WO2025012469A1 - Polymeric microparticles loaded with nanoparticles for the delivery of nucleic acids - Google Patents

Abstract

The present invention relates to microparticles of polymer, in particular an anionic or a cationic polymer, wherein are encapsulated lipidic nanoparticles (LNP) comprising nucleic acids such as siRNA. It also deals with a pharmaceutical comprising such microparticles and their use as a medicament, in particular for the treatment of chronic inflammatory bowel diseases (IBD).

Description

POLYMERIC MICROPARTICLES LOADED WITH NANOPARTICLES FOR THE DELIVERY OF NUCLEIC ACIDS

Field of the invention

The present invention relates to microparticles of polymer, in particular an anionic such as alginate or a cationic polymer, wherein are encapsulated lipidic nanoparticles (LNP) comprising nucleic acids such as siRNA. It also deals with a pharmaceutical comprising such microparticles and their use as a medicament, in particular for the treatment of chronic inflammatory bowel diseases (IBD).

Background of the invention

The inflammatory bowel diseases (IBD) include the ulcerative colitis (UC) and the Crohn’s disease (CD). The chronic inflammation in the gastrointestinal tract causes digestive disorders including diarrhea, rectal bleeding and abdominal pain as well as fever, weight loss and anemia ¹. While in the UC, inflammation is mainly observed in the colon, different parts of the intestine are affected in the CR. The current treatments are aminosalicylates, corticosteroids, immunomodulators and biotherapies ². Mesalazine is an aminosalicylate used for the local treatment of UC but is not recommended anymore for the CD due to the lack of efficacy. Corticosteroids (budesonide, prednisone) are administered for the treatment of IBD, especially the CR in first line and the UC after failure of aminosalicylates. Immunomodulators, especially azathioprine are recommended in case of refractory UC or to maintain the remission. Biotherapies including anti-TNF, anti-l L12-23 are also administered in the severe or refractory cases of IBD and as maintenance therapy for the remission ³ . These currents treatments faced numerous limitations in terms of efficiency and tolerability. Aminosalicylates induce very few side effects due to their local action but their efficiency is moderate. Until 16% of patients does not respond to corticosteroids in UC and CD. A resistance or a steroid dependance was also observed during the treatment ⁴. In addition to numerous adverse effects related to the immunomodulation (infections risks), azathioprine acts after 2 or 3 months of treatment. Primary and secondary non-responses are often observed with biotherapies due to immunogenicity and targeting issues. 30% and 40% of primary and secondary non-responders were observed with anti-TNF ⁵. In addition, to be efficient, a perfect compliance of the treatment regimen is necessary, but the administration routes (subcutaneous and intravenous) can be a limit and an inconvenience for the patient. As alternative to antibodies, nucleic acid-based strategies using small interfering RNA (siRNA) and antisense oligonucleotides were initiated to inhibit the expression of proinflammatory cytokines such as TNF. However, their benefit in preclinical and clinical trials was not clearly demonstrated for the treatment of IBD. The efficiency lack could be attributed to short plasma half life of nucleic acids after systemic administration ⁶ . Oral administration of nucleic acid then emerged to deliver nucleic acids especially siRNA, to the inflamed sites of the intestine for a better efficacy, tolerability and convenience for the patient. Lipo- polyplex or siRNA-loaded nanoparticles embedded in gastroresistant hydrogels were designed for the treatment of IBD ^7-9. However, their efficiency was limited due to major issues including a poor intracytoplasmic delivery of siRNA, a slow release from the hydrogel and a difficult scaling up due to complex formulations ¹⁰.

Thus, there was a need to develop a more effective treatment.

Summary of the invention

According to a first aspect, the present invention relates to microparticles of polymer wherein are encapsulated lipidic nanoparticles comprising nucleic acids, in particular microparticles of an anionic or cationic polymer, wherein are encapsulated lipidic nanoparticles comprising nucleic acids.

In a second aspect, the present invention deals with a process for manufacturing microparticles of anionic or cationic polymer wherein are encapsulated lipidic nanoparticles comprising nucleic acids.

A third aspect of the invention relates to a pharmaceutical composition comprising microparticles of polymer, in particular an anionic or a cationic polymer, wherein are encapsulated lipidic nanoparticles (LNP) comprising nucleic acids, in suspension in an additional pharmaceutically acceptable excipient.

A fourth aspect of the invention relates to microparticles of polymer, in particular an anionic or a cationic polymer wherein are encapsulated lipidic nanoparticles comprising nucleic acids, or a pharmaceutical composition comprising said microparticles, for use as a medicament, in particular for use in the treatment of chronic inflammatory bowel diseases (IBD).

Detailed description of the invention

The inventors have discovered that microparticles of polymer wherein are encapsulated lipidic nanoparticles (LNP) comprising nucleic acids were more effective for the treatment of inflammatory diseases. Thus, according to a first aspect, the present invention relates to microparticles of polymer wherein are encapsulated lipidic nanoparticles comprising nucleic acids. It means that nucleic acids are encapsulated into a LNP, and said LNP is itself encapsulated in a microparticle of polymer.

Said polymer may be an anionic polymer, a cationic polymer and a non-ionic polymer In particular said polymer is an anionic polymer or a cationic polymer.

In the sense of the invention, an anionic polymer is a polymer that has one or more monomer units that are covalently bound and bear a net negative charge.

Typically, an anionic polymer is alginate, pectin, carboxymethylcellulose (CMC), gellan gum, carrageenan gum or xanthan gum.

In the sense of the invention, a cationic polymer is a polymer that has one or more monomer units that are covalently bound and bear a net positive charge. Typically, a cationic polymer is chitosan or an acrylic copolymer with ammonium groups such as ethyl prop-2-enoate; methyl 2-methylprop-2-enoate;trimethyl-[2-(2-methylprop-2-enoyloxy)ethyl]azanium;chloride.

A non-ionic polymer is a polymer without any charge. Typically, a non-ionic polymer is Poly(D,L-lactic acid) (PLA), poly(D,L-lactic-co-glycolic acid) (PLGA) and poly(caprolactone) (PCL).

In particular, the present invention deals with alginate microparticles wherein are encapsulated lipidic nanoparticles which comprise nucleic acids.

Alginates are polysaccharides obtained from brown algae: kelp or wrack. Alginate is a linear polymer consisted of L-glucoronate (m) and D-mannuronate (n) residues connected via 1 ,4- glycosidic linkages. Alginate formula is as follows wherein m and n are integer higher than zero:

The inventors have discovered that these microparticles were more effective in treating inflammatory bowel diseases than systemic treatments based on monoclonal antibodies or immunosuppressants. The product acts directly on inflamed areas to inhibit the expression of pro-inflammatory cytokines. Selective distribution of microparticles in inflamed intestinal regions was demonstrated. Moreover, it allows a more efficient administration of nucleic acids by oral route. Local and targeted delivery is a less risky approach than systemic treatments and with fewer side effects. In addition, this route of treatment is more comfortable for the patient than parenteral administration and the patient can be autonomous in taking the treatment, thus avoiding expensive treatment costs as there is no need for hospitalization or trained personnel to perform the treatment administration.

Moreover, the formulation in the form of microparticles confers a better stability of the nucleic acids because the encapsulation protects the substances from chemical degradation by isolating them from the external environment (enzymes, acidic pH, bile salts, oxygen, light). It was demonstrated the possibility to freeze-dry the product in order to preserve it in dry form, thus avoiding storage at negative temperatures. Spray drying of microparticles was also envisaged to store them or to include them in solid dosage forms such as tablets and capsules. Moreover, the protective and muco-adhesive effect of alginate allows a prolonged retention on mucous membranes while preserving the activity of nucleic acids. This prolonged release at physiological pH (7.4) thus provides an extended action time of the RNA after administration, which allows a more effective local action and thus limits the number of administrations.

In particular, said microparticles of polymer, in particular an anionic or a cationic polymer, comprise 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 polymer, in particular an anionic or a cationic polymer by weight of the microparticles wherein are encapsulated lipidic nanoparticles comprising nucleic acids. In particular, said microparticles wherein are encapsulated lipidic nanoparticles comprising nucleic acids, essentially comprise polymer, in particular an anionic or a cationic polymer. In particular they essentially comprise alginate.

In particular, when the microparticle is made of an anionic polymer said microparticle may further comprise in addition of the anionic polymer, a multivalent cation such as calcium, barium, manganese, copper, aluminum and zinc or a cationic polyelectrolyte such as chitosan. In such an embodiment, said microparticles of anionic polymer comprise at least 0.01 wt%, in particular at least 0.1 wt%, in particular at least 1 wt% of multivalent cation by weight of the microparticles wherein are encapsulated lipidic nanoparticles comprising nucleic acids.

When the microparticle is made of a cationic polymer, said microparticle may further comprise in addition of the cationic polymer, a multivalent anion such as tripolyphosphate, pyrophosphate and sulfate or an anionic polyelectrolyte such as alginate, carrageenan, pectin, xanthan gum and hyaluronic acid. In such an embodiment, said microparticles of cationic polymer comprise at least 0.01 wt%, in particular at least 0.1 wt%, in particular at least 1 wt% of multivalent cation by weight of the microparticles wherein are encapsulated lipidic nanoparticles comprising nucleic acids.

In a particular embodiment, the mean hydrodynamic diameter of the microparticles is between 10 pm and 1mm, particularly between 50 pm and 500pm, more particularly between 100 pm and 200pm.

In particular, said lipidic nanoparticles which are encapsulated into the microparticles, are made of cholesterol, phospholipids, polyethylene glycol (PEG)-lipid (1 ,2-Dimyristoyl-rac- glycero-3-methoxypolyethylene glycol-2000) and an ionizable lipid with a positive charge at pH inferior to 6. In a particular embodiment, the lipidic nanoparticles contain molar lipid ratios of 30 - 40% for cholesterol, preferentially 38,5%, 5 - 20% for phospholipids, preferentially 10%, 0.5 - 3% for polyethylene glycol (PEG)-lipid (1 ,2-Dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000), preferentially 1.5% and 30 - 65% for the ionizable lipid, preferentially 50%.

The phospholipid may be phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylglycerols. It may be composed of saturated hydrocarbon chains such as distearoyl, dipalmitoyl, dimyristoyl and dilauroyl or unsaturated hydrocarbon chains such as dioleoyl.

Typically the ionizable lipid with a positive charge at pH inferior to 6 is 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-lso5-2DC18 (CAS 2412492-09-0), 98N12-5 (CAS 917572-74-8), C12-200 (CAS 1220890-25-4), CKK-E12 (CAS 1432494-65-9), 9A1 P9 (CAS 2760467-57-8), 7C1 , G0-C14 (CAS 1510653-27-6), L319 (CAS 1351586-50-9), 304-013 (CAS 1566559-80-5), OF-Deg-Lin (CAS 1853202-95-5), 306-O12B (CAS 2566523-06-4), 3060i10 (CAS 2322290-93-5), FTT5 (CAS 2328129-27-5), preferentially Dlin-MC3-DMA. Some of them are presented in Han et al. (2021) ¹¹.

In a particular embodiment, the mean hydrodynamic diameter of the lipidic nanoparticles (LNP) is between 30 nm and 250 nm, particularly between 50 nm and 200 nm, more particularly between 80 and 180 nm. The mean hydrodynamic diameter of the lipidic nanoparticles is necessarily inferior to the mean hydrodynamic diameter of the microparticles. The person skilled in the art perfectly knows how to choose a suitable hydrodynamic diameter of the lipidic nanoparticles for an effective encapsulation of the nanoparticles into the microparticles.

According to the present disclosure, the nucleic acids encapsulated into the lipidic nanoparticles may be a messenger RNA (mRNA), small interfering RNA (siRNA), an antisense oligonucleotide (ASO), a short hairpin RNA (shRNA) or a complementary DNA (cDNA).

In particular, the nucleic acid affects the expression of pro-inflammatory cytokines or integrins. The pro-inflammatory cytokine may be TNF-a, IL1 , IL6, IL8, IL10, IL17, CCL2, IL12 or IL23. The integrin may be a4p7 or a4pi .

In particular, the nucleic acid is a siRNA targeting TNFa, IL1 , IL6, IL8, IL10, IL17, CCL2, IL12 or IL23 or the integrins a4p7 or a4pi . In the present application, when it is said that a siRNA targets a protein such as a cytokine or an integrin, it means that the siRNA is designated to hybridize to the mRNA encoding said protein, leading to the destruction of said mRNA and thus to the decrease of the expression of the gene and the decrease of the level of the encoded protein.

In a particular embodiment, said microparticle is an alginate microparticle and the nucleic acids encapsulated into the LNP are siRNA targeting TNFa.

In a particular embodiment, the lipidic nanoparticles may contain different nucleic acids each targeting a different target. Said nanoparticles may comprise at least two different nucleic acids, in particular 2, 3 or 4 nucleic acids, each directed to different targets. Said targets are chosen among those listed above.

Said nanoparticles may further contain, in addition to nucleic acids, an active molecule. It allows to combine nucleic acids with an active molecule different from nucleic acids. Said active molecule may be a corticosteroid such as betamethasone, prednisolone, budesonide, a nonsteroidal anti-inflammatory drug such as mesalazine, olsalazine, 4-Aminosalicylic acid, sulfasalazine, or an immunosuppressant such as azathioprine, methotrexate, cyclosporine. Said active molecule may also be an antibody such as an anti-TNF antibody or an anti-cytokine antibody, for example an anti-IL-10. Said active molecule may also be an interleukin with protective properties such as IL22 or IL24.

The microparticles and nanoparticles may be conjugated to carbohydrates such as mannoserich compounds and antibodies C-type lectin receptors, Fc receptors and CD44 to target the immune cells including macrophages. They may also comprise or be grafted to therapeutic antibodies directed to a tumor necrosis factor, a pro-inflammatory cytokine, an integrin. For example, it may be an antibody directed to a pro-inflammatory cytokine chosen among the group consisting of: I L1 a, IL1 , IL6, ILS, IL10, IL17, CCL2, I L12 or IL23. For example, it may be an antibody directed to the integrin a4p7 or a4pi. For example, it may be an antibody directed to TNFa.

In a second aspect, the present invention deals with a process for manufacturing microparticles of anionic or cationic polymer wherein are encapsulated lipidic nanoparticles comprising nucleic acids, comprising the following steps:

The first phase of the process is:

I- Encapsulating nucleic acid in lipid nanoparticles comprising the substeps of:

1. Preparing the organic phase by dissolving lipids in an organic solvent for example absolute ethanol,

2. Preparing an aqueous phase by mixing the nucleic acids in an aqueous solution comprising a buffer such as citrate or acetate buffer,

3. Mixing the organic phase of substep 1.1 with the aqueous phase of substep 1.2 at a volume ratio between 1 :1 and 1 :3,

4. Recovering LNPs obtained at substep 1.3 by dialysis in an aqueous phase.

In a specific embodiment, at substep 1.1 the final lipid concentration is comprised between 25 and 50 mM. In particular, lipids include cholesterol, phospholipids, polyethylene glycol (PEG)- lipid (1 ,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000) and an ionizable lipid.

In a specific embodiment, at substep 1.2, the citrate or acetate buffer is at a concentration ranging from 10 to 100 mM. The buffer pH is inferior or equal to 6.

In a specific embodiment, at substep 1.3, the nitrogen-to-phosphate mole ratio (N:P ; nitrogen from the ionisable lipid and phosphate from the nucleic acid) is comprised between 3:1 and 9:1.

Between substep 1.3 and substep 1.4, a physiological buffer at pH 7.4 such as PBS may be added to the mixture, for example at a volume ratio of at least 1 :1.

In a specific embodiment, the dialysis at substep 1.4 is performed overnight at 4°C. The aqueous phase may be purified water, HEPES, 1-10% w/w sucrose, TRIS-HCI or a phosphate buffer.

Optionally, at the end of the process the LNPs may be concentrated by ultrafiltration of LNPs at 4°C and, if it is necessary, sterilized with a filter 0.45 or 0.22 pm. In one embodiment, the lipid nanoparticles comprising nucleic acids may be encapsulated in microparticles of an anionic polymer. The second phase of the process comprises:

II- Encapsulating lipid nanoparticles in microparticles of anionic polymer comprising the substeps of:

1. Preparing a solution of an anionic polymer, such as alginate, in water at concentration comprised between 1 and 10% w/v,

2. Adding the lipidic nanoparticles suspension obtained at substep 1.4 to the anionic polymer solution of substep 11.1 ,

3. Adding a citrate solution at pH inferior to 3 to the mixture of substep 11.2, in particular said pH is between 1.5 and 3, and said citrate solution is at a concentration between 20 mM and 150 mM, in particular at 100 mM,

4. Adding a calcium chloride stock solution, in particular to reach a final concentration in calcium chloride comprised between 15mM and 80mM,

5. Adding NaOH to reach a pH value between 7 and 8, in particular 7.4,

6. Centrifuging the solution obtained at substep 11.5, for example at 5000 rpm,

7. Collecting the microparticles.

At the end of the process, the supernatant may be replaced by water. Between substep 11.1 and 11.3 washings can be performed.

In a specific embodiment, the stock solution of calcium chloride is at concentrations comprised between 90 and 480 mM. In particular, citrate solution is at 100 mM and a pH inferior to 3.

The anionic polymer precipitation around the lipidic nanoparticles at step II.3 is allowed by the acidic pH and the raising of the pH which leads to ionic gelation. In a specific embodiment, said anionic polymer is alginate. Gelation with calcium is achieved by raising the pH with 1M sodium hydroxide to deprotonate the COOHs of alginate, inducing electrostatic interactions with Ca2+.

In another embodiment, the lipid nanoparticles comprising nucleic acids may be encapsulated in microparticles of a cationic polymer. The second phase of the process is:

II- Encapsulating lipid nanoparticles in microparticles of a cationic polymer comprising the substeps of:

1 . Preparing a solution of a cationic polymer, such as chitosan, in an acidic buffer such as an acetate buffer, for example at a pH comprised between 2 and 5, 2. Adding the lipidic nanoparticles suspension obtained at substep 1.4 to the cationic polymer solution,

3. Adding a buffer such as NaOH, TRIS-HCI, HEPES or phosphate solution to reach a pH superior to 5, in particular a pH between 5 and 8, leading to the cationic polymer precipitation around of the lipidic nanoparticles,

4. Adding an anionic polymer such as alginate, carrageenan, pectin, xanthan gum, hyaluronic acid or an anionic multivalent salt such as tripolyphosphate, Optionnally, adding acetic acid,

5. Centrifuging, for example at 5000 rpm, the solution obtained at substep 11.4,

6. Collecting the microparticles.

Substeps 11.3 and 11.4 can be reverted.

In a specific embodiment, the anionic polymer at substep 11.4 is added at a concentration between 1 et 10 % w/v.

Pharmaceutical compositions

A third aspect of the invention relates to a pharmaceutical composition comprising microparticles of polymer, in particular an anionic or a cationic polymer, wherein are encapsulated lipidic nanoparticles (LNP) comprising nucleic acids as described above, in suspension in an additional pharmaceutically acceptable excipient.

In particular, it is a pharmaceutical composition comprising microparticles of polymer, in particular an anionic or a cationic polymer, wherein are encapsulated lipidic nanoparticles (LNP) comprising nucleic acids as described above, in suspension in an additional pharmaceutically acceptable excipient.

As used herein, the term "pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, or formulation auxiliary of any type.

As will be appreciated by the skilled person in the art, the pharmaceutically acceptable excipients and/or carriers will be chosen based on the route of administration as described below, the location of the targeted tissue, the time course of delivery of the drug, etc.

There are numerous examples of excipients that can be added to the pharmaceutical composition. Examples include without limitation sucrose, mannitol, trehalose, buffers (tris- HCI, HEPES, phosphate). The pharmaceutical composition may be in the form of liquid or a solid form for oral administration. Solid forms include capsules, tablets, pills, powders, and granules.

Methods of use

A fourth aspect of the invention relates to microparticles of polymer, in particular an anionic or a cationic polymer, wherein are encapsulated lipidic nanoparticles comprising nucleic acids as described above, or a pharmaceutical composition as described above, for use as a medicament.

The invention relates to microparticles of polymer, in particular an anionic or a cationic polymer, wherein are encapsulated lipidic nanoparticles comprising nucleic acids as described above, or a pharmaceutical composition as described above, for use in a therapeutic method.

In particular, it relates to microparticles of polymer, in particular an anionic or a cationic polymer, wherein are encapsulated lipidic nanoparticles comprising nucleic acids as described above, or a pharmaceutical composition as described above, for use in the treatment of chronic inflammatory bowel diseases (IBD). These diseases are characterized by inflammation of the lining of the digestive tract. Typically, chronic inflammatory bowel diseases include Crohn's disease and ulcerative colitis (UC).

As used herein, the term "treatment" or "treat" refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.

In particular, the microparticles of polymer, in particular an anionic or a cationic polymer, wherein are encapsulated lipidic nanoparticles comprising nucleic acids as described above, or the pharmaceutical composition as described above, are administered to the patient by oral route.

They can be conveniently delivered in the form of liquids, capsules, tablets, pills, powders, and granules. In particular, said microparticles or composition thereof are administered to the patient in need in a dose expressed in mg of nucleic acids per kg which is comprised between 0.1 and 100 mg of nucleic acids per Kg, particularly between 0.5 and 50 mg of nucleic acids/Kg, more particularly between 3 and 10 mg of nucleic acids/Kg.

In a particular embodiment, said microparticles or composition thereof are administered to the patient in combination with at least one other active molecule. So, the administration of nanoparticles and the at least one other active principle can be simultaneous, sequential, or over a period of time.

In one embodiment, the at least one other active principle is selected from: a corticosteroid such as betamethasone, prednisolone, budesonide, a non-steroidal anti-inflammatory drug such as mesalazine, olsalazine, 4-Aminosalicylic acid, sulfasalazine, or an immunosuppressant such as azathioprine, methotrexate, cyclosporine. Said other active principle may also be an antibody such as an anti-TNF antibody or an anti-cytokine antibody, for example an anti-IL-10. Said other active principle may also be an interleukin with protective properties such as IL22 or IL24.

The at least one other active principle may be administrated orally, rectally, parenterally, intracisternally or intraperitoneally.

In particular, the invention relates to a method of treatment of chronic inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis (UC), comprising the administration of microparticles of polymer, in particular an anionic or a cationic polymer, wherein are encapsulated lipidic nanoparticles comprising nucleic acids as described above, or comprising the administration of a pharmaceutical composition as described above, to a patient in need.

Brief description of figures

Figure 1. Percentage of release of LNPs in alginate microparticles after 24 h of incubation at 37 °C in different solvent of suspension and different number of wash cycle via a dosage of cholesterol. A comparison of the percentage of liberation between the different number of wash cycle had been realized. Data are means ± SEM from triplicate of 3 independent formulations. ** p<0.01 in comparison with 1 wash cycle in each condition.

Figure 2. Percentage of viability on HepG2 cell line after 24 h of treatment by ONPATTRO® with different concentrations and different conditions: LNPs of ONPATTRO® only, ONPATTRO® in alginate microparticles resuspended in DPBS and diluted in medium without FCS, ONPATTRO® in alginate microparticles resuspended in SGF and diluted in medium without FCS and ONPATTRO® in alginate microparticles resuspended in SIF and diluted in medium without FCS. Negative control was medium without treatment. Data are means ± SEM from triplicate of 3 independent cultures. *** p<0.001 with respect to control.

Figure 3. Percentage of viability on THP-1 cell line after 24 h of treatment by LNPs loaded siRNA TNF-a with different concentrations and different conditions: LNPs loaded siRNA TNF- a only, LNPs loaded siRNA TNF-a in alginate microparticles resuspended in DPBS and diluted in medium without FCS and LNPs loaded siRNA TNF-a in alginate microparticles resuspended in SGF and diluted in medium without FCS. Negative control was medium without treatment. Data are means ± SEM from triplicate of 3 independent cultures.

Figure 4. Measurement of percentage of production of TTR protein by HepG2 cell lines after 24 h of treatment at different concentrations of ONPATTRO® with different conditions: ONPATTRO® only, ONPATTRO® in alginate microparticles resuspended in DPBS after 3 wash cycles and diluted in medium without FCS, ONPATTRO® in alginate microparticles preincubated in SGF after 3 wash cycles during 2 h then resuspended in DPBS and diluted in medium without FCS. Negative control was medium without treatment. Data are means ± SEM from triplicate of 3 independent cultures. *** p<0.001 with respect to control.

Figure 5. Measurement of percentage of production of TNF-a protein by THP-1 cell lines after 24 h of treatment at different concentrations of LNPs loaded siRNA TNF-a with different conditions: LNPs loaded siRNA TNF-a only, LNPs loaded siRNA TNF-a in alginate microparticles resuspended in DPBS after 1 or 2 wash cycles and diluted in medium without FCS, : LNPs loaded siRNA TNF-a in alginate microparticles pre-incubated in SGF during 2 h after 1 or 2 wash cycles then resuspended in DPBS and diluted in medium without FCS. Different controls had been studied in medium without FCS: siRNA only at different concentrations, LPS at 0.1 pg/mL, DPBS at 50:50 (v:v) and LNPs without siRNA, LNPs without siRNA in microparticles and empty microparticles with the same quantity than the biggest concentration of siRNA used for the corresponding condition. Negative control was medium without treatment. Data are means ± SEM from triplicate of 3 independent cultures. * p<0.05, ** p<0.01 and *** p<0.001 with respect to control.

Figure 6. The therapeutic index (colon mass/colon size) of the different groups of mice

Figure 7. Survival and clinical parameters, such as weight loss, rectal bleeding and diarrhoea monitored daily (death = 6 ; healthy = 0).

Figure 8. TNF-a expression compared to the colitis group in DSS model (experiment 2). siRNA TNF-a in free form or encapsulated in MPs and empty MPs were administered by the oral route. TNF- a levels in proximal (A) and distal (B) colons were determined by qRT-PCR. Each group was composed of 8 mice. * p<0.05, and *** p<0.001 with respect to colitis group.

Figure 9. Clinical score (assessment of survival and weight loss) of the different groups of mice according to the administered treatments. The negative control group not treated with TNBS corresponds to 0 (groups of 6) * p<0.05, and *** p<0.001 compared to the control (colitis group). (A) first experiment (B) second experiment.

Figure 10. Evolution of the mean body weights of each group compared to day 0 (groups of 6) (A) first experiment (B) second experiment.

Figure 11. Therapeutic index of the different groups of mice according to the administered treatments (groups of 6) * p<0.05, ** p<0.01 , and *** p<0.001 compared to the control (colitis group). (A) first experiment (B) second experiment.

Figure 12. Mouse colon after three consecutive days of different oral treatments. (A) first experiment (B) second experiment.

Figure 13. Measurement of the fluorescence intensity in intestinal sections after oral administration of fluoresceinamine labelled MPs in healthy mice and mice with TNBS-induced colitis.

Examples

I. Material

Sodium alginate, calcium chloride, citric acid, citrate, acetic acid, acetate, sodium hydroxide, potassium monobasic phosphate, pepsin, and pancreatin were obtained from Sigma Aldrich and kept in RNAse free conditions.

Lipids including: Dlin-MC3, mPEG-2000, DSPC and cholesterol were obtained from advantilipids. II. Methods

11.1. Simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) preparation

The composition of SGF and SIF was in accordance with the recommendations of the USP. SGF preparation required sodium chloride, pepsin, and hydrochloric acid. A solution of sodium chloride and pepsin was first prepared by adding 0,8g of sodium chloride and 1 ,28g of pepsin to 200 mL of ultrapure water under magnetic stirring for 5 minutes. Then, 2,8mL of 0,4N hydrochloric acid were added before adjusting the pH using hydrochloric acid and a pH meter to reach a pH between 1 and 2. Then, the solution was centrifugated for 10 minutes at 15000 g at 4°C. The supernatant was thus filtered through a 0,45|jm polycarbonate membrane.

SIF preparation requires monobasic potassium phosphate, sodium hydroxide and pancreatin. A solution was first prepared by adding 2,72 g of monobasic potassium phosphate, 15,4 mL of a 0,4N sodium hydroxide solution and 4 g of pancreatin in 150 mL of ultrapure water. The pH was adjusted using sodium hydroxide to reach a pH of 6,8. The volume is then adjusted to 200m L using ultrapure water. The solution was then centrifugated for 1 hour at 20 000g at 4°C before filtering it using a 0,45|jm polycarbonate membrane.

II. 2. Production of lipidic nanoparticles

Production of lipidic nanoparticles was performed from a lipidic and acidic aqueous phases. The lipidic phase contains four different ionizable lipids, DSPC, Cholesterol, PEGylated lipid, and an ionisable lipid with ratios of respectively 10%/38,5%/1 ,5%/50%. In this study D-Lin MC3 was used as ionizable lipid. The solvent used for this phase is absolute ethanol. The lipidic phase is prepared in two steps. First by adding DSPC, Cholesterol and PEG-DMG to absolute ethanol and mix in a 40°C water for 30 seconds in order to solubilise the lipids. Then, D-Lin MC3 is added to obtain the final lipidic solution. The acidic phase is composed of a buffer. Citric acid/citrate and acetic acid/acetate couples at pH inferior or equal to 6 were tested. siRNA or mRNA was then added in the acidic aqueous phase. In order to protect the RNA from degradation molecular biology graded water was used which is guaranteed to be contaminants and RNAse free. siRNA or mRNA was added in the acidic aqueous phase.

LNPs were obtained by nanoprecipitation consisting in the addition of the acidic solution of the acidic solution of RNA with the ethanolic solution of lipids. Mixing was performed by manual method or microfluidic method.

II.3. Production of alginate microparticles

To produce alginate microparticles, four solutions were prepared : an acidic buffer, an alginate solution (1 or 2%w/v), a hardening stock solution of CaCh (300 mM or 400 mM) and a NaOH (1 N) solution. For encapsulation, LNPs including Onpattro and RNA-loaded LNPs were added in the alginate solution. The alginate solution was added to the acidic buffer under magnetic stirring, leading to the alginate precipitation. Then the hardening CaCh solution is added and finally the NaOH to increase the pH at a physiological level. LNP-loaded alginate microparticles were then obtained. It then proceeded to wash them by first centrifuging them at 5000rpm for 5 minutes and then suspending them in ultrapure water. Washing cycles can be done between one and three times. An optional step consisting in the freeze-drying of microparticles can be performed for a long-term storage using sucrose as cryoprotective agent or other sugars and polyols.

11.4. Physicochemical characterisation of LNPs and alginate microparticles

The characterisation of the size and zeta potential of LNPs was done using a Malvern Zetasizer.

Laser granulometry was used to measure the size of the alginate microparticles using a Mastersizer 3000 from Malvern. This technique gives us three information about the size of the particles in the suspension. The D10 which corresponds to the 1^st decile size, the D50 which corresponds to the median size, and the D90 which corresponds to the 9^th percentile size.

11.5. RNA loading

In order to determine encapsulation efficiency in LNPs, the amount of RNA was measured in the samples after nanoprecipitation in 2 different conditions. With triton and without triton. RNA encapsulated inside the LNPs cannot be accessed by the reagent. Thus, the quantification without triton corresponds to the amount of unencapsulated siRNA. The triton leads to destabilization of the LNP membrane and then the RNA release. Qubit microRNA and ribogreen kits were used to detect and quantify the amount of RNA. The qubit microRNA was used to measure siRNA in a range between 0.2 and 150 ng/mL. The encapsulation efficiency (EE) was then calculated as follows :

11.6. LNP loading and liberation in microparticles

Microparticles were washed two times with ultrapure water and centrifuged to remove the free LNPs. After washing step, alginate microparticles were placed in different media to test their liberation in said media. After incubation, microparticles were centrifuged. The supernatant was preserved. The microparticles were then dissolved in a high concentrated phosphate buffer leading to the dissolution of the alginate. LNP amount was then assessed by quantification of the cholesterol using the Amplex cholesterol detection kit from Thermofischer. Cholesterol detection was done by adding 5pL of sample as well as 45pL of reaction buffer and 50pL of Reagent solution in a 96 well plate. The reagent solution contained reaction buffer, cholesterol oxidase, horseradish peroxidase, and cholesterol esterase. Standard curve was obtained using pure cholesterol. Samples were then incubated for 30 minutes at 37°C before being read. Reading used an absorption spectre between 530 and 560 nm and an emission spectre at 590 nm.

11.7. Viability assay

Cytotoxicity was tested on two different strands of THP1 cells. Cytotoxicity was measured using the MTT Promega test. With THP1 , empty alginate microparticles and LNPs were tested. Negative controls were done by adding only culture medium while positive control was performed with 0,1X triton in culture medium. 100 pL of each sample were added to the plate. Then 20pL of MTS were added to each well. The plate was then incubated between 1 and 4 hours at 37°C before reading using a spectrophotometer at a wavelength of 490nm.

11.8. siRNA transfection

Sandwich ELISA was used in order the measure the level of proteins in the samples which allows us to evaluate the inhibition of the protein expression and consequently the activity of siRNA. ELISA kits were obtained from Thermofisher.

For normalization of results, protein quantification using BCA assay was performed.

TTR quantifications

HepG2, a cell-line of human hepatocytes were seeded in a 96 well plate at 110 000 cells per well. At 24H post seeding, alginate microparticles previously incubated in DPBS, SGF then PDBS, SGF then SIF, and SIF alone were incubated with cells at concentration of 50pg/mL, 25pg/mL, 12,5pg/mL, 6,25pg/mL, 3,125pg/mL, and 1 ,57pg/mL for 24 hours. After treatment, TTR levels in hepatocytes were quantified to evaluate the activity of the Onpattro® released from alginate microparticles. For this measurement a coated sandwich ELISA plate from Invitrogen was used. The plate was read using a spectrophotometer at two different wavelengths. Respectively, 450 nm and 570 nm. The absorbance obtained with the 570nm reading was then subtracted from the 450 nm reading in order to reduce the background noise interference.

TNF

THP1 , a cell-line of human monocytes were seeded in 96 well plate at 10 000 cells. At 24h post seeding, cells were treated with phorbol 12-myristate 13-acetate (PMA) between 48 and 72 hours, leading to the differentiation of monocytes into macrophages. These macrophages are then able to react to lipid polysaccharides (LPS). LPS are surface antigen present on bacteria like Escherichia coli. The interaction between the macrophages and LPS will lead to the activation of the macrophages which will trigger an inflammatory response. This includes the secretion of many inflammatory mediators like cytokines or TNF.

Alginate microparticles previously incubated in DPBS, SGF then DPBS, SGF then SIF, and SIF alone were incubated with cells at concentrations of 233nanomol/mL (D1), 174,75nanomol/mL (D2), 131nanomol/mL (D3), 98,3nanomol/mL (D4) for 24 hours. After treatment, TNF- levels in THP1-derived macrophages were quantified to evaluate the activity of siRNA-loaded LNPs released from alginate microparticles. In this case an uncoated TNF ELISA plate was used. The plate was read using a spectrophotometer using two different wavelengths. Respectively, 490 nm and 570 nm. The absorbance obtained with the 570nm reading was then subtracted from the 490 nm reading in order to reduce the background noise interference.

II.9. In vivo evaluation

TNBS (2,4,6-Trinitrobenzenesulfonic acid) colitis

The efficiency of the LNPs in alginate microparticles was investigated with a TNBS model of colitis (62.5 mg/Kg TNBS). Male Swiss/CD-1 mice (average weight 25 g) were used. Colitis was induced with a rectal administration of a dose of 62.5 mg/Kg TNBS. Following colitis induction, animals were orally treated with 5 mg/kg of siRNA TNF alpha (administered volume: 100 pl of LNPs in Alginate microparticles) for three consecutive days. After TNBS induction at day 0, treatments were delivered at days 1 , 2 and 3. Mice were sacrificed at day 4. Two independent experiments were performed. Control animals (colitis control) were treated with normal saline.

DSS (dextran sulfate sodium) colitis

Experiment 1 : Bablc/jrj mice were used with a DSS model of colitis. Colitis were induced with 3.5% (w:v) of DSS in the drinking water during 10 days. Following colitis induction, animals were orally treated with 5 mg/kg of siRNA TNF alpha (administered volume: 100 pl of LNPs in Alginate microparticles) for three consecutive days. Control animals (colitis control) were treated with normal saline.

Experiment 2: Disease induction was performed in male C57BL/6J mice with 2% DSS in drinking water from DO to D5, with DSS solution renewal on D3. MPs containing siRNA TNF- a-loaded LNPs were orally and daily administered at 10mg/kg from day 3 to day 7. Mice were sacrificed at day 8.

Clinical evaluation

Survival and clinical parameters, such as weight loss, rectal bleeding and diarrhoea were monitored daily (death = 6 ; healthy = 0). After sacrifice the colon was weighed and measured to calculate the therapeutic index. Resected colon tissue samples were opened longitudinally, rinsed with ice-cold PBS to remove luminal content, and minced. TNF alpha concentration were determined using a Mouse ELISA. The samples were obtained after grinding in PBS.

III. Results

111.1. Synthesis of Lipidic Nanoparticles

Synthesis of lipidic nanoparticles (LNPs) used the manual method. The physicochemical characteristics of the LNPs have been measured to see if their production was reproducible.

LNPs with siRNA TNF-a

siRNA Encapsulation Ratio N:P 8:1 for in 79 ± 1 vitro studies (%) siRNA Encapsula vivo studies (%)

Table 1. Characteristics of LNPs produced in acetate buffer at 20 mM with a concentration of 18.75 nmol/mL of siRNA TNF-a

As it can see there is a low standard deviation regarding the size, PDI, zeta potential, and siRNA encapsulation of the microparticles. LNPs encapsulation in alginate microparticles

Table 2. Encapsulation efficiency of LNPs with TNF-a siRNA and ON PA TTRO® in alginate microparticles.

It is able to achieve a high encapsulation efficiency regardless of the type of LNPs used.

III.2. Synthesis of alginate microparticles

The critical production parameters of alginate microparticles using the acidic precipitation method was evaluated. Evaluation was done using laser granulometry. Three parameters were obtained, the first decile size (D10), the median size (D50), and the ninth decile size (D90).

% Alginate Calcium Encapsulation D10 (pm) D50 (pm) D90 (pm) Span chloride of LNPs in the

Stock Microparticles solution Concentration (mM)

1 % 300 \ 40 ± 8 162 ± 14 595 ± 57 3.4

1 % 400 \ 52 ± 11 148 ± 19 583 ± 113 3.6

2 % 300 \ 120 ± 38 437 ± 184 1385 ± 586 2.9

2 % 400 \ 94 ± 4 258 ± 44 707 ± 222 2.4

Table 2. Measurement of the size of alginate microparticles after 1 only wash in function of the percentage of alginate, the concentration of the calcium chloride stock solution and the encapsulation or not of LNPs in the microparticles

According to the results the concentration of calcium chloride added in the hardening solution does not affect size. Neither does the encapsulation of lipidic nanoparticles. However, the concentration of the alginate solution will affect it. An increase in the concentration of alginate in the alginate solution will lead to an increase in the size of alginate microparticles. Similarly, the effect of the number of wash cycles on the final size of alginate microparticles has been studied. Resuspension Solvent Cycles of wash D50 (pm) Span

SGF 1 149 ± 19 3.1

Water

168 ± 9

3.7

SGF 2 141 ± 19 2.8

Water

126 ± 10

3.6

SGF 3 113 ± 11 3.6

Table 3. Measurement of the size of alginate microparticles with LNPs inside in function of the solvent of resuspension and the number of washes

It appears that washing as a slight effect on the size of alginate microparticles. In effect, after 3 wash cycles the median size is around 40pm smaller than after one wash cycle. It was then set out to study other effects the number of wash cycles could have on the properties of alginate microparticles.

Dilution Solvent Number of washes Concentration of Calcium

(mg/L)

Water

967.28 ± 23.90

Water 2 420.00 ± 21.32

Water

304.31 ± 16.76

SGF 1 260.64 ± 45.69

Table 4. Concentration of Calcium in the alginate microparticles in function of the dilution solvent and the number of washes

Table 5. Measurement of the size of freeze-dried alginate microparticles with LNPs inside in function of the sucrose concentration prior to the freezing.

From sucrose concentrations above 1% w/w, the median diameters of freeze-dried alginate microparticles were in the same range as fresh microparticles, close to 50-60 pm.

The concentration of calcium in the solvent after 1 , 2 or 3 wash cycles was studied to study the variation in concentration. In water, there is a large reduction in the concentration of calcium between the first and second wash. However, the reduction is much smaller between the second and third wash. When it comes to SGF, even after a single wash there is lower concentration of calcium than in water even after 3 washes. This can be explained by the fact the the alginate molecules will be protonated in an acidic pH removing the links with the calcium. It was then investigated if this concentration of calcium could have an effect on the release of LNPs encapsulated in alginate microparticles.

As shown on Figure 1 , a higher number of wash cycles increases the liberation of LNPs in DPBS thanks to the reduction in the amount of calcium in the solvent. Also, SIF, thanks to the large amount of phosphate anions, will provoke the liberation of most of the LNPS regardless of the number of wash cycles. Also, it can see that in ultrapure water there is almost no liberation and no variation regarding the number of wash cycles. This seems to indicate that for the microparticles to dissolve, they need to lose some of their cross-linking agent, either by an acidic reaction or by chelation with anionic molecules. Furthermore, it can see that the liberation both in SIF and DPBS is greatly increased after an incubation period in SGF. This is coherent with the previous results showing that SGF reduces the concentration of calcium in the alginate microparticles. It seems that this reduction greatly increases the ability of the microparticles to dissolve. Overall, it has observed that the alginate microparticles are stable both in ultrapure water and SGF. And that an incubation period in SGF allows for a much greater release in other media like SIF and DPBS.

III.3. In vitro evaluation

As shown on Figure 2, viability remains around 100% with LNPs only or with LNPs in alginate microparticles resuspended in DPBS. However, with alginate microparticles resuspended in SGF at a 12.5pg/mL concentration it was obtained a viability around 30% which was quickly brought up to a 100% with a 6.2pg/mL concentration. Furthermore, with alginate microparticles resuspended in SIF the viability slowly increases when the concentration decreases. Going from approximately 50% at 12.5pg/mL up to 75% at 1 ,6pg/mL.

Similarly, to the previous experiment, Figure 3 shows a viability of 100% for LNPs alone and LNPs in alginate microparticles resuspended in DPBS. While there was a lower viability for LNPs in alginate microparticles resuspended in SGF this time at a concentration of 233nM. This shows us that the choice of siRNA does not affect viability at the used concentrations.

This experiment of Figure 4 was used as a proof of concept that LNPs could be encapsulated in alginate microparticles without affecting their efficacy. Which is why ONPATTRO® was used which is a well-known product with very stable characteristics and a long shelf-life thanks to its stability. When comparing efficacy between LNPs alone and LNPs-Alginate microparticles resuspended in DPBS it can see that there is a very similar reduction in TTR production regardless of ONPATTRO® concentration. Similarly, it was found a high efficacy of ONPATTRO ® encapsulated in alginate microparticles and resuspended first SGF and then in DPBS. This tells us that encapsulation in alginate microparticles does not affect the in-vitro efficacy of LNPs.

After obtaining the proof of concept that alginate microparticles do not affect the efficacy of LNPs using ONPATTRO®, it was studied the efficacy of the own LNPs in alginate microparticles. As it can be seen on Figure 5, siRNA alone is ineffective on its own to reduce the production of TNF-a. However, when siRNA is encapsulated in LNPs it is observed a frank dose-dependent reduction of the TNF-a production going from 20% with 250nM of siRNA to 55% with 105nM.

III.4. In vivo evaluation

III.4.1. In vivo evaluation in DSS and TNBS models

TNBS and DSS -induced colitis in mice were used to mimic Crohn’s disease and ulcerative colitis, respectively.

DSS model of colitis

In the experiment 1 , the level of TNF-a in the treated mouse was undetectable (background noise) after administration of the microparticles loaded with LNP by the oral route. In contrast, untreated mice had an average of 3048 pg/g colon of TNF-a.

In the experiment 2, colitis was induced with 2% DSS in drinking water from DO to D5. qPCR analysis was performed in proximal and distal colons. Results revealed a decrease of the TNF- a mRNA level in the distal colon (Figure 8), demonstrating the ability of MPs to affect the inflammatory response.

TNBS model of colitis

The administration by the oral route of LNP-loaded alginate microparticles reduced the clinical score of mice by a factor 3 compared with untreated groups and mice treated with empty microparticles and by a factor 2 with orally administered free NP. Histological observations demonstrated the lack of ulcerative necrotic lesions in the colon of treated animals similarly to healthy control.

In TNBS model, MPs (LNP siRNA MPs Oral route) administered by the oral route reduced both clinical score taking into account the survival rate and weight of animals (Figures 7 and 9) and therapeutical index from the ratio colon weight/size of animals (Figures 6 and 11). The effect was higher than siRNA-loaded LNPs demonstrating the interest of the gastroresistant property. After treatment with MPs, therapeutic index and colon pictures in Figure 12 were similar to healthy animals, demonstrating the complete recovery of animals. In the second experiment, animals start getting weight from Day 2, before the second administration to reach at Day 4, their initial weight before the TNBS- induction (Figure 10).

III.4.2. In vivo evaluation by confocal imaging

Labelling of alginate with fluoresceineamine and LNPs with cyanine was performed to evaluate the biodistribution of particles after oral administration in TNBS-induced colitis murine model.

Both confocal microscopy (photos not shown) and fluorescence measurements in intestinal segments in Figure 13 demonstrated the ability of MPs to reach specifically the inflamed colon. A very low fluorescent signal was observed in healthy colon due to the lack of targeting. LNPs were also observed in inflamed colon but not in healthy colon (photos not shown).

Bibliography

1. Seyedian, S. S., Nokhostin, F. & Malamir, M. D. 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, E. F. 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. TNFa 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 TNFa-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. Microparticles of polymer wherein are encapsulated lipidic nanoparticles (LNP) comprising nucleic acids.

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

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

4. Microparticles of polymer according to claim 1 or 2, wherein said cationic polymer is chitosan or acrylic copolymers with ammonium groups.

5. Microparticles according to any one of claims 1 to 4 characterized in that the mean hydrodynamic diameter of the microparticles is between 10 pm and 1mm, particularly between 50 pm and 500pm, more particularly between 100 pm and 200pm.

6. Microparticles according to any one of claims 1 to 5, wherein the LNP are made of cholesterol, phospholipids (such as DOPE, DSPC) and polyethylene glycol (PEG)-lipid (1 ,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000) and an ionizable lipid with a positive charge at pH inferior to 6.

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

8. Microparticles according to any one of claims 1 to 7 wherein the nucleic acids encapsulated into the LNP are: a messenger RNA (mRNA), small interfering RNA (siRNA), an antisense oligonucleotide (ASO) or a 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-a, IL1 , IL6, IL8, IL10, IL17, CCL2, IL12 or IL23, or integrins such as a4p7 or a4pi .

10. Microparticles according to any one of claims 1 to 9 wherein said polymer is alginate.

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

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 wherein said microparticles are administered to the patient by oral route.

14. Microparticles for use according to any one of claims 11 to 13 wherein said microparticles are administered in a dose comprised between 0.1 and 100 mg of nucleic acid per Kg, particularly between 0.5 and 50 mg of nucleic acid/Kg, more particularly between 3 and 10 mg of nucleic acid/Kg.

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

16. Process for manufacturing microparticles of anionic polymer wherein are encapsulated lipidic nanoparticles (LNP) which comprise nucleic acids, comprising:

I- Encapsulating nucleic acids in lipid nanoparticles comprising the substeps of:

1 . Preparing the organic phase by dissolving lipids in an organic,

2. Preparing an aqueous phase by mixing nucleic acids in an aqueous solution comprising a buffer,

3. Mixing the organic phase of substep 1.1 with the aqueous phase of substep 1.2 at a volume ratio between 1 :1 and 1 :3,

4. Recovering LNPs obtained at substep 1.3 by dialysis in an aqueous phase.

II- Encapsulating lipid nanoparticles in microparticles of anionic polymer comprising the substeps of:

1. Preparing a solution of an anionic polymer, such as alginate, in water at concentration comprised between 1 and 10% w/v,

2. Adding the lipidic nanoparticles suspension obtained at substep 1.4 to the anionic polymer solution of substep 11.1 , T1

3. Adding a citrate solution at pH inferior to 3 at a concentration between 20 mM and 150 mM, to the mixture of substep II.2,

4. Adding a calcium chloride stock solution,

5. Adding NaOH to reach a pH value between 7 and 8, 6. Centrifuging the solution obtained at substep II.5,

7. Collecting the microparticles.