Aqueous-based modified-released carrier system with polyelectrolytes and polyphenol

Abstract

The present disclosure relates to an aqueous-based system formed through the interfacial solidification of food-grade polymers, giving rise to core-shell capsules. The disclosure relates to a system able to retain molecules within a range of molecular weights from 100 Da to 10000 Da. The disclosure can also enable the entrance of such molecules in the core-shell systems. The processing of the system here disclosed is simple and rapid, in all-aqueous conditions, at temperature and pressure ranges considered as low energy consumption. The disclosure may find application in the pharmaceutical, medical, and agriculture industries, as well as on the generation of barrier-like devices for on-demand triggering of reactions for sensing, actuation, or diagnosis.

Description

Aqueous-based modified-released carrier system

[0001] The present invention relates to an aqueous-based carrier system formed through the interfacial solidification of polymers. More particularly, the disclosure relates to multifunctional materials that retain, control release and / or mediate the entrance of molecules. The invention also relates to a method of producing the aqueous-based system. The system of the present invention can be used in the pharmaceutical, biomedical and agriculture industry. The invention can be applied to biosensors and external factor-driven on-demand reactions.

[0002] Hydrogels are three-dimensional highly hydrated networks broadly applied in tissue engineering and regenerative medicine, which have also emerged as promising aqueous-based drug delivery systems (DDSs), with the potential to easily encapsulate hydrophilic drugs. The use of hydrogels processed in all-aqueous setups, free of organic solvents and other toxic molecules, has been appointed as a plausible way of circumventing the current environmental burden posed by the pharmaceutic industry, still highly dependent on classic organic solvent-based methods for the processing of DDSs. However, besides biopharmaceuticals such as antibodies and growth factors, most approved drugs are low molecular weight (LMW) molecules, with sizes below 1000 Da. Hydrogel size meshes, mostly after hydration, show average sizes higher than the size of LMW drugs. In addition to their hydrophilic nature, this size discrepancy leads to the low retention and rapid diffusion of these molecules from hydrogel meshes when those are placed in hydrated environments, such as blood and other body fluids.

[0003] Current strategies adopted to retain LMW molecules are typically based on drug-polymer interactions, through covalent conjugation or, most commonly, physical adsorption. Approaches explored so far for the modulation of drug release from matrices prepared in all-aqueous conditions has relied on the exploration of multi-compartmentalized hydrogel-in-hydrogel devices or, more often, on the exploration of the layer-by-layer (LbL) technology. The latter is a cost-effective and versatile bottom-up approach, able to form structures with different geometries, including fibers and capsules, in size ranges from nanometric to millimetric constructs. The LbL technologies rely on the alternated deposition of oppositely charged polymers, often in degradable or leachable templates, and are widely adopted to load and sustained release both hydrophilic and hydrophobic compounds. By tailoring the number of layers, it has been possible to control the release of drugs and, for charged and other interacting molecules, interactions with either the polycation or the polyanion layers have also showed to have important roles on regulating the drug release process. Despite such versatility, the LbL technique is multistep and time-consuming, and demands the prolonged immersion of the drug-loaded templates in aqueous environments, leading to the use of high volumes of water. Also, the diffusion of drugs from the template before the multilayer system is completely deposited may lead to low encapsulation efficiency, unless the overall environment is kept in equilibrium with excess of drug. Systems with low complexity and easy preparation are still in demand to enable the use of all-aqueous structures as effective retention and controlled release devices for the administration of LMW drugs, namely hydrophilic ones.

[0004] WO / 2005 / 063219 discloses gelatin capsules to encapsulate a small (206 Da) hydrophobic drug (ibuprofen).

[0005] US20220193200A1 discloses nanoparticles formed through complexation of tannic acid, a trivalent metallic ion, and a drug.

[0006] US2023201126A1 refers to inorganic materials and discloses a method of making a polymer deposit layer by layer.

[0007] Zakharova et al (2015) discloses a method for releasing hydrophobic molecules; the method is unsuitable for controlling the release of hydrophilic molecules.

[0008] EP1135021B1 discloses a sustained release dispersion of water insoluble beads each bead comprising a polymeric matrix comprised of a protein and a polysaccharide and containing at least one volatile hydrophobic component for release therefrom in atmospheric air.

[0009] US2011189240A1discloses a method for encapsulating cellular material; the method does not refer to the controlled release of small molecules.

[0010] CN115501173A discloses a natural polyphenol-based hierarchical porous hydrogel drug sustained release system and preparation method thereof; the system relies on a metallic ion that controls the release of the drug.

[0011] The invention solves this problem by providing a fully aqueous system that forms core-shell capsules through the interfacial solidification of food-grade polymers, specifically using alginate and ε-poly-L-lysine, followed by the addition of a polyphenol such as tannic acid. The system of the invention reduces the molecular weight cut-off (MWCO) of the polymeric mesh, effectively preventing the transport of small-sized molecules through the membrane. The process is simple, rapid, and conducted under mild conditions, making it energy-efficient and environmentally friendly. This innovation provides a versatile and efficient solution for the controlled permeability of small molecules, with potential applications in pharmaceutical, medical, and agricultural fields.

[0012] The invention addresses the technical problem of retaining and controlling the release of small molecules in aqueous environments by developing a fully aqueous system formed through the interfacial solidification of food-grade polymers. This system creates core-shell capsules that can retain molecules having a molecular weight within the range of 100 Da to 10000 Da. The key feature of the invention is the use of two oppositely charged polyelectrolytes to form a solid membrane upon complexation and add a polyphenol that closes the membrane thereby formed, preventing the transport of small-sized molecules across said membrane.

[0013] In a particular aspect of the present invention a membrane is formed by complexation by the addition of two oppositely charged polyelectrolytes, such as alginate and ε-poly-L-lysine, to which a polyphenol such as tannic acid is added to close the membrane, preventing the transport of small-sized molecules across said membrane. This approach allows for the controlled permeability of small molecules without relying on chemical interactions between the matrix and the encapsulated molecule, making the system versatile for various applications, including drug delivery, biosensors, and on-demand reactions.

[0014] The system is processed under mild conditions, i.e room temperature and pressure, and is rapidly formed, distinguishing it from existing methods like layer-by-layer technology, which are time-consuming and require prolonged immersion in aqueous environments. The invention's reliance on the molecular weight cut-off (MWCO) of the polymeric mesh, rather than chemical interactions, ensures effective retention and controlled release of both hydrophobic and hydrophilic drugs. Additionally, the system can be formed from polymers obtained from renewable sources, making it environmentally friendly and suitable for pharmaceutical, medical, and agricultural applications.

[0015] The invention utilizes an all-aqueous system, avoiding the use of organic solvents and toxic molecules. This makes it environmentally friendly and reduces the environmental burden typically associated with pharmaceutical processing.

[0016] Within the meaning of the invention, the term “all-aqueous” and “aqueous-based” means that the solvents used are exempt from organic solvents. The compositions according to the present invention are prepared based on inorganic aqueous solvents and may include any number of active ingredients and / or excipients, and can be in the form of a pharmaceutical dosage form, such as a capsule, a solution, a syrup, a suspension, a gel, etc., a fiber, a multibranched structure, a membrane or combinations thereof.

[0017] Within the meaning of the invention, the term “modified-release” means that the system is capable of retaining, retard and / or control the release of molecules across the form that the system takes, for example, across the walls of a capsule that encompasses said molecule.

[0018] The system operates under mild conditions, i.e room temperature and pressure, which translates to low energy consumption during processing. This is advantageous for large-scale applications where energy efficiency is crucial.

[0019] In a particular aspect, the invention allows for the rapid formation of core-shell capsules through interfacial complexation, significantly faster than traditional layer-by-layer (LbL) techniques. The entire process, including the addition of polyphenol, can be completed in just a few minutes.

[0020] The system can retain and control the release of small molecules, i.e molecules with molecular weight within the range of 100 Da to 10000 Da, based on the molecular weight cut-off (MWCO) of the polymeric mesh, rather than relying on chemical interactions. This ensures efficient encapsulation and controlled release of both hydrophilic and hydrophobic drugs.

[0021] The polymers used in the system are derived from renewable sources and are biodegradable, which enhances sustainability and reduces the dependency on animal sources.

[0022] The system can form structures with various shapes and sizes, including capsules and fibers, ranging from nanoscale to macroscale. This versatility allows for tailored applications in drug delivery, biosensors, and on-demand reactions.

[0023] The invention can be applied in multiple industries, including pharmaceuticals, medicine, agriculture, and diagnostics. It can be used for drug delivery, as sensors, and for external factor-driven on-demand reactions.

[0024] The present invention relates to an aqueous-based carrier system formed through the interfacial solidification of polymers. More particularly, the invention relates to multifunctional materials that are able to retain, control release and / or mediate the entrance of molecules, exhibiting potential to be applied on pharmaceutical, biomedical and agriculture fields. The disclosure further relates to biosensors and external factor-driven on-demand reactions.

[0025] The invention relates to an aqueous-based system formed through the interfacial complexation of two oppositely charged polyelectrolytes and a polyphenol. In a particular aspect of the invention the oppositely charged polyelectrolytes are alginate (ALG) and ε-poly-L-lysine (EPL) and the polyphenol is tannic acid. Upon complexation the oppositely charged polyelectrolytes and polyphenol creates a solid membrane with a molecular weight cut-off (MWCO) ranging from 100 Da to 10000 Da. The addition of polyphenol, such as tannic acid, helps to close the membrane, preventing the transport of small-sized molecules through it. The system is processed under mild conditions (room temperature and pressure) and is rapidly formed, with the complexation occurring in 5 minutes and the polyphenol addition taking 2 minutes.

[0026] The system distinguishes itself from prior art by being fully aqueous, processed in mild conditions, and capable of retaining both hydrophobic and hydrophilic drugs. The retention and release of molecules are driven by the MWCO of the mesh rather than chemical interactions between the matrix and the encapsulated molecule. The system can be formed from polymers obtained from renewable sources, and / or food-grade polymers, and can be designed in various forms, shapes and sizes such as a pharmaceutical dosage form, a capsule, a fiber, a multibranched structure, a membrane, or combinations thereof. The system can also incorporate ceramic or metallic nanoparticles to further close the mesh and can be used for drug delivery, biosensors, and external factor-driven on-demand reactions.

[0027] In an embodiment, the present disclosure takes advantage of the interfacial complexation of two oppositely charged polyelectrolytes to reduce the molecular weight cut-off (MWCO) of the polymeric mesh. The close closure of the membrane is guaranteed by the use of a polyphenol which helps preventing the transport of small-sized molecules through the membrane.

[0028] The present disclosure distinguishes itself from the systems of the prior art because it is fully aqueous and processed in mild conditions (room temperature and pressure), requiring low energy. Furthermore, the system herein disclosed is rapidly processed when compared to previous processes such as the layer-by-layer technique. The system herein disclosed is formed in quickly with the complexation step taking place in 5 minutes and the following stage, the addition of the polyphenol and consequent closure of the membrane, taking place in 2 minutes.

[0029] Additionally, the present disclosure distinguishes itself from the prior art because the retention and release in known systems typically relies on chemical interaction between the matrix and the encapsulated molecule. The retention and release from the system herein disclosed is mainly driven by the MWCO of the mesh, which is smaller than the molecular weight of the molecule that is retained.

[0030] Furthermore, the present disclosure distinguishes itself from the prior art because the systems known from the prior art depend on animal sources and can only retain hydrophobic drugs. The system herein disclosed, in turn, can be formed from polymers obtained from exclusively renewable sources and can retain both hydrophobic and hydrophilic drugs.

[0031] Also, the present disclosure distinguishes itself from the prior art because the system herein is capable of forming structures with shape and size versatility.

[0032] The present disclosure concerns an aqueous-based modified-release carrier system comprising two oppositely charged polyelectrolytes and a polyphenol wherein the oppositely charged polyelectrolytes are alginate (ALG) and ε-poly-L-lysine (EPL) and the polyphenol is selected from tannic acid, gallic acid, caffeic acid or ellagic acid.

[0033] In a particular aspect of the invention, the present disclosure concerns an aqueous-based modified-release carrier system that further comprises a ceramic nanoparticles, metallic nanoparticles, or combinations thereof.

[0034] In a particular aspect of the invention, the present disclosure concerns an aqueous-based modified-release carrier system is in the form of a pharmaceutical dosage form, a capsule, a fiber, a multibranched structure, a membrane, or combinations thereof.

[0035] In a particular aspect of the invention, the present disclosure concerns an aqueous-based modified-release carrier system that is in the form of a capsule wherein the dimension of the capsule ranges from 100 nm diameter to 100 mm diameter, in particular within the range of 2.5 to 3 mm.

[0036] In a particular aspect of the invention, the present disclosure concerns an aqueous-based modified-release carrier system comprising 2 wt% alginate (ALG), 0.75 wt% ε-poly-L-lysine (EPL) and a polyphenol in a range of 0.1 to 1.2% w / v.

[0037] In a most particular aspect of the invention, the present disclosure concerns an aqueous-based modified-release carrier comprising 2 wt% alginate (ALG), 0.75 wt% ε-poly-L-lysine (EPL) and 0.1, 0.2, 0.4, 0.8 or 1.2% w / v tannic acid.

[0038] In a most particular aspect of the invention, the present disclosure concerns an aqueous-based modified-release capsule comprising 2 wt% alginate (ALG), 0.75 wt% ε-poly-L-lysine (EPL) and 0.4% w / v tannic acid.

[0039] In another aspect of the invention, the present disclosure concerns an process of producing the aqueous-based modified-release carrier system herein described, the process comprising the steps of (i) dissolving or dispersing the alginate (ALG) and ε-poly-L-lysine (EPL) in aqueous solvents at a temperature ranging from 5ºC to 45ºC for 5 minutes, (ii) add tannic acid at pH 7 and agitate for 2 minutes, (iii) wash with acidic buffer until pH 4 is reached, and (iv) wash with alkaline buffer until pH 7 is reached.

[0040] In a particular aspect of the invention, the present disclosure concerns a process of producing the aqueous-based modified-release carrier system herein described, the process comprising an acidic buffer that is acetate buffer and comprising an alkaline buffer that is phosphate buffer saline.

[0041] In a particular aspect of the invention, the present disclosure concerns a process of producing the aqueous-based modified-release carrier system herein described, wherein the system is in the form of a capsule for carrying a pharmaceutical molecule.

[0042] In another aspect of the invention, the present disclosure concerns the use of the aqueous-based modified-release carrier system herein described for drug delivery, biosensoring, external factor-driven on-demand reaction, or diagnosis.

[0043] In a particular aspect of the invention, the system herein disclosed can be used for drug delivery.

[0044] In a particular aspect of the invention, the system herein disclosed can also be used as a sensor.

[0045] The present disclosure is also related to external factor-driven on-demand reactions.

[0046] Epsilon-poly-L-lysine is naturally produced by microbial synthesis and is biodegradable, water soluble and exhibits relatively low toxicity.

[0047] Alginate is a natural anionic polysaccharide consisting of β-D-mannuronic acid and α-L-guluronic acid monomers and is widely applied in the fabrication of biomaterials.

[0048] Tannic acid is a natural occurring polyphenol used for several medical applications owing to its antibacterial and antioxidant properties. Moreover, owing to the abundant hydroxyl groups and aromatic rings in its structure, this hydrolysable tannin can establish intermolecular interactions with several molecules, being also reported for drug delivery applications.

[0049] In an embodiment, the system is formed by any positively charged polyelectrolyte (cationic guar gum, chitosan, pectin, polyallylamine hydrochloride, polydiallyldimethylammonium chloride, polyethyleneimine, polylysine, poly(4-vinylpyridine), polyvinylamine, quaternised chitosan) and negatively charged polyelectrolyte (alginate, carboxymethyl cellulose, hyaluronic acid, poly(acrylic acid), poly(methacrylic acid), poly(styrene sulfonate), polyvinyl sulfonic acid).

[0050] In an embodiment, the present disclosure relates to a system formed through the interfacial complexation of epsilon-poly-L-lysine and alginate, followed by the addition of a polyphenol.

[0051] In an embodiment, the polyphenol is selected from tannic acid, gallic acid, caffeic acid or ellagic acid.

[0052] In a particular embodiment the present disclosure relates to a system formed through the interfacial complexation of 2 wt% alginate, 0.75 wt% ε-poly-L-lysine and a polyphenol in a range of 0.1 to 1.2% w / v, preferably 0.4% w / v of tannic acid.

[0053] In an embodiment, the system is formed by polymers with molecular weight ranging from 1000 to 8000000 Da.

[0054] In an embodiment, the system is formed by polymers crosslinked by irradiation (e.g., polyethylene, polyethylene terephthalate, polyisoprene, polypropylene, polyurethane, polyvinyl alcohol, polyvinyl chloride).

[0055] In an embodiment, the system is formed by polymers modified with click chemistry-enabling motifs such as for example alkene, alkyne, azide, epoxy, isocyanate and thiol.

[0056] In an embodiment, said system is formed via ionic interactions, covalent bonds, hydrogen bonds, van der Waals interactions, click chemistry or Schiff base.

[0057] In an embodiment, the closure of the mesh can be performed through the addition of ceramic nanoparticles (alumina (Al2O3), barium titanate (BaTiO3), cerium oxide (CeO2), copper oxide (CuO), hafnium oxide (HfO2), nanostructured zirconia (ZrO2), silica (SiO2), titanium dioxide (TiO2), yttrium oxide (Y2O3), zinc oxide (ZnO), zeolites) or metallic nanoparticles (aluminium (Al), cobalt (Co), copper (Cu), europium (Eu), gold (Au), iron oxide (Fe3O4and Fe2O3), nickel (Ni), palladium (Pd), platinum (Pt), samarium (Sm), silver (Ag), titanium (Ti)) to fill the voids of the mesh.

[0058] In an embodiment, the system can be designed as a pharmaceutical dosage form, a capsule, a solution, a syrup, a suspension, a gel, a fiber, fibers at macrometer scale, fibers at micrometer scale, fibers ranging from macro- to micrometer scale, a multibranched structure, a membrane or combinations thereof.

[0059] To promote an understanding of the principles by the embodiments of the present invention, reference will be made to the embodiments illustrated in the figures and to the language used to describe the same. It must be understood that there is no intention of limiting the scope of the present invention to the contents of the figures. Any alterations or later changes of the inventive features illustrated herein, and any additional application of the principles and embodiments of the invention shown, which would occur normally for one skilled in the art when reading this description, are considered as being within the scope of the claimed invention.Fig.1

[0060] a)Schematic representation of the formation of the capsules through polyelectrolyte complexation between alginate (ALG) and ε-poly-L-lysine (EPL).b)Schematic representation of the procedure followed to study the ability of pre-made capsules to prevent the permeation of small molecular weight molecules.c)Analysis of the permeability of the capsules to dyes varying the complexation time and phase II pH value.d)Encapsulation of methylene blue (MB, Mw=320 Da, positively charged). The complexation conditions to produce the capsules include a complexation time of 5 minutes and phase II pH value of 7 (left) or 5 (right). In the left side of each photo, there is a droplet of the medium at which the capsules were incubated with the dye; in the right side, there is a broken capsule and its dye release. Scale bar: 1 cm.e)Analysis of the permeability of the capsules to dyes after exposure to 0.04% TA, at either pH 4 or pH 7, for 60 minutes, varying the complexation time and phase II pH value.f)Chemical structure, charge, and molecular weight (Mw) of MB and Allura red AC (AR).g)Encapsulation of MB (Mw=320 Da, positively charged) (top) and AR (Mw=496 Da, negatively charged) (bottom). The complexation conditions to produce the capsules include phase II pH value of 5 and a complexation time of 5 minutes. Capsules were then soaked in 0.04% TA for 60 minutes and dipped in the respective dye. The control capsules were soaked in dH2O instead of TA, for 60 minutes. In the left side of each figure, there is a droplet of the medium at which the capsules were incubated with the dye; in the right side, there is a broken capsule and its content release. Scale bar: 1 cm.Fig.2

[0061] a)Analysis of the permeability of the capsules to dyes after exposure to 0.04% TA, at pH 7, for 5, 15 and 30 minutes, varying the complexation time and phase II pH value.b)Encapsulation of MB (Mw=320 Da, positively charged) (top) and AR (Mw=496 Da, negatively charged) (bottom). The complexation conditions to produce the capsules include phase II pH value of 5 and a complexation time of 5 minutes. Capsules were then soaked in 0.04% TA for 30 minutes and dipped in the respective dye. The control capsules were soaked in dH2O instead of TA, for 30 minutes. In the left side of each figure, there is a droplet of the medium at which the capsules were incubated with the dye; in the right side, there is a broken capsule and its content release. Scale bar: 1 cm.c)Analysis of the permeability of the capsules to dyes after exposure to TA solutions with different concentrations, for 2, 5, 10 and 15 minutes.d)Encapsulation of MB (Mw=320 Da, positively charged) (top) and AR (Mw=496 Da, negatively charged) (bottom). The complexation conditions to produce the capsules include phase II pH value of 5 and a complexation time of 5 minutes. Capsules were then soaked in 0.4% TA pH 7 for 2 minutes, washed with phosphate buffered saline (PBS), and dipped in the respective dye. The control capsules were soaked in dH2O instead of TA, for 2 minutes. In the left of each figure, there is a droplet of the medium at which the capsules were incubated with the dye; in the right, there is a broken capsule and its content release. Scale bar: 1 cm.e)Oxidation of the capsules with 10 M NaOH. i) Inner content of control capsules after exposure to NaOH. ii) Membrane of control capsules after exposure to NaOH. iii) Inner content of TA capsules after exposure to NaOH. iv) Membrane of TA capsules after exposure to NaOH.Fig.3

[0062] a)Schematicrepresentation ofthe procedure followed to test the dye release from the capsules.b)Release of AR in PBS (pH 7) and in acetate buffer (pH 4) from capsules without (control) and with TA, after exposure to each buffer for 30 min and overnight. Scale bar: 1 cm.c)Release of MB in PBS (pH 7) and in acetate buffer (pH 4) from capsules without (control) and with TA, after exposure to each buffer for 30 min and overnight. Scale bar: 1 cm.Fig.4

[0063] Dynamic light scattering (DLS) graphs obtained fora)a solution of 0.4% TA at pH 7,b)a solution of AR at pH 7,c)a mixture of 0.4% TA pH 7 and AR pH 7,d)a mixture of 0.4% TA pH 7 and MB pH 7. Each line refers to one replicate.Fig.5

[0064] Interaction of 0.4% TA pH 7 with MB (left) and AR (right), at pH 7.Fig.6

[0065] Interaction of TA with a) AR and b) MB. Results obtained after collecting the inner content of capsules, followed by oxidation with 10 M NaOH and 2 centrifugation steps (3689 x g for 5 min; 5902 x g for 10 min).Fig.7

[0066] Optimization of the post-treatment of alginate / EPL capsules with TA, to reach low permeability and dye-capsule binding. Results are representative of studies with AR and MB.Fig.8

[0067] Cross section images of control and TA-treated capsules.Fig.9

[0068] Visual permeability and dye-membrane interactions of different dyes after 1 hour of incubation with control and TA-treated membranes. A sample collected outside dye medium is shown as a reference for comparison with the released capsule content after rupture with a scalpel.Fig.10

[0069] Study of dye-membrane interactions and permeability with capsules treated with increasing concentrations of TA (0.1, 0.2, 0.4, 0.8, 1.2% w / v). Results for immersion at pH 7, for 1 hour.Fig.11

[0070] Schematic representation of the loading protocol for Method 1 - Saturation Approach.Fig.12

[0071] Schematic representation of the loading protocol for Method 2 - Weakening Approach.Fig.13

[0072] Release studies of different dyes using different loading methods (PBS at pH 7.2; acetate buffer at pH 4).Description of Description

[0073] The present disclosure provides an aqueous-based system, its processing method and use in drug delivery and barrier-like devices for on-demand triggering of reactions for sensing or diagnosis.Examples

[0074] EXAMPLE 1 – Preparation of core capsules

[0075] Core-shell capsules were processed by the promotion of the complexation between alginate (ALG) and ε-poly-L-lysine (EPL). Droplets of a solution of ALG (phase I) were dispersed in a larger volume bath of EPL (phase II) (Figure 1a) and complexation under agitation was left to occur for pre-determined times, in particular 2 minutes, 5 minutes, and 15 minutes. The capsules were then washed with a solution of acetate buffer (pH 4).

[0076] Concentrations of 0.75 wt% EPL and 2 wt% ALG were selected.

[0077] Permeation studies were performed with dyes dissolved in a buffer (PBS) at pH 7, and capsules were immersed in the solutions for the time needed to apparent equilibrium between the colour of the dye-laden buffer and the liquid volume in the interior of the capsules was achieved, that was 60 minutes in particular (Figure 1b, c, d).

[0078] The pH value of phase II – both acidic (pH 5) and neutral (pH 7) conditions - was also studied in combination with different complexation times, in particular 2 minutes, 5 minutes, and 15 minutes (Figure 1c). Regardless of the combination of complexation time and the pH value of phase II, methylene blue (MB; Mw=320 Da) uptake from the outside to the inside occurred within 60 minutes (Figure 1c,d).

[0079] Impermeable capsules to LMW molecules were obtained by adding tannic acid (TA) to the system after the complexation of polyelectrolytes.

[0080] EXAMPLE 2 – Post-treatment of capsules

[0081] A low concentration of 0.04% (w / v) – a value in the range of TA concentrations typically used in LbL-coating approaches – was selected to perform a post-treatment to capsules. The effect of adding TA solutions at pH values of 4 and 7 were tested (Figure 1e).

[0082] To complement results obtained with positively charged MB, Allura Red AC (AR) – a negatively charged food dye with red colour (496 Da) – was also tested (Figure 1f). Overall, the post-treatment of capsules with 0.04% TA for 60 minutes in agitation led to the apparent impermeabilization or achievement of very low passage of both positively (MB) or negatively (AR) charged small dyes (Figure 1e,g), as observed by the transparent or faint colour of the inner volumes of the capsules, as observed after membrane rupture (Figure 1g). Despite the overall apparent reduction of the MWCO conferred by TA, the interaction of the dyes with the membrane was altered according to the pH value used during TA adsorption (Figure 1g). While for capsules modified with TA at pH 4 the interaction of both dyes was observed, capsules modified with TA at pH 7 seemed capable of decreasing the adherence of the dyes (Figure 1g).

[0083] A shorter processing time of capsules was tested and the time of TA was reduced from 60 minutes to 5, 15 and 30 minutes (Figure 2a). For 0.04% TA, 30 minutes of exposure to TA prior to dye loading enabled the tailoring of the MWCO of capsules (5 minutes complexation; EPL at pH 5) (Figure 2b), with results similar to the ones obtained for 60 minutes (Figure 1g). Therefore, this condition was selected to perform further studies with increasing concentrations of TA, in particular 0.08%, 0.2% and 0.4%, and reduced times of post-modification step, in particular 2 minutes, 5 minutes, 10 minutes, and 15 minutes (Figure 2c). The exposure of the capsules to a TA solution of 0.4% (w / v), at pH 7, for 2 minutes (Figure 2d) enables the faster processing of capsules.

[0084] The presence of TA in the membranes was verified by immersing the capsules in a solution of 10 M NaOH (Figure 2e). The colour transition from colourless to a darker tonality in the TA capsules was instantaneous and proved the presence of this polyphenol.

[0085] Dye release studies were conducted by loading capsules with dye solutions, at pH 7 for 60 minutes, before the post-treatment with TA. The immersion of capsules for 60 minutes in dye solutions was enough to ensure a comparable colour observed for the inner and outer volumes, suggesting that an equilibrium was reached after that time. Dye-loaded capsules were then immersed in a 0.4% (w / v) solution of TA mixed with dye, in the same concentration as the solution initially used to load the TA-free capsules. The capsules loaded with dyes and post-treated with 0.4% TA and dye for 2 minutes were then briefly washed with either acidic buffer (acetate buffer, pH 4) or PBS (pH 7), depending on the respective solution used for the release assays (Figure 3a). The release of the dyes was registered after 30 minutes and an overnight period (Figure 3b,c). For AR, an apparently free diffusion of the molecule was observed in solutions both at pH 4 and 7 (Figure 3b). Upon the addition of TA, the release of the molecule from capsules at pH 7 seemed impaired, with neglectable release up to overnight incubation. At pH 4, however, the release of AR was comparable to the one in TA-free capsules.

[0086] The inner volume of capsules was treated with 10 M NaOH and a brown colour was observed, indicating the presence of the oxidized form of TA inside the capsules (Figure 2e).

[0087] The interaction of TA with the encapsulated cargo was assessed through dynamic light scattering (DLS) analysis () and through the mixture of TA with dye (Figures 5 and 6a). DLS analysis () corroborated the easily observable formation of aggregates when the contact of MB with TA is promoted (Figures 5 and 6b). At pH 4, the TA protective effect was lost for encapsulated AR, and a diffusion similar to the one of TA-free capsules was observed (Figure 3b).

[0088] The behaviour of the positively charged MB was assessed in a similar manner (Figure 3c). While at pH 7 the diffusion of MB to the medium was fast for TA-free capsules, at pH 4 the release of the dye was slower. A combined effect of dye chemistry and decrease of overall MWCO may be achieved for capsules immersed in acetate buffer. The addition of TA to the system enabled overall slower release profiles of MB from the capsules, regardless of the working pH, despite an apparently more striking effect at pH 7. The formation of MB-TA complexes at pH 7 is expected to have occurred, and may justify, at least partially, the higher retention of MB inside the TA-treated capsules.

[0089] EXAMPLE 3 – Permeability tests

[0090] Core-shell capsules were processed by the promotion of the complexation between alginate and ε-poly-L-lysine (EPL), both dissolved in phosphate buffer saline (PBS, pH 7.2), using a range of concentrations. Droplets of a solution of alginate at ~pH 7 (phase I) were dispersed in a larger volume bath of poly-L-lysine at pH 5 (phase II), allowing complexation under agitation to occur for 1 to 10 minutes. Droplets were dispersed at a constant flow using a syringe pump.

[0091] The test was conducted in capsules comprising 1% (w / v) alginate and 0.75% (w / v) EPL, with complexation time of 5 minutes. Capsules with diameters in the range of 2.5 to 3 mm were obtained.

[0092] Membrane permeability to dyes with different charges – methylene blue (MB - positively charged) and Allura red (AR - negatively charged) – was tested at pH 7. The effect of the post-treatment of capsules with different concentrations of TA and at different times was tested. Best results for both dyes (MB and AR) were obtained with 0.4% TA and 2 minutes of treatment ().

[0093] Tannic acid (TA) had the role of crosslinker of the membranes, capable of forming inter-TA bonds, as well as TA-membrane complexes which makes the capsules less permeable to dyes.

[0094] Scanning electron microscopy (SEM) images show an accumulation of TA in the outer region of the capsules, with some apparent aggregates, which corresponds to TA-TA microstructures (). The capsules are formed by a first layer of immediately reacted polyelectrolytes, followed by a tortuous diffused more dispersed inner region. The presence of TA in the membranes was confirmed by the immersion of capsules in 10 M NaOH, which immediately presented an oxidized brownish color.

[0095] The dependency of the system on the charge of dyes and their possible interactions with residual unreacted amine or carboxylic groups in the membranes, was tested with two dyes with zwitterionic properties (and reported neutral net charge at pH 7): fast green (FG) and methyl orange (MO).

[0096] The post-treatment of capsules with TA (0.4%, 2 min in agitation) in the MB and AR assays led to the impermeabilization of the capsules at pH 4, while dye affinity with the membrane was observed. At pH 7, no dye release after capsule burst, corresponding to a complete impermeabilization of the membrane and a significant adherence reduction of dye to the capsule membrane is observed. For MO and FG there was no significant difference between dye affinity towards the membrane at pH 4 or pH 7 post-treatment with TA. For FG, a noticeable decrease in permeability was observed at pH 7 after capsule disruption. For MO no changes in terms of permeability were observed.

[0097] An additional study with capsules treated with varying concentrations of TA, namely 0.1%, 0.2%, 0.4%, 0.8%, 1.2% (w / v) (). Dye interaction with the membrane and overall permeability was assessed for pH 7, after 1 hour of immersion. Even for the lowest TA concentration, MB, AR and FG were successfully prevented from permeating the capsule membrane. Additionally, MB interactions with the membrane showed a proportional decrease with increasing concentration of TA used to treat the capsules. FG showed a similar effect, although differences were more difficult to detect, suggesting that the interaction of FG with the membrane is not as sensitive to TA content as MB. An opposite effect was observed for AR and MO, with visually detected higher dye retention in the membranes for higher concentrations of TA. For MO, this result was particularly striking. Additional observational and DLS measurements (chromatography studies ongoing) to better characterize the affinity and tendency for the formation of aggregates between free dyes and solubilized TA). Of all dyes, MB immediately formed easily detected precipitates, while this phenomenon was not observed for the other dyes. As demonstrated in, TA is essentially bound to the polyelectrolyte complexed membrane, and not available in its free form to interact with its most favorable interacting dye (MB).

[0098] EXAMPLE 4 – Release tests

[0099] To improve the retention of small molecules a method for treating the system of the invention was developed. Tests show that after this further treatment the system is capable of maintaining a slow release of molecules.

[0100] The experiment started with the preparation of a system according to the invention in the form of capsules made from 2 wt% alginate (ALG), 0.75 wt% ε-poly-L-lysine (EPL) and 0.4% w / v of tannic acid.

[0101] In a first method – Method 1 following aSaturation Approach– a loading procedure based on the immersion of control (no TA) capsules in the dye solution until equilibrium was developed. The capsule was then “closed” with TA in the presence of the dyes (AR and MB used for optimization). This method is illustrated in.

[0102] The high affinity of some dyes (namely MB) with free TA, and the fact that MB precipitates upon the addition of TA to the reacting medium, suggests that the overall high loading efficiency and further slow / non-observable release of MB to the medium could be attributed to the formation of higher molecular weight MB-TA aggregates, and not to the ability of the membranes to retain the single molecule. Therefore, a second method was developed – Method 2 following aWeakening Approach– to enable the loading of dyes to the capsules after the immobilization of TA at the membrane. This method is illustrated in. In this method TA reacts with the alginate / EPL membrane becoming unavailable in its free form. This prevents the formation of significant amounts of dye-TA complexes. Therefore, the release of the free non-complexed small molecule is ensured. In Method 2, the capsule already modified with TA is “weakened” by exposure to pH 4 with an acidic buffer such as acetate buffer. This weakening corresponds to a partial loss of TA from the system, as previously illustrated in. Capsules are allowed to stay overnight in pH 4 and then return to a pH 7 with an alkaline buffer, such as a phosphate buffer, which closes the membrane again. Indeed, in an experiment conducted it was shown that AR entered more effectively at pH 4 in the TA-membrane, and that the release of the dye was slower at pH 7 when compared to a control sample.

[0103] Release studies of the loaded molecules were performed for both systems at pH 4 and pH 7, at 37ºC and in agitation. For Method 1, the TA treatment of the capsules enabled the achievement of a slower release of AR at pH 7 (), while in control samples a quicker burst release was observed. For pH 4, the treatment with TA did not lead to any significant effect. For FG both TA treatment and pH of the release media showed important effects. Treating capsules with TA treatment resulted in slower release profiles, both at pH 7 and 4. Also, slower release profiles were observed for samples in acidic environment. Also, in Method 1 the release of MO showed a slight sensitivity to pH (not shown), in a similar way as FG. Although not shown were, due to the high dye precipitation observed in the TA+dye medium, Method 1 also enabled the almost absolute retention of MB inside the capsules in TA-treated capsules at pH 7, similarly to the results shown to Method 2. While at pH 7 no release of MB was detected during the whole course of the experiment, at pH 4 both TA-treated and control capsules led to a controlled release profile of MB. For control samples at pH 7, a typical burst-release and subsequent stabilization of the release of MB at ~60 minutes was observed.

[0104] Release of FG with Method 2 was also studied. Results demonstrate the slower release of molecules in all conditions at pH 4, regardless of the presence of TA in the membranes.

[0105] While the examples described above illustrates a particular application of the system of the invention to capsules for the delivery of pharmaceutical, phytopharmaceutical or other small size molecules, it should be noted that the system may be processed in other forms or formats such as fibers with dimensions ranging from nanoscale to microscale, multibranched structures, membranes, or combinations or. The system of the invention may also be used in other settings and for other purposes other than delivering small molecules such as a sensor or for external factor-driven on-demand reactions.

[0106] The above-described embodiments are combinable. The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof.

[0107] As used in this description, the expression. “substantially” means that the real value is within an interval of about 10% of the desired value, variable or related limit, particularly within about 5% of the desired value, variable or related limit or particularly within about 1% of the desired value, variable or related limit.

[0108] Moreover, the term “or” is intended to mean an inclusive “or” rather than an exclusive “or.” That is unless specified otherwise, or clear from the context, the phrase “X employs A or B” is intended to mean any of the natural inclusive permutations. That is, the phrase “X employs A or B” is satisfied by any of the following instances: X employs A; X employs B; or X employs both A and B.

[0109] In addition, the articles “a” and “an” as used in this application and the appended claims should generally be construed to mean “one or more” unless specified otherwise or clear from the context to be directed to a singular form.

[0110] Further, as used herein, the term “exemplary” is intended to mean serving as an illustration or example of something and is not intended to indicate a preference.

[0111] The subject matter described above is provided as an illustration of the present invention and must not be interpreted to limit it. The terminology used to describe specific embodiments, according to the present invention, must not be interpreted to limit the invention. As used in this description, the definite and indefinite articles, in their singular form, aim to include in the interpretation the plural forms, unless the context of the description explicitly indicates the contrary. It will be understood that the expressions “comprise” and “include” when used in this description, specify the presence of the characteristics, the elements, the components, the steps, and the related operations, but do not exclude the possibility of other characteristics, elements, components, steps, and operations from being also contemplated.

[0112] All modifications, providing that they do not modify the essential features of the following claims, must be considered within the scope of protection of the present invention.

[0113] The invention can be applied to the following industries, fields, or areas of the economy:

[0114] The invention can be applied to the following industries, fields, or areas of the economy:

[0115] Medical Industry: The invention has applications in medical diagnostics and barrier-like devices for on-demand triggering of reactions for sensing or diagnosis.

[0116] Agriculture Industry: The invention can be applied in agricultural fields for controlled release of agrochemicals or other small molecules.

[0117] Biosensors: The invention can be used in the development of biosensors for various applications.

[0118] External Factor-Driven On-Demand Reactions: The invention can be utilized in systems that require on-demand triggering of reactions based on external factors.

Claims

Aqueous-based modified-release carrier systemcharacterised bycomprising two oppositely charged polyelectrolytes and a polyphenol wherein the oppositely charged polyelectrolytes are alginate (ALG) and ε-poly-L-lysine (EPL) and the polyphenol is selected from tannic acid, gallic acid, caffeic acid or ellagic acid.Aqueous-based modified-release carrier system according to the previous claimcharacterised byfurther comprising ceramic nanoparticles, metallic nanoparticles, or combinations thereof.Aqueous-based modified-release carrier system according to any of the previous claimscharacterised bythe fact the system is in the form of a pharmaceutical dosage form, a capsule, a fiber, a multibranched structure, a membrane, or combinations thereof.Aqueous-based modified-release carrier system according to claim 3characterised bythe fact that the dimension of the capsule ranges from 100 nm diameter to 100 mm diameter.Aqueous-based modified-release carrier system according to claim 4characterised bythe fact that the diameter of the capsule ranges from 2.5 to 3 mmAqueous-based modified-release carrier system according to any of the previous claimscharacterised bycomprising 2 wt% alginate (ALG), 0.75 wt% ε-poly-L-lysine (EPL) and a polyphenol in a range of 0.1 to 1.2% w / v.Aqueous-based modified-release carrier according to claim 6characterised bycomprising 2 wt% alginate (ALG), 0.75 wt% ε-poly-L-lysine (EPL) and 0.1, 0.2, 0.4, 0.8 or 1.2% w / v tannic acid.Aqueous-based modified-release capsulecharacterised bycomprising 2 wt% alginate (ALG), 0.75 wt% ε-poly-L-lysine (EPL) and 0.4% w / v tannic acid.Process of producing the aqueous-based modified-release carrier system of claims 1 to 8characterised bycomprising the step ofdissolving or dispersing the alginate (ALG) and ε-poly-L-lysine (EPL) in aqueous solvents at a temperature ranging from 5ºC to 45ºC for 5 minutes;add tannic acid at pH 7 and agitate for 2 minutes;wash with acidic buffer until pH 4 is reached; andwash with alkaline buffer until pH 7 is reached.Process of producing the aqueous-based modified-release carrier system according to claims 9characterised bythe fact that the acidic buffer is acetate buffer and the alkaline buffer is phosphate buffer saline.Process of producing the aqueous-based modified-release carrier system according to claims 9 or 10characterised bythe fact that the system is in the form of capsule for carrying a pharmaceutical molecule.Use of the aqueous-based modified-release carrier system of claims 1 to 11characterised bythe fact that the system is for drug delivery, biosensoring, external factor-driven on-demand reaction, or diagnosis.

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