PL215011B1 - Method for obtaining monodisperse pectin microgels using micro-flow system - Google Patents

Abstract

The method involves forming emulsion, and diffusing cross-linking ions in a continuous phase, where the continuous phase comprises oil, metal salt and acid. Nanoparticles are optionally contained in an aqueous phase i.e. dissipated pectin solution.

Description

The subject of the invention is a method for the preparation of monodisperse pectin microgels with the use of a microfluidic system. More specifically, the method of the invention enables the formulation of structure-controlled pectin microgels using microfluidic techniques to encapsulate nanoparticles.

Microfluidic techniques are a universal research tool used in science, in particular in biology, chemistry, medicine and pharmacy. They allow for operations on very small volumes of fluids (even picoliters), as well as for the production of monodisperse microparticles and microcapsules of controlled size and morphology.

Microcapsules are particles with a diameter of 1 to 1000 μm. They are carriers of microscopic amounts of substances, e.g. dyes or biologically active preparations. They consist of a nucleus and a thin shell. Their main application is the controlled release of medicinal substances at a specific place and time. The most important property of microcapsules is their small size that allows to build a huge surface on which the release of the encapsulated substance takes place.

Microcapsules are used in many industries [Thies, C. in Encyclopedia of Polymer Science and Technology (ed H. F. Mark) (John Wiley & Sons, Inc., 2005)]: pharmaceutical, food, cosmetic, paper, perfumery and others. Various formulations are encapsulated to:

i) improve their stability and durability; ii) improve their water solubility; iii) increase bioavailability; iv) mask the taste and / or smell; v) reduce their volatility; vi) be able to release them at a specific place and time; vii) extend their release process.

In the food industry, encapsulation is primarily aimed at masking the taste and smell, reducing hygroscopicity, protecting against oxidation and separating the ingredients of the product so that they do not react with each other. It is essential that the capsules are made of a non-toxic substance, preferably food-grade. In turn, the agricultural industry, the so-called agrochemistry, encapsulates, for example, plant protection products or pheromones. In this way, the volatility of the mixtures used, the toxicity to the environment and the amount used are limited. On the other hand, in the cosmetics industry, capsules are used in the production of creams, powders, hygiene products and deodorants (especially antiperspirants). Microcapsules are also widely used in biochemistry, medicine and pharmacy. They enclose microorganisms (e.g. yeast to accelerate fermentation processes [Bonin, S. Agro Przemysł 6, 20-23 (2008)]), cells (e.g. for transplantation purposes [Schmidt, JJ, Rowley, J. & Kong , HJ Journal of Biomedical Materials Research Part A 87A, 1113-1122 (2008)] to suppress the immune response of the recipient organism), active substances (e.g. drug encapsulation studies [Xu, Q., Hahsimoto, M., Dang, TT , Hoare, T., Kohane, DS, Whitesides, GM, Langer, R., Anderson, DG Small 5, 1575-1581 (2009); Yu, C.-Y., Cao, H., Zhang, X.- C., Zhou, F.-Z., Cheng,

S.-X., Zhang, X.-Z., and Zhuo, R.-X. Langmuir 25, 11720-11726 (2009)], enzymes [Um, E., Lee,

D.-S., Pyo, H.-B. & Park, J.-K. Microfluidics and Nanofluidics 5, 541-549 (2008)], hemoglobin [Schmidt, JJ, Rowley, J. & Kong, HJ Journal of Biomedical Materials Research Part A 87A, 1113-1122 (2008)] or other blood proteins [Liu, Z., Jiao, Y., Wang, Y., Zhou, C. & Zhang, Z. Advanced Drug Delivery Reviews 60, 1650-1662 (2008)]). In contrast, microcapsules with a gas core are used in the technique of medical ultrasound imaging as a contrast [Sirsi, S. R., Borden, M. A. Bubble Science, Engineering & Technology 1, 3-17 (2009)].

In most industries, the control of the size of the produced and used microcapsules is not a key issue. However, in the case of medicine and pharmacy, it is a critical factor. The size of the microcapsules determines their loading capacity and drug release time [Xu, Q., Hahsimoto, M., Dang, TT, Hoare, T., Kohane, DS, Whitesides, GM, Langer, R ., Anderson, DG Smali 5, 1575-1581 (2009)].

Drug-laden microcapsules are most often components of drug-controlled release systems. They enable the release of a pharmaceutical at a selected point, e.g. in the gastrointestinal tract, and control its release time. At the same time, they reduce the systemic toxicity of the drug, allowing to minimize the effective dose and apply it in the right place. An example is the popular drug aspirin. It is encapsulated to extend the time of release in the gastrointestinal tract (up to the entire night), and to protect the stomach walls from irritation. Also potassium chloride (KCI) preparations are encapsulated to avoid too high local concentrations which could irritate the gastric mucosa and cause bleeding.

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Due to their unique properties (e.g. thickening, stabilizing or gelling), biopolymers, such as hydrocolloids, are widely used in many industries (e.g. food or cosmetics) and in medicine. Particularly noteworthy are uronic acid gels (including alginate and pectic acid), whose very gentle gelling (ionic interactions) enables easy preparation of systems for trapping "sensitive chemical species or living organisms (microencapsulation). Alginates are one of the most frequently used biopolymers from the group of uronic acids to create microcapsules. The literature describes many different methods of creating alginate microgels using microfluidic systems of various geometries, both in laminar and multiphase flows. In general, the formation of microgels is always a two-step process. The first step is to give the polymer solution the desired shape (emulsion or fiber formation). Gelling then takes place, either chemically or physically, into or out of the system used to form the polymer. "Standard, spherical alginate gels are made by dispersing an aqueous solution of sodium alginate in a continuous phase and then initiating the gelling process. The continuous phase is most often cooking oil [Tan, W.-H. & Takeuchi, S. Advanced Materials 19, 2696-2701 (2007); Workman, V. L., Dunnett, S. B., Kille, P. & Palmer, D. D. Biomicrofluidics 1, 014105-014109 (2007); Liu, K., Ding, H.-J., Liu, J., Chen, Y. & Zhao, Χ.-Ζ. Langmuir 22, 9453-9457 (2006)] (due to non-toxicity), hexadecane [Choi, C.-H., Jung, J.-H., Rhee, YW, Kim, D.-P., Shim, S .-E. and Lee, C.-S. Biomedical Microdevices 9, 855-862 (2007)] (an easily available alkane that has emulsion stabilizing properties [Blythe, PJ, Morrison, BR, Mathauer, KA, Sudol, ED & El-Aasser, MS Langmuir 16, 898-904 ( 1999)]) or undecanol [Zhang, H., Tumarkin, E., Peerani, R., Nie, ZH, Sullan, RMA, Walker, GC, Kumacheva, E., Journal of the American Chemical Society 128, 12205-12210 (2006)] (because it dissolves metal salts better than other oils). To avoid coalescence, a surfactant (e.g. polyglycerol sesquistearate [Lin, Y.-H., Chen, C.- T., Huang, L. & Lee, G.-B. Biomedical Microdevices 9, 833-843 (2007)], Span85 [Chuah, AM, Kuroiwa, T., Kobayashi, I., Zhang, X. & Nakajima, M. Colloids and Surfaces A: Physicochemical and Engineering Aspects 351, 9-17 (2009) ], Span80 [Um, E., Lee, D.-S., Pyo, H.-B. & Park, J.-K. Microfluidics and Nanofluidics 5, 541-549 (2008)], lecithin [Tan, W . -H. & Takeuchi, S. Advanced Materials 19, 2696-2701 (2007)]). Also non-spherical forms [Liu, K., Ding, H.-J., Liu, J., Chen, Y. & Zhao, X.-Z. Langmuir 22, 9453-9457 (2006)], e.g. disks, sticks or fibers, are obtainable in microfluidic systems.

The methods of forming gels using microfluidic systems can be divided according to the point of gelation, as well as the method of delivery of the cross-linking agent. We distinguish:

i) gelation outside the microcircuit, ii) gelation inside the microcircuit, which can be further divided into:

- external - when the cross-linking agent is present in the continuous phase,

- internal - when the inactive cross-linking agent is present in the dissipated phase,

- methods with parallel supply of the cross-linking agent, i.e. where the cross-linking agent is fed with an additional stream or drop to the dispersed phase, iii) combined systems, i.e. those in which both gelation in the microcircuit and outside it are used.

All these methods are briefly outlined below.

The simplest way to formulate microgels is to cross-link in a gelling bath outside the microcircuit (Table 1). In this way, clogging of the channels with quick-gelling substances is avoided, but the disadvantage of this solution is frequent deformation of the particles obtained. An alternative could be gelling inside the microcircuits. We speak of external gelation inside microcircuits when the cross-linking agent in the active form is contained in the continuous phase and reaches the droplets of cross-linked polymer by diffusion (Table 2). The advantage of this method is the elimination of the problem of channel clogging. In addition, by controlling the gel time, liquid core capsules or solid gels can be easily obtained. A frequent disadvantage is the heterogeneity of the gel structures obtained, due to the fact that the gelling process depends on the diffusion of the cross-linking agent through the already formed gel network. Gels with a much more homogeneous structure are obtained by using internal cross-linking in the microcircuit (Table 2). In this method, the cross-linking agent is contained in the dispersed phase in its inactive form. Its activation occurs under the influence of environmental changes, e.g. changes in pH. A great disadvantage of this solution is the very frequent clogging of the droplet forming nozzle due to the rapid cross-linking of the polysaccharide. Similar problems occur when we introduce a cross-linking agent with an additional stream. On the other hand, when coalescence-induced gelling is used, difficulties can be encountered

Designing the appropriate microcircuit geometry (Table 2). Depending on the needs, you can also use combinations of the above-mentioned methods (combined methods; Table 3). The most common combinations are gelation in the microcircuit with gelling outside the microcircuit.

T a b e l a 1. Literature compilation on the production of alginate microgels in microfluidic systems with the use of gelation outside the microcircuit. (1 - Capretto, L., Mazzitelli, S., Luca, G. & Nastruzzi, C. Acta Biomaterialia 6, 429-435, (2010); 2 - Su, J., Zheng, Y., Wu, H. Lab on a chip 9, 996-1001, (2009); 3 - Chuah, AM, Kuroiwa, T., Kobayashi, I., Zhang, X. & Nakajima, M. Colloids and Surfaces A: Physicochemical and Engineering Aspects 351, 9-17, (2009); 4 - Huang, K.- S., Liu, Μ. -Κ., Wu, C.-H., Yen, Y.-T. and Lin, Y.-C. Journal of Micromechanics and Microengineering 17,1428 (2007); 5 - Huang, Κ.-S., Lai, TH., Lin, YC. Lab on a chip 6, 954-957, (2006); 6 - Capretto, L. , Mazzitelli, S., Balestra, C., Tosi, A., Nastruzzi, C. Lab on a chip 8, 617-621, (2008) .7 - Sugiura, S., Oda, T., Aoyagi, Y. , Satake, M., Ohkohchi, N., Nakajima, M. Lab on a chip 8, 1255-1257, (2008); 8 - Dohnal, J. & Stepanek, F. Powder Technology 200, 254-259, (2010 ); 9 - Rondeau, E. & CooperWhite, JJ Langmuir 24, 6937-6945, (2008).)

Continuous phase Diffuse phase Cross-linking bath Literature sunflower oil sodium alginate + agarose BaCI2 1 sodium alginate lack CaCl2 2 isoacetate + Span85 sodium alginate CaCl2 3 sunflower oil sodium alginate CaCl2 4 Oil sodium alginate CaCl2 5 sunflower oil sodium alginate BaCI2 6 sodium alginate lack CaCl2 7 air (piezoelectric method) sodium alginate CaCI2 (with viscosity modifications) 8 dimethyl carbonate sodium alginate CaCl2 9

T a b e l a 2. Literature compilation on the production of alginate microgels in microfluidic systems using gelation inside the microcircuit (1 - Zhang, H., Tumarkin,

E., Sullan, R. M. A., Walker, G. C. & Kumacheva, E. Macromolecular Rapid Communications 28, 527-538 (2007); 2 - Zhang, H., Tumarkin, E., Peerani, R., Nie, Z.H., Sullan, R.M.A., Walker, G.C., Kumacheva, E., Journal of the American Chemical Society 128, 12205-12210 (2006); 3 - Braschler, T., Johann, R., Heule, M., Metref, L. & Renaud, P. Lab on a chip 5, 553-559 (2005); 4 - Johann, R. M. & Renaud, P. Biointerphases 2, 73-79 (2007); 5 - Kim, C., Lee, K. S., Kim, Y. E., Lee, K.-J., Lee, S. H., Kim, T. S. and Kang, J. Y. Lab on a chip 9, 1294-1297, (2009); 6 - Ren, P.-W., Ju, X.-J., Xie, R. & Chu, L.-Y. Journal of Colloid and Interface Science 343, 392-395 (2010); 7 - Tan, W.-H. & Takeuchi, S. Advanced Materials 19, 2696-2701 (2007); 8 - Yuya, M., Wei-heong, T., Yukiko, T. & Shoji, T. Lab on a chip 9, 2217-2223 (2009); 9 - Workman, V. L., Dunnett, S. B., Kille, P. & Palmer, D. D. Biomicrofluidics 1, 014105-014109 (2007); 10 - Capretto, L., Mazzitelli, S., Balestra, C., Tosi, A., Nastruzzi, C. Lab on a chip 8, 617-621 (2008); 11 - Amici, E., Tetradis-Meris, G., de Torres, C. P. & Jousse,

F. Food Hydrocolloids 22, 97-104 (2008); 12 - Um, E., Lee, D.-S., Pyo, H.-B. & Park, J.-K. Microfluidics and Nanofluidics 5, 541-549 (2008); 13 - Liu, K., Ding, H.-J., Liu, J., Chen, Y. & Zhao, X.- Z. Langmuir 22, 9453-9457 (2006); 14 - Choi, C.-H., Jung, J.-H., Rhee, Y. W., Kim, D.-P., Shim, S.-E. and Lee, C.-S. Biomedical Microdevices 9, 855-862 (2007); 15 - Zhao, LB, Pan, L., Zhang, K., Guo, SS, Liu, W., Wang, Y., Chen, Y., Zhao, XZ, Chan, HLW Lab on a chip 9, 2981- 2986 (2009).)

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Method gelation Continuous phase Diffuse phase Additional channel Literature Outside undecanol + (CH3COO) 2Ca sodium alginate 1 undecanol + Cal2 sodium alginate 2 CaCl2 (laminar flow) sodium alginate EDTA - to stop the gelling process 3.4 oleic acid + CaCl2 sodium alginate 5 two: CaCl2, oil (multiple emulsion) two: sodium alginate, oil 6 inside oil + CH3COOH sodium alginate + CaCO3 1.7 corn oil + lecithin sodium alginate + CaCO3 GDL 8 corn oil + lecithin + CH3COOH sodium alginate + CaCO3 corn oil + CH3COOH 7 sunflower oil sodium alginate + CaCO3 sunflower oil + CH3COOH 9 sunflower oil + CH3COOH sodium alginate + BaCO3 10 Oil sodium alginate + CaCO3 GDL 11 Φ c c mineral oil + Span80 sodium alginate the CaCI2 stream 12 soybean oil sodium alginate CaCI2 drops (coalescence) 13 hexadecane + Span80 sodium alginate the CaCI2 stream 14 Oil sodium alginate + magnetic particles CaCI2 drops (coalescence) 15

Table 3. Literature summary on the production of alginate microgels in microfluidic systems using combined gelation methods (1 - Capretto, L., Mazzitelli, S., Balestra, C., Tosi, A., Nastruzzi, C. Lab on a chip 8 , 617-621 (2008); 2-Rondeau, E. & Cooper-White, JJ Langmuir 24, 6937-6945 (2008); 3-Shin, S.-J., Park, J.-Y., Lee, J.-Y., Park, H., Park, Y.-D., Lee, K.-B, Whang, C.-M. and Lee, S.-H. Langmuir 23, 9104-9108 (2007 ).)

Continuous phase Diffuse phase Cross-linking bath Literature sunflower oil sodium alginate + BaCl2 BaCI2 1 DMC combining 2 streams: sodium alginate solution and CaCl2 solution CaCl2 2 sodium alginate solution surrounded by concentric CaCl2 solution lack CaCl2 3

Pectin compounds, similarly to alginates, belong to the group of polyuronate hydrogels and are the main component of the cell walls of tall plants. Pectin compounds are non-toxic and fully biodegradable, therefore they have always been present in the human diet. They exhibit a wide spectrum of physiological activity: lower cholesterol levels [Sriamornsak, P. Journal of Silpakorn University 21-22, 206-228 (2001)], slow down glucose absorption [Voragen, A., Coenen,

G.-J., Verhoef, R. & Schols, H. Structural Chemistry 20, 263-275 (2009)], perform regulatory functions

PL 215 011 B1 in the small intestine (change of microbial and enzymatic activity, as well as local changes in the morphology of the intestinal wall, thanks to which the absorption of nutrients is improved) [Sriamornsak, P. Journal of Silpakorn University 21-22, 206-228 (2001)] , facilitate the excretion of toxic components with urine (e.g. bivalent heavy metals [Kohn, R. Carbohydrate Polymers 2, 273-275 (1982)]), have anti-carcinogenic properties [Nangia-Makker, P., Hogan, V., Honjo, Y ., Baccarini, S., Tait, L., Bresalier, R. and Raz, AJ Natl. Cancer Inst. 94, 1854-1862 (2002); Ovodov, Y. Russian Journal of Bioorganic Chemistry 35, 269-284 (2009)]. It should be emphasized that the FAO / WHO world standards do not impose restrictions on the maximum daily dose of pectin consumption. Pectins exhibit exceptional stability in the human digestive system - they are resistant to proteases and amylases. These enzymes are active in the upper gastrointestinal tract [Yu, C.-Y., Cao, H., Zhang, X.-C., Zhou, F.-Z., Cheng, S.-X., Zhang, X .-Z., And Zhuo, R.-X. Langmuir 25, 11720-11726 (2009)]. Only in the large intestine are pectinases present, which break down pectin compounds. For this reason, pectins are an excellent carrier for enteral drugs (e.g. for the treatment of colon cancer [Liu, L., Fishman, M. L., Kost, J. & Hicks, K. B. Biomaterials 24, 3333-3343 (2003)]). In addition, the compounds in question have many applications in the food industry (thickener replacing gelatin in jellies for vegans), cosmetics (thickener) and in agriculture (e.g. feed ingredient). Pectic substances find more and more new applications. A 2006 review [Liu, L., Fishman, M. & Hicks, K. Cellulose 14, 15-24 (2007)] describes the wide use of pectins and their derivatives in controlled drug delivery systems (colon, nasal, nasal leather). In turn, modified pectins are tested as a carrier material for DNA in gene therapies [Katav, T., Liu, LS, Traitel, T., Goldbart, R., Wolfson, M., Kost, J. Journal of Controlled Release 130, 183 -191 (2008)]. New ion-conducting SPE (solid polymer electrolyte) systems [Andrade, J. R., Raphael, E. & Pawlicka, A. Electrochimica Acta 54, 6479-6483 (2009)] based on pectins are also being developed. In turn, pectins cross-linked with divinylsulfonate have a chance to become synthetic bones with better mechanical properties than natural ones [Ichibouji, T., Miyazaki, T., Ishida, E., Sugino, A. & Ohtsuki, C. Materials Science and Engineering: C 29, 1765-1769 (2009)].

The structure of pectin compounds and the structure of their gels is well known and widely described in the literature. Depending on the plant species, the stage of its growth, as well as the tissue used and the method of isolation, pectin compounds of various structures are obtained. Approximately the pectin molecule consists of smooth and branched regions, and their distribution in the molecule is random (not blocky as with alginates). Smooth regions are composed primarily of 1,4-linked α-D-galacturonic acid (Gal) residues. Branched regions typically contain many different sugars, such as rhamnose, xylose, glucose, arabinose and galactose. The properties of the gel that will be formed from a particular type of pectin depend on the length and distribution of smooth and branched regions, as well as the length of the entire single chain. An equally important factor is the degree of esterification (methylation). The degree of methoxylation (DM) is defined as the amount of moles of methanol present in 100 moles of galacturonic acid and expressed as a percentage [Ovodov, Y. Russian Journal of Bioorganic Chemistry 35, 269-284 (2009)]. On the basis of this number, the division of pectic compounds into pectins was introduced: i) high-methoxylated pectins (HMP) in which more than 50% of the monomers have a methylated carboxyl group; ii) low-methoxylated pectins (LMP) where less than 50% of the monomers are methylated.

We can also distinguish low-methoxylated amidated pectins (LMA), which are formed during the chemical conversion of HMP in LMP in an alkaline environment, in reaction with ammonia. The mentioned types of pectins differ not only in structure, but also in the method of gelling (different conditions necessary for the formation of a gel). It is worth mentioning that an acidic pH environment is required for the gelation of pectins, in contrast to alginate compounds, which are unstable under these conditions. HMP gels are formed at a pH close to 3.0, in the presence of about 65% sugar (most often sucrose). The required pectin concentration is 0.3 - 2%. The gel network is formed as a result of the formation of hydrogen bonds between the carboxyl groups of one pectin chain and the hydroxyl groups of the adjacent chain. The addition of sugar additionally stabilizes the gel structure, taking part in the formation of hydrogen bonds. In turn, the high concentration of hydrogen ions prevents the dissociation of carboxyl groups. On the other hand, the structures of LMP and LMA gels are built in the presence of divalent metal ions, most often Ca ²⁺ , with a concentration of 0.01 - 0.1%. The process does not require the presence of other sugars and takes place in the pH range from 2.0 to 6.0. It is mainly described by the "egg-box model [Voragen, A., Coenen, G.-J., Verhoef, R. & Schols,

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H. Structural Chemistry 20, 263-275 (2009); Sriamornsak, P. Silpakorn University International Journal 3,

206-228 (2003)], although for amidated pectins one should also take into account the contribution of interactions between the -NH2 groups, which significantly change the properties of the obtained gel (e.g. the amount of ions needed to create a gel network from the amidated pectin solution is smaller than in the case of low methylated nonamidated pectins [Tho, I., Sande, SA & Kleinebudde, P. Chem. Eng. Sci. 60, 3899-3907 (2005)]).

Despite the many applications of pectin compounds (vide supra), there are not many reports in the literature on the preparation of pectin microparticles. One of the few articles on the preparation of pectin microspheres or microparticles is the work of Reis et al. [Reis, A. V., Guilherme, M. R., Paulino, A. T., Muniz, E. C., Mattoso, L. H. C. and Tambourgi, E. B. Langmuir 25, 2473-2478 (2009)]. The authors obtained hollow micro- and nanocapsules by using a two-step reaction. The first step was the modification of pectin with glycyl methacrylate (GMA). This modification was aimed at introducing additional groups to the pectin chain that could participate in the creation of additional intermolecular bonds. As a result, a more stable structure was created without significantly changing the properties of the gel. In the second stage, the obtained solution was mechanically emulsified (6000-12000 rpm) in the water / benzyl alcohol system in the presence of nitrogen gas (it ensured the formation of nucleus gas capsules) and the TEMED catalyst (Ν, Ν, Ν ', Ν'- tetramethylethyldiamine). After spray drying, the finished product was obtained. The produced capsules had diameters ranging from 0.16 to 24 μm and were characterized by polydispersity. It should be emphasized that multiple emulsions were also produced in an uncontrolled manner.

On the other hand, "classical gelation (cross-linking with divalent cations) of pectin microparticles was described in the work of Opanasopit et al. [Opanasopit, P., Apirakaramwong, A., Ngawhirunpat, T., Rojanarata, T. & Ruktanonchai, U. AAPS PharmSciTech 9, 67-74 (2008)], where pectin micro- and nanograins were used as a DNA carrier (potential application for gene therapy). The influence of several factors on the formation of microparticles was investigated: the type of cross-linking cation, its concentration, weight ratio (in relation to the pectin used), as well as the concentration and pH of the pectin solution. Among other things, the size and shape of the microparticles formed and their deoxyribonucleic acid charge. In order to obtain microspheres, the LMA pectin solution was dripped onto the solution of Ca ²⁺ , Mg ²⁺ or Mn ²⁺ ions. The load capacity test showed that the largest amount of DNA can be encapsulated in particles cross-linked with calcium ions. Moreover, based on the observation of the survival of human cells (Huh7, human liver cancer cells), no significant cytotoxicity of the obtained pectin microparticles was found. The conducted experiments confirm that pectin micro-grains can be successfully used as a carrier in gene therapies.

In turn, Yu et al. [Yu, C.-Y., Cao, H., Zhang, X.-C., Zhou, F.-Z., Cheng, S.-X., Zhang, X.-Z., and Zhuo, R .-X. Langmuir 25, 11720-11726 (2009)] created pectin nanocapsules with a nucleus filled with the pharmaceutical drug, 5-fluorouracil. The proposed procedure allowed for the production of grains and capsules with sizes ranging from 400 to 2600 nm. The loading capacity of 5-fluorouracil for all tested samples was higher than 12% (even up to 18% of the mass of the microparticle). This method has some disadvantages: sample preparation takes more than 50 hours and requires constant mixing and strict control of the temperature and viscosity of the solutions used. The elimination of the above-mentioned problem of dispersion control (requirement of monodispersity of particles), as well as ensuring precise control of the reaction conditions can be achieved by applying microfluidic techniques. However, there are few reports in the literature on the formation of emulsions or pectin microparticles, as opposed to alginate microparticles, in microchannels. Kawakatsu et al. in his works [Kawakatsu, T., Tragardh, G. & Tragardh, C. Journal of Food Engineering 50, 247-254 (2001); Kawakatsu, T., Tragardh, G. & Tragardh, C. Colloids and Surfaces A: Physicochemical and Engineering Aspects 189, 257-264 (2001)] described the formation of simple oil-in-pectin emulsions and multiple W / O / W emulsions and S / O / W (pectin-in-oil-in-water gel). For this purpose, a system with micro-terrace geometry (MT) was used. The proposed microtarracing technique enables the formulation of monodisperse particles, but does not give full control over the emulsification process carried out (it is difficult to control the size or morphology of the microgels obtained).

The invention relates to a method of formulating monodisperse pectin microparticles, for potential applications in the food and pharmaceutical industries, using microfluidic systems.

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According to the invention, a method for the preparation of monodisperse pectin microgels using a microfluidic system, comprising the steps of i) forming an emulsion and ii) gelling by diffusion into the dispersed phase of the cross-linking ions contained in the continuous phase, characterized in that the continuous phase comprises edible oil, in particular rapeseed oil, calcium salt and non-toxic acetic acid miscible with polar and nonpolar solvents, and the dispersed phase is an aqueous pectin solution optionally containing nanoparticles.

Preferably, virgin rapeseed oil is used as rapeseed oil.

Preferably, the calcium salt is calcium chloride or calcium carbonate.

Most preferably the calcium salt is calcium carbonate.

Preferably, the acetic acid is glacial acetic acid.

Preferably, the acid concentration in the continuous phase is at least 1.0 wt%, more preferably 10.0 wt%.

Preferably, the salt concentration in the continuous phase is from 0.05 wt% to 2.0 wt%, more preferably 0.4 wt%.

Preferably, the pectins are low esterified amidated pectins.

Preferably, the pectin has low, medium or high sensitivity to calcium ions, more preferably medium sensitivity to calcium ions.

Preferably, the concentration of the pectin solution is from 0.25 wt% to 1.0 wt%, most preferably 0.5 wt%.

Preferably, the process according to the invention further comprises the step of changing the oil-containing continuous phase.

Preferably, nanoparticles of different surface chemistry, preferably negatively charged surface, more preferably coated with SHC10H20COO ^- are encapsulated into the dispersed phase.

Preferably, there is a flow of the continuous phase and the disperse phase in the emulsification step.

According to the invention, it is preferred to use low esterified amidated pectins, natural polysaccharides which form stiff gels in the presence of metal cations. The gelation speed, structure and properties of gels are determined by the type or concentration of pectin or multivalent metal salt.

The method of the invention, in one preferred embodiment, further comprises the step of replacing said oil-containing continuous phase, i.e. replacing the acid-containing oil and the metal salt with pure oil. Oil change allows precise control of the diffusion time of the cross-linking agents from the organic phase to the drops containing the polysaccharide solution. As a result, better control of the size of the obtained droplets, high homogeneity and repeatability of the degree of cross-linking of the gummies is achieved.

Preferably, in said emulsion preparation step there is a flow of the continuous phase (oil with crosslinkers) and the dispersed phase (pectin solution), which in one embodiment of the invention are from 3 mL / h to 50 mL / h for the continuous phase, more preferably 6 mL / h and from 1 mL / h to 5 mL / h for the disperse phase, more preferably 1 mL / h.

The method according to the invention is characterized by simplicity, high repeatability and the possibility of producing monodisperse pectin microgels of a given size or morphology. The structure of the developed formulation system is simple, based on cheap equipment or materials as well as easily available and non-toxic substrates. This system allows full control over the size of the obtained pectin microparticles and can be a convenient tool for their production in industry (in particular, the pharmaceutical industry - drug controlled release systems). The current state of the art allows the preparation of pectin microgels. However, the known formulation methods do not guarantee monodispersion of the obtained particles or do not give full control over the emulsification process carried out (it is difficult to control the size or morphology of the microgels obtained).

Preferred Embodiments of the Invention.

The invention will now be illustrated in more detail in the preferred embodiments with reference to the accompanying drawings and tables in which:

Fig. 1 is a photomicrograph showing the production of 0.5% w / w aqueous pectin droplets in rapeseed oil in a flow-focusing microflow system.

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Volumetric flow rates expressed in mL / h (Qc for the continuous phase, Qd for the dispersed phase) are given in the figure. All channels in the system had the same cross-section with dimensions of 400 x 400 μm. The graph shows the dependence of the droplet size (length, L, normalized to the width of the channel, W) as a function of the ratio Qc / Qd;

Fig. 2 is a schematic diagram of a system for the preparation of monodisperse gel pectin microparticles. The operation of the system was based on: i) formulation of monodisperse droplets from the pectin solution, ii) diffusion of cross-linking agents from the continuous phase to the dispersed phase, iii) collection of the formed microparticles and secondary gelling;

Figs. 3 a-c. are photomicrographs illustrating the transition of pectin microparticles from the continuous phase to glycerin. The circles with clearly marked edges correspond to the microparticles in the continuous phase, while the poorly defined shapes correspond to the particles that passed into glycerin, d - shows a micrograph of the pectin gel network made with the use of a scanning electron microscope. The experiments were carried out using the microfluidic system shown in Fig. 2 with a 0.5% (w / w) pectin solution (Qd = 1 mL / h) and rapeseed oil with 0.4% CaCO3 and 4% CH3COOH (w / w) (Qc = 6 mL / h);

Fig. 4 schematically illustrates a modified system (additional module) for studying the influence of individual components of the reaction mixture on the structures of the pectin microgels obtained. The operation of the system was based on: i) formulation of monodisperse drops from the pectin solution, ii) diffusion of cross-linking agents from continuous to dispersed phase, iii) in situ extraction into the continuous phase without cross-linking agents, and iv) collecting the formed microparticles and secondary gelling;

Figure 5 shows photographs of (a) pectin gels and (bd) gels with encapsulated nanoparticles: (b) Ag / SHC11H22N ⁺ (CH3) 3, (c) Au / SHC11H22SO3 ^- , (d) Au / SHC10H20COO ^- . The result of qualitative research;

Fig. 6 illustrates the effect of crosslinking time. The graph shows the time profiles of decay of pectin microgels cross-linked at different times (1/2, 1, 3, 15, 24 h) and the release of gold nanoparticles from them in EDTA pC 3 solution. Gel microparticles were produced using a pectin solution with a concentration of 0.5 % and rapeseed oil with the addition of calcium carbonate (0.4%) and acetic acid (4%). A detailed description of the experiment is given in the text;

Figure 7 shows the absorbance values as a function of time measured in a suspension of pectin microgels at five different EDTA concentrations (pC 5; 4; 3.3; 3 and 2) and in water (blank). Microgels were obtained with the use of 0.5% pectin solution and rapeseed oil with the addition of calcium carbonate (0.4%) and acetic acid (4%). A detailed description of the experiment is given in the text;

Fig. 8 shows the transformed data of Fig. 7. Results were normalized to the absorbance value (Amax) of PCEDTA 2 at t = 7 h;

Figure 9 illustrates the temporal release profiles of AuNPs from microgels. The dispersed phase was a mixture of 0.5% pectin solution with AuNPs solution in a volume ratio of 2: 1 (P). The continuous phase was rapeseed oil with the addition of calcium carbonate (Ca) and acetic acid (H). The amounts of Ca and H are given in weight percent in the table. The detailed experimental procedure is given in the text. The points on the graphs reflect the mean absorbance values calculated for the two measurements;

Figure 10 illustrates the temporal release profiles of AuNPs from microgels. The dispersed phase was a mixture of 1% pectin solution with AuNPs solution in a 2: 1 volume ratio (P). The continuous phase was rapeseed oil with the addition of calcium carbonate (Ca) and acetic acid (H). The amounts of Ca and H are given in weight percent in the table. The detailed experimental procedure is given in the text. The points on the graphs reflect the mean absorbance values calculated for the two measurements;

Table 4 presents the results of studies on the solubility of calcium salts in glacial acetic acid and the miscibility of the obtained solutions (salt-acid) with rapeseed oil. Explanation of symbols: (++) - good, (+) - weak, (-) - insoluble or immiscible; Table 5 presents the selection of the type of nanoparticles for encapsulation in pectin gels. The experiment used nanoparticle solutions with a concentration of 1.5 mg / mL, 0.5% aqueous pectin solution and 0.5% CaCl2 solution. Photographs of the obtained gels are shown in Fig. 5;

P r z k ł a d 1

For the production of monodisperse pectin microgels, a simple flow-focusing microflow system with microchannel dimensions equal to 400 x 400 gm is used, performed according to the procedure described in [Ogończyk, D., Węgrzyn, J., Jankowski, P., Dąbrowski, B. & Garstecki, P. Lab on

PL 215 011 B1 a chip 10, 1324-1327 (2010)] and modified according to the procedure described in [P. Jankowski, D. Ogończyk, A. Kosiński, W. Lisowski and P. Garstecki (submitted)]. The following are used for production: 0.5% aqueous pectin solution (dispersed phase) and rapeseed oil with the addition of calcium carbonate and acetic acid in amounts of 0.4% and 4% by weight in relation to rapeseed oil (continuous phase). The continuous phase is prepared as follows: calcium carbonate is dissolved in glacial acetic acid, then the obtained solution is mixed with rapeseed oil and sonicated for 2 hours. The last step in the preparation of the continuous phase is its filtration using a simple syringe filter with a pore diameter of 1.2 ąm. The solutions prepared in the above-mentioned manner are placed in two syringes, which are then connected to the microcircuit with the use of polyethylene hoses. The syringes are placed in syringe pumps. The microcircuit connects to the receiver via a polyethylene discharge hose. The two phases are forced into the microcircuit with the aid of syringe pumps at the rates of 1 mL / h and 6 mL / h for the dispersed and continuous phases, respectively. The resulting microgels are collected in a receptacle containing water.

P r z k ł a d 2

For the production of monodisperse pectin microgels, a simple flow-focusing microflow system with microchannel dimensions equal to 400 x 400 μm is used, performed according to the procedure described in [Ogończyk, D., Węgrzyn, J., Jankowski, P., Dąbrowski, B. & Garstecki, P. Lab on a chip 10, 1324-1327 (2010)] and modified according to the procedure described in [P. Jankowski, D. Ogończyk, A. Kosiński, W. Lisowski and P. Garstecki (submitted)]. For production, the following are used: 0.5% aqueous pectin solution and 1.5 mg / mL aqueous solution of gold nanoparticles AU / SHC10H20COO ^- in a volume ratio of 2: 1 (dispersed phase) and rapeseed oil with the addition of calcium carbonate and acetic acid in amounts of 0 , 4% and 10% by weight in relation to rapeseed oil (continuous phase). The continuous phase is prepared as follows: calcium carbonate is dissolved in glacial acetic acid, then the obtained solution is mixed with rapeseed oil and sonicated for 2 hours. The last step in the preparation of the continuous phase is its filtration using a simple syringe filter with a pore diameter of 1.2 μm. The solutions prepared in the above-mentioned manner are placed in two syringes, which are then connected to the microcircuit with the use of polyethylene hoses. The syringes are placed in syringe pumps. The microcircuit connects to the receiver via a polyethylene discharge hose. By means of syringe pumps, both phases are forced into the microcircuit at the rates: 1 mL / h and 9 mL / h for the dispersed and continuous phases, respectively. The resulting microgels are collected in a receptacle containing water.

Summary of the results obtained in the above-mentioned examples of implementation

Controlling the size of monodisperse pectin microparticles in the microfluidic system

A flow-focusing microfluidic system with microchannel dimensions of 400 x 400 μm was used to produce drops of an aqueous pectin solution (Fig. 1). The control of volumetric flow rates of the continuous phase (rapeseed oil) allows to obtain monodisperse drops of an aqueous solution of pectin with a concentration of 0.5% by weight with a length ranging from half to four channel widths, i.e. 200-1600 μm, which corresponds to volumes in the range 15-150 pL. How was

-1/2, the obtained droplet size shown in Fig. 1 scales as L / W ~ (Qd / Qc) ^-1/2 , which does not correspond to the scaling for Newtonian fluids.

Optimization of the phase composition: continuous and dispersed for the formulation of pectin microgels

The aim of the invention was to develop a method of creating biocompatible pectin microgels. Accordingly, an edible oil, more preferably rapeseed oil, was used as the base for the continuous phase. The components of the continuous phase were also the cross-linking factors necessary for pectin gelation: hydrogen ions and cations of divalent metals. The choice of acetic acid (E260) was dictated by its wide industrial applications as well as its miscibility with both polar and nonpolar liquids, allowing it to easily diffuse from the continuous to the disperse phase. The choice of the metal salt was dictated by the low toxicity of the calcium salts as well as their very good solubility in acetic acid. Six different calcium salts (Table 4) were tested for solubility in acetic acid and the miscibility of the resulting solution with rapeseed oil. On the basis of the obtained results, it is most advantageous to use cross-linking agents for the preparation of pectin microgels: calcium carbonate dissolved in glacial acetic acid.

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Systems for the preparation of monodisperse gel microparticles in a microfluidic system

For the preparation of gel pectin microparticles, a microfluidic system was constructed, the diagram of which is presented in Fig. 2.

The idea of the presented system was as follows. In the microflow system, monodisperse droplets of the desired size were formulated (Fig. 1). The dispersed phase was a pectin solution with a given concentration, and the continuous phase was rapeseed oil with the addition of cross-linking agents (calcium salt and acetic acid). It has been experimentally verified that the addition of these factors has no major influence on the drop formation process compared to pure oil. The droplets formed in the microcircuit gelled as a result of contact with the continuous phase; due to diffusion of cross-linking agents into the dispersed phase. This gelling process is kinetic in nature, which means that it can be controlled over time. In turn, the gelation time can be controlled, for example, by setting the appropriate length of the gelation path or changing the volumetric flow velocities. The length of the gelation path was experimentally selected, which was 100 cm (a drainage polyethylene hose with an internal diameter of 0.76 mm) with a given total flow not exceeding 10 mL / h. The formed microparticles were collected into a reservoir of water where their further gelling took place. This process was due to: i) diffusion of cross-linking agents from the continuous phase to water in the receiver, ii) secondary diffusion of ions already enclosed in the microparticle (gelation inside the formed capsule). It should be noted that the microparticles accumulated at the oil-water interface. As the process progressed, the microgels passed from the oil phase to the water and fell by gravity to the bottom of the tank. The microgels produced in the system and an exemplary structure of the gel obtained in the above-mentioned method are shown in Fig. 3.

Due to the fact that further experiments consisted in examining the influence of individual components of the reaction mixtures on the structure of the obtained microgels, the proposed system (Fig. 2) was modified. An additional module was introduced which allowed the in situ extraction of pectin microparticles from the continuous phase containing the cross-linking agents into the continuous phase containing no additives (Fig. 4). This modification made it possible to obtain repeatability of measurements and eliminated the influence of diffusion of cross-linking agents from the continuous phase into the water in the receiver.

Encapsulation of nanoparticles in pectin gel microparticles

Potential applications of pectin microgels are, inter alia, systems for the controlled release of particles of active substances. Nanoparticles encapsulated in a pectin matrix were used as model substances. At the same time, the study of the processes of nanoparticle release from microgels was used to characterize the gel microstructures obtained under various conditions. Nanoparticles are characterized by very small sizes or intense colors, and the processes of their release can be monitored without the use of complicated equipment, e.g. using spectrophotometric techniques.

Selection of the appropriate type of nanoparticles for encapsulation

The next stage of the research was to encapsulate gold and silver nanoparticles with different surface chemistry in a pectin matrix (Table 5).

Pectic compounds are polyanions, therefore the type of encapsulated nanoparticles (their surface chemistry) affects the way of their gelation and the stability of the obtained gel structures. Encapsulation of positively charged NPs on the surface in the pectin matrix did not bring the expected results. The gelling process was extremely slow, as a result of which the obtained gels "blurred and underwent extremely large deformations (Fig. 5b). Probably the positive charge accumulated on the NPs surface to a large extent hindered (slowed down) the diffusion of ions and gel cross-linking (electrostatic interactions). Much better results were obtained with NPs with negatively charged surfaces. However, the type of coat turned out to be a key element. In the case of using gold nanoparticles coated with SHC11H22SO3^- gels with slightly worse performance were obtained (Table 5). The gelling process was prolonged, which caused the obtained gels to be distorted. Presumably, the rate of gelation is determined by a side reaction - the capture and attachment of calcium ions through the SHC11H22SO3 mantle^-. It was verified that as a result of the reaction SHC10H22SO3^- a precipitate forms with calcium ions. The best results were obtained for NPs coated with SHC10H20COO^-therefore, this type of nanoparticles was used to study gel structures obtained under various conditions. It should be noted that the charge of the coat of Au / SHC10H20COO nanoparticles it can be changed under the influence of pH (protonation of carboxyl groups in acidic environment), which can be significant when formulating microgels with the use of developed systems.

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Investigation of the influence of various factors on the structure of pectin microgels

The morphology of pectin gels is influenced, among others, by factors such as: type and concentration of a metal ion or pectin, pH of the environment, temperature and gel time.

The next stage of the research consisted in checking the effects of various factors on the structure of pectin microgels obtained in the developed microfluidic system (Fig. 4). The experiments used low-methylated amidated pectin with medium sensitivity to calcium ions, the gelling process of which is not dependent on the temperature. The gel time and the different content of the components of the reaction mixtures were tested: the amount of calcium salts and the concentration of H ⁺ ions or pectin. It should be mentioned that the release of substances from pectin matrices into pure water is a long-term process (time of the order of days or weeks). Therefore, in order to accelerate it, studies were carried out with the disodium salt of ethylenediaminetetraacetic acid (EDTA). EDTA effectively complexes calcium ions, destabilizing the structure of the gel and releasing encapsulated nanoparticles in a relatively short time, which greatly facilitates the conduct of experiments.

Gel time effect

The next experiments focused on checking the effect of cross-linking time of pectin microgels. The effect of the gelation time was checked using microgels with encapsulated gold Au / SHC10H20COO ^- gold nanoparticles, which were produced using a 0.5% aqueous solution of pectin and an aqueous solution of nanoparticles with a concentration of 1.5 mg / mL in a 2: 1 volume ratio (as the dispersed phase) and with rapeseed oil with the addition of 0.4% calcium carbonate and 4% acetic acid (as a continuous phase). The volumetric flow rates were 1 and 9 ml / h for the dispersed and continuous phases, respectively, and the volume of the droplets so produced was 30 pL. A portion of the analyzed microparticles (a single sample) was produced for 15 minutes and collected in 1.5 mL of water. As a result, the volume of a single tested sample was close to 1.75 mL (only the water phase was analyzed, while the oil phase was removed). After the desired gelation time (0.5 h, 1 h, 3 h, 15 h or 24 h), 10 mL of pC 3 EDTA solution was added to the sample. A spectrophotometer and a 3 cm wide cuvette (optical path) were used to monitor the release process. Absorption measurements were continuous, the time interval was 5 minutes, and the total measurement time was 60 minutes. The sample was mixed during the measurements.

It should be emphasized that the gelling process is kinetic, i.e. it is determined by the contact time of pectin solution drops with cross-linking agents and the time of secondary diffusion of ions enclosed in microparticles (gelation inside the formed capsules). The effect of time on the cross-linking process of pectin microgels is shown in Fig. 6.

Interpreting the obtained results, it can be concluded that the gel time is a very important factor. The obtained results clearly show that cross-linking takes place in two stages. It should be noted that the overall cross-linking process was long (hours), although the contact of the polymer drop with the cross-linking agents was relatively short (minutes). The time required for complete cross-linking for the 30 pL microgels was close to 10 h. This conclusion was drawn from the small signal differences in the time release profiles for 3 h, 15 h and 24 h (Fig. 6). A gel time of 12 h was selected for further experiments.

Effect of EDTA concentration

The rate of release of NPs from pectin matrices depends not only on the time of cross-linking, but also depends on the concentration of the ethylenediaminetetraacetic acid salt.

The EDTA effect was checked using microgels with encapsulated gold Au / SHC10H20COO ^- gold nanoparticles, which were produced using a 0.5% aqueous solution of pectin and an aqueous solution of nanoparticles with a concentration of 1.5 mg / mL in a 2: 1 volume ratio (as the dispersed phase) and from oil rapeseed with the addition of 0.4% calcium carbonate and 4% acetic acid (as a continuous phase). The volumetric flow rates were 1 and 9 ml / h for the dispersed and continuous phases, respectively, and the volume of the droplets so produced was 30 pL. An aliquot of the analyzed microparticles (a single sample) was produced for 10 minutes and collected in 1.0 mL of water. As a result, the volume of a single test sample was close to 1.17 mL (only the water phase was analyzed, while the oil phase was removed). The gelation time of a single sample was 12 h. Then 6.6 mL of EDTA solution at a given concentration (pC range 5 to 2) was added to the sample. A spectrophotometer and a 1 cm wide cuvette (optical path) were used to monitor the release process. Measurements were made after the time: 1 h, 3 h, 1 h from the addition of EDTA. The sample was agitated during the release process.

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The results of the experiments carried out with EDTA solutions of various concentrations (pC ranging from 5 to 2) and pectin microgels with encapsulated gold nanoparticles (gelling = 12 h) are shown in Fig. 7 and Fig. 8.

The obtained results confirmed that by controlling the EDTA concentration, the rate of nanoparticle release from pectin microgels can be easily controlled. For example, when using EDTA pC 2 solution, the complete release of NPs occurs in about 10 min (Fig. 7). On the other hand, the use of EDTA solutions with lower concentrations (pC <2) results in slower degradation of microgels and release of smaller amounts of NPs.

An EDTA solution with a concentration of 1.41 mM (pC 2.85) was selected for further studies on the effect of phase composition. This amount of chelating agent will enable the observation of differences in the degradation rate of microgels of different structure in the given experimental time of 45 minutes (testing the influence of various factors).

Influence of phase composition: continuous and dissipated

To investigate the effect of the composition of the continuous and dispersed phases on the obtained microgel structures, an appropriate procedure was needed that would ensure reliable and reproducible results. That is why it was so important to determine the optimal gel time and EDTA concentration in advance.

The effect of the phase composition was checked using microgels with encapsulated gold Au / SHC10H20COO ^- gold nanoparticles, which were produced using a 0.5% or 1% aqueous solution of pectin and an aqueous solution of nanoparticles with a concentration of 1.5 mg / mL in a 2: 1 volume ratio (as a phase dispersed ) and rapeseed oil with the addition of 0.05% or 0.4% or 2% calcium carbonate and 1% or 4% or 10% acetic acid (as a continuous phase). For the production of microgels, a modified microfluidic system was used, which ensured reproducible formulation conditions (scheme of the system is shown in Fig. 4). The volume flow rates were 1 and 9 mL / h for the dispersed and continuous phases, respectively, and the volume of the droplets so produced was 30 pL. A portion of the analyzed microparticles (a single sample) was produced for 15 minutes and collected in 1.5 mL of water. As a result, the volume of a single tested sample was close to 1.75 mL (only the water phase was analyzed, while the oil phase was removed). The gelation time of a single sample was 12 h. Then 10 mL of EDTA solution with a pC of 2.85 was added to the sample. A spectrophotometer and a 3 cm wide cuvette (optical path) were used to monitor the release process. Absorption measurements were continuous, the time interval was 3 minutes, and the total measurement time was 45 minutes. The sample was mixed during the measurements. For this purpose, a specially designed mixing system was used, which was controlled by a voltage regulator.

The study of the effect of phase composition consisted in testing: i) the concentration of pectin used (0.5% and 1%), ii) the concentration of acetic acid in rapeseed oil (1%, 4%, 10%) and iii) the amount of calcium carbonate added to the phase continuous (0.05%, 0.4%, 2%). The results of the experiments carried out are shown in Figs. 9 and 10.

By analyzing the obtained results, it can be concluded that by controlling the concentration of pectin, hydrogen ions or the amount of calcium salts, the structure of the obtained microgels can be controlled. The most important qualitative observation, resulting from the conducted experiments, concerned the key role of acetic acid in the formulation of stable pectic microgels. All microgels produced using low concentration acetic acid (1%) and 0.5% pectin solution were extremely unstable. Most of the AuNPs were released even before the addition of EDTA. On the other hand, the microparticles produced with 1% CH3COOH and 1% pectin solution were slightly more stable. In this case, the AuNPs were released approximately 15 minutes after the addition of the EDTA. Interestingly, for the analyzed microgels (produced with the use of 1% acid), no dependence of the release rate on the amount of calcium carbonate used was observed. It is also worth noting that these microparticles tended to stick together and form large agglomerates. On the other hand, microgels produced with the use of acetic acid with the highest concentration tested (10%) did not aggregate and remained stable even after adding the calcium complexing agent. Only in the case of the microbeads produced with the lowest amount of calcium carbonate (0.05%) some destabilization of the microparticles was observed (about 25% of the nanoparticles were released). The most interesting results were obtained for the average concentration of the acid used (4%). Microgels formulated with 0.5% pectin solution showed similar destabilization profiles, releasing all nanoparticles within about 20 minutes after adding EDTA. Interestingly, the microparticles produced from a 1% pectin solution degraded more slowly and the release rate was higher for particles prepared with more calcium.

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T a b e l a 4

Calcium salt Solubility in CH3COOH Miscibility with rapeseed oil Observations of final mixtures chloride ++ + oil cloudiness lactate ++ - delamination and loss of sediment acetate ++ - oil cloudiness and sediment loss carbonate + ++ good miscibility oxalate + - delamination and loss of sediment citrate - - immiscible

T a b e l a 5

NPs used NPs cargo Gelation - remarks Ag / SHCnH22N ⁺ (CH3) 3 (+) extremely slow; a cloud-shaped gel was finally obtained Au / SHC11H22SO3 ^- (-) free; capsules (gels with liquid nuclei) were obtained AU / SHC10H20COO (-) fast; homogeneous gels (fully gelled) were obtained

Patent claims

Claims (13)

Patent claims 1. The method of obtaining monodisperse pectin microgels with the use of a microfluidic system, comprising the steps of: i) preparation of an emulsion and ii) gelling by diffusion into the dispersed phase of cross-linking ions contained in the continuous phase, characterized in that the continuous phase contains edible oil, especially rapeseed oil, calcium salt and non-toxic acetic acid miscible with polar and nonpolar solvents, and the dispersed phase is an aqueous pectin solution optionally containing nanoparticles. 2. The method according to p. The process of claim 1, wherein the rapeseed oil is virgin rapeseed oil. 3. The method according to p. The process of claim 1 or 2, wherein the calcium salt is calcium chloride or calcium carbonate. 4. The method according to p. The process of claim 3, wherein the salt is calcium carbonate. 5. The method according to p. A process as claimed in any one of the preceding claims, wherein the acetic acid is glacial acetic acid. 6. The method according to p. The process of any of the preceding claims, characterized in that the acid concentration in the continuous phase is at least 1.0 wt.%, More preferably 10.0 wt.%. 7. The method according to p. The process of any one of claims 1-6, characterized in that the salt concentration in the continuous phase is from 0.05% by weight to 2.0% by weight, preferably 0.4% by weight. 8. The method according to p. A process as claimed in any one of the preceding claims, characterized in that the pectins are low esterified amidated pectins. 9. The method according to p. 8. The method of claim 8, characterized in that the pectin has low, medium or high sensitivity to calcium ions, preferably medium sensitivity to calcium ions. 10. The method according to p. 9. The method of claim 9, characterized in that the concentration of the pectin solution is from 0.25 wt% to 1.0 wt%, preferably 0.5 wt%. 11. The method according to p. The process of any one of claims 1 to 10, further comprising the step of changing the oil-containing continuous phase. 12. The method according to p. ^{A process as claimed in any one of the} preceding claims, characterized in that nanoparticles with different surface chemistry, preferably with a negatively charged surface, more preferably coated with SHC10H20COO - are encapsulated into the dispersed phase. 13. The method according to p. The process of any one of claims 1 to 12, characterized in that a flow of the continuous phase and the disperse phase takes place in the emulsion preparation step.