Method for preparing bioadhesive particles for immunosuppression, particles prepared using same, and local immunosuppression composition that is adhesive to surfaces of transplanted organ tissue, comprising particles

Abstract

The present invention relates to a method for preparing bioadhesive particles for immunosuppression, particles prepared using same, and a local immunosuppression composition comprising same. More specifically, the present invention relates to a method for preparing bioadhesive particles for immunosuppression, particles prepared using same, and a local immunosuppression composition comprising same, the method comprising the steps of: preparing a first solution including a biocompatible oil and a surfactant; preparing a second solution, which is a precursor solution including mussel adhesive protein, an immunosuppressive agent, a photocrosslinkable compound and a solvent; and photocrosslinking a mixture of the first solution and the second solution.

Description

Method for producing bioadhesive particles for immunosuppression, particles produced therefrom, and local immunosuppressive compositions for adhesiveness to the surface of transplanted organ tissues containing the same

[0001] The present invention relates to a method for producing bioadhesive particles for immunosuppression, particles produced therefrom, and a topical immunosuppressive composition comprising the same. More specifically, the present invention relates to a composition for producing bioadhesive particles loaded with an immunosuppressant based on a bioadhesive material such as a mussel adhesive protein, and for locally delivering such particles to a transplanted organ.

[0002] Immunosuppressants are pharmaceutical compounds that suppress the activity of the immune system. Immunosuppressants are commonly used in the treatment of autoimmune diseases and in the prevention and treatment of organ transplantation. After organ transplantation, the recipient's immune system almost always identifies the new organ as a foreign, potentially hostile substance due to differences in human leukocyte antigen (HLA) haplotypes between the donor and recipient. The recipient's immune system then attempts to eliminate the new organ by attacking and destroying its cellular components.

[0003] More specifically, the aforementioned organ transplantation technology involves transplanting allogeneic, xenogeneic, or artificially formed organs to achieve effective regeneration in cases where organ damage due to accidents or aging, or where replacement of tissues that are difficult to regenerate, is unavoidable. However, during the transplantation process, the immune system may recognize the transplanted organ as a foreign substance and initiate an immune response, potentially leading to transplant rejection, inflammation, damage, or even complete destruction of the transplanted organ. Therefore, additional treatment methods are needed to prevent immune responses in the transplanted area after the transplantation.

[0004] Currently, these immune responses are clinically controlled through the administration of immunosuppressive agents. Examples of immunosuppressive agents include calcineurin inhibitors such as cyclosporine and tacrolimus; corticosteroids; antimetabolites such as azathioprine and mycophenolate; and mTOR inhibitors such as sirolimus. Because these agents are often ineffective as monotherapy, combination therapies are often used to achieve effective suppression. However, because these agents act systemically, rather than locally at the transplant site, they can make transplant recipients more susceptible to infections and other diseases, potentially leading to serious infections in the recipient.

[0005] Furthermore, although macrolide immunosuppressants, such as tacrolimus, sirolimus, and everolimus, are highly active once absorbed into the organism or target tissue, their poor solubility and relatively large molecular size make them difficult to formulate and deliver to the site of action. On the other hand, for systemic therapy via oral or intravenous routes, immunosuppressants are typically presented in soluble formulations that contain significant amounts of solubilizing excipients, such as surfactants and organic solvents.

[0006] Therefore, if a technology is developed that can achieve local immunosuppression around the transplanted organ, it is expected to be widely applied in related fields.

[0007] One aspect of the present invention provides a method for preparing immunosuppressive bioadhesive particles that can be used in topical compositions.

[0008] Another aspect of the present invention provides an immunosuppressive particle manufactured by the method described above.

[0009] Another aspect of the present invention provides a topical immunosuppressive composition comprising bioadhesive particles for immunosuppression.

[0010] According to one embodiment of the present invention, a method for producing bioadhesive particles for immunosuppression is provided, comprising: preparing a first solution comprising a biocompatible oil and a surfactant; preparing a second solution, which is a precursor solution comprising a mussel adhesive protein, an immunosuppressant, a photocrosslinkable compound, and a solvent; and photocrosslinking a mixture of the first solution and the second solution.

[0011] According to another embodiment of the present invention, an immunosuppressive adhesive particle obtained by the present invention is provided.

[0012] According to another embodiment of the present invention, an adhesive topical immunosuppressive composition comprising the immunosuppressive adhesive particles of the present invention is provided.

[0013] The present invention enables localized, yet long-term, immunosuppression, which is expected to stably suppress immune rejection during xenotransplantation. The localized immunosuppression technology of the present invention can effectively prevent transplant rejection without systemic side effects, thereby increasing the success rate of organ transplantation. Furthermore, the immunosuppressant delivery formulation of the present invention can significantly reduce the preoperative preparation and additional treatment procedures during the surgical procedure, thereby significantly reducing medical time and costs. In particular, the development of a novel immunosuppressant delivery formulation with fewer side effects and longer duration through local immunosuppression is expected to have significant effects in the field of organ transplantation.

[0014] Figure 1 shows photographs of adhesive microgel particles of Example 1, exemplarily manufactured by the present invention, observed using a scanning electron microscope (a) and a confocal microscope (b), and the particle diameter distribution (c).

[0015] Figure 2 shows the results of analyzing the adhesive ability of microgel particles manufactured according to Example 1 using QCM (Quartz Crystal Microbalance).

[0016] Figure 3 shows photographs of microgel particles manufactured according to Example 1 before (a) and after (b) compression spraying using a confocal microscope, and (c) shows changes in the diameter of the particles.

[0017] Figure 4 is a photograph of the spray manufactured according to Manufacturing Example 1 observed under a fluorescence microscope before, 7 days after, and 14 days after spray surface coating on pig skin tissue.

[0018] Figure 5 is a photograph of a spray manufactured according to Manufacturing Example 1 locally sprayed on pig skin tissue, observed using an optical camera (left) and an optical microscope (right).

[0019] Figure 6 shows the loading efficiency of the immunosuppressive drug cyclosporine A in microgel particles manufactured according to Example 1, as analyzed using a UV absorbance analyzer.

[0020] Figure 7 shows the results of analyzing the release efficiency of the immunosuppressive drug cyclosporine A of microgel particles manufactured according to Example 1 using a UV absorbance analyzer. The results are (a) of measuring the released drug concentration using the UV absorbance analyzer and (b) of analyzing the expected date of complete release using the Higuchi model.

[0021] Figure 8(a) is a schematic diagram showing the spray application process in xenograft skin tissue transplantation, and Figures 8(b) and 8(c) show the results measured by an optical camera and FOBI (Fluorescence In Vivo Imaging System), respectively.

[0022] Figure 9 shows the results of ELISA analysis of blood collected from a xenograft transplantation model mouse, showing the concentration of IL-2 (a) and IFN-γ (b) at 1 and 3 weeks after transplantation surgery.

[0023] Figure 10 shows the results of analysis using a flow cytometer after collecting the spleen of a xenograft transplantation model mouse.

[0024] Figure 11 shows the results of analysis after collecting the transplanted skin tissue of a xenograft transplantation model mouse. It shows a photograph (a) observed with an optical microscope after H&E tissue staining and the results of measuring the inflammation area ratio (b).

[0025] Hereinafter, preferred embodiments of the present invention will be described with reference to the attached drawings. However, the embodiments of the present invention may be modified in various other forms, and the scope of the present invention is not limited to the embodiments described below.

[0026] The present invention relates to a spray-based long-term surface coating formulation technology comprising particles comprising a bioadhesive protein. The particles comprising the bioadhesive protein of the present invention may be sprayable adhesive micro-sized gel particles loaded with an immunosuppressive drug.

[0027] The method for producing the bioadhesive particles for immunosuppression of the present invention comprises the steps of: preparing a first solution containing a biocompatible oil and a surfactant; preparing a second solution, which is a precursor solution containing a mussel adhesive protein, an immunosuppressant, a photocrosslinkable compound, and a solvent; and photocrosslinking a mixture of the first solution and the second solution.

[0028] In each of the above steps, stirring may preferably be performed, for example, stirring may be performed at 500 to 1000 rpm. More preferably, the mixture of the first solution and the second solution may be stirred at 600 to 800 rpm to form micro-sized gel particles.

[0029] The biocompatible oil of the present invention may be at least one oil selected from the group consisting of mineral oil, oleic acid and Fluorinert FC 40 oil, for example, mineral oil.

[0030] Meanwhile, the surfactant that can be used in the present invention may be at least one nonionic surfactant selected from the group consisting of sorbitan monolaurate (Arlacel 20), sorbitan monopalmitate (Span-40), sorbitan monooleate (Span-80), sorbitan monostearate, and sorbitan tristearate, and for example, sorbitan monooleate (Span-80) can be used.

[0031] The above first solution may contain 1 to 10 wt% of a surfactant based on the total weight of the first solution, and preferably 3 to 5 wt% of a surfactant. If the content of the surfactant is less than the above range, there is a problem in that a stable emulsion is not formed, and if it exceeds the content, optimization problems due to changes in the viscosity of the solution and biotoxicity may occur.

[0032] The mussel adhesive protein that can be used in the present invention may be a protein derived from the byssus of a mussel. Preferably, the mussel adhesive protein includes, but is not limited to, a mussel adhesive protein derived from Mytilus edulis, Mytilus galloprovincialis, or Mytilus coruscus, or a variant thereof. For example, the mussel adhesive protein may be at least one selected from the group consisting of Mefp (Mytilus edulis foot protein)-1, Mgfp (Mytilus galloprovincialis foot protein)-1, Mcfp (Mytilus coruscus foot protein)-1, Mefp-2, Mefp-3, Mgfp-3, and Mgfp-5 derived from mussels. In addition, the mussel adhesive protein of the present invention may be selected from at least one of all mussel adhesive proteins described in WO2006 / 107183A1 and WO2005 / 092920A1, but is not limited thereto.

[0033] Meanwhile, the immunosuppressant that may be included in the second solution may be at least one selected from the group consisting of purine synthesis inhibitors such as azathioprine, folate antagonists such as methotrexate, macrolides, cyclosporine, tacrolimus, pimecrolimus, everolimus, sirolimus, deforolimus, everolimus, temsirolimus, zotarolimus, infliximab, etanercept, rituximab, tocilizumab, and abataccept, but is not limited thereto.

[0034] Meanwhile, the photocrosslinkable compound may include a photoreactive metal ligand and an electron acceptor.

[0035] In the present invention, the photoreactive metal ligand for providing a molecule that strongly absorbs visible light may be at least one selected from the group consisting of ruthenium (Ru(II)), palladium (Pd(II)), copper (Cu(II)), nickel (Ni(II)), manganese (Mn(II)), and iron (Fe(III)). For example, [Ru(II)bpy₃]Cl₂ is preferably used, but is not limited thereto.

[0036] In addition, the photocrosslinking compound may further include at least one selected from the group consisting of sodium persulfate, periodate, perbromate, perchlorate, vitamin B12, pentaamminechlorocobalt(Ⅲ), ammonium cerium(IV) nitrate, oxalic acid, and EDTA to provide an electron acceptor. For example, the photocrosslinking compound preferably uses sodium persulfate, but is not limited thereto.

[0037] More preferably, the photocrosslinkable compound is [Ru(bpy)3] 2+ A combination of sodium persulfate and sodium sulfate can be used, in which case, when irradiated with light containing blue light, for example, light with a wavelength of 400 to 500 nm, or 420 to 480 nm, an adhesive bioadhesive material in the form of gel particles can be formed.

[0038] More specifically, for the production of the immunosuppressive bioadhesive particles of the present invention, it is preferable to perform photocrosslinking while mixing the first solution and the second solution, for example, while mixing with stirring.

[0039] At this time, the mixture of the first solution and the second solution may be obtained by mixing the first solution and the second solution in a volume ratio of 5 to 15:1, for example, it may be obtained by mixing in a volume ratio of 8 to 12:1. If the first solution is included in an amount less than the above range, there is a problem that the droplets are not well separated during stirring, making it difficult to control the particle size, and if the first solution is included in an amount exceeding the above range, there is a problem that the precursor solution is not well separated during stirring, limiting particle generation.

[0040] The solvent that can be used in the method for manufacturing the immunosuppressive bioadhesive particles of the present invention may be at least one selected from the group consisting of a physiological saline solution such as PBS, an organic solvent, and a contrast agent, and may be used alone or in combination of two or more thereof. For example, the solvent of the present invention may be an aqueous solution containing 20 to 40 v / v% DMSO.

[0041] The second solution may contain 2 to 10 wt% of mussel adhesive protein, 0.01 to 5 wt% of immunosuppressant, and the remainder of a solvent, based on the total weight of the second solution. If the content of the mussel adhesive protein is less than the above range, the adhesiveness of the particles may be insufficient, and if it exceeds the above range, the particle size may increase or the particle shape may not be smoothly obtained. On the other hand, if the content of the immunosuppressant is less than the above range, the immunosuppressive effect may be insufficient, and if it exceeds the above range, the particle shape may not be smoothly obtained.

[0042] Meanwhile, the second solution may contain a photoreactive metal ligand in an amount of 0.1 to 5 mM, more specifically, 1 to 3 mM, and when the concentration of the photoreactive metal ligand is within the above range, a sufficient reaction speed required for photopolymerization is provided, and the subsequent initiator removal process can be carried out stably and effectively.

[0043] Additionally, the second solution may contain 1 to 20 mM of the electron acceptor, for example, 5 to 15 mM, and when the concentration of the electron acceptor is within the above range, stable micro-sized fine particles can be formed.

[0044] According to another aspect of the present invention, an immunosuppressive adhesive particle obtained by the method for producing the above-described immunosuppressive bioadhesive particle is provided.

[0045] The particle size obtained by the present invention may range from 1 to 200 μm, and the average particle diameter may be from 3 to 15 μm, for example, from 5 to 12 μm. When particles of such a size are formed, stable spraying is possible, and thus, the composition may be applied as a spray formulation.

[0046] According to another aspect of the present invention, a topical immunosuppressive composition comprising the immunosuppressive adhesive particles of the present invention is provided.

[0047] When using the local immunosuppressive composition of the present invention, the immunosuppressive drug loaded into the microparticles is continuously released while the particles are maintained, thereby shortening the dosing cycle.

[0048] At this time, the local immunosuppressive composition may contain 60 to 90 v / v% of the immunosuppressive adhesive particles and the remainder of the solvent based on the total volume of the composition, for example, 65 to 85 v / v% and the remainder of the solvent. If the amount of the immunosuppressive adhesive particles is less than the above range, a large amount of the composition tends to need to be applied to coat or apply the entire surface when applied to a target, and if it exceeds the above range, smooth spraying may not occur.

[0049] The topical immunosuppressive composition of the present invention may be a formulation for spraying. More specifically, the topical immunosuppressive composition of the present invention may be formulated as a spray for surface coating, and the solvent that may be used in this case may be, for example, an aqueous ethanol solution. Preferably, the spray composition may be prepared using a solvent obtained by mixing physiological saline and ethanol in a volume ratio of 1 to 5: 5 to 9.

[0050] The above spray method may be a compressed spray, and the compressed spray process can induce surface coating quickly and evenly. The drug delivery method based on the composition formulated as a spray formulation of the topical immunosuppressive composition of the present invention can maintain a stable immunosuppressive effect for a long period of time due to the high adhesiveness and retention power of the spray particles themselves.

[0051] That is, the organ to which the local immunosuppressive composition of the present invention is applied can be locally surface-coated with the local immunosuppressive composition of the present invention, thereby continuously releasing the loaded drug from the target organ, thereby enabling long-term drug action during organ transplantation.

[0052] Hereinafter, the present invention will be described in more detail through specific examples. The following examples are merely illustrative examples to aid understanding of the present invention and are not intended to limit the scope of the present invention.

[0053] Example

[0054] Example 1: Preparation of adhesive microgel particles loaded with immunosuppressive drugs

[0055] A 50 ml glass Erlenmeyer flask and a magnetic stirring bar were used as a batch reactor.

[0056] The first solution was prepared by mixing 3 wt% of the lipophilic surfactant Span 80 with mineral oil (Sigma Aldrich) based on the total weight of the first solution and stirring at 700 rpm in a glass Erlenmeyer flask.

[0057] Mussel adhesive protein is contained in an amount of 4 wt% (120 mg) based on the total weight of the second solution, and the immunosuppressant drug Cyclosporine A (CsA) is contained in an amount of 0.2 wt% (6 mg) based on the total weight of the second solution, and a polymerization reaction initiator [Ru(bpy)3] 2+ A second solution (referred to interchangeably as 'precursor solution') was prepared by dissolving 1 mM and 10 mM SPS in 3 ml of a mixed solvent containing 2 ml of DW (Distilled water) and 1 ml of DMSO (Dimethyl sulfoxide).

[0058] The first and second solutions were added in a volume ratio of 10:1 and stirred at 700 rpm for 10 minutes.

[0059] Afterwards, the mixture was exposed to blue light for 1 minute while continuously stirring at 700 rpm to induce photocrosslinking, thereby producing microgel particles, which were then obtained through centrifugation.

[0060] The microgel particles thus produced were observed using a scanning electron microscope (a) and a confocal microscope (b), and the results of measuring the diameter of the particles (c) are shown in Figure 1.

[0061] Meanwhile, the adhesive strength of the microgel particles thus obtained was measured using a QCM (Quartz Crystal Microbalance), and the results are shown in Fig. 2. As a result, as can be confirmed in Fig. 2, the particles were attached to the QCM sensor, and it was confirmed that the particles were well adhered even after the washing process.

[0062] Manufacturing Example 1: Manufacturing of a spray composition based on adhesive microgel particles

[0063] The adhesive microgel particles manufactured in Example 1 were concentrated in a 2 ml solution of PBS:ethanol (volume ratio 3:7) to a microgel volume ratio of 75 v / v% to manufacture a spray composition.

[0064] The manufactured spray composition was compressed and sprayed through a compression spray container, and the results of observing the particles before (a) and after (b) spraying using a confocal microscope and the change in diameter (c) are shown in Fig. 3.

[0065] Referring to the results of Fig. 3, it was confirmed that the microgel particles of the present invention were stably maintained without change in diameter even after the compression injection process.

[0066] Furthermore, to confirm the retention power of the spray particles, the spray composition was sprayed on pig skin tissue measuring 4 cm in width and 2 cm in length, dried for 2 minutes, and stirred in 10 ml of PBS for 2 weeks. The results were measured using a fluorescence microscope, and the results are shown in Fig. 4. As a result, it was confirmed that the surface coating state of the pig skin was stably maintained in a physiological underwater environment for 2 weeks.

[0067] In addition, the microgel particles were locally sprayed onto pig skin tissue and the coating of the desired area was measured using an optical camera (left photo of Fig. 5) and an optical microscope (right photo of Fig. 5), and this is shown in Fig. 5.

[0068] Experimental example

[0069] 1. Confirmation of immunosuppressive drug loading efficiency and release in adhesive microgel particles

[0070] (1) Confirmation of the efficacy of immunosuppressant drug loading

[0071] The adhesive microgel particles manufactured in Example 1 were freeze-dried for 1 day, then the gel particles were crushed with a mortar and pestle, mixed with distilled water (DW), and the absorbance was measured using a UV absorbance analyzer, which is shown in Figure 6.

[0072] The results were compared with the absorbance of cyclosporine A aqueous solutions at concentrations of 0.010%, 0.006%, and 0.004%. As a result, the loading efficiency of cyclosporine A drug in the adhesive microgel particles was calculated as (cyclosporine A inside the microgel / cyclosporine A contained in the initial precursor solution)*100, and it was confirmed that the drug was loaded with a loading efficiency of 62.4%.

[0073] (2) Confirm drug release efficiency

[0074] The microgel particles manufactured in Example 1 were mixed with 10 ml of PBS in a dialysis pack of MWCO 3.5 kD, placed in a conical tube containing 30 ml of PBS, and placed in an incubator at 37°C and stirred. 1 ml of the supernatant was extracted during the 3-week incubation period, and the released drug concentration was measured using a UV absorbance analyzer. The results are (a) and (b) of the Higuchi model. The results of analyzing the expected date of complete release through (b) are shown in Fig. 7. As a result, the expected period of time for the drug loaded on the adhesive microgel particles to be completely released was analyzed to be approximately 24 days.

[0075] 2. Observation of immune response following xenograft transplantation: Animal modeling and spray application

[0076] After euthanizing 6- to 8-week-old Sprague-Dawley rats, dorsal epidermal tissues were collected in 1 cm × 1 cm sized pieces. The collected tissues were washed twice consecutively in 5 ml of minimal essential medium (DMEM) containing fetal bovine serum (FBS) and antibiotics, and then stored in cold physiological saline solution (PBS).

[0077] The dorsal epidermal tissue of an anesthetized BALB / c mouse older than 6 weeks was incised into a 1 cm × 1 cm piece.

[0078] After spraying 1 ml of the adhesive microgel spray produced in Manufacturing Example 1 onto the rat epidermal tissue, it was dried for 2 minutes and then sutured to the incision site of the mouse using a suture, and the process is schematically shown in Fig. 8(a). Meanwhile, the results measured using an optical camera (b) and a Fluorescence In Vivo Imaging System (FOBI) (c) are shown in Fig. 8. As a result, it was confirmed that the rat tissue was successfully sutured and the sprayed adhesive microgel particles were maintained at the transplantation site.

[0079] 3. Tissue collection and immune factor analysis for immune response observation

[0080] A 4 cm incision was made in the skin below the ribs of the xenograft transplantation model mouse manufactured in the above 2., and the diaphragm and intercostal space were then incised. 0.3 to 0.5 ml of blood was collected from the left atrium using a 1 ml syringe. The collected blood was analyzed by ELISA and is shown in Fig. 9. The ELISA analysis results confirmed that the concentrations of IL-2 and IFN-γ in the blood of the untreated tissue (untreated group) were higher than those of the tissue collected from normal mice (normal group) at both the first and third weeks after tissue transplantation. In the case of tissues injected with only immunosuppressive drugs (CsA-only treatment group), the concentration of IL-2 in the blood was suppressed in the first week, but continuous release was not possible, and it was confirmed that it was similar to the negative group in the third week. In the case of tissues applied with adhesive microgel particle spray (MAP MP@CsA treatment group), the concentrations of IL-2 and IFN-γ in the blood were confirmed to be significantly suppressed until the third week.

[0081] In addition, the epidermis of the left hind leg of the xenograft transplantation model mouse manufactured in the above 2. was incised 1 cm above. After partially incising the dermis over the spleen, the connective tissue was incised to collect the spleen. The collected spleen tissue was finely crushed on a cell strainer and 3 ml of physiological saline was sprayed to obtain a physiological saline solution containing cells. The physiological saline solution in which the cells were dispersed was centrifuged at 2000 rpm for 3 minutes to separate only the sediment and then dispersed in a flow cytometry solution (90% physiological saline, 10% fetal bovine serum solution). The flow cytometry solution in which the cells were dispersed was diluted to a concentration of 500,000 cells per ml. After dissolving 0.001 wt% of CD3e and CD44-specific fluorescent dyes in the diluted cell-dispersed flow cytometry solution, the cells were stained in a darkroom for 30 minutes. The stained cells were centrifuged at 1500 rpm for 3 minutes and then washed three times consecutively by redispersing in 1 ml of flow cytometry solution. The washed cells were analyzed using a flow cytometer, and the T cell activation rate (CD44 fluorescence detection result, x-axis) compared to total T cells (CD3e fluorescence detection result, y-axis) is shown in Figure 10.

[0082] As a result, as can be confirmed in Fig. 10, compared to the tissues collected from normal mice (normal group), the tissues injected with only the immunosuppressive drug (CsA only treatment group) and the tissues not treated at all (negative group) showed a noticeably higher T cell activation tendency compared to the results of total T cell fluorescence detection. On the other hand, the tissues to which the adhesive microgel particle spray of Manufacturing Example 1 loaded with the immunosuppressive drug of the present invention was applied (MAP MPs@CsA treatment group) showed a similar T cell activation tendency to the normal group, and it was confirmed that a significant immunosuppressive effect was exhibited in terms of cell activity.

[0083] 4. Collection of transplanted xenograft tissue and analysis of inflammatory response

[0084] Skin tissues containing 0.5 cm of host tissue were collected from the transplant site of the xenograft model mouse manufactured in the above 2. Tissues from normal mice older than 6 weeks were also collected from the same location and size and stored in a 10 ml formalin solution. Afterwards, H&E tissue staining was performed on the collected tissues, and the results of observation under an optical microscope and analysis of the area ratio of inflammation using the Image J program are shown in Figure 11.

[0085] As a result, as can be seen in Fig. 11(a), it was confirmed that a severe inflammatory response occurred in the tissue injected with only an immunosuppressive drug (CsA-only treatment group) and the tissue that was not treated at all (untreated group) compared to the tissue collected from normal mice (normal group). On the other hand, it was confirmed that the inflammatory response was significantly improved in the tissue to which the adhesive microgel particle spray of Manufacturing Example 1 loaded with an immunosuppressive drug according to the present invention was applied (MAP MPs@CsA treatment group). Fig. 11(b) is a graph showing the results of measuring the inflammation area ratio.

[0086] Although the embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and it will be apparent to those skilled in the art that various modifications and variations are possible within a scope that does not depart from the technical spirit of the present invention described in the claims.

Claims

1. A step of preparing a first solution containing a biocompatible oil and a surfactant; A step of preparing a second solution, which is a precursor solution containing a mussel adhesive protein, an immunosuppressant, a photocrosslinking compound and a solvent; and A step of photocrosslinking a mixture of the first solution and the second solution; A method for producing an immunosuppressive bioadhesive particle comprising:

2. A method for producing bioadhesive particles for immunosuppression, wherein the biocompatible oil in claim 1 is at least one oil selected from the group consisting of mineral oil, oleic acid, and Fluorinert FC 40 oil.

3. A method for producing bioadhesive particles for immunosuppression, wherein in the first paragraph, the surfactant is at least one nonionic surfactant selected from the group consisting of sorbitan monolaurate (Arlacel 20), sorbitan monopalmitate (Span-40), sorbitan monooleate (Span-80), sorbitan monostearate, and sorbitan tristearate.

4. A method for producing an immunosuppressive bioadhesive particle, wherein the first solution comprises 1 to 10 wt% of a surfactant based on the total weight of the first solution.

5. A method for producing an immunosuppressive bioadhesive particle, wherein in paragraph 1, the mussel adhesive protein is at least one selected from the group consisting of Mefp-1, Mgfp-1, Mcfp-1, Mefp-2, Mefp-3, Mgfp-3, and Mgfp-5.

6. A method for producing an immunosuppressive bioadhesive particle in claim 1, wherein the immunosuppressant is at least one selected from the group consisting of azathioprine, methotrexate, a macrolide, cyclosporine, tacrolimus, pimecrolimus, everolimus, sirolimus, deforolimus, everolimus, temsirolimus, zotarolimus, infliximab, etanercept, rituximab, tocilizumab, and abatacept.

7. A method for producing an immunosuppressive bioadhesive particle, wherein the photocrosslinkable compound comprises a photoreactive metal ligand and an electron acceptor in the first paragraph.

8. A method for producing an immunosuppressive bioadhesive particle, wherein the solvent in paragraph 1 is an aqueous solution containing 20 to 40 v / v% of DMSO.

9. A method for producing an immunosuppressive bioadhesive particle in the first paragraph, wherein the second solution contains 2 to 10 wt% of a mussel adhesive protein, 0.01 to 5 wt% of an immunosuppressant, and the remainder of a solvent, based on the total weight of the second solution.

10. A method for producing an immunosuppressive bioadhesive particle in claim 7, wherein the second solution contains 0.1 to 5 mM of a photoreactive metal ligand and 1 to 20 mM of the electron acceptor.

11. A method for producing bioadhesive particles for immunosuppression, wherein the mixture of the first solution and the second solution in the first paragraph is obtained by mixing the first solution and the second solution in a volume ratio of 5 to 15:

1.

12. An immunosuppressive adhesive particle obtained by any one of claims 1 to 11.

13. An adhesive topical immunosuppressive composition comprising the immunosuppressive adhesive particles of claim 12.

14. In claim 13, the local immunosuppressive composition comprises 60 to 90 v / v% of immunosuppressive adhesive particles and the remainder of a solvent based on the total volume of the composition.

15. An adhesive local immunosuppressive composition in claim 13, wherein the local immunosuppressive composition is in a form for spraying.

Images