METHOD FOR FORMING COATED HYDROGEL BEADS

Abstract

The present invention provides a method of forming a coated hydrogel bead, wherein the hydrogel bead is coated via microfluidics.

Claims

1. A method for forming coated hydrogel microbeads, said method comprising: forming an emulsion comprising a carrier fluid and hydrogel microbeads as a discontinuous phase, and an emulsifying fluid as a continuous phase; and forming a coating on said hydrogel microbeads from the carrier fluid; wherein said carrier fluid comprises a coating agent and the emulsifying fluid is immiscible with the carrier fluid.

2. The method of claim 1, said method comprising, prior to forming the emulsion comprising the carrier fluid and hydrogel microbeads as a discontinuous phase, providing a mixture of the carrier fluid and hydrogel microbeads.

3. The method of claim 2 where said step of providing the mixture comprises forming said hydrogel microbeads via a method comprising: forming an emulsion, e.g. a first emulsion, comprising a hydrogel precursor composition as a discontinuous phase and a first emulsifying fluid as a continuous phase; and forming hydrogel microbeads from the hydrogel precursor composition; wherein the first emulsifying fluid is immiscible with the hydrogel precursor composition.

4. The method of claim 2 or claim 3 wherein the carrier fluid is a coating fluid and the step of providing the mixture of carrier fluid and hydrogel microbeads comprises combining the hydrogel beads with the coating fluid.

5. The method of claim 3 wherein the first emulsifying fluid is used as the carrier fluid, e.g. wherein the step of providing the mixture of carrier fluid and hydrogel microbeads comprises forming the hydrogel microbeads from the hydrogel precursor composition in the presence of the first emulsifying fluid.

6. The method of claim 5, said method comprising: providing a hydrogel precursor composition; providing a first emulsifying fluid, wherein the first emulsifying fluid is immiscible with the hydrogel precursor composition and comprises a coating agent; forming a first emulsion, said first emulsion comprising the hydrogel precursor composition as a discontinuous phase and the first emulsifying fluid as a continuous phase; forming hydrogel microbeads from the hydrogel precursor composition in the presence of the first emulsifying fluid; providing a second emulsifying fluid, wherein the second emulsifying fluid is immiscible with the first emulsifying fluid; forming a second emulsion, said second emulsion comprising the hydrogel microbeads and the first emulsifying fluid as a discontinuous phase and the second emulsifying fluid as a continuous phase; and forming a coating on said hydrogel microbeads from the first emulsifying fluid.

7. The method of any preceding wherein the step of forming an emulsion comprises contacting the two phases at an intersection of two microfluidic channels.

8. The method of any preceding claim wherein the coating is a polymer coating.

9. The method of any preceding claim wherein the carrier fluid comprises a lipophilic polymer and a hydrophobic solvent.

10. The method of any preceding claim wherein the hydrogel precursor composition or the hydrogel microbead comprises an encapsulated agent, preferably an encapsulated agent selected from pharmaceutical compounds or compositions, nutritional supplements, flavours, minerals, vitamins, small molecule drugs, proteins, therapeutic viruses, vaccines, therapeutic antibodies, nutritional supplements, carotene, biomarkers, tracer dyes, fluorescent dyes and combinations thereof.

11. A method for forming a coated hydrogel microbead (e.g. a method as claimed in any preceding claim) wherein the hydrogel microbead bead is coated via, and optionally also formed via, microfluidics.

12. A coated hydrogel microbead formed by the method of any preceding claim.

13. A coated hydrogel microbead (e.g. as claimed in claim 12) with a size in the range of 40 micrometres to 1000 micrometres across the largest dimension, and a coefficient of variance of between 2 and 9%, preferably wherein said coated hydrogel microbead has one or more encapsulated agents encapsulated in the hydrogel.

14. A controlled release composition comprising a coated hydrogel microbead according to claim 12 or claim 13.

15. A microfluidic system (e.g. for forming coated hydrogel microbeads by the method of any one of claims 1 to 11), the system comprising: a first microfluidic chip; and a second microfluidic chip; wherein the output of the first chip is connected to the input of the second chip.

Description

[0291] Certain preferred embodiments of the invention will now be described by way of the following non-limiting examples and with reference to the accompanying drawings in which:

[0292] FIGS. 1 and 2 show a hydrogel precursor composition being injected from the left into a first emulsifying fluid and forming droplets, in accordance with the microfluidic method of the present invention and Example 4.

[0293] FIGS. 3 and 4 show hydrogel beads formed in Example 4 using the microfluidic method of the present invention, after gelation, and prior to coating. FIG. 3 shows the bright-field (normal light) image of the beads. FIG. 4 shows the emission fluorescence coming from a dye encapsulated inside the hydrogel beads when excited by a laser.

[0294] FIG. 5 shows injection of a mixture of carrier fluid and hydrogel beads from the left into second emulsifying fluid, and the formation of droplets of carrier fluid containing a hydrogel bead, in accordance with the microfluidic method of the present invention and Example 5.

[0295] FIG. 6 is a schematic of the method of coating hydrogel beads. A carrier/coating fluid as herein described (1) which contains a hydrogel bead as herein described (2) can pass through a microfluidic channel (3). An emulsifying fluid (4) (referred to also as the second emulsifying fluid herein) that is immiscible with the coating fluid (1) can be brought into contact with the coating fluid (1) to form droplets of coating fluid which contain hydrogel beads (5). In one embodiment, as shown, the emulsifying fluid is brought into contact with the coating fluid (1) at a junction of microfluidic channels (3). The droplets of coating fluid which contain the hydrogel beads (5) are then subjected to a coating forming step (6). The step of forming the coating can be achieved by any methods known in the art, such as solvent evaporation, solvent extraction, UV exposure, temperature, a chemical reaction or a combination thereof. When the coating forming step has occurred, a coated hydrogel bead (7) is formed.

[0296] FIG. 7 shows coated hydrogel beads formed in Example 5 after evaporation of solvent from the carrier fluid.

[0297] FIG. 8 shows a coated hydrogel bead formed via Example 5 in accordance with the microfluidic method of the present invention.

[0298] FIGS. 7 and 8 show the emission fluorescence from the dye encapsulated inside the hydrogel beads cores when excited by laser. These images are overlays of the bright-field to show the coating (grey layer around the fluorescent cores) and the core (lighter fluorescent signal due to dextran RhodaminB trapped inside)

EXAMPLE 1—METHOD OF FORMING A HYDROGEL BEAD

[0299] A hydrogel precursor solution is prepared by mixing 8-arm PEG norbornene (example concentration: 20% w/v of solution), LAP (example concentration: 5 mM) and dithiol linker (e.g. dithiothreitol) in equimolar quantities with the PEG norbornene in an aqueous solvent (example: deionized water). An encapsulated agent can be added at this step.

[0300] This solution (hydrogel precursor composition) is then brought into contact with HFE with surfactant as emulsifying fluid, using a PDMS (polydimethylsiloxane, a silicon material) hydrophobic microfluidic chip with a flow focusing design (example size: nozzle of 30 μm width 50 μm height), to form a droplet of hydrogel precursor composition around 40 μm diameter size. The droplets are collected out of the chip in a larger volume of HFE in a vial and crosslink by exposing the vial and droplets to a UV source (365 nm wavelength, 3 min, placed at 10 cm of the vial). Hydrogel beads are formed.

EXAMPLE 2—METHOD OF MAKING A COATED HYDROGEL BEAD (GEL COATING)

[0301] Hydrogel beads previously formed are re-suspended in the suitable coating fluid, for example another hydrogel mixture made of 8-arm PEG acrylate and 8-arm PEG thiol in equimolar concentrations of acrylate and thiol. This mixture is reinjected in a hydrophobic PDMS microfluidic chip with a specific design to pack the hydrogel beads and a flow focusing design to form the droplet of coating fluid loaded with one hydrogel bead (flow focusing nozzle geometry superior to the hydrogel bead size, example: 50 μm width, 60 μm depth). HFE with surfactant is used as the (second) emulsifying fluid and flow rates are adapted to achieve the desired diameter of coating material. The droplets containing hydrogel beads are collected out of the chip in a vial and let to crosslink overnight (Michael addition between acrylate and thiol) to form core shell hydrogel structure with the active agent contained in the core geometry.

EXAMPLE 3—METHOD OF MAKING A COATED HYDROGEL BEAD (POLYMER COATING)

[0302] Hydrogel beads previously formed are re-suspended in a solution of PLGA (example 7-17 kDa molecular weight, 50:50 lactic:glycolic ratio) in DMC (dimethyl carbonate, a solvent) as the coating fluid. The mixture is reinjected in a hydrophilic microfluidic chip with a specific design to pack the hydrogel beads and a flow focusing design to form the droplet of coating fluid loaded with one hydrogel bead (flow focusing nozzle geometry superior to the hydrogel bead size, example: 50 μm width, 60 μm depth). Water with PVA (polyvinyl alcohol, a surfactant) is used as the (second) emulsifying agent and flow rates are adapted to achieve the desired diameter of coating material. The droplets containing hydrogel beads are collected out of the chip in a vial with large volume of water and DMC is extracted overnight by low agitation in the large volume of water. Hydrogel core-PLGA shell particles are obtained with the active agent contained in the core.

EXAMPLE 4—METHOD OF FORMING A HYDROGEL BEAD

[0303] Materials: [0304] Inner Phase (encapsulated agent): Dextran-RhodaminB (3.2 mg/mL in Pre-gel material) [0305] Pregel Material: 5% w/v PEG-Norbornene 8-arms (20 mM [NB] groups), 40 mM DTT (80 mM [SH] group) 1% w/v LAP initiator [0306] Outer Phase (emulsifying fluid 1): 10% w/v PLGA R502 (7-17 k Mw, 50;50, ester cap) in DMC (dimethylcarbonate) [0307] Microfluidic chip: Flow focusing, PDMS, 50 μm*25 μm nozzle

[0308] Method:

[0309] A hydrogel precursor composition was prepared by mixing the pre-gel material with the inner phase (encapsulated agent). The precursor composition was then brought into contact with the outer phase (emulsifying fluid 1, PLGA+DMC), in the microfluidic chip mentioned above. FIG. 1 shows droplets were formed at a throughput of ˜300 droplets per second.

[0310] The droplets were collected from the microfluidic chip into a vial/container while still in suspension with the emulsifying fluid (i.e. inside an Eppendorf in the PLGA+DMC phase) and then cross-linked to polymerise the hydrogel by exposure to 365 nm UV, for 3 minutes at maximum intensity to form hydrogel beads. The beads formed are shown in FIGS. 3 and 4. FIG. 4 shows the emission fluorescence coming from the dye encapsulated inside the hydrogel beads when excited by a laser.

EXAMPLE 5—METHOD OF COATING A HYDROGEL BEAD (REINJECTION/SECOND EMULSIFICATION)

[0311] Materials: [0312] Reinjection Phase (mixture of beads and carrier fluid): Hydrogel beads in 10% PLGA in DMC (from Example 4 first emulsification) [0313] Outer Phase (emulsifying fluid 2): 3% w/v PVA (9-10 k Mw 80% h) in DPBS [0314] Microfluidic chip: Flow focusing, PDMS, 100 μm*80 μm nozzle

[0315] Method:

[0316] The mixture of hydrogel beads in 10% PLGA in DMC formed in Example 4 was brought into contact with the outer phase (emulsifying fluid 2), in the microfluidic chip mentioned above. In this case, the 10% PLGA in DMC which functioned as an emulsifying fluid in the bead formation method of Example 4 is now being employed as the carrier fluid for the coating step. Droplets of carrier fluid were formed around beads as shown in FIGS. 5 and 6. After extracting the solvent (DMC) from the carrier fluid, a PGLA coating was formed around the hydrogel bead. Thus, core-shell particles were formed from hydrogel core template, as shown in FIGS. 7 and 8. FIGS. 7 and 8 show the emission fluorescence from the dye encapsulated inside the hydrogel beads cores when excited by laser. These images are overlays of the brightfield to show the coating (grey layer around the fluorescent cores) and the core (lighter fluorescent signal due to dextran RhodaminB trapped inside).

[0317] Examples 4 and 5 demonstrate that, by selecting the same (or similar) composition for the emulsifying fluid in the bead formation step (i.e. emulsifying fluid 1) as is to be used as the coating fluid in the coating step, it is not necessary to collect and wash the beads following polymerisation/gelation. The beads may be formed from droplets while still in the first emulsifying fluid, and the resultant mixture of first emulsifying fluid and beads can be injected unto the second emulsifying fluid to form droplets of first emulsifying fluid (now functioning as coating fluid) which contains beads.