a suspension. We submit that at least some of the particles in the suspension have virus (e.g. bacteriophage) bonded thereto.

Liquid media include water and organic solvents. The liquid medium may be non- polar, polar aprotic or polar protic.

Common non-polar organic solvents include pentane, cyclopentane, hexane, cyclohexane, benzene, toluene, 1,4-dioxane, chloroform, diethyl ether and dichloromethane . Common polar aprotic organic solvents include tetrahydrofuran (THF), ethyl acetate, acetone, dimethylformamide (DMF), acetonitrile, dimethyl sulfoxide (DMSO), and nitromethane.

Common protic organic solvents include formic acid, ethanol, methanol, acetic acid, and propanal.

The liquid medium may comprise water, i.e. the liquid medium may be an aqueous medium. Water is a polar protic solvent.

The mixture (the liquid medium comprising the particles and the virus (e.g. bacteriophage)) or the liquid medium may be described with reference to its pH. The mixture/liquid medium may have a pH of from 3 to 11, from 5 to 9, from 6 to 8 or around 7. The particles may comprise alumina particles and the mixture /liquid medium may have a pH of from 6 to 8. The examples demonstrate that stable alumina suspensions can be obtained at neutral pH.

The pH of the liquid medium may be adjusted by addition of acid (e.g. hydrochloric acid) or an alkali (e.g. ammonia).

The pH of the medium may be selected based on the isoelectric point (IEP) of the particles to be dispersed therein. In the absence of adsorbed species particle surfaces in aqueous suspension are generally assumed to be covered with surface hydroxyl species, M-OH (where M is a metal such as Al, Si, etc.). At pH values above the IEP, the predominate surface species is M-CT, while at pH values below the IEP, M-OH²⁺ species predominate. Alpha alumina has an isoelectric point (IEP) of around 8 to 9 in water at SATP. Silica has an isoelectric point of around 1.7 to 3.5 in water at SATP.

The mixture comprises the liquid medium, the particles and the virus (e.g. phage).

The mixture may comprise 0.5wt% or more, lwt% or more, 2wt% or more or 5wt% or more particles and/or the mixture may comprise 20wt% or less, 10wt% or less, 5wt% or less or 3wt% or less particles. The mixture may comprise higher concentrations than those obtained by conventional processes. The mixture may comprise 5 to 10wt% particles. The mixture may comprise 0.05wt% or more, 0.1wt% or more, 0.2wt% or more, 0.5wt% or more, lwt% or more, 3wt% or more or 5wt% or more virus (e.g. phage) and/or the mixture may comprise 10wt% or less, 5wt% or less, lwt% or less or 0.5wt% or less virus (e.g. phage).

The mixture can be described with reference to the ratio of particles to the virus (e.g. phage). The ratio by weight of particles to virus (e.g. phage) may be at least 50wt% particles to no more than 50wt% phage (e.g. virus); at least 70wt% particles to no more than 30wt% virus (e.g. phage) or at least 90wt% particles to no more than 10% virus (e.g. phage).

The invention will now be described, in a non-limiting fashion, with reference to the following figures:

Figure 1 is a schematic diagram of a bacteriophage;

Figure 2 shows images from a sedimentation experiment;

Figure 3 shows a TEM image of an alumina particle having a bacteriophage interacting with an outer surface thereof;

Figure 4 shows images from a sedimentation experiment (alumina);

Figure 5 shows plots of highlighted area over time (alumina with E4 phage (■), with M13 phage (A) and without phage (O));

Figure 6 shows images from a sedimentation experiment (calcium carbonate); and Figure 7 shows plots of highlighted area over time (calcium carbonate with E4 phage ( A), with M13 phage (D), with T4 phage (■) and without phage (O)).

MATERIALS

Particles

Alumina powder (A1₂0₃) having a particle size of lOOnm: 99.99% ultrafine, TM-DAR (Pred Materials International, Inc.)

Calcium carbonate (CaC0₃) having a particle size of lOOnm.

Bacteriophage

T4 phage - wild

M13 phage - wild E4 phage - mutated M13 phage - tetraglutamate fused to N terminals of P8. Glutamate is the conjugate base of the GAG amino acid and therefore contains a negatively charged carboxylate group and is polar.

Examples

Example 1 : 1% alumina slurry and T4 wild phage

Suspensions were prepared by the addition of lg of alumina powder to 100ml of de ionised (DI) water and then ball milling for 24 hours. The pH was then altered by the addition of hydrochloric acid and ammonia to achieve a range of pH 3, 5, 7, 9 and 11.

The medium used (tryptone soya broth (30g/L) in flasks / tryptone soya agar (40g/L)), pipette tips and DI water were prepared and sterilised by autoclaving at 121°C. The broth was then inoculated with E.coli at 36.5°C and left overnight to grow in a shaking incubator. Agar was prepared in petri dishes and then inoculated with E.coli and left in the standing incubator overnight at 36.5°C. Both agar and broth were inoculated with T4 phage and placed back into standing incubator and shaking incubator respectively and left overnight. The broth inoculated with T4 phage was observed for any changes. Agar inoculated with T4 phage was also observed for any plaque formation. T4 phage in agar was collected by rinsing the parts containing plaque over and then the liquid was collected. Any T4 phage not used was placed into Eppendorf tubes and frozen down and stored at -70°C.

For sedimentation analysis IOmI of T4 bacteriophage was added to each suspension at a 1 : 10 ratio in a 5ml protein tubes. The test and control samples were placed in an ultrasonic bath for 10 minutes before sedimentation was recorded at 0, 5, 10, 15, 20, 30 and 60 minutes and after 24 hours.

Figure 2 shows the results with the control samples (alumina only) on the left and the test samples (alumina and T4) on the right in each pair. The results after 5 minutes are shown on the top row and after 24 hours on the bottom row.

A dramatic difference is shown at pH 7. Without T4 phage, sedimentation of the alumina occurred within 5 minutes. With T4 phage, the alumina remained suspended at 5 minutes. Some signs of sedimentation took place at around 60 minutes, but some alumina remained suspended even at 24 hours.

For TEM analysis, IOmI of T4 was added to the suspensions with a pH of 3, 7 and 11 at a 1 : 1 ratio in a 5ml protein tube and placed in an ultrasonic bath for minutes. IOmI was then placed onto a parafilm strip inside a petri-dish, and a TEM carbon mesh was then floated on top for 1 minute before excess liquid was removed with filter paper. IOmI of 2% ammonium molybdate was then placed on the parafilm and the carbon mesh floated on top for a further 1 minute to allow for negative staining, excess stain removed via filter paper.

Figure 3 shows the interaction of T4 with a single alumina particle. There is clearly interaction between the outer surface of T4 bacteriophage and the alumina particle. Since the T4 bacteriophage is a wild-type virus, the interaction is not due peptides displayed on its surface.

Example 2: 2% alumina slurry and E4 mutated phage / Ml 3 wild phage

The sedimentation analysis was repeated at pH 7 with a 2% alumina slurry and 2.5 mΐ E4, 2.5m1 M13, 10m1 E4 and 10m1 M13. The amount of sedimentation was recorded at 0, 5, 10, 15, 30 and 60 minutes and the images are shown in figure 4.

The experiment was repeated on a larger scale (30ml) and the images were evaluated by Image J analysis for particle sedimentation. Highlighted area (cm²) is plotted against time as shown in figure 5. Aliquots of 100 pL 2% A1₂0₃ (w/v) suspensions mixed with (A) 2.5 pL, (B) 5 pL, (C) 10pL of (■) E4 (1012 PFU/mL), (▲) M13 (1012 PFU/mL) or (O) Tris-buffered saline in Eppendorf tubes placed statically in a rack at room temperature were imaged at 0, 5, 10, 15, 20, 30, 60, 120 mins. The opaque area of each image was highlighted, analysed using Image J and plotted against time period

As before, sedimentation of the control (alumina without phage) occurred quickly. Both E4 and M13 slowed down the sedimentation. E4 was more effective than M13. Example 3: 1% calcium carbonate slurry and T4 wild phage / M13 wild phage / E4 mutated phage

Example 1 was repeated with 1% calcium carbonate slurry and each of the T4, M13 and E4 phages. The results are shown in figures 6 and 7. Aliquots of 100 pL 2% Ca0₃ (w/v) suspensions mixed with (A) 50 pL or (B) 100 pL of T4 (_■), E4 (A ), M13 (D) or Tris-buffered saline (O) in Eppendorf tubes placed statically in a rack at room temperature were imaged at 0, 5, 10, 15, 20, 30, 60, 120 mins. The opaque area of each image was highlighted, analysed using Image J and plotted against time period.

Sedimentation of the control (calcium carbonate without phage) occurred quickly.

T4, M13 and E4 slowed down the sedimentation. T4 was most effective.

Claims

1. Use of a virus to disperse particles in a liquid medium.

2. The use of claim 1, wherein the particles have a particle size of lOOOnm or less.

3. The use claim 2, wherein the particles have a particle size of from lOnm to 200nm.

4. The use of any one of the preceding claims, wherein the virus is a bacteriophage.

5. The use of claim 4, wherein the bacteriophage is a phage of the Myoviridae or Inoviridae family.

6. The use of any one of claims 1 to 3, wherein the virus is an animal virus.

7. The use of claim 6, wherein the virus is an adenovirus or an insect-specific virus (ISV).

8. The use of any one of claims 1 to 3, wherein the virus is a plant virus.

9. The use of any one of the preceding claims, wherein the virus is a wild-type virus.

10. The use of any one of the preceding claims, wherein the particles are inorganic particles.

11. The use of claim 10, wherein the inorganic particles comprise an oxide, a nitride, a carbonate and/or a carbide.

12. The use of claim 11, wherein the particles comprise alumina and/or calcium carbonate.

13. The use of any one of the preceding claims, wherein the liquid medium is an aqueous medium.

14. The use of claim 13, wherein the liquid medium has a pH of from 5 to 9.

15. A process for dispersing particles in a liquid medium, the process comprising combining a virus and particles in a liquid medium to form a mixture having the particles suspended therein.

16. The process of claim 15, wherein combining the virus and the particles in a liquid medium comprises providing a first liquid medium comprising the particles; providing a second liquid medium comprising the virus; and

combining the first liquid medium and the second liquid medium to yield the mixture having the particles suspended therein.

17. The process of claim 16, wherein (i) the first liquid medium is an aqueous medium having a pH of from 5 to 9; (ii) the particles comprise alumina and / or calcium carbonate particles; and/or (iii) the virus is a bacteriophage of the Myoviridae and/or Inoviridae family

18. A composition comprising a plurality of particles and a plurality of virus; wherein at least one virus is bonded to an outer surface of a particle.

19. The composition of claim 18 comprising a liquid medium having the particles suspended therein.

20. The process of any one of claims 15 to 17 or the composition of claim 18 or 19, wherein the virus is a wild-type bacteriophage.

21. The process of any one of claims 15 to 17 or the composition of claim 18 or claim 19, wherein the ratio of particles to virus is at least 50wt% particles : no more than 50wt% virus.

22. The use of any one of claims 1 to 14, the process of any one of claims 15 to 17 or the process of claim 18 or 19, wherein the virus is a virion.