WO2015088299A1

WO2015088299A1 - Method for production of biopolymer-based droplets and particles in a microfluidic system

Info

Publication number: WO2015088299A1
Application number: PCT/LT2014/000013
Authority: WO
Inventors: Linas MAŽUTIS
Original assignee: Vilniaus Universitetas
Current assignee: Vilniaus Universitetas
Priority date: 2013-12-09
Filing date: 2014-12-03
Publication date: 2015-06-18
Anticipated expiration: 2016-06-09

Abstract

The present invention relates to micro-systems and methods for production of biopolymer-based droplets and particles. These particles can be used for encapsulation of biochemical, biological and/or therapeutic compounds. The encapsulated molecules can be released from particles into surrounding fluid over time and therefore potentially can be used as vesicles for drug transport and release. ˙

Description

METHOD FOR PRODUCTION OF BIOPOLYMER-BASED DROPLETS AND PARTICLES IN A MICROFLUIDIC SYSTEM

FIELD OF THE INVENTION

The present invention is directed to systems and methods for production of biopolymer- based droplets and particles for encapsulation and release of biochemical compounds such as drugs, antibodies or other type of therapeutics. BACKGROUND OF THE INVENTION

Drug delivery systems based on natural biodegradable polymers are attractive agents for improved delivery, stabilization and prolonged release of encapsulated drugs [1 , 2]. However, in many cases the techniques used for their production relies on mechanical steering [3] or spray-coagulation methods [4]. These methods, although efficient, produce polydisperse drug particles and give a poor control over the size and shape. In this respect droplet microfluidics provides a valuable tool for encapsulation of various biologicals and chemicals into highly monodisperse pico- and nano-liter volume droplets [5]. Numerous examples reported to date have exploited droplet microfluidics to produce single [6] and double emulsions [7, 8], as well as various types of particles composed of responsive polymers [9] and biodegradable materials [10, 1 1]. In this context, alginate, a natural polysaccharide derived from brown algae, is an attractive biomaterial for different applications in medical sciences [12, 13]. Alginate consists of mannuronic (M) and guluronic (G) acids arranged in different combinations such as blocks rich in either M or G unit, or blocks of alternating G and M units [14]. In the presence of divalent Ca cations the guluronic acids from nearby chains form ionic crosslink resulting in alginate hydrogel. The ratio of M and G units defines the physicochemical properties of the hydrogel [15]. Due to its excellent biocompatibility alginate-based hydrogels are highly promising candidates for use as drug delivery systems [12] or as biomedical implants [16]. Indeed, many efforts have been dedicated to produce different type of alginate particles. For example, alginate droplets can be produced using T-junction geometry but resulting bimodal distribution requires additional step of separation [17]. Alginate droplets can be also generated "on-chip", while inducing external gelation process "off- chip" [18]. It is possible to use capillary devices to produce alginate-based double emulsions [19, 20], Janus particles [21] and alginate beads carrying encapsulated cells [22]. However, the micro fluidic systems reported to date have generated alginate particles that are relatively large in size (~ 50-200 μηι in diameter), thereby reducing the scope of potential applications [23, 24]. For example, the use of microparticles as drug carriers requires their size to be reduced down to the cell dimensions (- 4-15 μηι). In particular, reduced size should facilitate the deeper penetration of particles into the diseased tissues and would less likely to cause clogging of blood vessels once particles are injected into the blood stream. Furthermore, hydrogel particles should remain dispersed in bodily fluids without adverse interaction with biomolecules, cells and tissues. Indeed, so far the production of smaller particles composed of biopolymers has been challenging due to relatively high viscosity of polymer solutions and poor control over the polymerization kinetics. The latter difficulty is associated with fast gelation process (which in case of alginate is triggered by Ca ions [25]), therefore complicating not only the production of monodisperse droplets but also the delivery of fluids and biological compounds into the droplets [20, 23].

SUMMARY OF THE INVENTION

Droplets and particles composed of natural biopolymers can be used as vesicles for encapsulation and delivery of drugs, therapeutics and other biochemical compounds. However, production of such vesicles is often restricted by the lack of micro-devices that would allow efficient and stable production of monodisperse droplets and particles. The present invention is directed to a novel microfluidic device for production of alginate particles of pancake shape having the size resembling to those of mammalian cells (~ 4- 15 μιη). The developed microdevice consists of three inlets for introduction of different fluids and a specially designed cross junction that facilitates the pinch-off process of viscous fluids into monodisperse droplets. Such microdevice is"capable of, but not limited to, for production of monodisperse biopolymer-based droplets and particles. To exemplify the operation and functionality of developed micro-device we showed the production of alginate particles carrying antibodies and delayed release of antibodies over time. These particles were biocompatible with blood and showed no signs of undesirable interactions with cells. The system and method of the present invention can be used to produce droplets, particles and other type of drug carrying vesicles. In one aspect, the invention comprises a microfluidic system for the production of biopolymer-based droplets. In one exemplary embodiment, the system comprises:

(i) an inlet for continuous phase (oil);

(ii) an inlet for the first fluid;

(iii) an inlet for the second fluid;

(iv) an inlet for the third fluid;

(v) specially designed nozzle that facilitated droplet pinch-off;

(vi) channel for droplet stabilization by surfactant, and;

(vii) droplet collection outlet.

In another aspect, the invention comprises the method for the formation of solid or semi-sold particles:

(i) Injection of a "polymer precursor" solution;

(ii) Injection of an "inducer" solution;

(iii) Injection of an "inert" solution that physically separates "polymer precursor" solution from the "inducer" solution;

(iv) Encapsulation of all three solutions into droplets;

(v) Polymerization (gelation) of particles inside droplets;

(vi) Droplet collection off-chip;

(vii) Particle release from emulsion.

Polymerization (gelation) herein, refers to the process in which a liquid form of "polymer precursor" is forming a solid or semi-sold hydrogel in the contact with "inducer". Typically, but not limited to, inducer can be divalent ions, chemical compounds, temperature, light, etc.

In one exemplary embodiment, the system further comprises fluid resistors to damp fluctuations arising during micro-device operation.

In one exemplary embodiment, the droplet generation occurs at cross-junction having a characteristic constriction that facilitates the break-up of fluid stream into monodisperse droplets.

In one exemplary embodiment, the micro-channels of each aqueous phase are merging into a single micro-channel upstream the flow-focusing junction. In one exemplary embodiment, the micro-channel of continuous phase is forming a flow-focusing junction with a micro-channel of aqueous phase.

In one exemplary embodiment, the invention comprises the method for the formation of monodisperse hydrogel droplets.

In one exemplary embodiment, the biochemical and biological compounds are introduced into droplets by dissolving them:

(i) In the "polymer precursor" solution;

(ii) In the "inducer" solution;

(iii) In the "inert" solution.

(iv) In all solutions.

In another exemplary embodiment, biochemical and biological compounds are dissolved in separate solutions. For example, biological compound A is dissolved in the "polymer precursor" solution and the biological compound B is dissolved in the "inducer" solution.

In one exemplary embodiment, the particles are released from the emulsion by braking emulsion (de-emulsification). Typically, but not limited to, emulsion can broken by chemical means, temperature or electric field by destabilizing the water-oil interface.

In one exemplary embodiment, the biochemical and biological compounds entrapped inside the hydrogel particle are released by dispersing particles into a fluid such as bodily fluids, blood, biological buffer and others, for example.

In one exemplary embodiment, the above methods are carried out but not limited to using a microfluidics system.

In one exemplary embodiment, the drops and particles have a size ranging from 1 to 100 μηι.

DESCRIPTION OF THE DRAWINGS

Figure 1: The design of a microfluidics device. The device contains an inlet for first fluid (1), the inlet for second fluid (2), the inlet for a third fluid (3) and the inlet for carrier oil (4). Droplets are generated at a flow-focussing junction (dashed square) and are collected at the outlet (5). The design also includes flow resistors (6) and passive filters (7). Turning now to dashed squares that represent a flow-focusing junction, a narrow constriction (8) is introduced at the flow-focusing junction to facilitate the breakup of viscous fluids. Channel for carrier oil (9) is coming from both sides of flow focusing junction. Three microfluidics channels (10, 1 1 and 12) are used to bring three fluids into a short (10-100 μπι long) single micro-channel that is placed upstream the nozzle. Droplets flowing in the channel (13) are stabilized by surfactant before they are collected off-chip.

Figure 2: Digital image of flow focusing junction during chip operation. Image showing alginate droplet production. The microfluidics channel (10) carries polymer precursor such as alginate solution, the microfluidics channel (1 1) carriers inert solution, such as water, the microfluidics channel (12) carriers inducer solution, such as 0.5% CaCl₂. All three fluids are flowing side-by-side and do not mix before they reach a nozzle (8) where the carrier oil coming from channel (9) brakes fluid stream into droplets. Droplets are then flowing inside the microchannel (13) before reaching the outlet.

Figure 3. Digital image of biopolymer-based droplets. Droplets were generated using microfludics chip indicated in Figure 1 and are composed of alginate 0.5 % (w/w) biopolymer. The size of droplets can be changed by varying the rates of the aqueous fluids and carrier oil, by changing the cross-section of a nozzle (#8 in Figure 1), or by changing the cross-section of a microfluidics channel (#13 in Figure 1).

Figure 4. Digital image of alginate particles dissolved in water. The size of particles is 16 ± 1 μιη.

Figure 5. Digital image of alginate particles dissolved in water. The size of particles is 3.5 ± 0.5 μηι.

Figure 6. Digital image of dried alginate particles.

Figure 7. Scanning electron microscopy image of alginate particles. Scale bar 10 μηι

Figure 8. Scanning electron microscopy image of alginate particles. Scale bar 10 μη

Figure 9. Alginate particles carrying encapsulated FITC labelled antibody dispersed in water.

Figure 10. The alginate particles from Figure 9 were dissolved in water (a) and in phosphate saline buffer, lx PBS (b) and the release kinetics of encapsulated FITC labelled antibody recorded over time. Figure 11. The alginate particles mixed with mouse blood do not show a visible sign of agglomeration, coagulation or any other undesirable effect thereby implying the biocompatibility of alginate particles.

DETAILED DESCRIPTION OF THE INVENTION

The present invention firstly provides a method for producing biopolymer-based droplets and particles that could be used for encapsulation of biochemical and biological compounds, drugs and pharmaceutics, and other molecules.

The encapsulated molecules can be released them into surrounding fluid over time. The term "microfluidic chip", as used herein, refers to a device, or chip, of only millimeters to a few square centimeters or tens of centimetres in size dealing with the handling of extremely small fluid volumes down to less than femto liters. Microfluidic chips are usually fabricated by using lithography-based technologies such as soft lithography.

In the method of the invention, the microfluidics chip comprises, but not limited to, following units:

(i) an inlet and microfluidic channel(s) for carrier oil;

(ii) an inlet and microfluidic channel(s) for the first fluid;

(iii) an inlet and microfluidic channel(s) for the second fluid;

(iv) an inlet and microfluidic channel(s) for the third fluid;

(v) a nozzle;

(vi) a microfluidics channel connecting the nozzle with the outlet, and

(vii) collection outlet.

In an embodiment, the fluids are introduced into the microfluidics chip via an inlet(s) and passes through the passive filter(s) and fluid resistor(s).

In a more particular embodiment, passive filters used in the chip of the invention are used to prevent microfluidic channels from clogging and act as solid support to avoid collapse of device structure. The fluid resistors damp fluctuation that might arise during device operation. These units may be well-known by the skilled person and their uses are illustrated in Figure 1. In an embodiment, the micro-channels of each fluid are merging into a single micro-channel upstream the flow-focusing junction where individual fluids meet but do not mix (Figure 2),

In an embodiment, the depth of the microfluidic channels are in the range from 1 μ^ηι to 100 μι^η, preferably in the range 10-20 μιη and more preferably in the range of 1 - 10 μιη.

In the method of the invention, the solid or semi-solid particles are produced inside the droplets by mixing two or more fluids. For example, Figure 2 shows the mixing of three fluids.

In the method of the invention, the droplets and/or particles are collected off-chip via outlet (Figure 3).

In an embodiment, the droplets are generated on the microfluidic chip comprising a flow- focusing junction (as illustrated in the dotted line box of Figure 1) allowing the production of droplets of different size. The droplet size can be controlled by adjusting the flow rates of aqueous phase and carrier oil and/or the cross-section of a nozzle and/or the cross-section of microfluidics channels.

In an embodiment, the droplets are generated at a frequency ranging from 0.01 Hz to 10 kHz, preferably from 0.1 kHz to 5 kHz, more preferably from 0.5 kHz to 2.5 kHz. A frequency of 1 kHz means that droplets are provided at a rate of 1000 droplets per second.

As used in this specification, the term "about" refers to a range of values ± 10% of the specified value. For example, "about 20" includes ± 10 % of 20, or from 18 to 22. Preferably, the term "about" refers to a range of values ± 5 % of the specified value.

In an embodiment, the droplets have a volume ranging from 0.01 pL to 1000 nL, preferably from 10 pL to 400 pL, more preferably from 1 pL to 10 pL, and even more preferably from 0.1 pL to 1 pL. In a particular embodiment, the droplets have a volume of 3 pL.

The droplets comprise a dispersed phase (for example, an aqueous solution) in a continuous immiscible phase. The particle is formed when the dispersed phase turns into a solid or semi-solid material (for example, when the polymer solution forms a hyrogel). As used in this specification, the term "material" refers to a range of biological an chemical compounds, mostly polymers, such as alginate, agarose, gelatin, collagen, polyethylene glycol, Polyvinyl alcohol and others. The entire droplet can turn into a hydrogel particle if a solution occupying the entire droplet volume forms a solid or semi-solid material.

Only a certain fraction of a droplet can turn into a hydrogel particle if only a fraction of a solution inside a droplet forms a solid or semi-solid material.

In an embodiment, the particles are produced by introducing biopolymer precursor solution, water and inducer solution into the droplets. As used in this specification, the term "biopolymer precursor" refers to chemical or biological material that is present in a liquid form and can form a solid or semi-solid material in the presence of "inducer". As used in this specification, the term "inducer" refers to a chemical or biological compound, light or temperature that induces the polymerization (gelation) of a biopolymer precursor solution.

In a particular embodiment, water is introduced between "polymer precursor" and "inducer" solutions to prevent premature polymerization (gelation) reaction (Figure 2).

In a more particular embodiment, "biopolymer precursor" is an alginate solution and "inducer" is a calcium chloride solution.

In particular, the alginate amount in a solution is in the range of 0.1-10% (w/w) preferably from 1 to 5% (w/w) and more preferably 0.5-1% (w/w).

In particular, the calcium chloride amount in a solution is in the range of 0.1-10% (w/w) preferably from 1 to 5% (w/w) and more preferably 0.5-1% (w/w).

In a first embodiment, the solid and semi-solid particle formation occurs inside the droplets flowing inside microfluidics chip before they reach the outlet.

In a second embodiment, the solid and semi-solid particle formation occurs inside the droplets after they reach the outlet.

Aqueous solutions within the droplets may comprise, for instance, various chemical compounds such as buffers, salts, carbohydrates, lipids, polymers, proteins, nucleic acids, cells or micro-organisms.

In a particular embodiment, the carrier oil used to generate droplets is a fluorinated oil and comprises a surfactant, a PFPE-PEG-PFPE (perfluoropolyether - polyethylene glycol - perfluoropolyether) tri-block copolymer. Said surfactant being present in the carrier oil at a concentration ranging from 0.05 % to 3 % (w/w), preferably ranging from 0.1 % to 1 % (w/w), more preferably ranging from 1% to 3% (w/w).

The method of the present invention is not limited by the type of surfactant or carrier oil used. The type of surfactant to be used will depend on the surfactant's effectiveness in facilitating droplet stabilization of the first and second sets of droplets, as well as the surfactant's effectiveness in stabilizing the interface of droplets, as well as the surfactant's compatibility with any reactants contained within the droplet. One of ordinary skill in the art will be able to select the appropriate surfactant, dispersed phase and carrier oil based on the desired properties of the droplets and reaction conditions used.

Surfactants, also named emulsifying agents, act at the water/oil interface to prevent (or at least to decay) separation of the phases. Many oils and surfactants (emulsifiers) can be used for the generation of droplets [26].

In an embodiment, the carrier oil is selected from the group consisting of fluorinated oil such as FC40 oil (3M^®), FC43 (3M^®), FC77 oil (3M^®), FC72 (3M^®), FC84 (3M^®), FC70 (3M^®), HFE-7500 (3M^®), HFE-7100 (3M^®), perfluorohexane, perfluorooctane, perfluorodecane, Galden-HT135 oil (Solvay Solexis), Galden-HT170 oil (Solvay Solexis), Galden-HTl 10 oil (Solvay Solexis), Galden-HT90 oil (Solvay Solexis), Galden-HT70 oil (Solvay Solexis), Galden PFPE liquids, Galden^® SV Fluids or H- Galden^® ZV Fluids; and hydrocarbon oils such as Mineral oils, Light mineral oil, Adepsine oil, Albolene, Cable oil, Baby Oil, Drakeol, Electrical Insulating Oil, Heat- treating oil, Hydraulic oil, Lignite oil, Liquid paraffin, Mineral Seal Oil, Paraffin oil, Petroleum, Technical oil, White oil, Silicone oils or Vegetable oils. In a particular embodiment, the carrier oil is a fluorinated oil. In a more particular embodiment, the carrier oil is selected from the group consisting of FC40 oil and HFE-7500 oil.

In an embodiment, the size of a particle is the same or smaller than the size of a droplet.

In a preferred embodiment, the depth of all channels on the microfluidic chip is the same and is in the range from 1 μιη to 1000 μηι, preferably in the range 50-500 μιτι and more preferably in the range of 20-300 μπι, and even more preferably in the range of 10- 100 μπι.

In a further embodiment, the method of the invention further comprises collecting droplets.

In another embodiment, the collected droplets are broken thereby releasing particles from droplets. This can be achieved by destabilizing the droplet water-oil interface using chemical means or using electro-coalescence, temperature, dilution, etc. In this particular embodiment, the droplet water-oil interface is destabilized by mixing the emulsion with chemical such as fluorinated octanol.

In one exemplary embodiment, the biochemical and biological molecules entrapped inside the particles are released by dispersing particles into a fluid such as bodily fluids, blood, biological buffer, water and/or other fluids.

The microfluidic chip of the invention may be fabricated by any method known by the skilled person such as soft lithography [27]. Fabrication of a microfluidic device is exemplified in the experimental section.

The following example is given for purposes of illustration and not by way of limitation.

EXAMPLE

Experimental Methods

Materials and reagents

Sodium alginate from brown algae (4-12 cP, 1% in H₂0) and CaCl₂ were from Sigma- Aldrich. Water used in experiments was deionized with Millipore Purification system (Milli-Q). Carrier oil was FC-40 (3M^®) with 3% (w/w) fluorosurfactant, l H,lH,2H,2H-PerfIuorooctan-l-ol (PFO) was from Fluorochem Ltd. FITC-labeled mouse IgG was from Jackson ImmunoResearch.

Microfluidics device fabrication and running

Rectangular microfluidic channels were fabricated using soft lithography by pouring poly(dimethylsiloxane) (PDMS, Sylgard 184, Dow Corning Corp.) onto a positive-relief silicon wafer (SILTRONIX) patterned with SU-8 photoresist (Microchem Corp). Curing agent was added to PDMS base to a final concentration of 10% (w/w), degassed and poured over the mould for crosslinking at 65°C for 12 hours. The structured PDMS layer was peeled off the mould and the inlet and outlet holes were punched with a 0.75 mm-diameter Harris Uni-Core biopsy punch (Electron Microscopy Sciences). The microchannels were sealed by bonding the PDMS to glass using an oxygen plasma (PlasmaPrep 2 plasma oven; GaLa Instrumente GmbH). The channels were treated with surface coating agent Aquapel to make it hydrophobic and subsequently flushed with nitrogen. Each of the fluids were injected into the PDMS channels via PTFE tubing (Fisher) connected to 1 raL syringes (Omnifix-F^®) and ONeolus needles (Terumo). The flow rates of liquids and oil were controlled by syringe pumps (PHD 2000, Harvard Apparatus). Microfluidic Device Operation

The flow regime in the microfluidics chip was laminar with low Capillary and Reynold numbers, Ca ~10^"4 and Re ~10^"2, respectively. The microfluidic device indicated in Figure 1 consist of four inlets containing passive filters used to trap dust, followed by fluid resistors used to damp fluid fluctuations arising during device operation. The liquids from each inlet flow into a central channel of 80 μιτι long and 30 μιη wide where flow is laminar, before being encapsulated into droplets. Emulsification of fluids occurs at flow- focusing junction having a short and narrow constriction of 10 μπι wide and 10 μιη long. The collection outlet is used to collect the droplets. The fluids were injected into the microfluidics chip via PTFE tubing (0.56 x 1.07 mm) connected to 1 mL syringes (Braun) and 25-gauge needles (Neolus). The flow rates of liquids and oil were controlled by syringe pumps (PHD 2000, Harvard Apparatus). The flow rate for aqueous phase was in the range of 20 - 50 μΐ/hr and for continuous phase 100 - 300 μΐ/hr. Emulsions were collected off-chip into a 1.5 mL tube. Droplet production was analyzed with a high-speed camera (Phantom V7.2) mounted on a Nikon Eclipse microscope.

Alginate particle release from droplets

To release alginate particles from emulsion PFO was added on top of emulsion to the final concentration of 10% (v/v) and incubated at room temperature for 5 min. The supernatant containing alginate particles was then transferred into a second tube and analyzed accordingly.

Results

Microfluidics chip design

Initially we tested alginate droplet production following principles as reported previously [20, 23], However, we could not recover alginate particles due to premature polymerization on-chip or due to inefficient gelation process. We also tested alginate droplet production using droplet fusion approach [21, 28] but this approach also lead to poor control over monodispersity of alginate particles. Due to these difficulties we aimed to create a simple and robust system that would allow production of alginate particles over long periods of time for encapsulation of biologically active molecules such as antibodies or drugs. Using soft lithography technique we created a microfluidic chip containing three aliquot inlets that merge into a single channel upstream the flow- focusing junction (FFJ) as shown in Figure 1 and Figure 2. The channel downstream the FFJ was 1000 μπι long to allow interface stabilization by surfactant before droplets collide and flow into the collection tube. During the course of droplet production we noticed that viscosity differences of three fluids trigger complex flow instabilities resulting in polydisperse droplets. To prevent this from happening we have incorporated fluid resistors for each aqueous phase R_f, = 19.2 mPa s m^"3), so that it is considerably higher than the resistance of collection channel (R = 3.2 mPa s m^" ), where the hydraulic resistance. In addition, we have utilized the nozzle (Figure 1, red dashed square) having constriction of 10 μιτι wide, which we found facilitated the pinch-off of viscous fluids into monodisperese droplets. To validate the functionality of the microfluidics device we tested alginate droplet production using different flow rates and alginate concentrations. Stable droplet production was achieved up to 2% (w/w) alginate, above which the viscosity of alginate solution became too high making droplet production process unreliable. To prevent premature hydrogel formation inside microfluidic chip, a stream of water was introduced between CaCl₂ and alginate solutions. However, fast diffusion of small Ca²⁺ ions through the thin layer of middle stream could induce premature alginate polymerization and eventually obscure droplet production. Diffusion and mixing between two laminar streams inside rectangular microfluidics channels has been reported previously [29]. We found that that when using a microchannel of 80 μιη long and the middle phase of >10 μιη thick was sufficient to prevent premature alginate polymerization on-chip.

Alginate droplet production

Having established the design of a microfluidic chip we tested production of alginate droplets by using different flow conditions. Stable alginate droplet production was achieved when the flow rate for continuous phase was at least two times higher than the total flow rate of dispersed phases. After series of optimization steps we found that when setting the total flow rate for dispersed phase at 100-150 μΙΤΙΐΓ and for continuous phase at 200-300 μί/ΐιτ, allowed reproducible alginate droplet production over the period of few hours. We tested droplet production using different CaCl₂ concentrations ranging from 0.1 to 5% (w/w) dissolved in water. At lowest concentrations tested (0.1%) produced alginate droplets remained in a liquid form and did not polymerize into the hydrogel particles. On another hand, at CaCl₂ concentrations between 2-5% gelation occurred during droplet pinch-off process inducing the formation of a pearl-like train (string) similarly to previous report [30]. In the middle regime (0.5-1.0%) alginate polymerization occurred within few milliseconds inside the droplets (after the pinch-off process) before their collection off-chip (Figure 3). In these conditions we could produce stable monodisperse droplets over extended periods of time. Therefore, using flow conditions 30 μΐ/hr for 1 % alginate, 30 μΙ7ητ for 0.5% CaCl₂ and 40 μΙ7ητ for water phase and 300 μΙ 1ΐΓ for carrier oil, we obtained hydrogel particles of pancake shape resembling the blood cells (Figure 4). Considering that the size of such particles approaches the dimensions of mammalian cells (~ 5 μηΐ thick and ~ 16 μπι wide) it could find useful applications in biomedicine and pharmaceutical sectors as the candidates for improved drug delivery and release. Moreover, we also obtained hydrogel particles of ~ 3-4 μη in diameter (Figure 5). The alginate particles could be dried to obtain a powder and then dissolved in a fluid (Figure 6). We also evaluated the size and the shape of particles using scanning electron microscopy (Figure 7 and Figure 8).

Encapsulation and release

To test the encapsulation and release of molecules from the alginate particles we used the FITC labelled antibody (Figure 9). Droplets carrying encapsulated IgG were > collected into 1.5 mL tube and after emulsification was complete alginate particles were released from the emulsion by mixing it with 20% PFO. The resulting particles were then dissolved in physiological buffer, phosphate buffered saline buffer (PBS) and pure water, and IgG release monitored under fluorescence microscope over time. Considering existing literature data, we expected that our hydrogel vesicles have the Young modulus of ~ 2 kPa and therefore the pore size of ~ 16 nm [31 , 32]. It is reasonable to assume that due to limited gel porosity the release of larger proteins (~ 20 nm in size) will be delayed in time. As expected, alginate vesicles dissolved in water retained IgG for over 36 hours, therefore providing a mean for prolonged release of encapsulated therapeutics (Figure 10). In contrast, antibody release in l x PBS was immediate, presumably due to ion pair formation between Ca²⁺ and P0₄ ^2" groups resulting in a loose hydrogel matrix and thus fast IgG release. In mammalian serum, however, the free calcium concentration is ~ 100 μg/mL [33]; a significant amount that would reduce the alginate hydrogel dissolution. Finally, we tested the biocompatibility of alginate particles within biological fluids by mixing alginate vesicles (n ~ 10³) carrying encapsulated antibodies with 10 \iL of blood extracted from the mouse. Alginate vesicles remained well dispersed in the blood with no signs of cell adherence to the surface of particles or causing undesirable blood clogging (Figure 11). These results indicate that alginate vesicles are biocompatible and might be well suited for future biomedical applications. Subsequent research is necessary for evaluation of release kinetics of antibodies from alginate particles into blood and our current efforts are dedicated to investigate this task. Lately, the concept of "polymeric artificial cells" has experienced interest not only as a mean to deliver the encapsulated drug compounds but also as efficient bioabsorbents of toxic byproducts in the blood [34].

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Claims

1. A method for producing hydrogel droplets and particles by using microfluidics system (device, chip).

2. The method according to claim 1, wherein the microfluidics chip comprises

(i) an inlet and micro fluidic channel(s) for carrier oil;

(ii) an inlet and microfiuidic channel(s) for the first fluid;

(iii) an inlet and microfiuidic channel(s) for the second fluid;

(iv) an inlet and microfiuidic channel(s) for the third fluid;

(v) a nozzle;

(vi) droplet and particle collection outlet;

(vii) a microfluidics channel connecting the nozzle with the outlet, and

3. The method according to claim 2, wherein the fluids are introduced into the microfluidics chip via an inlet(s) and passes through the passive filter(s) and fluid resistor(s).

4. The method according to claims 2 and 3, wherein the micro-channels of each fluid are merging into a single micro-channel.

5. The method according to claims 2 and 3, wherein the depth of micro-channels are in the range from 1 μηι to 100 μπι, preferably in the range 10-20 μπι and more preferably in the range of 1-10 μηι.

6. The method according to claims 2 and 3, wherein the width of micro-channels are in the range from 1 μηι to 1000 μπι, preferably in the range 50-500 μπι and more preferably in the range of 20-300 μη , and even more preferably in the range of 10-100 μηι.

7. The method according to claims 1 and 3, wherein the droplets are generated on the microfiuidic chip comprising a constriction and flow- focusing junction.

8. The method according to claims 1 and 3, wherein the droplet size is controlled by adjusting the flow rates of aqueous phase and carrier oil and/or by adjusting the cross- section of a nozzle and/or by adjusting the cross-section of microfluidics channels.

9. The method according to claim 1, wherein the droplet and/or particle size is in the range from 1 μηι to 100 μηι, preferably in the range 10-20 μηι and more preferably in the range of 1 - 10 μηι.

10. The method according to claim 1, wherein the droplets and particles are composed of natural biopolymers.

1 1. The method according to claim 10, wherein natural biopolymer is alginate.

12. The method according to any one claims 7 to 1 1 , wherein the solid or semi- solid particles are produced inside the droplets by mixing two, three or more fluids.

13. The method according to any one claims 7 to 12, wherein solid or semi-solid particles can be released from droplets by braking droplet water-oil interface.

14. The method according to claim 1 or any one claim from 7 to 12, wherein the particles carry encapsulated molecules such as drugs, antibodies, biological or chemical compounds, proteins, enzymes, DNA, RNA, cells, etc.

15. The method according to any one claim 14, wherein encapsulated molecules are released from particle into surrounding fluid such as bodily fluid, blood, water, buffer, etc.

16. The method according to claim 15, wherein the release of encapsulated molecules and compounds can be controlled by adjusting the pore size of hydrogel.