System for the oral release of proteins

Abstract

The present invention relates to a mucoadhesive patch for the buccal or sublingual delivery of proteins or polypeptides, comprising at least 100 bilayers, each consisting of a chitosan (CHI) layer and an anionic polysaccharide layer. The present invention also relates to a method for producing such a patch.

Description

[Technical Field]

[0001] This invention relates to a system in the form of a mucosal-adherent patch for the oral release of proteins. [Background technology]

[0002] Oral administration of pharmaceuticals is the most widely used route of administration. However, due to the high sensitivity of proteins to the action of gastrointestinal enzymes, the release of therapeutically targeted proteins via this route is difficult. The oral mucosa is an attractive alternative route of administration because it avoids the first-pass effect, has low enzymatic activity and a physiological pH range, is anatomically accessible, and is well angiogenic. Systems for oral release can therefore provide either local (mucosal) or systemic (transmucosal) release of proteins. [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] WO2005 / 052035 [Overview of the project] [Problems that the invention aims to solve]

[0004] There remains an unresolved need for mucosal adhesion systems with optimized penetration capabilities, which would contribute to a significant reduction in the therapeutic dose administered and better control of absorption. [Means for solving the problem]

[0005] To address this need, the inventors have developed a mucosal adhesive patch consisting of a combination of two polysaccharides, chitosan (CHI) and anionic polysaccharides such as hyaluronic acid (HyA).

[0006] The patch of this invention, which adheres to the oral mucosa or sublingual mucosa, is called an "intraoral" patch or a "sublingual" patch.

[0007] The present invention therefore provides a mucosal adhesive patch for the oral or sublingual release of a protein or polypeptide, comprising at least 100 bilayers, each composed of a chitosan (CHI) layer and a layer of an anionic polysaccharide or salt thereof having a molecular weight between 500 and 1000 kDa, wherein the layer in contact with the mucous membrane and the layer in contact with the oral or sublingual environment are composed of chitosan (CHI), and the protein or polypeptide is incorporated into the patch and / or adsorbed onto the surface of the patch.

[0008] In a preferred embodiment, the present invention provides an intraoral or sublingual mucosal adhesive patch comprising a protein or polypeptide, comprising 100 to 200 bilayers, each composed of a chitosan (CHI) layer and a layer of an anionic polysaccharide or salt thereof having a molecular weight between 500 and 1000 kDa, wherein the anionic polysaccharide is hyaluronic acid, and the layer in contact with the mucous membrane and the layer in contact with the intraoral or sublingual environment are composed of chitosan (CHI), and the protein or polypeptide is incorporated into the patch and / or adsorbed onto the surface of the patch, and is intended for intraoral or sublingual release.

[0009] The patch is self-supporting and dissolves in saliva in the presence of enzymes that can break down two polysaccharides.

[0010] The present invention further, i) A step of forming a multilayer film of CHI / anionic polysaccharide on the substrate by alternating layer deposition (layer by layer deposition method), ii) Steps to remove the multilayer film from the substrate, iii) The process of bringing the multilayer membrane into contact with the protein, thereby loading the protein into the multilayer membrane. The present invention relates to a method for producing the patch, which includes a step comprising the steps described above.

[0011] The present invention further relates to a patch according to the present invention, which can be obtained by the method described herein. [Brief explanation of the drawing]

[0012] [Figure 1] This figure shows the thickness and degradation profiles of (CHI / HyA)100 self-supporting membranes. (A) Membrane thickness depending on the change in molecular weight of HyA (660kDa or 1020kDa, corresponding to HyA.LW and HyA.HW, respectively), or stacking time: short cycle or long cycle. The statistical significance of the difference between the two groups was determined using an ANOVA test with one controlled factor. ns: no significant difference, ****: p<0.0001. (B) Degradation profiles of membranes immersed in artificial saliva, expressed as the percentage of mass loss depending on various membrane fabrication parameters. (C) Scanning electron microscope (SEM) images of short-cycle (CHI / HyA660)-CHI membranes immersed in artificial saliva for 30 minutes, 1 hour, or 3 hours. The scale bar corresponds to 44 μm. (D) Images of short-cycle (CHIFITC / HyA660)-CHIFITC membranes obtained by confocal microscopy after 30 minutes, 3 hours, 6 hours, or 24 hours in artificial saliva. The control condition corresponds to immersion of the membrane in acetate buffer for 24 hours. a, b, c, d, and e are the membrane surface, and the lower cross-sectional images are of the same membrane in the depth direction (z-section). The horizontal scale bar corresponds to 50 μm. [Figure 2] This figure shows that the membrane degradation products are not toxic to human epithelial cells. (A) HeLa cells and (B) Ho-1u-1 cells and the degradation products after 24-hour incubation. The membrane is (CHI / HyA)100 membrane, and membrane.HCl is (CHI / HyA)100 membrane treated with hydrochloric acid (HCl). The statistical significance of the difference between the two groups was determined using an ANOVA test with one controlled factor. ns: No significant difference. [Figure 3-1] (Figure 3A) This figure shows the control of sublingual mucosal inflammation by membrane treatment with HCl. Mucosal thickness (A) and recruitment of MHCII-positive (MHCII+) cells (B) were evaluated in the entire mucosa (epithelium and lamina propria) after application of the membrane or 1-fluoro-2,4-dinitrobenzene (DNFB). Untreated membranes (patches) were administered for 30 or 60 minutes, and membranes treated with HCl (patch HCl) or NaCl (patch NaCl) were applied for 30 minutes. Each value provided corresponds to the mean of at least three measurements from three independent individuals (N=3). Statistical significance of the difference between two groups was determined using an ANOVA test with one controlled factor. ns: no significant difference. *: p<0.05. (Figure 3B) This figure shows the control of sublingual mucosal inflammation by membrane treatment with HCl. Mobilization of MHCII-positive (MHCII+) cells (B) was evaluated in the entire mucosa (epithelium and lamina propria) after application of a membrane or 1-fluoro-2,4-dinitrobenzene (DNFB). Untreated membranes (patches) were administered for 30 or 60 minutes, and HCl-treated membranes (patch HCl) or NaCl-treated membranes (patch NaCl) were applied for 30 minutes. Each value provided corresponds to the mean of at least three measurements from three independent individuals (N=3). Statistical significance of the difference between two groups was determined using an ANOVA test with one controlled factor. ns: no significant difference. *: p<0.05. (Figure 3C) Image showing MHCII+ cell mobilization at the mucosal level, obtained by histological labeling. Scale bar: 170 μm. [Figure 3-2](Figure 3D) This figure shows the levels of the inflammatory cytokine IL-1β in the tongue and sublingual tissue of mice, measured 6 hours after membrane application. The statistical significance of the difference between the two groups was determined using an ANOVA test with one controlled factor. ns: no significant difference. *: p<0.05, ***: p<0.001, ****: p<0.0001. (Figure 3E) This figure shows the levels of the inflammatory cytokine IL-6 in the tongue and sublingual tissue of mice, measured 6 hours after membrane application. The statistical significance of the difference between the two groups was determined using an ANOVA test with one controlled factor. ns: no significant difference. *: p<0.05, ***: p<0.001, ****: p<0.0001. (Figure 3F) This figure shows the levels of the inflammatory cytokine TNF-α in the tongue and sublingual tissue of mice, measured 6 hours after membrane application. The statistical significance of the difference between the two groups was determined using an ANOVA test with one controlled factor. ns: no significant difference. *: p<0.05, ***: p<0.001, ****: p<0.0001. [Figure 4] This is a schematic diagram illustrating protein integration in layer-by-layer (LbL) assemblies due to pH changes. After functionalization by protein trapping, the LbL membrane becomes a bioactive patch ready for immediate application. CHI: Chitosan, HyA: Hyaluronic acid. [Figure 5]This figure shows the integration and release profiles of ovalbumin labeled with Alexa Fluor 647 (OVAAlexa F.647) from the membrane. (A) Levels of OVAAlexa F.647 integration into (CHI / HyA)100-CHI patches after incubation with 0.5 μg of protein in HCl and rinsing in various buffers. (B) Profile of OVAAlexa F.647 release from the patches over a 2-hour rinsing time in various solutions. Data correspond to three mean values ​​and standard errors (±SEM) from three independent experiments (N=3). (C, top) Confocal microscope image of the surface of the patch containing OVAAlexa F.647. Scale bar corresponds to 50 μm. (C, bottom) Optical section image of the patch in the depth direction along the (z) axis. Dotted lines represent the patch boundaries. (D) Fluorescence intensity of OVAAlexa F.647 and the patch (CHI.MW.FITC) along the arrow indicated in C (bottom). [Figure 6] This figure shows the retention time of patches on the sublingual mucosa. (A) Staining with hematoxylin of (CHI / HyA)100 patches in contact with the sublingual mucosa 20 minutes after patch administration. The scale bar corresponds to 100 μm. M = mucosa, P = patch. (B) Molecular fluorescence tomography of OVAAF647 in solution or incorporated into (CHI / HyA)100 patches. Fluorescence signals were detected 2, 10, or 30 minutes after administration. [Figure 7] This figure shows the tissue penetration of OVAAF647 after patch administration. Confocal microscopy of DAPI-labeled mouse tongue sections 2 minutes after administration of OVAAF647 in solution, or 2, 10, 30, and 60 minutes after administration of OVAAF647 incorporated in (CHI / HyA)100 patches. OVAAF647 was observed in the keratinized layer 10 minutes after administration (white arrow) and in the submucosa 30 minutes after administration (dashed arrow). Scale bar: 20 μm. Dotted lines correspond to mucosal boundaries. [Figure 8]This figure shows that the chemotactic ability of the cytokine CCL20 is maintained in the patch. In vitro chemotaxis of CCL20 released from the patch compared to CCL20 in solution. A concentration of 25 ng × mL-1 was used to evaluate the migration of DC2.4 cells. Growth medium, salivary enzymes only, and (CHI / HyA)100 membrane were used as negative controls. Statistical significance of the difference between the two groups was determined using an ANOVA test with one controlled factor. ns: no significant difference, *: p<0.05, **: p<0.01. [Figure 9] This figure shows the thickness and resolution profiles of (CHI / HyA)100-CHI self-supporting membranes or (VIS / HyA)100-VIS self-supporting membranes. (A) Membrane thickness according to the number of bilayers, i.e., 50, 100, or 200 bilayers with HyA as the final layer. Membrane thickness made from 100 bilayers, with the final layer made from CHI obtained from Sigma or Viscosan® (VIS) obtained from Flexichem. Each type of membrane was measured at least 20 times. Statistical significance between the two groups was determined using an ANOVA test with one controlled factor. ns: no significant difference, ****: p<0.0001. (B) Resolution profiles of membranes immersed in artificial saliva, expressed as a percentage of mass loss, depending on the different types of membranes made from CHI or VIS. Each value shown corresponds to the mean of three measurements from three independent experiments (N=3). [Figure 10]This figure shows the integration and release profiles of OVAAF647 from the membrane. (A) Levels of OVAAF647 integration into the (CHI / HyA)100-CHI patch after incubation with 0.5 μg of protein in NaCl and rinsing in various buffers. (B) Profile of OVAAF647 release from the patch over a 2-hour rinsing time in various solutions. Data correspond to the average of three values ​​from three independent experiments. (C, top) Confocal microscopy image of the surface of the patch containing OVAAF647. Scale bar corresponds to 50 μm. (C, bottom) Optical section image of the patch in the depth direction along the (z) axis. Dotted lines represent the patch boundaries. (D) Fluorescence intensity of OVAAF647 and the (CHIFITC) patch along the arrows shown in Figure C (bottom). [Figure 11] This figure shows a scheme illustrating the trapping of proteins or polypeptides due to the expansion or contraction of a multilayer membrane. [Modes for carrying out the invention]

[0013] As described in the examples provided below, the inventors have developed a mucosal adhesive patch for oral or sublingual release of proteins, which ensures better control of the dose of administered proteins and their absorption by the mucosa. The inventors have also demonstrated the non-toxic characteristics of this type of patch and the absence of mucosal inflammation after application of this type of patch.

[0014] The present invention relates to a mucosal adhesive patch for the oral or sublingual release of a protein or polypeptide, comprising at least 100 bilayers, each comprising a chitosan (CHI) layer and a layer of an anionic polysaccharide or salt thereof having a molecular weight between 500 and 1000 kDa, wherein the layer of the patch in contact with the mucosa and the layer in contact with the oral or sublingual environment are composed of chitosan (CHI), and the protein or polypeptide is incorporated into the patch and / or adsorbed onto the surface of the patch.

[0015] definition A "patch" refers to a multilayer adhesive system or multilayer adhesive membrane containing biologically active compounds such as proteins or polypeptides.

[0016] "Mucosal-adherent" means a patch, as defined in this application, that can adhere to a mucous membrane, preferably the oral mucosa or sublingual mucosa.

[0017] "Self-supporting" refers to a structure that does not require a support, whose stability is derived from its own inherent rigidity.

[0018] The term "target" refers to all humans or animals, preferably mammals, such as horses, sheep, cows, dogs, cats, etc.

[0019] Proteins and polypeptides A patch according to the present invention enables the release of any desired protein or polypeptide. "Protein" typically means a polypeptide consisting of at least 100 amino acids, or a polypeptide linked together. The protein preferably has a molecular weight between 20 and 200 kDa. More specifically, the protein may have a molecular weight of less than 70 kDa, or a high molecular weight, for example, 100 to 200 kDa.

[0020] Among the target proteins, the inventors may refer to, for example, antibodies (such as immunoglobulin G).

[0021] Polypeptides, which may be protein fragments, typically have 10 to 100 amino acids, more preferably 20 to 80 amino acids or 20 to 60 amino acids.

[0022] In a preferred embodiment, the protein or polypeptide is the allergen.

[0023] Allergens as defined in this invention include antigens that can stimulate an allergic reaction in a subject. Allergens may be contained in or derived from foods such as milk, eggs, sesame, wheat, soybeans, fish, seafood, peanuts, and nuts. Allergens may also be contained in or derived from non-food items such as dust mites, pollen, insect bites, animal hair, wool, and pharmaceuticals. Preferably, allergens are polypeptides or proteins that form all or part of an antigen recognizable by cells of the immune system, and with respect to these, the inventors aim to induce tolerance to the allergen.

[0024] In an example of a preferred embodiment, the protein delivered is ovalbumin. The inventors can also mention beta-lactoglobulin, alpha-lactalbumin, and casein among preferred food allergens.

[0025] The present invention therefore relates to an allergen for use in desensitization of a subject allergic to the allergen, which is administered in the form of a patch as described herein. Desensitization of a subject, also known as allergic immunotherapy, allows the subject to become resistant to a particular allergen over a long period of time.

[0026] Multilayer patch The patch according to the present invention comprises at least 100 bilayers, each consisting of a chitosan (CHI) layer and a layer of an anionic polysaccharide or salt thereof having a molecular weight between 500 and 1000 kDa, wherein the layer in contact with the mucous membrane and the layer in contact with the oral environment or sublingual environment are composed of chitosan (CHI).

[0027] The patch according to the present invention is formed from a multilayer adhesive film comprising at least 100 bilayers, each composed of chitosan and the anionic polysaccharide, wherein two layers located at two ends or periphery of the film are composed of chitosan (CHI).

[0028] According to a particular embodiment of the present invention, the patch comprises 100 to 200 CHI / anionic polysaccharide bilayers. According to a preferred embodiment, the patch comprises 100 or 200 CHI / anionic polysaccharide bilayers, and more preferably 100 CHI / anionic polysaccharide bilayers.

[0029] According to another specific embodiment of the present invention, the patch has a thickness of 10 to 20 μm, preferably 11 to 19 μm, 12 to 18 μm, 13 to 17 μm, 14 to 16 μm, and also more preferably about 15 μm.

[0030] Chitosan is a polysaccharide composed of a random distribution of β-(1-4) linked D-glucosamine (deacetylated units) and N-acetyl-D-glucosamine (acetylated units). It is produced by the chemical deacetylation (in an alkaline medium) or enzymatic deacetylation of chitin, a component of the exoskeleton of arthropods (crustaceans), the endoskeleton of cephalopods (squid, etc.), or the cell walls of fungi. This raw material is typically demineralized by treatment with hydrochloric acid, then deproteinized in the presence of sodium hydroxide or potassium hydroxide, and finally decolorized with an oxidizing agent. The degree of acetylation (DA) is the percentage of acetylated units to the total number of units. This can be determined by Fourier transform infrared spectroscopy (FTIR) or titration with a strong base.

[0031] Preferably, the chitosan selected in the present invention has a degree of deacetylation (DD) of 75% or more, preferably 78% or more. Chitosan has the advantage of being one of the few natural cationic polysaccharides that is easy to use and also has good adhesion properties.

[0032] Anionic polysaccharides are polymers of the carbohydrate family, composed of several monosaccharides linked together by glycosidic bonds. According to the present invention, the polysaccharide or its salt has a molecular weight between 500 and 1000 kDa. Among anionic polysaccharides, the inventors may refer to, for example, glycosaminoglycans (GAGs), fucoidans, alginates, carrageenans, and urbans.

[0033] Glycosaminoglycans (GAGs) are long-chain, linear polysaccharides found in almost all tissues. The basic unit of a GAG is a disaccharide consisting of a hexose (generally hexuronic acid) linked to a hexosamine. One of the characteristic features of these oligosaccharide chains is their remarkable heterogeneity. In fact, countless combinations arise from the varying lengths of the chains and modifications to their structure (sulfation, epimerization). Depending on the properties of the monosaccharides and the way the disaccharides are linked together, GAGs are classified into five major families: heparin (Hp) and heparan sulfate (HS), hyaluronic acid (HA), chondroitin sulfate (CS), dermatan sulfate, and keratan sulfate.

[0034] Fucoidan is a polysaccharide that contains fucose as a basic sugar. The name fucoidan comes from the Fucus genus (brown algae) in which it is produced.

[0035] Alginates are polysaccharides obtained from brown algae. Alginates are polymers formed from two monomers, mannuronic acid and guluronic acid, which are linked together by a β-1-4 bond.

[0036] Carrageenan is a polysaccharide (galactan) extracted from red algae. Three main categories are available on the market today: κ-carrageenan, ι-carrageenan, and λ-carrageenan, which differ in the number and position of sulfate groups, as well as the number of 3,6-anhydrogalactose crosslinks.

[0037] Urbanes are sulfated anionic polysaccharides extracted from Ulva-type green algae. Urbanes consist of two types: urbanobiuronate 3-sodium sulfate type A, which contains 3-rhamnose sulfate linked to glucuronic acid by a 1-4 type linkage, and urbanobiuronate 3-sodium sulfate type B, which contains 3-rhamnose sulfate linked to iduronic acid by a 1-4 type linkage.

[0038] According to a particular embodiment of the present invention, the anionic polysaccharide or salt thereof is selected from glycosaminoglycans (GAGs), fucoidans, alginates, carrageenans, and urbans. More specifically, the glycosaminoglycan (GAG) or salt thereof is selected from hyaluronic acid (HA), heparin (Hp), heparan sulfate (Hs), chondroitin sulfate (CS), dermatan sulfate (DS), and keratan sulfate (KS), preferably hyaluronic acid, and more preferably sodium hyaluronate.

[0039] Preferably, the anionic polysaccharide as defined above is hydrolyzable by salivary enzymes.

[0040] Manufacturing of mucosal adhesive patches Mucosal adhesive patches according to the present invention can be produced by an automated immersion process that assembles a positively charged layer (chitosan) and a negatively charged layer (anionic polysaccharide). This layer-by-layer (LbL) method is familiar to those skilled in the art and has been used in particular in international application WO2005 / 052035 for the preparation of multilayer films of crosslinked polymer electrolytes.

[0041] The multilayer film thus formed is then brought into contact with a protein or polypeptide to provide a mucosal adhesive patch loaded with the protein or polypeptide.

[0042] The present invention is therefore a method for manufacturing the patch described herein, i) A step of forming a multilayer film of CH / anionic polysaccharide by an alternating layering (LbL) method on a substrate, ii) Steps to remove the multilayer film from the substrate, iii) A step of bringing a multilayer film into contact with a protein or polypeptide, thereby loading the protein or polypeptide into the multilayer film. The present invention relates to a method that includes a process consisting of the following steps.

[0043] Step i) of this method, which consists of forming a multilayer film of CH / anionic polysaccharides, is carried out using the technique of alternating layering (LbL) on a substrate. This automated technique uses an immersion robot, for example, the DR-3 robot from Riegler & Kirstein GmbH. More specifically, the chitosan solution and the anionic polysaccharide (polyelectrolyte) solution are prepared in a buffer solution such as sodium acetate buffer solution. The pH of the solution can be adjusted to about 5.5 with sodium hydroxide (NaOH) and acetic acid (CH3COOH). Furthermore, the substrate used as a support in the immersion robot is prepared on an adhesive tape. A commonly used substrate is polypropylene. The substrate is then continuously immersed in the chitosan solution and the anionic polysaccharide solution, with at least one washing step, preferably two washing steps, in sodium acetate buffer solution, water, or buffered saline between pH 5 and 6, preferably in sodium acetate buffer solution. The formation of a CHI / anionic polysaccharide bilayer is made possible by a cycle comprising immersion of the substrate in a chitosan solution, at least one washing step, preferably one washing step, and subsequent immersion in an anionic polysaccharide solution and at least one washing step, preferably one washing step. This cycle is repeated the number of times necessary to obtain the desired number of bilayers. The immersion time and washing time may be varied. According to a particular embodiment, the immersion time of the substrate in the polyelectrolyte (CHI and anionic polysaccharide) solution is between 2 and 10 minutes, preferably between 2 and 8 minutes, and more preferably between 3 minutes (short cycle, SC) or 6 minutes (long cycle, LC). According to another particular embodiment, the washing time is between 1 and 10 minutes, preferably between 2 and 4 minutes, and more preferably between 2 minutes (short cycle, SC) or 4 minutes (long cycle, LC).

[0044] Step ii) of this method, which consists of removing the multilayer film from the substrate, particularly from polystyrene, may be accompanied by an optional prior drying step. The resulting multilayer film may then be cut to the required size.

[0045] Step iii) of this method makes it possible to load a protein or polypeptide into the multilayer membrane. More precisely, the loading of the protein or polypeptide into the multilayer membrane is achieved by passive diffusion. In particular, step iii) is carried out by dissolving the protein or polypeptide in a suitable buffer solution to prevent protein denaturation. According to a particular embodiment, the multilayer membrane is contacted with the protein or polypeptide in an acidic solution, preferably hydrochloric acid (HCl) with a pH between 2 and 4, preferably with a pH of about 3. According to another particular embodiment, the multilayer membrane is contacted with the protein or polypeptide in a saline solution, preferably a sodium chloride (NaCl) solution or potassium chloride (KCl) solution with a pH between 5 and 7, preferably with a pH of about 6.5.

[0046] Preferably, contact between the multilayer membrane and the protein or polypeptide is achieved by placing at least one droplet of a buffer solution containing the protein or polypeptide onto the surface of the multilayer membrane. The membrane is then optionally dried to provide a mucosal adhesive patch in which the protein or polypeptide is incorporated and / or adsorbed onto the surface of the patch.

[0047] Preferably, and especially in cases where the protein or polypeptide remains at least partially adsorbed on the surface, the resulting patch is polarized in the sense that the concentration of the protein or polypeptide is higher in the upper layer where the protein or polypeptide is placed compared to the concentration in the lower layer.

[0048] The method may also include an optional step (referred to as step iii-0) of equilibrating the multilayer membrane before contacting it with a protein or polypeptide. This equilibration step involves immersing the multilayer membrane obtained after step ii) in an acidic solution with a pH between 2 and 4, preferably an acidic solution consisting of hydrochloric acid (HCl), or in physiological saline with a pH between 5 and 7, preferably a physiological saline consisting of sodium chloride (NaCl). This optional equilibration step of the multilayer membrane at an acidic pH allows for expansion of the membrane in particular, thereby promoting the loading of proteins or polypeptides. The multilayer membrane loaded with proteins or polypeptides may shrink when immersed in a buffer solution, thus enabling the trapping of proteins or polypeptides into the patch.

[0049] According to a particular embodiment, the method of the present invention further includes an intermediate step, before step iii) is performed, which is to equilibrate the multilayer film removed according to step ii) with a hydrochloric acid (HCl) solution with a pH between 2 and 4, preferably with a pH of about 3, or a sodium chloride (NaCl) solution with a pH between 5 and 7, preferably with a pH of about 6.5.

[0050] A preferred manufacturing method according to the present invention is: i) A step of forming a multilayer film of CH / anionic polysaccharide by an alternating layering (LbL) method on a substrate, ii) Steps to remove the multilayer film from the substrate, iii-0) Before step iii) is carried out, equilibrate the multilayer film removed according to step ii) with an acidic solution having a pH between 2 and 4, preferably a pH of about 3, preferably an acidic solution consisting of hydrochloric acid (HCl), or with physiological saline having a pH between 5 and 7, preferably a pH of about 6.5, preferably a physiological saline consisting of sodium chloride (NaCl). iii) A step of bringing a multilayer film into contact with a protein or polypeptide, thereby loading the protein or polypeptide into the multilayer film. The process includes steps consisting of the following:

[0051] Load the patch The protein or polypeptide can be incorporated into the patch or adsorbed onto the surface of the patch, as described above.

[0052] Typically, proteins or polypeptides with molecular weights below 70 kDa or 80 kDa are preferably incorporated entirely or nearly entirely into the patch, i.e., preferably at least 90% protein is incorporated. High molecular weight proteins or polypeptides may be adsorbed entirely or partially onto the surface of the patch.

[0053] The amount of protein or polypeptide incorporated and / or adsorbed depends on the protein or polypeptide and the desired biological or pharmacological effect. This amount is, for example, 50 ng / cm³. 2 From 5 mg / cm³ 2 Preferably 10 ng / cm 2 From 1 mg / cm³ 2 , 1 μg / cm³ 2 From 1 mg / cm³ 2 It can change.

[0054] Applicable The patches obtained in this way can be stored at 4°C or room temperature before application.

[0055] Typically, the patch is 2-3 cm in size when applied to human subjects. 2 The patch is of a certain size. The patch can be applied, for example, to the inside of the cheek, the palate, the gums, or under the tongue. Sublingual application (i.e., application to the ventral side of the tongue) is particularly advantageous. Preferably, the patch is applied such that the chitosan layer on which the protein or polypeptide is placed during the manufacture of the patch becomes the layer that comes into contact with the mucous membrane.

[0056] The drawings and embodiments illustrate the present invention without limiting its scope. [Examples]

[0057] Manufacturing and testing of mucosal adhesive patches 1. Materials and Methods 1.1 Materials Chitosan (CHI) with a medium molecular weight was purchased from Sigma - Aldrich. Before use, CHI was purified by filtration and precipitation in water and ethanol, and then lyophilized to a final molecular weight of 770 kDa and a degree of deacetylation (DD) of 78%. At low pH (<6.5), CHI is a positively charged polyelectrolyte. This was compared with Viscosan® (VIS), another polysaccharide obtained from Flexichem with a different distribution of N - acetylated groups. LW Two types of sodium hyaluronate (HyA), 610 kDa (HyA HW ) and 1020 kDa (HyA

[0058] In all experiments, for the formation of the membranes, a polymer solution was prepared without prior preparation by dissolving in sodium acetate buffer (0.1 M CH3COOH, 0.15 M NaCl, pH 5.5, room temperature) at a polymer concentration of 1 mg / mL.

[0059] 1.2 Fluorescent chitosan 20 mL of medium - weight chitosan (CHI, 770 kDa, 10 mg×mL -1 in 0.1 mol×L acetic acid -1 ) was reacted with 20 mL of fluorescein isothiocyanate (FITC, 1 mg×mL -1 in dehydrated methanol) at room temperature for 3 hours without exposure to light. The pH was then raised to 10 to precipitate CHI, which was then centrifuged at 11000 g for 15 minutes and washed several times with water until no fluorescence was detected in the supernatant. CHI was then dissolved in 0.1 mol×L -1Dissolve in 20 mL of acetic acid, and remove unbound residues of FITC by dialysis (Spectrum, USA). This dialysis was performed in water in the dark for 3 days, and the water was changed daily. The amounts of CHI and FITC were determined by spectrophotometric method at 490 nm and 270 nm, respectively. Fluorescein-labeled CHI (CHI) of moderate mass was used. FITC ) to 0.1 mol × L -1 2 mg × mL in acetic acid -1 The mixture was divided into aliquots and stored at -20°C.

[0060] 1.3 Manufacturing of CHI / HyA films Self-supporting membranes (CHI / HyA) were produced by alternating layer-by-layer (LbL) stacking using an immersion robot (DR-3, Riegler & Kirstein GmbH). The membranes were manufactured using polystyrene substrates cleaned by sonication in ethanol and distilled water (5 minutes per solution). Between each stacking in the polymer solution, a washing step using sodium acetate buffer was performed to clean the substrates in CHI or Viscosan®, and in 0.2% (mass / volume) (HyA (610kDa)) concentrated in sodium acetate buffer (0.2M CH3COONa, 0.2M CH3COOH, pH 5.5, room temperature). LW Or 1020kDa HyA HW The films were continuously immersed in the solution. These immersions were repeated 100 times, with a loading time of 3 minutes for the naturally derived polymer and a washing step of 2 minutes. The films were then left to dry at room temperature. Finally, the films were easily removed from their respective substrates by simply pulling them with tweezers. Fluorescent film (CHI FITC / HyA) 100 -CHI FITC ) in sodium acetate buffer at pH 5.5, 2 mg × mL -1 In the chitosan solution, 0.5% CHI FITC Using this method, the preparation was carried out as described above, without exposure to light.

[0061] 1.4 LbL film thickness The thickness of the generated film was measured after drying and substrate removal. Film thickness was determined using a micrometer (High-Accuracy Digimatic Micrometer, Mitutoyo Corporation), and 20 measurements were taken at different locations in the center of the film.

[0062] 1.5 Research on artificial saliva and enzymatic degradation 1cm 2 The membrane was weighed before the experiment. Artificial saliva was prepared using α-amylase, hyaluronidase, and lysozyme, and dissolved in buffer (0.15 M NaCl, 20 mM HEPES, pH 6.5). All enzymes were administered in 100 μg × mL. -1 The final concentration was determined. The sample was immersed in artificial saliva and incubated at 37°C for 30 minutes, 1 hour, 3 hours, 6 hours, or 24 hours with slow stirring. After each incubation time, the membrane was dried at 37°C and weighed. The percentage of mass loss (WL) of the membrane under different conditions was determined from Equation 1, where Wi represents the initial dry mass of the membrane and Wf represents the mass of the dry membrane after each given time point. Three independent experiments were performed three times for each condition, and the average value was obtained as the percentage of mass loss. WL=(Wi-Wf) / Wi×100 (Equation 1)

[0063] For observation of the film using a confocal laser scanning microscope (CLSM), the fluorescent film was fixed to a glass plate and incubated in artificial saliva at 37°C for 30 minutes, 3 hours, 6 hours, or 24 hours with stirring. Degradation was stopped by rinsing with acetate buffer before observation using a CLSM with an LSM710 confocal microscope (Carl Zeiss SAS, France). All images were analyzed using Carl Zeiss Zen software and Image J software.

[0064] 1.6 Scanning Electron Microscopy (SEM) Morphological analysis of the samples (before and after decomposition) was performed using a scanning electron microscope (Merlin Compact VP, Zeiss) with an accelerating voltage of 5 kV. Both sides of the film were observed. Before observation, all specimens were coated with copper (Balzers MED 010).

[0065] 1.7 Cytotoxicity Test Immortalized Ho-1u-1 cells (a human cell line derived from squamous cell carcinoma cells collected from the floor of the mouth, obtained from GIMAP, Saint Etienne, France) were mixed with 10% (volume / volume) heat-inactivated fetal bovine serum (FBS) and 1% (volume / volume) penicillin / streptomycin, and D-glucose (4.5 g × L). -1 ), Pilvate (1 mmol x L -1 ), and L-glutamine (2 mmol × L -1 HeLa cells (human epithelial cell line derived from adenocarcinoma, ATCC® CCL-2) were cultured in Dulbecco's modified Eagle medium (DMEM), i.e., DMEM / Ham's F12 nutrient cocktail (1:1). D-glucose (4.5 g × L) containing 10% (vol / vol) heat-inactivated FBS and 1% (vol / vol) penicillin / streptomycin. -1 ), Pilvate (1 mmol x L -1 ), and L-glutamine (2 mmol × L -1 The cells were cultured in DMEM containing ). The cells were maintained at 37°C under a 5% CO2 atmosphere.

[0066] Two days before the cytotoxicity test, cells were seeded in a 96-well culture plate. Simultaneously, the (CHI / HyA)100-CHI membrane was finely cut and resized (3 cm per 1 mL of medium). 2 The membrane was then sterilized in 70% ethanol and by exposure to UV light. The membrane was then treated with 100 μg × mL of salivary enzymes (lysozyme, α-amylase, and hyaluronidase). -1The cells were incubated overnight at 37°C in a culture medium containing the product. The culture medium was then removed from the wells and replaced with a medium containing the degradation product. The cells were incubated at 37°C for 24 hours. Subsequently, methylthiazolyldiphenyl-tetrazolium bromide (MTT, 0.5 mg × mL) was added. -1 The cells were then incubated at 37°C for 3 hours with 10% (vol / vol) Triton X-100 and HCl (0.1 mol × L) in anhydrous isopropanol at 37°C and in the absence of light. -1 The cells were incubated overnight in a solubilizing solution containing ). Absorbance was measured at 570 nm and 690 nm (i-control Infinite® M1000 Pro, Tecan, Switzerland). A positive control was performed using 0.1% (vol / vol) sodium dodecyl sulfate (SDS), and a negative control was performed using cells only. Data were obtained as the average of three trials from three independent experiments.

[0067] 1.8 Mouse In vivo studies were conducted using 6- to 8-week-old female CB6F1 mice (Charles River Laboratories, France) and male SHK-1 mice (Charles River Laboratories, France) for tomography experiments. All animals were maintained under pathogen-free conditions. All animal studies were conducted in accordance with European Union guidelines and approved by regional and national ethics committees.

[0068] 1.9 Sublingual administration of membrane or solution The membrane was cut into small pieces to the size of a mouse tongue (2 mm x 7 mm) and sterilized with UV light. The membrane or liquid formulation was then administered to lightly anesthetized mice (4% isoflurane) via the sublingual route (ventral part of the tongue). After administration, light pressure was applied to the dorsal part of the tongue for 10 seconds (until the mouse woke up) to ensure contact between the membrane or solution and the mucous membrane. No other restraints were used. After recovery from anesthesia, the animals were allowed to drink water and groom themselves freely. Water was removed for 30 minutes after administration and returned for subsequent experiments.

[0069] 1.10 Histological analysis of mucosal swelling and recruitment of MHCII-positive cells Untreated membranes, membranes treated with HCl, or membranes treated with NaCl were administered for 30 or 60 minutes. A 12.5 μl volume of 0.5% 1-fluoro-2,4-dinitrobenzene (DNFB) solution was used as a positive control for inflammation. No procedures were performed on the negative control group. Each group contained three mice. Mice were agile throughout the entire treatment time, and water was removed for the first 30 minutes. At each time point, the tongue was placed in an OCT® matrix (Optimal Cutting Temperature, Tissue Tek) and stored at -80°C until frozen sectioning. In the frozen sectioning process, 6 μm thick slices were prepared using a cryostat (Leica) and fixed to glass plates with acetone at -20°C. For analysis of mucosal swelling, tongue slices were stained with hematoxylin (Gill formula, Vector) and images were captured using an inverted microscope (Nikon Ti-E). Mucosal swelling was analyzed over a 2 mm length extending from the ventral base. Segmental measurements were performed using the polygon tool in ImageJ software. For MHCII staining, tongue slices were first incubated with peroxidase blocking reagent (Dako), then with biotinylated rat anti-mouse antibody (BD pharmigen), developed using the Vectastain Elite ABC kit (Vector) and AEC peroxidase substrate (Vector), and finally counterstained with hematoxylin (Gill formula, Vector). The slices were examined using a Nikon Ti-E microscope. The number of MHCII-positive cells (MHCII+) was counted over a 2 mm length extending from the ventral base of the sublingual membrane using the ImageJ software's plug-in cell counter.

[0070] 1.11 Sample preparation for cytokine quantification Each group contained three mice. Two groups received either an untreated membrane or a membrane treated with HCl. The other three mouse groups received 10 μL of CHI (770 kDa, 2 mg × mL in acetate buffer, pH 5.5) via the sublingual route. -1 Sigma (USA), 10 μL HyA (610 kDa, 0.95 cm³ in acetate buffer, pH 5.5) 3 ×kg -1 , 2 mg × mL -1 The mice were administered either 10 μL of PBS or acetate buffer (pH 5.5) or a combination of these two polymers. The control group was administered either 10 μL of PBS or acetate buffer (pH 5.5) via the sublingual route as a negative control, or 12.5 μL of 0.5% (vol / vol) DNFB in chloroform as a positive control. Six hours after application of the membrane or solution, the tongues were excised, frozen in liquid nitrogen, and stored at -80°C. Briefly, the tongues were homogenized using scissors and then treated with RIPA buffer [Tris HCl (50 mmol × L)]. -1 ), NaCl(150 mmol × L -1 ), Triton X-100 (1%), sodium deoxycholate (0.5%), SDS (0.1%), EDTA (1 mmol × L) -1The preparations were incubated on ice for 2 hours in a cocktail of 1% protease inhibitors (Thermo Scientific). The preparations were then homogenized using a ball-type beater (2 × 5 minutes, 30 Hz, 4°C) (TissueLyzer II, Qiagen, Germany) and sonication (2 minutes, 60 Hz), followed by centrifugation at 10,000 rpm and 4°C for 10 minutes. The total protein concentration in the supernatant was then determined using the BCA protein assay kit (ThermoFisher Scientific, USA). Interleukin-1 beta (IL-1β), IL-6, and tumor necrosis factor alpha (TNF-α) in each sample were simultaneously quantified by electroluminescence using the Mesoscale Discovery system (Meso QuickPlex SQ 120, MSD, USA) (V-Plex Proinflammatory Panel 1 Mouse Kit, MSD, USA). Data were obtained as the average of two trials using three mice for each condition.

[0071] 1.12 Protein incorporation through pH-dependent expansion To evaluate the ability of membranes to incorporate proteins at different pH values, the membranes were tested in 1 mM HCl (pH 3-3.5), phosphate-buffered saline (PBS 1×, Gibco® by Life Technologies, Inc., pH 7.4), or sodium chloride buffer (NaCl 0.15 mol × L). -1 HEPES 0.02 mol × L -1 The film was equilibrated at room temperature for 1 hour at pH 6.5. Before the experiment, a 12 mm diameter membrane was sterilized by exposure to UV light for 20 minutes. After equilibrating the excess HCl, the PBS or NaCl buffer was removed, and the protein droplets were placed on the membrane. The protein used was 5 μg × mL in 1 mM HCl or NaCl buffer. -1 Alexa Fluor 647-labeled ovalbumin (OVA) (loading dose of 500 ng) AF647 ), or 500 ng × mL -1The cytokine was one of the following (loading doses of 50 ng): CCL20. The membrane was incubated overnight at 4°C. After rinsing with acetate buffer at pH 5.5 and drying under a stream of air, the functionalized membrane was called a "bioactive patch".

[0072] 1.13 Research on ovalbumin release Before the experiment, OVA AF647 The protein was incorporated into the membrane as described above. Protein release was monitored at various pH levels using pH 5.5 acetate buffer, Dulbecco pH 7.4 PBS (dPBS), or pH 6.5 artificial saliva as rinse solutions. Immediately after adding the rinse solution, the protein was removed and stored to assess the amount of unbound protein ("quick rinse", QR). OVA from the membrane. AF647 The OVAs were released at 10, 20, 30, 40, 60, 90, and 120 minutes into the video, over a period of two hours. AF647 The Alexa Fluor 647 was analyzed by fluorescence spectroscopy (Infinite M1000, Tecan) with the excitation / emission wavelengths fixed at 650 / 668 nm. Calibration curves of the labeled protein in dPBS, acetate buffer, HCl, and artificial saliva were recorded at the aforementioned wavelengths.

[0073] 1.14 Tomographic analysis of patch mucosal adhesion and protein retention time For tomographic analysis of protein retention time in the oral region of mice, groups of two mice were anesthetized for 5 minutes in a chamber containing 4% isoflurane. For solution administration, 10 μl of OVA was administered. AF647 The solution was placed on the base of the ventral side of the mouse's tongue. For patch administration, the patch was placed on the ventral side of the tongue. Each formulation contained 5 μg of OVA. AF647 The drug was administered. Mice were placed in an isoflurane-guided tomography chamber (FMT 4000, Perkin Elmer) with their heads facing inward, and images were acquired 2, 10, or 30 minutes after administration.

[0074] 1.15 Infiltration of ovalbumin in the sublingual mucosa OVA in the sublingual mucosa AF647 The penetration of the OVA was evaluated after administration of the liquid formulation (10 μl) or the (CHIFITC-HyA)100-CHI patch. AF647 The formulations were incorporated by incubation in HCl, as described in 1.12. Groups of two mice were administered various formulations and euthanized 2, 10, 30, or 60 minutes after administration. Sublingual mucosa (ventral surface of the tongue and floor of the mouth) was collected, embedded in OCT® matrix, and stored at -80°C. 40 μm sections were prepared, labeled with DAPI nuclear probes, and then observed using a confocal microscope (LSM 710, Zeiss, Germany).

[0075] 1.16 Chemotivity Test The chemotactic effect of the chemokine CCL20, delivered by the developed membrane, was tested in vitro using a mouse dendritic cell line (DC2.4, #SCC142, Millipore). Cells were cultured at 37°C and under a 5% CO2 atmosphere in RPMI medium (hereinafter referred to as GM) supplemented with a mixture of GlutaMAX, 10% FBS, 10 mM HEPES, 50 μM β-mercaptoethanol, and non-essential amino acids (1×). The chemotactic test was performed for 20 minutes in a cell culture insert ThinCert® (Greiner Bio-One, ref: 665 610) placed in a 12-well plate. Before the test, the membrane, chemokine CCL20, and chemokine CCL20 (500 ng × mL) were added. -1 The membrane loaded with the salivary enzyme solution (0.1 mg x mL dissolved in GM without FBS) is slowly stirred. -1 The α-amylase, hyaluronidase, and lysozyme were incubated at 37°C for 24 hours. The inserts were then placed in 2.0 × 10⁶ 12-well plates. 5Cells were seeded at individual cell / insert density and incubated at 37°C for 10 minutes. The lower chamber of a 12-well plate was then carefully filled with 1 mL of chemotactic solution or control solution and incubated at 37°C for 10 minutes. The positive control corresponded to dendritic cells (DCs) incubated in the presence of GM alone. Finally, the inner surface of each insert was swabbed using a cotton swab. After fixation (with 4% paraformaldehyde) and staining with DAPI, the samples were observed under an inverted microscope (Nikon Ti-E). The results obtained represent the average of three independent experiments.

[0076] 1.17 Statistical analysis All data were analyzed and entered into Graphpad Prism software version 7.0. The quantities shown represent the mean ± standard deviation (SD) of at least three trials, separate from the emitted data, which represent the mean ± standard error (SEM). p < 0.05 was considered statistically significant after Tukey's multiple comparison test following an ANOVA test with one controlled factor, or Dunnett's multiple comparison test for cytotoxicity tests.

[0077] 2.Results 2.1 Degradation of self-supporting membranes in artificial saliva (CHI / HyA) 100 Untreated films were prepared by varying three parameters: the molecular weight (MW) of HyA, the loading time of the polymer electrolyte and rinsing solution, and the properties of CHI. The effects of these parameters on film thickness were investigated first. (CHI / HyA) 100The growth of untreated membranes was shown to be linear for membranes consisting of 50 bilayers (4.5±1.39 μm), 100 bilayers (10.30±7.67 μm), and 200 bilayers (17.04±7.96 μm) (Figure 9). At least 50 bilayers were required for easy handling without any post-procedure treatment. No significant difference was observed in membrane thickness with respect to the MW of HyA (610 kDa or 1000 kDa) (Figure 1A). Significant differences were observed in membrane thickness with respect to loading time, i.e., long cycle (LC, 6 minutes with polyelectrolyte and 4 minutes with rinse buffer) and short cycle (SC, 3 minutes with polyelectrolyte and 2 minutes with rinse buffer), with an increase of approximately 32% when the membrane was fabricated under LC conditions (Figure 1A).

[0078] The effect of the conditions used to construct the membranes on their degradation was also investigated. Furthermore, since the membranes were intended for sublingual application, artificial saliva was generated. The artificial saliva consisted of a physiological solution containing three enzymes produced in human saliva: α-amylase, lysozyme, and hyaluronidase. The degradation of the generated membranes was monitored and quantified over 24 hours and expressed as a percentage of mass loss. Membranes constructed with Viscosan® degraded faster than CHI-based membranes, as expected due to the rapid biodegradability of Viscosan® (Figure 9B). The overall degradation profile of CHI-based membranes was not significantly affected by either the molecular weight of HyA or the loading time (Figure 1B), although a delay was observed in the first phase of the degradation process (first hour) for membranes constructed under LC conditions (insert in Figure 1B). After 24 hours, all membranes reached weight loss levels ranging from 75% to 95%.

[0079] To obtain morphological information regarding the degradation of these membranes, (CHI / HyA LW ) 100The membrane was observed using SEM and confocal microscopy. Images from these two techniques showed initial surface erosion at 30 minutes, followed by the formation of surface pores at 1 hour, and finally, deeper pores at 3 hours (Figures 1C and 1D). Enzymatic degradation of the membrane surface was also observed from 30 minutes of immersion in artificial saliva under hydration conditions, where the membrane expanded to approximately 30 μm (Figure 1D). No degradation was observed after 24 hours in acetate buffer (Figure 1D, control).

[0080] Since these membranes are designed for the administration of proteins via mucosa, the inventors selected membranes made with CHI rather than Viscosan® to ensure long-term diffusion of cargo proteins from the initial stages of degradation in contact with the mucosa. Membranes made under SC conditions were also selected to shorten the generation time, but HyA HW Since no difference in thickness or decomposition was observed compared to the other option, the 610kDa HyA LW Randomly selected. To ensure optimal mucosal adhesion, all membranes used in animal experiments had a first and last layer made of CHI. Therefore, in the following study, (CHI / HyA LW )100-CHI membrane is simply (CHI / HyA) 100 He was called [that].

[0081] 2.2 Cytotoxicity against human epithelial cells The cytotoxicity of the membrane degradation products against two human epithelial cell lines (HeLa and Ho-1u-1) was evaluated over a 24-hour incubation period. No toxicity was observed in either cell line, as approximately 100% viability remained (Figures 2A-2B). This result is explained by the known biocompatibility of the two unmodified polysaccharides used to construct the membrane. Combining the polymers did not affect their biological safety. Consequently, the membrane can be applied in vivo to the sublingual mucosa of mice without causing cytotoxic effects on tissue epithelial cells.

[0082] 2.3 Inflammation of the sublingual mucosa in mice The in vivo inflammatory response induced by the patch was evaluated in mice by assessing various key features of the inflammatory state. First, the swollen surface of the sublingual mucosa epithelium and lamina propria was observed under different conditions. Since 0.5% 1-fluoro-2,4-dinitrobenzene (DNFB) has been shown to induce inflammation when administered via the sublingual route [LeBorgne et al., 2006], it was used as a positive control for inflammation. An increase in mucosal thickness was observed 30 minutes after DNFB administration, and a slight decrease was observed after 2 hours (Figure 3A). Notably, the swelling of the lamina propria was maintained for 6 hours, while epithelial thickness had already decreased after 2 hours. (CHI / HyA) 100 To quantify membrane-induced swelling, untreated and HCl-treated membranes (incubated for 1 hour in 1 mM HCl, pH 3) were applied to the sublingual mucosa for 30 or 60 minutes. Since HCl treatment forms part of the protocol for protein incorporation, its effect on the inflammatory response was evaluated. Mucosal swelling after application of the untreated membrane was comparable to that induced by the DNFB-positive control at 2 hours, while the mucosa in contact with the HCl-treated membrane was significantly thinner. To correlate tissue swelling with immune cell infiltration, immunohistochemical staining for major histocompatibility complexes (MHCII) of class II was performed. MHCII is a major marker for antigen-presenting cells (APCs), particularly dendritic cells (DCs), B lymphocytes, and macrophages. The degree of MHCII+ cell infiltration in the mucosa significantly increased 30 minutes after administration of the untreated membrane (Figures 3B-3C) and returned to control levels after 60 minutes. Interestingly, as observed in the mucosal swelling (Figure 3A), the membrane treated with HCl for 30 minutes did not induce MHCII+ cell infiltration, and the infiltration remained at a similar level to that of the control.

[0083] To identify the mid-term inflammation profiles induced by untreated and HCl-treated membranes, the expression profiles of inflammatory cytokines were quantified 6 hours after patch administration. In all tested conditions (polymer in solution, or membranes with or without HCl treatment), the levels of IL-1β, IL-6, and TNF-α were similar to control levels, indicating that no inflammatory signaling was induced 6 hours after patch application.

[0084] 2.4 Integration / Release of the Model Protein Ovalbumin (CHI / HyA) as a system for protein diffusion 100 To evaluate the membrane, it was functionalized with the model protein ovalbumin and labeled with the fluorophore Alexa Fluor 647 (OVA). AF647 ). After equilibrating the membrane in 1 mM HCl, pH 3, OVA AF647 The OVA was incorporated into the membrane (according to the scheme in Figure 4). The membrane was rinsed with pH 5.5 acetate buffer (rinse buffer for the superimposed membrane), removing 98% of the OVA. AF647 While it became possible to incorporate OVA, rinsing with PBS (pH 7.4) slightly reduced its incorporation (94%) (Figure 5A). In acetate buffer, OVA was less than 7%. AF647 The difference was confirmed by the dynamics of release over two hours, as a certain amount was released from the patch compared to 31% in PBS (Figures 5A-5B). When immersed in artificial saliva, 35% of OVA was released after two hours. AF647 The OVA was released from the patch, with approximately 90% released 24 hours after immersion (data not shown). OVA at pH 3 before rinsing with acetate buffer. AF647The integration profile showed that approximately 80% of the proteins were integrated within 10 micrometers below the surface, corresponding to one-third of the total hydrated thickness (Figures 5C-5D). The functionalized membrane, therefore, is an asymmetric patch, with the protein-containing surface being the surface in contact with the mucosa. Certain proteins may be sensitive to acidic pH (denaturation, loss of activity, etc.), therefore OVA AF647 Incorporation was also performed in PBS at a physiological pH of 7.4 instead of HCl at pH 3. However, due to significant membrane swelling, the patch could not be manipulated or visualized after protein incorporation. Furthermore, due to structural instability, the degree of incorporation was less than 60% (data not shown). Conversely, in NaCl buffer (NaCl 0.15 mol × L), -1 HEPES 0.02 mol × L -1 A solution (pH 6.5) was used for protein incorporation. For membrane rinsing, the same solutions were tested (acetic acid buffer, PBS, and artificial saliva). As observed with loading at an acidic pH, rinsing with acetate buffer resulted in 94% protein incorporation, while rinsing with PBS resulted in 85% OVA incorporation. AF647 It was simply incorporated (Figure 10A). In the emission profile, OVA was less than 10%. AF647 In contrast, in PBS, approximately 36% of OVAs are released from the membrane. AF647 The rapid release of the protein confirmed that acetate buffer ensured protein retention inside the membrane (Figure 10B). Protein release reached 69% two hours after rinsing in artificial saliva. When proteins were incorporated with NaCl buffer, the distribution of proteins within the patch was found to be more homogeneous than with HCl, and did not show a depth-direction osmotic gradient along the Z-axis (Figures 10C, 10D).

[0085] 2.5 Mucosal adhesion and retention time of cargo proteins The mucosal adhesion of the patch was evaluated in vivo by visualizing the film remaining on the sublingual mucosa 20 minutes after administration (Figure 6A). OVA by molecular fluorescence imaging. AF647Monitoring revealed the dispersion rate of the protein after administration as a liquid formulation (Figure 6B). During the first few minutes of image acquisition, OVA AF647 It was already distributed along the gastrointestinal tract, and after 10 minutes, the signal was undetectable in the mouth. When administered simultaneously with a mucosal adhesive membrane, OVA AF647 It was detected in the mouth for at least 30 minutes, in the form of an enhanced signal.

[0086] 2.6 Presentation and infiltration of cargo proteins on mucous membranes OVA AF647 Tissue penetration was monitored by confocal microscopy. The signal intensity 2 minutes after administration was much weaker in liquid form than that indicated by the membrane (Figure 7). OVA AF647 10 minutes after patch administration, CHI FITC It penetrated the stratum corneum in a similar manner (white arrow, Figure 7). The patch was undetectable 30 minutes after application, but OVA AF647 It was located deep within the mucosa (dashed white arrow, Figure 7). Even if accumulation was detected in the epithelium (surface layer), OVA AF647 The signal was observed in the lamina propria and submucosa. The signal was barely detectable 60 minutes after administration. The decrease in the protein's fluorescence signal at 60 minutes after administration compared to 30 minutes after administration can be explained by protein clearance in the tissue and its absorption by immune cells such as APCs.

[0087] 2.7 Biological activity of incorporated proteins (CHI / HyA) 100To evaluate the functionality and bioactivity of the proteins incorporated into the membrane, the chemoattractant cytokine (CCL20) was loaded into the assembly and samples were taken after degradation of the membrane in artificial saliva. The cytokine CCL20 was used as a chemoattractant for mouse dendritic cells (DC2.4) to which the CCR6 receptor is bound (data not shown). Similar migration was observed with CCL20 in solution and CCL20 recovered after dissolution of the patch, indicating that the chemoattractant ability of the cytokine was preserved within the patch (Figure 8). The bioactivity of the protein was thus preserved by the patch, and the cytokine showed a significant chemoattractant effect on the targeted cells.

Example

[0088] (CHI / HyA) 100 Evaluation of Incorporation Rate in the Patch 1. Incorporation of Bovine Serum Albumin (BSA) Materials and Methods (CHI / HyA) 100 The membrane was cut into small pieces to form 1 cm 2 squares, which were incubated in either a pH 3 HCl buffer solution or a pH 6.5 NaCl buffer solution containing bovine serum albumin (BSA) at a concentration of 10 mg×mL -1 . The (CHI / HyA) 100 membrane squares were completely covered with a liquid volume of approximately 200 μl. Upon equilibration, each of them contained approximately 2 mg of absorbed BSA. The square (CHI / HyA) 100The membrane was incubated in a release buffer (acetate buffer, pH 5.5) without BSA, and the amount of protein released was measured for 4 hours using a bicinchoninic acid (BCA)-based protein assay kit. Measurements of the optical density (562 nm) were performed by taking samples of the release buffer at the starting point (t0), as well as 5 minutes, 10 minutes, 20 minutes, 30 minutes, 60 minutes, 120 minutes, and 240 minutes after incubation, and the release buffer was replaced after each sample collection. The measured values of the blank obtained in wells containing only the incubation solution were systematically subtracted. From the standard curve, the protein concentration per milliliter could be determined for each release time, and then the amount of BSA for 200 μl was referenced. Finally, the amount of protein incorporated into the membrane was revealed by subtracting the amount of BSA determined for 200 μl from the theoretical 2 mg of BSA initially absorbed.

[0089] Results (CHI / HyA) 100 1 cm of the membrane 2 The amount of BSA incorporated per centimeter of the membrane was determined by assaying the BSA released from membranes pre-incubated in concentrated BSA solutions of different pHs. Very similar release profiles were observed between BSA incorporated at pH 3 and BSA incorporated at pH 6.5, with 0.79 mg and 0.75 mg being released immediately (t0), respectively, and an additional 0.18 mg and 0.16 mg being released after 5 minutes, and again, 0.03 mg and 0.01 mg being released after 10 minutes. After 20 minutes of release, the BSA released between time t10 and time t20 was no longer detectable, indicating that release mainly occurs within 10 minutes. The total amount of BSA released measured corresponded to 1.01 mg for BSA incorporated at pH 3 and 0.92 mg for BSA incorporated at pH 6.5. As a result, the square (CHI / HyA) that initially absorbed approximately 2 mg of BSA 100The membrane still contained 1 mg of BSA 4 hours after rinsing, meaning it contained approximately 0.99 mg of BSA in the incorporation performed at pH 3 and approximately 1.08 mg in the incorporation performed at pH 6.5. The incorporation rate was therefore 50% under both conditions.

[0090] 2. (CHI / HyA) 100 Incorporation of immunoglobulins (IgG) into membranes Materials and methods (CHI / HyA) 100 Cut the membrane into small pieces, 1 cm 2 The square was prepared and incubated for 1 hour in either 200 μl of pH 3 HCl equilibrium solution (sample MbA) or 200 μl of pH 6.5 NaCl-Hepes equilibrium solution (sample MbB). The square MbA membrane was then subjected to 2 μg × mL -1 Incubate the donkey secondary antibody containing type G immunoglobulin (IgG, against goat IgG, conjugated to the fluorescent dye Alexa 633; ​​Invitrogen, molecular probe A21082-lot 73A2-1) in 150 μl of pH 3 HCl-based solution, and also incubate a square MbB membrane with 2 μg × mL -1 The IgG was incubated in 150 μl of a pH 6.5 NaCl-Hepes-based mixture containing the same IgG. Simultaneously, square dried (CHI / HyA) samples that had not undergone the equilibrium step were incubated. 100 The membranes (MbC and MbD) were incubated in the same incorporation solution as samples MbA and MbB. Incubation in the incorporation solution was carried out overnight at 4°C. The following day, the membranes were rinsed three times for 2 minutes each in acetate buffer (0.1 M CH3COOH, 0.1 M CH3COONa, pH 5.5), and then dried in a stream of light (PSM). Finally, the membranes were observed using a confocal fluorescence microscope (Zeiss, LSM 710) with a 20x objective lens.

[0091] result Membranes (Alexa 633nm) with and without a pre-equilibrium step at different pH levels were examined (images not provided). The presence of IgG was equally detectable under all conditions, and in principle, it was detectable on the plate wall and the opposite surface of membranes MbA, MbB, MbC, and MbD during incubation. However, the labeling of IgG was more evident in the MbA sample, indicating that the pre-equilibrium step only benefits integration at pH 3. Furthermore, the amount of IgG incorporated into the membrane was 1 cm³. 2 It can be estimated that this is approximately 400 ng per unit.

[0092] (References) TIFF0007842755000001.tif29158

Claims

1. A patch for oral or sublingual mucosal adhesion intended for the oral or sublingual release of proteins or polypeptides, comprising at least 100 bilayers, each composed of a chitosan (CHI) layer and a layer of an anionic polysaccharide or salt thereof having a molecular weight between 500 and 1000 kDa, wherein the layer in contact with the mucous membrane of the patch and the layer in contact with the oral or sublingual environment are composed of chitosan (CHI), and the protein or polypeptide is incorporated into the patch and / or adsorbed onto the surface of the patch.

2. The patch according to claim 1, comprising 100 to 200 CHI / anionic polysaccharide bilayers.

3. The patch according to claim 1, having a thickness of 10 to 20 μm.

4. The patch according to claim 1, wherein the anionic polysaccharide or salt thereof is selected from glycosaminoglycans (GAGs), fucoidan, alginate, carrageenan, and urban.

5. The patch according to claim 4, wherein the glycosaminoglycan (GAG) or a salt thereof is selected from hyaluronic acid (HA), heparin (Hp), heparan sulfate (Hs), chondroitin sulfate (CS), dermatan sulfate (DS), and keratan sulfate (KS).

6. The patch according to claim 1, wherein the allergen is a protein or polypeptide.

7. A composition comprising the allergen for use in desensitizing a subject allergic to the allergen, which is administered in the form of a patch as described in Claim 6.

8. A method for manufacturing a patch according to any one of claims 1 to 6, i) A step of forming a multilayer film of CHI / anionic polysaccharide by alternating layering (LbL) on a substrate, ii) Steps to remove the multilayer film from the substrate, iii) A step of bringing a multilayer film into contact with a protein or polypeptide, thereby loading the protein or polypeptide into the multilayer film. A method comprising the steps of:

9. The method according to claim 8, wherein the multilayer film is contacted with a protein or polypeptide in a hydrochloric acid (HCl) solution with a pH between 2 and 4 according to step iii).

10. The method according to claim 8, wherein the multilayer film is contacted with a protein or polypeptide in a sodium chloride (NaCl) solution between pH 5 and 7 according to step iii).

11. The method according to claim 8, further comprising an intermediate step of equilibrating the multilayer film removed according to step ii) with a hydrochloric acid (HCl) solution having a pH between 2 and 4, or a sodium chloride (NaCl) solution having a pH between 5 and 7, before step iii) is carried out.

Images