WO2015120861A1

WO2015120861A1 - Method for producing nano-embedded microparticles

Info

Publication number: WO2015120861A1
Application number: PCT/DK2015/050032
Authority: WO
Inventors: Jorrit Jeroen WATER; Pall Thor INGVARSSON; Adam Jun BOHR
Original assignee: University Of Copenhagen
Priority date: 2014-02-14
Filing date: 2015-02-13
Publication date: 2015-08-20

Abstract

The present invention relates to a rapid, high-throughput and continuous method for producing nano-embedded microparticles in powder form, thereby providing a cost- effective process which can be performed aseptically. The invention further relates to an apparatus for performing the method of the invention.

Description

Method for producing nano-embedded microparticles Field of invention The present invention relates to a rapid, high-throughput and continuous method for producing nano-embedded microparticles in powder form, thereby providing a cost- effective process which can be performed aseptically. The invention further relates to an apparatus for performing the method of the invention. Background of invention

Nanoparticles are considered attractive compounds for several industries, including the pharmaceutical industry, food industry and cosmetics industry, in particular for use as vehicles. Nanoparticles can be used as a drug delivery system to encapsulate or entrap an active compound or drug and nano-encapsulation or nano-entrapment may thus result in increased pharmacokinetic half-life. Encapsulation and entrapment may also result in increased stability of the active compound. Nanoparticles may be designed in order to control drug release at the target site. Bioavailability of the drug may also be increased, both locally and systemically.

Nanoparticles can be difficult to handle because of their small size, which also renders them less stable than microparticles. Collecting devices generally collect microparticles more efficiently than nanoparticles. In consequence, it can be desirable to embed nanoparticles in a carrier matrix to form nano-embedded microparticles (also referred to as Trojan particles, nanocomposite microparticles, nanoparticle-assembled capsules or nanoaggregates). Nano-embedded microparticles have a greater size than nanoparticles, thereby facilitating particle collection and potentially increasing physical stability of the particles. Thus it can be advantageous to produce nano-embedded microparticles containing sensitive or degradation-prone proteins. When delivered to the target site, the matrix is degraded at a speed that is dependent on the nature of the matrix and of the environment at the target site, allowing re-dispersion of the nanoparticles in the local environment depending on the matrix properties. Another advantage of nano-embedded microparticle powders is the increased shelf-life and the reduced production costs. Thus nano-embedded microparticles are attractive for various applications, including drug delivery applications. Duret et al., 2012, discloses a method for generating nano-embedded microparticles of intraconazole of a size comprised between 250 nm and 2 μηι. First, a surfactant is dissolved in a solution under stirring, before itraconazole is added. The resulting suspension is collected and is first homogenised using a high-speed homogeniser for 10 minutes. The resulting pre-homogenised suspension is collected and further homogenised using a high-pressure homogeniser (300 cycles). The homogenised suspension is then collected and resuspended in a large volume of a carrier solution. After resuspension, the resulting suspensions are collected and spray-dried, resulting in the formation of microparticles embedded with nanoparticles. The method disclosed in Duret et al. requires a surfactant and is not suited for high-throughput. The method involves numerous steps, takes a long time and is not continuous since the output of each step needs to be collected before proceeding to the next step. Furthermore, the method cannot conveniently be performed aseptically.

El-Sherbiny et al., 2012, discloses a method for generating curcumin-loaded PLGA nanoparticles embedded in a PEG-chitosan graft copolymer. The method involves numerous steps, some of which require high temperature or specific environments such as dry nitrogen atmosphere. The output of each step needs to be collected before proceeding to the next step. Therefore the method cannot be performed aseptically. The method is time-consuming and takes several days to generate nano-embedded microparticles. Large amounts of solvent are required, which makes the process costly. The produced particles may also comprise residual surfactant solvent. Because of the long reaction or stirring times involved, the method is only suited for producing nano- embedded microparticles from stable nanoparticles. The method is not continuous nor convenient for aseptic production. Cheow et al. 2011 , Azarmi et al. 2006, Sham et al. 2003, Chougule et al. 2007, Jensen et al. 2012, Grenha et al. 2005 and Muttil et al. 2010 report similar methods for the generation of nano-embedded microparticles. None of the above methods discloses a method which is fast, continuous and suitable for high-throughput production of nano-embedded microparticles. Because of the discontinuous nature of these methods, they cannot be carried out in a fully-integrated platform and thus cannot be performed aseptically. As a consequence a further step of sterilisation may be required; this is not only time-consuming, but can negatively affect stability of the microparticles. Thus fast, continuous and high-throughput methods for producing nano-embedded microparticles are needed, which can be performed aseptically in a fully-integrated platform. Such methods need to be fast enough that nano-embedded microparticles can be generated even from sensitive or unstable active compounds. Methods are also needed which do not require surfactants, stabilisers or solvents, of which traces may remain in the final product, thereby reducing the fraction of active compound, sometimes causing additional safety issues. mary of invention

The invention is directed to a continuous method for producing nano-embedded microparticles. The method comprises the steps of:

i) generating nanoparticles in one or more microfluidic devices or static

mixers, thereby generating a nanoparticle suspension;

ii) mixing said nanoparticle suspension with a matrix solution, thereby

obtaining a feed stream;

iii) drying said feed stream, thereby obtaining a dry powder comprising nano- embedded microparticles.

The invention further relates to an apparatus for performing the method of the invention, said apparatus comprising:

i) one or more microfluidic devices or static mixers;

ii) means for providing fluid to said one or more microfluidic devices or static mixers;

iii) means for providing a matrix solution;

iv) a junction for mixing the output from said one or more microfluidic devices or static mixers with said matrix solution;

v) means for performing liquid atomisation and drying of the output of said junction;

vi) a collecting device.

Surprisingly, the combination of microfluidics or static mixing and spray drying results in a method which is fast, can be performed continuously at least up to the collecting step, can be adapted to be high-throughput and can be performed aseptically, thereby reducing production costs and increasing production efficiency. The method disclosed here combines microfluidics at high flow rates (2-20 mL/min) combined with spray drying. One could expect such a fast production method to result in incomplete particle formation. However, the nano-embedded microparticles produced with the method of the invention have essentially the same size as nanoparticles produced by microfluidics only, showing that particle formation is indeed complete.

The method of the invention presents numerous advantages. One advantage is that the method is fast: the method can be performed so that an active compound is converted to a nanoparticle embedded in a microparticle in a matter of minutes or seconds. A visible or easily collectable powder output can be generated within 10 minutes of starting the method or less. Another advantage is that the method can be performed continuously at least up to the collection of the nano-embedded

microparticle powder, which can be performed continuously or discontinuously depending on the collection device employed. The method of the invention is suited for high-throughput production and is easily scaled up. The method of the invention involves very short reaction and/or mixing times, so that it is suited for producing nano- embedded microparticles containing unstable or sensitive active compounds. Also disclosed here is an apparatus that provides a fully-integrated platform for performing the method of the invention. Thus the method of the invention can be performed aseptically by sterilising the apparatus and/or feed materials before use, thereby further reducing production times and costs otherwise incurred.

Description of Drawings

Figure 1. Schematic drawing of a nano-embedded microparticle of the invention. NP: nanoparticle. M: matrix.

Figure 2. Schematic overview of an apparatus of the invention. (A): Combined microfluidic device and spray dryer. One or more syringes (1) provide the microfluidic device (2) with incoming liquid. The peristaltic pump (3) pumps the matrix solution from the container (4) for mixing with the nanoparticle solution exiting the microfluidic device (2) at the junction (5). The resulting feed stream is fed into the drying region (7) of the spray dryer via the nozzle or atomizer (6). The resulting particles are collected in the collecting device (8). Arrows show the direction of the flow. (B): Enlarged view of the microfluidic device (2), the peristaltic pump (3) and the junction (5). The nanoparticle solution (9) is mixed with the matrix solution (10) at the junction (5). The resulting solution is the feed stream (1 1). Arrows indicate the direction of the flows.

Figure 3. Representative scanning electron microscopy images of microparticle powders at different magnifications, obtained by the method of the invention. Particles from two samples are shown; the samples were prepared at different conditions. (A) Sample 1 , prepared at microfluidics flow rate, 3 ml/min; spray drying flow rate, 3 ml/min and matrix concentration (20 mg/ml) and. (B) Sample 2, prepared at microfluidics flow rate, 6 ml/min; spray drying flow rate, 3 ml/min and matrix concentration (50 mg/ml).

Figure 4. (A) Particle size measurement (left axis, diameter in nm) and PDI (right axis) of the nanogels before spray drying. Values represent the mean ± S.D. (n=3). (B) Zeta potential (Y-axis, in mV) measurement of the four formulations nanogel formulations before spray drying. Values represent the mean ± S.D. (n=3). (C) Comparison of particle size (Y-axis, diameter in nm) of nanogels and the NEMs with trehalose after re- dispersion of NEMs shown in the figure. Values represent the mean ± S.D. (n=3).

Figure 5. SEM images of the NEM powders with Trehalose demonstrate the size and shape of the particles.

Figure 6. This figure shows an overview of the particle preparation performance of both the size (Fig. 6A; Y-axis: size in nm) and polydispersity index (Fig. 6B; Y-axis: PDI) comparing PLGA particles with different amounts of sodium caprate (w/w) prepared with microfluidic mixing (PLGA concentration was fixed at 5 mg/ml and the same flow rates were used as above). (n=3, means ±SD).

Detailed description of the invention

Definitions

Active compound: An active compound as understood herein is any compound exerting a desired activity. Active compounds include, but are not limited to, aroma compounds, pharmaceutically active compounds, such as therapeutic compounds, cosmetic compounds.

Collecting device: Several types of particle collecting devices (or collectors) exist, the most widely used being the cyclone collector, in which centrifugal forces are used to separate the particles from the airstream. Collectors may be used in parallel, e.g. two collectors, in order to increase e.g. efficiency. The collecting device may be suitable for the collection step to occur in a continuous manner.

Continuous: As understood herein, the term 'continuous' refers to a process which can be run without interruption. In the present context, a continuous production method is to be understood as a method wherein all steps at least up to the collection step can be performed without pause or interruption, with an uninterrupted flow from one step to the next.

Dispersion: Dispersion as understood herein refers to chemical dispersion, i.e. a system in which particles are dispersed in a continuous phase of a different composition (or state). Generally, the particles dispersed in the liquid or solid matrix forming the dispersion medium are believed to be statistically distributed; in other words, a dispersion typically does not display a particular structure. Aerosols (liquid dispersed in a gas), emulsions (liquid dispersed in a liquid), foams (gas dispersed in a solid or in a liquid), solid aerosol (or dust; solid dispersed in a gas) and suspensions (solid dispersed in a liquid) are some examples of dispersions.

Dispersity: Dispersity is a measure of the heterogeneity of sizes of molecules or particles in a mixture. A collection of objects is called monodisperse or uniform if the objects have the same size, shape, or mass. A sample of objects with an inconsistent size, shape and mass distribution is called polydisperse or non-uniform.

Electrospinning: In this technique like in electrospraying, application of high voltage to a polymer solution can result in the formation of a cone-jet geometry. The term

'electrospinning' is used if the jet turns into very fine fibers instead of breaking into small droplets.

Electrospraying: Electrospraying is sometimes also referred to as electrohydrodynamic atomization. It is a process in which electricity is employed to disperse a liquid stream. High voltage is applied to a liquid supplied through an emitter (usually a glass or metallic capillary). Ideally the liquid reaching the emitter tip forms a Taylor cone, which in turn emits a liquid jet through its apex. Varicose waves on the surface of the jet lead to the formation of small and highly charged liquid droplets, which are radially dispersed due to Coulomb repulsion.

Encapsulation or micro-encapsulation: The term 'encapsulation' will be used herein interchangeably with the terms 'micro-encapsulation' and 'entrapment'. It refers to a process in which particles or droplets (the core, also referred to as internal phase) are surrounded or partly surrounded by a coating (or shell). In a relatively simple form, a microcapsule is a small sphere with a uniform coating. Capsules typically have diameters between few micrometres and few millimetres and are thus often called micro-capsules.

Hydrodynamic focusing: Hydrodynamic focusing occurs when multiple flows with substantially different flow rates come into contact. The most common configuration is a 3-inlet device that allows rapid mixing of the contents of a small core stream (also termed inner stream or focused stream) with a bulk sheath flow (also termed outer stream). The centre flow stream is pinched between two sheath streams, thereby shrinking the core stream width. Controlling the flow rate of the sheath streams allows control of the direction of the core stream. This doubles the area of the diffusion interface and greatly reduces the diffusion distances. In conventional microfluidic devices, hydrodynamic focusing is achieved through careful control of flow rates by multiple pumps and/or pressure sources. Hydrodynamic focusing is used to improve mixing efficiency and provide a horizontally uniform environment in the reaction. Hydrodynamic focusing may be two-dimensional (2D) or three-dimensional (3D). 2D focusing relies on sheath flows in one plane (horizontal or vertical) while 3D focusing relies on horizontal and vertical sheath flows.

Liquid atomization: Liquid atomization processes allow dispersion of a liquid stream. Such processes include spray drying, electrospraying, electrospinning, supercritical fluids assisted atomization, ultrasonic atomization, spray freeze-drying and melt congealing.

Mass median aerodynamic diameter (MMAD): The MMAD is the median of the distribution of airborne particle mass with respect to the aerodynamic diameter.

MMADs are usually accompanied by the geometric standard deviation (g or sigma g) which characterizes the variability of the particle size distribution.

Matrix solution: A matrix solution or carrier solution is to be construed as the solution which is mixed with the nanopartides prior to spray drying. The matrix may comprise stabilisers, surfactants, compounds altering taste, and other compounds. The matrix is such that the nanopartides are substantially stable (colloidal) in the matrix. The matrix may be such that it does not react with the nanopartides.

Melt congealing or spray congealing: In this process a liquid melt is atomized into a cooling chamber. A cold gas stream enters the chamber, typically in co-current configuration, contacting the droplets whereby solidification takes place. This involves the transformation of molten droplets from liquid to solid state with removal of energy from the droplets. The transition of a melt from a soft or fluid state to a rigid or solid state by cooling is called congealing. Hence, the spray congealing process can be described by four events: i) atomization of the melt into droplets , ii) contact of the droplets with the cold congealing gas, iii) solidification of the droplets into particles and iv) separation of the particles from the congealing gas.

Microfluidic device: A microfluidic device or microfluidic chip as understood herein is a device allowing manipulation of fluids and particles that are geometrically constrained at the micron scale. Such miniaturised devices allow high throughput manipulation and provide well-defined control over the cellular microenvironment due to precise fluid handling, for example via hydrodynamic focusing. Fluid enters the microfluidic device via one or more inlet channels. The manipulated fluid exits the microfluidic device via one or more outlet channels. Certain microfluidic devices enable hydrodynamic focusing; in other words, a first fluid can be constrained in an inner stream (focused stream) by a second fluid in an outer stream (or sheath flow).

Microparticle: A microparticle is a particle having a size in the range of 1 μηι to 999 μηι. Microvortex: A microvortex as understood herein is a vortex formed in the microfluidic device, at the intersection of the inlet channels. Microvortices favour convective mixing as compared to laminar flow, which favours diffusive (passive) mixing, while at the same time enabling flow focusing and thus preventing aggregation near the channel walls.

Nanocarrier: A nanocarrier is a carrier with a size ranging from 1 to 100 nm. It is used as a transport module for another substance, such as a drug. Commonly used nanocarriers include micelles, polymers, carbon-based materials, liposomes and other substances.

Nano-embedded microparticle: A nano-embedded microparticle as understood herein is a microparticle comprising nanoparticles embedded in a micro particles formed from a matrix.

Nanoparticle: A nanoparticle is a particle having a size in the range of 1 nm to 999 nm. There are several types of nanoparticles, and the term nanoparticle as used herein can refer to any of them. Examples of types of nanoparticles include, but are not limited to, nanocapsules, nanogels, nanospheres, nanocomplexes and colloids.

Particle yield: The particle yield as understood herein is equal to the amount of collected powder compared with the amount of solid material fed into a process.

Polvdispersity index (PDI): The polydispersity index (PDI) or heterogeneity index, or simply dispersity, as used herein is a measure of the distribution of molecular mass in a given sample. Redispersibility: Redispersibility is the ability of particles in a powder to redisperse in solution. Redispersibility is measured by measuring the size of the particles

resuspended in water; the smaller the resuspended particles are, the greater their redispersibility. Redispersibility of the nanoparticles embedded in the microparticles of the invention is defined as the ratio between the size of the nanoparticles after dispersion of the spray dried microparticles and the size of similar nanoparticles produced by microfluidics only.

Reynolds number: The Reynolds number (Re) is a dimensionless quantity that is used to help predict similar flow patterns in different fluid flow situations. It is defined as the ratio of inertial forces to viscous forces and consequently quantifies the relative importance of these two types of forces for given flow conditions. Reynolds numbers are used to characterize different flow regimes within a similar fluid, such as laminar or turbulent flow: laminar flow occurs at low Reynolds numbers, where viscous forces are dominant, and is characterized by smooth, constant fluid motion; turbulent flow occurs at high Reynolds numbers and is dominated by inertial forces, which tend to produce chaotic eddies, vortices and other flow instabilities.

Static mixer: A static mixer is a precision engineered device for the continuous mixing of fluid materials such as liquids or gas streams. Thus a static mixer can be used to mix liquids, to mix gas streams, to disperse gas into liquids or to blend immiscible liquids. There are many different designs and geometries used for static mixers in order to manipulate the fluids in a suitable way; flow division and radial mixing are two commonly used designs. Generally the energy needed for the mixing process comes from a loss in pressure as the fluids flow through the static mixer. Such mixing can take place at different orders of size and volume and can be used for high throughput industrial applications.

Spray freeze drying: This process involves spraying a liquid composition at very low temperatures (-90°C) and subsequent dehydration of the resulting frozen particles in a stream of cold, desiccated air.

Spray drying: Spray drying is a method of producing a dry powder by rapidly drying a liquid with a hot gas. The process has three essential steps: atomisation, where droplets are formed; drying gas and droplet contact, where the liquid feed is turned into droplets; and finally powder recovery, where the dried particles are separated from the drying gas stream.

Supercritical fluids assisted atomization: This process is based on the solubilization of supercritical carbon dioxide in a liquid solution, for example a liquid nanoparticle suspension; the ternary mixture is then sprayed through a nozzle, and micro particles are formed as a consequence.

Ultrasonic atomization: Ultrasonic atomization takes advantage of ultrasonic nozzles, which use high (20 kHz to 180 kHz) frequency vibration to produce narrow drop size distribution and low velocity spray from a low viscosity liquid.

Method for production of nano-embedded microparticles

In one aspect, the invention relates to a continuous method for producing a powder composition comprising nano-embedded microparticles, said method comprising the steps of:

i) generating nanoparticles in one or more microfluidic devices or static

mixers, thereby generating a nanoparticle suspension;

ii) mixing said nanoparticle suspension with a matrix solution, thereby

obtaining a feed stream;

In another aspect, the invention relates to an apparatus for performing the method of any one of the preceding claims, said apparatus comprising:

i) one or more microfluidic devices or static mixers;

iii) means for providing a matrix solution;

vi) a collecting device.

The method of the invention is a continuous method for production of nano-embedded microparticles.

In a first step of the method, a nanoparticle suspension is generated by mixing of a first fluid and a second fluid in one or more microfluidic devices or static mixers. One of the first and the second fluids may be a gas. One or both of the first and the second fluids may be a liquid. In some embodiments, the first fluid and the second fluid are liquids. The first liquid may comprise an active compound such as a drug, while the second liquid may contain a nanocarrier material in solution. The two liquids are typically miscible unless an emulsion is desired, whereby the nanocarrier material may be precipitated, crosslinked or complexed in the two liquids. The resulting mixture is a nanoparticle suspension in which the active compound is mixed with the nanocarrier material in nanoparticles. When one of the first and the second fluids is a gas and the other is a liquid, nanobubbles or microbubbles are generated.

In a second step, the nanoparticle solution flowing out of the microfluidic device or of the static mixer is immediately mixed with a matrix solution comprising water-soluble molecules such as sugars, salts or polymers. The second step is performed in continuation of the first step, without collecting the output of the first step. The nanoparticle suspension is continuously flowing directly into the matrix solution, without interruption.

In a third step, the combined matrix solution and nanoparticle suspension is dried to generate a dry powder of nano-embedded microparticles. The nano-embedded microparticles are microparticles of the matrix liquid in which the nanoparticles produced in the first step of the method are clustered.

The method of the invention is particularly useful for stabilising nanoparticles comprising unstable active compounds, such as unstable drugs or carriers. Unstable drugs or carriers are converted into nano-embedded microparticles in just a few seconds because the method is carried out without interruption, thereby greatly decreasing the time during which the active compound can react, can get degraded or otherwise can get negatively affected by the process. The method can be carried out so that a visible and collectable output is generated in a few minutes.

The method of the invention does not necessarily require organic solvents, stabilisers or surfactants, thereby increasing production yields. As a consequence, the fraction of active compound in a powder produced by the present method is greater than in a powder produced by traditional methods. In some embodiments, organic solvents may be used in the microfluidic devices or static mixers to prepare the nanoparticles e.g. via nanoprecipitation, where the solvent is subsequently removed via evaporation without an additional process.

Thus the method of the invention facilitates production and increases production yield of nanoparticles in a stabilised and easily collectable form.

Microfluidic device

In some embodiments, the present method for producing a dry powder composition comprising nano-embedded microparticles comprises the steps of:

i) generating nanoparticles in one or more microfluidic devices, thereby

generating a nanoparticle suspension;

ii) mixing said nanoparticle suspension with a matrix solution, thereby

obtaining a feed stream;

The microfluidic device used in the invention may be one or more microfluidic devices, such as two, three, four, five or more microfluidic devices. In one embodiment the one or more microfluidic devices is a single microfluidic device. When the one or more microfluidic device is two or more microfluidic devices, these may be arranged in parallel or in series. When arranged in parallel, the microfluidic devices collectively produce a larger output and can be used for further scale-up or for mixing several types of nanoparticles in a common stream. The microfluidic device is fed by at least two inlet channels and comprises at least one outlet channel. In one embodiment, the microfluidic device is fed by two or three inlet channels and comprises one outlet channel. The first inlet channel contains a first fluid and the second inlet channel contains a second fluid; the first and second fluids may be a liquid or a gas. The first and the second fluids may be miscible or immiscible. In some embodiments, the first and second fluids are liquids. In some embodiments, one or both of the first and second liquids comprises a dissolved species such as an active compound and the other liquid comprises a dissolved species such as a nanocarrier. Within the microfluidic device, the flows of the first and second liquids are such that mixing can occur between the liquids. Such mixing results in embedment of the dissolved species in the nanocarrier, thereby generating nanoparticles. Microfluidic devices suitable for the generation of nanoparticles according to the method of the invention can have any shape and dimension and are manufactured with materials known in the art. In some embodiments, the microfluidic device has three inlets having each a rectangular cross-section converging in an outlet channel also having a rectangular cross-section. In other embodiments, the cross-section of the inlet channels and/or of the outlet channel is square, circular or oval. In some embodiments, dimensions of rectangular inlet channels are such that their width and height are between 1 μηι and 1000 μηι and their length is between 1 and 20 mm. For example, in one embodiment, the inlet channels are 200 μηι wide, 400 μηι high and 10 mm long. In another embodiment with circular inlet channels, the diameter is between 1 μηι and 1000 μηι and the length is between 1 and 20 mm. In some embodiments, the dimensions of the outlet channel are such that the width and height are between 10 μηι and 3000 μηι and the length is between 1 mm and 50 mm. For example, in one embodiment, the outlet channel is 2000 μηι wide, 400 μηι high and 20 mm long. In another embodiment with a circular outlet channel, the diameter is between 10 μηι and 3000 μηι and the length is between 1 mm and 50 mm. In some embodiments, the outlet channel has dimensions similar to the dimensions of the inlet channel. Such devices give a lower output, which may be desirable when one of the materials used is precious, rare, or costly.

Several parameters may be adjusted to control the formation of nanoparticles. The direction and the flow rates of the first and second liquid may be adjusted. In some embodiments, the first and the second liquids may flow next to each other in a laminar flow. Low flow rates (10-1000 μΙ_/ηιίη) within the microfluidic device favour mixing by diffusion, while high flow rates (1-20 mL/min) favour turbulent flow and mixing by convective forces. In one embodiment, the flow rate of the first and/or the second liquid is between 0.1 and 1 mL/min. In another embodiment, the flow rate of the first and/or the second liquid is between 1 and 5 mL/min. In another embodiment, the flow rate of the first and/or the second liquid is between 5 and 10 mL/min. In another embodiment, the flow rate of the first and/or the second liquid is between 10 and 15 mL/min. In another embodiment, the flow rate of the first and/or the second liquid is between 15 and 20 mL/min. In some embodiments, complexation occurs between the species in the first and second liquids. In other embodiments, an emulsion of the first and the second liquids is formed or a precipitation of the dissolved nanocarrier species takes place when the liquids are mixed. The first and second liquids may be miscible or immiscible. One of the liquids may act as an anti-solvent to the species. A specific molecule,

electromagnetic radiation or a change in the temperature or pH of the environment may also be used as a stimulus or "reactant" to induce the formation or assembly of nanoparticles. Examples include, but are not limited to, a UV-mediated cross-linking process, an enzyme-driven process or a pH-induced precipitation. Further, in some cases unreacted groups of dissolved or precipitated species may be sorted away for instance via a side channel using chip geometry or using electrical or magnetic energy to exclude unwanted material in a continuous manner.

In another embodiment, the microfluidic device is fed by three inlet channels and comprises one outlet channel. A first liquid flows in the first inlet channel, a second liquid flows in the second inlet channel and a third liquid flows in the third inlet channel. Two of the first, second and third liquids may be identical. In some embodiments, a first liquid flows in the first and third inlet channels and a second liquid flows in the second inlet channel. In some embodiments, the microfluidic device allows the use of hydrodynamic flow focusing. Thus the first liquid flowing into the microfluidic device from the first and the third inlet channels may form a sheath flow (or outer flow) within the device, constraining the second liquid flowing into the microfluidic device from the second inlet channel and forming the focused flow (or inner flow). The flow rates in the first, second and third inlet channels may be adjusted so as to control the flow rates of the sheath flow and of the focused flow within the device. Control of the flow rates within the device may allow control of the extent of mixing. Thus control over the formation of nanoparticles may be achieved. In some embodiments, the inner flow rate is between 0.1 and 10 mL/min, such as between 0.1 and 5 mL/min, such as between 0.1 and 1 mL/min, such as about 0.3 mL/min, about 0.9 mL/min or about 1 mL/min. In some embodiments, the inner flow rate is between 0.1 mL and 10 mL/min, such as between 0.5 mL/min and 5 mL/min, such as 0.8 mL/min and 1.2 mL/min, such as 1 mL/min. In other embodiments, the inner flow rate is between 0.1 and 10 mL/min, such as between 0.15 and 5 mL/min, such as between 0.2 and 1 mL/min, such as between 0.25 and 0.5 mL/min, such as about 0.3 mL/min. In other embodiments, the inner flow rate is between 0.1 and 10 mL/min, such as between 2 and 9.8 mL/min, such as between 5 and 9.5 mL/min, such as between 8 and 9.2 mL/min, such as about 9 mL/min.

In some embodiments, the outer flow rate is between 1 mL/min and 25 mL/min, such as between 1 and 10 mL/min, such as between 1 and 5 mL/min, such as about 1 mL/min, about 4 mL/min, about 10 mL/min or about 20 mL/min. In some embodiments, the outer flow rate is between 1 and 25 mL/min, such as between 5 and 24 mL/min, such as between 10 and 23 mL/min, such as between 15 and 22 mL/min, such as between 18 and 21 mL/min, such as 20 mL/min. In other embodiments, the outer flow rate is between 1 and 25 mL/min, such as between 2 and 20 mL/min, such as between 5 and 15 mL/min, such as between 8 and 12 mL/min, such as 10 mL/min.

In some embodiments, the ratio R, where R is the flow rate of the focused flow divided by the flow rate of the sheath flow, is such that R<1. Preferably, the ratio R is such that formation of microvortices is favoured. Thus in some embodiments R is such that

0.1≤R≤0.2. Preferably, R=0.2. Microvortices enable convective mixing of the streams and flow focusing, thus preventing aggregation of solutes near the chamber walls. Preferably, the microvortices are symmetric but in some embodiments the system may benefit from asymmetric vortex formation. The formation of microvortices is favoured in turbulent systems, i.e. systems where inertial forces are dominant. Microvortices occur in systems with high Reynolds numbers, typically at high flow rates. In such systems, the Reynolds number is preferably such that 1≤Re≤200, the output flow rate is between 1 mL/min and 20 mL/min, and the flow ratio between the inner and outer liquid is greater than 5. In other embodiments, Re>200.

In other embodiments, mixing occurs passively, e.g. via diffusive forces. This is the case when the flows are laminar, i.e. the Reynolds number is low (Re~1), and the viscous forces are dominant, typically at low flow rates. For example, the flow rate is between 10 and 200 μί/ηιίη, such as between 10 and 100 μί/ηιίη, such as between 100 and 200 μί/ηιίη, such as between 10 and 50 μίΑτιίη, such as between 50 and 100 μΙ_/ηιίη, such as between 100 and 150 μΙ_/ηιίη, such as between 150 and 200 μΙ_/ηιίη. At low flow rates the output from multiple devices in parallel can be combined to form a higher system throughput. In some embodiments, the first liquid or the first compound dissolved therein is not soluble in the second liquid. In other embodiments, the first liquid or the first compound cannot be stably mixed with the second liquid or the second compound. Thus it is an advantage of the invention to allow formation of nanoparticles consisting of species which are highly reactive to one another, and which cannot normally be contacted without reacting. Thus one advantage of the invention is that such reactive species are in contact with one another only for a short period of time, thereby limiting the possibilities of cross-reaction. For such species it may be particularly advantageous that the flow rates of the streams within the microfluidic device are high and favour the formation of microvortices, thereby favouring rapid mixing with limited contact times.

Thus in a first step of the method of the invention nanoparticles are generated. The output of the first step may conveniently be scaled up by placing several microfluidic devices in parallel, or several duplicates of the microfluidic channel system within one device, thereby providing a linear increase in the total flow rate.

Static mixer

Although nanoparticles are mainly generated using microfluidics devices with geometries in the micro level, static mixers may also be employed as an alternative to produce the nanoparticles. Static mixers are easily produced from steel or resins and provides high throughput in a single device unit and resistance towards most organic solvents. Static mixers can be used in the present method to achieve microfluidic mixing.

Thus in some embodiments, the present method for producing a dry powder composition comprising nano-embedded microparticles comprises the steps of:

i) generating nanoparticles in one or more static mixers, thereby generating nanoparticle suspension;

ii) mixing said nanoparticle suspension with a matrix solution, thereby

obtaining a feed stream;

The static mixer used in the invention may be one or more static mixers, such as two, three, four, five or more static mixers. In one embodiment the one or more microfluidic devices is a single static mixer. When the one or more static mixer is two or more static mixer, these may be arranged in parallel or in series. When arranged in parallel, the static mixer collectively produce a larger output and can be used for further scale-up or for mixing several types of nanoparticles in a common stream.

The static mixer is fed by at least two inlet channels and comprises at least one outlet channel. In one embodiment, the static mixer is fed by two inlet channels and comprises one outlet channel. The first inlet channel contains a first fluid and the second inlet channel contains a second fluid; the first and second fluids may be a liquid or a gas. The first and the second fluids may be miscible or immiscible. In some embodiments, the first and second fluids are liquids. In some embodiments, one or both of the first and second liquids comprises a dissolved species such as an active compound and the other liquid comprises a dissolved species such as a nanocarrier. Within the static mixer, the flows of the first and second liquids are such that mixing can occur between the liquids.

In one embodiment, the static mixer comprises three inlet channels. Two of the inlet channels may contain a first fluid and the third channel a second fluid. In some embodiments, the first fluid is a liquid and the second fluid is a gas. In other embodiments, the three inlet channels contain a first, a second and a third fluid, where the first, second and third fluids are all different. The first, second and third fluids may all be liquids, or the first and second fluids may be a first liquid and a second liquid and the third fluid may be a gas. In another embodiment, the static mixer comprises four inlet channels. Three of the inlet channels may contain a first and a second fluid, for example two inlet channels contain a first fluid and the third inlet channel contains a second fluid, where the first and second fluids are liquids, while the fourth inlet channel may contain a third fluid such as a gas. In other embodiments, three of the four inlet channels contain a first, a second and a third fluid, where the first, second and third fluid are different, and the fourth inlet channel contains a fourth fluid such as a gas. In some embodiments, two of the four inlet channels contain a first fluid such as a first liquid and the other two inlet channels contain a second fluid such as a second liquid. One of the first and second fluids may also be a gas. Such mixing results in embedment of the dissolved species in the nanocarrier, thereby generating nanoparticles.

Static mixers suitable for the generation of nanoparticles according to the method of the invention can have any shape and dimension and are manufactured with materials known in the art. In some embodiments, the static mixer has two inlets having each a rectangular cross-section converging in an outlet channel also having a rectangular cross-section. In other embodiments, the cross-section of the inlet channels and/or of the outlet channel is square, circular or oval. In some embodiments, the static mixer is circular and the dimensions of the inlet channels are such that their diameter is between 100 μηι and 20 cm, such as between 1 mm and 19 cm, such as between 1 cm and 18 cm, such as between 2 cm and 17 cm, such as between 3 cm and 16 cm, such as between 4 cm and 15 cm, such as between 5 cm and 14 cm, such as between 6 cm and 13 cm, such as between 7 cm and 12 cm, such as between 8 cm and 11 cm, such as between 9 cm and 11.5 cm, such as 10 cm. For example, in one embodiment, the inlet channels have a diameter of 1.1 mm, the outlet channel has a diameter of 1.3 mm, the reaction chamber has a diameter of 6 mm. In other embodiments, the static mixer has a square or rectangular cross-section and the dimensions of the inlet channels are such that their weidth and/or height are between 100 μηι and 2 cm, such as between 200 μηι and 1.75 cm, such as between 500 μηι and 1.5 cm, such as between 750 μηι and 1 cm, such as between 1 mm and 75 mm, such as between 1.1 and 50 mm, such as between 1.2 and 25 mm, such as between 1.3 and 15 mm, such as between 1.4 and 10 mm, such as 1.5 mm. In some embodiments, the static mixer comprises a mixing chamber connecting the inlet channels with the outlet channels. The mixing chamber can have a rectangular or square cross-section or it can be funnel-shaped or cylindrical. The mixing chamber has dimensions such that its cross-section is between 1 mm and 10 cm in diameter or in height, such as between 50 mm and 9 cm, such as between 1 cm and 8 cm, such as between 2 cm and 7 cm, such as between 3 cm and 6 cm, such as between 4 cm and 5 cm. It will be understood that the length of the static mixer is such that mixing is efficiently achieved and typically depends on the other dimensions of the static mixer.

The one or more static mixers can also have dimensions that give a lower output, which may be desirable when one of the materials used is precious, rare, or costly. Several parameters may be adjusted to control the formation of nanoparticles. The direction and the flow rates of the liquids in each inlet channel may be adjusted. In some embodiments, the flow rate is between 1 and 200 mL/min, such as between 10 and 150 mL/min, such as between 25 and 140 mL/min, such as between 50 and 130 mL/min, such as between 75 and 120 mL/min, such as between 90 and 110 mL/min, such as 100 mL/min. In other embodiments, the flow rate is between 1 and 200 mL/min, such as between 10 and 150 mL/min, such as between 25 and 100 mL/min, such as between 30 and 80 mL/min, such as between 35 and 75 mL/min, such as between 40 and 70 mL/min, such as between 45 and 60 mL/min, such as 50 mL/min. When flow rates higher than 100 mL/min are used, the static mixers can be arranged in parallel in order to enhance the flow properties.

Thus in a first step of the method of the invention nanoparticles are generated. The output of the first step may conveniently be scaled up by placing several microfluidic devices in parallel, or simply several duplicates of the microfluidic channel system within one device, thereby providing a linear increase in the total flow rate.

Active compound

In some embodiments, the nanoparticles consist of a nanocarrier embedding an active compound, such as an active therapeutic compound. In one embodiment, the active compound is a small molecule drug, an oligonucleotide or a polynucleotide, a peptide, a metal oxide, a lipid or a substance which is a gas at room temperature or body temperature. In another embodiment, the active compound is a medical imaging contrast agent, or a molecule desirable for cosmetic preparations, such as an aroma compound, materials reflecting electromagnetic radiation and dyes. In another embodiment, the active compound is a molecule desirable for nutritional purposes such as a compound suited for preparing functional foods. The active compound may be, by way of example only, insulin or an antimicrobial peptide such as Novicidin. Nanocarriers

Suitable nanocarriers are known in the art. The choice of nanocarrier depends on the nature of the active compound to be encapsulated. Preferably, the active compound is not soluble in the nanocarrier. Examples of nanocarriers are polymers and polymer conjugates, such as chitosan or alginate, lipid-based carriers, polysaccharides, polymers of mono-, di- and polysaccharides. In some embodiments, the nanocarrier is hyaluronic acid, which may be modified with one or more aryl or alkyl succinic anhydrides. In another embodiment, the nanocarrier is a polymer such as chitosan or alginate. In another embodiment, the nanocarrier is poly(lactic-co-glycolic acid) (PLGA). Other suitable nanocarriers include responsive polymers, the properties of which can change depending on e.g. pH, temperature, ionic strength or exposure to electromagnetic radiation at a specific wavelength, as is known in the art. Nanocarriers also include minerals such as silicon dioxide, metal oxides and calcium carbonate, chelating agents, enzymes or peptides. Nanoparticles

The nanoparticles formed within the microfluidic device or static mixer have an average size between 1 and 999 nm. In one embodiment, the nanoparticles have an average size between 20 and 200 nm. In another embodiment, the nanoparticles have an average size between 50 and 200 nm, such as between 50 and 150 nm, such as between 50 and 100 nm, such as between 100 and 150 nm. In another embodiment, the nanoparticles have an average size between 100 and 200 nm, such as between 120 and 180 nm, such as between 140 and 160 nm. In yet another embodiment, the nanoparticles have an average size between 200 and 400 nm, such as between 200 and 300 nm, such as between 220 and 250 nm, such as 233 nm. In yet another embodiment, the nanoparticles have an average size of 280 nm. The desired average size depends on the purpose of the resulting nano-embedded micro particles and may be adjusted accordingly, for example by varying parameters such as the concentration of the nanocarrier or the flow rate in the one or more microfluidic devices. It will be understood that the nanoparticles may comprises more than one active compound and/or more than one nanocarrier.

In some embodiments, the nanoparticles formed in the microfluidic device or static mixer consist of an inner part and of an outer part, where the inner part comprises the first liquid and the outer part the second liquid, or where the inner part comprises the second liquid and the outer part comprises the first liquid. In embodiments where the first and the second liquids comprise an active compound, the inner part of the nanoparticles comprises the active compound of one of the first and second liquids, and the outer part the active compound of the other one of the first and second liquids. In embodiments where only one of the first and the second liquids comprises an active compound, the active compound is comprised within the outer part or within the inner part of the nanoparticles. Preferably, the active compound is comprised within the inner part of the nanoparticle. In most cases, however, the nanoparticles formed in the microfluidic device or static mixer are often particles where the active compound is dispersed with the nanocarrier within the nanoparticle. This can be in the form of a molecular dispersion or as a suspension of the active compound within the nanoparticle. Typically the active molecule(s) will be present both inside the particle and on the surface of the particles. The distribution of the different molecules within the nanoparticles depends on the properties of the molecules. A hydrophobic small molecule drug would for instance form a good dispersion within a nanoparticle composed of an amorphous hydrophobic polymer. In some embodiments, the nanoparticles are water-insoluble and are soluble in organic solvents. In other embodiments, the nanoparticles are partially water-soluble in a specific biological environment. In some embodiments the nanocarrier and active compound are soluble on their own but insoluble or partially soluble when together as a complex. Some materials for crystalline or semi-crystalline nanoparticles and others form amorphous nanoparticles.

It will be understood that several types of nanoparticles exist. The term nanoparticle as used herein may thus refer to any of these types, including, but not limited to, nanocapsules, nanogels, nanospheres, nanocomplexes and colloids.

Matrix solution

The one or more microfluidic devices each comprises at least one outlet channel allowing for exit of the nanoparticle solution. The nanoparticle solution results from the mixing within the one or more microfluidic devices as described above. If a microfluidic device comprises more than one outlet channel, the nanoparticle solutions contained within each outlet channel may be joined in one channel prior to mixing with the matrix solution, immediately downstream of the microfluidic device. Alternatively, the nanoparticle solutions within each outlet channel may each be mixed with the matrix solution downstream of the microfluidic device, and then joined into one channel. In both cases, a single feed stream is formed comprising the nanoparticle solution mixed with the matrix solution. In some embodiments, the matrix solution is water-soluble.

Likewise, the one or more static mixers each comprises at least one outlet channel allowing for exit of the nanoparticle solution. The nanoparticle solution results from the mixing within the one or more static mixers as described above. If a static mixer comprises more than one outlet channel, the nanoparticle solutions contained within each outlet channel may be joined in one channel prior to mixing with the matrix solution, immediately downstream of the microfluidic device. Alternatively, the nanoparticle solutions within each outlet channel may each be mixed with the matrix solution downstream of the static mixer, and then joined into one channel. In both cases, a single feed stream is formed comprising the nanoparticle solution mixed with the matrix solution. In some embodiments, the matrix solution is water-soluble. Mixing of the nanoparticle solution and of the matrix solution may occur in the downstream part of the one or more microfluidic devices or static mixers or at a junction downstream of the one or more microfluidic devices or static mixers. For example, the mixing occurs at a cross junction of the channel containing the nanoparticle solution and of the channel containing the matrix solution.

In embodiments where the one or more microfluidic device or static mixer is two or more devices arranged in parallel, the nanoparticle solution flowing in the outlet channels of the devices can be joined at a cross junction in order to form one common outlet flow before being mixed with the matrix solution. It will be understood that whether the nanoparticle solutions are joined before mixing with the matrix solution or whether the nanoparticle solutions are mixed with the matrix solution before being joined in one stream is of little relevance to the invention.

In other embodiments, the one or more microfluidic device or static mixers is two or more devices arranged in series. Such an arrangement may simply be a repetition of the same device, where the inner flow in each new device consists of the nanoparticle suspension exiting the previous device and the sheath flow consists of the same liquid as in the previous device. This may favour a higher degree of mixing. Such

arrangements may also be particularly relevant for embodiments where it is desirable to mix more than two fluids. For example, it may be desirable to coat the nanoparticles. In this case, a first device yields nanoparticles as described above. The nanoparticle suspension then flows in one of the inlet channels of a second device and forms the inner stream, while a coating solution flows in one of the other inlet channels of the second device and forms the sheath flow.

The matrix solution is such that it is capable of embedding the nanoparticles of the nanoparticle solution. Further, the matrix solution is such that when mixed with the nanoparticle solution it is capable of forming nano-embedded microparticles when spray-dried. The nature of the matrix solution should be determined considering the intended use of the nano-embedded microparticles. If the nanoparticles comprise an active compound such as a drug and the powder of nano-embedded microparticles is intended for administration in an organism, for example by inhalation or ingestion, the matrix solution should be such that it is degradable or soluble in the environment in which the administration is intended in order to allow release of the nanoparticles comprising the active compound. In one embodiment, the organism is a human organism. In some embodiments, the matrix solution may be chosen so that it counteracts potential side-effects or undesirable physical or chemical properties of the components within the nanoparticles. In some embodiments, the matrix solution is water-soluble. In other embodiments, the matrix solution is approved for oral, pulmonary or parenteral administration.

The matrix solution may be such that it can help control the release of the active compound within the nanoparticle. Thus a matrix which is slowly degradable in a specific environment, in which the active compound is to be released, may be advantageous in some embodiments. The matrix may also contain molecules which are used to stimulate release from the nanoparticles. In other embodiments, where a rapid release is desirable, the matrix solution is chosen so that it is rapidly degradable in the targeted environment. In some embodiments dissolution at a certain temperature or pH is desirable and a matrix material allowing such function is used. In further embodiments the matrix component may form interactions with the components of the nanoparticles, such as electrostatic interaction, in order to stabilize the nano-embedded microparticles.

Suitable matrix components include, but are not limited to, polymers, polysaccharides, saccharides, trehalose, inulin, lactose, sucrose, mannitol, amino acids, hypromellose (HPMC) and other cellulose-derived polymers, povidone (PVP), polyethylene glycol (PEG). Other suitable matrix components include amino acids, such as water-soluble amino acids. Matrix components that swell upon wetting are also useful for

redispersion of nanoparticles. The matrix concentration relative to the nanoparticles should be such that the resulting nanoparticles have the desired stability and redispersibility, for example the nanoparticle to matrix ratio is between 1 :2 and 1 :10, such as between 1 :2 and 1 :5, such as between 1 :2 and 1 :3, such as about 1 :2. In other embodiments, the nanoparticle to matrix ratio is between 2: 1 and 1 :20, such as between 1.5: 1 and 1 : 15, such as between 1 :2 and 1 :10, such as between 1 :25 and 1 :10, such as between 1 :2 and 1 :7, such as between 1 :25 and 1 :5, such as about 1 :5. In some embodiments, the nanoparticle to matrix ratio is between 2:1 and 1 :20, such as between 1 :1 and 1 : 15, such as between 1 :2 and 1 : 12, such as between 1 :2 and 1 :10, such as between 1 :3 and 1 :9, such as between 1 :4 and 1 :8, such as between 1 :5 and 1 :7.5, such as about 1 :7. In other cases the nanoparticles are stable on their own and the nanoparticle to matrix ratio is further reduced.

Without being bound by theory, it appears that the redispersibility is increased when the nanoparticle to matrix ratio increases. In some embodiments, the matrix is a saccharide, a disaccharide or a polysaccharide solution at a concentration between 1 and 2000 mg/mL, such as between 25 and 1500 mg/mL, such as between 50 and 1000 mg/mL, such as between 75 and 500 mg/mL, such as between 100 and 250 mg/mL, such as between 100 and 200 mg/mL, such as between 100 and 150 mg/mL, such as about 100 mg/mL.

In some embodiments, the matrix solution comprises inactive nanoparticles or a combination of inactive nanoparticles and dissolved excipients. In such embodiments a nanoaggregate is formed from the nanoparticles comprising an active compound, i.e. the active nanoparticles, and the inactive particles comprised in the matrix solution. The active nanoparticles are thus stabilised by the inactive nanoparticles.

The feed stream is the stream that results from the mixing of the nanoparticle solution with the matrix solution. The feed stream is fed into the spray dryer. In some embodiments, the matrix solution and the nanoparticle solution are contacted at a junction or intersection, and the mixing may occur from the junction until the feed stream passes through the nozzle of the spray dryer. Mixing can be controlled by controlling the flow rates of the incoming nanoparticle solution and matrix solution. The flow rate of the feed stream is between 1 mL/min and 10 mU min, such as between 1 mL/min and 5 mL/min, such as between 1 mL/min and 4 mL/min, such as between 2 mL/min and 4 mL/min, such as about 3 mL/min. The flow rate of the feed stream entering the spray dryer is such that it is compatible with spray drying. Preferably, the flow rate of the incoming matrix solution is adjusted relative to the flow rate of the incoming nanoparticle solution so that the resulting stream (the feed stream) has an appropriate nanoparticle concentration. For example, the feed stream has a nanoparticle concentration greater than 0.5 % w/v, such as greater than 0.75 % w/v, such as greater than 1 % w/v. This can be achieved if the ratio Rf between the flow rate of the nanoparticle solution and the flow rate of the matrix solution is between 10: 1 and 1 :10, such as between 1 :5 and 5: 1 , such as between 1 :2 and 2: 1 , such as about 1 : 1. It is an advantage of the invention that the feed stream is spray dried immediately after mixing of the nanoparticle suspension and the matrix solution, thereby limiting the time during which undesired reactions or degradation can occur. This is particularly relevant for unstable nanoparticles or unstable nanoparticle suspensions.

Drying

In a third step of the method of the invention, the nanoparticle solution mixed with the matrix solution is subjected to drying within a dryer. Such drying may comprise a step of liquid atomisation, which allows dispersion of the stream. In some embodiments, the drying and the liquid atomisation are both performed within the dryer, e.g. in a spray- dryer. In some embodiments, the liquid atomisation process is selected from the group comprising electrospraying, electrospinning, supercritical fluids assisted atomization, ultrasonic atomization, spray freeze-drying, spray-drying and melt congealing. In a preferred embodiment and as detailed below, the drying step is a spray-drying step.

Spray drying

After the nanoparticle solution and the matrix solution are mixed as described above, the feed stream is spray dried in order to generate a dry powder of nano-embedded microparticles. In the method of the invention, the feed stream is spray dried continuously, i.e. as it is being formed, without interruption between the formation of the feed stream and the spray drying.

Spray drying is a process allowing drying of a liquid with a hot gas, typically

atmospheric air or nitrogen, the latter being preferably used for drying flammable or oxygen-sensitive liquids.

A spray dryer consists of an inlet with a nozzle or atomizer, which disperses the feed stream into a drop-sized spray. Suitable nozzles are known to the skilled person, and include rotary disks, single-fluid high-pressure swirl nozzles, two-fluid nozzles, three- fluid nozzles and ultrasonic nozzles. The spray then enters a drying region wherein it is dried by the hot gas. The temperature of the gas depends on the boiling point of the solvents used and on the sensitivity of the materials to be spray dried to heat.

Generally, a temperature above the solvent boiling point is likely to yield hollow particles, while a temperature below the solvent boiling point is likely to yield compact particles. Thus the temperature of the gas may be adjusted by the skilled person in order to yield the desired microparticles.

The spray dryer may be single-effect, with only one drying gas blown typically from the upper part of the drying chamber, or multiple-effect, where the drying chamber further comprises a fluidised bed at its bottom. The spray dryer may be a small scale

(benchtop) spray dryer or a large scale spray dryer. Single-effect spray dryers generally result in powders with poor flowability. The fluidised bed of multiple-effect spray dryers allows agglomeration of the fine particles to obtain bigger particles having commonly a medium particle size within a range of 100 to 300 μηι, in the form of free- flowing powders. This is particularly relevant for producing nano-embedded

microparticles powders intended for preparing tablets or for oral administration.

The method of the invention may further comprise a collecting step. Collection of the powder may be continuous or discontinuous. Spray dryers suitable for the method of the invention may comprise a collecting region, such as a cyclone collector. The nano- embedded microparticles produced by the method of the invention may be collected in the collecting region. In some embodiments, collection of the microparticles is performed continuously. In other embodiments, the collecting region needs to be emptied manually or automatically at specific times, before it is full. Besides spray drying, other liquid atomization techniques, optionally combined with a drying step, may be used to convert the nanoparticle suspension into a dry powder of nano-embedded microparticles. Such techniques include, but are not limited to, electrospraying, electrospinning, supercritical fluids assisted atomization, ultrasonic atomization, spray freeze-drying and melt congealing. In all embodiments, the nanoparticle suspension is mixed with the matrix solution prior to liquid atomization. When the liquid atomization technique does not directly result in a dry powder, but instead produces a liquid or an aerosol, a drying step may be included in the method of the invention in order to yield a dry powder of nano-embedded microparticles. Drying methods are known to the skilled person and include, by way of example, liquid extraction, active drying and passive evaporative drying. Thus a dry powder of nano- embedded microparticles is produced. In some embodiments, the liquid atomization step is performed using a spray dryer with an atomization gas flow rate between 100 and 2000 L/h, such as between 150 and 1500 L/h, such as between 200 and 1000 L/h, such as between 250 and 750 L/h, such as between 300 and 500 L/h, such as between 400 and 500 L/h, such as 450 L/h. In some embodiments, the aspiration rate is 50% or more, such as 60% or more, such as 70% or more, such as 80% or more, such as 90% or more, such as 100%. Spray dryers with higher atomization gas flow rates may also be used, such as a spray dryer with an atomization gas flow rate between 2000 and 100000 L/h, such as between 4000 and 75000 L/h, such as between 10000 and 50000 L/h, such as between 20000 and 30000 L/h, such as 25000 L/h.

In some embodiments, the inlet temperature of the spray dryer is between 30 and 150°C, such as between 35 and 100°C, such as between 40 and 75°C, such as between 45 and 60°C, such as 50°C. In other embodiments, the inlet temperature of the spray dryer is between 30 and 150°C, such as between 40 and 140°C, such as between 50 and 130°C, such as between 60 and 125°C, such as between 70°C and 120°C, such as between 80°C and 1 10°C, such as between 90°C and 105°C, such as 100°C.

Nano-embedded microparticle powder The method of the invention provides dry powders of nano-embedded micro particles. The nano-embedded microparticles have a mass median aerodynamic diameter (MMAD) between 0.5 and 100 μηι. In a preferred embodiment, the nano-embedded microparticles have an MMAD between 1 and 100 μηι, such as between 1 and 75 μηι, such as between 1 and 50 μηι, such as between 1 and 25 μηι, such as between 5 and 10 μηι, such as between 6 and 9 μηι, such as 7 μηι or 8 μηι.

The nano-embedded microparticle powders of the invention can be such that the moisture content of the powder is low. In some embodiments, the moisture content is lower than 3%, such as lower than 2%, such as lower than 1 %. The moisture content can be measured by thermogravimetric analysis, and it can be controlled, as is known in the art, by parameters such as the residence time in the drying region. For example, the direction of the drying air relative to the direction of the spray may be varied. The drying air and the spray can thus be blown in a co-current flow, whereby a short residence time is achieved, or in a counter-current flow, whereby a longer residence time is achieved. In some cases a higher moisture content between 2-15% is desirable to stabilize the nanoparticles or components within the nanoparticles and a higher moisture content is used. Thus in some embodiments, the moisture content is between 2 and 15%, such as between 3 and 14%, such as between 4 and 13%, such as between 5 and 12%, such as between 6 and 1 1 %, such as between 7 and 10%, such as between 8 and 10%, such as 9%. In other embodiments, the moisture content is between 1 and 6%, such as between 2 and 5%, such as 3% or 4%.

In some embodiments, the nano-embedded microparticles of the invention are such that upon resuspension in a liquid such as water, the size of the nanoparticles is comprised between 50 and 200 nm, such as between 50 and 150 nm, such as between 50 and 100 nm, such as between 50 and 75 nm. Thus in some embodiments the nanoparticles embedding the microparticles of the invention have a high redispersibility, where redispersibility of the nanoparticles is defined as the ratio between the size of the nanoparticles after dispersion of the spray dried microparticles and the size of similar nanoparticles produced by microfluidics only. In preferred embodiments, the redispersibility is close to 1.

In some embodiments, the polydispersibility index (PDI) of the nanoparticles embedding the microparticles is low. When the nanoparticles comprise an active agent for which a controlled release is desired, it is important that the PDI is low. High PDI indicates a great size variability of the nanoparticles, which in turn can result in imprecise dosing of a drug at a target site. Thus in some embodiments, the PDI of the nanoparticles is lower than 0.5. In preferred embodiments, the PDI of the nanoparticles is lower than 0.4, such as lower than 0.3, such as lower than 0.2, such as lower than 0.1 or lower.

The dry powder comprising the nano-embedded microparticles may be used for numerous applications in medicine, therapeutic treatments, cosmetics, functional food production. The resulting dry powder may be formulated as is known in the art in a way suited for the purpose. For instance, the powder can be formulated for inhalation, for ingestion or for injection, by methods known in the art. In some embodiments, the powder is formulated as a tablet or capsule for oral administration. This can be particularly relevant for the medical field, such as for local therapy within the gastrointestinal tract or lungs, for increasing solubility or permeability of difficult drugs such as anticancer drugs and for delivery of fragile molecules such as peptide and protein drugs. Anticancer drugs are currently mainly administered intravenously but oral routes for delivering anticancer drugs can be developed using the nanoparticle based system.

A dry powder of nano-embedded microparticles can be produced with the method of the invention in a few minutes. It takes but a few seconds from the entry of an active compound into the microfluidic device until a nanoparticle of the same active compound is embedded in a microparticle powder and can be collected. Thus in some embodiments, the travel time of an active compound molecule is less than 10 minutes, such as less than 5 minutes, such as less than 1 minute, such as less than 45 seconds, such as less 30 seconds, such as less than 10 seconds. It will be understood that collection of the nano-embedded microparticles is facilitated when the amounts of powder to collect are sufficient to allow collection without losing microparticles. Such a collectable powder may be generated with the method of the invention in less than 10 minutes, such as less than 5 minutes, such as less than 1 minute, such as less than 45 seconds, such as less than 30 seconds, such as less than 10 seconds.

The present method thus presents numerous advantages. First, the method is faster than conventional methods, and thus it is well suited for generating nano-embedded microparticles even with unstable nanoparticles or unstable nanoparticle suspensions since the time during which the nanoparticles or nanoparticle suspensions are free (that is, not embedded in the matrix micro particles) is limited. Moreover, the method is continuous up to the collection point. The collection step may also be continuous. The method involves only few steps and only one collection step. Thus aseptic production is possible, since there need not be any interference from the user until the powder is collectable. Because the processing time is so short, there is no need to use a surfactant or a solvent, which results in a greater fraction of active compound in the final product, i.e. in a greater yield. The short processing time also results in the method being suitable for generating nano-embedded microparticles even with unstable nanoparticles or unstable nanoparticle suspensions.

Apparatus

Also provided herein is an apparatus for performing the method of the invention, said apparatus comprising:

i) one or more microfluidic devices or static mixers;

iii) means for providing a matrix solution;

vi) a collecting device.

Thus the apparatus of the invention is suitable for performing the method of the invention and for collecting the powder produced by said method. Moreover, such apparatus provides a fully integrated platform for performing the high-throughput method of the invention. In some embodiments, the apparatus of the invention comprises one or more microfluidic devices. The apparatus may comprise at least two microfluidic devices, such as three microfluidic devices, such as four microfluidic devices, such as five microfluidic devices, such as six microfluidic devices. In embodiments where the apparatus comprises at least two microfluidic devices, the devices may be arranged in parallel or in series. In embodiments where the apparatus comprises at least three microfluidic devices, a number of the devices may be arranged in parallel while the rest of the devices may be arranged in series. It will be understood that any combination of arrangements in parallel or in series is considered to be within the scope of the invention. For example, a first group of microfluidic devices may be arranged in series in order to produce, in a first step, nanoparticles as described above. In a second step, the nanoparticles exiting the first microfluidic device are mixed as described with a coating material in a second microfluidic device located downstream the first microfluidic device. In certain embodiments where it is desirable, the apparatus may comprise a second group of microfluidic devices, arranged in parallel of the first group, where the second group consists of a third and a fourth microfluidic devices, identical to the first and second microfluidic devices and resulting in identical coated

nanoparticles. The output streams of the first and second groups of microfluidic devices may be joined further downstream, for example at a junction, in order to form one joint outlet flow.

In other embodiments, the apparatus of the invention comprises one or more static mixers. The apparatus may comprise at least two static mixers, such as three static mixers, such as four static mixers, such as five static mixers, such as six static mixers. In embodiments where the apparatus comprises at least two static mixers, the devices may be arranged in parallel or in series. In embodiments where the apparatus comprises at least three static mixers, a number of the devices may be arranged in parallel while the rest of the devices may be arranged in series. It will be understood that any combination of arrangements in parallel or in series is considered to be within the scope of the invention. For example, a first group of static mixers may be arranged in series in order to produce, in a first step, nanoparticles as described above. In a second step, the nanoparticles exiting the first static mixer are mixed as described with a coating material in a second static mixer located downstream the first static mixer. In certain embodiments where it is desirable, the apparatus may comprise a second group of static mixers, arranged in parallel of the first group, where the second group consists of a third and a fourth static mixers, identical to the first and second static mixers and resulting in identical coated nanoparticles. The output streams of the first and second groups of static mixers may be joined further downstream, for example at a junction, in order to form one joint outlet flow. The apparatus of the invention further comprises means for providing fluid to said one or more devices (microfluidic devices or static mixers). Such means are known in the art. In some embodiments, the means for providing fluid to the one or more devices is a pump, for example a peristaltic pump. The fluid may be a gas or a liquid. The fluid may be contained in a suitable container connected to the means for providing the fluid, by means known in the art. For example, the connection is achieved by way of tubing. Suitable tubing is known in the art and may consist of e.g. silicone or silicone rubber. Suitable containers are known in the art. In some embodiments, the container is a flask or a bottle. In other embodiments, the container is a syringe.

The apparatus of the invention further comprises means for providing a matrix solution. Such means are known in the art. In some embodiments, the means for providing the matrix solution is a pump, for example a peristaltic pump. The matrix solution may be contained in a suitable container connected to the means for providing the matrix solution, by means known in the art. For example, the connection is achieved by way of tubing. Suitable tubing is known in the art and may consist of e.g. silicone or silicone rubber. Suitable containers are known in the art. In some embodiments, the container is a flask or a bottle. In other embodiments, the container is a syringe. Also provided herein is an apparatus comprising a junction wherein the outlet flow from the one or more devices, i.e. the one or more microfluidic devices or static mixers, is mixed with the matrix solution to form a mixed flow. The junction may be a cross junction or a Y junction. In some embodiments, the apparatus comprises a spray dryer which is fed with the mixed flow, also termed feed stream. In preferred embodiments, the feed stream is directly fed into the spray dryer via an inlet channel.

It will be understood that in embodiments with at least two devices, thus at least two output streams, the at least two output streams may be joined prior to being contacted with the matrix solution; alternatively, each of the at least two output streams may be mixed with a matrix solution to form at least two mixed flows. In the latter case, the at least two mixed flows are preferably joined at a junction, so as to form a single nanoparticle suspension, also termed feed stream. The at least two mixed flows are preferably joined prior to being fed into means for performing liquid atomization and drying of the feed stream. The apparatus further comprises a dryer for performing liquid atomization and drying of the feed stream. In other words, the dryer is a device that enables conversion of the feed stream into a dry powder of nano-embedded microparticles. Such devices include, but are not limited to, devices suitable for performing liquid atomization such as electrospraying, electrospinning, supercritical fluids assisted atomization, ultrasonic atomization, spray freeze-drying and melt congealing. In some embodiments, the nanoparticle suspension is mixed with the matrix solution prior to liquid atomization. When the liquid atomization technique does not directly result in a dry powder, but instead produces a liquid or an aerosol, a drying step may be included in the method of the invention in order to yield a dry powder of nano-embedded microparticles. Drying methods are known to the skilled person and include, by way of example, liquid extraction, active drying and passive evaporative drying. Thus the means for performing liquid atomization and drying of the nanoparticle suspension allows production of a dry powder of nano-embedded microparticles. In preferred

embodiments, the liquid atomizer and dryer means for performing liquid atomization and drying of the feed stream is a spray dryer.

The apparatus disclosed herein is suitable for performing the continuous method described above to produce a dry powder of nano-embedded microparticles. The apparatus also enables collection of the dry powder. Thus the apparatus of the invention comprises a collecting device. Collecting devices suited for the purpose are known in the art. For example, the collecting device may be a cyclone collector. The collecting device may be suited for continuous collection of the dry powder.

Alternatively, the collecting device may comprise a container such as a vial, said container having to be emptied manually or automatically at determined time intervals, before the container is full. Thus the apparatus of the invention allows for continuous production of a dry powder of nano-embedded microparticles and for continuous or semi-continuous collection of said powder. In some embodiments, the apparatus comprises a granulation device such as a fluidized bed, wherein the granulation device is connected to the dryer. The

nanoparticles can thus be obtained as granules.

The apparatus of the invention is suitable for producing a sterile powder of nano- embedded microparticles. Aseptic production can be achieved e.g. by sterilising the apparatus prior to production, thereby ensuring that the apparatus is sterile before use. In another embodiment, the apparatus is not sterilised prior to performing the method of the invention. If desirable, the resulting powder may be sterilised following collection, by methods known in the art. For example, the powder may be sterilised by filtration or heat treatment.

The dry powder produced by the fully integrated apparatus of the invention can be used as described above, for example the dry powder may be used for therapeutic or nutritional purposes. Thus it may be necessary for the dry powder to be sterile or essentially free of contaminating microorganisms. Sterilisation of the powder may be performed by methods known in the art, such as heat treatment or filtration. However such methods may be tedious, costly and time consuming. Moreover, some dry powders may be heat-sensitive, and others may comprise micropartides of such a size that filtration would not lead to efficient sterilisation. Thus it is an advantage that the apparatus of the invention can be sterilised prior to use, thereby enabling aseptic production of a sterile dry powder, without having to perform a further sterilisation step after collecting the powder.

Examples

Example 1. Preparation of nano-em bedded micropartides

Microfluidic device:

The microfluidic chip in this study made use of hydrodynamic flow-focusing and was fabricated from polydimethylsiloxane (PDMS) (SYLGARD 184, Dow Corning, Midland, Ml) by YongTea Kim from Georgia Tech University using soft-lithography. The microfluidic device used had three inlet channels with rectangular cross-sections with dimensions: width = 200 μηι, height = 400 μηι and length = 10 mm. These channels converged in an intersection forming a single outlet channel of rectangular cross- section with dimensions: width = 2000 μηι, height = 400 μηι, and length = 20 mm.

In such microfluidic devices, symmetric microvortices are created at the intersection of the inlets in the microfluidic chip resulting in rapid mixing of the fluids. Such devices exhibit focusing patterns that prevent aggregation of solutes near the channel walls (Kim et a/ 2012). The hydrodynamic flow-focusing chip was used in this study in order to achieve microfluidics driven mixing at high flow rates comparable and compatible with the liquid flow rates used in a lab-scale spray dryer. Microfluidics chips with a different geometry and mixing process could also be used to connect with a spray drier, and for those with a low flow rate several microfluidic chips could be arranged in parallel with a common inlet and outlet to achieve a higher total flow rate. In this study a lab scale spray dryer, Buchi B-290 (Buchi Labortechnik AG, Postfach, Switzerland) was used equipped with a dehumidifier, Buchi B-296 (Buchi Labortechnik AG). All samples were prepared by first obtaining a steady inlet and outlet temperature before atomizing the sample solutions. With reference to figure 2A and 2B, the combined microfluidics and spray drying process was performed mixing the nanoparticle solution (9) exiting the microfluidic device (2), with the matrix solution (10), thereby generating a feed stream (11) at a y- junction (5). The feed stream is then fed into the drying region of the spray-dryer via the nozzle (6) and collected in the collecting region (8).

In this study a model system based on the cationic anti-microbial peptide, Novicidin (Novozymes, Denmark) and the anionic polymer, hyaluronic acid (Novozymes, Denmark) was used to produce micro-embedded nanoparticles. Three gas-tight glass syringes (Hamilton) were filled with the solutions containing either Novicidin (inner stream, one inlet channel) or hyaluronic acid (outer stream, two inlet channels) and mounted on syringe pumps (Pump 11 elite, Harvard apparatus,

Cambridge, USA) and sprayed at different flow rates as indicated below: Sample 1 :

Hyaluronic acid 0.05 mg/mL, flow rate 1.35 mL/min.

Novicidin 0.25 mg/mL, flow rate 0.3 mL/min.

Sample 2:

Hyaluronic acid 0.05 mg/mL, flow rate 4.05 mL/min. Novicidin 0.25 mg/mL, flow rate 0.9 mL/min.

The flow rates in the outer streams were matched and set at a higher flow rate (1.35 or 4.05 ml/min) than the flow rate for the inner liquid stream. The microfluidics output was fed through a silicone tube and into a y-junction also made of silicone rubber.

The peristaltic pump of the spray dryer was used to mix in a sugar solution with the liquid output from the microfluidic device (nanoparticle solution). Aqueous solutions with trehalose at concentrations 20 mg/ml and 50 mg/ml (see Table 1) were mixed with the nanoparticle solution and spray dried under the following conditions: inlet temperature, 150°C; outlet temperature, 78 or 87°C; drying air flow rate, 439 L/hour; atomizing air flow rate, 600 L/hour; and feed flow rate: 3 mL/min). Resulting

microparticle powders were collected from collection vial and cyclone. Table 1. Conditions for each sample

Characterization of nano-embedded micro particles

Powder yield: The yield was measured by weighing the powder collected in the cyclone collector. Results are shown in table 2. Microparticle aerodynamic size measurements: The mass median aerodynamic diameter (M AD) of particles was measured by a time of flight principle with an Aerodynamic Particle Sizer 3321 (TSi incorporated, Shoreview, MN, USA). Results are shown in table 2.

Redispersibility and nanopartide size measurements: Redispersibiiity of nanopartides was studied by dispersing 10 mg of the spray-dried nano-embedded microparticles in water and measuring particle size via dynamic light scattering using a Zetasizer (Malvern instruments, UK). The presence of aggregates and the poiydispersity index (PDI) of the particles were determined and compared with particle size and size distribution of nanogels prepared using only the microfiuidics device. Results are shown in table 2.

Table 2. Characteristics of the nano-embedded microparticle powder for each sample.

*: value for nanopartides made with microfluidic device alone: 64 ± 8 nm.

Morphology of nano-embedded microparticles: Scanning electron microscopy of microparticles was performed using a XL 30 FEG. A thin layer of particles was spread out on carbon tape and mounted on metallic studs, sputter-coated with gold and viewed at an accelerating voltage of 2kV. Images representative of the whole sample were taken for each of the samples where possible (Figure 3). Figure 3A shows pictures for sample 1. Figure 3B shows pictures for sample 2. The particles observed were generally spherical with smooth surface. Some of the particles of sample 1 were attached to each other, while this was not the case for the particles of sample 2.

Conclusion

We demonstrate that the present method can be used to produce nano-embedded microparticles with good redispersibility. The yield is increased when the combined flow rate within the microfluidic device (inner flow + outer flow) is increased. Smaller microparticles were obtained when the flow rate of the outer stream was 3-fold the flow rate of the inner stream, while the size of the nanoparticles after redispersion was similar in both samples.

Example 2. High throughput production of nano-embedded microparticles by a combined microfluidics-spray drying technique.

Purpose of study: To prepare drug loaded nanocarriers with controlled size, narrow size distribution and high drug loading in a reproducible, high throughput process using microfluidics. To produce dry, nano-embedded microparticles intended for pulmonary delivery using microfluidics combined with spray drying with control of mean

aerodynamic diameter and nanoparticle loading/distribution. Incorporation of in-process monitoring in the microfluidics and spray drying setup to monitor the product quality via feedback loops. A simplistic approach is used in the project where 2-3 analysis techniques are incorporated along the particle preparation process to monitor properties such as particle size, drug loading, residual moisture and solution properties.

1) Preparation of nano-embedded particles (NEM) using the combined microfluidics - spray drying technology

2) One-step production of NEMs using a continuous process without the use of stabilizers or excessive organic solvents

3) High throughput production of dry, therapeutic nanoparticle formulation

4) In-process monitoring for NEM synthesis

A model system consisting of nanocarriers with low stability is used to demonstrate that the production of a dry powder of nanoparticles embedded in microparticles produced via our technology can be used to stabilize the nanocarrier system for a prolonged time. A model system for the nanocarriers consisting of the protein drug, insulin, and the polymer chitosan or alginate, is used, providing a relevant system for nanocarrier- based drug delivery. Preparation and characterization of nanoparticles with microfluidics device:

- Nanocarriers are prepared

- Characterization of particle size using zetasizer

- Measurement of drug encapsulation efficiency

- Optimization of particle size

- Structural characterization of insulin Preparation and characterization of nano-embedded microparticles:

- Matrix components using spray drier: inulin, lactose, sucrose, trehalose, mannitol, leucin

- Powder residual moisture is analysed using thermogravimetric analysis and near-infrared spectroscopy

- Redispersibility of NEMs are examined via suspension in aqueous medium

- Redispersion time is studied

- Nanoparticle size is measured using zetasizer and compared with size of nanoparticles prepared using microfluidic device alone

- Stability of NEMs is studied

- Characterization of mass median aerodynamic diameter of NEMs using impactor

- Characterization of geometric diameter and morphology using master sizer and scanning electron microscopy

Incorporation of in-process analysis:

- Incorporation of size analysis into microfluidics process (nanosi

zetasizer)

- Drug entrapment efficiency measurement using UV / HPLC

- Study on residual moisture using NIR

Example 3: Preparation of chitosan/TPP based nano-embedded microparticles

Preparation of chitosan loaded nano-embedded microparticles

Microfluidic mixing was used to prepare initial nanogel suspensions that were subsequently spray dried. This mixing process was carried out by feeding three liquid feeds into a static mixer, which was then connected with a spray dryer.

In this study a static mixer designed by Fang et al. was produced in high molecular weight poly lactic acid using a 3D printer (MakerBot Replicator) and used to produce the nanogels. The static mixer had inlet channels of 1.1 mm in width, outlet channel with diameter of 1.3 mm, and the reaction chamber was 6 mm in diameter and 1.5 mm in height as described by Fang et al. Also in this study a lab scale spray dryer, Buchi B-290, was used equipped with a dehumidifier, Buchi B-296. The combined static mixing and spray drying process was performed by mixing the nanoparticle suspension with the matrix solution and the combined feed stream was then fed into the spray dryer.

A model system based on the cationic polymer chitosan and the anionic components, alginate and sodium triphosphate (TPP), and combinations thereof, were used to produce nano-embedded microparticles.

Two gas-tight glass syringes were filled with the solutions containing either chitosan and TPP and/or alginate and the compositions shown in Table 3 were prepared. The compounds were dissolved in 1 % (w/v) acetic acid and had a final pH of 3.2.

Table 3: Composition of nanogel formulations

, _x. Chitosan ._x , _{A 1} . _t TPP Alginate

Formulation _{ί t}. Chitosan:(Alginate+TPP) _{ί t}.

concentration _t. , , . concentration concentration name , , , . ratio (w/w) , , , . (mg/mL)

(mg/mL) (mg/mL)

CT1 1 3: 1 1.5

CT2 0.5 3: 1 0.75

CTA 0.5 3: 1 0.60 0.15

CA 0.5 3: 1 0.75

The peristaltic pump of the spray drying setup was used to mix in a sugar solution of trehalose or mannitol with the nanogel suspension. Trehalose and mannitol were mixed with the nanosuspension at ratios of 10: 1 - 20: 1 (w/w) and spray dried at an inlet temperature of 1 10 °C, feed rate of 6 ml/min, atomization gas flow rate of 450 L/h. The resulting microparticle powders were collected from collection vial and cyclone.

Redispersibility and nanoparticle size measurements

Redispersibiiity of nanoparticies were studied by dispersing 10 mg of the spray-dried nano-embedded microparticles in water and measuring particle size and zeta potential via dynamic light scattering using a Zetasizer (Malvern instruments, UK). The presence of aggregates and the polydispersity index (PDI) of the particles were determined and compared with particle size and size distribution of nanogels prepared using the static mixer that were not spray dried. Results are shown in Figure 4A. The size and PDI of the nanogels before spray drying indicate that the particles prepared without TPP were larger than those prepared using TPP. Figure 4B shows the zeta potential of the nanogels before spray drying and Figure 4C shows the size comparison of the redispersed nanogels after spray drying. The nanogels were slightly larger after redispersion compared with before redispersion using trehalose.

Optimization of the spray drying inlet temperature

For the following nanogel formulation (CTA), with a trehalose matrix, the influence of the inlet temperature on the final NEMs morphology, moisture content, particle size (before and after spray drying) and MMAD was investigated as described below

(results figure 5 and table 4).

Morphology of nano-embedded microparticles

Scanning electron microscopy of microparticles was performed using a XL 30 FEG. A thin layer of particles was spread out on carbon tape and mounted on metallic studs, sputter-coated with gold and viewed at an accelerating voltage of 2kV. Images representative of the whole sample were taken for each of the samples (Figure 5). The particles observed were generally spherical with smooth surface. In case of an inlet temperature of 100 °C particles appeared to be attached to each other while for samples prepared at higher inlet temperature the particles were not attached.

Table 4: Effects of different inlet temperatures during production of CTA nanogel based NEMs

Formulations Moisture Particle size (nm) MMAD (|.im)

Content Before Spray After re(%) Drying dispersion

Conclusion

We demonstrate that the present method can be used to produce nano-embedded microparticles of chitosan and TPP with good redispersibility. The nanogel composition, the choice of the matrix component and the production parameters have an effect on the resulting NEMs.

Example 4. Preparation of PLGA-based nano-embedded microparticles

In this study a model system based on the polymer poly(lactic-co-glycolic acid) (PLGA) nanoparticles with and without sodium caprate surface as surface modifier was prepared into micro-embedded nanoparticles. In this study the same microfluidics chip and spray drying setup was used as in Example 1. Three gas-tight glass syringes (Hamilton) were filled with the solutions containing

PLGA dissolved in acetonitrile (inner stream, one inlet channel) and sodium caprate dissolved in deionized water (outer stream, two inlet channels) and mounted on syringe pumps (Pump 1 1 elite, Harvard apparatus, Cambridge, USA) and sprayed at different flow rates as indicated below. As an alternative to the syringe pumps, two sets of HPLC pumps were also used to drive the feed solutions into the microfluidics chip, resulting in a more continuous approach. The HPLC pumps and the syringe pumps resulted in nanoparticles in the same size range when using the same flow rates.

The flow rates in the outer streams were matched and set at a higher flow rate (10 ml/min) than the flow rate for the inner liquid stream (1 ml/min). The microfluidics output was characterized to investigate the nanoparticle production performance.

Measurement of PLGA nanoparticles using a zetasizer device showed a size range between 150 - 280 nm depending on the initial PLGA concentration (2.5-15 mg/ml) and the size increased as a function of the PLGA concentration. At PLGA

concentrations below 7 mg/ml a polydispersity index (PDI) below 0.10 was obtained.

PLGA nanoparticles were also prepared with incorporation of Sodium Caprate (C10NA) at different ratios and the size and PDI of the nanoparticles prepared are shown in Figure 6. The peristaltic pump of the spray dryer was used to mix in a sugar solution with the liquid output from the microfluidic device (nanoparticle suspension). Aqueous solutions with the sugars, mannitol, lactose, trehalose and inulin were used to test their stabilization and redispersibility of the PLGA nanoparticles. All sugar molecules were studied at nanoparticle:sugar ratios between 1 :1 and 1 :7 w/w. A PLGA feed with a concentration of 5 mg/ml was mixed with a sugar feed with a concentration of 100mg/ml. Spray drying was performed at an inlet temperature of 50°C, a feed rate of 3 ml/min, atomization gas flow rate of 450 L/h and 100% aspiration rate. The resulting microparticle powders were collected from collection vial and cyclone. Moisture content was studied using thermogravimetric analysis. The NEMs prepared showed different moisture content ranging between 2-10% depending on the sugar molecules used, with NEMs containing trehalose having the highest moisture content and with most formulations having moisture content at the lower side of the range (data not shown).

Redispersibility of nanoparticles was studied by dispersing 10 mg of the spray-dried nano-embedded microparticles in water and measuring particle size via dynamic light scattering using a Zetasizer. The presence of aggregates and the polydispersity index (PDI) of the particles were determined and compared with particle size and size distribution of nanoparticles prepared using only the microfluidics device. The particles showed partial to full redispersibility depending on the sugar molecules used in the matrix. Inulin showed the highest redispersibility even at low nanoparticle:sugar ratios while NEMs with mannitol were only partially redispersible even at high

nanoparticle:sugar ratios (data not shown).

We demonstrate that the present method can be used to produce nano-embedded microparticles from PLGA nanoparticles with good redispersibility. The yield was increased when the combined flow rate within the microfluidic device (inner flow + outer flow) was increased.

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El-Gendy, N., & Berkland, C. (2010). Combination nanoparticle agglomerates of fluticasone propionate and albuterol sulphate. In Respiratory drug delivery (pp. 819- 24).

Jensen, D. K., Jensen, L. B., Koocheki, S., Bengtson, L, Cun, D., Nielsen, H. M., & Foged, C. (2012). Design of an inhalable dry powder formulation of DOTAP-modified PLGA nanoparticles loaded with siRNA. Journal of Controlled Release, 757(1), 141- 148.

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A continuous method for producing a dry powder composition comprising nano- embedded microparticles, said method comprising the steps of:

generating nanoparticles in one or more microfluidic devices or static mixers, thereby generating a nanoparticle suspension;

mixing said nanoparticle suspension with a matrix solution, thereby obtaining a feed stream;

drying said feed stream, thereby obtaining a dry powder comprising nano- embedded microparticles.

The method of item 1 , wherein none of steps i) and ii) comprise a collection step.

The method of any one of the preceding items, wherein the drying step comprises a liquid atomisation step, such as spray-drying.

The method of any one of the preceding items, wherein the method further comprises a collection step after step iii).

The method of item 5, wherein the collection step can be performed in a continuous manner without interfering with the other steps.

The method of any one of the preceding items, wherein the method can be performed in less than 10 minutes, such as less than 5 minutes, such as less than 1 minute, such as less than 45 seconds, such as less 30 seconds, such as less than 10 seconds.

The method of any one of the preceding items, wherein the method can be performed aseptically.

The method of any one of the preceding items, wherein the one or more microfluidic devices or static mixers are arranged in parallel.

9. The method of any one of the preceding items, wherein the one or more

microfluidic devices or static mixers are arranged in series. 10. The method of any one of the preceding items, wherein the one or more microfluidic devices or static mixers comprise at least two inlet channels and at least one outlet channel.

1 1. The method of any one of the preceding items, wherein a fluid flows in each channel.

12. The method of any one of the preceding items, wherein the fluid is a liquid or a gas.

13. The method of any one of the preceding items, wherein at least one of the fluids flowing in the at least two inlet channels is a liquid.

14. The method of any one of the preceding items, wherein the at least two inlet channels is three inlet channels and the at least one outlet channel is one outlet channel.

15. The method of any one of the preceding items, wherein a first fluid flowing from one of the three inlet channels forms an inner stream and a second fluid flowing from the two other inlet channels forms an outer stream.

16. The method of any one of the preceding items, wherein the nanoparticles are formed by contacting at least one inner stream and at least one outer stream. 17. The method of any one of the preceding items, wherein the first fluid and the second fluid are miscible or immiscible.

18. The method of any one of the preceding items, wherein the outer stream is capable of hydrodynamically focusing the inner stream.

19. The method of any one of the preceding items, wherein the flow rates of the inner stream and of the outer stream favour the formation of microvortices at the intersection of the inlet channels. 20. The method of any one of the preceding items, wherein the flow rates of the inner stream and of the outer stream are comprised between 0.1 and 20 mL/min, such as between 0.1 and 1 mL/min, such as between 1 and 5 mL/min, such as between 5 and 10 mL/min, such as between 10 and 15 mL/min, such as between 15 and 20 mL/min. The method of any one of the preceding items, wherein the microvortices favour mixing of the inner stream and of the outer stream. The method of any one of the preceding items, wherein the outer stream and the inner stream are at least partly mixed via diffusion or convection. The method of any one of the preceding items, wherein the inner stream comprises at least one first compound and the outer stream comprises at least one second compound. The method of item 24, wherein the nanoparticles comprise the first compound in their inner part and the second compound in their outer part. The method of any one of the preceding items, wherein the nanoparticles are formed by complexation, by emulsion, by co-precipitation, or by cross-linking of the at least one first compound with the at least one second compound. The method of any one of the preceding items, wherein the ratio R defined as the flow rate of the inner stream divided by the flow rate of the outer stream is such that R≤1. The method of any one of the preceding items, wherein 0.01≤R≤0.3. The method of item 24, wherein the first compound is dispersed with the second compound within the nanoparticles. The method of any one of the preceding items, wherein the nanoparticles comprise at least one active compound and optionally a nanocarrier. The method of item 29, wherein the active compound is selected from the group consisting of a small molecule drug, an oligonucleotide, a polynucleotide, a peptide, a metal oxide, a lipid, a contrast agent, an aroma compound, and a compound suitable for functional foods. The method of any one of the preceding items, wherein the nanoparticles are water-insoluble and soluble in organic solvents. 32. The method of any one of the preceding items, wherein the nanoparticles are partially water-soluble in a specific biological environment.

33. The method of any one of the preceding items, wherein the matrix solution comprises a polymer, a polysaccharide or a disaccharide, or an amino acid.

34. The method of any one of the preceding items, wherein the matrix solution comprises at least one of the compounds selected from the group consisting of trehalose, inulin, lactose, sucrose, mannitol, amino acids, hypromellose (HPMC) and other cellulose-derived polymers, povidone (PVP) and

polyethylene glycol (PEG).

35. The method of any one of the preceding items, wherein the matrix solution comprises inactive nanoparticles or a combination of inactive nanoparticles and dissolved excipients.

36. The method of any one of the preceding items, wherein the nanoparticles are not soluble in the matrix solution. 37. The method of any one of the preceding items, wherein the matrix solution is water-soluble.

38. The method of any one of the preceding items, wherein the matrix solution is approved for oral administration.

39. The method of any one of the preceding items, wherein the ratio Rf, where Rf =(flow rate of the nanoparticle solution) / (flow rate of the matrix solution), is such that Rf is comprised between 10:1 and 1 : 10. 40. The method of any one of the preceding items, wherein the feed stream

comprising the nanoparticle suspension mixed with the matrix solution is spray dried after being directly fed into the inlet of a dryer.

41. The method of any one of the preceding items, wherein spray drying of the feed stream yields a powder of nano-embedded micro particles.

42. The method of item 41 , wherein the powder is substantially dry. 43. The method of any one of the preceding items, wherein the moisture content of the dry powder is lower than 3%, such as lower than 2%, such as lower than 1 %. 44. The method of any one of the preceding items, wherein the moisture content of the dry powder is between 2 and 15%, such as between 3 and 14%, such as between 4 and 13%, such as between 5 and 12%, such as between 6 and 1 1 %, such as between 7 and 10%, such as between 8 and 10%, such as 9%.

45. The method of any one of the preceding items, wherein the nano-embedded microparticles have a mass median aerodynamic diameter (MMAD) between 1 and 30 μηι.

46. The method of any one of the preceding items, wherein the nanoparticles have improved stability when embedded in the microparticles compared to their free form.

47. The method of any one of the preceding items, wherein the redispersibility of the nanoparticles embedded in the microparticles is close to 1.

48. The method of any one of the preceding items, wherein the polydispersity index (PDI) of the microparticles in the dry powder is lower than 0.3, such as lower than 0.2, such as lower than 0.1

49. An apparatus for performing the method according to any one of the preceding items, said apparatus comprising:

i) one or more microfluidic devices or static mixers;

iii) means for providing a matrix solution;

iv) a junction for mixing the output from said one or more microfluidic

devices or static mixers with said matrix solution;

v) a dryer for performing liquid atomisation and drying of the output of said junction;

vi) a collecting device. 50. The apparatus of item 49, wherein the one or more microfluidic devices or static mixers is at least two microfluidic devices or static mixers, such as three microfluidic devices or static mixers, such as four microfluidic devices or static mixers, such as five microfluidic devices or static mixers, such as six microfluidic devices or static mixers.

51. The apparatus of any one of items 49 to 50, wherein the means for providing fluid to said one or more microfluidic devices or static mixers is a continuously operating pump such as a peristaltic pump.

52. The apparatus of any one of items 49 to 51 , wherein the fluid is contained in a first container such as a syringe or a bottle.

53. The apparatus of any one of items 49 to 52, wherein the first container is

connected to the pump and to the one or more microfluidic devices or static mixers via tubing such as silicone or silicone rubber tubing.

54. The apparatus of any one of items 49 to 53, wherein the one or more

microfluidic devices or static mixers are arranged in parallel or in series.

55. The apparatus of any one of items 49 to 54, wherein the means for providing said matrix solution is a continuously operating pump such as a peristaltic pump. 56. The apparatus of any one of items 49 to 55, wherein the matrix solution is

contained in a second container such as a syringe or a bottle.

57. The apparatus of any one of items 49 to 56, wherein the second container is connected to the pump and to the junction via tubing such as silicone or silicone rubber tubing.

58. The apparatus of any one of items 49 to 57, wherein the dryer is a spray dryer.

59. The apparatus of any one of items 49 to 58, wherein the collecting device is a cyclone collector.

60. The apparatus of any one of items 49 to 59, wherein the apparatus can be sterilised prior to use. The apparatus of any one of items 49 to 60, further comprising a granulation device such as a fluidized bed, wherein the granulation device is connected to the dryer. A dry powder composition comprising nano-embedded microparticles obtainable by the method of any one of claims 1 to 48.

Claims

A continuous method for producing a dry powder composition comprising nano- embedded micro particles, said method comprising the steps of:

The method of claim 1 , wherein none of steps i) and ii) comprise a collection step.

The method of any one of the preceding claims, wherein the drying step comprises a liquid atomisation step, such as spray-drying.

The method of any one of the preceding claims, wherein the method further comprises a collection step after step iii).

The method of claim 4, wherein the collection step can be performed in a continuous manner without interfering with the other steps.

The method of any one of the preceding claims, wherein the method can be performed in less than 10 minutes, such as less than 5 minutes, such as less than 1 minute, such as less than 45 seconds, such as less 30 seconds, such as less than 10 seconds.

The method of any one of the preceding claims, wherein the method can be performed aseptically.

The method of any one of the preceding claims, wherein the one or more microfluidic devices or static mixers are arranged in parallel or in series.

9. The method of any one of the preceding claims, wherein the one or more microfluidic devices or static mixers comprise at least two inlet channels and at least one outlet channel.

10. The method of any one of the preceding claims, wherein the nanoparticles are formed by contacting at least one inner stream and at least one outer stream.

1 1. The method of any one of the preceding claims, wherein the flow rates of the inner stream and of the outer stream favour the formation of microvortices at the intersection of the inlet channels, said microvortices favouring mixing of the inner stream and of the outer stream.

12. The method of any one of the preceding claims, wherein the inner stream

comprises at least one first compound and the outer stream comprises at least one second compound.

13. The method of any one of the preceding claims, wherein the nanoparticles are formed by complexation, by emulsion, by co-precipitation, or by cross-linking of the at least one first compound with the at least one second compound.

14. The method of any one of the preceding claims, wherein the ratio R defined as the flow rate of the inner stream divided by the flow rate of the outer stream is such that R≤1.

15. The method of any one of the preceding claims, wherein 0.01≤R≤0.3.

16. The method of any one of the preceding claims, wherein the nanoparticles comprise at least one active compound and optionally a nanocarrier.

17. The method of any one of the preceding claims, wherein the matrix solution comprises a polymer, a polysaccharide or a disaccharide, or an amino acid.

18. The method of any one of the preceding claims, wherein the feed stream

19. The method of any one of the preceding claims, wherein the polydispersity index (PDI) of the microparticles in the dry powder is lower than 0.3, such as lower than 0.2, such as lower than 0.1

20. An apparatus for performing the method according to any one of the preceding claims, said apparatus comprising:

i) one or more microfluidic devices or static mixers;

iii) means for providing a matrix solution;

vi) a collecting device.

21. The apparatus of claim 20, further comprising a granulation device such as a fluidized bed, wherein the granulation device is connected to the dryer.

22. A dry powder composition comprising nano-embedded microparticles

obtainable by the method of any one of claims 1 to 19.