THIN FILM VORTEX FLUIDIC FABRICATION OF LIPOSOMES

PRIORITY DOCUMENTS

[0001] The present application claims priority from Australian Provisional Patent Application No. 2017902772 titled "Thin film vortex fluidic fabrication of liposomes" and filed on 14 July 2017, the content of which is hereby incorporated by reference in its entirety.

INCORPORATION BY REFERENCE

[0002] The following publication is referred to in the present application and its content is hereby incorporated by reference in its entirety:

• United States patent application US 2013/0289282

TECHNICAL FIELD

[0003] The present disclosure relates processes for producing liposomes, fabricating lipid based self- assembled arrays, self-assembled macromolecules, nano-emulsions, and incorporating bioactive molecules and other molecules of interest in these materials.

BACKGROUND

[0004] Phospholipids are the building block components of liposomes and are ubiquitous in nature. In water, these amphiphilic molecules tend to organize into a vesicular structure due to maximizing hydrophobic interactions and to entropically minimizing free energy. Fabricating liposomes with a target size between 50 nm to 200 nm is important for drug delivery applications through enhanced cellular uptake, but processing these liposomes for medical and industrial applications is challenging.1

Fabricating such liposomes for uptake of drug molecules, for example, involves multi-step procedures including dispersing the hydrophobic/hydrophilic drug dissolved into a suitable solvent to then prepare multi-lamellar vesicles (MLV) using the lipid thin film hydration technique and formation of Large Unilamellar Vesicles (LUVs) through rapid freeze and thawing, followed by repetitive extrusion through polycarbonate filters.2 Various improvements have been made in the field of microfluidics to reduce this multistep processing. Microfluidic methods such as electro formation & hydration, extrusion, pulse jetting, double emulsion templating, ice droplet hydration, transient membrane injection, droplet emulsion transfer and hydrodynamic focusingl ,2,3 have been used to fabricate liposomes with control over size and polydispersity compared to traditional batch-processes.3 Despite this control over size and polydispersity, processing liposomes for medical, industrial and bulk processing while addressing scalability remains a challenge.

[0005] The primary goal for approving liposomal formulations was to alter the therapeutic index, reducing the toxicity of the parent drug, improving the bio-distribution, rendering the formulation nontoxic and weakly immunogenic, optimizing the residence clear times of drugs from the body, and also providing enhanced permeability rates of the drug (EPR). The very first liposomal pharmaceutical product approved by the Food and Drug administration (FDA) was Doxil, introduced in 1995, which is a chemotherapy drug known as doxorubicin encapsulated into PEGylated liposomes for Kaposi's Sarcoma, ovarian and breast cancer treatments. ⁴ Depending on the type of phospholipids, the liposomes can be cationic, anionic or neutral at physiological pH, with surface charge playing an important role on the stability of the liposome, both in formulation and in serum. Incorporation of other lipophilic compounds such as cholesterol reduces the fluidity within the liposomal bilayers and also induces premature drug leakage from the liposomes. Thus introducing polymer conjugated on the surface of liposomes provides enhanced stability through improved steric repulsion and also prevents aggregation and subsequent endocytosis by the phagocytes. Apart from drug-delivery applications, liposomes are widely exploited in the field of nutraceuticals and cosmetics where the term "natural", "organic" or "no added preservatives" are the driving force for the consumer market. The active ingredient is encapsulated within the liposome and a controlled release system is obtained through them at the target site. ³ ' ⁶ ' ⁷ The primary aim behind targeting liposomes is associated with better therapeutic outcome of anti-cancer drugs, and drugs in general, in animal tumour models compared to non-targeted liposomes, preventing premature drug leakage at the healthy tissue sites, and thus providing a controlled release system at the targeted site.

[0006] There is thus a need to provide new methods for producing liposomes in a controlled manner.

SUMMARY

[0007] According to a first aspect there is provided a process for producing liposomes, the process comprising:

providing a lipid suspension;

introducing the lipid suspension to a thin film tube reactor comprising a tube with an inner hydrophobic functionalized surface, the tube having a longitudinal axis, wherein the angle of the longitudinal axis relative to the horizontal is between 0 degrees and about +90 and -90 degrees;

rotating the tube about the longitudinal axis at a predetermined rotational speed; and recovering the liposomes from the thin film tube reactor.

[0008] The process may be used to produce 70 - 200 nm liposomes with a polydispersity of 0.2 [0009] The process of the first aspect can be used as an in-situ process to encapsulate one or more functional molecules on the surface of the liposomes and/or within the liposomes. In this way, the liposomes can be used as effective nano-carriers with enhanced encapsulation efficiency. A wide variety of functional molecules can be encapsulated in this way including, but not limited to: drug molecules; bioactive compounds such as organic polyphenols including curcumin and resveratrol; nutraceutical molecules; quantum dots; metal nanoparticles; inorganic nanoparticles; proteins; peptides; RNA; DNA; and combinations thereof.

[0010] Therefore, according to a second aspect there is provided a process for producing liposomes having one or more functional molecules encapsulated therein or thereon, the process comprising:

providing a lipid suspension and a fluid containing the one or more functional molecules; introducing the lipid suspension and fluid containing the one or more functional molecules to a thin film tube reactor comprising a tube with an inner hydrophobic functionalized surface, the tube having a longitudinal axis, wherein the angle of the longitudinal axis relative to the horizontal is between 0 degrees and about +90 and -90 degrees;

rotating the tube about the longitudinal axis at a predetermined rotational speed; and recovering the liposomes having one or more functional molecules encapsulated therein or thereon from the thin film tube reactor.

[0011] The predetermined rotational speed may be selected by taking into consideration one or more other processing parameters in the thin film tube reactor. In certain embodiments of the first and second aspects, the predetermined rotational speed from about 2000 rpm to about 14000 rpm, such as about 9000 rpm.

[0012] In certain embodiments of the first and second aspects, the angle of the longitudinal axis relative to the horizontal is from about minus 45 to about plus 60 degrees.

[0013] According to a third aspect there is provided a process for producing hybrid liposomes from at least two different fluorophore tagged lipids, the process comprising:

providing a first liposome suspension wherein at least some of the lipids in the liposome comprise a first fluorophore tag;

providing a second preformed liposome suspension wherein at least some of the lipids in the liposome comprise a second fluorophore tag or providing a lipid suspension wherein at least some of the lipids in the lipid suspension comprise a second fluorophore tag

introducing each of the liposomes suspension( s) and/or lipid suspension to a thin film tube reactor comprising a tube with an inner hydrophobic functionalized surface, the tube having a longitudinal axis, wherein the angle of the longitudinal axis relative to the horizontal is between 0 degrees and about +90 and -90 degrees; rotating the tube about the longitudinal axis at a predetermined rotational speed; and recovering the fluorophore attached liposomes from the thin film tube reactor.

[0014] In certain embodiments of the third aspect, the first and/or second liposome suspension comprise 1 % fluorophore tagged lipid.

[0015] The process of the third aspect can be used for fusion of two different liposomes with reorganisation into the optimum/target size membrane. For example, it can be used for fusion of two different liposomes labelled with two different fluorophore dyes which are fluorescence resonance energy transfer (FRET) pairs in nature. It has also been shown that two different bilayers consisting of different head groups provide enhanced stability and rigidity to the liposomes. Engineering hybrids of two different liposomes can provide membrane stability and enhanced cellular uptake compared to liposomes processed from a single head group phospholipid chain. ⁸

[0016] Using the processes described herein, fluorophore dyes can be incorporated into liposomes by in- situ VFD processing and post VFD processing using batch processing (magnetic stirring). This also allows attaching functional moieties, such as folate, proteins, peptide or polysaccharide receptors, to the fluorescent markers. Optionally, the liposomes can be wrapped with a polymer such as polyethylene glycol (PEG) bearing receptors, protein/peptide chains, antibodies or fluorescent markers attached to the polymer, and the drug is released at the specific site and tracked through fluorescent markers ^{9 1ϋ} .

[0017] The processes described herein can also be used to produce lipid assemblies such as chiral ribbons and rods, by utilising the control (operating) parameters of the VFD, and other species present such calcium and magnesium ions. Calcium and magnesium ions can control the formation of the lipid bilayers, changing the nature of the phase, resulting in different lipid assemblies such as chiral ribbons, rods and nano-cochleates (scrolls of bilayer). Use of these in drug delivery and medical applications in general is limited by the lack of processing capabilities in generating them. ^{11 12} ' ¹³

[0018] The processes described herein can also be used to produce stable nano-emulsions for cosmetic and nutraceutical formulations. Oil/water emulsions are formed in the presence of surfactants such as phospholipids and albumin proteins. Colloidal dispersions of oil/water and surfactants have been widely used in industry as potent delivery systems in a number of pharmaceutical formulations ranging from food, cosmetics and point of care products which come in the form of micro emulsions ¹⁴ . These micro emulsions are not thermodynamically stable and quickly phase separate prominently across the formulation in the form of flocculation and coalescence through the phenomena of Ostwald ripening.

[0019] The processes described herein can also be used to produce liposomes encapsulated with proteins/enzymes for pharmaceutical industry wherein a controlled release of the enzyme is required ¹⁶ . [0020] The processes described herein can also be used for real-time monitoring self-assemblies under shear. This method can be used to study fabrication of self-assemblies under shear and monitor them realtime through Small Angle Neutron Scattering (SANS), but not limited to, and could include small angle neutron scattering (SAXS).

BRIEF DESCRIPTION OF DRAWINGS

[0021 ] Embodiments of the present invention will be discussed with reference to the accompanying figures wherein:

[0022] Figure 1 (left) Cross section showing the components of the VFD. (b) Average film thickness (mm) versus tilt in the VFD tube. Photographs showing film thickness of 3 mL of liquid in a 10 mm VFD tube at different rotational speeds ¹⁷ (right) Shows a milky lipid suspension before processing in the VFD which is illustrated, and the clear solution after processing in the VFD at a concentration of 1 mg/mL. The VFD processing was carried out at an optimised rotational speed of 9000 rpm and flow-rate of 0.1 mL/min.

[0023] Figure 2 shows a size distribution of liposomes processed through VFD at an optimised rotational speed of 9000 rpm and flow rate of 0.1 mL/min revealing 70 - 200 nm particle sizes, characterized using Nanoparticle Tracking Analysis.

[0024] Figure 3 shows a scanning electron microscopy image for the surface and morphology of the liposomes fabricated using the VFD at an optimised rotational speed of 9000 rpm and flow rate of O. lmL/min.

[0025] Figure 4 shows an atomic force microscopy image for surface and bilayer thickness of the liposomes fabricated using the VFD at an optimised rotational speed of 9000 rpm and flow rate of O. lmL/min.

[0026] Figure 5 shows small angle neutron scattering (SANS) data for liposomes fabricated using the VFD at an optimised rotational speed of 9000 rpm and flow rate of 0. lmL/min; the data is modelled with a with a lamellar model FFHG applied for fitting of the data, affording a calculated bilayer thickness 4.19 ± 0.2 nm.

[0027] Figure 6 shows real-time monitoring of small angle neutron scattering (SANS) data for phospholipid suspension in D ₂ 0 with a final concentration of lmg/mL in confined mode at 4 different speeds i.e. 2000, 4000, 6000 and 8000 rpm. [0028] Figure 7 shows real-time monitoring of small angle neutron scattering (SANS) data for phosphonated calixarenes in 50% phosphate buffer in D ₂ 0 with a final concentration of 1 mg/mL in confined mode at 4 different speeds i.e. 2000, 4000, 6000 and 8000 rpm.

[0029] Figure 8 shows real-time monitoring of small angle neutron scattering (SANS) data for SDS micelles at 2 wt% final concentration in confined mode at 4 different speeds i.e. 2000, 4000, 6000 and 8000 rpm.

[0030] Figure 9 shows SEM images of nano-ribbons and lipid rods generated in the VFD. The final concentration is 50 ug/mL in phosphate buffer in one jet-feed while the other jet feed is water. The optimized speed ranges from 6500 - 7000 rpm at an optimized flow-rate of 0.1 mL/min.

[0031] Figure 10 shows Confocal Images for Giant Unilamellar Liposomes (GUV) of 600 run in diameter. The processing parameters are processed at an optimized condition of 5000 rpm at an optimized flow-rate of 0.1 mL/min.

[0032] Figure 11 (Left) in-situ synthesis of curcumin & lipid nanoparticles in the VFD, of 100 - 200 nm in size. The optimized conditions for the VFD is 1 mL/min at 4000 rpm. (Right) Dynamic Light scattering (DLS) data for VFD processed Curcumin encapsulation nano-particles at 4000 rpm and flow- rate of 1 mL/min.

[0033] Figure 12 shows Dynamic Light scattering (DLS) data for VFD processed curcumin

encapsulation nano-particles at 6000 and 8000 rpm with flow-rate of 1 mL/min.

[0034] Figure 13 shows (left) UV-absorbance for curcumin nano-particles processed at three different speeds such as 4000 rpm, 6000 rpm, 8000 rpm. (right) Fluorescence spectrum for three different speeds such as 4000 rpm, 6000 rpm and 8000 rpm.

[0035] Figure 14 shows SEM images for curcumin nano-particles processed at 4000 rpm (top) SEM images for 6000 rpm and 8000 rpm (bottom) at an optimised flow-rate of 1 mL/min.

[0036] Figure 15 FT-IR spectrum for as received curcumin and nano-formulation of curcumin processed through VFD at an optimised speed of 4000 rpm and flow-rate of 1 mL/min.

[0037] Figure 16 XRD spectrum for as received curcumin and nano-formulation of curcumin processed through VFD at an optimised speed of 4000 rpm and flow-rate of 1 mL/min.

[0038] Figure 17 Size distribution data for 1 mole% NBD labelled fluorophore dye with the liposomes processed in the VFD at an optimised speed of 5000 rpm under confined mode (Green colour) vs unlabelled liposomes processed in VFD at an optimised speed of 9000 rpm under continuous flow with a flow-rate of 0. 1 mL/min (Blue colour).

[0039] Figure 18 Fluorescence spectroscopy data for NBD fluorophore dye before VFD processing (Blue colour) vs NBD fluorophore dye labelled liposomes processed in the VFD at an optimised speed of 5000 rpm under confined mode of operation of the VFD (Green colour).

[0040] Figure 19 Size distribution data for 1 mole% Rhodamine labelled fluorophore dye with the liposomes processed in the VFD (Red colour) vs unlabelled liposomes processed in VFD at an optimised speed of 5000 rpm under confined mode operation of the VFD (Blue colour).

[0041] Figure 20 Fluorescence Spectroscopy Data for Rhodamine fluorophore dye before VFD processing (Blue colour) vs Rhodamine fluorophore dye labelled liposomes processed in the VFD at an optimised speed of 5000 rpm under confined mode of operation of the VFD (Red colour).

[0042] Figure 21 AFM height-image for NBD-PE labelled fluorophore liposomes at an optimised speed of 5000 rpm under confined mode of operation of the VFD.

[0043] Figure 22 AFM height-image for Rh-PE labelled fluorophore liposomes at an optimised speed of 5000 rpm under confined mode of operation of the VFD.

[0044] Figure 23 AFM height-image for mixed liposomes of two differently labelled fluorophores at an optimised speed of 5000 rpm under confined mode of operation of the VFD.

[0045] Figure 24 AFM height-image and SEM images for PEG treated NBD-PE land Rh-PE liposomes. PEG treatment was through magnetic stirring on bench with the VFD processed liposomes at an optimised rotational speed of 9000 rpm and flow rate of 0.1 mL/min.

[0046] Figure 25 AFM height-image for mixed liposomes of two differently labelled fluorophores treated with PEG in VFD at an optimised rotational speed of 5000 rpm operating under confined mode.

[0047] Figure 26 FRET signal for Rh-PE fluorophore, mixed liposomes w/o PEG processed at an optimised rotational speed of 5000 rpm under confined mode and mixed liposomes treated with PEG processed at an optimised rotational speed of 5000 rpm under confined mode of operation of the VFD.

[0048] Figure 27 Fluorescence optical micro-graphs for liposomes labelled with fluorophore dyes processed though VFD at an optimised rotational speed of 5000 rpm under confined mode and VFD mixed liposomes with FRET pair fluorophores at an optimised rotational speed of 5000 rpm under confined mode of operation of the VFD. [0049] Figure 28 Images for fish-oil nano-emulsions processed through VFD at an optimised rotational speed of 8000 rpm under continuous at 0.1 mL/min, fish-oil (no VFD. no homogenization, fish-oil emulsions processed through homogenization at a speed of 13,500 rpm for 10 minutes at 25 °C).

[0050] Figure 29 SEM images for batch processed emulsions and VFD processed at an optimised rotational speed of 8000 rpm under continuous flow at 0.1 mL/min (left), TEM image for nano-emulsion processed through VFD processing at an optimised rotational speed of 8000 rpm under continuous flow at 0.1 mL/min (right).

[0051] Figure 30 Dynamic Light Scattering (DLS) data for fish-oil encapsulation through VFD processing at an optimised rotational speed of 8000 rpm under continuous flow at 0.1 mL/min.

[0052] Figure 31 AFM height-image for nano-emulsions processed through VFD processing at an optimised rotational speed of 8000 rpm under continuous flow at 0.1 mL/min.

[0053] Figure 32 Beta-carotene images for batch processed with homogenization at 13,500 rpm for 10 minutes at 25 °C and VFD processed at an optimised rotational speed of 8000 rpm under continuous flow- made at a flow-rate of 0.1 mL/min (left), DLS data for particle size-distribution.

[0054] Figure 33 Beta-carotene AFM height-images for particles processed through VFD processing an optimised rotational speed of 8000 rpm under continuous flow at 0.1 mL/min.

[0055] Figure 34 C-PC encapsulation through VFD an optimised rotational speed of 8000 rpm under continuous flow at 0.1 mL/min and no-VFD processing.

[0056] Figure 35 C-PC encapsulation AFM-height image for VFD processed particles at an optimised rotational speed of 8000 rpm under continuous flow at 0.1 mL/min.

DESCRIPTION OF EMBODIMENTS

[0057] Described herein is a process for producing liposomes. The process comprises providing a lipid suspension and introducing the lipid suspension to a thin film tube reactor comprising a tube with an inner hydrophobic functionalized surface, the tube having a longitudinal axis, wherein the angle of the longitudinal axis relative to the horizontal is between 0 degrees and about +90 and -90 degrees. The tube is rotated about the longitudinal axis at a predetermined rotational speed to form liposomes which are then recovered from the thin film tube reactor.

[0058] Advantageously, the applicant has found that 70 - 200 nm liposomes with polydispersity of 0.2 - 0.3 are formed in the process. [0059] The thin film tube reactor (device) used in the processes described herein is a vortex fluidic device (VFD). Details of the VFD are described in published United States patent application US 2013/0289282, the details of which are incorporated herein by reference. Briefly, the thin film tube reactor comprises a tube rotatable about its longitudinal axis by a motor. The tube is substantially cylindrical or comprises a portion that is tapered. The motor can be a variable speed motor for varying the rotational speed of the tube and can be operated in controlled set frequency and set change in speed. A generally cylindrical tube is particularly suitable but it is contemplated that the tube could also take other forms and could, for example, be a tapered tube, a stepped tube comprising a number of sections of different diameter, and the like. The tube can be made of any suitable material including glass, metal, plastic, ceramic, and the like. In certain embodiments, the tube is made from borosilicate or quartz. Optionally, the inner surface of the tube can comprise surface structures or aberrations. In embodiments, the tube is a pristine borosilicate NMR glass tube or a quartz tube of the same dimensions, which has an internal diameter typically 17.7± 0.013 mm.

[0060] The tube is situated on an angle of incline relative to the horizontal position above 0 degrees and less than 90 degrees and can take on negative values. In certain embodiments, the tube is situated on an angle of incline relative to the horizontal of between 10 degrees and +90 and -90 degrees. The angle of incline can be varied. In certain embodiments the angle of incline is 45 degrees. In certain other embodiments the angle of incline is 60 degrees. In still other embodiments, the angle of incline is -45 degrees, which can be effective in circumventing material adhering to the surface of the tube.

[0061] For the majority of the processes described herein, the angle of incline has been optimized to be 45 degrees relative to the horizontal position, which corresponds to the maximum cross vector of centrifugal force in the tube and gravity. In certain embodiments, the angle of incline is from about minus

45 to about plus 60 degrees. However, other angles of incline can be used including, but not limited to, 1 degree, 2 degrees, 3 degrees, 4 degrees, 5 degrees, 6 degrees, 7 degrees, 8 degrees, 9 degrees, 10 degrees, 1 1 degrees, 12 degrees, 13 degrees, 14 degrees, 15 degrees, 16 degrees, 17 degrees, 1 8 degrees, 19 degrees, 20 degrees, 21 degrees, 22 degrees, 23 degrees, 24 degrees, 25 degrees, 26 degrees, 27 degrees, 28 degrees, 29 degrees, 30 degrees, 31 degrees, 32 degrees, 33 degrees, 34 degrees, 35 degrees, 36 degrees, 37 degrees, 38 degrees, 39 degrees, 40 degrees, 41 degrees, 42 degrees, 43 degrees, 44 degrees,

46 degrees, 47 degrees, 48 degrees, 49 degrees, 50 degrees, 51 degrees, 52 degrees, 53 degrees, 54 degrees, 55 degrees, 56 degrees, 57 degrees, 58 degrees, 59 degrees, 60 degrees, 61 degrees, 62 degrees, 63 degrees, 64 degrees, 65 degrees, 66 degrees, 67 degrees, 68 degrees, 69 degrees, 70 degrees, 71 degrees, 72 degrees, 73 degrees, 74degrees, 75 degrees, 76 degrees, 77 degrees, 78 degrees, 79 degrees, 80 degrees, 81 degrees, 82 degrees, 83 degrees, 84 degrees, 85 degrees, 86 degrees, 87 degrees, 88 degrees, and 89 degrees. If necessary, the angle of incline can be adjusted so as to adjust the location of the vortex that forms in the rotating tube relative to the closed end of the tube. Optionally, the angle of incline of tube can be varied in a time-dependent way during operation for dynamic adjustment of the location and shape of the vortex. For a negative value of inclination the tune can be open at both ends with the liquid delivered from the highest end and collected at the bottom of the tube.

[0062] A spinning guide or a second set of bearings assists in maintaining the angle of incline and a substantially consistent rotation around the longitudinal axis of the tube.

[0063] Liposomes are widely utilized as delivery systems across number of drug delivery and pharmaceutical applications. Processing these liposomes is a multi-step procedure involving dissolving the drug into a suitable solvent to then prepare multi-lamellar vesicles (MLV) using the lipid film hydration technique and formation of Large Unilamellar Vesicles (LU Vs) through freeze and thawing, which is then followed by repetitive extrusion through polycarbonate filters. Various improvements have been made in the field of microfluidics to reduce this multistep processing. Microfluidic methods such as electro-formation and hydration, extrusion, pulse jetting, double emulsion templating, ice droplet hydration, transient membrane injection, droplet emulsion transfer and hydrodynamic focusing ^1,2 have been used to fabricate these liposomes with control over size and polydispersity compared to traditional batch (flask) processing, but such microfluidics are difficult to scale up for industrial production, and can suffer from clogging of the channels. In contrast, the processes described herein are directly scalable for a single thin film tube reactor or parallel arrays of thin film tube reactors, and the thin film tube reactor does not suffer from clogging.

[0064] The processes described herein have been developed for producing liposomes at an industrial scale level in a thin film vortex fluidic device, without the need for solvents such as chloroform and methanol, for which their use in medicine, cosmetics and food processing is prohibited or questionable. The processing is one step processing and produces liposomes with 70 - 200 nm in size with

polydispersity of 0.2 - 0.3.

[0065] In certain embodiments, the tube of the thin film tube reactor is glass tube having an inner surface functionalized with a hydrophobic coating. The hydrophobic coating may be formed from an alkyl silane, such as octadodecylsilane (ODTS), hexamethyldisilazane (HMDS) or octyltrichlorosilane (OTS-C8).

[0066] A lipid suspension is prepared by suspending the lipid or mixture of lipids in water, water soluble organic solvents, such as ethanol or immiscible solvents.

[0067] A range of lipids can be used to form liposomes using the processes described herein. In certain embodiments, the lipid is the membrane lipid l-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC) which is zwitter-ionic and amphiphilic in nature. Other suitable lipids may be selected from the group consisting of, but not limited to l,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn- glycero-3-phosphocholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), 1 ,2-distearoyl-sn-glycero- 3-phosphocholine (DSPC), l ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1 ,2-dimyristoyl-sn- glycero-3-phosphoethanolainine (DMPE), l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1 ,2-dimyristoyl-sn-glycero-3-phosphate (sodium salt) (DMPA'Na), l ,2-dipalmitoyl-sn-glycero-3-phosphate (sodium salt) (DPPA'Na), 1,2- dioleoyl-sn-glycero-3-phosphate (sodium salt) (DOPAvNa), l,2-diinyristoyl-sn-glycero-3-phospho-(l'- rac-glycerol) (sodium salt) (DMPG' a), l ,2-dipalmitoyl-sn-glycero-3-phospho-(T-rac-glycerol) (sodium salt) (DPPG'Na), l ,2-dioleoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (sodium salt) (DOPG'Na), 1,2- dimyristoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DMPS'Na), l,2-dipalmitoyl-sn-glycero-3- phospho-L-serine (sodium salt) (DPPS ^» Na), l ,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DOPS'Na), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(glutaryl) (sodium salt) (DOPE- Glutary l*(Na)2), Tetramyristoyl Cardiolipin*(Na)2, 1 ,2-distearoy l-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000] (sodium salt) (DSPE-mPEG-200ONa), 1 ,2-distearoyl-sn-glycero- 3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (sodium salt) (DSPE-mPEG-5000vNa), l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide (polyethylene glycol)-2000] (sodium salt) (DSPE- aleimide PEG-2000*Na), l ,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP'Cl).

[0068] Other lipids that can be used include phospholipid mimics, such as, but not limited to phosphonated calix-4-arenes.

[0069] Still other lipids that can be used include surfactants such as sodium dodecyl sulfate (SDS; also known as sodium lauryl sulfate (SLS)).

[0070] The lipid suspension is processed under shear in the thin film tube reactor whereupon the turbid suspension becomes clear with the formation of liposomes of 70 - 200 run in diameter. The size distribution of the liposomes can be characterised through Dynamic Light Scattering and Nanoparticle Tracking Analysis (NT A). Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM) imaging can also be used for morphology and size characterisation.

[0071] In cases where the lipid is not completely soluble/dispersed in water, the lipid can be dissolved in a buffer, for example a phosphate buffer, and processed in the thin film tube reactor, affording similar results, i.e. liposomes 70 - 200 nm in diameter. For example, this treatment may be required for some phospholipids.

[0072] The tube of the thin film tube reactor may be rotated at a predetermined rotational speed that may be selected by taking into consideration one or more other processing parameters in the thin film tube reactor. The predetermined rotational speed from about 2000 rpm to about 14000 rpm, such as about 9000 rpm. Optionally, the speed can be pulsed or varied in a controlled way.

[0073] The thin film tube reactor can be operated in a confined mode of operation for a finite amount of liquid in the tube. Alternatively, or in addition, the thin film tube reactor can be operated in a continuous mode of operation whereby jet feeds are set to deliver reactant fluids into the rapidly rotating tube, depending on the flow rate. Reactant fluids are supplied to the inner surface of the tube by way of at least one feed tube. Any suitable pump can be used to pump the reactant fluid from a reactant fluid source to the feed tube(s). In certain embodiments the flow rate of the jet feeds delivering liquids to the tube can be varied, from about 0.1 mL/min to about 10 mL/min.

[0074] In certain embodiments the jet feeds are positioned at different locations along the tube such that multiple processes can be carried out in a single VFD tube, and/or the outlet from one VFD tube can be delivered to a second VFD tube, and so on.

[0075] A collector may be positioned substantially adjacent to the opening of the tube and can be used to collect product exiting the tube. Fluid product exiting the tube may migrate under centrifugal force to the wall of the collector where it can exit through a product outlet.

[0076] Optionally, field effects can be applied to the tube, including light (LEDs and laser, and the like), magnetic, electric, and sonic to vary the nature of the resulting assembled phospholipid, and incorporation of other agents such as drugs and functional nanoparticles.

[0077] The processes of the present disclosure can be used to produce hybrid liposomes from at least two different fluorophore tagged lipids. The process may comprise providing a first liposome suspension wherein at least some of the lipids in the liposome comprise a first fluorophore tag and providing a second preformed liposome suspension wherein at least some of the lipids in the liposome comprise a second fluorophore tag or providing a lipid suspension wherein at least some of the lipids in the lipid suspension comprise a second fluorophore tag. Each of the liposomes suspension(s) and/or lipid suspensions are introduced to a thin film tube reactor comprising a tube with an inner hydrophobic functionalized surface, the tube having a longitudinal axis, wherein the angle of the longitudinal axis relative to the horizontal is between 0 degrees and about +90 and -90 degrees. The tube is rotated about the longitudinal axis at a predetermined rotational speed and the fluorophore attached liposomes are recovered from the thin film tube reactor.

[0078] Liposomes with fluorophore tagged lipids are utilised in the aforementioned processes. The fluorophore tagged lipids may comprise a first lipid comprising a first fluorophore tag and a second lipid comprising a second fluorophore tag. The first and second fluorophore tags may be FRET pairs. The fluorophore tagged lipids in these embodiments may be selected from the group consisting of: Rh-PE l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamin e rhodamine B sulfonyl) (ammonium salt), NBD-PE (-oleoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine-N-[tetra (ethylene glycol)]-N'-(7- nitro-2-l ,3benzoxadiazol-4-yl) (ammonium salt), l -palmitoyl-2-{ 12-[(7-nitro-2-l ,3-benzoxadiazol-4- yl)amino]dodecanoyl}-sn-glycero-3-phosphoethanolamine (Fatty acid labelled NBD-PE), 1,2-dioleoyl- sn-glycero-3-phosphoethanolamine-N-(l-pyrenesulfonyl) (ammonium salt) (Pyrene-PE), 1 ,2-dioleoyl-sn- glycero-3-phosphoethanolamine-N-(Cyanine 5) (PE-cy5), and l ,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-(Cyanine 7) (PE-cy7). For example, a first liposome suspension may be formed from l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamin e rhodamine B sulfonyl)

(ammonium salt) (Rh-PE) and a second preformed liposome suspension and/or the lipid suspension may be formed from oleoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine-N-[tetra (ethylene glycol)] -N'-(7- nitro-2-l,3benzoxadiazol-4-yl) (ammonium salt) (NBD-PE).

[0079] The processes of the present disclosure can also be used as an in-situ process to encapsulate one or more functional molecules on the surface of the liposomes and/or within the liposomes. In this way, the liposomes can be used as effective nano-carriers with enhanced encapsulation efficiency. A wide variety of functional molecules can be encapsulated in this way including, but not limited to: drug molecules; bioactive compounds such as organic polyphenols including curcumin and resveratrol; nutraceutical molecules; quantum dots; metal nanoparticles; inorganic nanoparticles; proteins; peptides; RNA; DNA; and combinations thereof.

[0080] In certain specific embodiments, paclitaxel, 5-fluorouracil, carboplatin, or combinations of these drugs can be encapsulated on the surface of the liposomes and/or within the liposomes. In other certain specific embodiments, gold and other metal or shell and alloy nanoparticles, inorganic nanoparticles such as superparamagnetic magnetite can be encapsulated on the surface of the liposomes and/or within the liposomes.

EXAMPLES

[0081 ] Experimental Details

[0082] Nanoparticle Tracking Analysis (NT A)

[0083] Data were taken in triplicate 60s videos to get a standard error for the data, for nanoparticle tracking analysis.

[0084] Scanning Electron Microscopy (SEM) & (AFM) [0085] Imaging of SEM was carried out on samples in 1 : 10 dilution drop-casted on silicon wafer, dried over-night and coated with platinum of 2.0 nm in thickness by using dual target sputter coater. The accelerating voltage used was 10:00 Kv and spot-size of 0.3, the same sample preparation was utilised for AFM imaging coated with platinum in order to access better adhesion with sticky surface of the lipid with the cantilever tip.

[0086] Small Angle Neutron Scattering (SANS)

[0087] SANS experiments were carried out at ANSTO on opal reactor instrument Bilby, the

phospholipid was dissolved in D2O at a concentration of 1 mg/mL i.e. 0.1 wt% D2O and processed in VFD and sample collected was characterized in a cuvette of 2mm at low q range.

[0088] Example 1 - Vortex Fluidic Device (VFD) production of liposomes

[0089] WD experiments were undertaken in the confined mode of operation of the device, where a set amount of liquid in a 20 mm OD (16.7 mm ID) tube was subjected to shear stress (Figure 1). Here, shearing in the film arises from the reciprocal action between the centrifugal and gravitational forces in association with Stewartstown/Ekman layers and less understood fluid dynamics. While the thin film is parabolic in shape, with a vortex to the base of the rotating tube, this becomes less apparent at high speeds where the thin film can spread along the entire tube.

[0090] A 1 -palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC) lipid suspension at a concentration of 25 μg/mL was subjected to shear in the confined mode, with the volume set at 1 mL and the processing time at 30 minutes, tilt angle Θ set at 45 degrees, at 25°C. Post shearing, the liquid was collected and the size of the particles characterised using DLS. The VFD can be operated at different rotational speeds, the lower rotational speeds for a 20 mm OD borosilicate glass tube start from 2000 rpm. This is the threshold for forming a vortex to the bottom of the tube with the film taking on a parabolic shape. As the rotational speed increases the liquid spreads out and the height of the thin film up the tube increases.

[0091 ] Rotational speed effects in confined mode on the size and polydispersity

[0092] The size of the liposomes generated at all the rotational speeds were between 100 to 300 nm in diameter with high PDI values from 0.5 - 0.6. After a number of processing operations at 9000 rpm, which is the upper end of operation of the current VFDs, the lipids became attached to the surface of the tube. This finding is a physisorption phenomenon where the lipids self-assemble via van-der Waals force and intermolecular hydrogen bonding with the hydrophilic surface of the tube. [0093] Another experiment was also carried out where the rotational speed was taken to 9000 rpm for 15 minutes and brought back to 4000 rpm for another 15 minutes, and the suspension of the liposomes was characterized using DLS. The resultant particle size was the same as that obtained for rotating exclusively at 5000 rpm. An intensity peak reappeared which confirms that the lipids are attached to the glass surface at 9000 rpm and spontaneous formation of liposomes are formed when the speed is lowered to 4000 rpm.

[0094] Continuous flow studies in hydrophobic tube

[0095] Due to the physical attachment of the lipids to the VFD glass tube, the surface of the VFD tube was functionalised and transformed from a hydrophilic to a hydrophobic surface using a silane derivative, and subsequent experiments were undertaken exclusively under continuous flow in a hydrophobic tube.

[0096] In the first step, the glass surface was treated with a piranha solution as a 3: 1 solution of H2SO4: H2O2 which is a strong oxidising agent and removes all the organic debris, exposing silanol groups (Si-OH) on the surface of the borosilicate glass. This surface treatment ensures that the surface is hydrophilic, which is mandatory for the covalently binding of silane to the surface of the glass, in making the surface then hydrophobic.

[0097] The silane derivative that was used for rendering the surface of the tube hydrophobic was a linear chain silane derivative, trichloro-octadodecylsilane (ODTS) of chemical formula CH3

(CH2)i7SiCb. Surface bound water was removed by heating the tube at 150°C in an oven, exposing the surface silanol groups. The silane condenses with silanol groups on the glass surface. The hydrophobicity of the tube was tested with a drop of water, and there was a significant increase in the apparent contact angle indicating successful attachment of the dodecyl moieties to the surface, as has been previously established.

[0098] Rotational speed effects in continuous flow & mechanism for formation of liposomes

[0099] The use of the VFD in the confined mode is a proven approach to gain insight into the optimal operating parameters in using the device in the continuous flow mode. The confined mode data had large deviations in the data, and this may reflect the problem of some of the liquid in the film being restricted to the wedge at the top of the liquid. This liquid will have different fluid dynamics compared to the bulk thin film, i.e. shear stress is not uniform along the length of the film.

[00100] The next set of experiments were carried out under continuous flow studies where the above issue is addressed, using hydrophobic tubes as the proven surface requirement for the confined mode of operation of the VFD. Here the liquid is delivered via jet feeds attached to syringe pumps to the bottom of the rapidly rotating tube, with the flow rate of the jet feeds controlled and the processed sample collected at the other end of the tube. Rotational speeds were studied from 5000 rpm to 9000 rpm at a low flow rate of 0.1 mL/min and the size of the particles (liposomes) screened/characterised using DLS.

[00101 ] For a flow rate of 0.1 mL/min. in a hydrophobic tube, the resulting liposomes range in diameter from 100 - 200 nm with a relatively low polydispersity which is equivalent to previously established batch processing of liposomes. The highest shear is expected at 9000 rpm where the film is thinner and this results in a more compact and tight distribution for the self-assembled liposomes. At this speed, 100 nm diameter particles are present with a low polydispersity of 0.3. Before processing the lipid suspension was turbid in nature, which indicates the presence of large colloidal particles. After VFD processing the solution became clear which is consistent with the formation of liposomes.

[00102] The use of the hydrophobic tubes results in more control over the formation of liposomes, as established for the confined mode of operation of the device. Such tubes will have less viscous drag in the fluid flow and this appears to control the self-assembly of the liposomes. For a hydrophilic tube there is higher PDl with less control over the self-assembly process, and presumably this relates to the more dominating viscous drag on the shear stress leading to less control over the self-assembly leading to bilayers which then form liposomes.

[00103] It is much more energetically favourable for the hydrocarbon chain to associate with other hydrocarbon carbons in reducing the surface area when in water, which is a free energy driven process with a decrease in entropy. The phospholipids arrange with their hydrophilic head group in contact with water in lowering the interfacial tension, but in a hydrophilic tube the expected additional drag will perturb the fluid dynamics, and presumably this is at the expense of controlling the self-assembly process.

[00104] Optimising the operating parameters of the VFD

[00105] The VFD has a number of parameters to vary, beyond those of conventional batch processing, and this enhances the versatility of the microfluidic platform. These include controlling the rotational speed, the flow rates, the tilt angle, surface effects (hydrophobic versus hydrophilic), as well as the expected controlling the concentration, temperature, and choice of solvent. A number of these parameters were systematically varied in tracking towards an optimised process, as discussed below. [00106] Optimisation of Tilt angle Θ

[00107] The effect of different tilt angles on the formation of the liposomes was studied from

0° to 75° under continuous flow mode, at flow rate of 0.1 mL/min and rotational speed 9000 rpm; 90° was not attempted as the processed sample would be difficult to collect. At 0° tilt, both the size and PDT have large error bars, where the shear comes exclusively from the viscous drag. This in accord with confined mode studies at 0° tilt showing no effect relative to batch processing. As the tilt angle increase there is a combined effect of centrifugal force and gravitational force which results in an increase in shear stress in the thin film. This is associated with an increase in residence time.

Increasing the tilt angle gives lower polydispersity and thus a more homogeneous population of liposomes.

[00108] Optimisation of flow-rates by DLS & NTA

[00109] A series of flow rates were varied from 0.1 mL/min to 1.0 mL/min at a rotational speed of 9000 rpm and tilt angle of 45° to establish the optimised flow, i.e. where the size distribution is compact and the size and the polydispersity are also optimal. Each flow rate, 0.1 to 1 mL/min, was for the device operating at 9000 rpm, and for a concentration of lipid at 25 μg/mL. The size and PDl was characterised using DLS. At lower flow rates the size and polydispersity is narrow up to 0.5 mL/min, thereafter it is steady. There is little change in polydispersity for the different flow rates.

[001 10] The VFD processed liposomes distribution of the particles was also studied using nanoparticle tracking analysis (NTA), for all the flow rates, i.e. 0.1 , 0.3, 0.5, 0.7, 1.0 mL/min. As the flow rate is increased, using a computer controlled syringe pump, the residence time increases, which is defined by the minimum time required for a small part of the processed sample to exit the rotating tube after being delivered to the base of the tube. The higher the flow rate, the shorter the processing time, which may affect the formation of the liposomes, and indeed this is reflected in the data from NTA, with reduced uniformity of the particles for increasing flow rates (Figure 2).

[001 1 1 ] An increase in flow rate is associated with an increase in volume of liquid in the VFD tube at any given time. This is associated with an increase in the thickness of the film and this will result in less average viscous drag. Presumably this is reflected in an increase in heterogeneous population of liposomes. The overall data suggests that low flow rates, from 0.1 mL/min to 0.3 mL/min are optimal for a narrow size distribution of the liposomes, at least for a concentration of 25 μg/mL and rotational speed of 9000 rpm.

[001 12] Concentration effects on liposome formation [001 13] The concentration of lipid suspension was varied from 25 μβ/ηιΐ. to 0.1 mg/mL to establish if this results is an any change in the self-assembly, for example, from the formation of liposomes or lipid rods, which are much more entropically favoured as the concentration

dramatically increases from the critical micellar concentration.

[001 14] The critical micellar concentration for the POPC lipid is 0.46 nM, and on increasing the concentration, there is further transition to larger and more ordered mesophase lyotropic liquid crystalline structures, which can form complex three-dimensional networks of interconnected surfaces. They can be arranged into ordered arrays of cylinders. However, this was not evident in the liquids processed using the VFD, i.e. there is control of the self-assembly in forming only liposomes in the microfluidic device, with one notable exception in forming tubules at a certain rotational speed. This arises from vibrationally induced faraday wave effect.

[001 15] Zeta-potential and stability of liposomes

[001 16] Zeta potentials of the solutions of liposomes generated in the VFD in Milli-Q water at a pH of 7.4 were measured using DLS. The Zeta potential was in the range - 15 to -25 mV which suggests that the liposomes are relatively stable, the higher the charge (±mV), the higher the repulsion.

[001 17] Characterisation using Scanning Electron Microscopy (SEM) & bilayer thickness using Atomic Force Microscopy (AFM)

[001 18] A fast Scan Ascyst peak force tapping mode AFM was used. The advantages of using this mode over the multimode AFM are that the peak force tapping mechanism decouples the cantilever response from the resonance, and automatically adjusts all the critical imaging parameters, direct interaction and a uniformly optimised fed back loop. The latter is controlled automatically for all samples. Scan Asyst algorithm is used to optimise the set-point the gain for the minimum force required to track samples and can lower the Z limit automatically which results in clearer images. The user is required to select the actual scan area, which can be achieved by input of appropriate values or simply by using a mouse and drawing a box inside the previous image acquired34.

Liposomes tend to collapse after drying, and this is expected on the silicon surface, Fig 33. The vertical thick ness from an AFM can then provide the thickness of the bilayer. If the thickness of the bilayer is around 5 nm then once the liposomes completely collapses, the total thickness across the bilayer should be double this. Fig. 33, assuming that the liposomes are uni-lamellar, ie. a single bilayer. For the sample studied, the measured thickness was 15.9 nm, but given that a 2.0 nm layer of platinum was deposited on the surface, the measured distance is in the ball park for a deflated unilamellar vesicle. To further investigate the hypothesis that the vesicles are uni-lamellar, and that they deflate on loss of solvent, further characterization was undertaken with the liposomes in solution. The results are shown in Figures 3 and 4.

[001 19] Following the process of this example, micelles were also formed from a phospholipid suspension (Figure 6), phosphonated calixarenes (Figure 7) and surfactants (Figure 8).

[00120] Example 2 - Vortex fluidic mediated membrane fusion of liposomes and FRET assay

[00121 ] VFD experiments were initially undertaken in the confined mode of operation of the device, where a set amount of liquid in a 20 mm OD (16.7 mm ID) tube was subjected to shear stress.

[00122] Processing liposomes

[00123] A lipid suspension was prepared at a concentration of 1 mg/mL, and was used in a VFD at room temperature, under continuous flow mode, with one jet feed delivering the liquid at a flow rate of 0.1 mL/min. The rotational speed was set at 9000 rpm and the tilt angle was 45°, using a hydrophobic tube.

[00124] Processing fluorophore labelled liposomes

[00125] After preparing liposomes under continuous flow mode, 1 mL of the resulting liposome solution was added to 1% of l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamin e rhodamine B sulfonyl) (ammonium salt) (Rh-PE). The mixture was then added to the VFD operating in the confined mode, using speeds of 5000 rpm, with a tilt angle of 45°. These processes were run for 15 minutes. The same operations were also performed in the presence of 1 % oleoyl-2-hydroxy-sn-glycero-3- phosphoethanolamine-N-[tetra (ethylene glycol)]-N'-(7-nitro-2-l ,3benzoxadiazol-4-yl) (ammonium salt) (NBD-PE) instead of 1% Rh-PE.

[00126] Mixing experiments for membrane fusion

[00127] In this processing, 1 mL of Rh-PE labelled liposomes were mixed with 1 mL of NBD-PE labelled liposomes using the confined mode of operation in the VFD, for 15 minutes, with a rotational speed of 5000 rpm, with a tilt angle of 45°, in a hydrophobic tube.

PEG stabilisation experiments [00129] 1 lnL of the 1%PEG of the liposomal concentration and 1 mL of Rh-PE liposomes (5 krpm) was mixed using a magnetic stirrer for 5 to 10 minutes, and similarly for NBD-PE liposome dye systems. The labelled liposomes Rh-PE with PEG (1 mL & 1 wt%), and 1 mL of labelled liposomes NBD-PE with PEG 1 wt%, were mixed together in a VFD in confined mode for 15 min, for a rotational speed of 5000 rpm, with a tilt angle of 45°, in a hydrophobic tube.

[00130] Mixing experiments for membrane fusion after PEG stabilisation

[00131] In this processing, 1 mL of Rh-PE labelled liposomes and 1% peg were mixed with 1 mL of NBD-PE labelled liposomes with 1% peg using the confined mode of operation in the VFD, for 15 minutes, with a rotational speed of 5000 rpm, with a tilt angle of 45°, in a hydrophobic tube.

[00132] Example 3 - Preparation of nano-particles containing the bio-active molecule curcumin

[00133] In this processing, the active molecule curcumin was solubilised at high alkaline pH of

12.0 and re-precipitated in presence of citric acid. This technique is also known as "anti-solvent" strategy. To this solution there was a further addition of a lipid suspension to stabilise the curcumin precipitates. This solution was introduced into the thin film reactor at a flow-rate of 1 mL/min. The final pH was maintained at 5.0. The final product was collected from the outlet (Figure 1 1).

[00134] Dynamic Light Scattering (DLS)

[00135] Particle size distribution and polydispersity index were measured by dynamic light scattering (Nano ZS90, Malvern instruments, Worcester, UK) technique at 25°C, using a He-Ne laser of 633 nm and a detector angle of 173°. Three independent measurements were performed for each sample. The Malvern zeta sizer instrument measured the time dependent fluctuations of light scattered based on the particle sizes. Samples were analyzed 24 hours after preparation (Figures 1 1 and 12).

[00136] Scanning electron microscopy (SEM)

[00137] Samples were analyzed using Inspect FEI F50 SEM (PS216). The spot size was 4.0, voltage was 10.0 Kv and magnification was at 50,000. The sample preparation was as follows: 20 of as-prepared sample was drop casted on silicon wafer and air dried overnight, followed by Platinum sputter coating of 2 nm in thickness, then observed under SEM with the above given parameters (Figure 14).

[00138] UV-visible spectrophotometer (Uv-vis) [00139] Ultraviolet-visible (UV-Vis) absorption spectra of curcumin were recorded on a CARY

Eclipse UV spectrometer with 1 cm optical path. The spectrum was recorded from 300 - 650 nm wavelength (Figure 13).

[00140] Fluorescence spectrophotometer

[00141 ] All the measurements were conducted on Vary cary eclipse fluorimeter. The excitation for curucmin was set as 425 nm and emission data was collected.

[00142] Fourier Transfer Infrared Spectroscopy (FTIR)

[00143] All the measurements were conducted on perkin-elmer FTIR spectrum 100. The wave number was set from 500 - 4000 cm-1. The lyophilised dry samples were analysed through FTIR with a control FTIR on curcumin powder for comparision (Figure 15).

[00144] X-ray diffraction (XRD)

[00145] All the measurements were conducted on bruker XRD, the lyophilised dry samples were analysed through XRD with a control XRD on curcumin powder for comparision (Figure 16).

[00146] Example 4 - Preparation of nano-particles containing the bio-active molecule beta- carotene

[00147] In this processing, beta-carotene was suspended in ethanol and introduced into the thin- film reactor which had an inner surface functionalised with hydrophobic coating from one jet-feed. The jet-feed had a magnetic stirrer to keep the suspension in motion. The lipid suspension was introduced into the thin-film reactor from another jet-feed. Both the jet feeds had a final flow-rate of 0.5 mL/min. The final product was collected from the outlet (Figure 32).

[00148] Dynamic Light Scattering (DLS)

[00149] Particle size distribution and polydispersity index were measured by dynamic light scattering (Nano ZS90, Malvern instruments, Worcester, UK) technique at 25°C, using a He-Ne laser of 633 nm and a detector angle of 173°. Three independent measurements were performed for each sample. The Malvern zeta sizer instrument measured the time dependent fluctuations of light scattered based on the particle sizes. Samples were analyzed 24 hours after preparation (Figure 32).

[00150] Atomic Force Microscopy (AFM) [00151] The samples were analyzed using an atomic force microscope (Nanoscope V, Multimode

SPM). Images were acquired using silicon probes in tapping mode in air with Scanner "J" from 500 nm-3 μιη scan sizes. The sample preparation for AFM was as follows: 20 uL of 1 : 10 diluted sample was drop casted on silicon wafer and air dried overnight, the sample was washed 3 times with Milli-Q water and images were taken under air AFM (Figure 33).

[00152] Example 5 - Preparation of nano-particles containing the bio-active protein C- phycocyanin

[00153] In this processing the protein c-phycocyanin and lipid were solubilised in the presence of a phosphate buffer. This solution was introduced into the thin film reactor at at a flow-rate of 0.1 mL/min at a pre-determined speed of 8000 rpm. The final product was collected from the outlet (Figure 34).

[00154] Atomic Force Microscopy (AFM)

[00155] The samples were analyzed using an atomic force microscope (Nanoscope V, Multimode

2 SPM). Images were acquired using silicon probes in tapping mode in air with Scanner "E" from 500 nm-3 μιη scan sizes. The sample preparation for AFM was as follows: 20 μΤ of 1 : 100 diluted sample was drop casted on silicon wafer and air dried overnight, the sample was washed 3 times with Milli-Q water and images were taken under AFM (Figure 35).

[00156] Example 6 - Vortex fluidic assisted fabrication of nano-emulsions

[00157] In this processing, nano-encapsulations were formulated using fish-oil acting as a bioactive ingredient, a mixture of non-ionic surfactant and water. Oil in water nano-encapsulations containing a 2% (w/v) dispersed phase at a surfactant to oil ratio of 2 mg/mL. Briefly, a lipid: oil suspension in water was premixed as an emulsion, then introduced in the VFD through jet-feeds with the rotational speed at 8000 rpm and a flow-rate of 0.1 mL/min at 25°C. The product was collected and sonicated for 20 minutes. Also, another homogenization method was applied in order to compare with the VFD formulation, the same concentrations as above were homogenized at 13,500 rpm for 10 minutes at 25°C and similarly sonicated for 20 minutes (Figure 28).

[00158] Dynamic Light Scattering (DLS)

[00159] Nano-encapsulation particle size distribution and polydispersity index were measured by dynamic light scattering (Nano ZS90, Malvern instruments, Worcester, UK) technique at 25°C, using a He-Ne laser of 633 nm and a detector angle of 173°. Three independent measurements were performed for each sample. The Malvern zeta sizer instrument measured the time dependent fluctuations of light scattered based on the particle sizes. Samples were analyzed 24 hours after preparation (Figure 30).

[00160] Scanning Electron Microsocpy (SEM)

[00161] The nano-encapsulation samples were analyzed using Inspect FEI F50 SEM (PS216).

The spot size was 2.0, voltage was 5.0 Kv and magnification was at 50,000. The sample preparation was as follows: 20 μΐ. of as-prepared sample? was drop casted on silicon wafer and air dried overnight, followed by Platinum sputter coating of 2 nm in thickness, then observed under SEM with the above given parameters (Figure 29).

[00162] Transmission Electron Microscopy (TEM)

[00163] The nano-encapsulation samples were analyzed using high resolution Transmission

Electron Microscope (FEI Titan Themis 80-200). The sample preparation was as follows: 20 μL· of sample was fixed on the carbon grids and left to air dry for an hour, the excess sample was removed by blotting paper, followed by staining of 2% uranyl acetate solution, then images were taken under TEM at 250x magnification.

[00164] Atomic Force Microscopy (AFM)

[00165] The nano-encapsulation samples were analyzed using an atomic force microscope

(Nanoscope V, Multimode 2 SPM). Images were acquired using silicon probes in tapping mode in air with Scanner "E" from 500 nm-3 μιη scan sizes. The sample preparation for AFM was as follows: 20 of 1 : 100 diluted sample was drop casted on silicon wafer and air dried overnight, the sample was washed 3 times with Milli-Q water and images were taken under AFM (Figure 31 ).

[00166] Real-time small angle neutron scattering (SANS)

[00167] Real-time SANS for SDS micelles under shear: A total 2wt% sodium dodecylsulphate

(SDS) was dissolved in D20. 1 mL of suspension is rotated in the thin film reactor at pre-determined speed of 2000, 4000, 6000, 8000 rpm in confined mode.

[00168] Real-time SANS for phosphonated calix-4-arene micelles under shear: A total 0. lwt% of phosphonated calix-4-arenes was dissolved in D20. 1 mL of suspension is rotated in the thin film reactor at pre-determined speed of 2000, 4000, 6000, 8000 rpm in confined mode. [00169] Real-time SANS for Phospholipid under shear: A total 0.1 wt% of phospholipid (POPC) was dissolved in D20. 1 mL of suspension is rotated in the thin film reactor at pre-determined speed of 2000, 4000, 6000, 8000 rpm in confined mode.

[00170] Throughout the specification and the claims that follow, unless the context requires otherwise, the words "comprise" and "include" and variations such as "comprising" and "including" will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

[00171] The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement of any form of suggestion that such prior art forms part of the common general knowledge.

[00172] It will be appreciated by those skilled in the art that the invention is not restricted in its use to the particular application described. Neither is the present invention restricted in its preferred embodiment with regard to the particular elements and/or features described or depicted herein. It will be appreciated that the invention is not limited to the embodiment or embodiments disclosed, but is capable of numerous rearrangements, modifications and substitutions without departing from the scope of the invention as set forth and defined by the following claims.

REFERENCES

[00173] [1] Kim, J. (2016). Liposomal drug delivery system. Journal of Pharmaceutical

Investigation, 46(4), 387-392.

[00174] [2] Fouladi, F., Steffen, K., & Mallik, S. (2017). Enzyme-Responsive Liposomes for the

Delivery of Anticancer Drugs. Bioconjugate Chemistry, 28(4), 857-868.

[00175] [3] Dario Carugo, Elisabetta Bottaro, Joshua Owen, Eleanor Stride, & Claudio Nastruzzi.

(2016). Liposome production by microfluidics: Potential and limiting factors. Scientific Reports, 6, Scientific Reports, 2016, Vol.6.

[00176] [4] Immordino, M. L., Dosio, F., & Cartel, L. (2006). Stealth liposomes: review of the basic science, rationale, and clinical applications, existing and potential. International Journal of

Nanomedicine, (3), 297-315.

[00177] [5] Gibbs, Bernard F., Kermasha, Selim, Alii, Inteaz, & Mulligan, Catherine N. (1999).

Encapsulation in the food industry: A review. International Journal of Food Sciences and Nutrition, 50(3), 213.

[00178] [6] [14] Zhang, Yumin, Yang, Cuihong, Wang, Weiwei, Liu, Jinjian, Liu, Qiang, Huang,

Fan,. Liu, Jianfeng. (2016). Co-delivery of doxorubicin and curcumin by pH-sensitive prodrug nanoparticle for combination therapy of cancer. Scientific Reports, 6( 1 ), 21225.

[00179] [7] Meng, J., Guo, F., Xu, H., Liang, W., Wang, C, & Yang, X. (2016). Combination

Therapy using Co-encapsulated Resveratrol and Paclitaxel in Liposomes for Drug Resistance Reversal in Breast Cancer Cells in vivo. Scientific Reports (Nature Publisher Group), 6, 22390.

[00180] [8] Yuko T. Sato, Kaori Umezaki, Shinichi Sawada, Sada-Atsu Mukai, Yoshihiro Sasaki,

Naozumi Harada, Kazunari Akiyoshi. (2016). Engineering hybrid exosomes by membrane fusion with liposomes. Scientific Reports, 6, Scientific Reports, 2016, Vol.6.

[00181] [9] Wu, Liu, & Lee. (2006). A folate receptor-targeted liposomal formulation for paclitaxel. International Journal of Pharmaceutics, 316( 1), 148-153.

[00182] [10] Zheng, Y., Tang, L., Mabardi, L., umari, S., & Irvine, D. (2017). Enhancing

Adoptive Cell Therapy of Cancer through Targeted Delivery of Small-Molecule Immunomodulators to Internalizing or Noninternalizing Receptors. ACS Nano, 11(3), 3089-3100. [00183] [11] Pavvar, A., Bothiraja, C, Shaikh, K, & Mali, A. (2015). An insight into cochleates, a potential drug delivery system. RSC Advances, 5(99), 81 188-81202.

[00184] [12] Sorrenti, A., Ilia, O., & Ortuno, R. (2013). Amphiphiles in aqueous solution: Well beyond a soap bubble. Chemical Society Reviews, 42(21 ), 8200-8219.

[00185] [13] Spector, M., Singh, A., Messersmith, P., & Schnur, J. (2001). Chiral Self-Assembly of Nanotubules and Ribbons from Phospholipid Mixtures. Nano Letters, 1(7), 375-378.

[00186] [14] Mcclements, D. (2012). Nanoemulsions versus microemulsions: Terminology, differences, and similarities. Soft Matter, 8(6), 1719-1729.

[00187] [15] Xing, H., Hwang, K., & Lu, Y. (2016). Recent Developments of Liposomes as

Nanocarriers for Theranostic Applications. Theranostics, 6(9), 1336-52.

[00188] [16] Hwang, Kim, Choo, Seong, Hien, & Lee. (2012). Effects of operating paraineters on the efficiency of liposomal encapsulation of enzymes. Colloids and Surfaces B: Biointerfaces, 94(C), 296- 303.

[00189] [17] Yasmin, L., Chen, X., Stubbs, ., & Raston, C. (2013). Optimising a vortex fluidic device for controlling chemical reactivity and selectivity. Scientific Reports, 3(1), 2282.