Aerosolization of apolipoprotein a1 nanoparticles enriched with alpha-1-antitrypsin for the treatment of pulmonary emphysema in patients suffering from alpha-1 antitrypsin deficiency

Abstract

The present invention relates to a novel method of treating pulmonary emphysema in patients suffering from alpha-1 antitrypsin deficiency (AATD), a genetic disorder that causes low levels of alpha-1 antitrypsin (AAT), a protein that protects the lungs from damage by neutrophil elastase. The invention consists of aerosolizing nanoparticles composed of apolipoprotein A1 enriched with AAT (A1NP). The invention aims to deliver these nanoparticles directly to the lungs, where they can interact with the alveolar surface and modulate the inflammatory and proteolytic processes that lead to emphysema. In particular, the inventors report that said nanoparticles are not cytotoxic, have anti-inflammatory and anti-elastase properties, can cross alveolar epithelial cells, and are not immunogenic in mice.

Description

[0001] AEROSOLIZATION OF APOLIPOPROTEIN Al NANOPARTICLES ENRICHED WITH ALPHA- 1 -ANTITRYPSIN FOR THE TREATMENT OF PULMONARY EMPHYSEMA IN PATIENTS SUFFERING FROM ALPHA-1 ANTITRYPSIN DEFICIENCY

[0002] FIELD OF THE INVENTION:

[0003] The present invention is in the field of medicine, in particularly pneumology.

[0004] BACKGROUND OF THE INVENTION:

[0005] Alpha- 1 antitrypsin deficiency (AATD) is a rare genetic disease affecting 1 in 1,600 to 2,000 people in Europe, for a total of approximately 125 000 affected individuals, including 74 000 severe cases with 10 000 in France. This disease is caused by mutation of the SERPINA1 gene coding for the alpha- 1- antitrypsin (AAT) protein. AAT is mainly synthesized in the liver and secreted into the circulation, where its main role is to protect lung tissue from neutrophil elastase-induced proteolysis. Point mutations (proteinase inhibitor genotype ZZ for instance) can lead to low plasma AAT levels (<l lpM) and predispose to pulmonary emphysema and liver disease. Emphysema is characterized by progressive and irreversible proteolytic degradation of alveolar tissue, leading to reduced respiratory function and increased risk of mortality. Despite the significant impact of AATD on patients' health, there is currently no effective treatment for the disease. The standard of care for AATD-related lung disease is intravenous administration of AAT. Studies have shown that, in addition to the cost and inconvenience of intravenous AAT treatment, only 2-3% of AAT enters the lungs. This therapy does not halt disease progression, but reduces the rate of decline in lung density and function. However, it has no effect on exacerbation rates or health status. Exacerbations are defined as an acute worsening of respiratory symptoms requiring additional treatment. These episodes have a negative impact on disease progression and quality of life. To improve patient care, AAT aerosolization has been explored. Moreover, there is a high level of patient interest in the development of a commercially available inhaled AAT replacement product. The inhaled route enables replacement therapy to reach the target site of action with higher AAT levels. Recently, the 1st randomized placebo-controlled clinical trial to investigate the effects of nebulized AAT concluded that, in AATD patients with frequent exacerbations, inhaled AAT for 50 weeks had no effect on time to 1 st exacerbation, but may have altered the pattern of episodes (trend towards improvement in forced expiratory volume in 1 second). This clinical trial showed that inhaled AAT was safe and feasible in a large cohort of patients. Now, a new formulation of AAT would considerably increase therapeutic benefits.

[0006] Apolipoprotein Al nanoparticles (AINPs), also known as reconstituted High-Density Lipoproteins (HDLs) can be used as therapeutic vectors. Native HDLs (plasma-isolated HDLs) are composed of lipids and proteins (70% of apolipoprotein Al "ApoAl"), responsible for transporting cholesterol from peripheral tissues to the liver. This pathway requires ApoAl to interact with membrane proteins such as ABCA1 (ATP -binding cassette Al) and SR-B1 (scavenger receptor class B type 1). In addition to cholesterol transport, native HDLs and reconstituted HDLs have antioxidant, anti-endotoxin and anti-inflammatory properties. These protective effects have led several research teams to work on reconstituted HDL-based therapies administered intravenously, to treat cardiovascular diseases, cancers and neurodegenerative diseases. It was demonstrated that native HDLs could be loaded with AAT and that i.v. injection of these nanoparticles significantly reduced elastase-induced pulmonary emphysema compared with HDLs or AAT alone in mice (Moreno, J. -A.; Ortega-Gomez, A.; Rubio-Navarro, A.; Louedec, L.; Ho-Tin-Noe, B.; Caligiuri, G.; Nicoletti, A.; Levoye, A.; Plantier, L.; Meilhac, O. High-Density Lipoproteins Potentiate Al -Antitrypsin Therapy in Elastase Induced Pulmonary Emphysema. Am. J. Respir. Cell Mol. Biol. 2014, 51 (4), 536-549). Notably, the multi- protective effects of HDLs are mainly mediated by ApoAl . Interactions of ApoAl with ABCA1 and SR-B1 have been shown to decrease neutrophil recruitment and activation. There is a growing interest in the cholesterol-independent signaling pathways associated with ABCA1 regulation which include the NF-KB, TLR4 / MyD88, JAK2 / STAT3, cAMP / PKA pathways involved in inflammatory responses. ABC Al's dual role in repressing inflammation and maintaining cholesterol homeostasis represents a promising therapeutic target for inflammatory lung diseases in the future.

[0007] SUMMARY OF THE INVENTION:

[0008] The present invention is defined by the claims. In particular, the present invention relates to the aerosolization of apolipoprotein Al nanoparticles enriched with alpha- 1 -antitrypsin for the treatment of pulmonary emphysema in patients suffering from alpha- 1 antitrypsin deficiency.

[0009] DETAILED DESCRIPTION OF THE INVENTION:

[0010] The present invention relates to a method of treating pulmonary emphysema in a patient suffering from alpha-1 antitrypsin deficiency comprising administering to the patient’s lungs a therapeutically effective amount of apolipoprotein Al nanoparticles enriched with alpha-1- antitrypsin by aerosolization.

[0011] As used herein, the term “patient” is interchangeable with the term “individual” or “subject”, and may refer to a subject to be treated by the methods disclosed herein. Typically, the patient suffers from alpha- 1 antitrypsin deficiency and is affected or likely to suffer from pulmonary emphysema. In some embodiments, the patient is a mammal. In some embodiments, the mammal is a human. In some embodiments, the patient is a human infant. In some embodiments, the patient is a human child. In some embodiments, the patient is a human adult.

[0012] As used herein, the term "alpha-1 antitrypsin" or “AAT” refers to a glycoprotein that belongs to the serine protease inhibitor (serpin) family and is primarily synthesized by the liver. Alpha- 1 antitrypsin inhibits various proteases, such as neutrophil elastase, cathepsin G, proteinase 3, and chymotrypsin, that are involved in inflammatory processes and tissue degradation. Alpha- 1 antitrypsin also has anti-inflammatory, anti-apoptotic, and immunomodulatory effects. Alpha-1 antitrypsin is the major component of the alpha-1 globulin fraction of blood plasma and its normal concentration ranges from 1.5 to 3.5 g / L. An exemplary amino acid sequence for human AAT is shown as SEQ ID NO:1.

[0013] SEQ ID NO : 1 >sp | P01009 | A1AT HUMAN Alpha- l-antitrypsin 0S=Homo sapiens OX=9606 GN=SERPINA1 PE=1 SV=3 MPSSVSWGILLLAGLCCLVPVSLAEDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFS LYRQLAHQSNSTNI FFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEI PEAQIHEGF QELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQ INDYVEKGTQGKIVDLVKELDRDTVFALVNYI FFKGKWERPFEVKDTEEEDFHVDQVTTV KVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAI FFLPDEGKLQHLENELTHDI ITKFL ENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKA VLTIDEKGTEAAGAMFLEAI PMSI PPEVKFNKPFVFLMIEQNTKSPLFMGKWNPTQK

[0014] As used herein, the term "alpha-1 antitrypsin deficiency" refers to a genetic disorder that causes a lack or dysfunction of alpha-l-antitrypsin (AAT), a protease inhibitor that protects the lungs from the damage caused by neutrophil elastase and other inflammatory enzymes. Alpha- 1 antitrypsin deficiency can lead to pulmonary emphysema, which is a chronic lung disease characterized by the destruction of the alveolar walls and the enlargement of the air spaces. Alpha- 1 antitrypsin deficiency is inherited in an autosomal codominant manner and is caused by mutations in the SERPINA1 gene, which encodes AAT. 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 patients 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 patient 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 patient beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during the treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular interval, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

[0015] As used herein, the term “therapeutically effective amount” is an amount that is effective to ameliorate a symptom of a disease. A therapeutically effective amount can be a “prophylactically effective amount” as prophylaxis can be considered therapy.

[0016] The method of the present invention is particularly suitable for reducing the number of exacerbations, which are episodes of acute worsening of respiratory symptoms, such as dyspnea, cough, sputum production, and wheezing. Exacerbations are a major cause of morbidity and mortality in patients with alpha- 1 antitrypsin deficiency and can accelerate the progression of pulmonary emphysema. By delivering AAT directly to the lungs via apolipoprotein Al nanoparticles, the method of the present invention can restore the protease- anti protease balance and prevent or reduce the inflammatory damage to the alveoli. Furthermore, the method can also improve the lung function, quality of life, and survival of patients with alpha-1 antitrypsin deficiency.

[0017] As used herein, the term "apolipoprotein Al nanoparticle" refers to a discoidal particle composed of a phospholipid bilayer and apolipoprotein Al (ApoAl), which is the major protein component of high-density lipoprotein (HDL) in plasma. ApoAl nanoparticles have a diameter of about 7 to 40 nanometers and can carry various bioactive molecules, such as alpha- 1- antitrypsin (AAT), within their core or on their surface. ApoAl nanoparticles have antiinflammatory and anti-oxidant properties and can modulate cholesterol transport and reverse cholesterol efflux. ApoAl nanoparticles can also penetrate the alveolar epithelium and deliver their cargo to the lung tissue.

[0018] Typically, the apolipoprotein Al nanoparticles of the present invention may be prepared by complexation of apoAl to phospholipids. Methods for obtaining apolipoprotein Al nanoparticles are disclosed in EP 1 425 031 and US 5,652,339. Typically, suitable lipids for the preparation of apolipoprotein Al nanoparticles are phospholipids, preferably phosphotidylcholine, for example l-palmitoyl-2-linoleoyl phosphatidylcholine (PC) or 1,2- dipalmitoyl PC. Other phospholipids may be used alone or in combination, including phosphatidylserines, phosphatidylethanolamines, phosphatidylinositols or sphingomyelins with acyl chains of different sizes. Optionally, apolipoprotein Al nanoparticles contain other lipids, for example cholesterol, cholesterol esters, triglycerides, or other lipids. The lipids may be synthetic, naturally occurring lipids or combinations thereof. Methods for preparing apolipoprotein Al nanoparticles are well known in the art and typically those described in Matz CE, Jonas A. J Biol Chem. 1982 Apr 25;257(8):4535-40 or Kim Y, Fay F, Cormode DP, Sanchez-Gaytan BL, Tang J, Hennessy EJ, Ma M, Moore K, Farokhzad OC, Fisher EA, Mulder WJ, Langer R, Fayad ZA. ACS Nano. 2013 Nov 26;7(l l):9975-83.

[0019] Typically, the apolipoprotein Al nanoparticles of the present invention have a molar ratio of phospholipid / apoAl from 2 to 250, preferably from 10 to 200, more preferably from 20 to 100, more preferably 20 to 50 and most preferably from 30 to 40. In some embodiments, the apolipoprotein Al nanoparticles of the present invention may optionally contain additional lipids such as cholesterol, cholesterol esters, triglycerides and / or sphingolipids, preferably in a molar ratio of lipid / apoAl up to 20.

[0020] In some embodiments, the apolipoprotein Al nanoparticles of the present invention are enriched with a AAT polypeptide having at least 90% of identity with amino acid sequence as set forth in SEQ ID NO: 1.

[0021] As used herein, the “percent identity” between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described below. The percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (Needleman, Saul B. & Wunsch, Christian D. (1970). "A general method applicable to the search for similarities in the amino acid sequence of two proteins". Journal of Molecular Biology. 48 (3): 443-53.). The percent identity between two nucleotide or amino acid sequences may also be determined using for example algorithms such as EMBOSS Needle (pair wise alignment; available at www.ebi.ac.uk). For example, EMBOSS Needle may be used with a BLOSUM62 matrix, a “gap open penalty” of 10, a “gap extend penalty” of 0.5, a false “end gap penalty”, an “end gap open penalty” of 10 and an “end gap extend penalty” of 0.5. In general, the “percent identity” is a function of the number of matching positions divided by the number of positions compared and multiplied by 100. For instance, if 6 out of 10 sequence positions are identical between the two compared sequences after alignment, then the identity is 60%. The % identity is typically determined over the whole length of the query sequence on which the analysis is performed. Two molecules having the same primary amino acid sequence or nucleic acid sequence are identical irrespective of any chemical and / or biological modification. According to the invention a first amino acid sequence having at least 70% of identity with a second amino acid sequence means that the first sequence has 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% of identity with the second amino acid sequence. In some embodiments, the apolipoprotein Al nanoparticles of the present invention are enriched with a AAT polypeptide having the amino acid sequence as set forth in SEQ ID NO: 1 that comprises one or more conservative mutations, preferably one or more conservatives substitutions.

[0022] As used herein, the term “mutation” has its general meaning in the art and refers to a substitution, deletion or insertion. The term "substitution" means that a specific amino acid residue at a specific position is removed and another amino acid residue is inserted into the same position.

[0023] As used herein, the term "conservative mutations" refers to amino acid modifications that do not significantly affect or alter the biologic function of the protein containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into a protein by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A “conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. Amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine. Amino acid substitutions may further be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and / or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine. Other groups of amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. Other families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0024] In some embodiments, the apolipoprotein Al nanoparticles of the present invention are enriched with a AAT polypeptide having the amino acid sequence as set forth in SEQ ID NO: 1.

[0025] The person skilled in the art would be aware of the conditions for carrying out the loading of the agent (i.e. the AAT polypeptide). Typically, the loading is performed by ultracentrifugation of the apolipoprotein Al nanoparticles that were previously incubated with an amount of the agent. For example, if the agent has a low affinity with the apolipoprotein Al nanoparticle, the apolipoprotein Al nanoparticle will be incubated with a higher concentration of said agent and for a longer time, than if the agent had a natural and high affinity for the apolipoprotein Al nanoparticle. In addition, the person skilled in the art is able to select the appropriate Molecular Weight Cutoff of the centrifugal device for carrying out the above mentioned filtrations. Another way to prepare the apolipoprotein Al nanoparticles of the present invention is by using microfluidics, as described in the EXAMPLE and Kim, YongTae, et al. "Single step reconstitution of multifunctional high-density lipoprotein-derived nanomaterials using microfluidics. "ACS nano 7.11 (2013): 9975-9983. Microfluidics is a technique that allows the manipulation of small volumes of fluids in microscale channels or devices. By controlling the flow rate, pressure, temperature and geometry of the microfluidic device, it is possible to generate uniform and monodisperse nanoparticles with precise control over their size, shape and composition. Microfluidics also offers advantages such as high throughput, low cost, reduced waste and enhanced stability of the nanoparticles.

[0026] As used herein, the term "aerosolization" refers to a process whereby a liquid formulation is converted to an aerosol.

[0027] Typically, aerosolization is performed by a nebulizer. As used herein, the term “nebulizer” or “aerosol generator” has its general meaning in the art and refers to a device that converts a liquid into an aerosol of a size that can be inhaled into the respiratory tract. Pneumonic, ultrasonic, electronic nebulizers, e.g., passive electronic mesh nebulizers, active electronic mesh nebulizers and vibrating mesh nebulizers are amenable for use with the invention if the particular nebulizer emits an aerosol with the required properties, and at the required output rate. The process of pneumatically converting a bulk liquid into small droplets is called atomization. The operation of a pneumatic nebulizer requires a pressurized gas supply as the driving force for liquid atomization. Ultrasonic nebulizers use electricity introduced by a piezoelectric element in the liquid reservoir to convert a liquid into respirable droplets. Various types of nebulizers are described in Respiratory Care, Vol. 45, No. 6, pp. 609-622 (2000), the disclosure of which is incorporated herein by reference in its entirety. For instance, the device can include a ventilator, optionally in combination with a mask, mouthpiece, mist inhalation apparatus, and / or a platform that guides users to inhale correctly and automatically deliver the drug (i.e. the apolipoprotein Al nanoparticle) at the right time in the breath. Representative aerosolization devices that can be used in accordance with the methods of the present invention include but are not limited to those described in U.S. Patent Nos. 6,357,671 ; 6,354,516; 6,241 ,159; 6,044,841 ; 6,041 ,776; 6,016,974; 5,823,179; 5,797,389; 5,660,166; 5,355,872; 5,284,133; and 5,277,175 and U.S. Published Patent Application Nos. 20020020412 and 20020020409. Using a jet nebulizer, compressed gas from a compressor or hospital airline is passed through a narrow constriction known as a jet. This creates an area of low pressure, and liquid medication from a reservoir is drawn up through a feed tube and fragmented into droplets by the air stream. Only the smallest drops leave the nebulizer directly, while the majority impact on baffles and walls and are returned to the reservoir. Consequently, the time required to perform jet nebulization varies according to the volume of the composition to be nebulized, among other factors, and such time can readily be adjusted by one of skill in the art.

[0028] Aerosolized apolipoprotein Al nanoparticles of the invention comprise droplets that are a suitable size for efficient delivery within the lung. Preferably, the formulation is effectively delivered to lung bronchi, more preferably to bronchioles, still more preferably to alveolar ducts, and still more preferably to alveoli. Thus, aerosol droplets are typically less than about 15 pm in diameter, and preferably less than about 10 pm in diameter, more preferably less than about 5 pm in diameter, and still more preferably less than about 2 pm in diameter. For efficient delivery to alveolar bronchi of a human subject, an aerosol composition preferably comprises droplets having a diameter of about 1 pm to about 5 pm. Droplet size can be assessed using techniques known in the art, for example cascade, impaction, laser diffraction, and optical pattemation. See McLean et al. (2000) Anal Chem 72:4796-804, Fults et al. (1991 ) J Pharm Pharmacol 43:726-8, and Vecellio None et al. (2001 ) J Aerosol Med 14: 107-14. The compositions of the present invention may comprise one or more pharmaceutically acceptable excipients, in particular selected from the group of an HFC / HFA propellant, a cosolvent, a bulking agent, a non-volatile component, a buffer / pH adjusting agent, a surfactant, a preservative, a complexing agent, or combinations thereof. Suitable propellants are those which, when mixed with the solvent(s), form a homogeneous propellant system in which a therapeutically effective amount of apolipoprotein Al nanoparticles can be dissolved. The HFC / HFA propellant must be toxicologically safe and must have a vapor pressure which is suitable to enable the apolipoprotein Al nanoparticle to be administered via a pressurized MDI. According to the present invention, the HFC / HFA propellants may comprise, one or more of 1,1,1,2-tetrafhioroethane (HFA-134(a)) and 1,1, 1,2, 3, 3, 3, -heptafluoropropane (HFA-227), HFC-32 (difluoromethane), HFC-143(a) (1,1,1 -trifluoroethane), HFC-134 (1, 1,2,2- tetrafluoroethane), and HFC-152a (1,1 -difluoroethane) or combinations thereof and such other propellants which may be known to the person having a skill in the art.

[0029] The invention will be further illustrated by the following Figures and examples. However, these examples and Figures should not be interpreted in any way as limiting the scope of the present invention.

[0030] FIGURES:

[0031] Figure 1: Physical characterization of Apolipoprotein Al nanoparticles. A1NP and A1NP- AAT are produced by microfluidic and characterized by dynamic light scattering to determine their size (A) and by electron microscopy to determine their shape (B). Results with A1NP show that productions are reproducible and make it possible to obtain nanoparticles of size between 7 to 12 nm, with a discoidal shape (open triangles). These results are comparable to CSL-111, which are apolipoprotein Al nanoparticles from CSL Behring, already used in several clinical trials.

[0032] Figure 2: Biological characterization of Apolipoprotein Al nanoparticles. A1NP are not cytotoxic for both endothelial and alveolar epithelial cell lines (HMEC-1 and A549 respectively) (A). A1NP (0.05mg / ml) significantly decrease TNF-a-induced IL-6 on HMEC-1 cells (B). A1NP-AAT display enhanced antineutrophil elastase activity compared with AAT or A1NP alone. Total amount of protein (pg) used for AAT (AAT) or A1NP (ApoAl) or A1NP- AAT (ApoAl + AAT) are shown (C). A1NP (0.05mg / ml) can cross alveolar epithelial cell lines (A549), cultured at the air-liquid interface in a time dependent manner (D). Transcytosis of A1NP is decreased when basolateral medium contains TNF-a. (A-D: n=3 independent experiments; ANOVA, Tukey’s multiple comparisons test; (E): n=3 PBS and 5 A1NP; Mann Whitney).

[0033] Figure 3: Physical characterization of Apolipoprotein Al nanoparticles. (A) AINPs are produced by microfluidic using a chip with 2 inlets for Apolipoprotein Al (ApoAl) injection at 0.8 mL / min and 1 inlet for phospholipids (POPC) injection at 0.1 mL / min. (B) AINPs are characterized by dynamic light scattering to determine their size (7-12 nm). Three independent productions are compared with CSL-111 nanoparticles. (C, D) Electron transmission microscopy is used to determine the shape of AINPs, compared with CSL-111 nanoparticles. White triangles indicate stacked disc-like structures, also known as “rolls”.

[0034] Figure 4: Apolipoprotein Al nanoparticles cytotoxicity and uptake assay. (A,B) MTT assays on endothelial (HMEC-1, A) and alveolar epithelial cell lines (A549, B), 24 hours after AINPs coincubation. (n=3-5 independent experiments; ANOVA; Tukey’s multiple comparison test; * p<0.05, ** p<0.01, **** p<0,0001). (C) AINPs [0.05mg / ml] are taken up by both cells after 6- and 24-hours incubation. Rabbit anti human ApoAl was used for labelling AINPs (green) and cell nuclei were stained with DAPI (blue). (D,E) ABCA1 -mediated uptake of AINPs in A549 cells with (D) a dot plot illustration for double staining with DilCis-AINPs and ABCA1 and (E) the mean fluorescence intensity of DilCis in ABCA1 negative and ABCA1 positive cells. (n=3 independent experiments; Paired t-test; * p < 0.05).

[0035] Figure 5: Anti-inflammatory properties of AINPs. AINPs [0.05 mg / ml] significantly decrease TNF-a-induced IL-6 at mRNA (A) and protein level (B) on HMEC-1 cells. (n=3-5 independent experiments; ANOVA; Tukey’s multiple comparison test; * p<0.05, ** p<0.01, **** p<0,0001).

[0036] Figure 6: Biodistribution of AINPs after aerosolization. (A) Experimental design for studying the biodistribution of AINPs administered intratracheally in aerosol form. (B) Determination of human ApoAl concentration in mouse plasma at different time points after aerosolization: Oh, 3h, 6h, 12h and 24h (n=6 for PBS, n=10 for AINPs). (C) AINPs labeled with DilC18 dye (red) reach the left and right lungs 6 hours after aerosolization and persist in both lungs for up to 24 hours. Representative illustration of 6 mice treated with PBS and 10 mice with AlNPs-DilC18. Figure 7: Cellular localization of AINPs after aerosolization. Immunofluorescence of lung sections from mice 6 hours after AINPs administration. ApoAl appears in green and cell nuclei are stained with DAPI (blue). (A) AGER (red), specific to type I pneumocytes. (B) SFTPC (orange), marker for type II pneumocytes. (C) EMCN (red), characteristic of pulmonary endothelial cells. Representative illustration of 10 mice with AlNPs-DilC18.

[0037] Figure 8: Passage of AINPs through A549 epithelial cells grown in air-liquid interface.

[0038] (A) Schema shows the experimental setup used to study AINPs migration through lung epithelial cells cultured in air / liquid interface (ALI). (B) Quantification of AINPs transcytosis under ALI conditions, measured by ELISA specific for human ApoAl, in the presence or absence of TNF-a in the basolateral medium. The cumulative concentration of AINPs in the basolateral medium is determined at different incubation times: 30 min, Ih, 2h, 4h and 6h. (C) Immunofluorescence analysis of ALI membrane after 6h of transcytosis with 0.5 mg / mL AINPs, comparing conditions with standard and TNF-a-supplemented basolateral medium. Phalloidin (green) marks the actin cytoskeleton, cell nuclei are stained blue, and AINPs appear in red. (D) Three-dimensional visualization of the ALI membrane under different experimental conditions.

[0039] Figure 9: FPLC purification of AINPs-AAT. 3 sets of AINPs-AAT purified of AAT-free quantity of proteins eluted (mAU) over time (min).

[0040] Figure 10: Western Blot illustration for AINPs-AAT quantification. Deposit 10 pg of AINPs-AAT, AAT range (500, 250, 100, 50, and 0 ng), and ApoAl range (15, 7.5, and 3.25 pg). Antibody revelation was used for AAT (left side), and ponceau red staining for ApoAl (right side). AAT: 52 kDa. ApoAl : 28 kDa.

[0041] Figure 11: Size distribution of AINPs-AAT. Production 1 in red, 2 in green, and 3 in blue. Size is expressed in nm relative to diameter (d. nm) — size distribution relation to the number (A) or the volume (B) in percent.

[0042] Figure 12: Effect of AINPs-AAT on elastase activity. Different quantities of AAT, AINPs, or AINPs-AAT were incubated with elastase and elastase-substrate. Data are means + / - SEM, n = 3, One Way ANOVA, Holm Sidak’s multiple comparison test. ****p<0.0001. Figure 13: Cytotoxicity assay of AINPs-AAT on HMEC-1. Endothelial cells were incubated for 24h with AINPs-AAT or not (0.015 to 0.5 mg / mL). Compared to the control by MTT assay (n=4) (A), and LDH assay (n=3) (B). Data are means + / - SEM, One-Way ANOVA, Tukey's multiple comparisons test. ****p<0.0001.

[0043] Figure 14: Anti-inflammatory effect of AINPs-AAT. Endothelial cells were stimulated with AINPs-AAT (0.016-0.1 mg / mL) + / - TNF-a (2.5 ng / mL). IL-6 secretion was quantified by ELISA (ng / mL). Control corresponds to unstimulated cells. Data are means + / - SEM, n=l.

[0044] Figure 15: Uptake of AINPs-AAT. Endothelial cells were stimulated with AINPs, AINPs- AAT, or AAT. Immunodetection was performed. DAPI (blue), ApoAl (red), AAT (green), and merge. A numerical zoom was performed in the grey square. All acquisitions were obtained with the same confocal parameters and have been treated with the same LUTs / parameters. Scale bar = 20 pm. n=l.

[0045] Figure 16: Blood passage kinetics of AINPs and AINPs-AAT. Mice were aerosolized with AINPs (n=5) or AINPs-AAT (n=5). Their blood was collected at 0, 6, 12, and 24 hours post aerosolization to quantify the nanoparticles in the blood by detecting human ApoAl (pg / mL). Data are means + / - SEM, 2-way ANOVA, Sidak's multiple comparisons test. * p<0.05.

[0046] Figure 17: AAT western blot in lungs. lOpg of protein extract (right superior lobe lysate) were separated on SDS-PAGE. Five mice exposed to AINPs are depicted on the left side (exposition time: 1 min). Five mice exposed to AINPs-AAT are depicted on the middle (exposition time: 1 min). Positive control (2pg of hAAT) is depicted on the right side (exposition time: 40 sec).

[0047] EXAMPLE 1:

[0048] Methods:

[0049] Production of Apolipoprotein Al nanoparticles

[0050] Apolipoprotein Al nanoparticles (AINPs) are produced using a microfluidic device (DARWIN Microfluidics), by mixing plasma isolated human Apolipoprotein Al (ApoAl) with phospholipids (POPC: phosphatidylcholine: 2-oleoyl-l-palmitoyl-sn-glycero-3- phosphocholine, 42773-500MG, Sigma). Alpha- 1 -anti -trypsin (AAT; Respreeza, CSL Behring, EU / 1 / 15 / 1006 / 003) enrichment is performed during the microfluidics process. Flow rates and molecule concentrations are as follows:

[0051] ApoAl [lmg / ml]; flow rate 0.8mL / min

[0052] AAT [0.6mg / ml]; flow rate 0.8mL / min

[0053] POPC [6mg / ml]; flow rate O.lmL / min

[0054] A1NP enriched in AAT are purified from free AAT by fast protein liquid chromatography (FPLC; size-exclusion chromatography, Superdex 200 increase 10 / 300 GL).

[0055] Physical characterization of Apolipoprotein Al nanoparticles

[0056] Particle size distribution is characterized by dynamic light scattering (DLS) analysis and their shape by electron microscopy.

[0057] Biological characterization of Apolipoprotein Al nanoparticles

[0058] Biological properties are assessed in two different cell lines. In addition to human endothelial cell line (HMEC-1), biological functions of AINPs are also measured in human alveolar epithelial cell line (A549) cultured at the air-liquid interface since epithelial cells will be the first cell type exposed to inhaled particles. A protocol for measuring the cytotoxicity of A1NP, ranging from 0.015 to lmg / ml, is first performed on HMEC-1 and A549 cells (MTT assay; M2128-5G, Sigma). Then, the following biological functions are evaluated:

[0059] Uptake by HMEC-1 and A549 cells: A1NP (0.05mg / ml) are incubated with HMEC-1 and A549 cells (submerged cultured) for 6 and 24 hours. Nanoparticle uptake is evaluated by immunofluorescence staining (anti ApoAl; 178422, Calbiochem) Anti-inflammatory assay on HMEC-1 cells: 90,000 HMEC-1 cells / well are plated in 12-well plate for 24 hours. Serum deprivation is performed for 3 hours before the stimulation with 2.5ng / ml of TNF-a, co-incubated with 0.05mg / ml of AINPs. mRNA are collected at 6h and supernatants at 16h to quantify IL-6 expression (Q-RT-PCR and ELISA respectively).

[0060] - Anti-elastase activity of A1NP is assessed by chromogenic assay (Calbiochem; 324696- 10MG). Briefly, elastase (from bronchoalveolar lavage) is co-incubated with substrate and AINPs, A1NP-AAT or AAT at different concentration.

[0061] - Transcytosis of A1NP through an epithelium: the dynamics of AINPs in the epithelium is measured on A549 cultured at the air-liquid interface. AINPs (0.05mg / ml) are placed at the apical side and basolateral medium is collected after 30 minutes and each hour for 6 hours, to quantify the amount of AINPs that migrated to the basolateral side (Human ApoAl ELISA, 3710-1HP-2 Mabtech).

[0062] Biodistribution of A1NP in the lung following aerosolization

[0063] Once characterized, A1NP are aerosolized intratracheally in C57BL / 6 mice, using a microaerosol sprayer model YAN 30012 (Yuyan Instruments), as authorized by the ethics committee. A 1stseries of experiments is carried out with AINPs, labeled with fluorescent phospholipids (DilC18 dye) to assess their pulmonary distribution at 6h, 24h and 48h. Each mouse receives 25 pl of A1NP (lOOpg ApoAl / 25pl) or PBS as control. To better characterize biodistribution, additional immunofluorescence detection of ApoAl, AAT and cellular markers are performed to identify the cell type capable of taking up A1NP (based on localization, morphology and markers, observed using a confocal microscope: F4 / 80 CDl lc for alveolar macrophages, AGER for type 1 pneumocytes, SFPTC for type 2 pneumocytes, EMCN for vascular endothelial cells, ACTA2 for myofibroblast, CSPG4 for pericytes, CALCA for neuroendocrine cells, MUC5B for goblet cells and TUBA1 A for ciliated cells).

[0064] Immunogenicity

[0065] Immunogenicity against human ApoAl is evaluated in C57BL / 6 mice after 2 A1NP aerosolizations separated by 11 days. The serum is collected 24 hours after the last exposure to quantify murine anti-human ApoAl antibodies.

[0066] Results:

[0067] A1NP productions by microfluidic are reproducible and make it possible to obtain nanoparticles of size between 7 to 12 nm (Figure 1A), with a discoidal shape (Figure IB). These results are comparable to CSL-111, which are ApoAl nanoparticles from CSL Behring, already used in several clinical trials. The same size distribution is observed with AAT enrichment (Figure 1A, right side).

[0068] Biological characterizations confirmed that A1NP (i) are not cytotoxic for both endothelial and epithelial cells (Figure 2A), (ii) can be taken up by human endothelial and epithelial cell lines (data not shown) and (iii) display anti-inflammatory properties (Figure 2B). Preliminary results show that A1NP-AAT are similar to AINPs and already demonstrate enhanced antineutrophil elastase activity (Figure 2C). Indeed, A1NP-AAT completely inhibit elastase activity with 65 pg of protein (ApoAl + AAT), whereas AAT alone requires ten times more protein to achieve similar inhibition. Interestingly, we also observed that AINPs have anti-elastase activity. These results suggest that not only does AAT retain its activity, but that its integration into A1NP enhances its anti-protease properties. This observation, which needs to be confirmed by further experiments, is in line with molecular modeling predictions which indicate that the hydrophobic loop of AAT's reactive center, the functional domain responsible for its protease inhibitor activity, is probably involved in lipid binding and that association with AINPs protects AAT from oxidative inactivation and enhances its antiprotease activity.

[0069] Interestingly, we found that AINPs can cross alveolar epithelial cell lines, cultured at the airliquid interface, in a time-dependent manner (Figure 2D). Transcytosis of AINPs is decreased when the basolateral medium contains TNF-a suggesting that lung inflammation may increase the bioavailability of aerosolized nanoparticles in alveoli.

[0070] Preliminary results on aerosolization of AINPs in mice show that this route of administration leads to a homogenous distribution of AINPs in the left and right lobes 6h following administration. Same observations were made at 24h while fluorescence began to diminish at 48h (data not shown). Additional staining show that AINPs may colocalize with some SFTPC positive cells, suggesting that type 2 pneumocytes can take up AINPs, but also other cell types (SFTPC negative cells) that will be further explored. During this preliminary animal experiments, we also evaluated if mice could develop an immune response against human ApoAl . Data show that A1NP treatment was not immunogenic for mice (Figure 2E).

[0071] EXAMPLE 2:

[0072] Methods:

[0073] AINPs production and characterization

[0074] AINPs are synthesized from plasma apolipoprotein Al (ApoAl) (plasma was obtained from the French blood national agency, EFS-LR agreement number #2018001378) and commercial phospholipids (2-oleoyl-l-palmitoyl-sn-glycero-3 -phosphocholine; 42773-500MG, Sigma). The production is carried out on a microfluidic chip with three input channels. The central inlet is for phospholipids prepared at a concentration of 6 mg / mL in ethanol at an injection flow rate of 0.1 mL / min (Darwin Microfluidics, SeryngeONE, Connection Kit 01). The other two inlets are used to inject ApoAl at a concentration of 1 mg / mL in TEN buffer (10 mM TRIS, ImM EDTA, 150 mM NaCl) at an injection rate of 0.8 mL / min. The solution obtained at the microfluidic chip outlet is centrifuged at 12,000 g for 15 minutes to sediment the aggregates. Then, they are concentrated and washed with a TEN buffer using a 10 kDa cut-off concentrator (Corning).

[0075] Dynamic light scattering characterization

[0076] AINPs were characterized by dynamic light scattering (DLS) spectroscopy as described previously1. AINPs were compared to CSL-111 (gently provided by CSL Berhing).

[0077] Electron microscopy characterization

[0078] Transmission electron microscopy analyses were conducted at the Center for Quantitative Imaging Lyon East (CIQLE, University of Lyon 1, Lyon, France). The morphological characteristics and size of the nanoparticles were assessed using negatively stained samples, imaged with a Gatan Orius 600 CCD camera (Gatan, USA) on a LaB6 JEOL JEM- 1400 transmission electron microscope (JEOL, Japan) operating at an accelerating voltage of 120 kV. For sample preparation, 300-mesh copper grids coated with a carbon film (Delta Microscopies, France) were glow-discharged for 30 seconds (Balzers SCD 040, Liechtenstein) to render the carbon surface hydrophilic, facilitating sample adhesion. Nanoparticle suspensions were subsequently deposited onto the treated grids, followed by negative staining using a 2% aqueous solution of uranyl acetate. After complete air drying, the grids were mounted onto a single-tilt holder and introduced into a JEOL JEM-2100 transmission electron microscope (JEOL, Japan), equipped with a cryo pole piece and operated at 120 kV. Image acquisition was performed using a Gatan SC600A CCD camera (Gatan, USA).

[0079] Cell Culture and stimulation

[0080] HMEC-1 cells, a human microvascular endothelial cell line (ATCC# CRL-3243), were cultured in MCDB 131 medium (P04-80057, Pan Biotech) supplemented with 10% FBS (ST30-3302, Pan Biotech), 100 units / mL Penicillin- lOOpg / mL Streptomycin (P06-07100, Pan Biotech), 250pg / mL Amphotericin-B (P06-01100, Pan Biotech), 10 ng / mL Epidermal Growth Factor (EGF) (E9644; Sigma-Aldrich, USA), 1 pg / mL Hydrocortisone (Sigma), and 10 mM L- Glutamine (Pan Biotech). A549 cells, a human alveolar epithelial cell line (ATCC# CCL-185), were routinely cultured in RPMI 1640 medium (P04-22100, Pan Biotech) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (ST30-3302, Pan Biotech) and 100 units / mL Penicillin- lOOpg / mL Streptomycin (P06-07100, Pan Biotech). Both cell lines were maintained in a humidified incubator at 37°C with 5% CO2 and subcultured when they reached 90% confluence. During stimulation with AINPs, the respective media were supplemented with 10% delipidated FBS for HMEC-1 and 5% delipidated FBS for A549.

[0081] AINPs Cytotoxicity

[0082] The cytotoxicity of AINPs on each cell line was evaluated using the 3-(4,5-dimethyl-thiazol- 2-yl)-2, 5 -diphenyl tetrazolium bromide (MTT) assay. 96-well plate were seeded with 10,000 cells / well. After 24hours of incubation, the medium was removed and the cells were stimulated with 100 pL of AINPs at concentrations ranging from 1 mg / mL to 0.015 mg / mL for 24 hours. lOpL of MTT reagent (M2128-5G, Sigma-Aldrich) diluted in PBS was added to each well five hours before the end, resulting in a final MTT concentration of 0.5 mg / mL. The plate was then centrifuged at 500g for 4 minutes at 25°C. The medium was removed and replaced with 200 pL of dimethyl sulfoxide (DMSO) (UD8050-B, Euromedex) to dissolve the formazan crystals. Absorbance was measured at 560 nm using a CLARIOStar Plus plate reader (BMG Labtech).

[0083] AINPs uptake on endothelial and epithelial cells

[0084] 8-well Labtek chamber slides were seeded with 50,000 cells / well for each cell line studied. Once cell confluence reached 80%, the wells were washed with IX PBS solution and were serum-deprived for 3 hours. Cells were stimulated with AINPs at a concentration of 0.05 mg / mL, or with their respective media for 6 hours and 24 hours. Cells were washed three times with PBS, then fixed with 4% paraformaldehyde (PF A) for 15 minutes. Slides were washed once with IX PBS for 5 minutes, then twice with IX PBS containing 0.05% Triton for 10 minutes. Cells were then blocked for 1 hour in PBS IX triton 0.05% BSA 2%. Primary antihuman ApoAl antibody (Calbiochem) was diluted 1 :500 in PBS IX triton 0.05% BSA 0.2% and incubated with the cells overnight at 4°C. After 3x 10-minute washes, the cells were incubated with DAPI (Ipg / mL, Sigma) mixed with an Alexa 488 goat anti-rabbit secondary antibody at 1 : 1000 dilution for 1 hour at room temperature. After 5x- 10-minute washes, the slides were mounted with fluorescent medium and images were captured with a confocal microscope (Nikon Eclipse Ti2).

[0085] Flow Cytometry

[0086] A total of 200,000 A549 epithelial cells were seeded in 6-well plates. After 24 hours, the culture medium was replaced with a lipid-depleted medium for an additional 24-hour period. Subsequently, cells were either stimulated or not with DilCis AINPs at concentration of 0.05 mg / mL for 4 hours, under lipid-free conditions. Following stimulation, cells were detached using Accutase (25-058-CI, Corning) and immediately placed on ice. Cell suspension was centrifuged at 1500 RPM for 5 minutes. The supernatant containing Accutase was discarded, and the resulting cell pellet was resuspended in FACS buffer (PBS supplemented with 0.1% BSA). After a second centrifugation, the supernatant was removed, and Fc receptor blocking reagent (anti-CD16 / CD32; 553141, BD Bioscience) was added at a concentration of 1 pg per 106cells. The suspension was incubated for 10 minutes at 4°C. Cells were then washed with FACS buffer and incubated with the human ABCA1 Alexa Fluor 488 conjugated antibody (NB100-2068G, NovusBio) at 1 pg per 106cells, for 30 minutes in dark at 4°C. Following a final wash with PBS, the cells were resuspended and analyzed by flow cytometry (Cytoflex, Beckman Coulter).

[0087] AINPs anti-inflammatory properties

[0088] The anti-inflammatory activity of AINPs was assessed on the TNF-a-stimulated HMEC-1 cell line by RT-qPCR and ELISA.

[0089] RT-qPCR

[0090] 50,000 cells were seeded in a 24-well plate. When the cells reached 70% confluence, they were washed and serum-deprived (0% FBS) for 3 hours. Cells were stimulated with AINPs at a concentration of 0.05 mg / mL for 6 hours, with or without addition of TNF-a. After stimulation, cells were lysed and RNA was extracted with RNeasy Plus Mini kit (74136, Qiagen) and quantified by Nanodrop (BMG Labtech). Reverse transcription was carried out using the NxGen M-MuLV reverse transcriptase (30222-1, Lucigen) according to the manufacturer’s standard protocol. Quantitative PCR was performed using the Blastaq Green 2x qPCR MasterMix (G892, Abm). The transcription levels of IL-6 were measured using the following primers: forward 5’-ACCCCCAGGAGAAGATTCCA-3’ (SEQ ID NO:2), reverse 5’- GCCTCTTTGCTGCTTTCACA-3’ (SEQ ID NO:3). The data were normalized against GAPDH (forward 5’-AGCCACATCGCTCAGACAC-3’ (SEQ ID NO:4), reverse 5’- GCCCAATACGACCAAATCC-3’(SEQ ID NO: 5)) and RNA polymerase II (RNApol2) (forward 5’-CGAGAAGGTCTCATTGACACAG-3’ (SEQ ID NO: 6), reverse 5’- ACCACCTGGTTGATGGAGTTCC-3’ (SEQ ID NO:7)).

[0091] ELISA

[0092] 70,000 cells / wells were seeded in a 12-well plate. When the cells reached 70% confluence, they were washed and serum-deprived (0% FBS) for 3 hours. Next, cells were stimulated with AINPs at a concentration of 0.05 mg / mL for 16 hours, with or without the addition of TNF-a. Supernatant was collected for IL-6 quantification using a human IL-6 ELISA Ready-SET-Go assay (Thermofisher). Absorbance was measured using a CLARIOstar plate reader (BMG Labtech) at 450-570 nm.

[0093] Aerosolization in mice

[0094] C57BL / 6 mice (8-10 weeks) were fed ad libitum with standard laboratory chow and water. All animal experiments were approved by the local ethics committee and Ministry of Higher Education and Research (APAFIS #53302-2025011413372952 v4). To visualize AINPs in the lung, the nanoparticles were first incubated with 200 pg of DilC18 at 37°C overnight with agitation at 250 RPM. Free DilC18 was removed by ultracentrifugation. The density of the AlNPs-DilC18 was adjusted to 1.23 g / mL, and the particles were then recovered through two sequential layers: the first layer consisted of KBr at a density of 1.21 g / mL and the second at 1.063 g / mL. Following ultracentrifugation at 252,000g for 18 hours at 4°C, the AlNPs-DilC18 were collected. A BCA protein assay (Sigma) was subsequently performed to determine the concentration of the nanoparticles. AlNPs-DilC18 were prepared at 8mg / ml. For intratracheal aerosolization of AINPs, mice were anesthetized with isoflurane (2.5%), followed by an intraperitoneal (i.p.) injection of ketamine (90 mg / kg) and xylazine (4.5 mg / kg) to deepen anesthesia. They were positioned on a panel inclined at 45° for intratracheal instillation, with a light source to visualize the tracheal orifice. Using a microsprayer aerosolizer, model YAN 30012 (Yuyan Instruments), a volume of 25 pL was administered between the vocal cords. Mice received either PBS solution or AlNPs-DilC18 (8 mg / ml). Blood was collected before and after aerosolization at 3, 6, 12 and 24 hours. 4 mice (1 PBS and 3 DHC18-A1NPS) were sacrificed at 12 hours and 4 mice (1 PBS and 3 DilC18-AlNPs) were sacrificed at 24 hours. After transcardiac perfusion with PBS and then 4% PF A, the lungs were then fixed overnight in 4% PF A, kept in 30% sucrose solution overnight, and then frozen in OCT at -80°C.

[0095] Immunostaining

[0096] Frozen sections (10 pm) were obtained using a cryostat (Leica CM1520; Leica Biosystems). OCT was eliminated with PBS and tissue section were incubated with DAPI (1 pg / mL) at RT for 20 minutes. Ibidi mounting medium was used to see fluorescence and images were observed on the Nanozoomer S60 digital slide scanner (Hamamatsu). To assess the cell types capable of capturing AINPs, co-labeling involving ApoAl and specific markers was performed. Tissue sections were first subjected to antigen unmasking in sodium citrate (pH 6), held at 80°C for 30 minutes. Once cooled to room temperature, the slides were washed with PBS, then blocked for 90 minutes in PBS 0.1% Triton - 2% BSA. Then, slides were incubated for a further 30 minutes in PBS IX, 0.1% Triton - 0.2% BSA with Fc Block at 0.025 mg / mL. Slides were then incubated overnight at 4°C with specific primary antibodies. For ApoAl detection, a mouse anti-human ApoAl primary antibody diluted 1 :200 was used. In parallel, specific antibodies were applied: a mouse anti-AGER rat antibody diluted 1 : 100 for type I pneumocytes (MAB 1179-500; R&D Systems), a mouse anti-SFTPC rabbit antibody diluted 1 :200 for type II pneumocytes (10774- 1AP; Proteintech), and a mouse anti-EMCN goat antibody diluted 1 : 100 for endothelial cells (AF4666-SP; R&D Systems). After five minutes washes with PBS 0.1% Triton, the slides were incubated with secondary antibodies coupled to suitable fluorochromes, including a donkey anti-mouse Alexa Fluor 488, a goat anti-rat Alexa Fluor 594, goat anti-rabbit Alexa Fluor 647, and donkey anti-goat Alexa Fluor 594, all diluted at 1 : 1000. After this step, the slides were washed three times with PBS 0.1% Triton, then incubated with DAPI at 1 pg / mL for 15 minutes for nuclei staining. Finally, after 3 washes in PBS 0.1% Triton, slides were mounted with IBIDI medium. Observations and acquisitions were made using a confocal microscope (Nikon Eclipse Ti2).

[0097] Air / liquid interface model

[0098] A total of 500,000 A549 cells were seeded into polycarbonate cell culture inserts with pore-size of 0.4 pm (PIHP01250; Millipore) pre-coated with 70 pg / mL of type I rat tail collagen (354236, Coming, USA). After 24 hours incubation, the medium in the apical compartment was removed and the medium at the basolateral side was replaced 3 times per week for 2 weeks2.

[0099] Transcytosis

[0100] To assess the potential of AINPs to cross a reconstituted epithelial barrier, the inserts were exposed to 0.05 mg / mL of AINPs. Basolateral media were collected after 30 minutes, 1 hour, 2 hours, 4 hours, and 6 hours and analyzed with a human ApoAl ELISA assay (3710-1HP-2, Mabtech). Absorbance was measured using a CLARIOstar plate reader (BMG Labtech) at 450- 570 nm. In another set of experiments, AINPs-exposed inserts were washed with PBS (top and bottom), then fixed with 4% paraformaldehyde (PF A) for 15 minutes on the top and the bottom. Inserts were washed twice with IX PBS and could be stored at 4°C for 1 week or used directly. Membranes of insert were cut and washed with IX PBS 0.5% Triton for 5 minutes. Cells were blocked with PBS IX triton 0.5% BSA 4% for 30 minutes. Primary anti-human ApoAl antibody (178422; Calbiochem) was diluted 1 :500 in PBS IX triton 0.5% BSA 1% and incubated 45 minutes at RT. After 2 washes with PBS IX, membranes were incubated with Alexa 594 goat anti -rabbit secondary antibody at 1 : 1000 dilution for 45 minutes at RT. After 2 washes with PBS IX, membranes were incubated with Alexa Fluor 488 anti-phalloidin antibody at dilution 1 :2000 and DAPI (Ipg / mL) for 45 minutes at RT. Membranes were mounted with fluorescent medium and images were captured with a confocal microscope (Nikon Eclipse Ti2).

[0101] Permeability assay

[0102] Following 6 hours of transcytosis, the inserts were retrieved and transferred into 300 pL of complete culture medium without phenol red (P04-16516, Pan Biotech). Subsequently, 100 pL of dextran labeled with fluorescein isothiocyanate (FITC-dextran) 70 kDa (Sigma) at a concentration of 1 mg / mL was applied to the apical compartment. The inserts were incubated at 37 °C for 40 minutes. Post-incubation, the basolateral medium was collected for each experimental condition. The fluorescence intensity was quantified using a spectrophotometer with excitation / emission wavelengths set at 490 ± 15 nm and 530 ± 30 nm (BMG Labtech). The concentration of FITC-dextran (70 kDa) in the basolateral compartment was determined using a standard calibration curve ranging from 0.25 mg / mL to 0.008 mg / mL. Result was expressed as the relative concentration, calculated as the ratio between the initial concentration applied at To and the concentration measured at T40 minutes (CUo / Cto).

[0103] Statistics

[0104] All statistical tests were performed on Graphpad Prism 5 software (Graphpad Software, San Diego, CA). Results were displayed as mean ± SEM values of repeated independent experiments. Statistical tests used were ordinary one-way ANOVA with Tukey’s multiple comparisons test or paired T test. Results were considered statistically significant when p<0.05.

[0105] Results:

[0106] While therapies based on apolipoprotein Al nanoparticles (AINPs) have not had the expected effects in cardiovascular diseases, their therapeutic potential in lung diseases has not been fully explored. Here, we used both physical and biological fields to report that anti-inflammatory AINPs can be homogeneously aerosolized in the lungs, paving the way for new therapeutic strategies for lung diseases. Production and physical characterization of AINPs

[0107] AINPs were reconstituted using a single-step, self-assembly method in a single layer, 3 -inlet microfluidic device (Figure 3A). As previously shown by Kim et al., this technique allows the production of reproducible and homogeneous batches of AINPs3. While Kim et al. performed their nanoparticles synthesis in phosphate buffer saline, we opted for reconstitution directly in a buffer designed to preserve AINPs by limiting their oxidation (TEN buffer). It is also worth noting that we used a five-fold higher concentration of ApoAl, enabling us to obtain nanoparticles of the expected majority size without the need for additional purification steps. Dynamic light scattering (DLS) analysis confirmed the reproducibility of these productions. These results indicate that the average size of AINPs was around 10 nm, like plasmatic HDL4and similar to that of CSL-111, an industrially produced apolipoprotein Al nanoparticle (Figure 3B). Structural comparison between AINPs and CSL-111 were observed under a transmission electron microscope (Figure 3C,D). This observation revealed that AINPs formed stacked disc-like structures (discoidal shape), also known as “rolls”, similar to those observed for CSL-111 and already described for plasmatic preP-HDL5. The overall morphology of AINPs was comparable to that of CSL-111 demonstrating the ability of our laboratory to generate biological nanoparticles comparable to pharmaceutical groups such as CSL Behring. CSL-111 and CSL-112 are nanoparticles made from human ApoAl and soy-derived phospholipids and have been evaluated in clinical trials6, making them a benchmark in terms of morphological and dimensional characteristics. Yet, in the context of coronary artery disease, these nanoparticles did not produce the expected atheromatous plaque reduction effect, underlining the need for optimizations to improve their therapeutic efficacy6. One proposed solution is to enrich these nanoparticles with bioactive molecules. For instance, Moreno et al. showed that high-density lipoproteins (HDL) enriched with alpha- 1 -antitrypsin, significantly reduced neutrophil elastase-induced pulmonary emphysema in mice, compared with unenriched HDL7. Thus, the use of AINPs as vectors for therapeutic molecules represents a promising approach requiring further investigation to optimize their therapeutic potential. Microfluidic offers significant advantages for this type of enrichment, whether of proteins, synthetic molecules or lipids8. Controlled flow rates and channel dimensions in the micrometer range promote molecule assembly, facilitating the incorporation of therapeutic compounds into nanoparticles, without using additional chemicals. AINPs are captured by cells without cytotoxicity

[0108] To assess the safety profile of AINPs, cell viability tests were conducted across a range of AINPs concentrations. HMEC-1 and A549 cells were incubated with AINPs at concentrations from 0.015 mg / mL to 1 mg / mL for 24 hours, followed by an MTT assay (Figure 4A,B). The results indicate no significant difference between stimulated and unstimulated cells, suggesting AINPs have no impact on the viability of either cell type. To visualize AINPs internalization into cells, confocal microscopy was performed on both HMEC-1 and A549 cells using anti- ApoAl antibodies. Observations show that after 6 hours, cells have internalized the AINPs, with an increase in labeling intensity observed after 24 hours (Figure 4C). In addition to MTT assay, these findings indicate that the nanoparticles do not alter cell morphology and integrity. It should be emphasized that nanoparticles not only bind to the cell surface, but are also internalized. Indeed, Silver et al demonstrated that hepatocytes incubated with HDL at 4°C only led to binding. At 37°C, results indicate an active uptake process9,10. Given that the uptake of HDL is mediated by the scavenger receptor class B type I (SR-BI) and the ATP binding cassette subfamily A member 1 (ABCA1)11, one may argue that AINPs internalization is also dependent on these receptors. Alveolar epithelial cells (type I and type II) as well as A549 cells do express ABCAl12,13but whether AINPs-mediated uptake is similar to endothelial cells11,14is unclear. To clarify the potential role of ABCA1 in AINPs uptake by A549 cells, we incubated cells with fluorescent AINPs (DilCis staining) for 6 hours and analyzed cells by flow cytometry. We observed that about 95% of A549 cells were positive for DilC18-AlNP. However, only approximately 27% of A549 cells were positive for ABC Al, but this population captured a higher amount of DilCis-AlNP, as evidenced by a significant increase in the mean fluorescence intensity of DilCis compared to ABC Al -negative A549 cells (Figure 4D,E and data not shown). This result confirms that a fraction of A549 cells do express ABCA1 in unstimulated condition and that ABCA1 may be involved in AINPs uptake. Interestingly, the dual role of ABCA1 in repressing inflammation while maintaining cholesterol homeostasis represents a promising therapeutic target for inflammatory lung diseases in the future13.

[0109] AINPs displays anti-inflammatory properties

[0110] Endothelial cells were stimulated with 2.5 ng / mL of TNF-a to induce an inflammatory response and co-stimulated with AINPs. Following 6 hours of incubation, we assessed the gene expression of the pro-inflammatory mediator IL-6 (Figure 5A). After 16 hours, IL-6 protein levels in the culture medium were quantified by ELISA (Figure 5B). Both IL-6 mRNA and protein levels increased about 3-fold under inflammatory conditions compared to untreated controls. However, treatment with AINPs significantly reduced IL-6 expression at both the transcriptional and protein levels, indicating an anti-inflammatory effect. These findings demonstrate that AINPs are biologically functional and exhibit anti-inflammatory activity in HMEC-1 cells under TNF-a-induced inflammatory conditions. This assay on endothelial cells is a classical hallmark to appreciate the anti-inflammatory properties of HDL and mimetics15. We did not explore the underlying mechanisms but one could be of particular interest. It has been shown that ApoAl binding to ABCA1 may trigger the expression of tristetraprolin, which subsequently promotes the degradation of inflammatory cytokine mRNA through its 3’-UTR AREs16.

[0111] AINPs are homogeneously distributed in the lung after aerosolization

[0112] To investigate the biodistribution of AINPs following their administration by aerosolization, AINPs were first incubated with DilCis to be detected by fluorescence (Figure 6A). Since the lung is closely linked to the capillary network, we also quantified the passage of AINPs in the bloodstream. AINPs were detected in the plasma at 6 hours post-administration, before decreasing at 12 hours and persisting at 24 hours (Figure 6B). At 6 hours post-administration, homogeneous red fluorescence was detected throughout the lung parenchyma of mice that had received DilCis-AINPs while no fluorescence was observed in control mice treated with PBS (Figure 6C). This uniform biodistribution of AINPs persisted at 24 hours, which is also consistent with AINPs plasma kinetics (Figure 6B). Interestingly, red fluorescence was also detected in the liver and kidneys at 24 hours (data not shown). These observations suggest that AINP-DilCis behave similarly to HDL, with elimination via hepatic and renal pathways. No significant variation in the body weight of the mice was observed throughout the experiment, supporting the absence of in vivo nanoparticle toxicity (data not shown). Moreover, additional experiments have demonstrated the absence of immunogenicity in mice given AINPs on days 0, 1 and 12 (data not shown). Surprisingly, other organs, such as the brain and the spleen, were also enriched in A1NP after passage into the bloodstream (data not shown), opening up therapeutic prospects targeting these organs. Although the lung remains the main organ targeted by aerosolization, this non-invasive route could also be considered for the treatment of pathologies characterized by chronic inflammation in peripheral organs. To further evaluate the precise localization of AINPs, we performed co-labeling between ApoAl and different cell types specific to lung tissue. We observed co-localization of ApoAl with cell-specific markers (AGER: type I pneumocytes; SFPTC: type II pneumocytes; EMCN: vascular endothelial cells) (Figure 7A,B,C and data not shown). These different structural lung cell types were able to take up and internalize AINPs, opening up interesting therapeutic perspectives. These include intracellular application of AINPs when enriched with therapeutic molecules that act intracellularly, such as siRNA. This AINP's broad spectrum of pulmonary penetration makes it the vector of choice for lung diseases.

[0113] The crossing of AINPs through an epithelium grown at an air-liquid interface is slower under inflammatory state.

[0114] Previous results in vivo suggest a progressive transfer of AINPs into the bloodstream. The nanoparticles may cross the alveolar barrier in the lung, being initially internalized by alveolar epithelial cells before reaching endothelial cells, allowing access to the vascular compartment. From a therapeutic perspective, this biological barrier could be in an inflammatory state. To further explore the passage of AINPs through an epithelium, we set up an air-liquid interface (ALI) model of lung epithelial cells using inserts (Figure 8A). According to previous characterization of ALI culture of A549 alveolar epithelial cells, this model reconstitutes epithelial layers with the expression of markers of both alveolar epithelial type I and type II cells17. After apical addition of AINPs, the inserts were incubated in either normal or inflammatory medium in the basolateral side, containing 5 ng / ml of TNF-a. A progressive passage of AINPs was observed over time in both experimental conditions. Surprisingly, we found a significant reduction in AINPs passage under inflammatory conditions (Figure 8B). In another set of experiments, we made sure that after the assay, the permeability of both unstimulated and TNF-a-stimulated epithelium was the same, ruling out the possibility that TNF-a may alter this parameter (data not shown). Confocal microscopy confirmed the presence of ApoAl in the cytoplasm of epithelial cells (Figure 8C,D and data not shown). Interestingly, it seems the amount of AINPs increased in the cytoplasm of TNF-a-stimulated epithelial cells. This result suggests that the inflammatory state of the epithelium could slow down the passage of AINPs through epithelial cells leading to an increase in the retention time of nanoparticles in the lung. A deeper understanding of the mechanisms involved in the transepithelial passage of AINPs would be relevant, to determine whether this is a process of transcytosis or other alternative mechanisms. ABCA1 is known to facilitate the interaction and internalization of pre-P HDL particles. This mechanism has been extensively characterized in endothelial cells14. Given that AINPs exhibit structural and functional similarities to pre-P HDL, one may argue that their cellular internalization is also mediated by ABCA1. Since ABCA1 is expressed by A549 and that TNF-a may decrease the expression of ABCA1 in another epithelial cell line (Caco-2)18, one may argue that TNF-a may decrease ABCA1 in A549 epithelial cell line leading to a decrease of AINPs transcytosis. Our flow cytometry results on ABCA1 expression showing an increase uptake in ABCA1 positive cells support a model in which ABCA1 partially mediates A1NP internalization, although additional receptors may contribute to uptake through other specific mechanisms. Consistent with existing literature, discoidal ApoAl particles have been shown to preferentially interact with ABCA1 to facilitate lipid acquisition14. In contrast, the SR-B1 receptor is known to recognize lipid-rich spherical HDL particles. The ABCG1 transporter also plays a role in lipid efflux and may be implicated in HDL trafficking19. It is interesting to note that Moreno et al. observed that intravenous injection of HDL in mice with elastase-induced emphysema led to increased HDL recruitment in the lungs compared to control mice20. This study suggests that A1NP may be preferentially recruited to inflamed tissues. Collectively, our data suggest that the microfluidically produced AINPs mimic the biological behavior of circulating discoidal HDL particles and may use similar uptake pathways.

[0115] CONCLUSION

[0116] In this study, we report the successful development of apolipoprotein Al -based nanoparticles (AINPs) produced via microfluidics and designed for pulmonary delivery. Our results demonstrate that AINPs possess favorable physicochemical characteristics, including a reproducible discoidal morphology and nanoscale dimensions (7-12 nm), comparable to clinically evaluated HDL mimetics such as CSL-111. In vitro, AINPs were efficiently internalized by both endothelial and alveolar epithelial cells, with uptake enhanced in ABC Al - positive populations, and displayed no cytotoxicity across a range of concentrations. Importantly, AINPs retained biological functionality, as evidenced by their significant antiinflammatory effects on TNF-a-stimulated endothelial cells, with a reduction in IL-6 expression at both transcript and protein levels.

[0117] Upon aerosolization in mice, AINPs exhibited homogeneous pulmonary biodistribution, reaching both lobes and persisting in lung tissue for up to 24 hours without triggering detectable immunogenicity. Furthermore, we confirmed their transcytosis across alveolar epithelial barriers both in vitro and in vivo, with delayed clearance under inflammatory conditions, potentially enhancing their therapeutic retention in inflamed lungs. This dual capacity for local action and systemic translocation highlights the versatility of AINPs not only as lung-targeted agents but also as systemic drug delivery vehicles following non-invasive administration. EXAMPLE 3:

[0118] Methods:

[0119] Production and purification of AINPs-AAT

[0120] Microfluidics. It allows precise control of reaction conditions, such as flow rate, temperature, and chemical composition. This is crucial for producing nanoparticles with specific characteristics, including size, shape, and size distribution3. To make our AAT-enriched nanoparticles, we used a 3-input chip. One input for phosphatidylcholine (POPC, 6 mg / mL, 42773, Sigma-Aldrich, USA) and 2 inputs for ApoAl purified from human plasma plus AAT (Respreeza 5000mg, CSL Behring, 35041 Marburg, Germany) (stock at 50 mg / mL for use at 2 mg / mL). The glass syringes (13.5 mm) containing plasmatic ApoAl (1 mg / mL) and AAT (2 mg / mL) were set to a flow rate of 0.80 mL / min, and the syringe (17.5 mm) containing POPC was set to a flow rate of 0.10 mL / min. The flow rate parameters were previously optimised and validated by APALAMA Laurine (PhD Student) as part of her thesis. Once coupling was complete, the collected nanoparticles were centrifuged at 12,000g for 30 min. The supernatant was recovered, washed in TEN buffer, and centrifuged at 6,000g for 30 minutes until 1 mL was reached. AINPs-AAT were then purified by Fast Protein Liquid Chromatography (FPLC, Akta pure, Cytiva / Danaher, Washington, USA), according to nanoparticle size, using a size exclusion chromatography, Superdex 200 Increase 10 / 300 GL column (Cytiva, Washington, USA) and filtered with a Minisart filter (0.2pm, SARTORIUS, Gottingen, Germany).

[0121] AAT and AINPs quantification

[0122] BCA. Firstly, a Bicinchoninic acid assay (BCA) was performed to determine the total protein required for western blot analysis. To do so, a range of BSA (1 mg / mL) from 0 to 20 pg and triplicate samples were made for each production of AINPs-AAT diluted at 1 / 4, i.e., 5 pL of sample in 15 pL of IX PBS. Next, 180 pL of BCA was added, and the plate was left at 37°C for 40 min. Finally, the sample was read at 562 nm using a CLARIOstar spectrometer (BMG Labtech, Ortenberg, Germany).

[0123] Western blot. 10 pg of total protein was denatured at 95°C for 5 min in IX loading blue and P-mercaptoethanol and then blotted onto a 12% SDS-PAGE acrylamide gel (40%, 29: 1, MFCD00080848, Sigma). Migration was performed at 150V for approximately Ih. The proteins in the gel were then transferred to a nitrocellulose membrane (A30779694, Amersham, Cytiva) at 50 mA for 1 h 30 min in ethanol-based transfer buffer. Once the transfer was complete, the membrane was placed in Ponceau red, and a photo of the membrane was taken using Amersham (Image Quant 800; Cytiva). The Ponceau red was then removed by successive washes of 0.1% Tween IX PBS, and the membrane was blocked for Ih in 0.1% Tween IX PBS, 5% milk. Once blocked, it was incubated with an anti-AAT antibody (1 / 5000, 5265- MSM2-P0, Thermo Fisher, Waltham, USA) prepared in 1% milk, 0.1% Tween IX PBS, O / N. The following day, the membrane was washed 4 times in 0.1% Tween IX PBS for 5 min and then incubated with a Goat Anti-Mouse-HRP antibody (1 / 1000) in 1% milk 0.1% Tween IX PBS for Ih. Once the incubation was complete, a series of 4 washes of 5 min in 0.1% Tween IX PBS was carried out, followed by revelation with ECL (#7003, Cell Signalling, Danvers, Massachusetts, USA) using Amersham. Quantification was performed using ImageJ.

[0124] Morphological determination

[0125] Dynamic Light Scattering. The size of AAT-enriched nanoparticles was determined using dynamic light scattering (DLS) as described by Rosanaly et al1.

[0126] Anti-elastase activity

[0127] The anti-elastase activity of AINPs and AINP-AATs was assessed using a chromogenic elastase substrate. 50 pL of human elastase (100 nM; Elastin Products Company, Inc., Owensville, Missouri, USA 65066) prepared in sodium acetate (50 mM) and NaCl (200 mM, [pH 5.5]) was incubated with a chromogenic substrate, MeOSuc-AAPV-AMC (100 pg / mL, Calbiochem, Darmstadt, Germany) in 25 mM Tris-HCl [pH 7.5], 125 pL final volume. AAT alone (O.Ol-lOOpg, Respreeza), AINPs and AINPs-AAT (50,100pg) were brought into contact with elastase, and 50 pL of substrate was added. Substrate hydrolysis was monitored over 50 30-second cycles at 37°C at 460 nm using the CLARIOstar Plus spectrometer. Using the fluorescence intensity obtained over 50 cycles, we calculate the "slope" via the spectrometer application. The slope represents the enzymatic reaction rate. Values were processed on Prism GraphPad to express our results as a percentage.

[0128] Cell culture

[0129] Adenocarcinoma human alveolar basal epithelial cells (A549, ATCC# CCL-185) were maintained in RPMI 1640 medium (P04-22100, Pan-Biotech, Germany) supplemented with 1% Penicillin-streptomycin (P06-07100, Pan-Biotech), 1% fungizone (P06-01100, Pan-Biotech), and 10% foetal bovine serum (FBS; P30-3401, Pan-Biotech) at 37°C in an atmosphere with 5% CO2. Human Microvascular Endothelial Cell-1 (HMEC-1, ATCC# CRL-3243) cells were cultured in MCDB 131 (P04-80057; Pan-Biotech) supplemented with 1% penicillin-streptomycin, 10 mM L-glutamine (MS023E100E, Biosera, Cholet, France), 1% fungizone, 10 ng / mL Human Epidermal Growth Factor (hEGF; E9644, Sigma-Aldrich, USA), 1 pg / mL hydrocortisone (H0135-1MG, Sigma), and 10% FBS (Pan-Biotech). They were maintained at 37°C in a 5% CO2 atmosphere.

[0130] Viability and cytotoxicity

[0131] Mitochondrial metabolic activity was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide (MTT) assay. HMEC-1 cells were seeded at 1.104cells / well for 24 hours. AINP-AATs were washed by centrifugation for 10 min at 14,000g at 4°C to replace the TEN buffer storage solution by sterile IX PBS. The medium was then removed and replaced with 100 pL of A1NP-AAT (0.5 mg / mL to 0.015 mg / mL) and / or medium alone 10% FBS, 0% FBS and 5% delipidated FBS (S00PH2000M, Biosera) (the delipidated FBS is to increase the avidity of HMECs to AINPs-AAT) as a control condition for 24h. One hour and 45 minutes before the end of the experiment, LDH lysis buffer was added to one column as a positive control for LDH release. 45 min after the medium was recovered for an LDH assay and 10 pL of MTT (M2128-5G; Sigma-Aldrich, USA) at 5 mg / mL diluted in sterile IX PBS was filtered and then added to the new 100 pL of each well for a final MTT concentration of 0.5 mg / mL for 1 hour. At the end, the plate was then centrifuged at 600 g for 4 min at 25°C. Then we added 200 pL of Dimethyl sulphoxide (DMSO, UD8050-B; Euromedex, France) to each well, which will allow the dissolution of the formazan crystals from the MTT present in the metabolism of the mitochondria of metabolically living cells. Absorbance was measured at 560 nm on a CLARIOstar spectrometer.

[0132] LDH release. 50 pL of each sample was placed in a 96-well plate. The LDH range was between 10 U / mL and 0.08 U / mL. Next, 50 pL of Tris buffer 10X (Sigma Aldrich, Darmstadt, Germany), 50 pL of Li -Lactate 50 nM (440469; Sigma Aldrich, Darmstadt, Germany), and 50 pL of mix reagent 4X were added to the range and the samples. The mix reagent was composed of lodonitrotetrazolium chloride (INT) at 33 mg / mL (18377; Sigma Aldrich), Phenazine methosulfate (PMS) at 9 pg / mL (P9625; Sigma Aldrich), 3 -Nicotinamide adenine dinucleotide sodium salt (NAD) at 3.7 mg / mL (N0632; Sigma Aldrich). The plate was stirred gently for 30 seconds, and then we waited 5 minutes before taking the reading at 490 nm on the CLARIOstar Plus spectrometer. Uptake

[0133] Cell culture and test. Day 1 : 15,000 cells per well were seeded on a Labtech (PEZGS0816, Merck KGaA, Darmstadt, Germany). On day 2, AINPs, AINP-AATs, and AATs were washed by centrifugation for 10 min at 14,000g at 4°C to replace the TEN buffer storage solution by sterile IX PBS. They were then resuspended in 5% delipidated FBS medium and HC. Meanwhile, the cells were serum deprived with MCDB131, 0% FBS, and HC medium for 3h. After this time, the cells were stimulated with 300pL of 5% delipidated FBS medium and HC for the control condition and with 300pL of AINPs (0.05 mg / mL), AINPs-AAT (0.05 mg / mL), or AAT (1.8 pg / mL) for 6h. Then the supernatant was removed, and the cells were rinsed in IX PBS and then fixed in 4% PFA for 15 min. They were then washed with IX PBS and processed.

[0134] Slide processing. To carry out antibody labelling, slides containing cells were treated as follows: 2 washes of 10 minutes in PBS IX Triton 0.1%, followed by a one-hour block in PBS IX Triton 0.1% BSA 2%. The slides were then incubated O / N with a primary antibody to ApoAl (Rabbit, 1 / 500; Calbiochem) and an antibody to AAT (Mouse, 1 / 5 000) at 4°C in a humidity chamber. The following day, 2 washes of 10 min with 0.1% Triton IX PBS were performed, and the slides were incubated for 1 hour with Donkey anti-rabbit Fluor Alexa 488 (DAR, 1 / 1 000, Invitrogen, USA), Donkey anti-mouse Fluor Alexa 594 (DAM, 1 / 1 000, Invitrogen) and DAPI (1 / 500). The slides were washed with PBS IX Triton 0.1%, 4 times for 10 min. Once the washes were complete, 3 drops of mounting medium (Ibidi, 50001, Grafelfing, Germany) were added, and the slides were sealed with a coverslip (Corning, 2980- 246, USA) and varnish. The slides were left to dry for 24 hours before being used for confocal microscopy (Nikon Eclipse Ti2 fluorescent microscope; Nikon Instruments Inc., Netherlands).

[0135] Anti-inflammatory test

[0136] We seeded HMEC-1 cells in a 24-well plate at 70,000 cells per well. 24 hours later, AINPs, AINP-AATs, and AATs were washed by centrifugation for 10 min at 14,000g at 4°C to replace the TEN buffer storage solution with sterile IX PBS. Then we deprived the cells of FBS and placed them in MCDB131 medium, 0% FBS, IX hydrocortisone, for 3 hours. This was followed by stimulation with MCDB 131, 5% FBS delipidated for the control condition, AINPs (0.05 mg / mL), AINPs-AAT (0.05 mg / mL), and AAT (1.8 pg / mL) + / - TNF-a (2.5 ng / mL) for 16 hours. The supernatants were recovered for an IL-6 ELISA. Animal experiments

[0137] Intratracheal aerosolization was performed on sixteen-week-old female C57BL / 6 mice (APAFIS #53302-2025011413372952 v4). Briefly, the mice were anesthetized in an induction chamber with isoflurane (2.5%). Once asleep, they received an intraperitoneal dose of Ketamine (90 mg / kg) / Xylazine (4.5mg / mL) mixture. For the treatment, animals were suspended on the plexiglass backboard in the supine position by their incisors. They received an aerosolization of 50 pL using a Micro Sprayer Aerosolizer (Yan30012; Shanghai Yuyan Instruments Co., China) of AINPs (1 mg ApoAl; n=5) or AINPs-AAT (1 mg ApoAl, 30 pg AAT; n=5) between vocal cords. The mice were returned to their cage, on a heating mat, until they woke up. The plasma was collected at 0 hours, 6 hours, 12 hours, and 24 hours. At 24 hours, mice were sacrificed, and organs were collected after 4% paraformaldehyde (PF A) injection in the left ventricle. The right superior lobe of lungs was snap frozen in liquid nitrogen for protein extraction. After the dissection, the lung, brain, heart, liver, kidney, and spleen were collected and placed into 4% PFA solution for 24 hours. The organs were washed with IX PBS, then treated with PBS 30% sucrose, and rinsed again in IX PBS before their inclusion in OCT (CellPath, United Kingdom).

[0138] ELISA

[0139] IL-6. Carried out according to the kit protocol (eBioscience, San Diego, CA 92121, USA).

[0140] ApoAl. Carried out according to the kit protocol (3710-1HP-1, Mabtech, Stockholm, Sweden). Sample dilution (mouse plasma): 1 / 1000.

[0141] Statistical analysis

[0142] All the results are expressed as + / - SEM from 1 to 4 independent experiments. Statistical tests were performed on GraphPad Prism 6 software (Prism software, GraphPad, USA). Appropriate statistical tests were used according to the sample distribution (normal test), the number of conditions compared, and whether the samples were paired or unpaired. Statistically significant differences were considered for a p-value < 0.05.

[0143] Results:

[0144] Purification of AINPs-AAT

[0145] Once the AINPs-AAT nanoparticles were produced, we ran our production through an FPLC (Fast Protein Liquid Chromatography) to separate the free AAT from the ApoAl nanoparticles enriched in AAT. To guarantee a degree of reproducibility in production, we produced 3 independent batches. As described in Figure 9, which shows the amount of eluted protein recovered as a function of time, there is no difference between each production run, and the profiles of each production run overlap.

[0146] AAT quantification in AINPs

[0147] To determine the quantity of AAT present in our ApoAl nanoparticles, we carried out Western blots to separate the AAT from the ApoAl in the nanoparticles. Using a ponceau red staining and antibody labelling method, we were able to calculate the quantity of AAT in pg per mg of ApoAl (Figure 10). On average, AAT enrichment was 47 pg of AAT per 1 mg of ApoAl with a 1 :0.031 enrichment ratio (Table 1).

[0148] Size of AINPs-AAT

[0149] Then, we wanted to determine whether these enriched nanoparticles retained their size, which is supposed to be identical to that of nascent HDL, i.e., between 7 and 12 nm, which was confirmed by DLS (Figure 11A,B).

[0150] Anti-elastase activity

[0151] Once the morphology and purity had been validated, we moved on to the biological tests. We used anti -elastase activity as our first test to validate the functionality of AAT after enrichment. As we can see from this graph, we have 100% elastase activity in the control condition. This activity progressively decreases when increasing AAT quantity. With AINPs-AAT, the elastase activity was significantly reduced compared with AINPs alone and the control. There was also no difference in activity between AINPs-AAT at 50 and 100 pg and AAT alone at 50 and 100 pg (Figure 12).

[0152] Effect and uptake of AINPs-AAT on endothelial cells

[0153] Cytotoxicity. To assess the cytotoxicity of our nanoparticles, we performed MTT tests. To do so, we stimulated our HMEC-1 cells with concentrations ranging from 1 mg / mL to 0.015 mg / mL. We can see (Figure 13A) that there was no significant difference between the concentrations tested and the control (unstimulated). To support the MTT results, we performed an LDH assay on cell supernatants from the MTT. We can see a significantly lower percentage of cytotoxicity with the different concentrations of AINPs-AAT compared to CTRL+, 100% cell death (Figure 13B). Anti-inflammatory. To determine whether our nanoparticles are anti-inflammatory, we stimulated HMEC-1 with AINPs-AAT at different concentrations (0.016-0.1 mg / mL) + / - TNF- a (2.5 ng / mL). As shown in Figure 14, IL-6 secretion appears to be decreased when endothelial cells are stimulated with our enriched nanoparticles compared with the TNF-a control. We can also note that AINPs-AAT would be able to reduce the basal inflammatory state of the cells.

[0154] Uptake. To determine whether endothelial cells take up our nanoparticles, we stimulated HMEC-1 with AINPs, AINPs-AAT, or AAT alone. As shown in Figure 15, the control showed no ApoAl or AAT labelling. Under AINPs and AINPs-AAT conditions, we can observe ApoAl and AAT labelling. In the stimulated condition with AAT alone, we can only see AAT labelling.

[0155] AINPs and AINPs-AAT kinetics in mice

[0156] After assessing the effects of nanoparticles in vitro, we decided to evaluate their biodistribution in wild-type mice (C57BL / 6) after aerosolization, with a particular focus on the kinetics of blood passage of + / - enriched nanoparticles. Results show a significant decrease in the concentration of AINPs-AAT passing into the bloodstream of mice compared with mice administered AINPs alone (Figure 16).

[0157] Biodistribution and quantification of AINPs-AAT and AINPs

[0158] Preliminary observations suggest that AINPs-AAT quantity in lung tissue is higher compared to AINPs. Regarding AAT detection in lungs, a western blot analysis on right superior lobe of mice exposed to AINPs-AAT confirmed the presence of AAT in these mice unlike mice exposed to AINPs (Figure 17).

[0159] TABLES:

[0160] Mean 1 2 7.(17 382. S 47 4 1 :0.03'’ Table 1: AAT enrichment in AINPs. 3 sets of AINPs-AAT production were used. The initial and final ratio corresponds to mokmol. Quantification was performed using ImageJ.

[0161] REFERENCES:

[0162] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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Claims

CLAIMS:

1. A method of treating pulmonary emphysema in a patient suffering from alpha- 1 antitrypsin deficiency comprising administering to the patient’s lungs a therapeutically effective amount of apolipoprotein Al nanoparticles enriched with alpha- 1 -antitrypsin by aerosolization.

2. The method according to claim 1 for reducing the number of exacerbations.

3. The method according to claim 1 for improving the lung function, quality of life, and survival of patients with alpha- 1 antitrypsin deficiency.

4. The method according to any one of claims 1 to 3 wherein the apolipoprotein Al nanoparticles are enriched with a AAT polypeptide having at least 90% of identity with amino acid sequence as set forth in SEQ ID NO: 1.

5. The method according to any one of claims 1 to 3 wherein the apolipoprotein Al nanoparticles are enriched with a AAT polypeptide having the amino acid sequence as set forth in SEQ ID NO: 1 that comprises one or more conservative mutations, preferably one or more conservatives substitutions.

6. The method according to any one of claims 1 to 3 wherein the apolipoprotein Al nanoparticles are enriched with a AAT polypeptide having the amino acid sequence as set forth in SEQ ID NO: 1.

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