Polymer microparticles carrying nanoparticles for nucleic acid delivery

Abstract

The present invention relates to polymer microparticles, more particularly to anionic or cationic polymer microparticles, that encapsulate lipid nanoparticles (LNPs) containing nucleic acids such as siRNA. The present invention also deals with pharmaceuticals comprising such microparticles, and the use of such microparticles as pharmaceuticals, more particularly for the treatment of chronic inflammatory bowel disease (IBD).

Description

[Technical Field]

[0001] The present invention relates to polymer microparticles, more particularly to anionic polymers such as alginates or cationic polymers, that encapsulate lipid nanoparticles (LNPs) containing nucleic acids such as siRNA. The present invention also deals with pharmaceuticals containing such microparticles and the use of such microparticles as pharmaceuticals, more particularly for the treatment of chronic inflammatory bowel disease (IBD). [Background technology]

[0002] Inflammatory bowel disease (IBD) includes ulcerative colitis (UC) and Crohn's disease (CD). Chronic inflammation of the gastrointestinal tract causes digestive disorders including diarrhea, rectal bleeding, and abdominal pain, as well as fever, weight loss, and anemia. 1 In ulcerative colitis (UC), inflammation is primarily observed in the colon, whereas in complete remission (CR), various parts of the intestine are affected. Current treatments include aminosalicylates, corticosteroids, immunomodulators, and biological therapies. 2 Mesalazine is an aminosalicylate used for the topical treatment of UC, but it is no longer recommended for CD due to its lack of efficacy. Corticosteroids (budesonide, prednisone) are administered for the treatment of IBD, particularly as first-line treatment for complete remission and for the treatment of UC after aminosalicylates have failed. Immunomodulatory agents, especially azathioprine, are recommended in cases of refractory UC or to maintain remission. Biological therapies, including anti-TNF and anti-IL12-23, are also administered in severe or refractory cases of IBD and as maintenance therapy for remission. 3 These current treatments face numerous limitations in terms of efficiency and tolerability. Aminosalicylates induce few side effects because their action is local, but their efficiency is moderate. In UC and CD, up to 16% of patients do not respond to corticosteroids. Resistance or steroid dependence has also been observed during treatment. 4In addition to numerous adverse effects associated with immunomodulation (risk of infection), azathioprine takes effect two or three months after treatment. Due to immunogenicity and targeting issues, primary and secondary failures are often observed with biological therapies. With anti-TNF, primary and secondary failures were observed in 30% and 40% of patients. 5 In addition, while complete adherence to the treatment regimen is necessary for efficiency, the routes of administration (subcutaneous and intravenous) are limited and may be inconvenient for the patient.

[0003] Nucleic acid-based strategies using small interfering RNAs (siRNAs) and antisense oligonucleotides have been initiated as an alternative to antibodies to inhibit the expression of pro-inflammatory cytokines such as TNF. However, their benefits in preclinical and clinical trials have not been clearly demonstrated for the treatment of IBD. The lack of efficiency may be due to the short plasma half-lives of nucleic acids after systemic administration. 6 Subsequently, for better efficacy, tolerability, and convenience for patients, oral administration of nucleic acids, particularly siRNA, emerged, delivering nucleic acids to the inflamed site in the intestines. Lipopolyplexes or siRNA-laden nanoparticles embedded in gastric-resistant hydrogels were designed for the treatment of IBD. 7~9 However, their efficiency was limited due to major problems including insufficient intracytoplasmic delivery of siRNA, slow release from hydrogels, and difficulties in scaling up due to complex formulation designs. 10 . [Overview of the project] [Problems that the invention aims to solve]

[0004] Therefore, it was necessary to develop more effective treatments. [Means for solving the problem]

[0005] According to a first aspect, the present invention relates to polymer microparticles containing nucleic acid-containing lipid nanoparticles, and more particularly to anionic or cationic polymer microparticles containing nucleic acid-containing lipid nanoparticles.

[0006] In a second embodiment, the present invention relates to a method for producing microparticles of anionic or cationic polymers in which lipid nanoparticles containing nucleic acids are encapsulated.

[0007] A third aspect of the present invention relates to a pharmaceutical composition comprising polymer microparticles, more particularly anionic or cationic polymer microparticles, containing nucleic acid-containing lipid nanoparticles (LNPs), suspended in an additional pharmaceutically acceptable excipient.

[0008] A fourth aspect of the present invention relates to polymer microparticles containing nucleic acid-containing lipid nanoparticles, more particularly to anionic or cationic polymer microparticles, or to pharmaceutical compositions containing such microparticles, for use as pharmaceuticals, and more particularly for use in the treatment of chronic inflammatory bowel disease (IBD). [Modes for carrying out the invention]

[0009] The inventors discovered that polymer microparticles containing nucleic acid-containing lipid nanoparticles (LNPs) were more effective in treating inflammatory diseases.

[0010] Accordingly, according to a first aspect, the present invention relates to polymer microparticles in which lipid nanoparticles containing nucleic acids are encapsulated. This means that the nucleic acids are encapsulated in LNPs, and the LNPs themselves are encapsulated in polymer microparticles.

[0011] The polymer may be an anionic polymer, a cationic polymer, or a nonionic polymer.

[0012] More specifically, the polymer is an anionic polymer or a cationic polymer.

[0013] In the intent of the present invention, an anionic polymer is a polymer having one or more monomer units that are covalently bonded and carry a net negative charge.

[0014] Typically, anionic polymers include alginates, pectins, carboxymethylcellulose (CMC), gellan gum, carrageenan gum, or xanthan gum.

[0015] In the intent of the present invention, a cationic polymer is a polymer having one or more monomer units that are covalently bonded and carry a net positive charge. Typically, cationic polymers are chitosan, or acrylic copolymers having ammonium groups such as ethylpropa-2-enoate; methyl-2-methylpropa-2-enoate; trimethyl-[2-(2-methylpropa-2-enoyloxy)ethyl]azanium; and chloride.

[0016] Nonionic polymers are polymers that do not possess any electric charge. Typically, nonionic polymers include poly(D,L-lactic acid) (PLA), poly(D,L-lactic acid-glycolic acid copolymer) (PLGA), and poly(caprolactone) (PCL).

[0017] More specifically, the present invention deals with alginate microparticles containing lipid nanoparticles that include nucleic acids.

[0018] Alginates are polysaccharides obtained from brown algae such as kelp or wrack. Alginates are linear polymers consisting of L-gluronate (m) and D-mannuronate (n) residues linked via 1,4-glycosidic linkages. The alginate formula is as follows, where m and n are integers greater than 0:

[0019] [ka]

[0020] The inventors have found that these microparticles were more effective in the treatment of inflammatory bowel disease than systemic treatment based on monoclonal antibodies or immunosuppressive drugs. This formulation acts directly on the inflamed sites to inhibit the expression of pro-inflammatory cytokines. Selective distribution of the microparticles in the inflamed intestinal regions has been demonstrated.

[0021] Furthermore, this enables more efficient administration of nucleic acids via the oral route. Local and targeted delivery is a less risky approach than systemic treatment and is associated with fewer side effects. In addition, this treatment route is less painful for the patient than parenteral administration, and the patient can be autonomous in undergoing the treatment, and thus avoid high treatment costs as no hospitalization or trained personnel are required to administer the treatment.

[0022] Furthermore, the formulation in the form of microparticles confers better stability to nucleic acids by isolating the substance from the external environment (enzymes, acidic pH, bile salts, oxygen, light) by encapsulation, such that the substance is protected from chemical degradation. It has been demonstrated that the formulation can be lyophilized in order to store the formulation in dry form and thus avoid storage at sub-zero temperatures. Spray drying of the microparticles is also envisaged for storing the microparticles or including them in solid dosage forms such as tablets and capsules.

[0023] Furthermore, the protective and mucoadhesive action of alginate preserves the activity of the nucleic acids while allowing for long-term retention on the mucosa. Thus, this long-term release at physiological pH (7.4) results in an extended action time of the RNA after administration, which allows for a more effective local action and thus limits the number of administrations.

[0024] Specifically, the microparticles of the polymer, specifically the microparticles of an anionic or cationic polymer, are at least 50 wt%, at least 60 wt%, at least 70 wt%, at least 80 wt%, at least 85 wt%, at least 90 wt%, at least 95 wt%, at least 99 wt%, preferably between at least 50 wt% and 99 wt% of the polymer, specifically an anionic or cationic polymer, based on the mass of the microparticles encapsulating the lipid nanoparticles containing nucleic acids. Specifically, the microparticles encapsulating the lipid nanoparticles containing nucleic acids essentially contain a polymer, specifically an anionic or cationic polymer. Specifically, they essentially contain alginate.

[0025] Specifically, when the microparticles are composed of an anionic polymer, the microparticles may further contain polyvalent cations such as calcium ions, barium ions, manganese ions, copper ions, aluminum ions, and zinc ions, or cationic polyelectrolytes such as chitosan, in addition to the anionic polymer. In such embodiments, the microparticles of the anionic polymer contain at least 0.01 wt%, specifically at least 0.1 wt%, specifically at least 1 wt% of polyvalent cations based on the mass of the microparticles encapsulating the lipid nanoparticles containing nucleic acids.

[0026] When the microparticles are composed of a cationic polymer, the microparticles may further contain polyvalent anions such as tripolyphosphate ions, pyrophosphate ions, and sulfate ions, or anionic polyelectrolytes such as alginate, carrageenan, pectin, xanthan gum, and hyaluronic acid, in addition to the cationic polymer. In such embodiments, the microparticles of the cationic polymer contain at least 0.01 wt%, specifically at least 0.1 wt%, specifically at least 1 wt% of polyvalent anions based on the mass of the microparticles encapsulating the lipid nanoparticles containing nucleic acids.

[0027] In certain embodiments, the average hydrodynamic diameter of the microparticles is between 10 μm and 1 mm, more specifically between 50 μm and 500 μm, and more specifically between 100 μm and 200 μm.

[0028] More specifically, the lipid nanoparticles encapsulated in the microparticles consist of cholesterol, phospholipids, polyethylene glycol (PEG) lipids (1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene glycol-2000), and ionized lipids having a positive charge at a pH of less than 6. In certain embodiments, the lipid nanoparticles contain lipid molar ratios of 30-40%, preferably 38.5%, for cholesterol, 5-20%, preferably 10%, for polyethylene glycol (PEG) lipids (1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene glycol-2000), preferably 1.5%, and for ionized lipids, preferably 30-65%, preferably 50%.

[0029] Phospholipids may be phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylglycerol. Phospholipids may be composed of saturated hydrocarbon chains such as distearoyl, dipalmitoyl, dimyristoyl, and dilauroyl, or unsaturated hydrocarbon chains such as dioleoyl.

[0030] Typically, ionizable lipids having a positive charge at a pH below 6 are Dlin-MC3-DMA (CAS 1224606-06-7), SM-102 (CAS 2089251-47-6), ALC-0315 (CAS 2036272-55-4), Acuitas A9 (CAS 2036272-50-9), Arcturus 4C CH3 (CAS 2230647-30-8), Genevant CL1 (CAS 1450888-71-7), LP01 (CAS 1799316-64-5), OF-02 (CAS 1883431-67-1), A18-Iso5-2DC18 (CAS 2412492-09-0), 98N12-5 (CAS 917572-74-8), C12-200 (CAS 1220890-25-4), cKK-E12 (CAS 1432494-65-9), 9A1P9 (CAS 2760467-57-8), 7C1, G0-C14 (CAS 1510653-27-6), L319 (CAS 1351586-50-9), 304-O13 (CAS 1566559-80-5), OF-Deg-Lin (CAS 1853202-95-5), 306-O12B (CASs 2566523-06-4), 306Oi10 (CAS 2322290-93-5), FTT5 (CAS 2328129-27-5), and preferably Dlin-MC3-DMA. Some of these are shown by Han et al. (2021) 11 as shown in

[0031] In certain embodiments, the average hydrodynamic diameter of the lipid nanoparticles (LNP) is between 30 nm and 250 nm, specifically between 50 nm and 200 nm, and more specifically between 80 and 180 nm. The average hydrodynamic diameter of the lipid nanoparticles is necessarily less than the average hydrodynamic diameter of the microparticles. One of ordinary skill in the art is fully aware of how to select the hydrodynamic diameter of lipid nanoparticles suitable for the effective encapsulation of nanoparticles into microparticles.

[0032] According to this disclosure, the nucleic acids encapsulated in the lipid nanoparticles may be messenger RNA (mRNA), small interfering RNA (siRNA), antisense oligonucleotide (ASO), short hairpin RNA (shRNA), or complementary DNA (cDNA).

[0033] More specifically, nucleic acids affect the expression of pro-inflammatory cytokines or integrins. Pro-inflammatory cytokines may be TNF-α, IL1, IL6, IL8, IL10, IL17, CCL2, IL12, or IL23. Integrins may be α4β7 or α4β1.

[0034] In detail, the nucleic acids are siRNAs that target TNFα, IL1, IL6, IL8, IL10, IL17, CCL2, IL12, or IL23, or integrin α4β7 or α4β1. In this application, when an siRNA is said to target a cytokine or protein such as an integrin, it means that the siRNA is designed to hybridize to the mRNA encoding the protein, resulting in the disruption of the mRNA, and therefore a decrease in the expression of the gene and a decrease in the level of the encoded protein.

[0035] In certain embodiments, the microparticles are alginate microparticles, and the nucleic acid encapsulated in the LNP is TNFα-targeting siRNA.

[0036] In certain embodiments, the lipid nanoparticles may contain different nucleic acids, each targeting a different target. The nanoparticles may contain at least two different nucleic acids, more specifically two, three, or four nucleic acids, each targeting a different target. The targets are selected from those listed above.

[0037] The aforementioned nanoparticles may further contain an active molecule in addition to nucleic acids. This allows nucleic acids to be used in combination with active molecules other than nucleic acids. The active molecule may be a corticosteroid such as betamethasone, prednisolone, or budesonide; a nonsteroidal anti-inflammatory drug such as mesalazine, orsalazine, 4-aminosalicylic acid, or sulfasalazine; or an immunosuppressant such as azathioprine, methotrexate, or cyclosporine. The active molecule may also be an anti-TNF antibody or an anti-cytokine antibody, such as an antibody against IL-10. The active molecule may also be an interleukin having protective properties, such as IL-22 or IL-24.

[0038] Microparticles and nanoparticles can be conjugated with carbohydrates such as mannose-rich compounds, as well as antibodies targeting immune cells, including macrophages, such as type C lectin receptors, Fc receptors, and CD44. They may also contain or be grafted onto therapeutic antibodies targeting tumor necrosis factor, pro-inflammatory cytokines, and integrins. For example, a therapeutic antibody may target a pro-inflammatory cytokine selected from the group consisting of IL1a, IL1, IL6, IL8, IL10, IL17, CCL2, IL12, or IL23. For example, a therapeutic antibody may target integrin α4β7 or α4β1. For example, a therapeutic antibody may target TNFα.

[0039] Preparation of nanoparticles In a second embodiment, the present invention relates to a method for producing microparticles of anionic or cationic polymers containing nucleic acid-containing lipid nanoparticles, the first step of which is: I- This is a process of encapsulating nucleic acids in lipid nanoparticles. 1. A lower step of preparing an organic phase by dissolving lipids in an organic solvent, such as anhydrous ethanol. 2. A lower step of preparing an aqueous phase by mixing nucleic acids in an aqueous solution containing a buffer such as a citrate buffer or an acetate buffer. 3. A lower step in which the organic phase of lower step I.1 is mixed with the aqueous phase of lower step I.2 in a volume ratio between 1:1 and 1:3. 4. A sub-step in which the LNP obtained in sub-step I.3 is recovered by dialysis in an aqueous phase. Process That is the case.

[0040] In a specific embodiment, the final lipid concentration in the lower step I.1 is between 25 and 50 mM. Specifically, the lipids include cholesterol, phospholipids, polyethylene glycol (PEG) lipids (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000), and ionized lipids.

[0041] In a specific embodiment, in step I.2, the citrate or acetate buffer solution has a concentration in the range of 10 to 100 mM. The pH of the buffer solution is 6 or less.

[0042] In a specific embodiment, in the lower step I.3, the nitrogen-to-phosphate molar ratio (N:P; nitrogen derived from ionized lipids and phosphate derived from nucleic acids) is between 3:1 and 9:1.

[0043] Between steps I.3 and I.4, a physiological buffer with a pH of 7.4, such as PBS, may be added to the mixture in a volume ratio of at least 1:1.

[0044] In a specific embodiment, the dialysis in the lower step I.4 is carried out overnight at 4°C. The aqueous phase may be purified water, HEPES, 1-10 w / w% sucrose, TRIS-HCl, or phosphate buffer.

[0045] Optionally, at the end of the procedure, the LNPs may be concentrated by ultrafiltration of the LNPs at 4°C and, if necessary, sterilized using a 0.45 or 0.22 μm filter.

[0046] In one embodiment, lipid nanoparticles containing nucleic acids may be encapsulated in microparticles of anionic polymer. The second phase of the method is II- This is a process of encapsulating lipid nanoparticles in microparticles of anionic polymer. 1. A lower step of preparing an aqueous solution of anionic polymers such as alginates at a concentration between 1 and 10 w / v%, 2. A lower step in which the lipid nanoparticle suspension obtained in lower step I.4 is added to the anionic polymer solution in lower step II.1. 3. A sub-step in which a citric acid solution with a pH of less than 3 is added to the mixture of sub-step II.2, more specifically, the pH is between 1.5 and 3, and the citric acid solution has a concentration between 20 mM and 150 mM, more specifically, 100 mM, sub-step 4. Add calcium chloride stock solution to achieve a final concentration of calcium chloride between 15 mM and 80 mM in the subsequent steps. 5. Add NaOH to bring the pH value between 7 and 8, specifically to 7.4, in the following steps. 6. A lower step in which the solution obtained in lower step II.5 is centrifuged at, for example, 5000 rpm. 7. Sub-process for recovering microparticles Process Includes.

[0047] At the end of the procedure, the supernatant may be replaced with water. Washing may be performed between sub-step II.1 and sub-step II.3.

[0048] In a specific embodiment, the stock solution of calcium chloride is at a concentration between 90 and 480 mM. More specifically, the citric acid solution is 100 mM and has a pH of less than 3.

[0049] The precipitation of the anionic polymer around the lipid nanoparticles in step II.3 is made possible by an acidic pH and an increase in pH that induces ionic gelation. In a specific embodiment, the anionic polymer is an alginate. Gelation with calcium is achieved by increasing the pH with 1 M sodium hydroxide to deprotonate the COOH group of the alginate and induce electrostatic interaction with Ca2+.

[0050] In another embodiment, the nucleic acid-containing lipid nanoparticles may be encapsulated in cationic polymer microparticles. The second step of the method is II- This is a process of encapsulating lipid nanoparticles in cationic polymer microparticles. 1. A lower step of preparing a solution of a cationic polymer such as chitosan in an acidic buffer such as acetate buffer at a pH between 2 and 5, 2. A lower step in which the lipid nanoparticle suspension obtained in lower step I.4 is added to the cationic polymer solution. 3. A buffer solution such as NaOH, TRIS-HCl, HEPES, or phosphoric acid solution is added to bring the pH above 5, specifically between 5 and 8, to induce a precipitate of cationic polymer around the lipid nanoparticles. 4. Add an anionic polymer such as alginate, carrageenan, pectin, xanthan gum, or hyaluronic acid, or an anionic polyvalent salt such as tripolyphosphate, and optionally add acetic acid in a subsequent step. 5. A lower step in which the solution obtained in lower step II.4 is centrifuged at, for example, 5000 rpm. 6. Sub-process for recovering microparticles Process That is the case.

[0051] Sub-processes II.3 and II.4 can be performed in reverse order.

[0052] In a specific embodiment, the anionic polymer in the lower step II.4 is added at a concentration between 1 and 10 w / v%.

[0053] Pharmaceutical composition A third aspect of the present invention relates to a pharmaceutical composition comprising polymer microparticles, more particularly anionic or cationic polymer microparticles, encapsulating lipid nanoparticles (LNPs) containing nucleic acids as described above, suspended in an additional pharmaceutically acceptable excipient.

[0054] More specifically, it is a pharmaceutical composition comprising polymer microparticles, more specifically anionic or cationic polymer microparticles, encapsulating lipid nanoparticles (LNPs) containing nucleic acids as described above, suspended in additional pharmaceutically acceptable excipients.

[0055] As used herein, the terms “pharmaceutically acceptable” or “pharmaceutically acceptable” mean molecular entities and compositions that, when administered appropriately to mammals, particularly humans, do not cause any adverse reactions, allergic reactions, or other undesirable reactions. A pharmaceutically acceptable carrier or excipient means any type of filler, diluent, or compounding aid, whether non-toxic, solid, semi-solid, or liquid.

[0056] As will be understood by those skilled in the art, pharmaceutically acceptable excipients and / or carriers may be selected based on the route of administration, the location of the targeted tissue, the time course of drug delivery, etc., as described below.

[0057] There are many examples of excipients that can be added to pharmaceutical compositions. Examples include, but are not limited to, sucrose, mannitol, trehalose, and buffers (tris-HCl, HEPES, phosphate).

[0058] Pharmaceutical compositions may be in liquid or solid form for oral administration. Examples of solid forms include capsules, tablets, pills, powders, and granules.

[0059] How to use A fourth aspect of the present invention relates to polymer microparticles containing lipid nanoparticles containing nucleic acids as described above, more particularly to anionic or cationic polymer microparticles, or to pharmaceutical compositions as described above, for use as pharmaceuticals.

[0060] The present invention relates to polymer microparticles containing nucleic acid-containing lipid nanoparticles as described above, more particularly to anionic or cationic polymer microparticles, or to pharmaceutical compositions as described above, for use in therapeutic methods.

[0061] More specifically, the present invention relates to polymer microparticles containing nucleic acid-containing lipid nanoparticles as described above, more particularly to anionic or cationic polymer microparticles, or to pharmaceutical compositions as described above, for use in the treatment of chronic inflammatory bowel disease (IBD). These diseases are characterized by inflammation of the inner wall of the gastrointestinal tract. Typical examples of chronic inflammatory bowel diseases include Crohn's disease and ulcerative colitis (UC).

[0062] As used herein, the terms “treatment” or “to treat” include both prophylactic or preventive treatments and curative or disease-modifying treatments, including treatments for patients at risk of or suspected of having a disease, and for patients diagnosed with a disease or medical condition, and including the suppression of clinical relapses. Treatments may be administered to subjects with a medical disability or who may eventually develop a medical disability, in order to prevent, treat, delay the onset of, reduce the severity of, or improve one or more symptoms of a disability or recurrent disability, or to extend the subject’s survival beyond the survival expected in the absence of such treatment.

[0063] More specifically, polymer microparticles containing nucleic acid-containing lipid nanoparticles as described above, more specifically, anionic or cationic polymer microparticles, or pharmaceutical compositions as described above are administered to patients via oral route.

[0064] These can be easily delivered in the form of liquids, capsules, tablets, pills, powders, and granules.

[0065] More specifically, the microparticles or compositions thereof are administered to patients in need at doses expressed as mg of nucleic acid per kg, which range from 0.1 to 100 mg of nucleic acid per kg, more specifically from 0.5 to 50 mg of nucleic acid / kg, and more specifically from 3 to 10 mg of nucleic acid / kg.

[0066] In certain embodiments, the microparticles or compositions thereof are administered to a patient in combination with at least one other active molecule. Therefore, the administration of the nanoparticles and at least one other active ingredient may be simultaneous, sequential, or over a period of time.

[0067] In one embodiment, at least one other active ingredient is selected from corticosteroids such as betamethasone, prednisolone, and budesonide; nonsteroidal anti-inflammatory drugs such as mesalazine, orsalazine, 4-aminosalicylic acid, and sulfasalazine; or immunosuppressants such as azathioprine, methotrexate, and cyclosporine. The other active ingredient may also be an anti-TNF antibody or an anti-cytokine antibody, such as an antibody against IL-10. The other active ingredient may also be an interleukin having protective properties, such as IL-22 or IL-24.

[0068] At least one other active ingredient may be administered orally, rectally, parenterally, intracisionally, or intraperitoneally.

[0069] More specifically, the present invention relates to a method for treating chronic inflammatory bowel disease (IBD), such as Crohn's disease and ulcerative colitis (UC), comprising administering to a patient in need polymer microparticles containing lipid nanoparticles such as nucleic acids as described above, more specifically anionic or cationic polymer microparticles, or administering a pharmaceutical composition such as described above. [Brief explanation of the drawing]

[0070] [Figure 1] This graph shows the percentage release of LNPs in alginate microparticles after 24-hour incubation at 37°C and different wash cycles in suspensions of different solvents, mediated by the amount of cholesterol added. It compares the percentage of release between different wash cycles. Data are the mean ± SEM of three independent formulations. **p ≤ 0.01 (compared to one wash cycle under each condition). [Figure 2] This graph shows the survival percentage in HepG2 cell lines after 24 hours of treatment with different concentrations of ONPATTRO® and different conditions: ONPATTRO® LNP only, resuspended in DPBS and diluted in FCS-free medium in alginate microparticles; ONPATTRO® resuspended in SGF and diluted in FCS-free medium in alginate microparticles; and ONPATTRO® resuspended in SIF and diluted in FCS-free medium in alginate microparticles. The negative control was untreated medium. Data are the mean ± SEM of three consecutive cultures. ***p ≤ 0.001 (compared to control). [Figure 3]This graph shows the survival percentage in THP-1 cell lines after 24 hours of treatment with different concentrations of LNP-loaded siRNA TNF-α and under different conditions: LNP-loaded siRNA TNF-α alone, LNP-loaded siRNA TNF-α in alginate microparticles resuspended in DPBS and diluted in FCS-free medium, and LNP-loaded siRNA TNF-α in alginate microparticles resuspended in SGF and diluted in FCS-free medium. The negative control was untreated medium. Data are the mean ± SEM of triplicate data from three independent cultures. [Figure 4] This graph shows the percentage of TTR protein production in HepG2 cell lines after 24 hours of treatment with different concentrations of ONPATTRO® under different conditions: ONPATTRO® only, ONPATTRO® in alginate microparticles diluted in FCS-free medium after three wash cycles and resuspending in DPBS, and ONPATTRO® in alginate microparticles diluted in FCS-free medium after three wash cycles and pre-incubating in SGF for 2 hours, then resuspending in DPBS and diluting in FCS-free medium. The negative control was untreated medium. Data are the mean ± SEM of three consecutive cultures. ***p ≤ 0.001 (compared to control). [Figure 5]The graph shows the percentage of TNF-α protein production by THP-1 cell lines after 24 hours of treatment with different concentrations of LNP-loaded siRNA TNF-α: LNP-loaded siRNA TNF-α only, LNP-loaded siRNA TNF-α in alginate microparticles diluted in FCS-free medium after one or two wash cycles and resuspending in DPBS, and LNP-loaded siRNA TNF-α in alginate microparticles diluted in FCS-free medium after one or two wash cycles and pre-incubating in SGF for 2 hours, then resuspending in DPBS and diluting in FCS-free medium. Various controls were studied in FCS-free medium: different concentrations of siRNA only, 0.1 μg / mL LPS, 50:50 (v:v) DPBS, and LNP without siRNA, LNP without siRNA in microparticles, and the same amount of empty microparticles as the maximum concentration of siRNA used for the corresponding conditions. The negative control was untreated medium. The data are the mean ± SEM of triplicate cultures from three independent cultures: *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001 (compared to control). [Figure 6] This graph shows the therapeutic index (colon mass / colon size) for different mouse groups. [Figure 7] This table shows clinical parameters such as survival, weight loss, rectal bleeding, and diarrhea, which were monitored daily (death = 6, healthy = 0). [Figure 8] This graph shows TNF-α expression compared to the colitis group in the DSS model (Experiment 2). siRNA TNF-α in free form or encapsulated in MPs, along with empty MPs, were administered orally. TNF-α levels in the proximal (A) and distal (B) colon were determined by qRT-PCR. Each group consisted of 8 mice. *p≦0.05, and ***p≦0.001 (compared to the colitis group). [Figure 9]This graph shows the clinical scores (evaluation of survival and weight loss) of different mouse groups according to the treatment they received. The negative control group, which was not treated with TNBS, corresponds to 0 (group of 6 mice). *p≦0.05, and ***p≦0.001 (compared to the control (colitis group)). (A) Experiment 1, (B) Experiment 2. [Figure 10] This graph shows the change in the average weight of each group compared to day 0 (group of 6 animals). (A) Experiment 1, (B) Experiment 2. [Figure 11] This graph shows the treatment index for different mouse groups according to the treatment administered (group of 6 mice): *p≦0.05, **p≦0.01, and ***p≦0.001 (compared to the control group (colitis group)). (A) Experiment 1, (B) Experiment 2. [Figure 12] These images show the colons of mice after different oral treatments over three consecutive days. (A) Experiment 1, (B) Experiment 2. [Figure 13] This figure shows the measured fluorescence intensity in intestinal sections after oral administration of fluorescein amine-labeled MP to healthy mice and mice with TNBS-induced colitis. [Examples]

[0071] I. Materials Sodium alginate, calcium chloride, citric acid, citrate, acetic acid, acetate, sodium hydroxide, potassium dihydrogen phosphate, pepsin, and pancreatin were obtained from Sigma Aldrich and maintained under RNAse-free conditions.

[0072] Dlin-MC3, mPEG-2000, DSPC, and lipids including cholesterol were obtained from Advantilipids.

[0073] II. Method II.1. Preparation of simulated gastric juice (SGF) and simulated intestinal juice (SIF) The compositions of SGF and SIF followed USP recommendations. The preparation of SGF required sodium chloride, pepsin, and hydrochloric acid. First, a solution of sodium chloride and pepsin was prepared by adding 0.8 g of sodium chloride and 1.28 g of pepsin to 200 mL of ultrapure water under magnetic stirring for 5 minutes. Next, 2.8 mL of 0.4 N hydrochloric acid was added, and then the pH was adjusted to between 1 and 2 using hydrochloric acid and a pH meter. The solution was then centrifuged at 15000 g for 10 minutes at 4°C. The supernatant was then filtered through a 0.45 μm polycarbonate membrane.

[0074] The preparation of SIF required potassium dihydrogen phosphate, sodium hydroxide, and pancreatin. First, the solution was prepared by adding 2.72 g of potassium dihydrogen phosphate, 15.4 mL of 0.4 N sodium hydroxide solution, and 4 g of pancreatin to 150 mL of ultrapure water. The pH was adjusted to 6.8 using sodium hydroxide. The volume was then adjusted to 200 mL using ultrapure water. The solution was then centrifuged at 20,000 g for 1 hour at 4°C, and subsequently filtered through a 0.45 μm polycarbonate membrane.

[0075] II.2. Fabrication of Lipid Nanoparticles Lipid nanoparticles were prepared using a lipid phase and an acidic aqueous phase. The lipid phase contained four different ionized lipids, namely DSPC, cholesterol, PEGylated lipids, and ionized lipids, in ratios of 10% / 38.5% / 1.5% / 50%, respectively. In this study, D-Lin MC3 was used as the ionized lipid. The solvent used for this phase was anhydrous ethanol. The lipid phase was prepared in two steps. First, DSPC, cholesterol, and PEG-DMG were added to anhydrous ethanol and mixed in water at 40°C for 30 seconds to solubilize the lipids. Then, D-Lin MC3 was added to obtain the final lipid solution. The acidic phase consisted of a buffer. Citrate / citrate pairs and acetate / acetate pairs with a pH of 6 or less were tested. Then, siRNA or mRNA was added to the acidic aqueous phase. To protect the RNA from degradation, molecular biology grade water, guaranteed to be free of impurities and RNAse, was used. siRNA or mRNA was added to the acidic aqueous phase.

[0076] LNPs were obtained by nanoprecipitation consisting of the addition of an acidic solution of RNA and an ethanol solution of lipids. Mixing was performed by manual methods or by microfluidics.

[0077] II.3. Preparation of Alginate Microparticles Four solutions were prepared to produce alginate microparticles: an acidic buffer, an alginate solution (1 or 2 w / v%), a curing CaCl2 stock solution (300 mM or 400 mM), and a NaOH (1N) solution. For mounting, LNPs containing Onpattro and RNA-loaded LNPs were added to the alginate solution. The alginate solution was added to the acidic buffer under magnetic stirring to induce alginate precipitation. Then, the curing CaCl2 solution was added, and finally NaOH was added to increase the pH to a physiological level. LNP-loaded alginate microparticles were then obtained. Subsequently, they were washed by first centrifuging at 5000 rpm for 5 minutes, and then suspending in ultrapure water. The washing cycle can be performed between 1 and 3 times. An optional step consisting of lyophilization of the microparticles can be performed for long-term storage using sucrose or other sugars and polyols as cryoprotectants.

[0078] II.4. Physicochemical Characterization of LNPs and Alginate Microparticles The size and zeta potential characteristics of LNPs were evaluated using a Malvern Zetasizer.

[0079] Laser particle size analysis was used to measure the size of alginate microparticles using a Malvern Mastersizer 3000. This technique allows the inventors to obtain three types of information about the particle size in the suspension: D10, corresponding to the first decile diameter; D50, corresponding to the median diameter; and D90, corresponding to the ninth percentile diameter.

[0080] II.5. RNA Loading To determine the encapsulation efficiency in LNPs, the amount of RNA was measured in the sample after nanoprecipitation under two different conditions: with and without triton. RNA encapsulated within LNPs cannot be approached by reagents. Therefore, quantification without triton corresponds to the amount of unencapsulated siRNA. Triton causes destabilization of the LNP membrane and subsequent RNA release. The amount of RNA was detected and quantified using Qubit microRNA and the ribogreen kit. siRNA was measured using Qubit microRNA in the range of 0.2–150 ng / mL. The encapsulation efficiency (EE) was then calculated as follows:

[0081] II.6. Loading and release of LNPs in microparticles The microparticles were washed twice with ultrapure water and centrifuged to remove free LNPs. After the washing step, the alginate microparticles were placed in different culture media to test their release into the media. After incubation, the microparticles were centrifuged. The supernatant was saved. The microparticles were then dissolved in high-concentration phosphate buffer to induce alginate dissolution. The amount of LNPs was then evaluated by quantification of cholesterol using a Thermofischer Amplex cholesterol detection kit. Cholesterol detection was performed by adding 5 μL of sample, 45 μL of reaction buffer, and 50 μL of reagent solution to a 96-well plate. The reagent solution contained reaction buffer, cholesterol oxidase, horseradish peroxidase, and cholesterol esterase. A standard curve was obtained using pure cholesterol. The sample was then incubated at 37°C for 30 minutes and read. The readings were obtained using the absorption spectrum between 530 and 560 nm and the emission spectrum at 590 nm.

[0082] II.7. Survival Assay Cytotoxicity was tested for two different strains of THP1 cells. Cytotoxicity was measured using the MTT Promega test. In THP1 cells, empty alginate microparticles and LNPs were tested. A negative control was established by adding only culture medium, and a positive control was established using 0.1x triton in culture medium. 100 μL of each sample was added to a plate. Then, 20 μL of MTS was added to each well. The plate was then incubated at 37°C for 1–4 hours, after which readings were taken at a wavelength of 490 nm using a spectrophotometer.

[0083] II.8. siRNA Transfection The inventors measured the levels of protein in the sample using a sandwich ELISA, which allows them to evaluate the inhibition of protein expression and therefore the activity of siRNA. The ELISA kit was obtained from Thermofisher.

[0084] To normalize the results, protein quantification was performed using a BCA assay.

[0085] Quantitative determination of TTR Human hepatocyte cell line HepG2 was seeded at a rate of 110,000 cells per well in a 96-well plate. 24 hours after seeding, alginate microparticles pre-incubated in DPBS, SGF followed by PDBS, SGF followed by SIF, and SIF alone were incubated with the cells for 24 hours at concentrations of 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3.125 μg / mL, and 1.57 μg / mL. After treatment, TTR levels in hepatocytes were quantified to evaluate the activity of Onpattro® released from the alginate microparticles. Invitrogen coated sandwich ELISA plates were used for this measurement. The plates were read at two different wavelengths using a spectrophotometer: 450 nm and 570 nm. The absorbance obtained at 570 nm was then subtracted from the 450 nm reading to reduce background noise interference.

[0086] TNF 10,000 human monocyte cells of the THP1 cell line were seeded in 96-well plates. 24 hours after seeding, the cells were treated with phorbol 12-myristate 13-acetate (PMA) for 48–72 hours to induce differentiation of monocytes into macrophages. These macrophages were then able to react to lipid polysaccharides (LPS), a surface antigen found in bacteria such as Escherichia coli. The interaction between macrophages and LPS can trigger macrophage activation, which can lead to an inflammatory response. This includes the secretion of many inflammatory mediators, such as cytokines or TNF.

[0087] Alginate microparticles, pre-incubated in DPBS, SGF followed by DPBS, SGF followed by SIF, and SIF alone, were incubated with cells for 24 hours at concentrations of 233 nanomol / mL (D1), 174.75 nanomol / mL (D2), 131 nanomol / mL (D3), and 98.3 nanomol / mL (D4). After treatment, TNF levels in THP1-derived macrophages were quantified to evaluate the activity of siRNA-borne LNPs released from the alginate microparticles. Uncoated TNF ELISA plates were used. The plates were read at two different wavelengths using a spectrophotometer: 490 nm and 570 nm. The absorbance obtained at 570 nm was then subtracted from the 490 nm reading to reduce background noise interference.

[0088] II.9. In vivo evaluation TNBS (2,4,6-trinitrobenzenesulfonic acid) colitis The efficiency of LNPs in alginate microparticles was investigated using a TNBS model of colitis (62.5 mg / kg TNBS). Male Swiss / CD-1 mice (average body weight 25 g) were used. Colitis was induced by rectal administration of 62.5 mg / kg TNBS.

[0089] After inducing colitis, the animals were orally treated with siRNA TNF alfa at a dose of 5 mg / kg (dosage: 100 μl of LNP in alginate microparticles) for three consecutive days. Treatment was performed on days 1, 2, and 3, following TNBS induction on day 0. The mice were sacrificed on day 4. Two independent experiments were conducted. Control animals (colitis controls) were treated with physiological saline.

[0090] DSS (dextran sulfate sodium) colitis Experiment 1: A DSS model of colitis, the Babelc / jrj mouse, was used. Colitis was induced over 10 days using 3.5% (w:v) DSS in drinking water. After colitis induction, the animals were orally treated with 5 mg / kg siRNA TNF alfa (dosage: 100 μl of LNP in alginate microparticles) for 3 consecutive days. Control animals (colitis controls) were treated with physiological saline.

[0091] Experiment 2: Disease induction was performed in male C57BL / 6J mice using 2% DSS in drinking water from day D0 to day D5, with the DSS solution being replaced with fresh solution on day D3. MP containing siRNA TNF-α-loaded LNPs was orally administered at a dose of 10 mg / kg daily from day 3 to day 7. The mice were sacrificed on day 8.

[0092] Clinical evaluation Survival and clinical parameters such as weight loss, rectal bleeding, and diarrhea were monitored daily (death = 6, healthy = 0). After sacrifice, the colon was weighed and measured to calculate the therapeutic index. The resected colon tissue sample was incised longitudinally, rinsed with ice-cold PBS to remove luminal contents, and then shredded. TNF-alpha concentration was determined using mouse ELISA. After grinding in PBS, the sample was obtained.

[0093] III. Results III.1 Synthesis of Lipid Nanoparticles Lipid nanoparticles (LNPs) were synthesized using manual methods. The physicochemical characteristics of the LNPs were measured to confirm the reproducibility of their synthesis.

[0094] [Table 1]

[0095] As can be seen, the standard deviations for size, PDI, zeta potential, and siRNA encapsulation of microparticles are low.

[0096] [Table 2]

[0097] High encapsulation efficiency can be achieved regardless of the type of LNP used.

[0098] III.2. Synthesis of Alginate Microparticles The key fabrication parameters of alginate microparticles using the acid precipitation method were evaluated. The evaluation was performed using laser particle size analysis. Three parameters were obtained: the first decyl diameter (D10), the median diameter (D50), and the ninth decyl diameter (D90).

[0099] [Table 3]

[0100] The results showed that the concentration of calcium chloride added to the curing solution did not affect the size. The same was true for the encapsulation of lipid nanoparticles. However, the concentration of the alginate solution may affect the size. Increasing the concentration of alginate in the alginate solution may result in an increase in the size of alginate microparticles. Similarly, the effect of the number of washing cycles on the final size of alginate microparticles was also studied.

[0101] [Table 4]

[0102] Washing is thought to have only a slight effect on the size of alginate microparticles. In fact, after three washing cycles, the median diameter is approximately 40 μm smaller than after one washing cycle. We then began to study other possible effects that the number of washing cycles may have on the properties of alginate microparticles.

[0103] [Table 5]

[0104] [Table 6]

[0105] At sucrose concentrations exceeding 1 w / w%, the median diameter of the freeze-dried alginate microparticles was within the same range as the microparticles immediately after preparation, approximately 50–60 μm.

[0106] The concentration of calcium in the solvent was studied after one, two, or three washing cycles to investigate the variation in concentration. In the case of water, a significant decrease in calcium concentration was observed between the first and second washing cycles. However, the decrease between the second and third washing cycles was much smaller in comparison. In the case of SGF, even after one washing cycle, a lower calcium concentration was observed than after three washing cycles in water. This can be explained by the fact that alginate molecules can protonate at acidic pH and discouple from calcium. Next, it was investigated whether this calcium concentration could affect the release of LNPs encapsulated in alginate microparticles.

[0107] As shown in Figure 1, a higher number of wash cycles increases the release of LNPs into DPBS due to the decrease in the amount of calcium in the solvent. Furthermore, SIF, due to its large amount of phosphate anions, can cause the release of most LNPs regardless of the number of wash cycles. In the case of ultrapure water, it can be observed that there is little release and variation with respect to the number of wash cycles. This suggests that for microparticles to dissolve, they must lose some crosslinking agent either through an acidic reaction or chelation with anionic molecules. Moreover, it can be observed that the release into both SIF and DPBS increases significantly after the incubation period in SGF. This is consistent with previous results showing that SGF reduces the calcium concentration in alginate microparticles. This reduction is thought to greatly increase the solubility of the microparticles. Overall, alginate microparticles were observed to be stable in both ultrapure water and SGF. It was also observed that the incubation period in SGF allows for much greater release into other media such as SIF and DPBS.

[0108] III.3. In vitro evaluation As shown in Figure 2, the survival rate remained at approximately 100% for LNPs alone or for LNPs in alginate microparticles resuspended in DPBS. However, for alginate microparticles resuspended in SGF, a survival rate of approximately 30% was obtained at a concentration of 12.5 μg / mL, and at a concentration of 6.2 μg / mL, the survival rate quickly increased to 100%. Furthermore, for alginate microparticles resuspended in SIF, the survival rate increased gradually as the concentration decreased, rising from approximately 50% at 12.5 μg / mL to 75% at 1.6 μg / mL.

[0109] As in previous experiments, Figure 3 shows the 100% viability of LNP alone and LNP in alginate microparticles resuspended in DPBS. However, a lower viability was observed for LNP in alginate microparticles resuspended in SGF at a concentration of 233 nM. This indicates to the inventors that the selection of siRNA does not affect the viability at the concentration used.

[0110] The experiment shown in Figure 4 was used as a proof of concept that LNP can be encapsulated in alginate microparticles without affecting its efficacy. Therefore, ONPATTRO®, a well-known formulation with very stable characteristics and a long shelf life due to that stability, was used. When comparing the efficacy between LNP alone and LNP-alginate microparticles resuspended in DPBS, it was confirmed that a very similar decrease in TTR production was observed regardless of the ONPATTRO® concentration. Similarly, high efficacy was found in ONPATTRO® encapsulated in alginate microparticles and resuspended first in SGF and then in DPBS. This demonstrates to the inventors that encapsulation in alginate microparticles does not affect the in vitro efficacy of LNP.

[0111] After obtaining proof of concept that alginate microparticles do not affect the efficacy of LNPs using ONPATTRO®, we studied the efficacy of LNPs themselves in alginate microparticles. As shown in Figure 5, siRNA alone does not have the effect of reducing TNF-α production. However, when siRNA is encapsulated in LNPs, a clear dose-dependent decrease in TNF-α production is observed, from 20% with 250 nM siRNA to 55% with 105 nM siRNA.

[0112] III.4. In-person evaluation III.4.1. In vivo evaluation in DSS and TNBS models We used TNBS and DSS-induced colitis in mice to mimic Crohn's disease and ulcerative colitis, respectively.

[0113] DSS model of colitis In Experiment 1, TNF-α levels in treated mice were undetectable (background noise) after oral administration of microparticles loaded with LNPs. In contrast, untreated mice had an average colonic TNF-α level of 3048 pg / g.

[0114] In Experiment 2, colitis was induced from D0 to D5 using 2% DSS in drinking water. qPCR analysis was performed in the proximal and distal colon. The results revealed a decrease in TNF-α mRNA levels in the distal colon (Figure 8), demonstrating that MP can influence the inflammatory response.

[0115] TNBS model for colitis Oral administration of LNP-loaded alginate microparticles reduced the clinical score of mice by one-third compared to untreated mice and mice treated with empty microparticles, and by half compared to oral administration of free NPs. Histological observations demonstrated the absence of ulcerative necrotic lesions in the colon of treated animals, similar to healthy controls.

[0116] In the TNBS model, MP administered orally (LNP siRNA MP oral route) reduced both the animal's survival rate and clinical score (Figures 7 and 9), taking body weight into account, and the therapeutic index from the animal's colon mass / size ratio (Figures 6 and 11). The effect was greater than that of siRNA-loaded LNP, demonstrating the importance of gastric tolerance characteristics. After treatment with MP, the therapeutic index and colon images in Figure 12 were similar to those of healthy animals, demonstrating complete recovery of the animals. In the second experiment, the animals began to gain body weight from day 2 before the second dose, reaching their initial body weight before TNBS induction on day 4 (Figure 10).

[0117] III.4.2. In vivo evaluation using confocal imaging We labeled alginates with fluoresceinamine and LNPs with cyanine, and evaluated the in-vivo distribution of particles after oral administration in a mouse model of TNBS-induced colitis.

[0118] Both confocal microscopy observations (images not shown) and fluorescence measurements of intestinal segments in Figure 13 demonstrated that MPs can specifically reach the inflamed colon. In the healthy colon, very low fluorescence signals were observed due to the lack of targeting. In addition, LNPs were observed in the inflamed colon but not in the healthy colon (images not shown).

[0119] (References) 1. Seyedian, SS, Nokhostin, F. & Malamir, MD A review of the diagnosis, prevention, and treatment methods of inflammatory bowel disease. J Med Life 12, 113-122 (2019). 2. Nakase, H. Optimizing the Use of Current Treatments and Emerging Therapeutic Approaches to Achieve Therapeutic Success in Patients with Inflammatory Bowel Disease. Gut Liver 14, 7-19 (2020). 3. Stange, EF Current and future aspects of IBD research and treatment: The 2022 perspective. Frontiers in Gastroenterology 1, (2022). 4. Manz, M. et al. Therapy of steroid-resistant inflammatory bowel disease. Digestion 86 Suppl 1, 11-15 (2012). 5. Goll, R. et al. Pharmacodynamic mechanisms behind a refractory state in inflammatory bowel disease. BMC Gastroenterol 22, 464 (2022). 6. Marafini, I. & Monteleone, G. Inflammatory bowel disease: new therapies from antisense oligonucleotides. Ann Med 50, 361-370 (2018). 7. Yavvari, P. S. et al. A nanogel based oral gene delivery system targeting SUMOylation machinery to combat gut inflammation. Nanoscale 11, 4970-4986 (2019). 8. Xiao, B. et al. TNFα gene silencing mediated by orally targeted nanoparticles combined with interleukin-22 for synergistic combination therapy of ulcerative colitis. J Control Release 287, 235-246 (2018). 9. Laroui, H. et al. Fab’-bearing siRNA TNFα-loaded nanoparticles targeted to colonic macrophages offer an effective therapy for experimental colitis. J Control Release 186, 41-53 (2014). 10. Shinn, J. et al. Oral Nanomedicines for siRNA Delivery to Treat Inflammatory Bowel Disease. Pharmaceutics 14, 1969 (2022). 11. Han et al. An ionizable lipid toolbox for RNA delivery. Nature communications. 12:7233 (2021).

Claims

1. Polymer microparticles containing lipid nanoparticles (LNPs) that include nucleic acids.

2. Microparticles of the polymer according to claim 1, wherein the polymer is an anionic polymer or a cationic polymer.

3. Microparticles of the polymer according to claim 1 or 2, wherein the anionic polymer is alginate, pectin, carboxymethylcellulose (CMC), gellan gum, carrageenan gum, or xanthan gum.

4. Microparticles of the polymer according to claim 1 or 2, wherein the cationic polymer is chitosan or an acrylic copolymer having an ammonium group.

5. A microparticle according to any one of claims 1 to 4, characterized in that the average hydrodynamic diameter of the microparticle is between 10 μm and 1 mm, more specifically between 50 μm and 500 μm, and more specifically between 100 μm and 200 μm.

6. The microparticle according to any one of claims 1 to 5, wherein the LNP is composed of cholesterol, phospholipids (e.g., DOPE, DSPC), polyethylene glycol (PEG) lipids (1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene glycol-2000), and ionized lipids having a positive charge at a pH of less than 6.

7. The microparticle according to any one of claims 1 to 6, characterized in that the average hydrodynamic diameter of the LNP is between 30 nm and 250 nm, more specifically between 50 nm and 200 nm, and more specifically between 80 nm and 180 nm.

8. The microparticle according to any one of claims 1 to 7, wherein the nucleic acid encapsulated in the LNP is messenger RNA (mRNA), small interfering RNA (siRNA), antisense oligonucleotide (ASO), or short hairpin RNA (shRNA).

9. Microparticles according to any one of claims 1 to 8, wherein the nucleic acid affects the expression of pro-inflammatory cytokines such as TNF-α, IL1, IL6, IL8, IL10, IL17, CCL2, IL12, or IL23, or integrins such as α4β7 or α4β1.

10. The microparticles according to any one of claims 1 to 9, wherein the polymer is alginate.

11. Microparticles according to any one of claims 1 to 10 for use as a pharmaceutical.

12. Microparticles according to any one of claims 1 to 10 for use in the treatment of chronic inflammatory bowel diseases such as ulcerative colitis or Crohn's disease.

13. Microparticles for use according to claim 12, administered to a patient via an oral route.

14. Microparticles for use according to any one of claims 11 to 13, administered in doses comprising between 0.1 and 100 mg of nucleic acid per kg, more specifically between 0.5 and 50 mg of nucleic acid / kg, and more specifically between 3 and 10 mg of nucleic acid / kg.

15. Microparticles for use according to any one of claims 11 to 14, administered simultaneously, sequentially, or over a period of time in combination with at least one other active molecule.

16. A method for producing microparticles of anionic polymer containing lipid nanoparticles (LNPs) containing nucleic acids, I- This is a process of encapsulating nucleic acids in lipid nanoparticles.

1. A lower step of preparing an organic phase by dissolving lipids in an organic solvent.

2. A lower step of preparing an aqueous phase by mixing nucleic acids in an aqueous solution containing a buffering agent.

3. A lower step in which the organic phase of lower step I.1 is mixed with the aqueous phase of lower step I.2 in a volume ratio between 1:1 and 1:

3.

4. A sub-step in which the LNP obtained in sub-step I.3 is recovered by dialysis in an aqueous phase. Processes including II- This is a process of encapsulating lipid nanoparticles in microparticles of anionic polymer.

1. A lower step of preparing an aqueous solution of anionic polymers such as alginates at a concentration between 1 and 10 w / v%, 2. A lower step in which the lipid nanoparticle suspension obtained in lower step I.4 is added to the anionic polymer solution in lower step II.

1.

3. A citric acid solution with a pH of less than 3 and a concentration between 20 mM and 150 mM is added to the mixture from step II.

2.

4. Sub-step in which calcium chloride stock solution is added, 5. A lower step involves adding NaOH to adjust the pH to a value between 7 and 8.

6. A sub-step in which the solution obtained in sub-step II.5 is centrifuged.

7. Sub-process for recovering microparticles Process Methods that include...

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