WO2021161064A1

WO2021161064A1 - Core/shell microcapsules and a method for the fabrication of core/shell microcapsules

Info

Publication number: WO2021161064A1
Application number: PCT/IB2020/051059
Authority: WO
Inventors: Izabela Osica; Wojciech ŚWIĘSZKOWSKI; Emilia Choińska; Leszek Stobiński
Original assignee: Politechnika Warszawska
Priority date: 2020-02-11
Filing date: 2020-02-11
Publication date: 2021-08-19

Abstract

Core/shell microcapsules according to the invention are characterised in that they consist of a polyester porous core comprising a biologically active substance and hydrogel shell encompassing said core prepared from an aqueous solution of alginate at a concentration of 0.01% (w/v) to 30.0% (w/v), with the addition of graphene oxide in the amount of 0.01% (w/v) to 10.0% (w/v) in relation to the alginate solution volume. Polyester porous core is made from an emulsion obtained from an aqueous solution of a biologically active substance and a polyester solution prepared in an organic solvent. Polyester is selected from the group consisting of: lactic acid polymers, glycolic acid polymers, ε-caproic acid polymers and their copolymers. The solvent for the biologically active substance comprises a physiological saline solution or biologically acceptable buffer, and the ratio of organic phase to aqueous phase is from 1: 1 to 20: 1.

Description

Core/shell microcapsules and a method for the fabrication of core/shell microcapsules

The present invention relates to core/shell microcapsules based on polyesters and polysaccharides, with the addition of graphene oxide flakes, as bioactive substances delivery systems, with the ability to modulate the release rate and release on demand.

Tissue engineering is an intensively developing field of technical sciences, which main goal is to produce functional substitutes for damaged tissues or entire organs. The proposed tissue engineering solutions that combine the knowledge of bioengineering and materials science meet the problems of modem regenerative medicine, such as: (i) lack of donors for tissue transplants, (ii) patient burden associated with autogenous transplants, (iii) complications associated with allogeneic or xenogeneic transplants, (iv) lack of artificial prostheses integration into the patient's body.

Tissue engineering is based on the manipulation of live cells placed on artificial scaffolds made of novel biomaterials and providing a support for the forming spatial structure. The proper development of the tissue formed by this method requires the use of specific environmental stimuli, i.e. bioactive signalling molecules, including growth factors and hormones.

Biologically active factors, necessary to ensure the proper course of the cell differentiation process and subsequent their fusion with the host tissue, delivered directly are also associated with the risk of complications. Many currently used bioactive agents are characterized by pleiotropy, i.e. multidirectional mode of action. Therefore, a direct, uncontrolled administration of these factors is associated with a high risk of adverse effects. For example, in the case of BMP-2 factor, which is involved in regulation of the growth and differentiation of osteoblasts and chondroblasts, its uncontrolled diffusion into nearby tissues can cause undesired ectopic bone formation, native bone resorption and soft tissue edema.

Therefore, development of methods in which signalling molecules will be delivered at the proper time and concentration and protected against degradation, is one of the most important research directions in the field of regenerative medicine. Nowadays various delivery systems of bioactive factors, mainly based on polymeric matrices, are being developed (micro-, nanocapsules and spheres, micro- and nanofibers, hydrogels, scaffolds, lipid nanospheres, and combined systems), where the incorporation occurs in physical (adsorption, encapsulation) or chemical manner (with the formation of chemical bonds between structures and the matrix). To date, the best strategy for clinical application is enclosing the active factors within microspheres (encapsulation), especially using the nano- and micro-encapsulation technique, often integrated with bioscaffolds.

Microencapsulation is a process of coating or closing a material or mixture of materials within a shell (a membrane), which is a specific material or system. Typically, these materials differ in physicochemical properties, therefore the microencapsulation results in a composite product - microcapsules, those are composed of a core and shell with different material characteristics. There are many microencapsulation methods that can be divided into two main groups: chemical processes (e.g. coacervation, interfacial or in situ polymerization) and mechanical or physical processes (e.g. spray drying, microfluidic encapsulation, centrifugal extrusion, nozzle vibration technology). One of the most promising methods is a commercially available technology combining coaxial extrusion with nozzle vibration and electrostatic scattering. Devices available on the market have the ability to produce microcapsules in sterile conditions and on a semi-industrial scale.

The biomaterials most commonly used in encapsulation are natural and synthetic resorbable polymer materials, among which two groups should be distinguished: polysaccharides, including alginate, chitosan; and aliphatic polyesters such as polylactide (PLA), polyglycolide (PGA), polylactide-glycolide copolymer (PLGA). Standard approach uses various types of combinations of these polymers in order to extend the release time of a given factor.

Novel approach in the field of delivery systems for bioactive substances aims to achieve sustained release, as well as to take control over the release of the factor encapsulated in the microcapsules. With the exclusive use of the above polymers, this control is limited, and therefore the effect of various types of additives is widely studied to achieve so-called on- demand release. This release can occur under the influence of various external stimuli. Depending on the additives used, most often nanoparticles, different release stimuli are used, e.g. magnetic field when using magnetic nanoparticles, electrical stimuli for electroreactive particles, and light (mainly in the infrared range) for particles with specific optical properties. This last of the above described activation techniques shows the least invasiveness and is the most widely studied method. Furthermore, laser methods are already widely used in medical practice, e.g. for dermatology.

Near-infrared (IR) lasers with a wavelength (750 - 1200 nm) possess a greater tissue penetration depth than lasers operating in the visible or ultraviolet range. Therefore, IR lasers are a good choice as a stimulus for the release of factors from microcapsules previously applied to a target site requiring deep penetration (e.g. under the subcutaneous tissue). Release by means of an IR laser is based on a photothermal effect, which refers to the phenomenon of absorption of electromagnetic radiation by a selected material, which is excited, resulting in thermal energy (heat) release. Many release systems that utilize various phenomena occurring in the matrix of material under locally-increased temperature are being developed; for example it can be a change in the conformation of polymer chains in the so-called "polymer brushes". The release mechanism used in the present invention is based on the following effect: nanoparticles with specific photosensitive properties introduced into the polymer matrix by light radiation absorption lead to a local heating of material, which in turn accelerates its degradation and causes faster release of bioactive agents.

Scientists investigate various nanoparticles that strongly absorb light for their use in polymer microcapsules for release via IR. Most promising and widely studied particles include metal nanoparticles (mainly gold), carbon nanotubes and graphene. Due to their composition and structure, carbon nanomaterials show the highest biological compatibility and interaction harmony with living matter. Therefore, both carbon nanotubes and graphene prevail in the latest scientific reports in the field of nanomedicine. Although often used graphene possesses a potential to be applied in the present invention due to its unique light absorption capacity, it also has drawbacks. One of them is graphene hydrophobic nature, which causes its poor dispersion in most of the fluids that are friendly to the biological environment. This feature significantly limits the medical use of graphene, due to its easy agglomeration in body fluids, e.g. serum or saline. However, there is another graphene form that is more friendly to living organisms, i.e. graphene oxide (GO). GO exhibits properties very similar to graphene, while it has a hydrophilic character, thus it is much more biocompatible and exhibits less toxicity than graphene. Therefore, GO provides broad possibilities for the design of biocompatible nanocoposite materials with interesting properties. Over the past five years, several papers relating to microcapsules with the addition of graphene oxide were published. The microcapsules described in two different publications, which basically represent the same idea (Luo, Q., et al. (2016). Carbon, 106, 125-131; Dong, L., et al. (2017). Ultrasonics sonochemistry, 36, 437-445.), are prepared in the emulsification process (oil-water), using GO water dispersion and agents dissolved in organic liquid. However, those microcapsules preclude the encapsulation of hydrophilic substances. This problem was somehow solved by using another fabrication method - spray drying (Li, W., et al. (2014). Chemical Communications, 50 (100), 15867-15869.), where the active agent is directly placed in the GO water dispersion, which, however, causes the ingredient to be found in the microcapsule shell, not inside. The use of inorganic spherical particles as templates for the resulting GO-containing coating was another proposed solution (Kurapati, R., et al. (2013). Chemical Communications, 49 (7), 734-736.). However, the proposed system allowed encapsulation of positively charged factors only. Another work relates to microcapsules obtained using the layer-by-layer (LbL) technique on melamine resin template spheres (Deng, L., et al. (2016). ACS applied materials & interfaces, 8 (11), 6859-6868.). Unfortunately, this is a time-consuming method, moreover, it completely eliminates the possibility of active agent encapsulation during the production process, which further extends the process of preparing ready-for-use microcapsules. In addition, all of the above described microcapsules consist only of a thin shell (GO and a shell integrating additive, e.g. a cross-linking small molecule agent, so-called linker), they are hollow, so they do not retain their spherical structure either at the stage of removing the templates or later during the drying process. The "collapse" phenomenon of microcapsules is obviously undesirable, as it can negatively affect the release process, its lesser predictability, and also hinder subsequent introduction into the scaffolds.

A good solution is the use of microfluidic systems that allow obtaining monodisperse, mechanically strong microcapsules with thicker shell walls (Kaufman, G., et al. (2017). ACS applied materials & interfaces, 9 (50), 44192-44198.). This system allows encapsulation of hydrophilic factors. The shell is cross-linked by an interfacial reaction of epoxides applied to GO flakes and macromolecular silicone fluid functionalized with amine groups. GO flakes form a chemically cross-linked film on the surface of water droplets in oil generated by the microfluidic device (w/o/w emulsion). In another microfluidic-based manufacturing system, the presented microcapsules have a double GO film, located inside and outside of hydrogel shell (o/w/o emulsion) (Byun, A., et al. (2015). Chemical Communications, 51 (64), 12756- 12759.). In this system, shell is a hydrogel based on MPC (2-ethylphosphorylcholine methacrylate) cross-linked with ultraviolet light using an addition of photoinitiator (Darocur 1173). In both systems, GO flakes accumulate between the aqueous and oily phase, because GO acts as a stabilizer in Pickering emulsions. In this case, modification of the microcapsule shell permeability is not entirely possible without adversely affecting the mechanical stability of the microcapsules. Moreover, microfluidic methods can only be used on a laboratory scale due to their low efficiency.

International patent application WO2016199167 A4 discloses in a wide range of claims core/shell microcapsule compositions modified with nanomaterials for active agent- controlled release, including the use of graphene oxide as the nanomaterial in the composition and drugs as the released active agents. However, the composition described in this patent application is only fulfilled when applied to water-insoluble active agents, which significantly limits its use. Many biologically active agents are soluble only in aqueous solutions. Moreover, described method of producing microcapsules allows the use of only hydrophobic polymers soluble in organic liquids, including polyurea, polyurethane, polyesters and polyamides.

Three of the works discussed above, present the results for factors released by means of infrared radiation, demonstrating the effectiveness of this method for given compositions with graphene oxide. IR lasers emitting 808 and 1064 nm wavelengths were used in these studies.

There are no examples in the literature of core/shell microcapsules with the addition of GO that could represent a solution to the problem of the microcapsule structure stability. This problem was solved by the present invention.

The present invention relates to the use of graphene oxide flakes dispersed in a hydrogel applied to a shell of microcapsules used for delivery of active agents. Active agents are incorporated within a porous core of the microcapsule, wherein said core is obtained on the basis of an emulsion containing an aqueous solution of the bioactive agent and a polymer solution prepared in an organic solvent.

Core/shell microcapsules modified with graphene oxide according to the present invention are characterised in that they consist of a polyester porous core comprising a biologically active substance surrounded by hydrogel shell prepared from an aqueous solution of alginate at a concentration of 0.01% (w/v) to 30.0% (w/v), with the addition of graphene oxide in the amount of 0.01% (w/v) to 10.0% (w/v) in relation to the alginate solution volume. The polyester porous core is made from an emulsion obtained from an aqueous solution of a biologically active substance and a polyester solution prepared in an organic solvent, and wherein the polyester is selected from the group consisting of: lactic acid polymers (PLA), glycolic acid polymers (PGA), e-caproic acid polymers (PCL) and their copolymers, the solvent for the biologically active substance comprises a physiological saline solution or biologically acceptable buffer, and the ratio of organic phase to aqueous phase is from 1: 1 to 20: 1.

Preferably, the solvent for polyester is a hydrophobic organic solvent, including: toluene, ethylene acetate, trichloroethylene, diisopropyl ether, pentane, methyl ethyl ketone, methyl-t-butyl ether, hexane, heptane, diethyl ether, dichloromethane, 1,2-dichloroethane, cyclohexane, chloroform, carbon tetrachloride, butyl acetate, n-butanol, benzene.

Preferably, the core is made of a polyester solution with the addition a (w/o) emulsion stabilizing surfactant. Preferably said surfactant is a non-ionic surfactant at a concentration of 0.01% to 30.0%. Preferably, the non-ionic surfactant is non-ionic sorbitan monooleate.

Preferably, alginate is sodium alginate.

Preferably, the buffering solution is selected from the group comprising: citric acid/phosphate buffer, saline solution (PBS), acetate buffer, barbital buffer, borate buffer, Britton-Robinson buffer, cacodylate buffer, citrate buffer, collidine buffer, formate buffer, maleate buffer, Mcllvaine buffer, phosphate buffer, Prideaux-Ward buffer, succinate buffer, citrate-phosphate-borate (Teorella-Stanhagen) buffer, MES (2-(N15morpholino) ethanesulfonic acid), BIS-TRIS (bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane), ADA (N-(2-Acetamido)iminodiacetic acid), ACES (N-(carbamoylmethyl)-2- aminoethanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), MOPSO (3- (N-morpholino)-2-hydroxypropanesulfonic acid), BIS-TRIS PROPANE (l,3-bis(tris (hydroxymethyl)methylamino)propane), BES (N,N-bis(2-hydroxyethyl)-20 2- aminoethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), TES (N-tris (hydroxymethyl)methyl-2-aminoethanesulfonic acid), HEPES (N-(2-hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid), DIPSO (3-(N,N-bis(2-hydroxyethyl)amino)-2- hydroxypropanesulfonic acid), MOBS (4-(N-morpholine)butanesulphonic acid), TAPSO (3- (N-tris(hydroxymethyl)methylamino)-2-hydroxy-propanesulfonic acid), 25 tris (hydroxymethylaminomethane), HEPPSO (N-(2-hydroxyethyl)piperazine-N'-(2- hydroxypropane sulfonic acid), POPSO (piperazine-N,N'-bis(2-hydroxypropanesulfonic acid), TEA (triethanolamine), EPPS (N-(2-hydroxyethyl)piperazine-N'-(3-propanesulfonic acid), TRICINE (N-tris(hydroxymethyl)methylglycine), GLY-GLY (glycylglycine), BICINE (N,N- bis(2-hydroxyethyl)glycine), HEPBS (N-(2-hydroxyethyl-30-yl)piperazine-N'-(4- butanesulfonic acid)), TAPS (N-tris acid (hydroxymethyl) methyl-3-aminopropanesulfonic or AMPD (2-amino-2-methyl- 1 ,3-propanediol).

Preferably, the aqueous solution of the active substance comprises bovine serum albumin, human serum albumin, lysozyme or ovalbumin as an additional stabilizing factor.

Preferably, the size of microcapsules ranges from 1 to 1000 pm.

Microcapsules according to the present invention can be used for delivery of any substance intended for biomedical use. Preferably, the active substance is selected from: growth factors, antibiotics, anticancer agents, anti-inflammatory agents, hormones, enzymes, vitamins, cytokines. Particularly preferably, the active substance is: bone morphogenetic proteins, hepatocyte growth factors, keratinocyte growth factor, oncostatin M, dexamethasone, supplements for neuronal cell culture, basic fibroblast growth factors, vascular endothelial growth factors, nicotinamide, interleukin 6.

A method of producing core/shell microcapsules modified with graphene oxide, with coaxial extrusion technology using nozzle vibration technology and electrostatic dispersion of formed droplets according to the present invention is characterised in that an aqueous alginate solution at a concentration of 0.01% (w/v) to 30.0% (w/v) with the addition of graphene oxide in the amount of 0.01% (w/v) to 10.0% (w/v) in relation to the volume of alginate solution, and an emulsion from an aqueous solution of a biologically active substance and a solution of polyester in an organic solvent are prepared, wherein the polyester is selected from the group consisting of: lactic acid polymers, glycolic acid polymers, e-caproic acid polymers and their copolymers, and the solvent of the biologically active substance is a saline solution or a biologically acceptable buffer, and the ratio of organic phase to aqueous phase is from 1:1 to 20:1, preferably 10:1. Afterwards microcapsules are formed from said aqueous solution and said emulsion, next microcapsules are crosslinked in an aqueous solution of calcium chloride or strontium chloride at a concentration of 1 mM to 1 M (preferably 100 mM) for the period in the range of 0.1 hours to 24 hours (preferably 3 hours). Afterwards microcapsules are rinsed with water, frozen and freeze-dried. Preferably, the solvent for polyester is a hydrophobic organic solvent, including: toluene, ethylene acetate, trichloroethylene, diisopropyl ether, pentane, methyl ethyl ketone, methyl-t-butyl ether, hexane, heptane, diethyl ether, dichloromethane, 1,2-dichloroethane, cyclohexane, chloroform, carbon tetrachloride, butyl acetate, n-butanol, benzene.

Preferably, the core is made of a polyester solution with the addition a (w/o) emulsion stabilizing surfactant. Preferably said surfactant is a non-ionic surfactant at a concentration of 0.01% to 30.0%. Preferably the non-ionic surfactant is non-ionic sorbitan monooleate.

Preferably, the crosslinking solution comprises a surfactant in an amount of 0.01% to 10.0%, which maintains the spherical shape of the structure of crosslinked microcapsules. Polyethylene glycol octylphenyl ethers are preferably used.

Preferably, an aqueous solution of the biologically active substance comprises an addition of bovine serum albumin, human serum albumin, lysozyme or ovalbumin.

Any substance intended for biomedical application can be used as the bioactive substance in the microcapsules according to the present invention.

Preferably, the active substance is selected from: growth factors, antibiotics, anti cancer agents, anti-inflammatory agents, hormones, enzymes, drugs, vitamins, cytokines. Particularly preferably, the active substance is: bone morphogenetic proteins, hepatocyte growth factors, keratinocyte growth factor, oncostatin M, dexamethasone, supplements for neuronal cell culture, basic fibroblast growth factors, vascular endothelial growth factors, nicotinamide, interleukin 6.

An aqueous solution of sodium alginate with graphene oxide as well as an emulsion of the active substance and polyester are extruded by means of syringe pumps to the vibrating head and then into concentrically arranged nozzles, wherein the emulsion is directed to the inner nozzle and the aqueous solution is directed to the outer nozzle. During the coaxial extrusion process, a formation of a droplet chain (and later microcapsules) from a continuous stream occurs by dividing the stream by vibration. Next, the droplet chain is dissipated by electrostatic forces acting in the electrostatic field generated by the built-in electrode located at the nozzles outlet. Electrostatic interactions cause a dispersion of the created droplets, preventing their coalescence. The vibration frequency as well as the voltage at the electrode are set depending on the liquids used and the diameter of the nozzles. Microcapsules according to present invention are preferably produced using nozzles with a diameter of 80 mih and 200 mih for the inner and outer nozzles respectively. Microcapsules according to the present invention are prepared using the flow rates ranging from 0.1 to 20.0 ml/min for the external liquid and the flow rates range from 0.05 to 10.0 ml/min for the internal liquid.

In order to form an emulsion directly used as the core liquid, the organic and aqueous phases are homogenized by ultrasounds. The polymer solution of a given concentration, preferably freshly prepared, is placed in an ice bath (in order to prevent temperature degradation of the active substance during the homogenization step), then a homogenizer tip is placed in the solution, and the active substance solution is dropped after the activation of the ultrasounds. The emulsion creation process is very fast and relatively safe for the encapsulated active substance, with appropriately selected parameters such as amplitude, short work time of homogenizer, and pulse working mode.

PBS -based emulsion produced in this way is characterized by good stability (20 hours at a laboratory temperature of about 22 °C), whereas a lower stability is provided when using HEPES buffer (3 hours at a temperature of about 22 °C). The emulsion stability strongly depends on the active agent used and the type of buffering solution. Microcapsules according to the present invention are preferably produced immediately after preparation of the core emulsion.

A suspension of graphene oxide in an alginate solution, for example NaAlg, can be obtained by two methods. The first of these includes a preparation of an aqueous NaAlg solution as a first step, which is followed by the addition of an appropriate amount of GO water suspension in order to achieve an appropriate, desired final concentration of GO flakes. The second method includes a preparation of a specific final concentration of aqueous GO suspension as a first step, followed by the addition of an appropriate amount of solid NaAlg in order to achieve the specified NaAlg concentration in the solution. The first method uses an ultrasonic homogenizer of a certain amplitude operated for a short time in pulse mode to provide a dispersion of GO flakes. In the second method, a GO dispersion is obtained during rapid mixing of ingredients on a magnetic stirrer for a long period of time (up to 12 hours). Both methods allow to obtain a satisfactory GO dispersion in NaAlg solution. Uniform dispersion of GO flakes has been confirmed in imaging studies of microcapsule surfaces prior to the drying process using atomic force microscope (AFM). The obtained suspensions in the given concentration ranges show very good stability (stable even for several weeks at 4 °C), without the use of additional stabilizers. Microcapsules of the present invention are preferably made from a freshly prepared GO suspension in NaAlg. Both the NaAlg and GO concentrations have an impact on the rheological parameters of the liquid used for shell formation, which is significant for the microcapsule production process. Lower or higher concentrations impede or preclude carrying out the microcapsule production process.

Microcapsules according to the invention, obtained in the method according to the invention, have a polymeric core with a porous structure, containing a bioactive substance, and a polysaccharide shell (membrane) based on an aqueous sodium alginate solution with the addition of graphene oxide flakes. Thanks to the addition of graphene oxide flakes, the microcapsules according to the invention allow release on demand and modulation of the active substance release rate by changing the permeability of the shell. Infrared radiation emitted by the laser with a wavelength of 1064 nm is used as a stimulus. Deposition of the active substance in the polymer porous core ensures the stability of the encapsulated factor.

Microcapsules according to the inventions comprise resorbable biopolymers, including hydrogels, while said microcapsules can be used to deliver active agents that are soluble in aqueous solutions. However, the invention can also be applied for the delivery of water- insoluble agents that can be directly added to the polymer solution in the organic solvent.

Microcapsules are produced using a commercially available device - an encapsulator, which is based on coaxial extrusion using nozzle vibration technology and electrostatic scattering of formed droplets.

The present invention ensures a satisfying use of practically any agent, and is oriented primarily to bioactive agents such as antibiotics, growth factors, anti-cancer agents, and anti inflammatory agents that require prolonged or stimulated release.

Microcapsules according to the present invention can be applied directly to the transplant site as a backfill, by post-surgery injection, or introduced into the hydrogel scaffold.

The invention has been verified by in vitro studies using a bovine serum albumin, BSA, CAS: 9048-46-8, which is a model encapsulated factor often used in this type of research. Also, the on-demand release tests were carried out using an infrared radiation laser with a wavelength of 1064 nm. The following are two embodiments of the invention that present model combinations of the composition of the ingredients used to make the microcapsules and the selected manufacturing parameters.

The following examples, which include preferred embodiments of the microcapsule compositions and the selected manufacturing parameters.

Fig. 1 shows microcapsules obtained according to the composition and method of production given in Example 1, i.e. PLGA / CaAlg (core / shell) with the addition of 0.05% (w/v) GO flakes in the membrane, with the model active agent BSA in the HEPES solution.

Fig. 2 shows microcapsules obtained according to the composition and method of production given in Example 2, i.e. PLGA / SrAlg (core / shell) with the addition of 0.25% (w/v) GO flakes in the membrane, with the model active agent BSA in PBS solution.

Fig. 3 shows microcapsules obtained according to the composition and method of production given in Example 2., PLGA / SrAlg (core / shell) with BSA in PBS solution, modified by different GO flake content in the membrane, (a) 0%; (b) 0.05%; (c) 0.1%; (d) 0.25% (w/v) respectively. Microcapsules macro images are inserted in the upper left corner.

Fig. 4 shows SEM images of microcapsules obtained according to the composition and method of production given in Example 2., PLGA / SrAlg (core / shell) with BSA in PBS solution, modified by different GO flake content in the membrane (a) 0%; (b) 0.05%; (c) 0.1%; (d) 0.25% (w/v) respectively, after the freeze-drying process. Macro images of microcapsules after the lyophilization process are inserted in the upper left comer.

Fig. 5 shows confocal images of microcapsules with fluorescein derivative (FITC)- loaded BSA protein.

Fig. 6 shows the cumulative release curve of BSA protein from microcapsules with different GO content. Release was carried out in a PBS solution at 37 °C to reflect the human body conditions.

Fig. 7 shows the release curves of BSA from microcapsules prepared with 0.25% (w/v) GO addition. BSA release curve from microcapsules incubated at 37°C (grey line) and with the additional use of an IR laser (black line). The laser was used twice during the incubation process. The microcapsules were irradiated for 30 minutes with an infrared laser (1064 nm) with a power density of 1.1 W/cm . Example 1. - microcapsules cross-linked with calcium chloride according to "GO05" composition

Initially, w/o emulsion was formed by adding 1.0 mL of BSA solution in phosphate buffered saline (10 mg/mL) to 10 mL of a 6% (w/v) PLGA solution in dichloromethane. In order to stabilize the emulsion, Span 80 surfactant (4% (w/v)) was added to the PLGA solution. Emulsification was carried out in an ice bath using the ultrasonic homogenizer (Sonicator Sonics Vibra Cell VC-505) operating at 30% amplitude for 90 seconds in a pulse mode set to 5: 3 seconds ("on": "off"). The BSA solution was added dropwise to the PLGA solution in DCM during the first 30 seconds of the homogenization process.

Polymer solution that forms the shell of the microcapsules was obtained by dispersion of graphene oxide flakes in 1.15% (w/v) aqueous sodium alginate solution. The amount of 2.08 mg of 1.2% GO water suspension (w/v) was added to the 47.92 ml of NaAlg solution, followed by homogenization using the ultrasonic homogenizer (Sonicator Sonics Vibra Cell VC-505), wherein the process parameters were as follows: amplitude 30%, time 180 seconds, pulse mode 5: 3 seconds ("on": "off"). As a result, a GO suspension was prepared with a final concentration of 0.05% (w/v) in an aqueous NaAlg solution with final concentration of 1.1% (w/v). Freshly prepared core and shell liquids were used for the manufacturing of microcapsules with using of encapsulator which has fulfilled the abovementioned description (Encapsulator B-390, BUCHI, Switzerland), using internal and external nozzles with 80 and 200 pm diameter respectively, equipped with two syringe pumps. The liquids were placed in appropriate syringes and injected into the vibrating chamber with set, empirically determined flow rates, next, liquids were extruded through coaxial nozzles forming a chain of droplets falling into the calcium chloride solution (100 mM) with the addition of Triton X-100 (0.1% (v/v)), under constant magnetic stirring.

The following optimal parameter values were selected based on the selection of encapsulation parameters for the prepared liquids according to the GO05 "composition a) Internal (core) liquid flow rate: 0.4 ml/min, b) External liquid (shell) flow rate: 4 ml/min, c) Vibration frequency: 1000 Hz, d) Voltage: 1000 V e) Distance of the nozzle from the level of crosslinking solution: 20 cm.

Microcapsules were held in a calcium chloride solution for 3 hours to complete gelation process and evaporation of the organic solvent from the core. Next, the microcapsules were washed several times with demineralized water and immediately frozen in liquid nitrogen (- 196 °C), and finally freeze-dried for 24 hours at the condenser temperature of -70 °C.

Morphology of microcapsules after crosslinking process was observed using optical microscope (Bresser Biolux NV). Fig. 1 shows microcapsules obtained according to the composition and method of production given in Example 1, i.e. PLGA / CaAlg (core / shell) with the addition of 0.05% (w/v) GO flakes in the membrane. BSA in HEPES solution was used as the model active agent. The average size of the microcapsules is 450 pm, wherein the core size is about 100 pm. The 20x magnification shows that the core of the microcapsule does not come into direct contact with the membrane, between the core and membrane there is a space created due to solvent evaporation, which creates a “cavity” with a diameter of 220 pm. In most cases, the core of the microcapsules prepared according to Example 1 is not located in the central position. Probably the core is "pushed" towards the direction of the solvent evaporation. Example 2. - microcapsules cross-linked with strontium chloride according to "G025" composition

The w/o core emulsion was prepared identically as described in Example 1.

The liquid used for the shell of microcapsules was obtained by dispersion of graphene oxide flakes in 1.39% (w/v) NaAlg aqueous solution. An amount of 10.42 mg of 1.2% GO aqueous suspension (w/v) was added to 39.58 ml of NaAlg solution, followed by homogenization using the ultrasonic homogenizer (Sonicator Sonics Vibra Cell VC-505), using following process parameters: amplitude 30%, time 180 seconds, pulse mode 5: 3 seconds ("on": "off"). As a result, a GO suspension was prepared with a final concentration of 0.25% (w / v) in an aqueous NaAlg solution with a final concentration of 1.1% (w / v).

Freshly prepared liquids for the core and shell were used to prepare microcapsules using the encapsulator (Encapsulator B-390, BUCHI, Switzerland) as described in Example 1, with identical process parameters.

Microcapsules were held in strontium chloride solution for 3 hours to complete gelation process and evaporation of the organic solvent from the core. Next, microcapsules were washed several times with demineralized water and immediately frozen in liquid nitrogen (- 196 °C), and finally freeze-dried for 24 hours at a condenser temperature of -70 ⁰ C. Morphology of microcapsules after the crosslinking process was observed using optical microscope (Bresser Biolux NV). Fig. 2 shows microcapsules obtained according to the composition and method of production given in Example 2, i.e. PLGA / SrAlg (core / shell) with the addition of 0.25% (w/v) GO flakes in the membrane. BSA in PBS solution was used as the model active agent. The average microcapsule size is 450 pm, with the approx core size of 80 pm. The 20x magnification shows that the microcapsule core is in contact with the membrane, which is probably due to slower evaporation of the solvent from the inside of the microcapsule due to the lower permeability of the membrane. The core is also centrally located, which can be associated with both slower evaporation of the solvent and increased viscosity of the liquid used on the membrane.

Example 3.

A series of microcapsules based on the composition of Example 2, with a different GO content (0.05; 0.1; 0.25% (w/v)) was prepared to demonstrate the possibility of release rate modulation by changing the GO flake content of the microcapsule membrane. For comparative purposes, microcapsules of identical composition and production parameters, but without the addition of graphene oxide, were prepared.

Release studies of the BSA model protein were conducted in vitro, by placing approximately 2 mg of microcapsules in a sealed tube containing 1 ml of PBS (pH 7.4). Tubes were incubated at 37 °C with horizontal shaking (100 rpm). At various time intervals, 300 pi of PBS was removed and the same amount of fresh buffer was added to maintain the incubation test conditions. The BSA concentration in the collected solution samples was determined using a set of reagents for protein determination (QuantiPro ™ BCA, for a protein in concentration range of 0.5-30 pg/ml), by recording absorbance at 560 nm using a UV-Vis spectrophotometer (FLUOstar Omega). Calculations of BSA concentration at individual time intervals were carried out on the basis of data obtained from the calibration curve for BSA standard samples in PBS. The absorbance for the calibration curve was measured together with tested samples. The BSA release profile from the microcapsules was presented as a cumulative release percentage versus incubation time curve, based on data collected from ten samples for each type of microcapsules.

Morphology of microcapsules before and after the freeze-drying process was observed using an optical microscope (Bresser Biolux NV) and a scanning electron microscope (SEM, Phenom ProX) respectively. Microcapsules according to the present invention obtained according to example 2, are shown in Fig. 3. The increase in GO content is directly observable by changing the colour of the microcapsules, transparent for microcapsules without addition of GO to dark brown for microcapsules with 0.25% (w/v) GO content. As the GO content in the membrane increases, the core of the microcapsules takes a central location. Furthermore, small increase in the microcapsule and core diameters can also be observed. A visible effect of the GO addition for the microcapsules is observed after the freeze-drying process, which allows their spherical structure to be preserved (Fig. 4). This given example shows that a small addition of GO (0.1%) causes a significant improvement in the shell stiffness, thereby stabilizing the structure of the microcapsules.

To illustrate the location of the encapsulated factor, microcapsules with a BSA protein designated fluorescein derivative (FITC) were also prepared. Micro structure images were taken using a confocal microscope (Leica TCS SP8). It is clearly visible that BSA is found in the core of the microcapsules, from where its subsequent release will occur (Fig. 5).

Results of the release assay for an exemplary series of microcapsules according to the invention are summarized in Fig. 6. A reduction of the protein release rate from microcapsules with the addition of GO is clearly visible. During approx. 200 hours, over 20% more BSA is released from microcapsules without the addition of GO than from microcapsules with 0.25% (w/v) GO. The complete release of the encapsulated substance from microcapsules without GO occurs after 300 hours of incubation. During this time, less than 60% BSA is released from microcapsules with 0.25% (w / v) of GO. Graphene-modified graphene oxide microcapsules show clearly reduced permeability. GO flake dispersion creates hard-permeable barriers in the material and extends the path of the substance penetrating the material. By using the right amount of GO flakes, the release rate of a given factor can be adjust

Preliminary laser studies of the BSA protein release were carried out using an Nd:YAG laser operating at an infrared wavelength of 1064 nm. During the incubation at 37 °C, microcapsules were exposed twice to laser light for 30 min with a laser power of 110 mW (laser beam diameter was 0.1 cm ). PBS samples (300 pi) were taken just before the microcapsules were exposed to the laser, 2 and 24 hours after exposure to light, respectively. The appropriate amount of fresh PBS solution was added each time. Fig. 7 shows laser release results, which indicate that "on demand" protein release is possible. A clear increase in BSA concentration in the solution is visible just in two hours after exposure to the laser.

Claims

1. Core/shell microcapsules modified with graphene oxide, characterised in that they consist of a polyester porous core comprising a biologically active substance and hydrogel shell encompassing said core prepared from an aqueous solution of alginate at a concentration of 0.01% (w/v) to 30.0% (w/v), with the addition of graphene oxide in the amount of 0.01% (w/v) to 10.0% (w/v) in relation to the alginate solution volume, whereby the polyester porous core is made from an emulsion obtained from an aqueous solution of a biologically active substance and a polyester solution prepared in an organic solvent, and wherein the polyester is selected from the group consisting of: lactic acid polymers, glycolic acid polymers, e-caproic acid polymers and their copolymers, the solvent for the biologically active substance comprises a physiological saline solution or biologically acceptable buffer, and the ratio of organic phase to aqueous phase is from 1: 1 to 20: 1.

2. Microcapsules according to claim 1, characterised in that the alginate is sodium alginate.

3. Microcapsules according to claim 1, characterised in that the solvent for polyester is preferably a hydrophobic organic solvent.

4. Microcapsules according to claim 1 or 3, characterised in that the solvent for polyester is: toluene, ethylene acetate, trichloroethylene, diisopropyl ether, pentane, methyl ethyl ketone, methyl-t-butyl ether, hexane, heptane, diethyl ether, dichloromethane, 1,2-dichloroethane, cyclohexane, chloroform, carbon tetrachloride, butyl acetate, n- butanol, benzene.

5. Microcapsules according to claim 1, characterised in that the core is made of a polyester solution with the addition of a non-ionic surfactant at a concentration of 0.01% to 30.0%.

6. Microcapsules according to claim 5, characterised in that the non-ionic surfactant is non-ionic sorbitan monooleate.

7. Microcapsules according to claim 1, characterised in that the aqueous solution of the active substance comprises bovine serum albumin, human serum albumin, lysozyme or ovalbumin as an additional stabilizing factor.

8. Microcapsules according to claim 1, characterised in that their size is in the range from 1 to 1000 pm.

9. Microcapsules according to claim 1, characterised in that the biologically active substance is selected from: growth factors, antibiotics, anticancer agents, anti inflammatory agents, hormones, enzymes, drugs, vitamins, cytokines.

10. A method of producing core/shell microcapsules modified with graphene oxide, with coaxial extrusion technology using nozzle vibration technology and electrostatic dispersion of formed droplets, characterised in that an aqueous alginate solution at a concentration of 0.01% (w/v) to 30.0% (w/v) with the addition of graphene oxide in the amount of 0.01% (w / v) to 10.0% (w / v) in relation to the volume of alginate solution, and an emulsion from an aqueous solution of a biologically active substance and a solution of polyester in an organic solvent are prepared, wherein the polyester is selected from the group consisting of: lactic acid polymers, glycolic acid polymers, e- caproic acid polymers and their copolymers, and the solvent of the biologically active substance is a saline solution or a biologically acceptable buffer, and the ratio of organic phase to aqueous phase is from 1: 1 to 20: 1, afterwards microcapsules are formed from said aqueous solution and said emulsion, next microcapsules are crosslinked in an aqueous solution of calcium chloride or strontium chloride at a concentration of 1 mM to 1 M for the period in the range of 0.1 hours to 24 hours, afterwards microcapsules are rinsed with water, frozen and lyophilized.

11. The method according to claim 10, characterised in that the alginate is sodium alginate.

12. The method according to claim 10, characterised in that the solvent for polyester is a hydrophobic organic solvent.

13. The method according to claim 10 or 11, characterised in that the solvent for polyester is: toluene, ethylene acetate, trichloroethylene, diisopropyl ether, pentane, methyl ethyl ketone, methyl-t-butyl ether, hexane, heptane, diethyl ether, dichloromethane, 1,2-dichloroethane, cyclohexane, chloroform, carbon tetrachloride, butyl acetate, n- butanol, benzene.

14. The method according to claim 10, characterised in that the core is made of a polyester solution with the addition of a nonionic surfactant at a concentration of 0.01% to 30.0%.

15. The method according to claim 14, characterised in that the surfactant is nonionic sorbitan monooleate.

16. The method according to claim 10, characterised in that the aqueous solution of the biologically active substance comprises an addition of bovine serum albumin, human serum albumin, lysozyme or ovalbumin.

17. The method according to claim 10, characterised in that the biologically active substance is selected from: growth factors, antibiotics, anticancer agents, anti inflammatory agents, hormones, enzymes, drugs, vitamins, cytokines.

18. The method according to claim 10, characterised in that the surfactant is added in an amount of 0.01% to 10.0%.

19. The method according to claim 18, characterised in that the surfactant is polyethylene glycol octylphenyl ethers.

20. The method according to claim 10, characterised in that the freezing of the microcapsules is carried out in liquid nitrogen and the lyophilization is carried out for at least 24 hours.